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

Data on the construction of a recombinant HEK293 cell line overexpressing hERG potassium channel and examining the presence of hERG mRNA and protein expression

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Yi Fan Teah

2017-10-01

Full Text Available The data presented in this article are related to the research article entitled “The effects of deoxyelephantopin on the cardiac delayed rectifier potassium channel current (IKr and human ether-a-go-go-related gene (hERG expression” (Y.F. Teah, M.A. Abduraman, A. Amanah, M.I. Adenan, S.F. Sulaiman, M.L. Tan [1], which the possible hERG blocking properties of deoxyelephantopin were investigated. This article describes the construction of human embryonic kidney 293 (HEK293 cells overexpressing HERG potassium channel and verification of the presence of hERG mRNA and protein expression in this recombinant cell line.

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.

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

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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...

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

Dynamics of hERG closure allow novel insights into hERG blocking by small molecules.

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Schmidtke, Peter; Ciantar, Marine; Theret, Isabelle; Ducrot, Pierre

2014-08-25

Today, drug discovery routinely uses experimental assays to determine very early if a lead compound can yield certain types of off-target activity. Among such off targets is hERG. The ion channel plays a primordial role in membrane repolarization and altering its activity can cause severe heart arrhythmia and sudden death. Despite routine tests for hERG activity, rather little information is available for helping medicinal chemists and molecular modelers to rationally circumvent hERG activity. In this article novel insights into the dynamics of hERG channel closure are described. Notably, helical pairwise closure movements have been observed. Implications and relations to hERG inactivation are presented. Based on these dynamics novel insights on hERG blocker placement are presented, compared to literature, and discussed. Last, new evidence for horizontal ligand positioning is shown in light of former studies on hERG blockers.

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.

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.

In Silico Predictions of hERG Channel Blockers in Drug Discovery

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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 ...

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.

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

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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.

Effects of Tannic Acid, Green Tea and Red Wine on hERG Channels Expressed in HEK293 Cells.

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Xi Chu

Full Text Available Tannic acid presents in varying concentrations in plant foods, and in relatively high concentrations in green teas and red wines. Human ether-à-go-go-related gene (hERG channels expressed in multiple tissues (e.g. heart, neurons, smooth muscle and cancer cells, and play important roles in modulating cardiac action potential repolarization and tumor cell biology. The present study investigated the effects of tannic acid, green teas and red wines on hERG currents. The effects of tannic acid, teas and red wines on hERG currents stably transfected in HEK293 cells were studied with a perforated patch clamp technique. In this study, we demonstrated that tannic acid inhibited hERG currents with an IC50 of 3.4 μM and ~100% inhibition at higher concentrations, and significantly shifted the voltage dependent activation to more positive potentials (Δ23.2 mV. Remarkably, a 100-fold dilution of multiple types of tea (green tea, oolong tea and black tea or red wine inhibited hERG currents by ~90%, and significantly shifted the voltage dependent activation to more positive potentials (Δ30.8 mV and Δ26.0 mV, respectively. Green tea Lung Ching and red wine inhibited hERG currents, with IC50 of 0.04% and 0.19%, respectively. The effects of tannic acid, teas and red wine on hERG currents were irreversible. These results suggest tannic acid is a novel hERG channel blocker and consequently provide a new mechanistic evidence for understanding the effects of tannic acid. They also revealed the potential pharmacological basis of tea- and red wine-induced biology activities.

Effects of Tannic Acid, Green Tea and Red Wine on hERG Channels Expressed in HEK293 Cells

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Xu, Bingyuan; Li, Wenya; Lin, Yue; Sun, Xiaorun; Ding, Chunhua; Zhang, Xuan

2015-01-01

Tannic acid presents in varying concentrations in plant foods, and in relatively high concentrations in green teas and red wines. Human ether-à-go-go-related gene (hERG) channels expressed in multiple tissues (e.g. heart, neurons, smooth muscle and cancer cells), and play important roles in modulating cardiac action potential repolarization and tumor cell biology. The present study investigated the effects of tannic acid, green teas and red wines on hERG currents. The effects of tannic acid, teas and red wines on hERG currents stably transfected in HEK293 cells were studied with a perforated patch clamp technique. In this study, we demonstrated that tannic acid inhibited hERG currents with an IC50 of 3.4 μM and ~100% inhibition at higher concentrations, and significantly shifted the voltage dependent activation to more positive potentials (Δ23.2 mV). Remarkably, a 100-fold dilution of multiple types of tea (green tea, oolong tea and black tea) or red wine inhibited hERG currents by ~90%, and significantly shifted the voltage dependent activation to more positive potentials (Δ30.8 mV and Δ26.0 mV, respectively). Green tea Lung Ching and red wine inhibited hERG currents, with IC50 of 0.04% and 0.19%, respectively. The effects of tannic acid, teas and red wine on hERG currents were irreversible. These results suggest tannic acid is a novel hERG channel blocker and consequently provide a new mechanistic evidence for understanding the effects of tannic acid. They also revealed the potential pharmacological basis of tea- and red wine-induced biology activities. PMID:26625122

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

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.

hERG trafficking inhibition in drug-induced lethal cardiac arrhythmia.

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Nogawa, Hisashi; Kawai, Tomoyuki

2014-10-15

Acquired long QT syndrome induced by non-cardiovascular drugs can cause lethal cardiac arrhythmia called torsades de points and is a significant problem in drug development. The prolongation of QT interval and cardiac action potential duration are mainly due to reduced physiological function of the rapidly activating voltage-dependent potassium channels encoded by human ether-a-go-go-related gene (hERG). Structurally diverse groups of drugs are known to directly inhibit hERG channel conductance. Therefore, the ability of acute hERG inhibition is routinely assessed at the preclinical stages in pharmaceutical testing. Recent findings indicated that chronic treatment with various drugs not only inhibits hERG channels but also decreases hERG channel expression in the plasma membrane of cardiomyocytes, which has become another concern in safety pharmacology. The mechanisms involve the disruption of hERG trafficking to the surface membrane or the acceleration of hERG protein degradation. From this perspective, we present a brief overview of mechanisms of drug-induced trafficking inhibition and pathological regulation. Understanding of drug-induced hERG trafficking inhibition may provide new strategies for predicting drug-induced QT prolongation and lethal cardiac arrhythmia in pharmaceutical drug development. Copyright © 2014 Elsevier B.V. All rights reserved.

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

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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...

The effects of deoxyelephantopin on the cardiac delayed rectifier potassium channel current (IKr) and human ether-a-go-go-related gene (hERG) expression.

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Teah, Yi Fan; Abduraman, Muhammad Asyraf; Amanah, Azimah; Adenan, Mohd Ilham; Sulaiman, Shaida Fariza; Tan, Mei Lan

2017-09-01

Elephantopus scaber Linn and its major bioactive component, deoxyelephantopin are known for their medicinal properties and are often reported to have various cytotoxic and antitumor activities. This plant is widely used as folk medicine for a plethora of indications although its safety profile remains unknown. 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. The hERG potassium channel is an important antitarget in cardiotoxicity evaluation. This study investigated the effects of deoxyelephantopin on the current, mRNA and protein expression of hERG channel in hERG-transfected HEK293 cells. The hERG tail currents following depolarization pulses were insignificantly affected by deoxyelephantopin in the transfected cell line. Current reduction was less than 40% as compared with baseline at the highest concentration of 50 μM. The results were consistent with the molecular docking simulation and hERG surface protein expression. Interestingly, it does not affect the hERG expression at both transcriptional and translational level at most concentrations, although higher concentration at 10 μM caused protein accumulation. In conclusion, deoxyelephantopin is unlikely a clinically significant hERG channel and I kr blocker. Copyright © 2017 Elsevier Ltd. All rights reserved.

Improved functional expression of recombinant human ether-a-go-go (hERG K+ channels by cultivation at reduced temperature

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Hamilton Bruce

2007-12-01

Full Text Available Abstract Background HERG potassium channel blockade is the major cause for drug-induced long QT syndrome, which sometimes cause cardiac disrhythmias and sudden death. There is a strong interest in the pharmaceutical industry to develop high quality medium to high-throughput assays for detecting compounds with potential cardiac liability at the earliest stages of drug development. Cultivation of cells at lower temperature has been used to improve the folding and membrane localization of trafficking defective hERG mutant proteins. The objective of this study was to investigate the effect of lower temperature maintenance on wild type hERG expression and assay performance. Results Wild type hERG was stably expressed in CHO-K1 cells, with the majority of channel protein being located in the cytoplasm, but relatively little on the cell surface. Expression at both locations was increased several-fold by cultivation at lower growth temperatures. Intracellular hERG protein levels were highest at 27°C and this correlated with maximal 3H-dofetilide binding activity. In contrast, the expression of functionally active cell surface-associated hERG measured by patch clamp electrophysiology was optimal at 30°C. The majority of the cytoplasmic hERG protein was associated with the membranes of cytoplasmic vesicles, which markedly increased in quantity and size at lower temperatures or in the presence of the Ca2+-ATPase inhibitor, thapsigargin. Incubation with the endocytic trafficking blocker, nocodazole, led to an increase in hERG activity at 37°C, but not at 30°C. Conclusion Our results are consistent with the concept that maintenance of cells at reduced temperature can be used to boost the functional expression of difficult-to-express membrane proteins and improve the quality of assays for medium to high-throughput compound screening. In addition, these results shed some light on the trafficking of hERG protein under these growth conditions.

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.

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

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

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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.

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.

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.

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.

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

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

Science.gov (United States)

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.

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.

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.

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

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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.

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

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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.

Endocytosis of hERG Is Clathrin-Independent and Involves Arf6

Science.gov (United States)

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

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...

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

Science.gov (United States)

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.

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

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....

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.

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...

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...

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...

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...

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

Science.gov (United States)

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.

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.

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

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

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.

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.

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.

Science.gov (United States)

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.

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

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...

The human ether-a-go-go-related gene (hERG) current inhibition selectively prolongs action potential of midmyocardial cells to augment transmural dispersion.

Science.gov (United States)

Yasuda, C; Yasuda, S; Yamashita, H; Okada, J; Hisada, T; Sugiura, S

2015-08-01

The majority of drug induced arrhythmias are related to the prolongation of action potential duration following inhibition of rapidly activating delayed rectifier potassium current (I(Kr)) mediated by the hERG channel. However, for arrhythmias to develop and be sustained, not only the prolongation of action potential duration but also its transmural dispersion are required. Herein, we evaluated the effect of hERG inhibition on transmural dispersion of action potential duration using the action potential clamp technique that combined an in silico myocyte model with the actual I(Kr) measurement. Whole cell I(Kr) current was measured in Chinese hamster ovary cells stably expressing the hERG channel. The measured current was coupled with models of ventricular endocardial, M-, and epicardial cells to calculate the action potentials. Action potentials were evaluated under control condition and in the presence of 1, 10, or 100 μM disopyramide, an hERG inhibitor. Disopyramide dose-dependently increased the action potential durations of the three cell types. However, action potential duration of M-cells increased disproportionately at higher doses, and was significantly different from that of epicardial and endocardial cells (dispersion of repolarization). By contrast, the effects of disopyramide on peak I(Kr) and instantaneous current-voltage relation were similar in all cell types. Simulation study suggested that the reduced repolarization reserve of M-cell with smaller amount of slowly activating delayed rectifier potassium current levels off at longer action potential duration to make such differences. The action potential clamp technique is useful for studying the mechanism of arrhythmogenesis by hERG inhibition through the transmural dispersion of repolarization.

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....

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.

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...

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.

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.

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

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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.

Cloning, functional expression, and characterization of a PKA-activated gastric Cl- channel.

Science.gov (United States)

Malinowska, D H; Kupert, E Y; Bahinski, A; Sherry, A M; Cuppoletti, J

1995-01-01

cDNA encoding a Cl- channel was isolated from a rabbit gastric library, sequenced, and expressed in Xenopus oocytes. The predicted protein (898 amino acids, relative molecular mass 98,433 Da) was overall 93% similar to the rat brain ClC-2 Cl- channel. However, a 151-amino acid stretch toward the COOH-terminus was 74% similar to ClC-2 with six amino acids deleted. Two new potential protein kinase A (PKA) phosphorylation sites (also protein kinase C phosphorylation sites) were introduced. cRNA-injected Xenopus oocytes expressed a Cl- channel that was active at pHtrans 3 and had a linear current-voltage (I-V) curve and a slope conductance of 29 +/- 1 pS at 800 mM CsCl. A fivefold Cl- gradient caused a rightward shift in the I-V curve with a reversal potential of +30 +/- 3 mV, indicating anion selectivity. The selectivity was I- > Cl- > NO3-. The native and recombinant Cl- channel were both activated in vitro by PKA catalytic subunit and ATP. The electrophysiological and regulatory properties of the cloned and the native channel were similar. The cloned protein may be the Cl- channel involved in gastric HCl secretion.

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.

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

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

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

Molecular Cloning and Sequencing of Hemoglobin-Beta Gene of Channel Catfish, Ictalurus Punctatus Rafinesque

Science.gov (United States)

: Hemoglobin-y gene of channel catfish , lctalurus punctatus, was cloned and sequenced . Total RNA from head kidneys was isolated, reverse transcribed and amplified . The sequence of the channel catfish hemoglobin-y gene consists of 600 nucleotides . Analysis of the nucleotide sequence reveals one o...

Molecular cloning of a K+ channel from the malaria parasite Plasmodium falciparum

DEFF Research Database (Denmark)

Ellekvist, Peter; Ricke, Christina Høier; Litman, Thomas

2004-01-01

In most living cells, K(+) channels are important for the generation of the membrane potential and for volume regulation. The parasite Plasmodium falciparum, which causes malignant malaria, must be able to deal with large variations in the ambient K(+) concentration: it is exposed to high...... concentrations of K(+) when inside the erythrocyte and low concentrations when in plasma. In the recently published genome of P. falciparum, we have identified a gene, pfkch1, encoding a potential K(+) channel, which to some extent resembles the big-conductance (BK) K(+) channel. We have cloned the approximately...

Accidental cloning of a single-photon qubit in two-channel continuous-variable quantum teleportation

International Nuclear Information System (INIS)

Ide, Toshiki; Hofmann, Holger F.

2007-01-01

The information encoded in the polarization of a single photon can be transferred to a remote location by two-channel continuous-variable quantum teleportation. However, the finite entanglement used in the teleportation causes random changes in photon number. If more than one photon appears in the output, the continuous-variable teleportation accidentally produces clones of the original input photon. In this paper, we derive the polarization statistics of the N-photon output components and show that they can be decomposed into an optimal cloning term and completely unpolarized noise. We find that the accidental cloning of the input photon is nearly optimal at experimentally feasible squeezing levels, indicating that the loss of polarization information is partially compensated by the availability of clones

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}. �

Asymmetric quantum cloning machines

International Nuclear Information System (INIS)

Cerf, N.J.

1998-01-01

A family of asymmetric cloning machines for quantum bits and N-dimensional quantum states is introduced. These machines produce two approximate copies of a single quantum state that emerge from two distinct channels. In particular, an asymmetric Pauli cloning machine is defined that makes two imperfect copies of a quantum bit, while the overall input-to-output operation for each copy is a Pauli channel. A no-cloning inequality is derived, characterizing the impossibility of copying imposed by quantum mechanics. If p and p ' are the probabilities of the depolarizing channels associated with the two outputs, the domain in (√p,√p ' )-space located inside a particular ellipse representing close-to-perfect cloning is forbidden. This ellipse tends to a circle when copying an N-dimensional state with N→∞, which has a simple semi-classical interpretation. The symmetric Pauli cloning machines are then used to provide an upper bound on the quantum capacity of the Pauli channel of probabilities p x , p y and p z . The capacity is proven to be vanishing if (√p x , √p y , √p z ) lies outside an ellipsoid whose pole coincides with the depolarizing channel that underlies the universal cloning machine. Finally, the tradeoff between the quality of the two copies is shown to result from a complementarity akin to Heisenberg uncertainty principle. (author)

Cloning and functional expression of a plant voltage-dependent chloride channel.

Science.gov (United States)

Lurin, C; Geelen, D; Barbier-Brygoo, H; Guern, J; Maurel, C

1996-01-01

Plant cell membrane anion channels participate in basic physiological functions, such as cell volume regulation and signal transduction. However, nothing is known about their molecular structure. Using a polymerase chain reaction strategy, we have cloned a tobacco cDNA (CIC-Nt1) encoding a 780-amino acid protein with several putative transmembrane domains. CIC-Nt1 displays 24 to 32% amino acid identity with members of the animal voltage-dependent chloride channel (CIC) family, whose archetype is CIC-0 from the Torpedo marmorata electric organ. Injection of CIC-Nt1 complementary RNA into Xenopus oocytes elicited slowly activating inward currents upon membrane hyperpolarization more negative than -120 mV. These currents were carried mainly by anions, modulated by extracellular anions, and totally blocked by 10 mM extracellular calcium. The identification of CIC-Nt1 extends the CIC family to higher plants and provides a molecular probe for the study of voltage-dependent anion channels in plants. PMID:8624442

Neuronal fast activating and meningeal silent modulatory BK channel splice variants cloned from rat

DEFF Research Database (Denmark)

Poulsen, Asser Nyander; Jansen-Olesen, Inger; Olesen, Jes

2011-01-01

The big conductance calcium-activated K(+) channel (BK) is involved in regulating neuron and smooth muscle cell excitability. Functional diversity of BK is generated by alpha-subunit splice variation and co-expression with beta subunits. Here, we present six different splice combinations cloned...... and RCK2 (4 aa at SS1) and upstream of the calcium "bowl" (27 aa at SS4). Two other truncated variants, X2(92) and X2(188), lacking the intracellular C-terminal (stop downstream of S6), were cloned from cerebral vascular/meningeal tissue. They appear non-functional as no current expression was observed...

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.

Hypoosmotic cell swelling as a novel mechanism for modulation of cloned HCN2 channels

DEFF Research Database (Denmark)

Calloe, Kirstine; Elmedyb, Pernille; Olesen, Søren-Peter

2005-01-01

This work demonstrates cell swelling as a new regulatory mechanism for the cloned hyperpolarization-activated, cyclic nucleotide-gated channel 2 (HCN2). HCN2 channels were coexpressed with aquaporin1 in Xenopus laevis oocytes and currents were monitored using a two-electrode voltage-clamp. HCN2...... channels were activated by hyperpolarization to -100 mV and the currents were measured before and during hypoosmotic cell swelling. Cell swelling increased HCN2 currents by 30% without changing the kinetics of the currents. Injection of 50 nl intracellular solution resulted in a current increase of 20......%, indicating that an increase in cell volume also under isoosmotic conditions may lead to activation of HCN2. In the absence of aquaporin1 only negligible changes in oocyte cell volume occur during exposure to hypoosmotic media and no significant change in HCN2 channel activity was observed during perfusion...

Fluorescence-tracking of activation gating in human ERG channels reveals rapid S4 movement and slow pore opening.

Directory of Open Access Journals (Sweden)

Zeineb Es-Salah-Lamoureux

2010-05-01

Full Text Available hERG channels are physiologically important ion channels which mediate cardiac repolarization as a result of their unusual gating properties. These are very slow activation compared with other mammalian voltage-gated potassium channels, and extremely rapid inactivation. The mechanism of slow activation is not well understood and is investigated here using fluorescence as a direct measure of S4 movement and pore opening.Tetramethylrhodamine-5-maleimide (TMRM fluorescence at E519 has been used to track S4 voltage sensor movement, and channel opening and closing in hERG channels. Endogenous cysteines (C445 and C449 in the S1-S2 linker bound TMRM, which caused a 10 mV hyperpolarization of the V((1/2 of activation to -27.5+/-2.0 mV, and showed voltage-dependent fluorescence signals. Substitution of S1-S2 linker cysteines with valines allowed unobstructed recording of S3-S4 linker E519C and L520C emission signals. Depolarization of E519C channels caused rapid initial fluorescence quenching, fit with a double Boltzmann relationship, F-V(ON, with V((1/2 (,1 = -37.8+/-1.7 mV, and V((1/2 (,2 = 43.5+/-7.9 mV. The first phase, V((1/2 (,1, was approximately 20 mV negative to the conductance-voltage relationship measured from ionic tail currents (G-V((1/2 = -18.3+/-1.2 mV, and relatively unchanged in a non-inactivating E519C:S620T mutant (V((1/2 = -34.4+/-1.5 mV, suggesting the fast initial fluorescence quenching tracked S4 voltage sensor movement. The second phase of rapid quenching was absent in the S620T mutant. The E519C fluorescence upon repolarization (V((1/2 = -20.6+/-1.2, k = 11.4 mV and L520C quenching during depolarization (V((1/2 = -26.8+/-1.0, k = 13.3 mV matched the respective voltage dependencies of hERG ionic tails, and deactivation time constants from -40 to -110 mV, suggesting they detected pore-S4 rearrangements related to ionic current flow during pore opening and closing.THE DATA INDICATE: 1 that rapid environmental changes occur at the

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.

Molecular Basis of Cardiac Delayed Rectifier Potassium Channel Function and Pharmacology.

Science.gov (United States)

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.

Cell swelling activates cloned Ca(2+)-activated K(+) channels: a role for the F-actin cytoskeleton

DEFF Research Database (Denmark)

Jorgensen, Nanna K; Pedersen, Stine F; Rasmussen, Hanne B

2003-01-01

Cloned Ca(2+)-activated K(+) channels of intermediate (hIK) or small (rSK3) conductance were expressed in HEK 293 cells, and channel activity was monitored using whole-cell patch clamp. hIK and rSK3 currents already activated by intracellular calcium were further increased by 95% and 125......%, respectively, upon exposure of the cells to a 33% decrease in extracellular osmolarity. hIK and rSK3 currents were inhibited by 46% and 32%, respectively, by a 50% increase in extracellular osmolarity. Cell swelling and channel activation were not associated with detectable increases in [Ca(2+)](i), evidenced...... by population and single-cell measurements. In addition, inhibitors of IK and SK channels significantly reduced the rate of regulatory volume decrease (RVD) in cells expressing these channels. Cell swelling induced a decrease, and cell shrinkage an increase, in net cellular F-actin content. The swelling...

Information-theoretic limitations on approximate quantum cloning and broadcasting

Science.gov (United States)

Lemm, Marius; Wilde, Mark M.

2017-07-01

We prove quantitative limitations on any approximate simultaneous cloning or broadcasting of mixed states. The results are based on information-theoretic (entropic) considerations and generalize the well-known no-cloning and no-broadcasting theorems. We also observe and exploit the fact that the universal cloning machine on the symmetric subspace of n qudits and symmetrized partial trace channels are dual to each other. This duality manifests itself both in the algebraic sense of adjointness of quantum channels and in the operational sense that a universal cloning machine can be used as an approximate recovery channel for a symmetrized partial trace channel and vice versa. The duality extends to give control of the performance of generalized universal quantum cloning machines (UQCMs) on subspaces more general than the symmetric subspace. This gives a way to quantify the usefulness of a priori information in the context of cloning. For example, we can control the performance of an antisymmetric analog of the UQCM in recovering from the loss of n -k fermionic particles.

APETx4, a Novel Sea Anemone Toxin and a Modulator of the Cancer-Relevant Potassium Channel KV10.1

Directory of Open Access Journals (Sweden)

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

A Gateway MultiSite recombination cloning toolkit.

Directory of Open Access Journals (Sweden)

Lena K Petersen

Full Text Available The generation of DNA constructs is often a rate-limiting step in conducting biological experiments. Recombination cloning of single DNA fragments using the Gateway system provided an advance over traditional restriction enzyme cloning due to increases in efficiency and reliability. Here we introduce a series of entry clones and a destination vector for use in two, three, and four fragment Gateway MultiSite recombination cloning whose advantages include increased flexibility and versatility. In contrast to Gateway single-fragment cloning approaches where variations are typically incorporated into model system-specific destination vectors, our Gateway MultiSite cloning strategy incorporates variations in easily generated entry clones that are model system-independent. In particular, we present entry clones containing insertions of GAL4, QF, UAS, QUAS, eGFP, and mCherry, among others, and demonstrate their in vivo functionality in Drosophila by using them to generate expression clones including GAL4 and QF drivers for various trp ion channel family members, UAS and QUAS excitatory and inhibitory light-gated ion channels, and QUAS red and green fluorescent synaptic vesicle markers. We thus establish a starter toolkit of modular Gateway MultiSite entry clones potentially adaptable to any model system. An inventory of entry clones and destination vectors for Gateway MultiSite cloning has also been established (www.gatewaymultisite.org.

Prediction of Thorough QT study results using action potential simulations based on ion channel screens.

Science.gov (United States)

Mirams, Gary R; Davies, Mark R; Brough, Stephen J; Bridgland-Taylor, Matthew H; Cui, Yi; Gavaghan, David J; Abi-Gerges, Najah

2014-01-01

Detection of drug-induced pro-arrhythmic risk is a primary concern for pharmaceutical companies and regulators. Increased risk is linked to prolongation of the QT interval on the body surface ECG. Recent studies have shown that multiple ion channel interactions can be required to predict changes in ventricular repolarisation and therefore QT intervals. In this study we attempt to predict the result of the human clinical Thorough QT (TQT) study, using multiple ion channel screening which is available early in drug development. Ion current reduction was measured, in the presence of marketed drugs which have had a TQT study, for channels encoded by hERG, CaV1.2, NaV1.5, KCNQ1/MinK, and Kv4.3/KChIP2.2. The screen was performed on two platforms - IonWorks Quattro (all 5 channels, 34 compounds), and IonWorks Barracuda (hERG & CaV1.2, 26 compounds). Concentration-effect curves were fitted to the resulting data, and used to calculate a percentage reduction in each current at a given concentration. Action potential simulations were then performed using the ten Tusscher and Panfilov (2006), Grandi et al. (2010) and O'Hara et al. (2011) human ventricular action potential models, pacing at 1Hz and running to steady state, for a range of concentrations. We compared simulated action potential duration predictions with the QT prolongation observed in the TQT studies. At the estimated concentrations, simulations tended to underestimate any observed QT prolongation. When considering a wider range of concentrations, and conventional patch clamp rather than screening data for hERG, prolongation of ≥5ms was predicted with up to 79% sensitivity and 100% specificity. This study provides a proof-of-principle for the prediction of human TQT study results using data available early in drug development. We highlight a number of areas that need refinement to improve the method's predictive power, but the results suggest that such approaches will provide a useful tool in cardiac safety

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

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.

Synthesis and biological evaluation of pyrrolidine derivatives as novel and potent sodium channel blockers for the treatment of ischemic stroke.

Science.gov (United States)

Seki, Maki; Tsuruta, Osamu; Tatsumi, Ryo; Soejima, Aki

2013-07-15

A novel series of pyrrolidine derivatives as Na(+) channel blockers was synthesized and evaluated for their inhibitory effects on neuronal Na(+) channels. Structure-activity relationship (SAR) studies of a pyrrolidine analogue 2 led to the discovery of 5e as a potent Na(+) channel blocker with a low inhibitory action against human ether-a-go-go-related gene (hERG) channels. Compound 5e showed remarkably neuroprotective activity in a rat transient middle cerebral artery occlusion (MCAO) model, suggesting that 5e would act as a neuroprotectant for ischemic stroke. Copyright © 2013 Elsevier Ltd. All rights reserved.

Characterization of potassium channel modulators with QPatch automated patch-clamp technology: system characteristics and performance

DEFF Research Database (Denmark)

Kutchinsky, Jonatan; Friis, Søren; Asmild, Margit

2003-01-01

Planar silicon chips with 1-2-microm etched holes (average resistance: 2.04 +/- 0.02 MOmega in physiological buffer, n = 274) have been developed for patch-clamp recordings of whole-cell currents from cells in suspension. An automated 16-channel parallel screening system, QPatch 16, has been deve......-response relationship characterizations of verapamil and rBeKm-1 blockage of hERG currents provided IC(50) values similar to values reported in the literature....

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...

Micro- and nanofabrication methods for ion channel reconstitution in bilayer lipid membranes

Science.gov (United States)

Tadaki, Daisuke; Yamaura, Daichi; Arata, Kohei; Ohori, Takeshi; Ma, Teng; Yamamoto, Hideaki; Niwano, Michio; Hirano-Iwata, Ayumi

2018-03-01

The self-assembled bilayer lipid membrane (BLM) forms the basic structure of the cell membrane and serves as a major barrier against ion movement. Ion channel proteins function as gated pores that permit ion permeation across the BLM. The reconstitution of ion channel proteins in artificially formed BLMs represents a well-defined system for investigating channel functions and screening drug effects on ion channels. In this review, we will discuss our recent microfabrication approaches to the formation of stable BLMs containing ion channel proteins as a potential platform for next-generation drug screening systems. BLMs formed in a microaperture having a tapered edge exhibited highly stable properties, such as a lifetime of ∼65 h and tolerance to solution changes even after the incorporation of the human ether-a-go-go-related gene (hERG) channel. We also explore a new method of efficiently incorporating human ion channels into BLMs by centrifugation. Our approaches to the formation of stable BLMs and efficient channel incorporation markedly improve the experimental efficiency of BLM reconstitution systems, leading to the realization of a BLM-based high-throughput platform for functional assays of various ion channels.

Patients with Long QT Syndrome Due to Impaired hERG-encoded Kv11.1 Potassium Channel Have Exaggerated Endocrine Pancreatic and Incretin Function Associated with Reactive Hypoglycemia

DEFF Research Database (Denmark)

Hyltén-Cavallius, Louise; Iepsen, Eva W; Wewer Albrechtsen, Nicolai J

2017-01-01

Background -Loss-of-function mutations in hERG (encoding the Kv11.1 voltage-gated potassium channel) cause long QT syndrome (LQT2) due to prolonged cardiac repolarization. However, Kv11.1 is also present in pancreatic α and β cells and intestinal L and K cells, secreting glucagon, insulin, and th...

Trade-off capacities of the quantum Hadamard channels

International Nuclear Information System (INIS)

Bradler, Kamil; Hayden, Patrick; Touchette, Dave; Wilde, Mark M.

2010-01-01

Coding theorems in quantum Shannon theory express the ultimate rates at which a sender can transmit information over a noisy quantum channel. More often than not, the known formulas expressing these transmission rates are intractable, requiring an optimization over an infinite number of uses of the channel. Researchers have rarely found quantum channels with a tractable classical or quantum capacity, but when such a finding occurs, it demonstrates a complete understanding of that channel's capabilities for transmitting classical or quantum information. Here we show that the three-dimensional capacity region for entanglement-assisted transmission of classical and quantum information is tractable for the Hadamard class of channels. Examples of Hadamard channels include generalized dephasing channels, cloning channels, and the Unruh channel. The generalized dephasing channels and the cloning channels are natural processes that occur in quantum systems through the loss of quantum coherence or stimulated emission, respectively. The Unruh channel is a noisy process that occurs in relativistic quantum information theory as a result of the Unruh effect and bears a strong relationship to the cloning channels. We give exact formulas for the entanglement-assisted classical and quantum communication capacity regions of these channels. The coding strategy for each of these examples is superior to a naieve time-sharing strategy, and we introduce a measure to determine this improvement.

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...

Molecular cloning and analysis of zebrafish voltage-gated sodium channel beta subunit genes: implications for the evolution of electrical signaling in vertebrates

Directory of Open Access Journals (Sweden)

Zhong Tao P

2007-07-01

Full Text Available Abstract Background Action potential generation in excitable cells such as myocytes and neurons critically depends on voltage-gated sodium channels. In mammals, sodium channels exist as macromolecular complexes that include a pore-forming alpha subunit and 1 or more modulatory beta subunits. Although alpha subunit genes have been cloned from diverse metazoans including flies, jellyfish, and humans, beta subunits have not previously been identified in any non-mammalian species. To gain further insight into the evolution of electrical signaling in vertebrates, we investigated beta subunit genes in the teleost Danio rerio (zebrafish. Results We identified and cloned single zebrafish gene homologs for beta1-beta3 (zbeta1-zbeta3 and duplicate genes for beta4 (zbeta4.1, zbeta4.2. Sodium channel beta subunit loci are similarly organized in fish and mammalian genomes. Unlike their mammalian counterparts, zbeta1 and zbeta2 subunit genes display extensive alternative splicing. Zebrafish beta subunit genes and their splice variants are differentially-expressed in excitable tissues, indicating tissue-specific regulation of zbeta1-4 expression and splicing. Co-expression of the genes encoding zbeta1 and the zebrafish sodium channel alpha subunit Nav1.5 in Chinese Hamster Ovary cells increased sodium current and altered channel gating, demonstrating functional interactions between zebrafish alpha and beta subunits. Analysis of the synteny and phylogeny of mammalian, teleost, amphibian, and avian beta subunit and related genes indicated that all extant vertebrate beta subunits are orthologous, that beta2/beta4 and beta1/beta3 share common ancestry, and that beta subunits are closely related to other proteins sharing the V-type immunoglobulin domain structure. Vertebrate sodium channel beta subunit genes were not identified in the genomes of invertebrate chordates and are unrelated to known subunits of the para sodium channel in Drosophila. Conclusion The

Digging into Lipid Membrane Permeation for Cardiac Ion Channel Blocker d-Sotalol with All-Atom Simulations.

Science.gov (United States)

DeMarco, Kevin R; Bekker, Slava; Clancy, Colleen E; Noskov, Sergei Y; Vorobyov, Igor

2018-01-01

Interactions of drug molecules with lipid membranes play crucial role in their accessibility of cellular targets and can be an important predictor of their therapeutic and safety profiles. Very little is known about spatial localization of various drugs in the lipid bilayers, their active form (ionization state) or translocation rates and therefore potency to bind to different sites in membrane proteins. All-atom molecular simulations may help to map drug partitioning kinetics and thermodynamics, thus providing in-depth assessment of drug lipophilicity. As a proof of principle, we evaluated extensively lipid membrane partitioning of d-sotalol, well-known blocker of a cardiac potassium channel K v 11.1 encoded by the hERG gene, with reported substantial proclivity for arrhythmogenesis. We developed the positively charged (cationic) and neutral d-sotalol models, compatible with the biomolecular CHARMM force field, and subjected them to all-atom molecular dynamics (MD) simulations of drug partitioning through hydrated lipid membranes, aiming to elucidate thermodynamics and kinetics of their translocation and thus putative propensities for hydrophobic and aqueous hERG access. We found that only a neutral form of d-sotalol accumulates in the membrane interior and can move across the bilayer within millisecond time scale, and can be relevant to a lipophilic channel access. The computed water-membrane partitioning coefficient for this form is in good agreement with experiment. There is a large energetic barrier for a cationic form of the drug, dominant in water, to cross the membrane, resulting in slow membrane translocation kinetics. However, this form of the drug can be important for an aqueous access pathway through the intracellular gate of hERG. This route will likely occur after a neutral form of a drug crosses the membrane and subsequently re-protonates. Our study serves to demonstrate a first step toward a framework for multi-scale in silico safety pharmacology

Digging into Lipid Membrane Permeation for Cardiac Ion Channel Blocker d-Sotalol with All-Atom Simulations

Directory of Open Access Journals (Sweden)

Kevin R. DeMarco

2018-02-01

Full Text Available Interactions of drug molecules with lipid membranes play crucial role in their accessibility of cellular targets and can be an important predictor of their therapeutic and safety profiles. Very little is known about spatial localization of various drugs in the lipid bilayers, their active form (ionization state or translocation rates and therefore potency to bind to different sites in membrane proteins. All-atom molecular simulations may help to map drug partitioning kinetics and thermodynamics, thus providing in-depth assessment of drug lipophilicity. As a proof of principle, we evaluated extensively lipid membrane partitioning of d-sotalol, well-known blocker of a cardiac potassium channel Kv11.1 encoded by the hERG gene, with reported substantial proclivity for arrhythmogenesis. We developed the positively charged (cationic and neutral d-sotalol models, compatible with the biomolecular CHARMM force field, and subjected them to all-atom molecular dynamics (MD simulations of drug partitioning through hydrated lipid membranes, aiming to elucidate thermodynamics and kinetics of their translocation and thus putative propensities for hydrophobic and aqueous hERG access. We found that only a neutral form of d-sotalol accumulates in the membrane interior and can move across the bilayer within millisecond time scale, and can be relevant to a lipophilic channel access. The computed water-membrane partitioning coefficient for this form is in good agreement with experiment. There is a large energetic barrier for a cationic form of the drug, dominant in water, to cross the membrane, resulting in slow membrane translocation kinetics. However, this form of the drug can be important for an aqueous access pathway through the intracellular gate of hERG. This route will likely occur after a neutral form of a drug crosses the membrane and subsequently re-protonates. Our study serves to demonstrate a first step toward a framework for multi-scale in silico safety

Activation of human ether-a-go-go-related gene potassium channels by the diphenylurea 1,3-bis-(2-hydroxy-5-trifluoromethyl-phenyl)-urea (NS1643)

DEFF Research Database (Denmark)

Hansen, Rie Schultz; Diness, Thomas Goldin; Christ, Torsten

2005-01-01

The cardiac action potential is generated by a concerted action of different ion channels and transporters. Dysfunction of any of these membrane proteins can give rise to cardiac arrhythmias, which is particularly true for the repolarizing potassium channels. We suggest that an increased repolari......The cardiac action potential is generated by a concerted action of different ion channels and transporters. Dysfunction of any of these membrane proteins can give rise to cardiac arrhythmias, which is particularly true for the repolarizing potassium channels. We suggest that an increased......M. Application of NS1643 also resulted in a prolonged postrepolarization refractory time. Finally, cardiomyocytes exposed to NS1643 resisted reactivation by small depolarizing currents mimicking early afterdepolarizations. In conclusion, HERG channel activation by small molecules such as NS1643 increases...

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.

Experimental reversion of the optimal quantum cloning and flipping processes

International Nuclear Information System (INIS)

Sciarrino, Fabio; Secondi, Veronica; De Martini, Francesco

2006-01-01

The quantum cloner machine maps an unknown arbitrary input qubit into two optimal clones and one optimal flipped qubit. By combining linear and nonlinear optical methods we experimentally implement a scheme that, after the cloning transformation, restores the original input qubit in one of the output channels, by using local measurements, classical communication, and feedforward. This nonlocal method demonstrates how the information on the input qubit can be restored after the cloning process. The realization of the reversion process is expected to find useful applications in the field of modern multipartite quantum cryptography

Sinusoidal voltage protocols for rapid characterisation of ion channel kinetics.

Science.gov (United States)

Beattie, Kylie A; Hill, Adam P; Bardenet, Rémi; Cui, Yi; Vandenberg, Jamie I; Gavaghan, David J; de Boer, Teun P; Mirams, Gary R

2018-03-24

Ion current kinetics are commonly represented by current-voltage relationships, time constant-voltage relationships and subsequently mathematical models fitted to these. These experiments take substantial time, which means they are rarely performed in the same cell. Rather than traditional square-wave voltage clamps, we fitted a model to the current evoked by a novel sum-of-sinusoids voltage clamp that was only 8 s long. Short protocols that can be performed multiple times within a single cell will offer many new opportunities to measure how ion current kinetics are affected by changing conditions. The new model predicts the current under traditional square-wave protocols well, with better predictions of underlying currents than literature models. The current under a novel physiologically relevant series of action potential clamps is predicted extremely well. The short sinusoidal protocols allow a model to be fully fitted to individual cells, allowing us to examine cell-cell variability in current kinetics for the first time. Understanding the roles of ion currents is crucial to predict the action of pharmaceuticals and mutations in different scenarios, and thereby to guide clinical interventions in the heart, brain and other electrophysiological systems. Our ability to predict how ion currents contribute to cellular electrophysiology is in turn critically dependent on our characterisation of ion channel kinetics - the voltage-dependent rates of transition between open, closed and inactivated channel states. We present a new method for rapidly exploring and characterising ion channel kinetics, applying it to the hERG potassium channel as an example, with the aim of generating a quantitatively predictive representation of the ion current. We fitted a mathematical model to currents evoked by a novel 8 second sinusoidal voltage clamp in CHO cells overexpressing hERG1a. The model was then used to predict over 5 minutes of recordings in the same cell in response to

Proapoptotic Role of Potassium Ions in Liver Cells

Directory of Open Access Journals (Sweden)

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.

Evolving regulatory paradigm for proarrhythmic risk assessment for new drugs.

Science.gov (United States)

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.

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....

Molecular analysis of a thylakoid K+channel

International Nuclear Information System (INIS)

1999-01-01

The work undertaken sought to use a novel probe to identify and clone plant ion (K) channels. It was also proposed that in vitro biochemical studies of cation transport across purified preparations of thylakoid membrane be employed to characterize a putative K channel in this membrane system. Over the last several years, an enormous data base of partially-sequenced mRNAs and numerous genomes (including those of plants) has evolved and provides a powerful alternative to this brute-force approach to identify and clone cDNAs encoding physiologically important membrane proteins such as channels. The utility of searching genetic databases for relevant sequences, in addition to the difficulty of working with membrane proteins, led to changes in research focus during the granting period. During the course of the funding period, work was finished up which documented the presence of a K channel in the thylakoid membrane and demonstrated that K fluxes through this channel were required for optimal photosynthetic activity, likely due to the requirement for charge balancing of proton flux

Molecular analysis of a thylakoid K+channel

Energy Technology Data Exchange (ETDEWEB)

NONE

1999-09-10

The work undertaken sought to use a novel probe to identify and clone plant ion (K) channels. It was also proposed that in vitro biochemical studies of cation transport across purified preparations of thylakoid membrane be employed to characterize a putative K channel in this membrane system. Over the last several years, an enormous data base of partially-sequenced mRNAs and numerous genomes (including those of plants) has evolved and provides a powerful alternative to this brute-force approach to identify and clone cDNAs encoding physiologically important membrane proteins such as channels. The utility of searching genetic databases for relevant sequences, in addition to the difficulty of working with membrane proteins, led to changes in research focus during the granting period. During the course of the funding period, work was finished up which documented the presence of a K channel in the thylakoid membrane and demonstrated that K fluxes through this channel were required for optimal photosynthetic activity, likely due to the requirement for charge balancing of proton flux.

Molecular analysis of a thylakoid K+ channel

Energy Technology Data Exchange (ETDEWEB)

NONE

2000-05-01

The work undertaken during the prior granting period sought to use a novel probe to identify and clone plant ion (K) channels. It was also proposed that in vitro biochemical studies of cation transport across purified preparations of thylakoid membrane be employed to characterize a putative K channel in this membrane system. Over the last several years (including those of the previous grant period), an enormous data base of partially-sequenced mRNAs and numerous genomes (including those of plants) has evolved and provides a powerful alternative to this brute-force approach to identify and clone cDNAs ending physiologically important membrane proteins such as channels. The utility of searching genetic databases for relevant sequences, in addition to the difficulty of working with membrane proteins, led to changes in research focus during the prior granting period, and has resulted in the identification of a new class of plant ion channels, which will be the focus of research during the proposed new granting period.

Molecular cloning and functional expression of the K+ channel KV7.1 and the regulatory subunit KCNE1 from equine myocardium

DEFF Research Database (Denmark)

Pedersen, Philip Juul; Thomsen, Kirsten B.; Flak, Jon B.

2017-01-01

To characterize equine KV7.1/KCNE1 currents and compare them to human KV7.1/KCNE1 currents to determine whether KV7.1/KCNE1 plays a similar role in equine and human hearts. Methods mRNA encoding KV7.1 and KCNE1 was isolated from equine hearts, sequenced, and cloned into expression vectors. The channel subunits...... were heterologously expressed in Xenopus laevis oocytes or CHO-K1 cells and characterized using voltage-clamp techniques. Results Equine KV7.1/KCNE1 expressed in CHO-K1 cells exhibited electrophysiological properties that are overall similar to the human orthologs; however, a slower deactivation...

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.

Gambierol, a toxin produced by the dinoflagellate Gambierdiscus toxicus, is a potent blocker of voltage-gated potassium channels☆

Science.gov (United States)

Cuypers, Eva; Abdel-Mottaleb, Yousra; Kopljar, Ivan; Rainier, Jon D.; Raes, Adam L.; Snyders, Dirk J.; Tytgat, Jan

2008-01-01

In this study, we pharmacologically characterized gambierol, a marine polycyclic ether toxin which is produced by the dinoflagellate Gambierdiscus toxicus. Besides several other polycyclic ether toxins like ciguatoxins, this scarcely studied toxin is one of the compounds that may be responsible for ciguatera fish poisoning (CFP). Unfortunately, the biological target(s) that underlies CFP is still partly unknown. Today, ciguatoxins are described to specifically activate voltage-gated sodium channels by interacting with their receptor site 5. But some dispute about the role of gambierol in the CFP story shows up: some describe voltage-gated sodium channels as the target, while others pinpoint voltage-gated potassium channels as targets. Since gambierol was never tested on isolated ion channels before, it was subjected in this work to extensive screening on a panel of 17 ion channels: nine cloned voltage-gated ion channels (mammalian Nav1.1–Nav1.8 and insect Para) and eight cloned voltage-gated potassium channels (mammalian Kv1.1–Kv1.6, hERG and insect ShakerIR) expressed in Xenopus laevis oocytes using two-electrode voltage-clamp technique. All tested sodium channel subtypes are insensitive to gambierol concentrations up to 10 μM. In contrast, Kv1.2 is the most sensitive voltage-gated potassium channel subtype with almost full block (>97%) and an half maximal inhibitory concentration (IC50) of 34.5 nM. To the best of our knowledge, this is the first study where the selectivity of gambierol is tested on isolated voltage-gated ion channels. Therefore, these results lead to a better understanding of gambierol and its possible role in CFP and they may also be useful in the development of more effective treatments. PMID:18313714

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.

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

Directory of Open Access Journals (Sweden)

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.

Molecular cloning, functional expression and subcellular localization of two putative vacuolar voltage-gated chloride channels in rice (Oryza sativa L.).

Science.gov (United States)

Nakamura, Atsuko; Fukuda, Atsunori; Sakai, Shingo; Tanaka, Yoshiyuki

2006-01-01

We isolated two cDNA clones (OsCLC-1 and OsCLC-2) homologous to tobacco CLC-Nt1, which encodes a voltage-gated chloride channel, from rice (Oryza sativa L. ssp. japonica, cv. Nipponbare). The deduced amino acid sequences were highly conserved (87.9% identity with each other). Southern blot analysis of the rice genomic DNA revealed that OsCLC-1 and OsCLC-2 were single-copy genes on chromosomes 4 and 2, respectively. OsCLC-1 was expressed in most tissues, whereas OsCLC-2 was expressed only in the roots, nodes, internodes and leaf sheaths. The level of expression of OsCLC-1, but not of OsCLC-2, was increased by treatment with NaCl. Both genes could partly substitute for GEF1, which encodes the sole chloride channel in yeast, by restoring growth under ionic stress. These results indicate that both genes are chloride channel genes. The proteins from both genes were immunochemically detected in the tonoplast fraction. Tagged synthetic green fluorescent protein which was fused to OsCLC-1 or OsCLC-2 localized in the vacuolar membranes. These results indicate that the proteins may play a role in the transport of chloride ions across the vacuolar membrane. We isolated loss-of-function mutants of both genes from a panel of rice mutants produced by the insertion of a retrotransposon, Tos17, in the exon region, and found inhibition of growth at all life stages.

Ginseng gintonin activates the human cardiac delayed rectifier K+ channel: involvement of Ca2+/calmodulin binding sites.

Science.gov (United States)

Choi, Sun-Hye; Lee, Byung-Hwan; Kim, Hyeon-Joong; Jung, Seok-Won; Kim, Hyun-Sook; Shin, Ho-Chul; Lee, Jun-Hee; Kim, Hyoung-Chun; Rhim, Hyewhon; Hwang, Sung-Hee; Ha, Tal Soo; Kim, Hyun-Ji; Cho, Hana; Nah, Seung-Yeol

2014-09-01

Gintonin, a novel, ginseng-derived G protein-coupled lysophosphatidic acid (LPA) receptor ligand, elicits [Ca(2+)]i transients in neuronal and non-neuronal cells via pertussis toxin-sensitive and pertussis toxin-insensitive G proteins. The slowly activating delayed rectifier K(+) (I(Ks)) channel is a cardiac K(+) channel composed of KCNQ1 and KCNE1 subunits. The C terminus of the KCNQ1 channel protein has two calmodulin-binding sites that are involved in regulating I(Ks) channels. In this study, we investigated the molecular mechanisms of gintonin-mediated activation of human I(Ks) channel activity by expressing human I(Ks) channels in Xenopus oocytes. We found that gintonin enhances IKs channel currents in concentration- and voltage-dependent manners. The EC50 for the I(Ks) channel was 0.05 ± 0.01 μg/ml. Gintonin-mediated activation of the I(Ks) channels was blocked by an LPA1/3 receptor antagonist, an active phospholipase C inhibitor, an IP3 receptor antagonist, and the calcium chelator BAPTA. Gintonin-mediated activation of both the I(Ks) channel was also blocked by the calmodulin (CaM) blocker calmidazolium. Mutations in the KCNQ1 [Ca(2+)]i/CaM-binding IQ motif sites (S373P, W392R, or R539W)blocked the action of gintonin on I(Ks) channel. However, gintonin had no effect on hERG K(+) channel activity. These results show that gintonin-mediated enhancement of I(Ks) channel currents is achieved through binding of the [Ca(2+)]i/CaM complex to the C terminus of KCNQ1 subunit.

Cloning

Science.gov (United States)

Cloning describes the processes used to create an exact genetic replica of another cell, tissue or organism. ... named Dolly. There are three different types of cloning: Gene cloning, which creates copies of genes or ...

What is Cloning?

Science.gov (United States)

Donate Home Cloning What is Cloning What is Cloning Clones are organisms that are exact genetic copies. ... clones made through modern cloning technologies. How Is Cloning Done? Many people first heard of cloning when ...

Optimal processing of reversible quantum channels

Energy Technology Data Exchange (ETDEWEB)

Bisio, Alessandro, E-mail: alessandro.bisio@unipv.it [QUIT Group, Dipartimento di Fisica, INFN Sezione di Pavia, via Bassi 6, 27100 Pavia (Italy); D' Ariano, Giacomo Mauro; Perinotti, Paolo [QUIT Group, Dipartimento di Fisica, INFN Sezione di Pavia, via Bassi 6, 27100 Pavia (Italy); Sedlák, Michal [Department of Optics, Palacký University, 17. Listopadu 1192/12, CZ-771 46 Olomouc (Czech Republic); Institute of Physics, Slovak Academy of Sciences, Dúbravská Cesta 9, 845 11 Bratislava (Slovakia)

2014-05-01

We consider the general problem of the optimal transformation of N uses of (possibly different) unitary channels to a single use of another unitary channel in any finite dimension. We show how the optimal transformation can be fully parallelized, consisting in a preprocessing channel followed by a parallel action of all the N unitaries and a final postprocessing channel. Our techniques allow to achieve an exponential reduction in the number of the free parameters of the optimization problem making it amenable to an efficient numerical treatment. Finally, we apply our general results to find the analytical solution for special cases of interest like the cloning of qubit phase gates.

Pharmacologic Approach to Defective Protein Trafficking in the E637K-hERG Mutant with PD-118057 and Thapsigargin.

Directory of Open Access Journals (Sweden)

Haiyan Mao

Full Text Available Treatment of LQT2 is inadequate. Many drugs which can pharmacologically rescue defective protein trafficking in LQT2 also result in potent blockade of HERG current, negating their therapeutic benefit. It is reported that PD-118057 and thapsigargin can rescue LQT2 without hERG channel blockade, but the precise mechanism of action is unknown. Furthermore, the effect of PD-118057 and thapsigargin on the dominant negative E637K-hERG mutant has not been previously investigated.IN THIS STUDY, WE INVESTIGATED: (a the effect of PD-118057 and thapsigargin on the current amplitudes of WT-hERG and WT/E637K-hERG channels; (b the effect of PD-118057 and thapsigargin on the biophysical properties of WT-hERG and WT/E637K-hERG channels; (c whether drug treatment can rescue channel processing and trafficking defects of the WT/E637K-hERG mutant.The whole-cell Patch-clamp technique was used to assess the effect of PD-118057 and thapsigargin on the electrophysiological characteristics of the rapidly activating delayed rectifier K(+ current (Ikr of the hERG protein channel. Western blot was done to investigate pharmacological rescue on hERG protein channel function.In our study, PD-118057 was shown to significantly enhance both the maximum current amplitude and tail current amplitude, but did not alter the gating and kinetic properties of the WT-hERG channel, with the exception of accelerating steady-state inactivation. Additionally, thapsigargin shows a similar result as PD-118057 for the WT-hERG channel, but with the exception of attenuating steady-state inactivation. However, for the WT/E637K-hERG channel, PD-118057 had no effect on either the current or on the gating and kinetic properties. Furthermore, thapsigargin treatment did not alter the current or the gating and kinetic properties of the WT/E637K-hERG channel, with the exception of opening at more positive voltages.Our findings illustrate that neither PD-118057 nor thapsigargin play a role in correcting

Clone DB: an integrated NCBI resource for clone-associated data

Science.gov (United States)

Schneider, Valerie A.; Chen, Hsiu-Chuan; Clausen, Cliff; Meric, Peter A.; Zhou, Zhigang; Bouk, Nathan; Husain, Nora; Maglott, Donna R.; Church, Deanna M.

2013-01-01

The National Center for Biotechnology Information (NCBI) Clone DB (http://www.ncbi.nlm.nih.gov/clone/) is an integrated resource providing information about and facilitating access to clones, which serve as valuable research reagents in many fields, including genome sequencing and variation analysis. Clone DB represents an expansion and replacement of the former NCBI Clone Registry and has records for genomic and cell-based libraries and clones representing more than 100 different eukaryotic taxa. Records provide details of library construction, associated sequences, map positions and information about resource distribution. Clone DB is indexed in the NCBI Entrez system and can be queried by fields that include organism, clone name, gene name and sequence identifier. Whenever possible, genomic clones are mapped to reference assemblies and their map positions provided in clone records. Clones mapping to specific genomic regions can also be searched for using the NCBI Clone Finder tool, which accepts queries based on sequence coordinates or features such as gene or transcript names. Clone DB makes reports of library, clone and placement data on its FTP site available for download. With Clone DB, users now have available to them a centralized resource that provides them with the tools they will need to make use of these important research reagents. PMID:23193260

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...

TRP channels: an overview

DEFF Research Database (Denmark)

Pedersen, Stine Falsig; Owsianik, Grzegorz; Nilius, Bernd

2005-01-01

The TRP ("transient receptor potential") family of ion channels now comprises more than 30 cation channels, most of which are permeable for Ca2+, and some also for Mg2+. On the basis of sequence homology, the TRP family can be divided in seven main subfamilies: the TRPC ('Canonical') family......, the TRPV ('Vanilloid') family, the TRPM ('Melastatin') family, the TRPP ('Polycystin') family, the TRPML ('Mucolipin') family, the TRPA ('Ankyrin') family, and the TRPN ('NOMPC') family. The cloning and characterization of members of this cation channel family has exploded during recent years, leading...... to a plethora of data on the roles of TRPs in a variety of tissues and species, including mammals, insects, and yeast. The present review summarizes the most pertinent recent evidence regarding the structural and functional properties of TRP channels, focusing on the regulation and physiology of mammalian TRPs....

Comparative effects of sodium channel blockers in short term rat whole embryo culture

Energy Technology Data Exchange (ETDEWEB)

Nilsson, Mats F, E-mail: Mats.Nilsson@farmbio.uu.se [Department of Pharmaceutical Biosciences, Uppsala University (Sweden); Sköld, Anna-Carin; Ericson, Ann-Christin; Annas, Anita; Villar, Rodrigo Palma [AstraZeneca R and D Södertälje (Sweden); Cebers, Gvido [AstraZeneca R and D, iMed, 141 Portland Street, Cambridge, MA 02139 (United States); Hellmold, Heike; Gustafson, Anne-Lee [AstraZeneca R and D Södertälje (Sweden); Webster, William S [Department of Anatomy and Histology, University of Sydney (Australia)

2013-10-15

This study was undertaken to examine the effect on the rat embryonic heart of two experimental drugs (AZA and AZB) which are known to block the sodium channel Nav1.5, the hERG potassium channel and the L-type calcium channel. The sodium channel blockers bupivacaine, lidocaine, and the L-type calcium channel blocker nifedipine were used as reference substances. The experimental model was the gestational day (GD) 13 rat embryo cultured in vitro. In this model the embryonic heart activity can be directly observed, recorded and analyzed using computer assisted image analysis as it responds to the addition of test drugs. The effect on the heart was studied for a range of concentrations and for a duration up to 3 h. The results showed that AZA and AZB caused a concentration-dependent bradycardia of the embryonic heart and at high concentrations heart block. These effects were reversible on washout. In terms of potency to cause bradycardia the compounds were ranked AZB > bupivacaine > AZA > lidocaine > nifedipine. Comparison with results from previous studies with more specific ion channel blockers suggests that the primary effect of AZA and AZB was sodium channel blockage. The study shows that the short-term rat whole embryo culture (WEC) is a suitable system to detect substances hazardous to the embryonic heart. - Highlights: • Study of the effect of sodium channel blocking drugs on embryonic heart function • We used a modified method rat whole embryo culture with image analysis. • The drugs tested caused a concentration dependent bradycardia and heart block. • The effect of drugs acting on multiple ion channels is difficult to predict. • This method may be used to detect cardiotoxicity in prenatal development.

Comparative effects of sodium channel blockers in short term rat whole embryo culture

International Nuclear Information System (INIS)

Nilsson, Mats F; Sköld, Anna-Carin; Ericson, Ann-Christin; Annas, Anita; Villar, Rodrigo Palma; Cebers, Gvido; Hellmold, Heike; Gustafson, Anne-Lee; Webster, William S

2013-01-01

This study was undertaken to examine the effect on the rat embryonic heart of two experimental drugs (AZA and AZB) which are known to block the sodium channel Nav1.5, the hERG potassium channel and the L-type calcium channel. The sodium channel blockers bupivacaine, lidocaine, and the L-type calcium channel blocker nifedipine were used as reference substances. The experimental model was the gestational day (GD) 13 rat embryo cultured in vitro. In this model the embryonic heart activity can be directly observed, recorded and analyzed using computer assisted image analysis as it responds to the addition of test drugs. The effect on the heart was studied for a range of concentrations and for a duration up to 3 h. The results showed that AZA and AZB caused a concentration-dependent bradycardia of the embryonic heart and at high concentrations heart block. These effects were reversible on washout. In terms of potency to cause bradycardia the compounds were ranked AZB > bupivacaine > AZA > lidocaine > nifedipine. Comparison with results from previous studies with more specific ion channel blockers suggests that the primary effect of AZA and AZB was sodium channel blockage. The study shows that the short-term rat whole embryo culture (WEC) is a suitable system to detect substances hazardous to the embryonic heart. - Highlights: • Study of the effect of sodium channel blocking drugs on embryonic heart function • We used a modified method rat whole embryo culture with image analysis. • The drugs tested caused a concentration dependent bradycardia and heart block. • The effect of drugs acting on multiple ion channels is difficult to predict. • This method may be used to detect cardiotoxicity in prenatal development

Recombinational Cloning Using Gateway and In-Fusion Cloning Schemes

Science.gov (United States)

Throop, Andrea L.; LaBaer, Joshua

2015-01-01

The comprehensive study of protein structure and function, or proteomics, depends on the obtainability of full-length cDNAs in species-specific expression vectors and subsequent functional analysis of the expressed protein. Recombinational cloning is a universal cloning technique based on site-specific recombination that is independent of the insert DNA sequence of interest, which differentiates this method from the classical restriction enzyme-based cloning methods. Recombinational cloning enables rapid and efficient parallel transfer of DNA inserts into multiple expression systems. This unit summarizes strategies for generating expression-ready clones using the most popular recombinational cloning technologies, including the commercially available Gateway® (Life Technologies) and In-Fusion® (Clontech) cloning technologies. PMID:25827088

"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…

Unified Approach to Universal Cloning and Phase-Covariant Cloning

OpenAIRE

Hu, Jia-Zhong; Yu, Zong-Wen; Wang, Xiang-Bin

2008-01-01

We analyze the problem of approximate quantum cloning when the quantum state is between two latitudes on the Bloch's sphere. We present an analytical formula for the optimized 1-to-2 cloning. The formula unifies the universal quantum cloning (UQCM) and the phase covariant quantum cloning.

Regulation of cloned, Ca2+-activated K+ channels by cell volume changes

DEFF Research Database (Denmark)

Grunnet, Morten; MacAulay, Nanna; Jorgensen, Nanna K

2002-01-01

Ca2+-activated K+ channels of big (hBK), intermediate (hIK) or small (rSK3) conductance were co-expressed with aquaporin 1 (AQP1) in Xenopus laevis oocytes. hBK channels were activated by depolarization, whereas hIK and rSK3 channels were activated by direct injection of Ca2+ or Cd2+ into the ooc...

Enzyme free cloning for high throughput gene cloning and expression

NARCIS (Netherlands)

de Jong, R.N.; Daniëls, M.; Kaptein, R.; Folkers, G.E.

2006-01-01

Structural and functional genomics initiatives significantly improved cloning methods over the past few years. Although recombinational cloning is highly efficient, its costs urged us to search for an alternative high throughput (HTP) cloning method. We implemented a modified Enzyme Free Cloning

Novel Two-Step Classifier for Torsades de Pointes Risk Stratification from Direct Features

Directory of Open Access Journals (Sweden)

Jaimit Parikh

2017-11-01

Full Text Available While pre-clinical Torsades de Pointes (TdP risk classifiers had initially been based on drug-induced block of hERG potassium channels, it is now well established that improved risk prediction can be achieved by considering block of non-hERG ion channels. The current multi-channel TdP classifiers can be categorized into two classes. First, the classifiers that take as input the values of drug-induced block of ion channels (direct features. Second, the classifiers that are built on features extracted from output of the drug-induced multi-channel blockage simulations in the in-silico models (derived features. The classifiers built on derived features have thus far not consistently provided increased prediction accuracies, and hence casts doubt on the value of such approaches given the cost of including biophysical detail. Here, we propose a new two-step method for TdP risk classification, referred to as Multi-Channel Blockage at Early After Depolarization (MCB@EAD. In the first step, we classified the compound that produced insufficient hERG block as non-torsadogenic. In the second step, the role of non-hERG channels to modulate TdP risk are considered by constructing classifiers based on direct or derived features at critical hERG block concentrations that generates EADs in the computational cardiac cell models. MCB@EAD provides comparable or superior TdP risk classification of the drugs from the direct features in tests against published methods. TdP risk for the drugs highly correlated to the propensity to generate EADs in the model. However, the derived features of the biophysical models did not improve the predictive capability for TdP risk assessment.

A Seminar on Human Cloning: Cloning in Reproductive Medicine

OpenAIRE

Illmensee, Karl

2001-01-01

This review article summarizes the historical development of mammalian cloning, presents current advances and presumed risk factors in the field of reproductive cloning, discusses possible clinical applications of therapeutic and diagnostic cloning and outlines prospective commercial trends in pharmacytical cloning. Predictable progress in biotechnology and stem cell engineering should prove to be advantageous for patients' health and for novel benefits in reproductive and regenerative medicine.

CO-independent modification of K+ channels by tricarbonyldichlororuthenium(II) dimer (CORM-2).

Science.gov (United States)

Gessner, Guido; Sahoo, Nirakar; Swain, Sandip M; Hirth, Gianna; Schönherr, Roland; Mede, Ralf; Westerhausen, Matthias; Brewitz, Hans Henning; Heimer, Pascal; Imhof, Diana; Hoshi, Toshinori; Heinemann, Stefan H

2017-11-15

Although toxic when inhaled in high concentrations, the gas carbon monoxide (CO) is endogenously produced in mammals, and various beneficial effects are reported. For potential medicinal applications and studying the molecular processes underlying the pharmacological action of CO, so-called CO-releasing molecules (CORMs), such as tricabonyldichlororuthenium(II) dimer (CORM-2), have been developed and widely used. Yet, it is not readily discriminated whether an observed effect of a CORM is caused by the released CO gas, the CORM itself, or any of its intermediate or final breakdown products. Focusing on Ca 2+ - and voltage-dependent K + channels (K Ca 1.1) and voltage-gated K + channels (Kv1.5, Kv11.1) relevant for cardiac safety pharmacology, we demonstrate that, in most cases, the functional impacts of CORM-2 on these channels are not mediated by CO. Instead, when dissolved in aqueous solutions, CORM-2 has the propensity of forming Ru(CO) 2 adducts, preferentially to histidine residues, as demonstrated with synthetic peptides using mass-spectrometry analysis. For K Ca 1.1 channels we show that H365 and H394 in the cytosolic gating ring structure are affected by CORM-2. For Kv11.1 channels (hERG1) the extracellularly accessible histidines H578 and H587 are CORM-2 targets. The strong CO-independent action of CORM-2 on Kv11.1 and Kv1.5 channels can be completely abolished when CORM-2 is applied in the presence of an excess of free histidine or human serum albumin; cysteine and methionine are further potential targets. Off-site effects similar to those reported here for CORM-2 are found for CORM-3, another ruthenium-based CORM, but are diminished when using iron-based CORM-S1 and absent for manganese-based CORM-EDE1. Copyright © 2017 Elsevier B.V. All rights reserved.

Cloning of observables

OpenAIRE

Ferraro, Alessandro; Galbiati, Matteo; Paris, Matteo G. A.

2005-01-01

We introduce the concept of cloning for classes of observables and classify cloning machines for qubit systems according to the number of parameters needed to describe the class under investigation. A no-cloning theorem for observables is derived and the connections between cloning of observables and joint measurements of noncommuting observables are elucidated. Relationships with cloning of states and non-demolition measurements are also analyzed.

Cloning of observables

International Nuclear Information System (INIS)

Ferraro, Alessandro; Galbiati, Matteo; Paris, Matteo G A

2006-01-01

We introduce the concept of cloning for classes of observables and classify cloning machines for qubit systems according to the number of parameters needed to describe the class under investigation. A no-cloning theorem for observables is derived and the connections between cloning of observables and joint measurements of noncommuting observables are elucidated. Relationships with cloning of states and non-demolition measurements are also analysed. (letter to the editor)

Cloning and expression of ligand-gated ion-channel receptor L2 in central nervous system

International Nuclear Information System (INIS)

Houtani, Takeshi; Munemoto, Yumi; Kase, Masahiko; Sakuma, Satoru; Tsutsumi, Toshiyuki; Sugimoto, Tetsuo

2005-01-01

An orphan receptor of ligand-gated ion-channel type (L2, also termed ZAC according to the presence of zinc ion for channel activation) was identified by computer-assisted search programs on human genome database. The L2 protein shares partial homology with serotonin receptors 5HT3A and 5HT3B. We have cloned L2 cDNA derived from human caudate nucleus and characterized the exon-intron structure as follows: (1) The L2 protein has four transmembrane regions (M1-M4) and a long cytoplasmic loop between M3 and M4. (2) The sequence is conserved in species including chimpanzee, dog, cow, and opossum. (3) Nine exons form its protein-coding region and especially exon 5 corresponds to a disulfide bond region on the amino-terminal side. Our analysis using multiple tissue cDNA panels revealed that at least two splicing variants of L2 mRNA are present. The cDNA PCR amplification study revealed that L2 mRNA is expressed in tissues including brain, pancreas, liver, lung, heart, kidney, and skeletal muscle while 5HT3A mRNA could be detected in brain, heart, placenta, lung, kidney, pancreas, and skeletal muscle, and 5HT3B mRNA in brain, kidney, and skeletal muscle, suggesting different significance in tissue expression of these receptors. Regional expression of L2 mRNA and protein was examined in brain. The RT-PCR studies confirmed L2 mRNA expression in hippocampus, striatum, amygdala, and thalamus in adult brain. The L2 protein was immunolocalized by using antipeptide antibodies. Immunostained tissue sections revealed that L2-like immunoreactivity was dominantly expressed in the hippocampal CA3 pyramidal cells and in the polymorphic layer of the dentate gyrus. We analyzed the expression of L2 protein in HEK293 cells using GFP fusion protein reporter system. Western blots revealed that L2 protein confers sugar chains on the extracellular side. In transfected HEK293 cells, cellular membranes and intracellular puncta were densely labeled with GFP, suggesting selective dispatch to the

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 Clone Factory

Science.gov (United States)

Stoddard, Beryl

2005-01-01

Have humans been cloned? Is it possible? Immediate interest is sparked when students are asked these questions. In response to their curiosity, the clone factory activity was developed to help them understand the process of cloning. In this activity, students reenact the cloning process, in a very simplified simulation. After completing the…

Multipartite asymmetric quantum cloning

International Nuclear Information System (INIS)

Iblisdir, S.; Gisin, N.; Acin, A.; Cerf, N.J.; Filip, R.; Fiurasek, J.

2005-01-01

We investigate the optimal distribution of quantum information over multipartite systems in asymmetric settings. We introduce cloning transformations that take N identical replicas of a pure state in any dimension as input and yield a collection of clones with nonidentical fidelities. As an example, if the clones are partitioned into a set of M A clones with fidelity F A and another set of M B clones with fidelity F B , the trade-off between these fidelities is analyzed, and particular cases of optimal N→M A +M B cloning machines are exhibited. We also present an optimal 1→1+1+1 cloning machine, which is an example of a tripartite fully asymmetric cloner. Finally, it is shown how these cloning machines can be optically realized

Different protein kinase C isoenzymes mediate inhibition of cardiac rapidly activating delayed rectifier K+ current by different G-protein coupled receptors.

Science.gov (United States)

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.

(Patho)physiological implications of the novel epithelial Ca2+ channels TRPV5 and TRPV6.

NARCIS (Netherlands)

Nijenhuis, T.; Hoenderop, J.G.J.; Nilius, B.; Bindels, R.J.M.

2003-01-01

The epithelial Ca(2+) channels TRPV5 and TRPV6 constitute the apical Ca(2+) entry mechanism in active Ca(2+) (re)absorption. These two members of the superfamily of transient receptor potential (TRP) channels were cloned from the vitamin-D-responsive epithelia of kidney and small intestine and

Human therapeutic cloning (NTSC): applying research from mammalian reproductive cloning.

Science.gov (United States)

French, Andrew J; Wood, Samuel H; Trounson, Alan O

2006-01-01

Human therapeutic cloning or nuclear transfer stem cells (NTSC) to produce patient-specific stem cells, holds considerable promise in the field of regenerative medicine. The recent withdrawal of the only scientific publications claiming the successful generation of NTSC lines afford an opportunity to review the available research in mammalian reproductive somatic cell nuclear transfer (SCNT) with the goal of progressing human NTSC. The process of SCNT is prone to epigenetic abnormalities that contribute to very low success rates. Although there are high mortality rates in some species of cloned animals, most surviving clones have been shown to have normal phenotypic and physiological characteristics and to produce healthy offspring. This technology has been applied to an increasing number of mammals for utility in research, agriculture, conservation, and biomedicine. In contrast, attempts at SCNT to produce human embryonic stem cells (hESCs) have been disappointing. Only one group has published reliable evidence of success in deriving a cloned human blastocyst, using an undifferentiated hESC donor cell, and it failed to develop into a hESC line. When optimal conditions are present, it appears that in vitro development of cloned and parthenogenetic embryos, both of which may be utilized to produce hESCs, may be similar to in vitro fertilized embryos. The derivation of ESC lines from cloned embryos is substantially more efficient than the production of viable offspring. This review summarizes developments in mammalian reproductive cloning, cell-to-cell fusion alternatives, and strategies for oocyte procurement that may provide important clues facilitating progress in human therapeutic cloning leading to the successful application of cell-based therapies utilizing autologous hESC lines.

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

Coding sequence of human rho cDNAs clone 6 and clone 9

Energy Technology Data Exchange (ETDEWEB)

Chardin, P; Madaule, P; Tavitian, A

1988-03-25

The authors have isolated human cDNAs including the complete coding sequence for two rho proteins corresponding to the incomplete isolates previously described as clone 6 and clone 9. The deduced a.a. sequences, when compared to the a.a. sequence deduced from clone 12 cDNA, show that there are in human at least three highly homologous rho genes. They suggest that clone 12 be named rhoA, clone 6 : rhoB and clone 9 : rhoC. RhoA, B and C proteins display approx. 30% a.a. identity with ras proteins,. mainly clustered in four highly homologous internal regions corresponding to the GTP binding site; however at least one significant difference is found; the 3 rho proteins have an Alanine in position corresponding to ras Glycine 13, suggesting that rho and ras proteins might have slightly different biochemical properties.

Ethical issues in animal cloning.

Science.gov (United States)

Fiester, Autumn

2005-01-01

The issue of human reproductive cloning has recently received a great deal attention in public discourse. Bioethicists, policy makers, and the media have been quick to identify the key ethical issues involved in human reproductive cloning and to argue, almost unanimously, for an international ban on such attempts. Meanwhile, scientists have proceeded with extensive research agendas in the cloning of animals. Despite this research, there has been little public discussion of the ethical issues raised by animal cloning projects. Polling data show that the public is decidedly against the cloning of animals. To understand the public's reaction and fill the void of reasoned debate about the issue, we need to review the possible objections to animal cloning and assess the merits of the anti-animal cloning stance. Some objections to animal cloning (e.g., the impact of cloning on the population of unwanted animals) can be easily addressed, while others (e.g., the health of cloned animals) require more serious attention by the public and policy makers.

Optimally cloned binary coherent states

DEFF Research Database (Denmark)

Mueller, C. R.; Leuchs, G.; Marquardt, Ch

2017-01-01

their quantum-optimal clones. We analyze the Wigner function and the cumulants of the clones, and we conclude that optimal cloning of binary coherent states requires a nonlinearity above second order. We propose several practical and near-optimal cloning schemes and compare their cloning fidelity to the optimal...

Hybrid sequencing approach applied to human fecal metagenomic clone libraries revealed clones with potential biotechnological applications.

Science.gov (United States)

Džunková, Mária; D'Auria, Giuseppe; Pérez-Villarroya, David; Moya, Andrés

2012-01-01

Natural environments represent an incredible source of microbial genetic diversity. Discovery of novel biomolecules involves biotechnological methods that often require the design and implementation of biochemical assays to screen clone libraries. However, when an assay is applied to thousands of clones, one may eventually end up with very few positive clones which, in most of the cases, have to be "domesticated" for downstream characterization and application, and this makes screening both laborious and expensive. The negative clones, which are not considered by the selected assay, may also have biotechnological potential; however, unfortunately they would remain unexplored. Knowledge of the clone sequences provides important clues about potential biotechnological application of the clones in the library; however, the sequencing of clones one-by-one would be very time-consuming and expensive. In this study, we characterized the first metagenomic clone library from the feces of a healthy human volunteer, using a method based on 454 pyrosequencing coupled with a clone-by-clone Sanger end-sequencing. Instead of whole individual clone sequencing, we sequenced 358 clones in a pool. The medium-large insert (7-15 kb) cloning strategy allowed us to assemble these clones correctly, and to assign the clone ends to maintain the link between the position of a living clone in the library and the annotated contig from the 454 assembly. Finally, we found several open reading frames (ORFs) with previously described potential medical application. The proposed approach allows planning ad-hoc biochemical assays for the clones of interest, and the appropriate sub-cloning strategy for gene expression in suitable vectors/hosts.

Hybrid sequencing approach applied to human fecal metagenomic clone libraries revealed clones with potential biotechnological applications.

Directory of Open Access Journals (Sweden)

Mária Džunková

Full Text Available Natural environments represent an incredible source of microbial genetic diversity. Discovery of novel biomolecules involves biotechnological methods that often require the design and implementation of biochemical assays to screen clone libraries. However, when an assay is applied to thousands of clones, one may eventually end up with very few positive clones which, in most of the cases, have to be "domesticated" for downstream characterization and application, and this makes screening both laborious and expensive. The negative clones, which are not considered by the selected assay, may also have biotechnological potential; however, unfortunately they would remain unexplored. Knowledge of the clone sequences provides important clues about potential biotechnological application of the clones in the library; however, the sequencing of clones one-by-one would be very time-consuming and expensive. In this study, we characterized the first metagenomic clone library from the feces of a healthy human volunteer, using a method based on 454 pyrosequencing coupled with a clone-by-clone Sanger end-sequencing. Instead of whole individual clone sequencing, we sequenced 358 clones in a pool. The medium-large insert (7-15 kb cloning strategy allowed us to assemble these clones correctly, and to assign the clone ends to maintain the link between the position of a living clone in the library and the annotated contig from the 454 assembly. Finally, we found several open reading frames (ORFs with previously described potential medical application. The proposed approach allows planning ad-hoc biochemical assays for the clones of interest, and the appropriate sub-cloning strategy for gene expression in suitable vectors/hosts.

The topsy-turvy cloning law.

Science.gov (United States)

Brassington, Iain; Oultram, Stuart

2011-03-01

In debates about human cloning, a distinction is frequently drawn between therapeutic and reproductive uses of the technology. Naturally enough, this distinction influences the way that the law is framed. The general consensus is that therapeutic cloning is less morally problematic than reproductive cloning--one can hold this position while holding that both are morally unacceptable--and the law frequently leaves the way open for some cloning for the sake of research into new therapeutic techniques while banning it for reproductive purposes. We claim that the position adopted by the law has things the wrong way around: if we accept a moral distinction between therapeutic and reproductive cloning, there are actually more reasons to be morally worried about therapeutic cloning than about reproductive cloning. If cloning is the proper object of legal scrutiny, then, we ought to make sure that we are scrutinising the right kind of clone.

Academic Cloning.

Science.gov (United States)

Sikula, John P.; Sikula, Andrew F.

1980-01-01

The authors define "cloning" as an integral feature of all educational systems, citing teaching practices which reward students for closely reproducing the teacher's thoughts and/or behaviors and administrative systems which tend to promote like-minded subordinates. They insist, however, that "academic cloning" is not a totally…

A single-copy galK promoter cloning vector suitable for cloning strong promoters

DEFF Research Database (Denmark)

Dandanell, Gert; Court, Donald L.; Hammer, Karin

1986-01-01

We report the construction of lambda galK promoter cloning vectors for cloning and characterization of strong promoters. This phage, which contains a unique HindIII cloning site, was applied to the cloning and analysis of transcription initiations of the regulatory region of the deo-operon of...

Changes in the gut microbiota of cloned and non-cloned control pigs during development of obesity: gut microbiota during development of obesity in cloned pigs.

Science.gov (United States)

Pedersen, Rebecca; Andersen, Anders Daniel; Mølbak, Lars; Stagsted, Jan; Boye, Mette

2013-02-07

Obesity induced by a high-caloric diet has previously been associated with changes in the gut microbiota in mice and in humans. In this study, pigs were cloned to minimize genetic and biological variation among the animals with the aim of developing a controlled metabolomic model suitable for a diet-intervention study. Cloning of pigs may be an attractive way to reduce genetic influences when investigating the effect of diet and obesity on different physiological sites. The aim of this study was to assess and compare the changes in the composition of the gut microbiota of cloned vs. non-cloned pigs during development of obesity by a high-fat/high-caloric diet. Furthermore, we investigated the association between diet-induced obesity and the relative abundance of the phyla Firmicutes and Bacteroidetes in the fecal-microbiota. The fecal microbiota from obese cloned (n = 5) and non-cloned control pigs (n= 6) was investigated biweekly over a period of 136 days, by terminal restriction fragment length polymorphism (T-RFLP) and quantitative real time PCR (qPCR). A positive correlation was observed between body-weight at endpoint and percent body-fat in cloned (r=0.9, Pmicrobiota between the cloned pigs or between cloned and non-cloned control pigs. Body-weight correlated positively with the relative abundance of Firmicutes in both cloned (r=0.37; Pgut microbiota in neither the obese nor the lean state. Diet-induced obesity was associated with an increase in the relative abundance of Firmicutes over time. Our results suggest that cloned pigs are not a more suitable animal model for gut microbiota-obesity related studies than non-cloned pigs. This study is the first to evaluate if cloned pigs provide a better animal model than conventional pigs in diet-intervention, obesity and gut microbiota research.

Quantum cloning machines and the applications

Energy Technology Data Exchange (ETDEWEB)

Fan, Heng, E-mail: hfan@iphy.ac.cn [Beijing National Laboratory for Condensed Matter Physics, Institute of Physics, Chinese Academy of Sciences, Beijing 100190 (China); Collaborative Innovation Center of Quantum Matter, Beijing 100190 (China); Wang, Yi-Nan; Jing, Li [School of Physics, Peking University, Beijing 100871 (China); Yue, Jie-Dong [Beijing National Laboratory for Condensed Matter Physics, Institute of Physics, Chinese Academy of Sciences, Beijing 100190 (China); Shi, Han-Duo; Zhang, Yong-Liang; Mu, Liang-Zhu [School of Physics, Peking University, Beijing 100871 (China)

2014-11-20

No-cloning theorem is fundamental for quantum mechanics and for quantum information science that states an unknown quantum state cannot be cloned perfectly. However, we can try to clone a quantum state approximately with the optimal fidelity, or instead, we can try to clone it perfectly with the largest probability. Thus various quantum cloning machines have been designed for different quantum information protocols. Specifically, quantum cloning machines can be designed to analyze the security of quantum key distribution protocols such as BB84 protocol, six-state protocol, B92 protocol and their generalizations. Some well-known quantum cloning machines include universal quantum cloning machine, phase-covariant cloning machine, the asymmetric quantum cloning machine and the probabilistic quantum cloning machine. In the past years, much progress has been made in studying quantum cloning machines and their applications and implementations, both theoretically and experimentally. In this review, we will give a complete description of those important developments about quantum cloning and some related topics. On the other hand, this review is self-consistent, and in particular, we try to present some detailed formulations so that further study can be taken based on those results.

Quantum cloning machines and the applications

International Nuclear Information System (INIS)

Fan, Heng; Wang, Yi-Nan; Jing, Li; Yue, Jie-Dong; Shi, Han-Duo; Zhang, Yong-Liang; Mu, Liang-Zhu

2014-01-01

No-cloning theorem is fundamental for quantum mechanics and for quantum information science that states an unknown quantum state cannot be cloned perfectly. However, we can try to clone a quantum state approximately with the optimal fidelity, or instead, we can try to clone it perfectly with the largest probability. Thus various quantum cloning machines have been designed for different quantum information protocols. Specifically, quantum cloning machines can be designed to analyze the security of quantum key distribution protocols such as BB84 protocol, six-state protocol, B92 protocol and their generalizations. Some well-known quantum cloning machines include universal quantum cloning machine, phase-covariant cloning machine, the asymmetric quantum cloning machine and the probabilistic quantum cloning machine. In the past years, much progress has been made in studying quantum cloning machines and their applications and implementations, both theoretically and experimentally. In this review, we will give a complete description of those important developments about quantum cloning and some related topics. On the other hand, this review is self-consistent, and in particular, we try to present some detailed formulations so that further study can be taken based on those results

[Nuclear transfer and therapeutic cloning].

Science.gov (United States)

Xu, Xiao-Ming; Lei, An-Min; Hua, Jin-Lian; Dou, Zhong-Ying

2005-03-01

Nuclear transfer and therapeutic cloning have widespread and attractive prospects in animal agriculture and biomedical applications. We reviewed that the quality of oocytes and nuclear reprogramming of somatic donor cells were the main reasons of the common abnormalities in cloned animals and the low efficiency of cloning and showed the problems and outlets in therapeutic cloning, such as some basic problems in nuclear transfer affected clinical applications of therapeutic cloning. Study on isolation and culture of nuclear transfer embryonic stem (ntES) cells and specific differentiation of ntES cells into important functional cells should be emphasized and could enhance the efficiency. Adult stem cells could help to cure some great diseases, but could not replace therapeutic cloning. Ethics also impeded the development of therapeutic cloning. It is necessary to improve many techniques and reinforce the research of some basic theories, then somatic nuclear transfer and therapeutic cloning may apply to agriculture reproduction and benefit to human life better.

Probabilistic cloning of equidistant states

International Nuclear Information System (INIS)

Jimenez, O.; Roa, Luis; Delgado, A.

2010-01-01

We study the probabilistic cloning of equidistant states. These states are such that the inner product between them is a complex constant or its conjugate. Thereby, it is possible to study their cloning in a simple way. In particular, we are interested in the behavior of the cloning probability as a function of the phase of the overlap among the involved states. We show that for certain families of equidistant states Duan and Guo's cloning machine leads to cloning probabilities lower than the optimal unambiguous discrimination probability of equidistant states. We propose an alternative cloning machine whose cloning probability is higher than or equal to the optimal unambiguous discrimination probability for any family of equidistant states. Both machines achieve the same probability for equidistant states whose inner product is a positive real number.

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.

Quantum cloning and signaling

International Nuclear Information System (INIS)

Simon, C.; Weihs, G.; Zeilinger, A.

1999-01-01

We discuss the close connections between cloning of quantum states and superluminal signaling. We present an optimal universal cloning machine based on stimulated emission recently proposed by the authors. As an instructive example, we show how a scheme for superluminal communication based on this cloning machine fails. (Authors)

Recombination-assisted megaprimer (RAM) cloning

Science.gov (United States)

Mathieu, Jacques; Alvarez, Emilia; Alvarez, Pedro J.J.

2014-01-01

No molecular cloning technique is considered universally reliable, and many suffer from being too laborious, complex, or expensive. Restriction-free cloning is among the simplest, most rapid, and cost-effective methods, but does not always provide successful results. We modified this method to enhance its success rate through the use of exponential amplification coupled with homologous end-joining. This new method, recombination-assisted megaprimer (RAM) cloning, significantly extends the application of restriction-free cloning, and allows efficient vector construction with much less time and effort when restriction-free cloning fails to provide satisfactory results. The following modifications were made to the protocol:•Limited number of PCR cycles for both megaprimer synthesis and the cloning reaction to reduce error propagation.•Elimination of phosphorylation and ligation steps previously reported for cloning methods that used exponential amplification, through the inclusion of a reverse primer in the cloning reaction with a 20 base pair region of homology to the forward primer.•The inclusion of 1 M betaine to enhance both reaction specificity and yield. PMID:26150930

Human cloning and child welfare.

Science.gov (United States)

Burley, J; Harris, J

1999-01-01

In this paper we discuss an objection to human cloning which appeals to the welfare of the child. This objection varies according to the sort of harm it is expected the clone will suffer. The three formulations of it that we will consider are: 1. Clones will be harmed by the fearful or prejudicial attitudes people may have about or towards them (H1); 2. Clones will be harmed by the demands and expectations of parents or genotype donors (H2); 3. Clones will be harmed by their own awareness of their origins, for example the knowledge that the genetic donor is a stranger (H3). We will show why these three versions of the child welfare objection do not necessarily supply compelling reasons to ban human reproductive cloning. The claim that we will develop and defend in the course of our discussion is that even if it is the case that a cloned child will suffer harms of the type H1-H3, it is none the less permissible to conceive by cloning so long as these cloning-induced welfare deficits are not such as to blight the existence of the resultant child, whoever this may be. PMID:10226914

Automated Cell-Cutting for Cell Cloning

Science.gov (United States)

Ichikawa, Akihiko; Tanikawa, Tamio; Matsukawa, Kazutsugu; Takahashi, Seiya; Ohba, Kohtaro

We develop an automated cell-cutting technique for cell cloning. Animal cells softened by the cytochalasin treatment are injected into a microfluidic chip. The microfluidic chip contains two orthogonal channels: one microchannel is wide, used to transport cells, and generates the cutting flow; the other is thin and used for aspiration, fixing, and stretching of the cell. The injected cell is aspirated and stretched in the thin microchannel. Simultaneously, the volumes of the cell before and after aspiration are calculated; the volumes are used to calculate the fluid flow required to aspirate half the volume of the cell into the thin microchannel. Finally, we apply a high-speed flow in the orthogonal microchannel to bisect the cell. This paper reports the cutting process, the cutting system, and the results of the experiment.

Social behavior and kin discrimination in a mixed group of cloned and non cloned heifers (Bos taurus).

Science.gov (United States)

Coulon, M; Baudoin, C; Abdi, H; Heyman, Y; Deputte, B L

2010-12-01

For more than ten years, reproductive biotechnologies using somatic cell nuclear transfer have made possible the production of cloned animals in various domestic and laboratory species. The influence of the cloning process on offspring characteristics has been studied in various developmental aspects, however, it has not yet been documented in detail for behavioral traits. Behavioral studies of cloned animals have failed to show clear inter-individual differences associated with the cloning process. Preliminary results showed that clones favor each other's company. Preferential social interactions were observed among cloned heifers from the same donor in a mixed herd that also included cloned heifers and control heifers produced by artificial insemination (AI). These results suggest behavioral differences between cloned and non-cloned animals and similarities between clones from the same donor. The aim of the present study was to replicate and to extend these previous results and to study behavioral and cognitive mechanisms of this preferential grouping. We studied a group composed of five cloned heifers derived from the same donor cow, two cloned heifers derived from another donor cow, and AI heifers. Cloned heifers from the same donor were more spatially associated and interacted more between themselves than with heifers derived from another donor or with the AI individuals. This pattern indicates a possible kin discrimination in clones. To study this process, we performed an experiment (using an instrumental conditioning procedure with food reward) of visual discrimination between images of heads of familiar heifers, either related to the subjects or not. The results showed that all subjects (AI and cloned heifers) discriminated between images of familiar cloned heifers produced from the same donor and images of familiar unrelated heifers. Cattle discriminated well between images and used morphological similarities characteristic of cloned related heifers. Our

Hyperpolarization moves S4 sensors inward to open MVP, a methanococcal voltage-gated potassium channel.

Science.gov (United States)

Sesti, Federico; Rajan, Sindhu; Gonzalez-Colaso, Rosana; Nikolaeva, Natalia; Goldstein, Steve A N

2003-04-01

MVP, a Methanococcus jannaschii voltage-gated potassium channel, was cloned and shown to operate in eukaryotic and prokaryotic cells. Like pacemaker channels, MVP opens on hyperpolarization using S4 voltage sensors like those in classical channels activated by depolarization. The MVP S4 span resembles classical sensors in sequence, charge, topology and movement, traveling inward on hyperpolarization and outward on depolarization (via canaliculi in the protein that bring the extracellular and internal solutions into proximity across a short barrier). Thus, MVP opens with sensors inward indicating a reversal of S4 position and pore state compared to classical channels. Homologous channels in mammals and plants are expected to function similarly.

Optimally cloned binary coherent states

Science.gov (United States)

Müller, C. R.; Leuchs, G.; Marquardt, Ch.; Andersen, U. L.

2017-10-01

Binary coherent state alphabets can be represented in a two-dimensional Hilbert space. We capitalize this formal connection between the otherwise distinct domains of qubits and continuous variable states to map binary phase-shift keyed coherent states onto the Bloch sphere and to derive their quantum-optimal clones. We analyze the Wigner function and the cumulants of the clones, and we conclude that optimal cloning of binary coherent states requires a nonlinearity above second order. We propose several practical and near-optimal cloning schemes and compare their cloning fidelity to the optimal cloner.

Three concepts of cloning in human beings.

Science.gov (United States)

Cui, Ke-Hui

2005-07-01

Human cloning, organ cloning and tissue cloning are various types of cloning that occur at different levels with different methodologies. According to three standards of terminology for an embryo (fertilization through germ cells, development in the uterus and having the potential to produce a human life), tissue cloning and type I organ cloning will not produce an embryo. In contrast, human cloning and type II organ cloning will produce an embryo. Thus, only non-germinal tissue cloning and type I organ cloning are beyond the ethical question and will not change human beings as a species. Using cloned tissues to make new tissues or organs is promising for the future of medicine.

Local cloning of CAT states

International Nuclear Information System (INIS)

Rahaman, Ramij

2011-01-01

In this Letter we analyze the (im)possibility of the exact cloning of orthogonal three-qubit CAT states under local operation and classical communication (LOCC) with the help of a restricted entangled state. We also classify the three-qubit CAT states that can (not) be cloned under LOCC restrictions and extend the results to the n-qubit case. -- Highlights: → We analyze the (im)possibility of exact cloning of orthogonal CAT states under LOCC. → We also classify the set of CAT states that can(not) be cloned by LOCC. → No set of orthogonal CAT states can be cloned by LOCC with help of similar CAT state. → Any two orthogonal n-qubit GHZ-states can be cloned by LOCC with help of a GHZ state.

Effects of donor fibroblast cell type and transferred cloned embryo number on the efficiency of pig cloning.

Science.gov (United States)

Li, Zicong; Shi, Junsong; Liu, Dewu; Zhou, Rong; Zeng, Haiyu; Zhou, Xiu; Mai, Ranbiao; Zeng, Shaofen; Luo, Lvhua; Yu, Wanxian; Zhang, Shouquan; Wu, Zhenfang

2013-02-01

Currently, cloning efficiency in pigs is very low. Donor cell type and number of cloned embryos transferred to an individual surrogate are two major factors that affect the successful rate of somatic cell nuclear transfer (SCNT) in pigs. This study aimed to compare the influence of different donor fibroblast cell types and different transferred embryo numbers on recipients' pregnancy rate and delivery rate, the average number of total clones born, clones born alive and clones born healthy per litter, and the birth rate of healthy clones (=total number of healthy cloned piglets born /total number of transferred cloned embryos). Three types of donor fibroblasts were tested in large-scale production of cloned pigs, including fetal fibroblasts (FFBs) from four genetically similar Western swine breeds of Pietrain (P), Duroc (D), Landrace (L), and Yorkshire (Y), which are referred to as P,D,LY-FFBs, adult fibroblasts (AFBs) from the same four breeds, which are designated P,D,L,Y-AFBs, and AFBs from a Chinese pig breed of Laiwu (LW), which is referred to as LW-AFBs. Within each donor fibroblast cell type group, five transferred cloned embryo number groups were tested. In each embryo number group, 150-199, 200-249, 250-299, 300-349, or 350-450 cloned embryos were transferred to each individual recipient sow. For the entire experiment, 92,005 cloned embryos were generated from nearly 115,000 matured oocytes and transferred to 328 recipients; in total, 488 cloned piglets were produced. The results showed that the mean clones born healthy per litter resulted from transfer of embryos cloned from LW-AFBs (2.53 ± 0.34) was similar with that associated with P,D,L,Y-FFBs (2.72 ± 0.29), but was significantly higher than that resulted from P,D,L,Y-AFBs (1.47 ± 0.18). Use of LW-AFBs as donor cells for SCNT resulted in a significantly higher pregnancy rate (72.00% vs. 59.30% and 48.11%) and delivery rate (60.00% vs. 45.93% and 35.85%) for cloned embryo recipients, and a

Lessons learned from cloning dogs.

Science.gov (United States)

Kim, M J; Oh, H J; Kim, G A; Park, J E; Park, E J; Jang, G; Ra, J C; Kang, S K; Lee, B C

2012-08-01

The aim of this article is to review dog cloning research and to suggest its applications based on a discussion about the normality of cloned dogs. Somatic cell nuclear transfer was successfully used for production of viable cloned puppies despite limited understanding of in vitro dog embryo production. Cloned dogs have similar growth characteristics to those born from natural fertilization, with no evidence of serious adverse effects. The offspring of cloned dogs also have similar growth performance and health to those of naturally bred puppies. Therefore, cloning in domestic dogs can be applied as an assisted reproductive technique to conserve endangered species, to treat sterile canids or aged dogs, to improve reproductive performance of valuable individuals and to generate disease model animals. © 2012 Blackwell Verlag GmbH.

Consumers' attitudes toward consumption of cloned beef. The impact of exposure to technological information about animal cloning.

Science.gov (United States)

Aizaki, Hideo; Sawada, Manabu; Sato, Kazuo

2011-10-01

Novel food technologies, such as cloning, have been introduced into the meat production sector; however, their use is not widely supported by many consumers. This study was designed to assess whether Japanese consumers' attitudes toward consumption of cloned beef (specifically, beef derived from bovine embryo and somatic cell-cloned cattle) would change after they were provided with technological information on animal cloning through a web-based survey. The results revealed that most respondents did not discriminate between their attitudes toward the consumption of the two types of cloned beef, and that most respondents did not change their attitudes toward cloned beef after receiving the technological information. The respondents' individual characteristics, including their knowledge about the food safety of cloned beef and their basic knowledge about animal cloning, influenced the likelihood of a change in their attitudes after they received the information. In conclusion, some consumers might become less uncomfortable about the consumption of cloned beef by the straightforward provision of technological information about animal cloning; however, most consumers are likely to maintain their attitudes. Copyright © 2011 Elsevier Ltd. All rights reserved.

Deterministic Assisted Clone of an Arbitrary Two- and Three-qubit States via Multi-qubit Brown State

Science.gov (United States)

Hou, Kui; Zhu, Cheng-Jie; Yang, Ya-Ping

2017-08-01

We present two schemes for deterministic assisted clone(DAC) of an unknown two- and three-qubit entangled states with assistance via muti-qubit Brown state. In the schemes, the sender wish to teleport an unknown original entangled state which from the state preparer, and then create a perfect copy of the unknown state at her place. The DAC schemes include two stages. The first stage requires teleportation with Bell-state measurements via a five-qubit Brown state(or seven-qubit Brown state) as the quantum channel. In the second stage, to help the sender realize the quantum cloning, the state preparer performs projective measurements on their own particles which from the sender, then the sender can acquire a perfect copy of the unknown state by means of some appropriate unitary operations. Furthermore, the total success probability for assisted cloning a perfect copy of the unknown state can reach 1 in our schemes.

FastCloning: a highly simplified, purification-free, sequence- and ligation-independent PCR cloning method

Directory of Open Access Journals (Sweden)

Lu Jia

2011-10-01

Full Text Available Abstract Background Although a variety of methods and expensive kits are available, molecular cloning can be a time-consuming and frustrating process. Results Here we report a highly simplified, reliable, and efficient PCR-based cloning technique to insert any DNA fragment into a plasmid vector or into a gene (cDNA in a vector at any desired position. With this method, the vector and insert are PCR amplified separately, with only 18 cycles, using a high fidelity DNA polymerase. The amplified insert has the ends with ~16-base overlapping with the ends of the amplified vector. After DpnI digestion of the mixture of the amplified vector and insert to eliminate the DNA templates used in PCR reactions, the mixture is directly transformed into competent E. coli cells to obtain the desired clones. This technique has many advantages over other cloning methods. First, it does not need gel purification of the PCR product or linearized vector. Second, there is no need of any cloning kit or specialized enzyme for cloning. Furthermore, with reduced number of PCR cycles, it also decreases the chance of random mutations. In addition, this method is highly effective and reproducible. Finally, since this cloning method is also sequence independent, we demonstrated that it can be used for chimera construction, insertion, and multiple mutations spanning a stretch of DNA up to 120 bp. Conclusion Our FastCloning technique provides a very simple, effective, reliable, and versatile tool for molecular cloning, chimera construction, insertion of any DNA sequences of interest and also for multiple mutations in a short stretch of a cDNA.

Cloning, killing, and identity.

Science.gov (United States)

McMahan, J

1999-01-01

One potentially valuable use of cloning is to provide a source of tissues or organs for transplantation. The most important objection to this use of cloning is that a human clone would be the sort of entity that it would be seriously wrong to kill. I argue that entities of the sort that you and I essentially are do not begin to exist until around the seventh month of fetal gestation. Therefore to kill a clone prior to that would not be to kill someone like you or me but would be only to prevent one of us from existing. And even after one of us begins to exist, the objections to killing it remain comparatively weak until its psychological capacities reach a certain level of maturation. These claims support the permissibility of killing a clone during the early stages of its development in order to use its organs for transplantation. PMID:10226909

Cloning-free CRISPR

NARCIS (Netherlands)

Arbab, Mandana; Srinivasan, Sharanya; Hashimoto, Tatsunori; Geijsen, Niels; Sherwood, Richard I.

2015-01-01

We present self-cloning CRISPR/Cas9 (scCRISPR), a technology that allows for CRISPR/Cas9-mediated genomic mutation and site-specific knockin transgene creation within several hours by circumventing the need to clone a site-specific single-guide RNA (sgRNA) or knockin homology construct for each

Animal cloning: problems and prospects.

Science.gov (United States)

Wells, D N

2005-04-01

An efficient animal cloning technology would provide many new opportunities for livestock agriculture, human medicine, and animal conservation. Nuclear cloning involves the production of animals that are genetically identical to the donor cells used in a technique known as nuclear transfer (NT). However, at present it is an inefficient process: in cattle, only around 6% of the embryos transferred to the reproductive tracts of recipient cows result in healthy, longterm surviving clones. Of concern are the high losses throughout gestation, during birth and in the post-natal period through to adulthood. Many of the pregnancy losses relate to failure of the placenta to develop and function correctly. Placental dysfunction may also have an adverse influence on postnatal health. These anomalies are probably due to incorrect epigenetic reprogramming of the donor genome following NT, leading to inappropriate patterns of gene expression during the development of clones. Whilst some physiological tests on surviving clones suggest normality, other reports indicate a variety of post-natal clone-associated abnormalities. This variability in outcome may reflect species-specific and/or cloning methodological differences. Importantly, to date it appears that these clone-associated phenotypes are not transmitted to offspring following sexual reproduction. This indicates that they represent epigenetic errors, rather than genetic errors, which are corrected during gametogenesis. Whilst this needs confirmation at the molecular level, it provides initial confidence in the first application of NT in agriculture, namely, the production of small numbers of cloned sires from genetically elite bulls, for natural mating, to effectively disseminate genetic gain. In addition to the animal welfare concerns with the technology, the underlying health of the animals and the consequential effect on food safety are critical aspects that require investigation to gain regulatory and consumer

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

DEFF Research Database (Denmark)

Larsen, Anders Peter; Olesen, Søren-Peter

2010-01-01

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...

Reversibility of continuous-variable quantum cloning

International Nuclear Information System (INIS)

Filip, Radim; Marek, Petr; Fiurasek, Jaromir

2004-01-01

We analyze a reversibility of optimal Gaussian 1→2 quantum cloning of a coherent state using only local operations on the clones and classical communication between them and propose a feasible experimental test of this feature. Performing Bell-type homodyne measurement on one clone and anticlone, an arbitrary unknown input state (not only a coherent state) can be restored in the other clone by applying appropriate local unitary displacement operation. We generalize this concept to a partial reversal of the cloning using only local operations and classical communication (LOCC) and we show that this procedure converts the symmetric cloner to an asymmetric cloner. Further, we discuss a distributed LOCC reversal in optimal 1→M Gaussian cloning of coherent states which transforms it to optimal 1→M ' cloning for M ' < M. Assuming the quantum cloning as a possible eavesdropping attack on quantum communication link, the reversibility can be utilized to improve the security of the link even after the attack

Human cloning. Fact or fiction

International Nuclear Information System (INIS)

Abushama, Mandy D.; Ahmed, Badreldeen I.

2003-01-01

Cloning is the production of one or more individual plants or animals that are genetically identical to other plant, animal or human. Scientists even demonstrated that they were able to clone frog tadpoles from frog embryonic cells using nuclear transfer.Many animals have been cloned from adult cells using nuclear transfer. Somatic cell nuclear transfer which refers to the transfer of the nucleous from a somatic cell to an egg cell. Article further deals with benefits and misuses of human cloning

Local cloning of two product states

International Nuclear Information System (INIS)

Ji Zhengfeng; Feng Yuan; Ying Mingsheng

2005-01-01

Local quantum operations and classical communication (LOCC) put considerable constraints on many quantum information processing tasks such as cloning and discrimination. Surprisingly, however, discrimination of any two pure states survives such constraints in some sense. We show that cloning is not that lucky; namely, probabilistic LOCC cloning of two product states is strictly less efficient than global cloning. We prove our result by giving explicitly the efficiency formula of local cloning of any two product states

Structured Review of Code Clone Literature

NARCIS (Netherlands)

Hordijk, W.T.B.; Ponisio, Laura; Wieringa, Roelf J.

2008-01-01

This report presents the results of a structured review of code clone literature. The aim of the review is to assemble a conceptual model of clone-related concepts which helps us to reason about clones. This conceptual model unifies clone concepts from a wide range of literature, so that findings

Discovery of a new class of potential multifunctional atypical antipsychotic agents targeting dopamine D3 and serotonin 5-HT1A and 5-HT2A receptors: design, synthesis, and effects on behavior

DEFF Research Database (Denmark)

Butini, Stefania; Gemma, Sandra; Campiani, Giuseppe

2009-01-01

with a low affinity for dopamine D(2) receptors (to minimize extrapyramidal side effects), serotonin 5-HT(2C) receptors (to reduce the risk of obesity under chronic treatment), and for hERG channels (to reduce incidence of torsade des pointes). Pharmacological and biochemical data, including specific c...

Biophysical characterization of the short QT mutation hERG-N588K reveals a mixed gain-and loss-of-function

DEFF Research Database (Denmark)

Grunnet, M.; Diness, T.G.; Hansen, R.S.

2008-01-01

The short QT syndrome is a newly discovered pro-arrhythmic condition, which may cause ventricular fibrillation and sudden death. Short QT can originate from the apparent gain-of-function mutation N588K in the hERG potassium channel that conducts repolarising I(Kr) current. The present study...

Human cloning: can it be made safe?

Science.gov (United States)

Rhind, Susan M; Taylor, Jane E; De Sousa, Paul A; King, Tim J; McGarry, Michelle; Wilmut, Ian

2003-11-01

There are continued claims of attempts to clone humans using nuclear transfer, despite the serious problems that have been encountered in cloning other mammals. It is known that epigenetic and genetic mechanisms are involved in clone failure, but we still do not know exactly how. Human reproductive cloning is unethical, but the production of cells from cloned embryos could offer many potential benefits. So, can human cloning be made safe?

Probabilistic cloning and deleting of quantum states

International Nuclear Information System (INIS)

Feng Yuan; Zhang Shengyu; Ying Mingsheng

2002-01-01

We construct a probabilistic cloning and deleting machine which, taking several copies of an input quantum state, can output a linear superposition of multiple cloning and deleting states. Since the machine can perform cloning and deleting in a single unitary evolution, the probabilistic cloning and other cloning machines proposed in the previous literature can be thought of as special cases of our machine. A sufficient and necessary condition for successful cloning and deleting is presented, and it requires that the copies of an arbitrarily presumed number of the input states are linearly independent. This simply generalizes some results for cloning. We also derive an upper bound for the success probability of the cloning and deleting machine

Effective and efficient model clone detection

DEFF Research Database (Denmark)

Störrle, Harald

2015-01-01

Code clones are a major source of software defects. Thus, it is likely that model clones (i.e., duplicate fragments of models) have a significant negative impact on model quality, and thus, on any software created based on those models, irrespective of whether the software is generated fully...... automatically (“MDD-style”) or hand-crafted following the blueprint defined by the model (“MBSD-style”). Unfortunately, however, model clones are much less well studied than code clones. In this paper, we present a clone detection algorithm for UML domain models. Our approach covers a much greater variety...... of model types than existing approaches while providing high clone detection rates at high speed....

Wildlife conservation and reproductive cloning.

Science.gov (United States)

Holt, William V; Pickard, Amanda R; Prather, Randall S

2004-03-01

Reproductive cloning, or the production of offspring by nuclear transfer, is often regarded as having potential for conserving endangered species of wildlife. Currently, however, low success rates for reproductive cloning limit the practical application of this technique to experimental use and proof of principle investigations. In this review, we consider how cloning may contribute to wildlife conservation strategies. The cloning of endangered mammals presents practical problems, many of which stem from the paucity of knowledge about their basic reproductive biology. However, situations may arise where resources could be targeted at recovering lost or under-represented genetic lines; these could then contribute to the future fitness of the population. Approaches of this type would be preferable to the indiscriminate generation of large numbers of identical individuals. Applying cloning technology to non-mammalian vertebrates may be more practical than attempting to use conventional reproductive technologies. As the scientific background to cloning technology was pioneered using amphibians, it may be possible to breed imminently threatened amphibians, or even restore extinct amphibian species, by the use of cloning. In this respect species with external embryonic development may have an advantage over mammals as developmental abnormalities associated with inappropriate embryonic reprogramming would not be relevant.

Properties and function of KCNQ1 K+ channels isolated from the rectal gland of Squalus acanthias.

Science.gov (United States)

Kerst, G; Beschorner, U; Unsöld, B; von Hahn, T; Schreiber, R; Greger, R; Gerlach, U; Lang, H J; Kunzelmann, K; Bleich, M

2001-10-01

KCNQ1 (KVLQT1) K+ channels play an important role during electrolyte secretion in airways and colon. KCNQ1 was cloned recently from NaCl-secreting shark rectal glands. Here we study the properties and regulation of the cloned sKVLQT1 expressed in Xenopus oocytes and Chinese hamster ovary (CHO) cells and compare the results with those obtained from in vitro perfused rectal gland tubules (RGT). The expression of sKCNQ1 induced voltage-dependent, delayed activated K+ currents, which were augmented by an increase in intracellular cAMP and Ca2+. The chromanol derivatives 293B and 526B potently inhibited sKCNQ1 expressed in oocytes and CHO cells, but had little effect on RGT electrolyte transport. Short-circuit currents in RGT were activated by alkalinization and were decreased by acidification. In CHO cells an alkaline pH activated and an acidic pH inhibited 293B-sensitive KCNQ1 currents. Noise analysis of the cell-attached basolateral membrane of RGT indicated the presence of low-conductance (<3 pS) K+ channels, in parallel with other K+ channels. sKCNQ1 generated similar small-conductance K+ channels upon expression in CHO cells and Xenopus oocytes. The results suggest the presence of low-conductance KCNQ1 K+ channels in RGT, which are probably regulated by changes in intracellular cAMP, Ca2+ and pH.

Voltage-gated calcium channels of Paramecium cilia.

Science.gov (United States)

Lodh, Sukanya; Yano, Junji; Valentine, Megan S; Van Houten, Judith L

2016-10-01

Paramecium cells swim by beating their cilia, and make turns by transiently reversing their power stroke. Reversal is caused by Ca 2+ entering the cilium through voltage-gated Ca 2+ (Ca V ) channels that are found exclusively in the cilia. As ciliary Ca 2+ levels return to normal, the cell pivots and swims forward in a new direction. Thus, the activation of the Ca V channels causes cells to make a turn in their swimming paths. For 45 years, the physiological characteristics of the Paramecium ciliary Ca V channels have been known, but the proteins were not identified until recently, when the P. tetraurelia ciliary membrane proteome was determined. Three Ca V α1 subunits that were identified among the proteins were cloned and confirmed to be expressed in the cilia. We demonstrate using RNA interference that these channels function as the ciliary Ca V channels that are responsible for the reversal of ciliary beating. Furthermore, we show that Pawn (pw) mutants of Paramecium that cannot swim backward for lack of Ca V channel activity do not express any of the three Ca V 1 channels in their ciliary membrane, until they are rescued from the mutant phenotype by expression of the wild-type PW gene. These results reinforce the correlation of the three Ca V channels with backward swimming through ciliary reversal. The PwB protein, found in endoplasmic reticulum fractions, co-immunoprecipitates with the Ca V 1c channel and perhaps functions in trafficking. The PwA protein does not appear to have an interaction with the channel proteins but affects their appearance in the cilia. © 2016. Published by The Company of Biologists Ltd.

Islamic perspectives on human cloning.

Science.gov (United States)

Sadeghi, Mahmoud

2007-01-01

The present paper seeks to assess various views from Islamic jurists relating to human cloning, which is one of the controversial topics in the recent past. Taking Islamic jurisprudence principles, such as the rule of necessity for self preservation and respect for human beings, the rule of la darar wa la dirar ('the necessity to refrain from causing harm to oneself and others') and the rule of usr wa haraj, one may indicate that if human cloning could not be prohibited, as such, it could still be opposed because it gives way to various harmful consequences, which include family disorder, chaos in the clone's family relationships, physical and mental diseases for clones and suffering of egg donors and surrogate mothers. However with due attention to the fact that the reasons behind the prohibition of abortion only restrict the destruction of human embryos in their post-implantation stages, human cloning for biomedical research and exploitation of stem cells from cloned embryos at the blastocyst stage for therapeutic purposes would be acceptable.

Quantum cloning machines for equatorial qubits

International Nuclear Information System (INIS)

Fan Heng; Matsumoto, Keiji; Wang Xiangbin; Wadati, Miki

2002-01-01

Quantum cloning machines for equatorial qubits are studied. For the case of a one to two phase-covariant quantum cloning machine, we present the networks consisting of quantum gates to realize the quantum cloning transformations. The copied equatorial qubits are shown to be separable by using Peres-Horodecki criterion. The optimal one to M phase-covariant quantum cloning transformations are given

Update on the First Cloned Dog and Outlook for Canine Cloning.

Science.gov (United States)

Jang, Goo; Lee, ByeongChun

2015-10-01

As man's best friend, dogs have an important position in human society. Ten years ago, we reported the first cloned dog, and his birth has raised various scientific issues, such as those related to health, reproduction, and life span. He has developed without any unique health issues. In this article, we summarize and present perspectives on canine cloning.

Therapeutic and reproductive cloning: a critique.

Science.gov (United States)

Bowring, Finn

2004-01-01

This article is a critical examination of the science and ethics of human cloning. It summarises the key scientific milestones in the development of nuclear transplantation, explains the importance of cloning to research into the medical potential of embryonic stem cells, and discusses the well-worn distinction between 'therapeutic' and 'reproductive' cloning. Suggesting that this distinction will be impossible to police, it goes on to consider the ethics of full human cloning. It is concluded that it represents an unacceptable form of parental despotism, and that the genetic engineering and cloning of future human beings will fracture the foundations of modern humanism.

Novel cloning machine with supplementary information

International Nuclear Information System (INIS)

Qiu Daowen

2006-01-01

Probabilistic cloning was first proposed by Duan and Guo. Then Pati established a novel cloning machine (NCM) for copying superposition of multiple clones simultaneously. In this paper, we deal with the novel cloning machine with supplementary information (NCMSI). For the case of cloning two states, we demonstrate that the optimal efficiency of the NCMSI in which the original party and the supplementary party can perform quantum communication equals that achieved by a two-step cloning protocol wherein classical communication is only allowed between the original and the supplementary parties. From this equivalence, it follows that NCMSI may increase the success probabilities for copying. Also, an upper bound on the unambiguous discrimination of two nonorthogonal pure product states is derived. Our investigation generalizes and completes the results in the literature

Artificial cloning of domestic animals.

Science.gov (United States)

Keefer, Carol L

2015-07-21

Domestic animals can be cloned using techniques such as embryo splitting and nuclear transfer to produce genetically identical individuals. Although embryo splitting is limited to the production of only a few identical individuals, nuclear transfer of donor nuclei into recipient oocytes, whose own nuclear DNA has been removed, can result in large numbers of identical individuals. Moreover, clones can be produced using donor cells from sterile animals, such as steers and geldings, and, unlike their genetic source, these clones are fertile. In reality, due to low efficiencies and the high costs of cloning domestic species, only a limited number of identical individuals are generally produced, and these clones are primarily used as breed stock. In addition to providing a means of rescuing and propagating valuable genetics, somatic cell nuclear transfer (SCNT) research has contributed knowledge that has led to the direct reprogramming of cells (e.g., to induce pluripotent stem cells) and a better understanding of epigenetic regulation during embryonic development. In this review, I provide a broad overview of the historical development of cloning in domestic animals, of its application to the propagation of livestock and transgenic animal production, and of its scientific promise for advancing basic research.

[Scientific ethics of human cloning].

Science.gov (United States)

Valenzuela, Carlos Y

2005-01-01

True cloning is fission, budding or other types of asexual reproduction. In humans it occurs in monozygote twinning. This type of cloning is ethically and religiously good. Human cloning can be performed by twinning (TWClo) or nuclear transfer (NTClo). Both methods need a zygote or a nuclear transferred cell, obtained in vitro (IVTec). They are under the IVTec ethics. IVTecs use humans (zygotes, embryos) as drugs or things; increase the risk of malformations; increase development and size of abnormalities and may cause long-term changes. Cloning for preserving extinct (or almost extinct) animals or humans when sexual reproduction is not possible is ethically valid. The previous selection of a phenotype in human cloning violates some ethical principles. NTClo for reproductive or therapeutic purposes is dangerous since it increases the risk for nucleotide or chromosome mutations, de-programming or re-programming errors, aging or malignancy of the embryo cells thus obtained.

The ethics of human reproductive cloning.

Science.gov (United States)

Strong, Carson

2005-03-01

This article addresses the question of whether human reproductive cloning could be ethically justifiable in at least some cases involving infertile couples who would choose cloning as a way to have a genetically related child. At present, the risk of congenital anomalies constitutes a compelling argument against human reproductive cloning. The article explores whether reproductive cloning could be ethically justifiable if, at some future time, cloning becomes possible without an elevated risk of anomalies. It is argued that freedom to use cloning is a form of procreative freedom and, as such, deserves respect. All of the objections that have been raised against human reproductive cloning fall under three main categories: those that appeal to the interests of the child, those based on consequences for society, and those arising from teleological views. Objections that appeal to the child's interests are, in turn, of two main kinds: consequentialist and deontological. All of these types of objections are examined, and it is found that each involves serious problems that prevent it from being a reasonable objection in the context of the infertility cases considered. It is concluded that human reproductive cloning would be ethically justifiable in at least some cases involving infertile couples, provided that it could be performed without an elevated risk of anomalies.

Therapeutic cloning: The ethical limits

International Nuclear Information System (INIS)

Whittaker, Peter A.

2005-01-01

A brief outline of stem cells, stem cell therapy and therapeutic cloning is given. The position of therapeutic cloning with regard to other embryonic manipulations - IVF-based reproduction, embryonic stem formation from IVF embryos and reproductive cloning - is indicated. The main ethically challenging stages in therapeutic cloning are considered to be the nuclear transfer process including the source of eggs for this and the destruction of an embryo to provide stem cells for therapeutic use. The extremely polarised nature of the debate regarding the status of an early human embryo is noted, and some potential alternative strategies for preparing immunocompatible pluripotent stem cells are indicated

Economical quantum cloning in any dimension

International Nuclear Information System (INIS)

Durt, Thomas; Fiurasek, Jaromir; Cerf, Nicolas J.

2005-01-01

The possibility of cloning a d-dimensional quantum system without an ancilla is explored, extending on the economical phase-covariant cloning machine for qubits found in Phys. Rev. A 60, 2764 (1999). We prove the impossibility of constructing an economical version of the optimal universal 1→2 cloning machine in any dimension. We also show, using an ansatz on the generic form of cloning machines, that the d-dimensional 1→2 phase-covariant cloner, which optimally clones all balanced superpositions with arbitrary phases, can be realized economically only in dimension d=2. The used ansatz is supported by numerical evidence up to d=7. An economical phase-covariant cloner can nevertheless be constructed for d>2, albeit with a slightly lower fidelity than that of the optimal cloner requiring an ancilla. Finally, using again an ansatz on cloning machines, we show that an economical version of the 1→2 Fourier-covariant cloner, which optimally clones the computational basis and its Fourier transform, is also possible only in dimension d=2

No-cloning theorem on quantum logics

International Nuclear Information System (INIS)

Miyadera, Takayuki; Imai, Hideki

2009-01-01

This paper discusses the no-cloning theorem in a logicoalgebraic approach. In this approach, an orthoalgebra is considered as a general structure for propositions in a physical theory. We proved that an orthoalgebra admits cloning operation if and only if it is a Boolean algebra. That is, only classical theory admits the cloning of states. If unsharp propositions are to be included in the theory, then a notion of effect algebra is considered. We proved that an atomic Archimedean effect algebra admitting cloning operation is a Boolean algebra. This paper also presents a partial result, indicating a relation between the cloning on effect algebras and hidden variables.

Phase-covariant quantum cloning of qudits

International Nuclear Information System (INIS)

Fan Heng; Imai, Hiroshi; Matsumoto, Keiji; Wang, Xiang-Bin

2003-01-01

We study the phase-covariant quantum cloning machine for qudits, i.e., the input states in a d-level quantum system have complex coefficients with arbitrary phase but constant module. A cloning unitary transformation is proposed. After optimizing the fidelity between input state and single qudit reduced density operator of output state, we obtain the optimal fidelity for 1 to 2 phase-covariant quantum cloning of qudits and the corresponding cloning transformation

Effect of TH-lines and clones on the growth and differentiation of B cell clones in microculture.

Science.gov (United States)

Kotloff, D B; Cebra, J J

1988-02-01

Antibody isotype expression by B cell clones was analyzed using in vitro microcultures containing low numbers of hapten-gelatin-enriched B cells and higher numbers of hemocyanin-specific helper T cell lines or clones. Twenty-eight to sixty-three percent of clones grown in microculture with haptenated hemocyanin and T cells from established lines expressed IgG and/or IgA isotypes in random mixtures, almost always accompanied by IgM. Helper T cells from hemocyanin-specific clones also supported the expression of non-IgM isotypes by the B cell clones, suggesting that a single specificity of T cell can provide sufficient growth and differentiation factors for the display of isotype switching. A positive correlation between the antibody output of clones and the expression of non-IgM isotypes indicated that the switching process may be associated with cell division. Although memory B cells that give clones expressing IgG and/or IgA in the absence of IgM are also enriched on haptenated gelatin, they are not stimulable under conditions of this microculture assay.

Human reproductive cloning: a conflict of liberties.

Science.gov (United States)

Havstad, Joyce C

2010-02-01

Proponents of human reproductive cloning do not dispute that cloning may lead to violations of clones' right to self-determination, or that these violations could cause psychological harms. But they proceed with their endorsement of human reproductive cloning by dismissing these psychological harms, mainly in two ways. The first tactic is to point out that to commit the genetic fallacy is indeed a mistake; the second is to invoke Parfit's non-identity problem. The argument of this paper is that neither approach succeeds in removing our moral responsibility to consider and to prevent psychological harms to cloned individuals. In fact, the same commitment to personal liberty that generates the right to reproduce by means of cloning also creates the need to limit that right appropriately. Discussion of human reproductive cloning ought to involve a careful and balanced consideration of both the relevant aspects of personal liberty - the parents' right to reproductive freedom and the cloned child's right to self-determination.

Changes in the gut microbiota of cloned and non-cloned control pigs during development of obesity: gut microbiota during development of obesity in cloned pigs

DEFF Research Database (Denmark)

Pedersen, Rebecca; Andersen, Anders Daniel; Mølbak, Lars

2013-01-01

Background Obesity induced by a high-caloric diet has previously been associated with changes in the gut microbiota in mice and in humans. In this study, pigs were cloned to minimize genetic and biological variation among the animals with the aim of developing a controlled metabolomic model...... suitable for a diet-intervention study. Cloning of pigs may be an attractive way to reduce genetic influences when investigating the effect of diet and obesity on different physiological sites. The aim of this study was to assess and compare the changes in the composition of the gut microbiota of cloned vs....... non-cloned pigs during development of obesity by a high-fat/high-caloric diet. Furthermore, we investigated the association between diet-induced obesity and the relative abundance of the phyla Firmicutes and Bacteroidetes in the fecal-microbiota. The fecal microbiota from obese cloned (n = 5) and non...

Probabilistic cloning of three symmetric states

International Nuclear Information System (INIS)

Jimenez, O.; Bergou, J.; Delgado, A.

2010-01-01

We study the probabilistic cloning of three symmetric states. These states are defined by a single complex quantity, the inner product among them. We show that three different probabilistic cloning machines are necessary to optimally clone all possible families of three symmetric states. We also show that the optimal cloning probability of generating M copies out of one original can be cast as the quotient between the success probability of unambiguously discriminating one and M copies of symmetric states.

BIOETHICS AND HUMAN CLONING

Directory of Open Access Journals (Sweden)

Željko Kaluđerović

2011-12-01

Full Text Available In this paper the authors analyze the process of negotiating and beginning of the United Nations Declaration on Human Cloning as well as the paragraphs of the very Declaration. The negotiation was originally conceived as a clear bioethical debate that should have led to a general agreement to ban human cloning. However, more often it had been discussed about human rights, cultural, civil and religious differences between people and about priorities in case of eventual conflicts between different value systems. In the end, a non-binding Declaration on Human Cloning had been adopted, full of numerous compromises and ambiguous formulations, that relativized the original intention of proposer states. According to authors, it would have been better if bioethical discussion and eventual regulations on cloning mentioned in the following text had been left over to certain professional bodies, and only after the public had been fully informed about it should relevant supranational organizations have taken that into consideration.

The First Human Cloned Embryo.

Science.gov (United States)

Cibelli, Jose B.; Lanza, Robert P.; West, Michael D.; Ezzell, Carol

2002-01-01

Describes a process known as parthenogenesis which produces cloned, early-stage embryos and human embryos generated only from eggs. Speculates that this technology puts therapeutic cloning within reach. (DDR)

Automated cloning methods.; TOPICAL

International Nuclear Information System (INIS)

Collart, F.

2001-01-01

Argonne has developed a series of automated protocols to generate bacterial expression clones by using a robotic system designed to be used in procedures associated with molecular biology. The system provides plate storage, temperature control from 4 to 37 C at various locations, and Biomek and Multimek pipetting stations. The automated system consists of a robot that transports sources from the active station on the automation system. Protocols for the automated generation of bacterial expression clones can be grouped into three categories (Figure 1). Fragment generation protocols are initiated on day one of the expression cloning procedure and encompass those protocols involved in generating purified coding region (PCR)

Skewed X-inactivation in cloned mice

International Nuclear Information System (INIS)

Senda, Sho; Wakayama, Teruhiko; Yamazaki, Yukiko; Ohgane, Jun; Hattori, Naka; Tanaka, Satoshi; Yanagimachi, Ryuzo; Shiota, Kunio

2004-01-01

In female mammals, dosage compensation for X-linked genes is accomplished by inactivation of one of two X chromosomes. The X-inactivation ratio (a percentage of the cells with inactivated maternal X chromosomes in the whole cells) is skewed as a consequence of various genetic mutations, and has been observed in a number of X-linked disorders. We previously reported that phenotypically normal full-term cloned mouse fetuses had loci with inappropriate DNA methylation. Thus, cloned mice are excellent models to study abnormal epigenetic events in mammalian development. In the present study, we analyzed X-inactivation ratios in adult female cloned mice (B6C3F1). Kidneys of eight naturally produced controls and 11 cloned mice were analyzed. Although variations in X-inactivation ratio among the mice were observed in both groups, the distributions were significantly different (Ansary-Bradley test, P < 0.01). In particular, 2 of 11 cloned mice showed skewed X-inactivation ratios (19.2% and 86.8%). Similarly, in intestine, 1 of 10 cloned mice had a skewed ratio (75.7%). Skewed X-inactivation was observed to various degrees in different tissues of different individuals, suggesting that skewed X-inactivation in cloned mice is the result of secondary cell selection in combination with stochastic distortion of primary choice. The present study is the first demonstration that skewed X-inactivation occurs in cloned animals. This finding is important for understanding both nuclear transfer technology and etiology of X-linked disorders

Long QT 1 mutation KCNQ1A344V increases local anesthetic sensitivity of the slowly activating delayed rectifier potassium current

DEFF Research Database (Denmark)

Siebrands, Cornelia C; Binder, Stephan; Eckhoff, Ulrike

2006-01-01

BACKGROUND: Anesthesia in patients with long QT syndrome (LQTS) is a matter of concern. Congenital LQTS is most frequently caused by mutations in KCNQ1 (Kv7.1), whereas drug-induced LQTS is a consequence of HERG (human ether-a-go-go-related gene) channel inhibition. The aim of this study was to i...

Species-specific challenges in dog cloning.

Science.gov (United States)

Kim, G A; Oh, H J; Park, J E; Kim, M J; Park, E J; Jo, Y K; Jang, G; Kim, M K; Kim, H J; Lee, B C

2012-12-01

Somatic cell nuclear transfer (SCNT) is now an established procedure used in cloning of several species. SCNT in dogs involves multiple steps including the removal of the nuclear material, injection of a donor cell, fusion, activation of the reconstructed oocytes and finally transfer to a synchronized female recipient. There are therefore many factors that contribute to cloning efficiency. By performing a retrospective analysis of 2005-2012 published papers regarding dog cloning, we define the optimum procedure and summarize the specific feature for dog cloning. © 2012 Blackwell Verlag GmbH.

Mechanism of action of a novel human ether-a-go-go-related gene channel activator

DEFF Research Database (Denmark)

Casis, Oscar; Olesen, Søren-Peter; Sanguinetti, Michael C

2005-01-01

depolarizations, NS1643 enhanced the magnitude of wild-type hERG current in a concentration- and voltage-dependent manner with an EC(50) of 10.4 microM at -10 mV. The fully activated current-voltage relationship revealed that the drug increased outward but not inward currents, consistent with altered inactivation...

Assessment of the genetic diversity of natural rubber tree clones of the SINCHI Institutes clone collection, using of morphological descriptors

International Nuclear Information System (INIS)

Quesada Mendez, Isaac; Quintero Barrera, Lorena; Aristizabal, Fabio A; Rodriguez Acuna, Olga

2011-01-01

Genetic diversity of natural rubber clones of the in SINCHI Institute’s clone collection was assessed. Clones of Hevea brasiliensis (Willd. ex Adr. De Juss.) Muell.Arg., Hevea spp. (H. brasiliensis x H. benthamiana), and three more species of Hevea genus are a part of the collection. Seventy-two materials were characterized with twenty-eight morphological descriptors. They were later used to generate a similarity matrix through the analysis of multi-categorical variables, and to obtain clusters based on the matrix. A low variability between clones of H. brasiliensis and H. spp. was observed, presumably because of the direct descendants of most of the materials from crosses of parental PB 80, PB 5/51, PB 49 and Tjir, exception made of clone GU 1410. Clustering between some materials product of exclusive cross of PB series, a group between clones descendants of parental clones PB 86, and clustering between descendants of parental clones PB 5/51, were observed. Clones from other species of Hevea differ from this big group.

Unified universal quantum cloning machine and fidelities

Energy Technology Data Exchange (ETDEWEB)

Wang Yinan; Shi Handuo; Xiong Zhaoxi; Jing Li; Mu Liangzhu [School of Physics, Peking University, Beijing 100871 (China); Ren Xijun [School of Physics and Electronics, Henan University, Kaifeng 4750011 (China); Fan Heng [Institute of Physics, Chinese Academy of Sciences, Beijing 100190 (China)

2011-09-15

We present a unified universal quantum cloning machine, which combines several different existing universal cloning machines together, including the asymmetric case. In this unified framework, the identical pure states are projected equally into each copy initially constituted by input and one half of the maximally entangled states. We show explicitly that the output states of those universal cloning machines are the same. One importance of this unified cloning machine is that the cloning procession is always the symmetric projection, which reduces dramatically the difficulties for implementation. Also, it is found that this unified cloning machine can be directly modified to the general asymmetric case. Besides the global fidelity and the single-copy fidelity, we also present all possible arbitrary-copy fidelities.

Modulation of voltage-gated Na+ and K+ channels by pumiliotoxin 251D: a "joint venture" alkaloid from arthropods and amphibians.

Science.gov (United States)

Vandendriessche, Thomas; Abdel-Mottaleb, Yousra; Maertens, Chantal; Cuypers, Eva; Sudau, Alexander; Nubbemeyer, Udo; Mebs, Dietrich; Tytgat, Jan

2008-03-01

Certain amphibians provide themselves with a chemical defense by accumulating lipophilic alkaloids into skin glands from dietary arthropods. Examples of such alkaloids are pumiliotoxins (PTXs). In general, PTXs are known as positive modulators of voltage-gated sodium channels (VGSCs). Unlike other PTXs, PTX 251D does not share this characteristic. However, mice and insect studies showed that PTX 251D is highly toxic and to date the basis of its toxicity remains unknown. In this work, we searched for the possible target of PTX 251D. The toxin was therefore made synthetically and tested on four VGSCs (mammalian rNa(v)1.2/beta(1), rNa(v)1.4/beta(1), hNa(v)1.5/beta(1) and insect Para/tipE) and five voltage-gated potassium channels (VGPCs) (mammalian rK(v)1.1-1.2, hK(v)1.3, hK(v)11.1 (hERG) and insect Shaker IR) expressed heterologously in Xenopus laevis oocytes, using the two-electrode voltage clamp technique. PTX 251D not only inhibited the Na(+) influx through the mammalian VGSCs but also affected the steady-state activation and inactivation. Interestingly, in the insect ortholog, the inactivation process was dramatically affected. Additionally, PTX 251D inhibited the K(+) efflux through all five tested VGPCs and slowed down the deactivation kinetics of the mammalian VGPCs. hK(v)1.3 was the most sensitive channel, with an IC(50) value 10.8+/-0.5 microM. To the best of our knowledge this is the first report of a PTX affecting VGPCs.

Survival of Skin Graft between Transgenic Cloned Dogs and Non-Transgenic Cloned Dogs

Science.gov (United States)

Kim, Geon A; Oh, Hyun Ju; Kim, Min Jung; Jo, Young Kwang; Choi, Jin; Park, Jung Eun; Park, Eun Jung; Lim, Sang Hyun; Yoon, Byung Il; Kang, Sung Keun; Jang, Goo; Lee, Byeong Chun

2014-01-01

Whereas it has been assumed that genetically modified tissues or cells derived from somatic cell nuclear transfer (SCNT) should be accepted by a host of the same species, their immune compatibility has not been extensively explored. To identify acceptance of SCNT-derived cells or tissues, skin grafts were performed between cloned dogs that were identical except for their mitochondrial DNA (mtDNA) haplotypes and foreign gene. We showed here that differences in mtDNA haplotypes and genetic modification did not elicit immune responses in these dogs: 1) skin tissues from genetically-modified cloned dogs were successfully transplanted into genetically-modified cloned dogs with different mtDNA haplotype under three successive grafts over 63 days; and 2) non-transgenic cloned tissues were accepted into transgenic cloned syngeneic recipients with different mtDNA haplotypes and vice versa under two successive grafts over 63 days. In addition, expression of the inserted gene was maintained, being functional without eliciting graft rejection. In conclusion, these results show that transplanting genetically-modified tissues into normal, syngeneic or genetically-modified recipient dogs with different mtDNA haplotypes do not elicit skin graft rejection or affect expression of the inserted gene. Therefore, therapeutically valuable tissue derived from SCNT with genetic modification might be used safely in clinical applications for patients with diseased tissues. PMID:25372489

Knowledge and attitudes toward human cloning in Israel.

Science.gov (United States)

Barnoy, Sivia; Ehrenfeld, Malka; Sharon, Rina; Tabak, Nili

2006-04-01

The success of mammal cloning in 1997 has brought the issue of human cloning into public discussion. Human cloning has several aspects and potential applications for use in both reproductive and non-reproductive matters. The aim of this study was to evaluate the knowledge and attitudes toward human cloning in Israel. Data from 120 respondents (68 health professionals and 52 non-health professionals), all Jewish, Hebrew speaking with at least 15 years of education each, were collected using two questionnaires that dealt with knowledge and attitudes toward human cloning. Results showed that although health professionals had significantly more knowledge that non-health professionals, all respondents had poor knowledge about cloning. No difference in attitudes was found between the groups. Most respondents opposed human cloning, but more positive attitudes toward non-reproductive cloning were found. The results are discussed in the context of the deficit model. The findings indicate a need to provide information about human cloning to allow people to form their attitudes based on factual knowledge.

Pharmacological correction of long QT-linked mutations in KCNH2 (hERG) increases the trafficking of Kv11.1 channels stored in the transitional endoplasmic reticulum.

Science.gov (United States)

Smith, Jennifer L; Reloj, Allison R; Nataraj, Parvathi S; Bartos, Daniel C; Schroder, Elizabeth A; Moss, Arthur J; Ohno, Seiko; Horie, Minoru; Anderson, Corey L; January, Craig T; Delisle, Brian P

2013-11-01

KCNH2 encodes Kv11.1 and underlies the rapidly activating delayed rectifier K(+) current (IKr) in the heart. Loss-of-function KCNH2 mutations cause the type 2 long QT syndrome (LQT2), and most LQT2-linked missense mutations inhibit the trafficking of Kv11.1 channels. Drugs that bind to Kv11.1 and block IKr (e.g., E-4031) can act as pharmacological chaperones to increase the trafficking and functional expression for most LQT2 channels (pharmacological correction). We previously showed that LQT2 channels are selectively stored in a microtubule-dependent compartment within the endoplasmic reticulum (ER). We tested the hypothesis that pharmacological correction promotes the trafficking of LQT2 channels stored in this compartment. Confocal analyses of cells expressing the trafficking-deficient LQT2 channel G601S showed that the microtubule-dependent ER compartment is the transitional ER. Experiments with E-4031 and the protein synthesis inhibitor cycloheximide suggested that pharmacological correction promotes the trafficking of G601S stored in this compartment. Treating cells in E-4031 or ranolazine (a drug that blocks IKr and has a short half-life) for 30 min was sufficient to cause pharmacological correction. Moreover, the increased functional expression of G601S persisted 4-5 h after drug washout. Coexpression studies with a dominant-negative form of Rab11B, a small GTPase that regulates Kv11.1 trafficking, prevented the pharmacological correction of G601S trafficking from the transitional ER. These data suggest that pharmacological correction quickly increases the trafficking of LQT2 channels stored in the transitional ER via a Rab11B-dependent pathway, and we conclude that the pharmacological chaperone activity of drugs like ranolazine might have therapeutic potential.

Reproductive cloning combined with genetic modification.

Science.gov (United States)

Strong, C

2005-11-01

Although there is widespread opposition to reproductive cloning, some have argued that its use by infertile couples to have genetically related children would be ethically justifiable. Others have suggested that lesbian or gay couples might wish to use cloning to have genetically related children. Most of the main objections to human reproductive cloning are based on the child's lack of unique nuclear DNA. In the future, it may be possible safely to create children using cloning combined with genetic modifications, so that they have unique nuclear DNA. The genetic modifications could be aimed at giving such children genetic characteristics of both members of the couple concerned. Thus, cloning combined with genetic modification could be appealing to infertile, lesbian, or gay couples who seek genetically related children who have genetic characteristics of both members. In such scenarios, the various objections to human reproductive cloning that are based on the lack of genetic uniqueness would no longer be applicable. The author argues that it would be ethically justifiable for such couples to create children in this manner, assuming these techniques could be used safely.

Human cloning and 'posthuman' society.

Science.gov (United States)

Blackford, Russell

2005-01-01

Since early 1997, when the creation of Dolly the sheep by somatic cell nuclear transfer was announced in Nature, numerous government reports, essays, articles and books have considered the ethical problems and policy issues surrounding human reproductive cloning. In this article, I consider what response a modern liberal society should give to the prospect of human cloning, if it became safe and practical. Some opponents of human cloning have argued that permitting it would place us on a slippery slope to a repugnant future society, comparable to that portrayed in Aldous Huxley's novel, Brave New World. I conclude that, leaving aside concerns about safety, none of the psychological or social considerations discussed in this article provides an adequate policy justification for invoking the state's coercive powers to prevent human cloning.

U.S. consumers attitudes toward farm animal cloning.

Science.gov (United States)

Brooks, Kathleen R; Lusk, Jayson L

2011-10-01

In January 2008, the United States Food and Drug Administration concluded "meat and milk from cattle, swine, and goat clones or their offspring are as safe to eat as food we eat from those species now" (U.S. FDA, 2010). However, cloning remains a very controversial topic. A web-based survey administered by Knowledge Networks was used to determine U.S. consumers' awareness of and attitudes toward meat and milk from cloned cattle. Findings reveal consumers do not differentiate much between products from cloned animals and products from non-cloned animals. Overall consumers are concerned that animal cloning is an unnatural process and that it will lead to human cloning. Copyright © 2011 Elsevier Ltd. All rights reserved.

Local circulating clones of Staphylococcus aureus in Ecuador.

Science.gov (United States)

Zurita, Jeannete; Barba, Pedro; Ortega-Paredes, David; Mora, Marcelo; Rivadeneira, Sebastián

The spread of pandemic Staphylococcus aureus clones, mainly methicillin-resistant S. aureus (MRSA), must be kept under surveillance to assemble an accurate, local epidemiological analysis. In Ecuador, the prevalence of the USA300 Latin American variant clone (USA300-LV) is well known; however, there is little information about other circulating clones. The aim of this work was to identify the sequence types (ST) using a Multiple-Locus Variable number tandem repeat Analysis 14-locus genotyping approach. We analyzed 132 S. aureus strains that were recovered from 2005 to 2013 and isolated in several clinical settings in Quito, Ecuador. MRSA isolates composed 46.97% (62/132) of the study population. Within MRSA, 37 isolates were related to the USA300-LV clone (ST8-MRSA-IV, Panton-Valentine Leukocidin [PVL] +) and 10 were related to the Brazilian clone (ST239-MRSA-III, PVL-). Additionally, two isolates (ST5-MRSA-II, PVL-) were related to the New York/Japan clone. One isolate was related to the Pediatric clone (ST5-MRSA-IV, PVL-), one isolate (ST45-MRSA-II, PVL-) was related to the USA600 clone, and one (ST22-MRSA-IV, PVL-) was related to the epidemic UK-EMRSA-15 clone. Moreover, the most prevalent MSSA sequence types were ST8 (11 isolates), ST45 (8 isolates), ST30 (8 isolates), ST5 (7 isolates) and ST22 (6 isolates). Additionally, we found one isolate that was related to the livestock associated S. aureus clone ST398. We conclude that in addition to the high prevalence of clone LV-ST8-MRSA-IV, other epidemic clones are circulating in Quito, such as the Brazilian, Pediatric and New York/Japan clones. The USA600 and UK-EMRSA-15 clones, which were not previously described in Ecuador, were also found. Moreover, we found evidence of the presence of the livestock associated clone ST398 in a hospital environment. Copyright © 2016 Sociedade Brasileira de Infectologia. Published by Elsevier Editora Ltda. All rights reserved.

Local cloning of entangled states

International Nuclear Information System (INIS)

Gheorghiu, Vlad; Yu Li; Cohen, Scott M.

2010-01-01

We investigate the conditions under which a set S of pure bipartite quantum states on a DxD system can be locally cloned deterministically by separable operations, when at least one of the states is full Schmidt rank. We allow for the possibility of cloning using a resource state that is less than maximally entangled. Our results include that: (i) all states in S must be full Schmidt rank and equally entangled under the G-concurrence measure, and (ii) the set S can be extended to a larger clonable set generated by a finite group G of order |G|=N, the number of states in the larger set. It is then shown that any local cloning apparatus is capable of cloning a number of states that divides D exactly. We provide a complete solution for two central problems in local cloning, giving necessary and sufficient conditions for (i) when a set of maximally entangled states can be locally cloned, valid for all D; and (ii) local cloning of entangled qubit states with nonvanishing entanglement. In both of these cases, we show that a maximally entangled resource is necessary and sufficient, and the states must be related to each other by local unitary 'shift' operations. These shifts are determined by the group structure, so need not be simple cyclic permutations. Assuming this shifted form and partially entangled states, then in D=3 we show that a maximally entangled resource is again necessary and sufficient, while for higher-dimensional systems, we find that the resource state must be strictly more entangled than the states in S. All of our necessary conditions for separable operations are also necessary conditions for local operations and classical communication (LOCC), since the latter is a proper subset of the former. In fact, all our results hold for LOCC, as our sufficient conditions are demonstrated for LOCC, directly.

Quantum cloning of mixed states in symmetric subspaces

International Nuclear Information System (INIS)

Fan Heng

2003-01-01

Quantum-cloning machine for arbitrary mixed states in symmetric subspaces is proposed. This quantum-cloning machine can be used to copy part of the output state of another quantum-cloning machine and is useful in quantum computation and quantum information. The shrinking factor of this quantum cloning achieves the well-known upper bound. When the input is identical pure states, two different fidelities of this cloning machine are optimal

Cloning of a quantum measurement

International Nuclear Information System (INIS)

Bisio, Alessandro; D'Ariano, Giacomo Mauro; Perinotti, Paolo; Sedlak, Michal

2011-01-01

We analyze quantum algorithms for cloning of a quantum measurement. Our aim is to mimic two uses of a device performing an unknown von Neumann measurement with a single use of the device. When the unknown device has to be used before the bipartite state to be measured is available we talk about 1→2 learning of the measurement, otherwise the task is called 1→2 cloning of a measurement. We perform the optimization for both learning and cloning for arbitrary dimension d of the Hilbert space. For 1→2 cloning we also propose a simple quantum network that achieves the optimal fidelity. The optimal fidelity for 1→2 learning just slightly outperforms the estimate and prepare strategy in which one first estimates the unknown measurement and depending on the result suitably prepares the duplicate.

Cloning of a quantum measurement

Energy Technology Data Exchange (ETDEWEB)

Bisio, Alessandro; D' Ariano, Giacomo Mauro; Perinotti, Paolo; Sedlak, Michal [QUIT Group, Dipartimento di Fisica ' ' A. Volta' ' and INFN, via Bassi 6, I-27100 Pavia (Italy); QUIT Group, Dipartimento di Fisica ' ' A. Volta' ' via Bassi 6, I-27100 Pavia (Italy) and Institute of Physics, Slovak Academy of Sciences, Dubravska cesta 9, SK-845 11 Bratislava (Slovakia)

2011-10-15

We analyze quantum algorithms for cloning of a quantum measurement. Our aim is to mimic two uses of a device performing an unknown von Neumann measurement with a single use of the device. When the unknown device has to be used before the bipartite state to be measured is available we talk about 1{yields}2 learning of the measurement, otherwise the task is called 1{yields}2 cloning of a measurement. We perform the optimization for both learning and cloning for arbitrary dimension d of the Hilbert space. For 1{yields}2 cloning we also propose a simple quantum network that achieves the optimal fidelity. The optimal fidelity for 1{yields}2 learning just slightly outperforms the estimate and prepare strategy in which one first estimates the unknown measurement and depending on the result suitably prepares the duplicate.

Human embryo cloning prohibited in Hong Kong.

Science.gov (United States)

Liu, Athena

2005-12-01

Since the birth of Dolly (the cloned sheep) in 1997, debates have arisen on the ethical and legal questions of cloning-for-biomedical-research (more commonly termed "therapeutic cloning") and of reproductive cloning using human gametes. Hong Kong enacted the Human Reproductive Technology Ordinance (Cap 561) in 2000. Section 15(1)(e) of this Ordinance prohibits the "replacing of the nucleus of a cell of an embryo with a nucleus taken from any other cell," i.e., nucleus substitution. Section 15(1)(f) prohibits the cloning of any embryo. The scope of the latter, therefore, is arguably the widest, prohibiting all cloning techniques such as cell nucleus replacement, embryo splitting, parthenogenesis, and cloning using stem cell lines. Although the Human Reproductive Technology Ordinance is not yet fully operative, this article examines how these prohibitions may adversely impact on basic research and the vision of the Hong Kong scientific community. It concludes that in light of recent scientific developments, it is time to review if the law offers a coherent set of policies in this area.

Cloning the entanglement of a pair of quantum bits

International Nuclear Information System (INIS)

Lamoureux, Louis-Philippe; Navez, Patrick; Cerf, Nicolas J.; Fiurasek, Jaromir

2004-01-01

It is shown that any quantum operation that perfectly clones the entanglement of all maximally entangled qubit pairs cannot preserve separability. This 'entanglement no-cloning' principle naturally suggests that some approximate cloning of entanglement is nevertheless allowed by quantum mechanics. We investigate a separability-preserving optimal cloning machine that duplicates all maximally entangled states of two qubits, resulting in 0.285 bits of entanglement per clone, while a local cloning machine only yields 0.060 bits of entanglement per clone

DNA cloning: a practical approach. Volume 1

Energy Technology Data Exchange (ETDEWEB)

Glover, D M [ed.

1985-01-01

This book is written for the advanced molecular biologist who needs a detailed discussion of cloning technology. Topics of discussion include: genomic library cloning (size of a genomic library, screening methods, chromosome walking, host cell genetics, and general features of bacteriophage Iambda); use of gt10 and gt11 cDNA lambda vectors and general cDNA cloning; RNase H-Pol I cDNA synthesis; method of detecting fusion proteins produced in bacteria; pEMBL family of double-stranded plasmid vectors that can be used to generate single strands; Escherichia coli transformation; production of mutations in cloned sequences; and cloning in gram negative bacteria.

Human cloning: Eastern Mediterranean Region perspective.

Science.gov (United States)

Abdur Rab, M; Khayat, M H

2006-01-01

Recent advances in genomics and biotechnology have ushered in a new era in health development. Therapeutic cloning possesses enormous potential for revolutionizing medical and therapeutic techniques. Cloning technology, however, is perceived as having the potential for reproductive cloning, which raises serious ethical and moral concerns. It is important that the Islamic countries come to a consensus on this vital issue. Developing science and technology for better health is a religious and moral obligation. There is an urgent need for Muslim scholars to discuss the issue of stem cell research and cloning rationally; such dialogue will not only consider the scientific merits but also the moral, ethical and legal implications.

Radiation-induced aneusomic clones in bone marrow of rats

International Nuclear Information System (INIS)

Kohno, Sei-Ichi; Ishihara, Takaaki

1976-01-01

Wistar rats 3 months old were given a single whole-body X-irradiation with 700 R. They were killed 9.3 months, on average, after irradiation. From the bone marrows of the 23 irradiated rats, 54 clones of cells with radiation-induced chromosome abnormalities ranging from 3.3 to 78.3% in size were obtained. Karyotype analysis at the banding level showed that 43 out of the 54 clones had balanced chromosome constitutions and that the remaining 11 clones were unbalanced. The 43 balanced clones consisted of 33 clones with reciprocal translocations, 6 with inversions and 4 with both translocations and inversions. The 11 unbalanced clones were made up of 7 aneuploid clones and 4 pseudo-diploid clones. Of the 54 clones, 15 were large with frequencies of more than 25%. Contrary to general belief that cells with unbalanced chromosome constitutions have less capacity to proliferate than those with balanced ones, 8 of the 15 large clones, especially all, except 1, of the largest 6 clones were unbalanced, either aneuploid or pseudo-diploid

Subtype-specific, bi-component inhibition of SK channels by low internal pH

DEFF Research Database (Denmark)

Peitersen, Torben; Jespersen, Thomas; Jorgensen, Nanna K

2006-01-01

The effects of low intracellular pH (pH(i) 6.4) on cloned small-conductance Ca2+-activated K+ channel currents of all three subtypes (SK1, SK2, and SK3) were investigated in HEK293 cells using the patch-clamp technique. In 400 nM internal Ca2+ [Ca2+]i, all subtypes were inhibited by pH(i) 6...

Human Cloning

National Research Council Canada - National Science Library

Johnson, Judith A; Williams, Erin D

2006-01-01

.... Scientists in other labs, including Harvard University and the University of California at San Francisco, intend to produce cloned human embryos in order to derive stem cells for medical research...

Cloning and characterization of BmK86, a novel K+-channel blocker from scorpion venom

International Nuclear Information System (INIS)

Mao, Xin; Cao, Zhijian; Yin, Shijin; Ma, Yibao; Wu, Yingliang; Li, Wenxin

2007-01-01

Scorpion venom represents a tremendous hitherto unexplored resource for understanding ion channels. BmK86 is a novel K + -channel toxin gene isolated from a cDNA library of Mesobuthus martensii Karsch, which encodes a signal peptide of 22 amino acid residues and a mature toxin of 35 residues with three disulfide bridges. The genomic sequence of BmK86 consists of two exons disrupted by an intron of 72 bp. Comparison with the other scorpion toxins BmK86 shows low sequence similarity. The GST-BmK86 fusion protein was successfully expressed in Escherichia coli. The fusion protein was cleaved by enterokinase and the recombinant BmK86 was purified by HPLC. Using whole-cell patch-clamp recording, the recombinant BmK86 was found to inhibit the potassium current of mKv1.3 channel expressed in COS7 cells. These results indicated that BmK86 belongs to a representative member of a novel subfamily of α-KTxs. The systematic number assigned to BmK86 is α-KTx26.1

"Goodbye Dolly?" The ethics of human cloning.

Science.gov (United States)

Harris, J

1997-01-01

The ethical implications of human clones have been much alluded to, but have seldom been examined with any rigour. This paper examines the possible uses and abuses of human cloning and draws out the principal ethical dimensions, both of what might be done and its meaning. The paper examines some of the major public and official responses to cloning by authorities such as President Clinton, the World Health Organisation, the European parliament, UNESCO, and others and reveals their inadequacies as foundations for a coherent public policy on human cloning. The paper ends by defending a conception of reproductive rights of "procreative autonomy" which shows human cloning to be not inconsistent with human rights and dignity. PMID:9451604

Chorioallantoic placenta defects in cloned mice

International Nuclear Information System (INIS)

Wakisaka-Saito, Noriko; Kohda, Takashi; Inoue, Kimiko; Ogonuki, Narumi; Miki, Hiromi; Hikichi, Takafusa; Mizutani, Eiji; Wakayama, Teruhiko; Kaneko-Ishino, Tomoko; Ogura, Atsuo; Ishino, Fumitoshi

2006-01-01

Somatic cell nuclear transfer technology has been applied to produce live clones successfully in several mammalian species, but the success rates are very low. In mice, about half of the nuclear transfer embryos undergo implantation, but very few survive to term. We undertook detailed histological analyses of placentas from cloned mouse embryos generated from cumulus cells at 10.5 dpc of pregnancy, by which stage most clones have terminated their development. At 10.5 dpc, the extraembryonic tissues displayed several defined histological patterns, each reflecting their stage of developmental arrest. The most notable abnormality was the poor development of the spongiotrophoblast layer of diploid cells. This is in contrast to the placental hyperplasia frequently observed in somatic clones at 12.5 dpc or later stages. A variety of structural abnormalities were also observed in the embryos. Both placental and embryonic defects likely contribute to the low success rate of the mouse clones

Public perceptions of farm animal cloning in Europe

DEFF Research Database (Denmark)

Lassen, Jesper

This report presents a picture of European opinion on farm animal cloning. In the report, both agricultural and biomedical applications of farm animal cloning are considered. With the arrival of Dolly, animal cloning became an integral part of the biotech debate, but this debate did not isolate...... animal cloning as a single issue....

Lovely clone of coconuts

Energy Technology Data Exchange (ETDEWEB)

Branton, R.; Blake, J.

1983-05-01

It has taken over 10 years research and development to clone oil palms and coconut palms successfully. Unilever has recently built a tissue culture factory in England with a potential capacity for producing half a million clonal oil palms a year for export. Research on the cloning of coconut palms is reported here. Cloned palms may increase yields from oil palms by 20 to 30 percent and yields from coconut could be as high as five-fold over unselected stock. Improved yields would not only increase the yield of oil and copra but also the harvests of husk and shell which are immense potential sources of energy; the 1978 Philippine harvest of over 12 million nuts is equivalent in terms of energy to 3.8 billion litres of petrol (31 x 10/sup 12/ kcal).

Ethical issues regarding human cloning: a nursing perspective.

Science.gov (United States)

Dinç, Leyla

2003-05-01

Advances in cloning technology and successful cloning experiments in animals raised concerns about the possibility of human cloning in recent years. Despite many objections, this is not only a possibility but also a reality. Human cloning is a scientific revolution. However, it also introduces the potential for physical and psychosocial harm to human beings. From this point of view, it raises profound ethical, social and health related concerns. Human cloning would have an impact on the practice of nursing because it could result in the creation of new physiological and psychosocial conditions that would require nursing care. The nursing profession must therefore evaluate the ethics of human cloning, in particular the potential role of nurses. This article reviews the ethical considerations of reproductive human cloning, discusses the main reasons for concern, and reflects a nursing perspective regarding this issue.

[Mystery and problems of cloning].

Science.gov (United States)

Nikitin, V A

2010-01-01

The attention of investigators is attracted to the fact that, in spite of great efforts in mammalian cloning, advances that have been made in this area of research are not great, and cloned animals have developmental pathologies often incompatible with life and/or reproduction ability. It is yet not clear what technical or biological factors underlie this, and how they are connected or interact with each other, which is more realistic strategically. There is a great number of articles dealing with the influence of cloning with the nuclear transfer on genetic and epigenetic reprogramming of donor cells. At the same time we can see the practical absence of analytical investigations concerning the technology of cloning as such, its weak points, and possible sources of cellular trauma in the course of microsurgery of nuclear transfer or twinning. This article discusses step by step several nuclear transfer techniques and the methods of dividing early preimplanted embryos for twinning with the aim to reveal possible sources of cell damage during micromanipulation that may have negative influence on the development of cloned organisms. Several new author's technologies based on the study of cell biophysical characteristics are described, which allow one to avoid cellular trauma during manipulation and minimize the possibility of cell damage at any rate.

Quantum cloning without external control

International Nuclear Information System (INIS)

Chiara, G. de; Fazio, R.; Macchiavello, C.; Montangero, S.; Palma, G.M.

2005-01-01

Full text: In this work we present an approach to quantum cloning with unmodulated spin networks. The cloner is realized by a proper design of the network and a choice of the coupling between the qubits. We show that in the case of phase covariant cloner the XY coupling gives the best results. In the 1 → 2 cloning we find that the value for the fidelity of the optimal cloner is achieved, and values comparable to the optimal ones in the general N → M case can be attained. If a suitable set of network symmetries are satisfied, the output fidelity of the clones does not depend on the specific choice of the graph. We show that spin network cloning is robust against the presence of static imperfections. Moreover, in the presence of noise, it outperforms the conventional approach. In this case the fidelity exceeds the corresponding value obtained by quantum gates even for a very small amount of noise. Furthermore we show how to use this method to clone qutrits and qudits. By means of the Heisenberg coupling it is also possible to implement the universal cloner although in this case the fidelity is 10 % off that of the optimal cloner. (author)

Antisense oligonucleotides suppress cell-volume-induced activation of chloride channels.

Science.gov (United States)

Gschwentner, M; Nagl, U O; Wöll, E; Schmarda, A; Ritter, M; Paulmichl, M

1995-08-01

Cell volume regulation is an essential feature of most cells. After swelling in hypotonic media, the simultaneous activation of potassium and chloride channels is believed to be the initial, time-determining step in cell volume regulation. The activation of both pathways is functionally linked and enables the cells to lose ions and water, subsequently leading to cell shrinkage and readjustment of the initial volume. NIH 3T3 fibroblasts efficiently regulate their volume after swelling and bear chloride channels that are activated by decreasing extracellular osmolarity. The chloride current elicited in these cells after swelling is reminiscent of the current found in oocytes expressing an outwardly rectifying chloride current termed ICln. Introduction of antisense oligodeoxynucleotides complementary to the first 30 nucleotides of the coding region of the ICln channel into NIH 3T3 fibroblasts suppresses the activation of the swelling-induced chloride current. The experiments directly demonstrate an unambiguous link between a volume-activated chloride current and a cloned protein involved in chloride transport.

Generation of phase-covariant quantum cloning

International Nuclear Information System (INIS)

Karimipour, V.; Rezakhani, A.T.

2002-01-01

It is known that in phase-covariant quantum cloning, the equatorial states on the Bloch sphere can be cloned with a fidelity higher than the optimal bound established for universal quantum cloning. We generalize this concept to include other states on the Bloch sphere with a definite z component of spin. It is shown that once we know the z component, we can always clone a state with a fidelity higher than the universal value and that of equatorial states. We also make a detailed study of the entanglement properties of the output copies and show that the equatorial states are the only states that give rise to a separable density matrix for the outputs

Dose dependency of the frequency of micronucleated binucleated clone cells and of division related median clone sizes difference. Pt. 2

International Nuclear Information System (INIS)

Hagemann, G,; Kreczik, A.; Treichel, M.

1996-01-01

Following irradiation of the progenitor cells the clone growth of CHO cells decreases as a result of cell losses. Lethally acting expressions of micronuclei are produced by heritable lethal mutations. The dependency of the frequency of micronucleated binucleated clone cells and of the median clone sizes difference on the radiation dose was measured and compared to non-irradiated controls. Using the cytokinesis-block-micronucleus-method binucleated cells with micronuclei were counted as ratio of all binucleated cells within a clone size distribution. This ratio (shortened: micronucleus yield) was determined for all clone size distributions, which had been exposed to different irradiation doses and incubation times. The micronucleus yields were compared to the corresponding median clone sizes differences. The micronucleus yield is linearly dependent on the dose and is independent of the incubation time. The same holds true for the division related median clone sizes difference, which as a result is also linearly dependent on the micronucleus yield. Due to the inevitably errors of the cell count of micronucleated binucleated cells, an automatic measurement of the median clone sizes differences is the preferred method for evaluation of cellular radiation sensitivity for heritable lethal mutations. This value should always be determined in addition, if clone survival fractions are used as predictive test because it allows for an estimation of the remission probability of surviving cells. (orig.) [de

KCa2 and KCa3 channels in learning and memory processes, and neurodegeneration

Directory of Open Access Journals (Sweden)

Els F. E. Kuiper

2012-06-01

Full Text Available Calcium-activated potassium (KCa channels are present throughout the central nervous system as well as many peripheral tissues. Activation of KCa channels is essential for maintenance of the neuronal membrane potential and was shown to underlie the afterhyperpolarization (AHP that regulates action potential firing and limits the firing frequency of repetitive action potentials. Different subtypes of KCa channels were anticipated on the basis of their physiological and pharmacological profiles, and cloning revealed two well defined but phylogenetic distantly related groups of channels. The group subject of this review includes both the small-conductance KCa2 channels (KCa2.1, KCa2.2, and KCa2.3 and the intermediate-conductance (KCa3.1 channel. These channels are activated by submicromolar intracellular Ca2+ concentrations and are voltage independent. Of all KCa channels only the KCa2 channels can be potently but differentially blocked by the bee-venom apamin. In the past few years modulation of KCa channel activation revealed new roles for KCa2 channels in controlling dendritic excitability, synaptic functioning and synaptic plasticity. Furthermore, KCa2 channels appeared to be involved in neurodegeneration, and learning and memory processes. In this review, we focus on the role of KCa2 and KCa3 channels in these latter mechanisms with emphasis on learning and memory, Alzheimer’s disease and on the interplay between neuroinflammation and different neurotransmitters/neuromodulators, their signalling components and KCa channel activation.

Biodiversity versus cloning

International Nuclear Information System (INIS)

Jaramillo T, Jose Hernan

1998-01-01

The announcement has been made on the cloning of mice in these days and he doesn't stop to miss, because the world lives a stage where conscience of the protection is creating that should be given to the biodiversity. It is known that alone we won't subsist and the protection of the means and all that contains that environment is of vital importance for the man. But it is also known that the vegetables and animal transgenic that they come to multiply the species have appeared that we prepare. The transgenic has been altered genetically, for substitution of one or more genes of other species, inclusive human genes. This represents an improvement compared with the investigations that gave origin to the cloning animal. But it is necessary to notice that to it you arrived through the cloning. This year 28 million hectares have been sowed in cultivations of transgenic seeds and there is around 700 bovine transgenic whose milk contains a necessary protein in the treatment of the man's illnesses

Quantitative discrimination of Aggregatibacter actinomycetemcomitans highly leukotoxic JP2 clone from non-JP2 clones in diagnosis of aggressive periodontitis.

Science.gov (United States)

Yoshida, Akihiro; Ennibi, Oum-Keltoum; Miyazaki, Hideo; Hoshino, Tomonori; Hayashida, Hideaki; Nishihara, Tatsuji; Awano, Shuji; Ansai, Toshihiro

2012-10-11

Aggregatibacter actinomycetemcomitans is the etiological agent of periodontitis, and there is a strong association between clone JP2 and aggressive periodontitis in adolescents of African descent. The JP2 clone has an approximately 530-bp deletion (∆530) in the promoter region of the lkt/ltx gene, which encodes leukotoxin, and this clone has high leukotoxic activity. Therefore, this clone is very important in aggressive periodontitis. To diagnose this disease, culture methods and conventional PCR techniques are used. However, quantitative detection based on qPCR for the JP2 clone has not been developed due to genetic difficulties. In this study, we developed a qPCR-based quantification method specific to the JP2 clone. Based on our analysis of the DNA sequence of the lkt/ltx gene and its flanking region, we designed a reverse primer specific for the ∆530 deletion border sequence and developed a JP2-specific PCR-based quantification method using this primer. We also analyzed the DNA sequence of the ∆530 locus and found it to be highly conserved (97-100%) among 17 non-JP2 strains. Using the ∆530 locus, we designed a qPCR primer-probe set specific to non-JP2 clones. Next, we determined the numbers of JP2 and non-JP2 clone cells in the periodontal pockets of patients with aggressive periodontitis. The JP2-specific primers specifically amplified the genomic DNA of the A. actinomycetemcomitans JP2 clone and did not react with other bacterial DNA, whereas the non-JP2 specific primers reacted only with A. actinomycetemcomitans non-JP2 clones. Samples from the 88 periodontal sites in the 11 patients with aggressive periodontitis were analyzed. The bacterial cell numbers in 88 periodontal sites ranged from 0 to 4.8 × 10(8) (mean 1.28 × 10(7)) for JP2 clones and from 0 to 1.6 × 10(6) for non-JP2 clones (mean 1.84 × 10(5)). There were significant differences in the JP2 cell number between a clinical attachment level (CAL) ≤6 mm and a level ≥7 mm (p clones. This

Cloning and superluminal signaling£

Indian Academy of Sciences (India)

Cloning; cloning fidelity; superluminal signaling; state discrimination. PACS No. 03.65.Bz. 1. .... The possibility of superluminal signaling in quantum mechanics stems from the concept .... quantum mechanics and relativity [13]. .... [13] A Shimony, in Foundations of quantum mechanics in the light of new technology edited by.

Cloning Mice.

Science.gov (United States)

Ogura, Atsuo

2017-08-01

Viable and fertile mice can be generated by somatic nuclear transfer into enucleated oocytes, presumably because the transplanted somatic cell genome becomes reprogrammed by factors in the oocyte. The first somatic cloned offspring of mice were obtained by directly injecting donor nuclei into recipient enucleated oocytes. When this method is used (the so-called Honolulu method of somatic cell nuclear transfer [SCNT]), the donor nuclei readily and completely condense within the enucleated metaphase II-arrested oocytes, which contain high levels of M-phase-promoting factor (MPF). It is believed that the condensation of the donor chromosomes promotes complete reprogramming of the donor genome within the mouse oocytes. Another key to the success of mouse cloning is the use of blunt micropipettes attached to a piezo impact-driving micromanipulation device. This system saves a significant amount of time during the micromanipulation of oocytes and thus minimizes the loss of oocyte viability in vitro. For example, a group of 20 oocytes can be enucleated within 10 min by an experienced operator. This protocol is composed of seven parts: (1) preparing micropipettes, (2) setting up the enucleation and injection micropipettes, (3) collecting and enucleating oocytes, (4) preparing nucleus donor cells, (5) injecting donor nuclei, (6) activating embryos and culturing, and (7) transferring cloned embryos. © 2017 Cold Spring Harbor Laboratory Press.

Construction of an infectious plasmid clone of Muscovy duck parvovirus by TA cloning and creation of a partially attenuated strain.

Science.gov (United States)

Yen, T-Y; Li, K-P; Ou, S-C; Shien, J-H; Lu, H-M; Chang, P-C

2015-01-01

Muscovy duck parvovirus (MDPV) infection is a highly contagious and fatal disease of Muscovy ducklings. The infectious clone methodology is a valuable tool to study the pathogenic mechanisms of viruses, but no infectious clone of MDPV is yet available. In this study, a plasmid clone containing the full-length genome of MDPV was constructed using the TA cloning methodology. This MDPV clone was found to be infectious after transfection of primary Muscovy duck embryo fibroblast cells and passage in embryonated Muscovy duck eggs. Site-directed mutagenesis showed that the K75N mutation in the VP1 protein of MDPV resulted in the partial attenuation of the virus. The availability of an MDPV infectious clone can facilitate investigation of the pathogenic mechanisms of MDPV and development of vaccines against diseases caused by MDPV.

Quantum cloning machines and their implementation in physical systems

International Nuclear Information System (INIS)

Wu Tao; Ye Liu; Fang Bao-Long

2013-01-01

We review the basic theory of approximate quantum cloning for discrete variables and some schemes for implementing quantum cloning machines. Several types of approximate quantum clones and their expansive quantum clones are introduced. As for the implementation of quantum cloning machines, we review some design methods and recent experimental results. (topical review - quantum information)

Cloning of the human TASK-2 (KCNK5) promoter and its regulation by chronic hypoxia

International Nuclear Information System (INIS)

Brazier, Stephen P.; Mason, Helen S.; Bateson, Alan N.; Kemp, Paul J.

2005-01-01

The tandem P domain potassium channel family includes five members of the acid-sensing subfamily, TASK. TASK channels are active at resting potential and are inhibited by extracellular protons, suggesting they function as acid sensors and control excitability/ion homeostasis. Indeed, TASK-2 (KCNK5) has been shown to control excitability, volume regulation, bicarbonate handling, and apoptosis in a variety of tissues. With such diverse functions being ascribed to TASK-2, it is important to understand long-term as well as short-term regulation of this important channel. Thus, we have cloned the TASK-2 promoter, demonstrated that its transcriptional activity is dependent upon pO 2 , shown that deletion of overlapping consensus binding sites for NF-κB/Elk-1 ablates this O 2 sensitivity, and proved that Elk-1 binds preferentially to this site. Furthermore, the consequences of chronic hypoxia on natively expressed TASK-2 are decreased steady-state mRNA and cell depolarization showing that TASK-2 contributes to the excitability of this important lung cell type

Why Clone?

Science.gov (United States)

... than expected. Could we really clone dinosaurs? In theory? Yes. You would need: A well-preserved source ... it raises a number of ethical, legal, and social challenges that need to be considered. The vast ...

Therapeutic cloning in the mouse

Science.gov (United States)

Mombaerts, Peter

2003-01-01

Nuclear transfer technology can be applied to produce autologous differentiated cells for therapeutic purposes, a concept termed therapeutic cloning. Countless articles have been published on the ethics and politics of human therapeutic cloning, reflecting the high expectations from this new opportunity for rejuvenation of the aging or diseased body. Yet the research literature on therapeutic cloning, strictly speaking, is comprised of only four articles, all in the mouse. The efficiency of derivation of embryonic stem cell lines via nuclear transfer is remarkably consistent among these reports. However, the efficiency is so low that, in its present form, the concept is unlikely to become widespread in clinical practice. PMID:12949262

Association of Cocaine- and Amphetamine-Regulated Transcript (CART) Messenger RNA Level, Food Intake, and Growth in Channel Catfish

Science.gov (United States)

Cocaine-and Amphetamine-Regulated Transcript (CART) is a potent hypothalamic anorectic peptide in mammals and fish. We hypothesized that increased food intake is associated with changes in expression of CART mRNA within the brain of channel catfish. Objectives were to clone the CART gene, examine ...

Cloning Mice and Men: Prohibiting the Use of iPS Cells for Human Reproductive Cloning

OpenAIRE

Lo, Bernard; Parham, Lindsay; Alvarez-Buylla, Arturo; Cedars, Marcelle; Conklin, Bruce; Fisher, Susan; Gates, Elena; Giudice, Linda; Halme, Dina Gould; Hershon, William; Kriegstein, Arnold; Kwok, Pui-Yan; Wagner, Richard

2010-01-01

The use of iPSCs and tetraploid complementation for human reproductive cloning would raise profound ethical objections. Professional standards and laws that ban human reproductive cloning by somatic cell nuclear transfer should be revised to also forbid it by other methods, such as iPSCs via tetraploid complementation.

Human cloning: category, dignity, and the role of bioethics.

Science.gov (United States)

Shuster, Evelyne

2003-10-01

Human cloning has been simultaneously a running joke for massive worldwide publicity of fringe groups like the Raelians, and the core issue of an international movement at the United Nations in support of a treaty to ban the use of cloning techniques to produce a child (so called reproductive cloning). Yet, even though debates on human cloning have greatly increased since the birth of Dolly, the clone sheep, in 1997, we continue to wonder whether cloning is after all any different from other methods of medically assisted reproduction, and what exactly makes cloning an 'affront to the dignity of humans.' Categories we adopt matter mightily as they inform but can also misinform and lead to mistaken and unproductive decisions. And thus bioethicists have a responsibility to ensure that the proper categories are used in the cloning debates and denounce those who try to win the ethical debate through well-crafted labels rather than well-reasoned argumentations. But it is as important for bioethicists to take a position on broad issues such as human cloning and species altering interventions. One 'natural question' would be, for example, should there be an international treaty to ban human reproductive cloning?

[Cloning and law in Hungary].

Science.gov (United States)

Julesz, Máté

2015-03-01

Reproductive human cloning is prohibited in Hungary, as in many other countries. Therapeutic human cloning is not prohibited, just like in many other countries. Stem cell therapy is also allowed. Article III, paragraph (3) of the Hungarian basic law (constitution) strictly forbids total human cloning. Article 1 of the Additional Protocol to the Oviedo Convention, on the Prohibition of Cloning Human Beings (1998) stipulates that any intervention seeking to create a human being genetically identical to another human being, whether living or dead, is prohibited. In Hungary, according to Article 174 of the Criminal Code, total human cloning constitutes a crime. Article 180, paragraph (3) of the Hungarian Act on Health declares that embryos shall not be brought about for research purposes; research shall be conducted only on embryos brought about for reproductive purposes when this is authorized by the persons entitled to decide upon its disposal, or when the embryo is damaged. Article 180, paragraph (5) of the Hungarian Act on Health stipulates that multiple individuals who genetically conform to one another shall not be brought about. According to Article 181, paragraph (1) of the Hungarian Act on Health, an embryo used for research shall be kept alive for not longer than 14 days, not counting the time it was frozen for storage and the time period of research.

Emotional reactions to human reproductive cloning.

Science.gov (United States)

May, Joshua

2016-01-01

Extant surveys of people's attitudes towards human reproductive cloning focus on moral judgements alone, not emotional reactions or sentiments. This is especially important given that some (especially Leon Kass) have argued against such cloning on the ground that it engenders widespread negative emotions, like disgust, that provide a moral guide. To provide some data on emotional reactions to human cloning, with a focus on repugnance, given its prominence in the literature. This brief mixed-method study measures the self-reported attitudes and emotions (positive or negative) towards cloning from a sample of participants in the USA. Most participants condemned cloning as immoral and said it should be illegal. The most commonly reported positive sentiment was by far interest/curiosity. Negative emotions were much more varied, but anxiety was the most common. Only about a third of participants selected disgust or repugnance as something they felt, and an even smaller portion had this emotion come to mind prior to seeing a list of options. Participants felt primarily interested and anxious about human reproductive cloning. They did not primarily feel disgust or repugnance. This provides initial empirical evidence that such a reaction is not appropriately widespread. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

Cloning of low dose radiation induced gene RIG1 by RACE based on non-cloned cDNA library

International Nuclear Information System (INIS)

Luo Ying; Sui Jianli; Tie Yi; Zhang Yuanping; Zhou Pingkun; Sun Zhixian

2001-01-01

Objective: To obtain full-length cDNA of radiation induced new gene RIG1 based on its EST fragment. Methods: Based on non-cloned cDNA library, enhanced nested RACE PCR and biotin-avidin labelled probe for magnetic bead purification was used to obtain full-length cDNA of RIG1. Results: About 1 kb of 3' end of RIG1 gene was successfully cloned by this set of methods and cloning of RIG1 5' end is proceeding well. Conclusion: The result is consistent with the design of experiment. This set of protocol is useful for cloning of full-length gene based on EST fragment

Quantitative discrimination of Aggregatibacter actinomycetemcomitans highly leukotoxic JP2 clone from non-JP2 clones in diagnosis of aggressive periodontitis

Directory of Open Access Journals (Sweden)

Yoshida Akihiro

2012-10-01

Full Text Available Abstract Background Aggregatibacter actinomycetemcomitans is the etiological agent of periodontitis, and there is a strong association between clone JP2 and aggressive periodontitis in adolescents of African descent. The JP2 clone has an approximately 530-bp deletion (∆530 in the promoter region of the lkt/ltx gene, which encodes leukotoxin, and this clone has high leukotoxic activity. Therefore, this clone is very important in aggressive periodontitis. To diagnose this disease, culture methods and conventional PCR techniques are used. However, quantitative detection based on qPCR for the JP2 clone has not been developed due to genetic difficulties. In this study, we developed a qPCR-based quantification method specific to the JP2 clone. Methods Based on our analysis of the DNA sequence of the lkt/ltx gene and its flanking region, we designed a reverse primer specific for the ∆530 deletion border sequence and developed a JP2-specific PCR-based quantification method using this primer. We also analyzed the DNA sequence of the ∆530 locus and found it to be highly conserved (97–100% among 17 non-JP2 strains. Using the ∆530 locus, we designed a qPCR primer–probe set specific to non-JP2 clones. Next, we determined the numbers of JP2 and non-JP2 clone cells in the periodontal pockets of patients with aggressive periodontitis. Results The JP2-specific primers specifically amplified the genomic DNA of the A. actinomycetemcomitans JP2 clone and did not react with other bacterial DNA, whereas the non-JP2 specific primers reacted only with A. actinomycetemcomitans non-JP2 clones. Samples from the 88 periodontal sites in the 11 patients with aggressive periodontitis were analyzed. The bacterial cell numbers in 88 periodontal sites ranged from 0 to 4.8 × 108 (mean 1.28 × 107 for JP2 clones and from 0 to 1.6 × 106 for non-JP2 clones (mean 1.84 × 105. There were significant differences in the JP2 cell number between a clinical attachment level

Performance of new Hevea clones from IAC 400 series Perfomance de novos clones de Hevea da série IAC 400

Directory of Open Access Journals (Sweden)

Paulo de Souza Gonçalves

2007-06-01

Full Text Available The Hevea breeding program of Instituto Agronômico de Campinas (IAC has completed clonal evaluation on the following series: IAC 100, IAC 200 and IAC 300. The performance of 22 clones of Hevea brasiliensis (Willd. ex Adr. de Juss. Muell.-Arg., evolved at IAC, over a period of eleven years was evaluated in the Western Central part of the São Paulo State, Brazil. Among these 22 new clones, six were intraspecific hybrid clones (IAC 400, IAC 404, IAC 405, IAC 406, IAC 410, IAC 412 and the remaining are primary those resulted from selected ortets within half-sib progenies. An old popular clone RRIM 600, of Malaysian origin, was used as the control. The trial was laid out in a randomized block design with three replications. Yield performance over a period of four years, mean girth at the 11th year, girth increment before tapping and on tapping, thermal property of natural rubber produced, bark thickness, number of latex vessel rows in seven year virgin bark, percentage incidence of tapping panel dryness, wind damage and diseases like leaf and panel anthracnose have been observed. Sixty one percent of the clones were superior in relation to the control for yield. The clone IAC 400 recorded the highest yield (97.40 g tree-1 tap-1 over four years of tapping, followed by IAC 411 (78.87 tree-1 tap-1, whereas the control clone RRIM 600 recorded 50.86 g tree-1 tap-1. All selected clones were vigorous in growth. Girth increment of these clones was average to above average. Except for IAC 423, other clones had thick virgin bark at opening ranging from 4.84 mm for IAC 401 to 6.38 mm for IAC 416. The natural rubbers from IAC clones have shown good thermal stability up to 300ºC and no differences in the thermal behavior among rubber from clones of the IAC series and the clone RRIM 600 were found in inert atmosphere.O programa de melhoramento de Hevea do Instituto Agronômico de Campinas (IAC completou a avaliação dos clones da série IAC 100, IAC 200 e IAC

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.

The etomidate analog ET-26 HCl retains superior myocardial performance: Comparisons with etomidate in vivo and in vitro.

Science.gov (United States)

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.

Optimal cloning of mixed Gaussian states

International Nuclear Information System (INIS)

Guta, Madalin; Matsumoto, Keiji

2006-01-01

We construct the optimal one to two cloning transformation for the family of displaced thermal equilibrium states of a harmonic oscillator, with a fixed and known temperature. The transformation is Gaussian and it is optimal with respect to the figure of merit based on the joint output state and norm distance. The proof of the result is based on the equivalence between the optimal cloning problem and that of optimal amplification of Gaussian states which is then reduced to an optimization problem for diagonal states of a quantum oscillator. A key concept in finding the optimum is that of stochastic ordering which plays a similar role in the purely classical problem of Gaussian cloning. The result is then extended to the case of n to m cloning of mixed Gaussian states

[Telomere lengthening by trichostatin A treatment in cloned pigs].

Science.gov (United States)

Xie, Bing-Teng; Ji, Guang-Zhen; Kong, Qing-Ran; Mao, Jian; Shi, Yong-Qian; Liu, Shi-Chao; Wu, Mei-Ling; Wang, Juan; Liu, Lin; Liu, Zhong-Hua

2012-12-01

Telomeres are repeated GC rich sequences at the end of chromosomes, and shorten with each cell division due to DNA end replication problem. Previously, reprogrammed somatic cells of cloned animals display variable telomere elongation. However, it was reported that the cloned animals including Dolly do not reset telomeres and show premature aging. In this study, we investigated telomere function in cloned or transgenic cloned pigs, including the cloned Northeast Min pigs, eGFP, Mx, and PGC1α transgenic cloned pigs, and found that the telomere lengths of cloned pigs were significantly shorter than the nuclear donor adult fibroblasts and age-matched noncloned pigs (Pstage for 24 h. Consistent with previous reports, the developmental rate of SCNT embryos to the blastocyst stage was significantly increased compared with those of the control group (16.35% vs. 27.09%, 21.60% vs. 34.90%, Plengthen the telomere lengths of cloned pigs.

Trichostatin A (TSA) improves the development of rabbit-rabbit intraspecies cloned embryos, but not rabbit-human interspecies cloned embryos.

Science.gov (United States)

Shi, Li-Hong; Miao, Yi-Liang; Ouyang, Ying-Chun; Huang, Jun-Cheng; Lei, Zi-Li; Yang, Ji-Wen; Han, Zhi-Ming; Song, Xiang-Fen; Sun, Qing-Yuan; Chen, Da-Yuan

2008-03-01

The interspecies somatic cell nuclear transfer (iSCNT) technique for therapeutic cloning gives great promise for treatment of many human diseases. However, the incomplete nuclear reprogramming and the low blastocyst rate of iSCNT are still big problems. Herein, we observed the effect of TSA on the development of rabbit-rabbit intraspecies and rabbit-human interspecies cloned embryos. After treatment with TSA for 6 hr during activation, we found that the blastocyst rate of rabbit-rabbit cloned embryos was more than two times higher than that of untreated embryos; however, the blastocyst rate of TSA-treated rabbit-human interspecies cloned embryos decreased. We also found evident time-dependent histone deacetylation-reacetylation changes in rabbit-rabbit cloned embryos, but not in rabbit-human cloned embryos from fusion to 6 hr after activation. Our results suggest that TSA-treatment does not improve blastocyst development of rabbit-human iSCNT embryos and that abnormal histone deacetylation-reacetylation changes in iSCNT embryos may account for their poor blastocyst development. (c) 2008 Wiley-Liss, Inc.

Inverse fusion PCR cloning.

Directory of Open Access Journals (Sweden)

Markus Spiliotis

Full Text Available Inverse fusion PCR cloning (IFPC is an easy, PCR based three-step cloning method that allows the seamless and directional insertion of PCR products into virtually all plasmids, this with a free choice of the insertion site. The PCR-derived inserts contain a vector-complementary 5'-end that allows a fusion with the vector by an overlap extension PCR, and the resulting amplified insert-vector fusions are then circularized by ligation prior transformation. A minimal amount of starting material is needed and experimental steps are reduced. Untreated circular plasmid, or alternatively bacteria containing the plasmid, can be used as templates for the insertion, and clean-up of the insert fragment is not urgently required. The whole cloning procedure can be performed within a minimal hands-on time and results in the generation of hundreds to ten-thousands of positive colonies, with a minimal background.

Combinations of probabilistic and approximate quantum cloning and deleting

International Nuclear Information System (INIS)

Qiu Daowen

2002-01-01

We first construct a probabilistic and approximate quantum cloning machine (PACM) and then clarify the relation between the PACM and other cloning machines. After that, we estimate the global fidelity of the approximate cloning that improves the previous estimation for the deterministic cloning machine; and also derive a bound on the success probability of producing perfect multiple clones. Afterwards, we further establish a more generalized probabilistic and approximate cloning and deleting machine (PACDM) and discuss the connections of the PACDM to some of the existing quantum cloning and deleting machines. Finally the global fidelity and a bound on the success probability of the PACDM are obtained. Summarily, the quantum devices established in this paper improve and also greatly generalize some of the existing machines

Reproductive cloning in humans and therapeutic cloning in primates: is the ethical debate catching up with the recent scientific advances?

Science.gov (United States)

Camporesi, S; Bortolotti, L

2008-09-01

After years of failure, in November 2007 primate embryonic stem cells were derived by somatic cellular nuclear transfer, also known as therapeutic cloning. The first embryo transfer for human reproductive cloning purposes was also attempted in 2006, albeit with negative results. These two events force us to think carefully about the possibility of human cloning which is now much closer to becoming a reality. In this paper we tackle this issue from two sides, first summarising what scientists have achieved so far, then discussing some of the ethical arguments in favour and against human cloning which are debated in the context of policy making and public consultation. Therapeutic cloning as a means to improve and save lives has uncontroversial moral value. As to human reproductive cloning, we consider and assess some common objections and failing to see them as conclusive. We do recognise, though, that there will be problems at the level of policy and regulation that might either impair the implementation of human reproductive cloning or make its accessibility restricted in a way that could become difficult to justify on moral grounds. We suggest using the time still available before human reproductive cloning is attempted successfully to create policies and institutions that can offer clear directives on its legitimate applications on the basis of solid arguments, coherent moral principles, and extensive public consultation.

Meat and milk compositions of bovine clones

Science.gov (United States)

Tian, X. Cindy; Kubota, Chikara; Sakashita, Kunihito; Izaike, Yoshiaki; Okano, Ryoichi; Tabara, Norio; Curchoe, Carol; Jacob, Lavina; Zhang, Yuqin; Smith, Sadie; Bormann, Charles; Xu, Jie; Sato, Masumi; Andrew, Sheila; Yang, Xiangzhong

2005-01-01

The technology is now available for commercial cloning of farm animals for food production, but is the food safe for consumers? Here, we provide data on >100 parameters that compare the composition of meat and milk from beef and dairy cattle derived from cloning to those of genetic- and breed-matched control animals from conventional reproduction. The cloned animals and the comparators were managed under the same conditions and received the same diet. The composition of the meat and milk from the clones were largely not statistically different from those of matched comparators, and all parameters examined were within the normal industry standards or previously reported values. The data generated from our match-controlled experiments provide science-based information desired by regulatory agencies to address public concerns about the safety of meat and milk from somatic animal clones. PMID:15829585

High-dimensional quantum cloning and applications to quantum hacking.

Science.gov (United States)

Bouchard, Frédéric; Fickler, Robert; Boyd, Robert W; Karimi, Ebrahim

2017-02-01

Attempts at cloning a quantum system result in the introduction of imperfections in the state of the copies. This is a consequence of the no-cloning theorem, which is a fundamental law of quantum physics and the backbone of security for quantum communications. Although perfect copies are prohibited, a quantum state may be copied with maximal accuracy via various optimal cloning schemes. Optimal quantum cloning, which lies at the border of the physical limit imposed by the no-signaling theorem and the Heisenberg uncertainty principle, has been experimentally realized for low-dimensional photonic states. However, an increase in the dimensionality of quantum systems is greatly beneficial to quantum computation and communication protocols. Nonetheless, no experimental demonstration of optimal cloning machines has hitherto been shown for high-dimensional quantum systems. We perform optimal cloning of high-dimensional photonic states by means of the symmetrization method. We show the universality of our technique by conducting cloning of numerous arbitrary input states and fully characterize our cloning machine by performing quantum state tomography on cloned photons. In addition, a cloning attack on a Bennett and Brassard (BB84) quantum key distribution protocol is experimentally demonstrated to reveal the robustness of high-dimensional states in quantum cryptography.

RESEARCH ARTICLE Molecular cloning and functional ...

Indian Academy of Sciences (India)

Navya

2016-11-25

Nov 25, 2016 ... Molecular cloning and functional characterization of two novel ... Currently, many variants of HMW-GSs have been cloned from bread wheat .... SDS sedimentation tests were conducted using the methods described by Gao et ...

Willow yield is highly dependent on clone and site

DEFF Research Database (Denmark)

Ugilt Larsen, Søren; Jørgensen, Uffe; Lærke, Poul Erik

2014-01-01

Use of high-yielding genotypes is one of the means to achieve high yield and profitability in willow (Salix spp.) short rotation coppice. This study investigated the performance of eight willow clones (Inger, Klara, Linnea, Resolution, Stina, Terra Nova, Tora, Tordis) on five Danish sites......, differing considerably in soil type, climatic conditions and management. Compared to the best clone, the yield was up to 36 % lower for other clones across sites and up to 51 % lower within sites. Tordis was superior to other clones with dry matter yields between 5.2 and 10.2 Mg ha−1 year−1 during the first...... 3-year harvest rotation, and it consistently ranked as the highest yielding clone on four of the five sites and not significantly lower than the highest yielding clone on the fifth site. The ranking of the other clones was more dependent on site with significant interaction between clone and site...

Cloning mice and men: prohibiting the use of iPS cells for human reproductive cloning.

Science.gov (United States)

Lo, Bernard; Parham, Lindsay; Alvarez-Buylla, Arturo; Cedars, Marcelle; Conklin, Bruce; Fisher, Susan; Gates, Elena; Giudice, Linda; Halme, Dina Gould; Hershon, William; Kriegstein, Arnold; Kwok, Pui-Yan; Wagner, Richard

2010-01-08

The use of iPSCs and tetraploid complementation for human reproductive cloning would raise profound ethical objections. Professional standards and laws that ban human reproductive cloning by somatic cell nuclear transfer should be revised to also forbid it by other methods, such as iPSCs via tetraploid complementation. Copyright 2010 Elsevier Inc. All rights reserved.

Endangered wolves cloned from adult somatic cells.

Science.gov (United States)

Kim, Min Kyu; Jang, Goo; Oh, Hyun Ju; Yuda, Fibrianto; Kim, Hye Jin; Hwang, Woo Suk; Hossein, Mohammad Shamim; Kim, Joung Joo; Shin, Nam Shik; Kang, Sung Keun; Lee, Byeong Chun

2007-01-01

Over the world, canine species, including the gray wolf, have been gradually endangered or extinct. Many efforts have been made to recover and conserve these canids. The aim of this study was to produce the endangered gray wolf with somatic cell nuclear transfer (SCNT) for conservation. Adult ear fibroblasts from a female gray wolf (Canis lupus) were isolated and cultured in vitro as donor cells. Because of limitations in obtaining gray wolf matured oocytes, in vivo matured canine oocytes obtained by flushing the oviducts from the isthmus to the infundibulum were used. After removing the cumulus cells, the oocyte was enucleated, microinjected, fused with a donor cell, and activated. The reconstructed cloned wolf embryos were transferred into the oviducts of the naturally synchronized surrogate mothers. Two pregnancies were detected by ultrasonography at 23 days of gestation in recipient dogs. In each surrogate dog, two fetal sacs were confirmed by early pregnancy diagnosis at 23 days, but only two cloned wolves were delivered. The first cloned wolf was delivered by cesarean section on October 18, 2005, 60 days after embryo transfer. The second cloned wolf was delivered on October 26, 2005, at 61 days postembryo transfer. Microsatellite analysis was performed with genomic DNA from the donor wolf, the two cloned wolves, and the two surrogate female recipients to confirm the genetic identity of the cloned wolves. Analysis of 19 microsatellite loci confirmed that the cloned wolves were genetically identical to the donor wolf. In conclusion, we demonstrated live birth of two cloned gray wolves by nuclear transfer of wolf somatic cells into enucleated canine oocyte, indicating that SCNT is a practical approach for conserving endangered canids.

Clone tag detection in distributed RFID systems

Science.gov (United States)

Kamaludin, Hazalila; Mahdin, Hairulnizam

2018-01-01

Although Radio Frequency Identification (RFID) is poised to displace barcodes, security vulnerabilities pose serious challenges for global adoption of the RFID technology. Specifically, RFID tags are prone to basic cloning and counterfeiting security attacks. A successful cloning of the RFID tags in many commercial applications can lead to many serious problems such as financial losses, brand damage, safety and health of the public. With many industries such as pharmaceutical and businesses deploying RFID technology with a variety of products, it is important to tackle RFID tag cloning problem and improve the resistance of the RFID systems. To this end, we propose an approach for detecting cloned RFID tags in RFID systems with high detection accuracy and minimal overhead thus overcoming practical challenges in existing approaches. The proposed approach is based on consistency of dual hash collisions and modified count-min sketch vector. We evaluated the proposed approach through extensive experiments and compared it with existing baseline approaches in terms of execution time and detection accuracy under varying RFID tag cloning ratio. The results of the experiments show that the proposed approach outperforms the baseline approaches in cloned RFID tag detection accuracy. PMID:29565982

Clone tag detection in distributed RFID systems.

Science.gov (United States)

Kamaludin, Hazalila; Mahdin, Hairulnizam; Abawajy, Jemal H

2018-01-01

Although Radio Frequency Identification (RFID) is poised to displace barcodes, security vulnerabilities pose serious challenges for global adoption of the RFID technology. Specifically, RFID tags are prone to basic cloning and counterfeiting security attacks. A successful cloning of the RFID tags in many commercial applications can lead to many serious problems such as financial losses, brand damage, safety and health of the public. With many industries such as pharmaceutical and businesses deploying RFID technology with a variety of products, it is important to tackle RFID tag cloning problem and improve the resistance of the RFID systems. To this end, we propose an approach for detecting cloned RFID tags in RFID systems with high detection accuracy and minimal overhead thus overcoming practical challenges in existing approaches. The proposed approach is based on consistency of dual hash collisions and modified count-min sketch vector. We evaluated the proposed approach through extensive experiments and compared it with existing baseline approaches in terms of execution time and detection accuracy under varying RFID tag cloning ratio. The results of the experiments show that the proposed approach outperforms the baseline approaches in cloned RFID tag detection accuracy.

[Human cloning in Muslim and Arab law].

Science.gov (United States)

Aldeeb Abu-Sahlieh, Sami A

2009-01-01

Cloning is a modern medical procedure that Muslim religious authorities treat en resorting to the general principles established by classical Muslim law based on the Koran and the Sunnah of Muhhamad as the messenger of God. In this regard, human beings are not capable of deciding what is or what is not lawful without resorting to divine norms. Cloning clashes with several principles. Firstly, the principle of the respect for life in relation to surpernumeraries, but Muslim authors are not in unanimous agreement on the determination of the moment at which life begins. Secondly, is the respect of progeny: cloning could only take place between a married couple. But even if these two principles are respected, cloning poses two major problems: the diversity of species expounded by the Koran and the Sunnah and a lack of interest. Which explains the quasi-unanimous opposition of Muslim writings regarding cloning.

Telomeres and the ethics of human cloning.

Science.gov (United States)

Allhoff, Fritz

2004-01-01

In search of a potential problem with cloning, I investigate the phenomenon of telomere shortening which is caused by cell replication; clones created from somatic cells will have shortened telomeres and therefore reach a state of senescence more rapidly. While genetic intervention might fix this problem at some point in the future, I ask whether, absent technological advances, this biological phenomenon undermines the moral permissibility of cloning.

Gaussian cloning of coherent states with known phases

International Nuclear Information System (INIS)

Alexanian, Moorad

2006-01-01

The fidelity for cloning coherent states is improved over that provided by optimal Gaussian and non-Gaussian cloners for the subset of coherent states that are prepared with known phases. Gaussian quantum cloning duplicates all coherent states with an optimal fidelity of 2/3. Non-Gaussian cloners give optimal single-clone fidelity for a symmetric 1-to-2 cloner of 0.6826. Coherent states that have known phases can be cloned with a fidelity of 4/5. The latter is realized by a combination of two beam splitters and a four-wave mixer operated in the nonlinear regime, all of which are realized by interaction Hamiltonians that are quadratic in the photon operators. Therefore, the known Gaussian devices for cloning coherent states are extended when cloning coherent states with known phases by considering a nonbalanced beam splitter at the input side of the amplifier

[The discrete horror of cloning].

Science.gov (United States)

Guibourg, Ricardo A

2009-01-01

The author raises the topic of cloning after the decision of the Argentine government, which concerned for the "dignity of the human person", passed a decree of need and urgency, No. 200/97 (Annex), prohibiting cloning experiments with human beings. Therefore, considering that the topic is so terribly urgent and necessary, the author feels it is timely to consider it.

Technological Literacy and Human Cloning. Resources in Technology.

Science.gov (United States)

Baird, Steven L.

2002-01-01

Discusses how technology educators can deal with advances in human genetics, specifically, cloning. Includes a definition and history of cloning, discusses its benefits, and looks at social concerns and arguments for and against human cloning. Includes classroom activities and websites. (Contains 10 references.) (JOW)

Genetic superiority of exotic clones over indigenous clones for quantitative and qualitative traits

International Nuclear Information System (INIS)

Khan, I.A.; Khatri, A.; Ahmad, M.; Siddiqui, N.A.; Dahar, M.H.; Khanzada, M.H.; Nizamani, G.S.

1997-01-01

Seventeen exotic sugar cane clones along with two local checks (BL4 and L116) were planted for three consecutive years (1989-90 to 1991-92) and evaluated for cane yield, yield components (plant height, cane girth, stalks per stool, stool weight), fibre, sucrose and sugar yield. Two exotic clones AEC82-1026 and AEC86-329 proved to be significantly (p< 0.05) superior in cane yield (130.62 and 114.87 t/ha respectively) and sugar yield 18.10 and 19.33 t/ha respectively) to both checks, cane and sugar yield of BL4 were 100.73 and 12.69 t/ha and that of L116 were 74.19 11.03 t/ha respectively. Cane and sugar yields were positively (P<0.01) correlated with plant height, cane girth and weight per stool. These promising clones would be subjected to extensive studies for cane yield in different parts of Sindh province. (author)

Preservation and Reproduction of Microminipigs by Cloning Technology.

Science.gov (United States)

Enya, Satoko; Kawarasaki, Tatsuo; Otake, Masayoshi; Kangawa, Akihisa; Uenishi, Hirohide; Mikawa, Satoshi; Nishimura, Takashi; Kuwahawa, Yasushi; Shibata, Masatoshi

Microminipigs have been maintained in small populations of closed colonies, involving risks of inbreeding depression and genetic drift. In order to avoid these risks, we assessed the applicability of cloning technology. Male and female clones were produced from a stock of cryopreserved somatic cells, obtaining offspring by means of natural mating. Phenotypic and genotypic characteristics of original microminipigs, clones and their offspring were analyzed and recorded. Clones presented characteristics similar to those of the cell-stock data. Although the body weight of clones tended to be heavier than that of the cell-stock data, body weights of their offspring were similar to those of previous reports. Thus, cloned microminipigs have the potential to be a valuable genetic resource for reproduction and breeding. Our proposed methodology might be useful to provide a large number of animals with adequate quality from a limited population with sufficient genetic diversity. Copyright © 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

[Product safety analysis of somatic cell cloned bovine].

Science.gov (United States)

Hua, Song; Lan, Jie; Song, Yongli; Lu, Chenglong; Zhang, Yong

2010-05-01

Somatic cell cloning (nuclear transfer) is a technique through which the nucleus (DNA) of a somatic cell is transferred into an enucleated oocyte for the generation of a new individual, genetically identical to the somatic cell donor. It could be applied for the enhancement of reproduction rate and the improvement of food products involving quality, yield and nutrition. In recent years, the United States, Japan and Europe as well as other countries announced that meat and milk products made from cloned cattle are safe for human consumption. Yet, cloned animals are faced with a wide range of health problems, with a high death rate and a high incidence of disease. The precise causal mechanisms for the low efficiency of cloning remain unclear. Is it safe that any products from cloned animals were allowed into the food supply? This review focuses on the security of meat, milk and products from cloned cattle based on the available data.

Generating West Nile Virus from an Infectious Clone.

Science.gov (United States)

Vandergaast, Rianna; Fredericksen, Brenda L

2016-01-01

WNV infectious clones are valuable tools for elucidating WNV biology. Nevertheless, relatively few infectious WNV clones have been generated because their construction is hampered by the instability of flaviviral genomes. More recently, advances in cloning techniques as well as the development of several two-plasmid WNV infectious clone systems have facilitated the generation of WNV infectious clones. Here we described a protocol for recovering WNV from a two-plasmid system. In this approach, large quantities of these constructs are digested with restriction enzymes to produce complementary restriction sites at the 3' end of the upstream fragment and the 5' end of the downstream fragment. These fragments are then annealed to produce linear template for in vitro transcription to synthesize infectious RNA. The resulting RNA is transfected into cells and after several days WNV is recovered in the culture supernatant. This method can be used to generate virus from infectious clones encoding high- and low-pathogenicity strains of WNV, as well as chimeric virues.

Restriction enzyme body doubles and PCR cloning: on the general use of type IIs restriction enzymes for cloning.

Science.gov (United States)

Tóth, Eszter; Huszár, Krisztina; Bencsura, Petra; Kulcsár, Péter István; Vodicska, Barbara; Nyeste, Antal; Welker, Zsombor; Tóth, Szilvia; Welker, Ervin

2014-01-01

The procedure described here allows the cloning of PCR fragments containing a recognition site of the restriction endonuclease (Type IIP) used for cloning in the sequence of the insert. A Type IIS endonuclease--a Body Double of the Type IIP enzyme--is used to generate the same protruding palindrome. Thus, the insert can be cloned to the Type IIP site of the vector without digesting the PCR product with the same Type IIP enzyme. We achieve this by incorporating the recognition site of a Type IIS restriction enzyme that cleaves the DNA outside of its recognition site in the PCR primer in such a way that the cutting positions straddle the desired overhang sequence. Digestion of the PCR product by the Body Double generates the required overhang. Hitherto the use of Type IIS restriction enzymes in cloning reactions has only been used for special applications, the approach presented here makes Type IIS enzymes as useful as Type IIP enzymes for general cloning purposes. To assist in finding Body Double enzymes, we summarised the available Type IIS enzymes which are potentially useful for Body Double cloning and created an online program (http://group.szbk.u-szeged.hu/welkergr/body_double/index.html) for the selection of suitable Body Double enzymes and the design of the appropriate primers.

Evaluación de patógenos en clones de lulo (Solanum quitoense Lam. Pathogenity evaluation on Solanum quitoense Lam. Clones

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Consuelo Montes Rojas

2010-04-01

Full Text Available En el noroccidente de Popayán, Colombia, se evaluó la presencia de plagas causadas por patógenos en 42 clones de lulo (Solanum quitoense Lam.. Los clones fueron plantados en bolsas plásticas, donde se desarrollaron por 3 semanas antes de ser trasplantados al campo. Se utilizó un diseño de bloques completos al azar con cuatro repeticiones, la parcela útil estuvo conformada por 6 plantas, las cuales se sembraron a ‘tresbolillo’ a 2.5 m entre surcos y 2 m entre plantas. Para determinar el efecto de las plagas en el cultivo, se calculó el porcentaje de incidencia y severidad del ataque. La incidencia se evaluó como porcentaje de plantas afectadas, y la severidad como porcentaje de tejido afectado por el patógeno. Las enfermedades más limitantes para los 42 clones fueron: gota (Phytophthora infestans que provocó una mortalidad de plantas superior a 40%; fusarium (Fusarium oxysporum que se presentó en 12 de los clones evaluados; antracnosis (Colletotrichum sp. que afectó 21 clones, los cuales se clasificaron entre tolerantes y medianamente tolerantes; y mancha clorótica (Cladosporium sp. que afectó 21 clones, clasificados como susceptibles. Los clones PL19, PL24, PL11, PL35 fueron medianamente tolerantes. Se seleccionaron por supervivencia los clones: JY E1 (52.2%, PH E 1 (45.8%, VM E2 (45.8%; por supervivencia y por tolerancia a Fusarium oxysporum los clones PL35, PL11, PL24, PL8, PL19, 120052, 120043, ORE1, AGE1. Los clones SER 7, SER 15, SER 9, SEC 31, SEC 27 presentaron alta mortalidad pero se seleccionaron por ser medianamente tolerantes a gota, tolerantes a antracnosis y medianamente resistentes a nematodos, con buen vigor y producción.Presence of plant disease caused by pathogens on 42 clones of Solanum quitoense Lam. were evaluated in the north-western region of Popayán, Colombia. The seed of the clons were planted in plastic bags during three weeks and afterwards transplanted to the field. The statistical design

Elephant grass clones for silage production

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Rerisson José Cipriano dos Santos

2013-02-01

Full Text Available Ensiling warm-season grasses often requires wilting due to their high moisture content, and the presence of low-soluble sugars in these grasses usually demands the use of additives during the ensiling process. This study evaluated the bromatological composition of the fodder and silage from five Pennisetum sp. clones (IPA HV 241, IPA/UFRPE Taiwan A-146 2.114, IPA/UFRPE Taiwan A-146 2.37, Elephant B, and Mott. The contents of 20 Polyvinyl chloride (PVC silos, which were opened after 90 days of storage, were used for the bromatological analysis and the evaluation of the pH, nitrogen, ammonia, buffer capacity, soluble carbohydrates, and fermentation coefficients. The effluent losses, gases and dry matter recovery were also calculated. Although differences were observed among the clones (p < 0.05 for the concentrations of dry matter, insoluble nitrogen in acid detergents, insoluble nitrogen in neutral detergents, soluble carbohydrates, fermentation coefficients, and in vitro digestibility in the forage before ensiling, no differences were observed for most of these variables after ensiling. All of the clones were efficient in the fermentation process. The IPA/UFRPE TAIWAN A-146 2.37 clone, however, presented a higher dry matter concentration and the best fermentation coefficient, resulting in a better silage quality, compared to the other clones.

Mitigating prolonged QT interval in cancer nanodrug development for accelerated clinical translation.

Science.gov (United States)

Ranjan, Amalendu P; Mukerjee, Anindita; Helson, Lawrence; Vishwanatha, Jamboor K

2013-12-14

Cardiac toxicity is the foremost reason for drug discontinuation from development to clinical evaluation and post market surveillance [Fung 35:293-317, 2001; Piccini 158:317-326 2009]. The Food and Drug Administration (FDA) has rejected many potential pharmaceutical agents due to QT prolongation effects. Since drug development and FDA approval takes an enormous amount of time, money and effort with high failure rates, there is an increased focus on rescuing drugs that cause QT prolongation. If these otherwise safe and potent drugs were formulated in a unique way so as to mitigate the QT prolongation associated with them, these potent drugs may get FDA approval for clinical use. Rescuing these compounds not only benefit the patients who need them but also require much less time and money thus leading to faster clinical translation. In this study, we chose curcumin as our drug of choice since it has been shown to posses anti-tumor properties against various cancers with limited toxicity. The major limitations with this pharmacologically active drug are (a) its ability to prolong QT by inhibiting the hERG channel and (b) its low bioavailability. In our previous studies, we found that lipids have protective actions against hERG channel inhibition and therefore QT prolongation. Results of the manual patch clamp assay of HEK 293 cells clearly illustrated that our hybrid nanocurcumin formulation prevented the curcumin induced inhibition of hERG K+ channel at concentrations higher than the therapeutic concentrations of curcumin. Comparing the percent inhibition, the hybrid nanocurcumin limited inhibition to 24.8% at a high curcumin equivalent concentration of 18 μM. Liposomal curcumin could only decrease this inhibition upto 30% only at lower curcumin concentration of 6 μM but not at 18 μM concentration. Here we show a curcumin encapsulated lipopolymeric hybrid nanoparticle formulation which could protect against QT prolongation and also render increased

Multi-parameter in vitro toxicity testing of crizotinib, sunitinib, erlotinib, and nilotinib in human cardiomyocytes

International Nuclear Information System (INIS)

Doherty, Kimberly R.; Wappel, Robert L.; Talbert, Dominique R.; Trusk, Patricia B.; Moran, Diarmuid M.; Kramer, James W.; Brown, Arthur M.; Shell, Scott A.; Bacus, Sarah

2013-01-01

Tyrosine kinase inhibitors (TKi) have greatly improved the treatment and prognosis of multiple cancer types. However, unexpected cardiotoxicity has arisen in a subset of patients treated with these agents that was not wholly predicted by pre-clinical testing, which centers around animal toxicity studies and inhibition of the human Ether-à-go-go-Related Gene (hERG) channel. Therefore, we sought to determine whether a multi-parameter test panel assessing the effect of drug treatment on cellular, molecular, and electrophysiological endpoints could accurately predict cardiotoxicity. We examined how 4 FDA-approved TKi agents impacted cell viability, apoptosis, reactive oxygen species (ROS) generation, metabolic status, impedance, and ion channel function in human cardiomyocytes. The 3 drugs clinically associated with severe cardiac adverse events (crizotinib, sunitinib, nilotinib) all proved to be cardiotoxic in our in vitro tests while the relatively cardiac-safe drug erlotinib showed only minor changes in cardiac cell health. Crizotinib, an ALK/MET inhibitor, led to increased ROS production, caspase activation, cholesterol accumulation, disruption in cardiac cell beat rate, and blockage of ion channels. The multi-targeted TKi sunitinib showed decreased cardiomyocyte viability, AMPK inhibition, increased lipid accumulation, disrupted beat pattern, and hERG block. Nilotinib, a second generation Bcr-Abl inhibitor, led to increased ROS generation, caspase activation, hERG block, and an arrhythmic beat pattern. Thus, each drug showed a unique toxicity profile that may reflect the multiple mechanisms leading to cardiotoxicity. This study demonstrates that a multi-parameter approach can provide a robust characterization of drug-induced cardiomyocyte damage that can be leveraged to improve drug safety during early phase development. - Highlights: • TKi with known adverse effects show unique cardiotoxicity profiles in this panel. • Crizotinib increases ROS, apoptosis, and

Synthesis and characterisation of NS13558: a new important tool for addressing KCa1.1 channel function ex vivo

DEFF Research Database (Denmark)

Bentzen, Bo Hjorth; Andersen, Rune Wederkinck; Olesen, Søren-Peter

2009-01-01

Pharmacological activation of the large-conductance Ca(2+)-activated K(+) channel (KCa1.1) in the cardiac inner mitochondrial membrane has been found to protect the heart against ischemia reperfusion injuries. However, there are concerns about the selectivity of the pharmacological tools used...... to modulate the channel. Here, we address this issue by synthesising a methylated analogue of the tool KCa1.1 channel activator NS11021. The compound (NS13558) is designed as a structurally closely related and biologically inactive analogue of NS11021. NS13558 did not elicit any significant opening of cloned...... human KCa1.1 channels, but maintained comparable biological activity towards other cardiac ion channels as compared to NS11021. In isolated perfused rat hearts subjected to ischemia-reperfusion, infarct size was reduced from 29% in control to 7% in NS11021 treated hearts. In comparison, the inactive...

Challenges in regulating farm animal cloning

DEFF Research Database (Denmark)

Gunning, Jennifer; Hartlev, Mette; Gamborg, Christian

Report from the project Cloning in Public: A specific support action within the 6th framework programme, priority 5: Food quality and safety......Report from the project Cloning in Public: A specific support action within the 6th framework programme, priority 5: Food quality and safety...

The science and technology of farm animal cloning

DEFF Research Database (Denmark)

Gjerris, Mickey; Vajta, Gábor

, goats, horses, cats, etc. have been cloned with the somatic cell nuclear transfer technique. Although the technology still has relatively low success rates and there seems to be substantial problems with the welfare of some of the cloned animals, cloning is used both within basic research...... include the production of genetically identical animals for research purposes, and also the creation of genetically modified animals. In the agricultural sector, cloning can be used as a tool within farm animal breeding. We do not intend to give an exhaustive review of the all the literature available...

Production of cloned mice and ES cells from adult somatic cells by nuclear transfer: how to improve cloning efficiency?

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Wakayama, Teruhiko

2007-02-01

Although it has now been 10 years since the first cloned mammals were generated from somatic cells using nuclear transfer (NT), most cloned embryos usually undergo developmental arrest prior to or soon after implantation, and the success rate for producing live offspring by cloning remains below 5%. The low success rate is believed to be associated with epigenetic errors, including abnormal DNA hypermethylation, but the mechanism of "reprogramming" is unclear. We have been able to develop a stable NT method in the mouse in which donor nuclei are directly injected into the oocyte using a piezo-actuated micromanipulator. Especially in the mouse, only a few laboratories can make clones from adult somatic cells, and cloned mice are never successfully produced from most mouse strains. However, this technique promises to be an important tool for future research in basic biology. For example, NT can be used to generate embryonic stem (NT-ES) cell lines from a patient's own somatic cells. We have shown that NT-ES cells are equivalent to ES cells derived from fertilized embryos and that they can be generated relatively easily from a variety of mouse genotypes and cell types of both sexes, even though it may be more difficult to generate clones directly. In general, NT-ES cell techniques are expected to be applied to regenerative medicine; however, this technique can also be applied to the preservation of genetic resources of mouse strain instead of embryos, oocytes and spermatozoa. This review describes how to improve cloning efficiency and NT-ES cell establishment and further applications.

Clone Detection for Graph-Based Model Transformation Languages

DEFF Research Database (Denmark)

Strüber, Daniel; Plöger, Jennifer; Acretoaie, Vlad

2016-01-01

and analytical quality assurance. From these use cases, we derive a set of key requirements. We describe our customization of existing model clone detection techniques allowing us to address these requirements. Finally, we provide an experimental evaluation, indicating that our customization of ConQAT, one......Cloning is a convenient mechanism to enable reuse across and within software artifacts. On the downside, it is also a practice related to significant long-term maintainability impediments, thus generating a need to identify clones in affected artifacts. A large variety of clone detection techniques...... has been proposed for programming and modeling languages; yet no specific ones have emerged for model transformation languages. In this paper, we explore clone detection for graph-based model transformation languages. We introduce potential use cases for such techniques in the context of constructive...

Photonic quantum simulator for unbiased phase covariant cloning

Science.gov (United States)

Knoll, Laura T.; López Grande, Ignacio H.; Larotonda, Miguel A.

2018-01-01

We present the results of a linear optics photonic implementation of a quantum circuit that simulates a phase covariant cloner, using two different degrees of freedom of a single photon. We experimentally simulate the action of two mirrored 1→ 2 cloners, each of them biasing the cloned states into opposite regions of the Bloch sphere. We show that by applying a random sequence of these two cloners, an eavesdropper can mitigate the amount of noise added to the original input state and therefore, prepare clones with no bias, but with the same individual fidelity, masking its presence in a quantum key distribution protocol. Input polarization qubit states are cloned into path qubit states of the same photon, which is identified as a potential eavesdropper in a quantum key distribution protocol. The device has the flexibility to produce mirrored versions that optimally clone states on either the northern or southern hemispheres of the Bloch sphere, as well as to simulate optimal and non-optimal cloning machines by tuning the asymmetry on each of the cloning machines.

Post-death cloning of endangered Jeju black cattle (Korean native cattle): fertility and serum chemistry in a cloned bull and cow and their offspring.

Science.gov (United States)

Kim, Eun Young; Song, Dong Hwan; Park, Min Jee; Park, Hyo Young; Lee, Seung Eun; Choi, Hyun Yong; Moon, Jeremiah Jiman; Kim, Young Hoon; Mun, Seong Ho; Oh, Chang Eon; Ko, Moon Suck; Lee, Dong Sun; Riu, Key Zung; Park, Se Pill

2013-12-17

To preserve Jeju black cattle (JBC; endangered native Korean cattle), a pair of cattle, namely a post-death cloned JBC bull and cow, were produced by somatic cell nuclear transfer (SCNT) in a previous study. In the present study, we examined the in vitro fertilization and reproductive potentials of these post-death cloned animals. Sperm motility, in vitro fertilization and developmental capacity were examined in a post-death cloned bull (Heuk Oll Dolee) and an extinct nuclear donor bull (BK94-13). We assessed reproductive ability in another post-death cloned cow (Heuk Woo Sunee) using cloned sperm for artificial insemination (AI). There were no differences in sperm motility or developmental potential of in vitro fertilized embryos between the post-death cloned bull and its extinct nuclear donor bull; however, the embryo development ratio was slightly higher in the cloned sperm group than in the nuclear donor sperm group. After one attempt at AI, the post-death cloned JBC cow became pregnant, and gestation proceeded normally until day 287. From this post-death cloned sire and dam, a JBC male calf (Heuk Woo Dolee) was delivered naturally (weight, 25 kg). The genetic paternity/maternity of the cloned JBC bull and cow with regard to their offspring was confirmed using International Society for Animal Genetics standard microsatellite markers. Presently, Heuk Woo Dolee is 5 months of age and growing normally. In addition, there were no significant differences in blood chemistry among the post-death cloned JBC bull, the cow, their offspring and cattle bred by AI. This is the first report showing that a pair of cattle, namely, a post-death cloned JBC bull and cow, had normal fertility. Therefore, SCNT can be used effectively to increase the population of endangered JBC.

Cloning: Past, Present, and the Exciting Future. Breakthroughs in Bioscience.

Science.gov (United States)

Di Berardino, Marie A.

This document explores the history of cloning by focusing on Dolly the Sheep, one of the first large animal clonings. The disadvantages and advantages of transgenic clones are discussed as well as the future implications of cloning from the perspective of human health. (Contains 10 resources.) (YDS)

Transfer of experimental autoimmune thyroiditis with T cell clones

International Nuclear Information System (INIS)

Romball, C.G.; Weigle, W.O.

1987-01-01

We have investigated three T lymphocyte clones isolated from CBA/CaJ mice primed with mouse thyroid extract (MTE) in adjuvant. All three clones are L3T4+, Ig-, and Lyt2- and proliferate to MTE, mouse thyroglobulin (MTG) and rat thyroid extract. Clones A7 and B7 transfer thyroiditis to irradiated (475 rad) syngeneic mice, but not to normal recipients. The thyroid lesion induced by the B7 clone is characterized by the infiltration of both mononuclear and polymorphonuclear cells. The thyroiditis is transient in that lesions are apparent 7 and 14 days after transfer, but thyroids return to normal by day 21. Clone B7 showed helper activity for trinitrophenyl-keyhole limpet hemocyanin-primed B cells in vitro when stimulated with trinitrophenyl-MTG and also stimulated the production of anti-MTG antibody in recipient mice. Clone A7 induced thyroid lesions characterized by infiltration of the thyroid with mononuclear cells, with virtually no polymorphonuclear cell infiltration. This clone has shown no helper activity following stimulation with trinitrophenyl-MTG. The third clone (D2) proliferates to and shows helper activity to MTG, but fails to transfer thyroiditis to syngeneic, irradiated mice. On continuous culture, clone B7 lost its surface Thy. The loss of Thy appears unrelated to the ability to transfer thyroiditis since subclones of B7 with markedly different percentages of Thy+ cells transferred disease equally well

The evaluation of growth dynamics of Lonicera kamtschatica clones

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Ján Matuškovič

2007-01-01

Full Text Available Artickle deals with the evaluation of growth dynamics of selected set of clones Lonicera kamtschatica in the conditions of Nitra. We measured the growth of the shrubs twice a year (in spring and autumn during 2003–2005. Within all clones 5 shrubs were evaluated. On the basis of the obtained results we can claim the highest increase of height in case of LKL 21 followed by clones LKL 16 and LKL 5. The lowest growth increase was typical for LKL 58 and LKL 66.In term of statistical evaluation the year can be considered as a statistically significant factor forming a growth intensity of clones during 2003–2005. The effect of year on growing processes is strong (ε2 = 0.96 while the participation of year with clone influenced the growth increase in medium size (ε2 = 0.42. LKL 21 and LKL 58 in comparison with other clones are the most disperatable in term of growth increase. Within mentioned clones statistically significant differences were recorded in 7 evaluated pairs. In the same way LKL 42 is very different from another clones as well. On the basis of all provided analysis the tested clones from point of wiev perspectivity of planting can be set up in the following order: LKL 21, LKL 16, LKL 5, LKL 42, LKL 49, LKL 96, LKL 6, LKL 60, LKL 66 and LKL 58.

Duration of gestation in pregnant dogs carrying cloned fetuses.

Science.gov (United States)

Kim, Min Jung; Oh, Hyun Ju; Park, Jung Eun; Kim, Geon A; Park, Eun Jung; Jo, Young Kwang; Lee, Byeong Chun

2013-01-15

The aim of this study was to investigate gestation duration and the physiologic characteristics of pregnant dogs bearing cloned fetuses, especially in the prepartum period. A retrospective study was performed to compare gestation duration in females pregnant with cloned (somatic cell nuclear transfer) fetuses (cloned group) with those bearing noncloned fetuses (control group), and effects of litter size, birth weight, and breed of somatic cell donors on gestation duration in the cloned group were evaluated. Clinical delivery onset signs associated with serum progesterone concentration and rectal temperature were also compared in both groups. The gestation duration calculated from day of ovulation was significantly longer in the cloned (62.8 ± 0.3 days) versus the control group (60.9 ± 0.5 days; P dogs bearing cloned fetuses might be because of the smaller litter size in this group. Also, the weaker drop in serum progesterone levels in the prepartum period in cloned dog pregnancies indicates that the parturition signaling process might be altered resulting in longer gestation periods. Copyright © 2013 Elsevier Inc. All rights reserved.

The Shiite Pluralistic Position on Human Cloning

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Sayyid Hasan Islami Ardekani

2012-01-01

Full Text Available With regard to human cloning or artificial human reproduction – and contrary to the opinions of Sunni scholars - Shiite thinkers have not held a unified position. After having surveyed a number of Shiite fatwas and analyses on the subject, this essay will classify them into four groups. The first group states that we are granted absolute permission to engage in human cloning; while the second group believes that there is limited permission; the third group argues that cloning as such is primarily permitted but because of its consequences and secondary grounds it is prohibited and unlawful; and the fourth group is of the view that cloning as such and by itself is prohibited and unlawful. In what follows, the author has examined these four views, ending in support of the permission theory.

Towards Clone Detection in UML Domain Models

DEFF Research Database (Denmark)

Störrle, Harald

2013-01-01

Code clones (i.e., duplicate fragments of code) have been studied for long, and there is strong evidence that they are a major source of software faults. Anecdotal evidence suggests that this phenomenon occurs similarly in models, suggesting that model clones are as detrimental to model quality...... as they are to code quality. However, programming language code and visual models have significant differences that make it difficult to directly transfer notions and algorithms developed in the code clone arena to model clones. In this article, we develop and propose a definition of the notion of “model clone” based...... we believe that our approach advances the state of the art significantly, it is restricted to UML models, its results leave room for improvements, and there is no validation by field studies....

Personality consistency analysis in cloned quarantine dog candidates

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Jin Choi

2017-01-01

Full Text Available In recent research, personality consistency has become an important characteristic. Diverse traits and human-animal interactions, in particular, are studied in the field of personality consistency in dogs. Here, we investigated the consistency of dominant behaviours in cloned and control groups followed by the modified Puppy Aptitude Test, which consists of ten subtests to ascertain the influence of genetic identity. In this test, puppies are exposed to stranger, restraint, prey-like object, noise, startling object, etc. Six cloned and four control puppies participated and the consistency of responses at ages 7–10 and 16 weeks in the two groups was compared. The two groups showed different consistencies in the subtests. While the average scores of the cloned group were consistent (P = 0.7991, those of the control group were not (P = 0.0089. Scores of Pack Drive and Fight or Flight Drive were consistent in the cloned group, however, those of the control group were not. Scores of Prey Drive were not consistent in either the cloned or the control group. Therefore, it is suggested that consistency of dominant behaviour is affected by genetic identity and some behaviours can be influenced more than others. Our results suggest that cloned dogs could show more consistent traits than non-cloned. This study implies that personality consistency could be one of the ways to analyse traits of puppies.

Cloning and joint measurements of incompatible components of spin

International Nuclear Information System (INIS)

Brougham, Thomas; Andersson, Erika; Barnett, Stephen M.

2006-01-01

A joint measurement of two observables is a simultaneous measurement of both quantities upon the same quantum system. When two quantum-mechanical observables do not commute, then a joint measurement of these observables cannot be accomplished directly by projective measurements alone. In this paper we shall discuss the use of quantum cloning to perform a joint measurement of two components of spin associated with a qubit system. We introduce cloning schemes which are optimal with respect to this task. The cloning schemes may be thought to work by cloning two components of spin onto their outputs. We compare the proposed cloning machines to existing cloners

Características físicas e sensoriais de clones de batata-doce Physical and sensorial characteristics of sweetpotato clones

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Adriana Dias Cardoso

2007-12-01

Full Text Available Com o objetivo de avaliar propriedades físicas e sensoriais de clones de batata-doce em Vitória da Conquista - BA foi realizado este experimento, composto por 16 clones oriundos de Janaúba- G, Viçosa - MG, Bom Jardim de Minas - MG, Gurupi - TO, Santo Antônio da Platina - PR, Holambra II - SP, Vitória da Conquista - BA e Condeúba - BA. Utilizou-se o delineamento em blocos casualizados, com 16 tratamentos e 3 repetições. Avaliaram-se as características sensoriais: aparência, umidade, doçura, coloração da polpa, dificuldade de deglutição das raízes tuberosas e as características físicas: tempo de cozimento e peso específico. Os dados foram submetidos à análise de variância e teste de Scott-Knott a 5% de probabilidade, entretanto, as características sensoriais foram obtidas apenas em valores de porcentagem. O clone 25 apresentou as melhores características sensoriais e o clone 7 apresentou melhor tempo de cozimento.The aim of this experiment was to evaluate the physical and sensorial characteristics of sweetpotato clones in Vitória da Conquista, Bahia State, Brazil. Sixteen clones were analyzed, originating from Janaúba, MG, Viçosa, MG; Bom Jardim de Minas, MG; Gurupi, TO; Santo Antônio da Platina, PR; Holambra II, SP; Vitória da Conquista, BA; and Condeúba, BA. One utilized randomized blocks with 16 treatments and three repetitions. The following characteristics were analyzed: aspect, humidity, sweetness, color, deglutition difficulty, cooking and specific gravity of the storage roots. The data were submitted to variance analysis using a ScottKnott test with 5% probability. Clone 25 presented the best sensorial characteristics, and clone 7 presented the best cooking time.

Procreative liberty, enhancement and commodification in the human cloning debate.

Science.gov (United States)

Shapshay, Sandra

2012-12-01

The aim of this paper is to scrutinize a contemporary standoff in the American debate over the moral permissibility of human reproductive cloning in its prospective use as a eugenic enhancement technology. I shall argue that there is some significant and under-appreciated common ground between the defenders and opponents of human cloning. Champions of the moral and legal permissibility of cloning support the technology based on the right to procreative liberty provided it were to become as safe as in vitro fertilization and that it be used only by adults who seek to rear their clone children. However, even champions of procreative liberty oppose the commodification of cloned embryos, and, by extension, the resulting commodification of the cloned children who would be produced via such embryos. I suggest that a Kantian moral argument against the use of cloning as an enhancement technology can be shown to be already implicitly accepted to some extent by champions of procreative liberty on the matter of commodification of cloned embryos. It is in this argument against commodification that the most vocal critics of cloning such as Leon Kass and defenders of cloning such as John Robertson can find greater common ground. Thus, I endeavor to advance the debate by revealing a greater degree of moral agreement on some fundamental premises than hitherto recognized.

Should we clone human beings? Cloning as a source of tissue for transplantation.

Science.gov (United States)

Savulescu, J

1999-01-01

The most publicly justifiable application of human cloning, if there is one at all, is to provide self-compatible cells or tissues for medical use, especially transplantation. Some have argued that this raises no new ethical issues above those raised by any form of embryo experimentation. I argue that this research is less morally problematic than other embryo research. Indeed, it is not merely morally permissible but morally required that we employ cloning to produce embryos or fetuses for the sake of providing cells, tissues or even organs for therapy, followed by abortion of the embryo or fetus. PMID:10226910

TA-GC cloning: A new simple and versatile technique for the directional cloning of PCR products for recombinant protein expression.

Science.gov (United States)

Niarchos, Athanasios; Siora, Anastasia; Konstantinou, Evangelia; Kalampoki, Vasiliki; Lagoumintzis, George; Poulas, Konstantinos

2017-01-01

During the last few decades, the recombinant protein expression finds more and more applications. The cloning of protein-coding genes into expression vectors is required to be directional for proper expression, and versatile in order to facilitate gene insertion in multiple different vectors for expression tests. In this study, the TA-GC cloning method is proposed, as a new, simple and efficient method for the directional cloning of protein-coding genes in expression vectors. The presented method features several advantages over existing methods, which tend to be relatively more labour intensive, inflexible or expensive. The proposed method relies on the complementarity between single A- and G-overhangs of the protein-coding gene, obtained after a short incubation with T4 DNA polymerase, and T and C overhangs of the novel vector pET-BccI, created after digestion with the restriction endonuclease BccI. The novel protein-expression vector pET-BccI also facilitates the screening of transformed colonies for recombinant transformants. Evaluation experiments of the proposed TA-GC cloning method showed that 81% of the transformed colonies contained recombinant pET-BccI plasmids, and 98% of the recombinant colonies expressed the desired protein. This demonstrates that TA-GC cloning could be a valuable method for cloning protein-coding genes in expression vectors.

Counterfactual quantum cloning without transmitting any physical particles

Science.gov (United States)

Guo, Qi; Zhai, Shuqin; Cheng, Liu-Yong; Wang, Hong-Fu; Zhang, Shou

2017-11-01

We propose a counterfactual 1 →2 economical phase-covariant cloning scheme. Compared with the existing protocols using flying qubits, the main difference of the presented scheme is that the cloning can be achieved without transmitting the photon between the two parties. In addition, this counterfactual scheme does not need to construct controlled quantum gates to perform joint logical operations between the cloned qubit and the blank copy. We also numerically evaluate the performance of the present scheme in the practical experiment, which shows this cloning scheme can be implemented with a high success of probability and the fidelity is close to the optimal value in the ideal asymptotic limit.

Public perceptions of animal cloning

DEFF Research Database (Denmark)

Jelsøe, Erling; Vincentsen, Ulla; Andersen, Ida-Elisabeth

What was from the outset meant to be a survey testing predefined categories of ethical positions related to new biotechnologies with animal cloning as an example was subsequently developed into a process of broader involvement of groups of citizens in the issue. The survey was conducted at meetings...... in four different cities in Denmark. The participants were introduced to animal cloning and after that they filled out the questionnaire. Finally, the issue was discussed in focus groups. The process as a whole was run in a dialogue oriented way. Through the information they received in combination...... with reflecting on the survey questions the participants were well prepared for discussions in the focus groups. This approach made it possible, on the one hand to get a measure of the citizen's perceptions of the ethical aspects of animal cloning, but also to go deeper into their own thoughts of the issue...

Towards an understanding of British public attitudes concerning human cloning.

Science.gov (United States)

Shepherd, Richard; Barnett, Julie; Cooper, Helen; Coyle, Adrian; Moran-Ellis, Jo; Senior, Victoria; Walton, Chris

2007-07-01

The ability of scientists to apply cloning technology to humans has provoked public discussion and media coverage. The present paper reports on a series of studies examining public attitudes to human cloning in the UK, bringing together a range of quantitative and qualitative methods to address this question. These included a nationally representative survey, an experimental vignette study, focus groups and analyses of media coverage. Overall the research presents a complex picture of attitude to and constructions of human cloning. In all of the analyses, therapeutic cloning was viewed more favourably than reproductive cloning. However, while participants in the focus groups were generally negative about both forms of cloning, and this was also reflected in the media analyses, quantitative results showed more positive responses. In the quantitative research, therapeutic cloning was generally accepted when the benefits of such procedures were clear, and although reproductive cloning was less accepted there was still substantial support. Participants in the focus groups only differentiated between therapeutic and reproductive cloning after the issue of therapeutic cloning was explicitly raised; initially they saw cloning as being reproductive cloning and saw no real benefits. Attitudes were shown to be associated with underlying values associated with scientific progress rather than with age, gender or education, and although there were a few differences in the quantitative data based on religious affiliation, these tended to be small effects. Likewise in the focus groups there was little direct appeal to religion, but the main themes were 'interfering with nature' and the 'status of the embryo', with the latter being used more effectively to try to close down further discussion. In general there was a close correspondence between the media analysis and focus group responses, possibly demonstrating the importance of media as a resource, or that the media reflect

Calcium Activated K+ Channels in The Electroreceptor of the Skate Confirmed by Cloning. Details of Subunits and Splicing

Science.gov (United States)

King, Benjamin L.; Shi, Ling Fang; Kao, Peter; Clusin, William T.

2015-01-01

Elasmobranchs detect small potentials using excitable cells of the ampulla of Lorenzini which have calcium-activated K+ channels, first described in l974. A distinctive feature of the outward current in voltage clamped ampullae is its apparent insensitivity to voltage. The sequence of a BK channel α isoform expressed in the ampulla of the skate was characterized. A signal peptide is present at the beginning of the gene. When compared to human isoform 1 (the canonical sequence), the largest difference was absence of a 59 amino acid region from the S8-S9 intracellular linker that contains the strex regulatory domain. The ampulla isoform was also compared with the isoform predicted˜ in late skate embryos where strex was also absent. The BK voltage sensors were conserved in both skate isoforms. Differences between the skate and human BK channel included alternative splicing. Alternative splicing occurs at seven previously defined sites that are characteristic for BK channels in general and hair cells in particular. Skate BK sequences were highly similar to the Australian ghost shark and several other vertebrate species. Based on alignment of known BK sequences with the skate genome and transcriptome, there are at least two isoforms of Kcnma1α expressed in the skate. One of the β subunits (β4), which is known to decrease voltage sensitivity, was also identified in the skate genome and transcriptome and in the ampulla. These studies advance our knowledge of BK channels and suggest further studies in the ampulla and other excitable tissues. PMID:26687710

Cocoa Clone Resistant to Phytophthora Palmivora Pod Borer (CPB) in South Sulawesi

OpenAIRE

Sartika Dewi, Vien

2017-01-01

Helopeltis sp. is one of the main pest in cacao plants. Helopeltis sp. Able to decreasing the production of cacao about 50-60%. This research aims to understand the development of Helopeltis sp. investation in five types of clone cocoa. Collected data have done every week for six weeks in five types of clone cocoa which are clone GBT, clone M01, clone 45, clone s2 and clone BB. Every clone chosen 15 pod sampeles fruit with different size of pod following 5-10cm, 11-13cm and ripe pod which use...

Heterogeneity in induced thermal resistance of rat tumor cell clones

International Nuclear Information System (INIS)

Tomasovic, S.P.; Rosenblatt, P.L.; Heitzman, D.

1983-01-01

Four 13762NF rat mammary adenocarcinoma clones were examined for their survival response to heating under conditions that induced transient thermal resistance (thermotolerance). Clones MTC and MTF7 were isolated from the subcutaneous locally growing tumor, whereas clones MTLn2 and MTLn3 were derived from spontaneous lung metastases. There was heterogeneity among these clones in thermotolerance induced by either fractionated 45 0 C or continuous 42 0 C heating, but the order of sensitivity was not necessarily the same. The clones developed thermal resistance at different rates and to different degrees within the same time intervals. There was heterogeneity between clones isolated from within either the primary site or metastatic lesions. However, clones derived from metastatic foci did not intrinsically acquire more or less thermotolerance to fractionated 45 0 C or continuous 42 0 C heating than did clones from the primary tumor. Further, there was no apparent relationship between any phenotypic properties that conferred more or less thermotolerance in vitro and any phenotypic properties that conferred enhanced metastatic success of these same clones by spontaneous (subcutaneous) or experimental (intravenous) routes in vivo. These tumor clones also differ in their karyotype, metastatic potential, cell surface features, sensitivity to x-irradiation and drugs, and ability to repair sublethal radiation damage. These results provide further credence to the concept that inherent heterogeneity within tumors may be as important in therapeutic success as other known modifiers of outcome such as site and treatment heterogeneity

Stability of the JP2 clone of Aggregatibacter actinomycetemcomitans.

Science.gov (United States)

Haubek, D; Ennibi, O-K; Vaeth, M; Poulsen, S; Poulsen, K

2009-09-01

The JP2 clone of Aggregatibacter actinomycetemcomitans is strongly associated with aggressive periodontitis. To obtain information about colonization dynamics of the JP2 clone, we used PCR to examine its presence in 365 Moroccan juveniles from whom periodontal plaque samples were collected at baseline and after one and two years. Periodontal attachment loss was measured at baseline and at the two-year follow-up. At baseline, 43 (12%) carriers of the JP2 clone were found. Nearly half (44 %) of these were persistently colonized with the clone. The relative risk for the development of aggressive periodontitis, adjusted for the concomitant presence of other genotypes of A. actinomycetemcomitans, was highest for individuals continuously infected by the JP2 clone (RR = 13.9; 95% CI, 9.0 to 21.4), indicating a relationship between infectious dose and disease, which further substantiates the evidence for the JP2 clone as a causal factor in aggressive periodontitis.

Cloning of Plasmodium falciparum by single-cell sorting.

Science.gov (United States)

Miao, Jun; Li, Xiaolian; Cui, Liwang

2010-10-01

Malaria parasite cloning is traditionally carried out mainly by using the limiting dilution method, which is laborious, imprecise, and unable to distinguish multiply-infected RBCs. In this study, we used a parasite engineered to express green fluorescent protein (GFP) to evaluate a single-cell sorting method for rapidly cloning Plasmodium falciparum. By dividing a two-dimensional scattergram from a cell sorter into 17 gates, we determined the parameters for isolating singly-infected erythrocytes and sorted them into individual cultures. Pre-gating of the engineered parasites for GFP allowed the isolation of almost 100% GFP-positive clones. Compared with the limiting dilution method, the number of parasite clones obtained by single-cell sorting was much higher. Molecular analyses showed that parasite isolates obtained by single-cell sorting were highly homogenous. This highly efficient single-cell sorting method should prove very useful for cloning both P. falciparum laboratory populations from genetic manipulation experiments and clinical samples. Copyright 2010 Elsevier Inc. All rights reserved.

Cloning of Plasmodium falciparum by single-cell sorting

Science.gov (United States)

Miao, Jun; Li, Xiaolian; Cui, Liwang

2010-01-01

Malaria parasite cloning is traditionally carried out mainly by using the limiting dilution method, which is laborious, imprecise, and unable to distinguish multiply-infected RBCs. In this study, we used a parasite engineered to express green fluorescent protein (GFP) to evaluate a single-cell sorting method for rapidly cloning Plasmodium falciparum. By dividing a two dimensional scattergram from a cell sorter into 17 gates, we determined the parameters for isolating singly-infected erythrocytes and sorted them into individual cultures. Pre-gating of the engineered parasites for GFP allowed the isolation of almost 100% GFP-positive clones. Compared with the limiting dilution method, the number of parasite clones obtained by single-cell sorting was much higher. Molecular analyses showed that parasite isolates obtained by single-cell sorting were highly homogenous. This highly efficient single-cell sorting method should prove very useful for cloning both P. falciparum laboratory populations from genetic manipulation experiments and clinical samples. PMID:20435038

A strategy for clone selection under different production conditions.

Science.gov (United States)

Legmann, Rachel; Benoit, Brian; Fedechko, Ronald W; Deppeler, Cynthia L; Srinivasan, Sriram; Robins, Russell H; McCormick, Ellen L; Ferrick, David A; Rodgers, Seth T; Russo, A Peter

2011-01-01

Top performing clones have failed at the manufacturing scale while the true best performer may have been rejected early in the screening process. Therefore, the ability to screen multiple clones in complex fed-batch processes using multiple process variations can be used to assess robustness and to identify critical factors. This dynamic ranking of clones' strategy requires the execution of many parallel experiments than traditional approaches. Therefore, this approach is best suited for micro-bioreactor models which can perform hundreds of experiments quickly and efficiently. In this study, a fully monitored and controlled small scale platform was used to screen eight CHO clones producing a recombinant monoclonal antibody across several process variations, including different feeding strategies, temperature shifts and pH control profiles. The first screen utilized 240 micro-bioreactors were run for two weeks for this assessment of the scale-down model as a high-throughput tool for clone evaluation. The richness of the outcome data enable to clearly identify the best and worst clone as well as process in term of maximum monoclonal antibody titer. The follow-up comparison study utilized 180 micro-bioreactors in a full factorial design and a subset of 12 clone/process combinations was selected to be run parallel in duplicate shake flasks. Good correlation between the micro-bioreactor predictions and those made in shake flasks with a Pearson correlation value of 0.94. The results also demonstrate that this micro-scale system can perform clone screening and process optimization for gaining significant titer improvements simultaneously. This dynamic ranking strategy can support better choices of production clones. Copyright © 2011 American Institute of Chemical Engineers (AIChE).

Optimal multicopy asymmetric Gaussian cloning of coherent states

International Nuclear Information System (INIS)

Fiurasek, Jaromir; Cerf, Nicolas J.

2007-01-01

We investigate the asymmetric Gaussian cloning of coherent states which produces M copies from N input replicas in such a way that the fidelity of each copy may be different. We show that the optimal asymmetric Gaussian cloning can be performed with a single phase-insensitive amplifier and an array of beam splitters. We obtain a simple analytical expression characterizing the set of optimal asymmetric Gaussian cloning machines and prove the optimality of these cloners using the formalism of Gaussian completely positive maps and semidefinite programming techniques. We also present an alternative implementation of the asymmetric cloning machine where the phase-insensitive amplifier is replaced with a beam splitter, heterodyne detector, and feedforward

Optimal multicopy asymmetric Gaussian cloning of coherent states

Science.gov (United States)

Fiurášek, Jaromír; Cerf, Nicolas J.

2007-05-01

We investigate the asymmetric Gaussian cloning of coherent states which produces M copies from N input replicas in such a way that the fidelity of each copy may be different. We show that the optimal asymmetric Gaussian cloning can be performed with a single phase-insensitive amplifier and an array of beam splitters. We obtain a simple analytical expression characterizing the set of optimal asymmetric Gaussian cloning machines and prove the optimality of these cloners using the formalism of Gaussian completely positive maps and semidefinite programming techniques. We also present an alternative implementation of the asymmetric cloning machine where the phase-insensitive amplifier is replaced with a beam splitter, heterodyne detector, and feedforward.

Bengal Bay clone ST772-MRSA-V outbreak: conserved clone causes investigation challenges.

Science.gov (United States)

Blomfeldt, A; Larssen, K W; Moghen, A; Haugum, K; Steen, T W; Jørgensen, S B; Aamot, H V

2017-03-01

The Bengal Bay clone, ST772-MRSA-V, associated with multi-drug resistance, Panton-Valentine leukocidin (PVL) and skin and soft tissue infections, is emerging worldwide. In Norway, a country with low prevalence of meticillin-resistant Staphylococcus aureus (MRSA), increased occurrence of ST772-MRSA-V has also caused hospital outbreaks. The conserved nature of this clone challenged the outbreak investigations. To evaluate the usefulness of S. aureus protein A (spa) typing, multiple-locus variable number tandem repeat fingerprinting/analysis (MLVF/MLVA) and pulsed-field gel electrophoresis (PFGE) when investigating outbreaks with a conserved MRSA clone. A panel of 25 MRSA isolates collected in 2004-2014, consisting of six hospital outbreak isolates and 19 sporadic isolates, were analysed using spa typing, polymerase chain reaction detection of genes encoding PVL, MLVF/MLVA and PFGE. All isolates were ST772-MRSA-V-t657 and resistant to erythromycin, gentamicin and norfloxacin, and 88% were PVL positive. PFGE could not discriminate between the isolates (≥85% similarity). MLVF resolved five types [Simpson's index of diversity (SID)=0.56], MLVA resolved six types (SID=0.66), and both methods separated the hospital isolates into two defined outbreaks. MLVF/MLVA could not discriminate all epidemiologically unlinked cases and identical genotypes originated from a timespan of 10 years. MLVA was regarded as most suitable due to its higher discriminatory power and ability to provide unambiguous profiles. However, the Bengal Bay clone may require higher resolution methods for exact demarcation of outbreaks due to low diversity among isolates. Copyright © 2016 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.

TA-GC cloning: A new simple and versatile technique for the directional cloning of PCR products for recombinant protein expression.

Directory of Open Access Journals (Sweden)

Athanasios Niarchos

Full Text Available During the last few decades, the recombinant protein expression finds more and more applications. The cloning of protein-coding genes into expression vectors is required to be directional for proper expression, and versatile in order to facilitate gene insertion in multiple different vectors for expression tests. In this study, the TA-GC cloning method is proposed, as a new, simple and efficient method for the directional cloning of protein-coding genes in expression vectors. The presented method features several advantages over existing methods, which tend to be relatively more labour intensive, inflexible or expensive. The proposed method relies on the complementarity between single A- and G-overhangs of the protein-coding gene, obtained after a short incubation with T4 DNA polymerase, and T and C overhangs of the novel vector pET-BccI, created after digestion with the restriction endonuclease BccI. The novel protein-expression vector pET-BccI also facilitates the screening of transformed colonies for recombinant transformants. Evaluation experiments of the proposed TA-GC cloning method showed that 81% of the transformed colonies contained recombinant pET-BccI plasmids, and 98% of the recombinant colonies expressed the desired protein. This demonstrates that TA-GC cloning could be a valuable method for cloning protein-coding genes in expression vectors.

Early selection of Eucalyptus clones in retrospective nursery test ...

African Journals Online (AJOL)

Within the framework of the eucalyptus breeding programme in the Congo, two retrospective tests were conducted using mature clones in the field and young cuttings under nursery conditions with two hybrids: 13 clones of Eucalyptus tereticornis* Eucalyptus grandis for the test TC 82-1B and 17 clones of Eucalyptus ...

[Human cloning and the protection of women's interests].

Science.gov (United States)

Canabes, Marcela Ahumada

2008-01-01

The Human Cloning, both therapeutic and full birth cloning, involves and affects women in a special way. The United Nation's Declaration on the Cloning of Human Beings includes a special clause referred to them. Also the Spanish law does it. This works pretend to analyse the meaning of the inclusion of women's interests in this document. At the same time, I will consider the foundations and the importance of the reference to the women.

Experimental demonstration of continuous variable cloning with phase-conjugate inputs

DEFF Research Database (Denmark)

Sabuncu, Metin; Andersen, Ulrik Lund; Leuchs, G.

2007-01-01

We report the first experimental demonstration of continuous variable cloning of phase-conjugate coherent states as proposed by Cerf and Iblisdir [Phys. Rev. Lett. 87, 247903 (2001)]. In contrast to this proposal, the cloning transformation is accomplished using only linear optical components......, homodyne detection, and feedforward. As a result of combining phase conjugation with a joint measurement strategy, superior cloning is demonstrated with cloning fidelities reaching 89%....

Cardiovascular Safety Pharmacology of Sibutramine.

Science.gov (United States)

Yun, Jaesuk; Chung, Eunyong; Choi, Ki Hwan; Cho, Dae Hyun; Song, Yun Jeong; Han, Kyoung Moon; Cha, Hey Jin; Shin, Ji Soon; Seong, Won-Keun; Kim, Young-Hoon; Kim, Hyung Soo

2015-07-01

Sibutramine is an anorectic that has been banned since 2010 due to cardiovascular safety issues. However, counterfeit drugs or slimming products that include sibutramine are still available in the market. It has been reported that illegal sibutramine-contained pharmaceutical products induce cardiovascular crisis. However, the mechanism underlying sibutramine-induced cardiovascular adverse effect has not been fully evaluated yet. In this study, we performed cardiovascular safety pharmacology studies of sibutramine systemically using by hERG channel inhibition, action potential duration, and telemetry assays. Sibutramine inhibited hERG channel current of HEK293 cells with an IC50 of 3.92 μM in patch clamp assay and increased the heart rate and blood pressure (76 Δbpm in heart rate and 51 ΔmmHg in blood pressure) in beagle dogs at a dose of 30 mg/kg (per oral), while it shortened action potential duration (at 10 μM and 30 μM, resulted in 15% and 29% decreases in APD50, and 9% and 17% decreases in APD90, respectively) in the Purkinje fibers of rabbits and had no effects on the QTc interval in beagle dogs. These results suggest that sibutramine has a considerable adverse effect on the cardiovascular system and may contribute to accurate drug safety regulation.

Cloning animals by somatic cell nuclear transfer – biological factors

Science.gov (United States)

Tian, X Cindy; Kubota, Chikara; Enright, Brian; Yang, Xiangzhong

2003-01-01

Cloning by nuclear transfer using mammalian somatic cells has enormous potential application. However, somatic cloning has been inefficient in all species in which live clones have been produced. High abortion and fetal mortality rates are commonly observed. These developmental defects have been attributed to incomplete reprogramming of the somatic nuclei by the cloning process. Various strategies have been used to improve the efficiency of nuclear transfer, however, significant breakthroughs are yet to happen. In this review we will discuss studies conducted, in our laboratories and those of others, to gain a better understanding of nuclear reprogramming. Because cattle are a species widely used for nuclear transfer studies, and more laboratories have succeeded in cloning cattle than any other specie, this review will be focused on somatic cell cloning of cattle. PMID:14614770

Time-Efficient Cloning Attacks Identification in Large-Scale RFID Systems

Directory of Open Access Journals (Sweden)

Ju-min Zhao

2017-01-01

Full Text Available Radio Frequency Identification (RFID is an emerging technology for electronic labeling of objects for the purpose of automatically identifying, categorizing, locating, and tracking the objects. But in their current form RFID systems are susceptible to cloning attacks that seriously threaten RFID applications but are hard to prevent. Existing protocols aimed at detecting whether there are cloning attacks in single-reader RFID systems. In this paper, we investigate the cloning attacks identification in the multireader scenario and first propose a time-efficient protocol, called the time-efficient Cloning Attacks Identification Protocol (CAIP to identify all cloned tags in multireaders RFID systems. We evaluate the performance of CAIP through extensive simulations. The results show that CAIP can identify all the cloned tags in large-scale RFID systems fairly fast with required accuracy.

Cloning and adoption: a reply to Levy and Lotz.

Science.gov (United States)

Strong, Carson

2008-02-01

In previous articles I discussed the ethics of human reproductive cloning, focusing on a possible future scenario in which reproductive cloning can be accomplished without an elevated risk of anomalies to the children who are created. I argued that in such a scenario it would be ethically permissible for infertile couples to use cloning as a way to have genetically related children and that such use should not be prohibited. In 'Reproductive Cloning and a (Kind of) Genetic Fallacy', Neil Levy and Mianna Lotz raise objections to my conclusions. They disagree with the view, for which I argued, that some couples can have defensible reasons for desiring genetically related children. They also offer several new arguments against reproductive cloning, including an argument that it would diminish the number of adoptions, thereby adversely affecting the welfare of children who need to be adopted. In this paper I point out that Levy and Lotz's criticisms misconstrue my arguments and that there are serious problems with their arguments for prohibiting infertile couples from using cloning, including their argument from adoption.

[The status of human cloning in the international setting].

Science.gov (United States)

Rey del Castillo, Javier

2006-01-01

The General Assembly of the United Nations submitted a Declaration on Human Cloning in March 2005. The text of such Declaration was the result of a difficult and long process, taking more than three years. Being a Declaration instead of a Resolution, it has not legal capability in inforcing United Nations members to act according to its recommendations. This article begins with an explanation of several terms referred to cloning. Different countries' legislation on cloning is analyzed. Positions of the same countries at the Convention of the United Nations are as well analyzed. Comparing both countries' views shows that national legislation on cloning is independent and orientated by some countries' particular interests and biological and ethical views on these issues. Future developments on human cloning and its applications will be shared among all countries, both the ones currently allowing and supporting "therapeutic" cloning and the ones now banning it. In such case, it would be important to reach agreements on these issues at an international level. The article discusses possible legislative developments and offers some proposals to reach such agreements.

Early selection of elite clones of an ornamental bromeliad in vitro Seleção precoce in vitro de clones elite de uma bromélia ornamental

Directory of Open Access Journals (Sweden)

Candida Elisa Manfio

2010-07-01

Full Text Available Orthophytum grossiorum is a typical bromeliad from Atlantic forestry threatened of extinction. The objectives of this research were to select O. grossiorum clones with ornamental values easy to propagate in vitro, and establish in vitro propagation protocols for these clones. The project was developed in three steps: germination and in vitro selection of seedlings responsive to BAP (6-benzylaminopurine, selection of clones with ornamental values, and establishment of protocol for in vitro propagation of the selected clones. In the first step only 18.33% of plantlets germinated in vitro were responsive to BAP. These plantlets were selected and replicated in vitro several times, each replicated plantlet constituting a clone. In the second step these clones were established ex vitro and surveyed for ornamental attributes. Five out of 11 clones were selected in this step. These clones presented distinct phenotypic traits and were considered of high ornamental quality. In the third step a protocol for in vitro propagation was developed for each selected clone.Orthophytum grossiorum é uma bromélia ameaçada de extinção típica de Mata Atlântica. Os objetivos deste trabalho foram selecionar clones de O. grossiorum com potencial ornamental e de fácil propagação in vitro e estabelecer protocolo de propagação in vitro para esses clones. O trabalho foi desenvolvido em três etapas: germinação e em seleção in vitro de plântulas responsivas a BAP (6-benzylaminopurine, seleção de clones com valores ornamentais e estabelecimento de protocolo para propagação in vitro dos clones selecionados. Na primeira etapa, foi observado que apenas 18.33% das plântulas germinadas in vitro eram responsivas a BAP. Essas plântulas foram selecionadas e reproduzidas em in vitro, e cada plântula selecionada e reproduzida constituiu um clone. Na segunda etapa, esses clones foram estabelecidos ex vitro e selecionados em relação aos atributos ornamentais

Biotechnology. Perseverance leads to cloned pig in Japan.

Science.gov (United States)

Pennisi, E; Normile, D

2000-08-18

Low success rates and unpredictable results have plagued cloning researchers, particularly those trying to clone pigs. Now, on page 1188, Japanese researchers offer the first scientific report of a cloned pig, named Xena, raising hopes that pigs could one day provide an unlimited supply of organs for transplantation thanks to their close physiological relationship to humans. But this week those hopes were dealt a blow by more evidence suggesting that pig retroviruses can infect human cells.

[Media, cloning, and bioethics].

Science.gov (United States)

Costa, S I; Diniz, D

2000-01-01

This article was based on an analysis of three hundred articles from mainstream Brazilian periodicals over a period of eighteen months, beginning with the announcement of the Dolly case in February 1997. There were two main objectives: to outline the moral constants in the press associated with the possibility of cloning human beings and to identify some of the moral assumptions concerning scientific research with non-human animals that were published carelessly by the media. The authors conclude that there was a haphazard spread of fear concerning the cloning of human beings rather than an ethical debate on the issue, and that there is a serious gap between bioethical reflections and the Brazilian media.

Dogs cloned from adult somatic cells.

Science.gov (United States)

Lee, Byeong Chun; Kim, Min Kyu; Jang, Goo; Oh, Hyun Ju; Yuda, Fibrianto; Kim, Hye Jin; Hossein, M Shamim; Shamim, M Hossein; Kim, Jung Ju; Kang, Sung Keun; Schatten, Gerald; Hwang, Woo Suk

2005-08-04

Several mammals--including sheep, mice, cows, goats, pigs, rabbits, cats, a mule, a horse and a litter of three rats--have been cloned by transfer of a nucleus from a somatic cell into an egg cell (oocyte) that has had its nucleus removed. This technology has not so far been successful in dogs because of the difficulty of maturing canine oocytes in vitro. Here we describe the cloning of two Afghan hounds by nuclear transfer from adult skin cells into oocytes that had matured in vivo. Together with detailed sequence information generated by the canine-genome project, the ability to clone dogs by somatic-cell nuclear transfer should help to determine genetic and environmental contributions to the diverse biological and behavioural traits associated with the many different canine breeds.

Statement on Human Cloning

Science.gov (United States)

... as our understanding of this technology advances. Support Stem Cell Research (including Research Cloning) AAAS supports stem cell research, including the use of nuclear transplantation techniques (also ...

High-Throughput Cloning and Expression Library Creation for Functional Proteomics

Science.gov (United States)

Festa, Fernanda; Steel, Jason; Bian, Xiaofang; Labaer, Joshua

2013-01-01

The study of protein function usually requires the use of a cloned version of the gene for protein expression and functional assays. This strategy is particular important when the information available regarding function is limited. The functional characterization of the thousands of newly identified proteins revealed by genomics requires faster methods than traditional single gene experiments, creating the need for fast, flexible and reliable cloning systems. These collections of open reading frame (ORF) clones can be coupled with high-throughput proteomics platforms, such as protein microarrays and cell-based assays, to answer biological questions. In this tutorial we provide the background for DNA cloning, discuss the major high-throughput cloning systems (Gateway® Technology, Flexi® Vector Systems, and Creator™ DNA Cloning System) and compare them side-by-side. We also report an example of high-throughput cloning study and its application in functional proteomics. This Tutorial is part of the International Proteomics Tutorial Programme (IPTP12). Details can be found at http://www.proteomicstutorials.org. PMID:23457047

Clip, connect, clone

DEFF Research Database (Denmark)

Fujima, Jun; Lunzer, Aran; Hornbæk, Kasper

2010-01-01

using three mechanisms: clipping of input and result elements from existing applications to form cells on a spreadsheet; connecting these cells using formulas, thus enabling result transfer between applications; and cloning cells so that multiple requests can be handled side by side. We demonstrate...

Main: Clone Detail [KOME

Lifescience Database Archive (English)

Full Text Available Clone Detail Mapping Pseudomolecule data detail Detail information Mapping to the T...IGR japonica Pseudomolecules kome_mapping_pseudomolecule_data_detail.zip kome_mapping_pseudomolecule_data_detail ...

Piglets born from handmade cloning, an innovative cloning method without micromanipulation

DEFF Research Database (Denmark)

Du, Y.; Kragh, P.M.; Zhang, Y.

2007-01-01

Porcine handmade cloning (HMC), a simplified alternative of micromanipulation based traditional cloning (TC) has been developed in multiple phases during the past years, but the final evidence of its biological value, births of piglets was missing. Here we report the first births of healthy piglets......) of HMC reconstructed embryos developed to blastocysts with an average cell number of 77 ± 3 (n = 26) after 7 days in vitro culture (IVC). According to our knowledge, this is the highest in vitro developmental rate after porcine somatic cell nuclear transfer (SCNT). A total of 416 blastocysts from HMC......, mixed with 150 blastocysts from TC using a cell line from a different breed were transferred surgically to nine synchronized recipients. Out of the four pregnancies (44.4%) two were lost, while two pregnancies went to term and litters of 3 and 10 piglets were delivered by Caesarean section, with live...

Fundamental resource-allocating model in colleges and universities based on Immune Clone Algorithms

Science.gov (United States)

Ye, Mengdie

2017-05-01

In this thesis we will seek the combination of antibodies and antigens converted from the optimal course arrangement and make an analogy with Immune Clone Algorithms. According to the character of the Algorithms, we apply clone, clone gene and clone selection to arrange courses. Clone operator can combine evolutionary search and random search, global search and local search. By cloning and clone mutating candidate solutions, we can find the global optimal solution quickly.

Cloning Expeditions: Risky but Rewarding

Science.gov (United States)

2013-01-01

In the 1980s, a good part of my laboratory was using the then-new recombinant DNA techniques to clone and characterize many important cell surface membrane proteins: GLUT1 (the red cell glucose transporter) and then GLUT2 and GLUT4, the red cell anion exchange protein (Band 3), asialoglycoprotein receptor subunits, sucrase-isomaltase, the erythropoietin receptor, and two of the subunits of the transforming growth factor β (TGF-β) receptor. These cloned genes opened many new fields of basic research, including membrane insertion and trafficking of transmembrane proteins, signal transduction by many members of the cytokine and TGF-β families of receptors, and the cellular physiology of glucose and anion transport. They also led to many insights into the molecular biology of several cancers, hematopoietic disorders, and diabetes. This work was done by an exceptional group of postdocs and students who took exceptionally large risks in developing and using novel cloning technologies. Unsurprisingly, all have gone on to become leaders in the fields of molecular cell biology and molecular medicine. PMID:24061478

Towards Clone Detection in UML Domain Models

DEFF Research Database (Denmark)

Störrle, Harald

2010-01-01

Code clones - that is, duplicate fragments of code - have been studied for a long time. There is strong evidence that code clones are a major source of software faults. Anecdotal evidence suggests that this phenomenon is not restricted to code, but occurs in models in a very similar way. So it is...

Analysis of an epigenetic argument against human reproductive cloning.

Science.gov (United States)

Nordgren, Anders

2006-08-01

Human reproductive cloning is a much disputed ethical issue. This technology is often condemned as being contrary to human dignity. However, there are also risk arguments. An ethical argument that is often put forward by scientists but seldom developed in more detail focuses on health risks in animal cloning. There is a high risk that animal clones exhibit abnormalities and these are increasingly believed to be due to errors in epigenetic reprogramming. The argument is that human reproductive cloning should not be carried out because human clones are also likely to exhibit abnormalities due to inappropriate epigenetic reprogramming. Different versions of this epigenetic argument are analysed, a categorical version and a non-categorical. The non-categorical version is suggested to be more well-considered. With regard to policy making on human reproductive cloning, the categorical version can be used to prescribe a permanent ban, while the non-categorical version can be used to prescribe a temporary ban. The implications of the precautionary principle--as interpreted in the European Union--are investigated. The conclusion is that it seems possible to support a temporary ban by reference to this principle.

Probabilistic quantum cloning of a subset of linearly dependent states

Science.gov (United States)

Rui, Pinshu; Zhang, Wen; Liao, Yanlin; Zhang, Ziyun

2018-02-01

It is well known that a quantum state, secretly chosen from a certain set, can be probabilistically cloned with positive cloning efficiencies if and only if all the states in the set are linearly independent. In this paper, we focus on probabilistic quantum cloning of a subset of linearly dependent states. We show that a linearly-independent subset of linearly-dependent quantum states {| Ψ 1⟩,| Ψ 2⟩,…,| Ψ n ⟩} can be probabilistically cloned if and only if any state in the subset cannot be expressed as a linear superposition of the other states in the set {| Ψ 1⟩,| Ψ 2⟩,…,| Ψ n ⟩}. The optimal cloning efficiencies are also investigated.

Experimental continuous-variable cloning of partial quantum information

DEFF Research Database (Denmark)

Sabuncu, Metin; Leuchs, Gerd; Andersen, Ulrik Lund

2008-01-01

The fidelity of a quantum transformation is strongly linked with the prior partial information of the state to be transformed. We illustrate this interesting point by proposing and demonstrating the superior cloning of coherent states with prior partial information. More specifically, we propose...... two simple transformations that under the Gaussian assumption optimally clone symmetric Gaussian distributions of coherent states as well as coherent states with known phases. Furthermore, we implement for the first time near-optimal state-dependent cloning schemes relying on simple linear optics...

Twelve years before the quantum no-cloning theorem

Science.gov (United States)

Ortigoso, Juan

2018-03-01

The celebrated quantum no-cloning theorem establishes the impossibility of making a perfect copy of an unknown quantum state. The discovery of this important theorem for the field of quantum information is currently dated 1982. I show here that an article published in 1970 [J. L. Park, Found. Phys. 1, 23-33 (1970)] contained an explicit mathematical proof of the impossibility of cloning quantum states. I analyze Park's demonstration in the light of published explanations concerning the genesis of the better-known papers on no-cloning.

An accurate clone-based haplotyping method by overlapping pool sequencing.

Science.gov (United States)

Li, Cheng; Cao, Changchang; Tu, Jing; Sun, Xiao

2016-07-08

Chromosome-long haplotyping of human genomes is important to identify genetic variants with differing gene expression, in human evolution studies, clinical diagnosis, and other biological and medical fields. Although several methods have realized haplotyping based on sequencing technologies or population statistics, accuracy and cost are factors that prohibit their wide use. Borrowing ideas from group testing theories, we proposed a clone-based haplotyping method by overlapping pool sequencing. The clones from a single individual were pooled combinatorially and then sequenced. According to the distinct pooling pattern for each clone in the overlapping pool sequencing, alleles for the recovered variants could be assigned to their original clones precisely. Subsequently, the clone sequences could be reconstructed by linking these alleles accordingly and assembling them into haplotypes with high accuracy. To verify the utility of our method, we constructed 130 110 clones in silico for the individual NA12878 and simulated the pooling and sequencing process. Ultimately, 99.9% of variants on chromosome 1 that were covered by clones from both parental chromosomes were recovered correctly, and 112 haplotype contigs were assembled with an N50 length of 3.4 Mb and no switch errors. A comparison with current clone-based haplotyping methods indicated our method was more accurate. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Caracterização isoenzimática e morfológica de clones e introduções de alho Morphological and electrophoretic characterization of garlic clones

Directory of Open Access Journals (Sweden)

Walter José Siqueira

1985-01-01

Full Text Available Em virtude do grande número de denominações locais para clones de alho, nem sempre correspondentes a materiais distintos, conduziu-se o presente estudo objetivando a caracterização e classificação de 72 clones e introduções de alho (Allium sativum L., e um clone de alho-rei (A. ampeloprasum L.. Isso foi feito analisando as isoenzimas alcooldesidrogenase (ADH, esterase (EST, peroxidase (PRX e fosfoglucoisomerase (PGI através da técnica de eletroforese horizontal em gel de amido hidrolisado de batata. Verificou-se que os clones nacionais e introduzidos se enquadram nos grupos aqui denominados DIKA ou CJLB, respectivamente para os padrões de ADH, EST, PRX e PGI. Entretanto, os padrões CILB, CJKB e CIKB foram observados em alguns clones estrangeiros, sugerindo sua maior variabilidade em relação aos nacionais. O alho-rei apresentou padrões diferentes dos encontrados na espécie A. sativum L. A associação dos resultados da técnica de eletroforese de isoenzinas com a caracterização morfológica da parte aérea, bulbos, bulbilhos, coloração externa dos bulbos e bulbilhos e ciclo cultural, permitiu a classificação dos clones nacionais de alho em 19 grupos distintos.Since there exist different local names for the same garlic (Allium sativum L. clones, it was made an attempt to distinguish them by the morphology, cycle and isozyme electrophoresis. The isozyme analysis of alcoholdehydrogenase, esterase, peroxydase and phosphoglucoisomerase separated the Brazilian clones in two groups. The foreign clones had different band patterns adding other three more groups. Morphology of bulbs and clones allowed the separation of clones into eight groups; top morphology into ten and cycle length into three. Morphology, cycle and electrophoresis together characterized the seventy two analysed clones into nineteen distinct groups.

Calcium activated K⁺ channels in the electroreceptor of the skate confirmed by cloning. Details of subunits and splicing.

Science.gov (United States)

King, Benjamin L; Shi, Ling Fang; Kao, Peter; Clusin, William T

2016-03-01

Elasmobranchs detect small potentials using excitable cells of the ampulla of Lorenzini which have calcium-activated K(+) channels, first described in 1974. A distinctive feature of the outward current in voltage clamped ampullae is its apparent insensitivity to voltage. The sequence of a BK channel α isoform expressed in the ampulla of the skate was characterized. A signal peptide is present at the beginning of the gene. When compared to human isoform 1 (the canonical sequence), the largest difference was absence of a 59 amino acid region from the S8-S9 intra-cellular linker that contains the strex regulatory domain. The ampulla isoform was also compared with the isoform predicted in late skate embryos where strex was also absent. The BK voltage sensors were conserved in both skate isoforms. Differences between the skate and human BK channel included alternative splicing. Alternative splicing occurs at seven previously defined sites that are characteristic for BK channels in general and hair cells in particular. Skate BK sequences were highly similar to the Australian ghost shark and several other vertebrate species. Based on alignment of known BK sequences with the skate genome and transcriptome, there are at least two isoforms of Kcnma1α expressed in the skate. One of the β subunits (β4), which is known to decrease voltage sensitivity, was also identified in the skate genome and transcriptome and in the ampulla. These studies advance our knowledge of BK channels and suggest further studies in the ampulla and other excitable tissues. Copyright © 2015 Elsevier B.V. All rights reserved.

Cloning of the human androgen receptor cDNA

International Nuclear Information System (INIS)

Govindan, M.V.; Burelle, M.; Cantin, C.; Kabrie, C.; Labrie, F.; Lachance, Y.; Leblanc, G.; Lefebvre, C.; Patel, P.; Simard, J.

1988-01-01

The authors discuss how in order to define the functional domains of the human androgen receptor, complementary DNA (cDNA) clones encoding the human androgen receptor (hAR) have been isolated from a human testis λgtll cDNA library using synthetic oligonnucleotide probes, homologous to segments of the human glucocorticoid, estradiol and progesterone receptors. The cDNA clones corresponding to the human glucocorticoid, estradiol and progesterone receptors were eliminated after cross-hybridization with their respective cDNA probes and/or after restriction mapping of the cDNA clones. The remaining cDNA clones were classified into different groups after analysis by restriction digestion and cross-hybridization. Two of the largest cDNA clones from each group were inserted into an expression vector in both orientations. The linearized plasmids were used as templates in in vitro transcription with T7 RNA polymerase. Subsequent in vitro translation of the purified transcripts in rabbit reticulocyte lysate followed by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) permitted the characterization of the encoded polyeptides. The expressed proteins larger than 30,000 Da were analyzed for their ability to bind tritium-labelled dihydrotestosterone ([ 3 H] DHT) with high affinity and specificity

Implementing phase-covariant cloning in circuit quantum electrodynamics

Energy Technology Data Exchange (ETDEWEB)

Zhu, Meng-Zheng [School of Physics and Material Science, Anhui University, Hefei 230039 (China); School of Physics and Electronic Information, Huaibei Normal University, Huaibei 235000 (China); Ye, Liu, E-mail: yeliu@ahu.edu.cn [School of Physics and Material Science, Anhui University, Hefei 230039 (China)

2016-10-15

An efficient scheme is proposed to implement phase-covariant quantum cloning by using a superconducting transmon qubit coupled to a microwave cavity resonator in the strong dispersive limit of circuit quantum electrodynamics (QED). By solving the master equation numerically, we plot the Wigner function and Poisson distribution of the cavity mode after each operation in the cloning transformation sequence according to two logic circuits proposed. The visualizations of the quasi-probability distribution in phase-space for the cavity mode and the occupation probability distribution in the Fock basis enable us to penetrate the evolution process of cavity mode during the phase-covariant cloning (PCC) transformation. With the help of numerical simulation method, we find out that the present cloning machine is not the isotropic model because its output fidelity depends on the polar angle and the azimuthal angle of the initial input state on the Bloch sphere. The fidelity for the actual output clone of the present scheme is slightly smaller than one in the theoretical case. The simulation results are consistent with the theoretical ones. This further corroborates our scheme based on circuit QED can implement efficiently PCC transformation.

Entangled cloning of stabilizer codes and free fermions

Science.gov (United States)

Hsieh, Timothy H.

2016-10-01

Though the no-cloning theorem [Wooters and Zurek, Nature (London) 299, 802 (1982), 10.1038/299802a0] prohibits exact replication of arbitrary quantum states, there are many instances in quantum information processing and entanglement measurement in which a weaker form of cloning may be useful. Here, I provide a construction for generating an "entangled clone" for a particular but rather expansive and rich class of states. Given a stabilizer code or free fermion Hamiltonian, this construction generates an exact entangled clone of the original ground state, in the sense that the entanglement between the original and the exact copy can be tuned to be arbitrarily small but finite, or large, and the relation between the original and the copy can also be modified to some extent. For example, this Rapid Communication focuses on generating time-reversed copies of stabilizer codes and particle-hole transformed ground states of free fermion systems, although untransformed clones can also be generated. The protocol leverages entanglement to simulate a transformed copy of the Hamiltonian without having to physically implement it and can potentially be realized in superconducting qubits or ultracold atomic systems.

Human reproductive cloning and reasons for deprivation.

Science.gov (United States)

Jensen, D A

2008-08-01

Human reproductive cloning provides the possibility of genetically related children for persons for whom present technologies are ineffective. I argue that the desire for genetically related children is not, by itself, a sufficient reason to engage in human reproductive cloning. I show this by arguing that the value underlying the desire for genetically related children implies a tension between the parent and the future child. This tension stems from an instance of a deprivation and violates a general principle of reasons for deprivation. Alternative considerations, such as a right to procreative autonomy, do not appear helpful in making the case for human reproductive cloning merely on the basis of the desire for genetically related children.

Cloning the interleukin 1 receptor from human T cells

International Nuclear Information System (INIS)

Sims, J.E.; Acres, R.B.; Grubin, C.E.; McMahan, C.J.; Wignall, J.M.; March, C.J.; Dower, S.K.

1989-01-01

cDNA clones of the interleukin 1 (IL-1) receptor expressed in a human T-cell clone have been isolated by using a murine IL-1 receptor cDNA as a probe. The human and mouse receptors show a high degree of sequence conservation. Both are integral membrane proteins possessing a single membrane-spanning segment. Similar to the mouse receptor, the human IL-1 receptor contains a large cytoplasmic region and an extracellular, IL-1 binding portion composed of three immunoglobulin-like domains. When transfected into COS cells, the human IL-1 receptor cDNA clone leads to expression of two different affinity classes of receptors, with K a values indistinguishable from those determined for IL-1 receptors in the original T-cell clone. An IL-1 receptor expressed in human dermal fibroblasts has also been cloned and sequenced and found to be identical to the IL-1 receptor expressed in T cells

The Three Major Spanish Clones of Penicillin-Resistant Streptococcus pneumoniae Are the Most Common Clones Recovered in Recent Cases of Meningitis in Spain

Science.gov (United States)

Enright, Mark C.; Fenoll, Asunción; Griffiths, David; Spratt, Brian G.

1999-01-01

One hundred six isolates of Streptococcus pneumoniae recovered in Spain from patients with meningitis in 1997 and 1998 were characterized by multilocus sequence typing. A heterogeneous collection of genotypes was associated with meningitis in Spain: 65 different sequence types were resolved and, even at a genetic distance of 0.43, there were 37 distinct lineages. Thirty-eight percent of the isolates, including all isolates of serotypes 6B, 9V, 14, and 23F, were resistant to penicillin, and 24% of the isolates were members of the three major Spanish penicillin-resistant or multidrug-resistant clones of serotypes 6B, 9V, and 23F or serotype variants of these clones. These three clones (MICs, 1 to 2 μg of penicillin/ml) were the most common clones associated with pneumococcal meningitis in Spain during 1997 and 1998. Only two of the other clones associated with meningitis were penicillin resistant (MICs, 0.12 to 0.5 μg/ml). One of the two most prevalent penicillin-susceptible clones causing meningitis (serotype 3) has not been detected outside of Spain, whereas the other (serotype 18C) has been recovered from patients with meningitis in the United Kingdom, The Netherlands, and Denmark. The prevalence of meningitis caused by isolates of the three major Spanish penicillin-resistant or multiply antibiotic-resistant clones, which are now globally distributed, is disturbing and clearly establishes their ability to cause life-threatening disease. PMID:10488179

Human Cloning

Science.gov (United States)

2006-07-20

Human Fertilization and Embryology Authority (HFEA). A team of scientists headed by Alison Murdoch at the University of Newcastle received permission...not yet reported success in isolating stem cells from a cloned human embryo. A research team headed by Ian Wilmut at the University of Edinburgh...research group, headed by Douglas Melton and Kevin Eggan, submitted their proposal to a Harvard committee composed of ethicists, scientists and public

A novel approach to sequence validating protein expression clones with automated decision making

Directory of Open Access Journals (Sweden)

Mohr Stephanie E

2007-06-01

Full Text Available Abstract Background Whereas the molecular assembly of protein expression clones is readily automated and routinely accomplished in high throughput, sequence verification of these clones is still largely performed manually, an arduous and time consuming process. The ultimate goal of validation is to determine if a given plasmid clone matches its reference sequence sufficiently to be "acceptable" for use in protein expression experiments. Given the accelerating increase in availability of tens of thousands of unverified clones, there is a strong demand for rapid, efficient and accurate software that automates clone validation. Results We have developed an Automated Clone Evaluation (ACE system – the first comprehensive, multi-platform, web-based plasmid sequence verification software package. ACE automates the clone verification process by defining each clone sequence as a list of multidimensional discrepancy objects, each describing a difference between the clone and its expected sequence including the resulting polypeptide consequences. To evaluate clones automatically, this list can be compared against user acceptance criteria that specify the allowable number of discrepancies of each type. This strategy allows users to re-evaluate the same set of clones against different acceptance criteria as needed for use in other experiments. ACE manages the entire sequence validation process including contig management, identifying and annotating discrepancies, determining if discrepancies correspond to polymorphisms and clone finishing. Designed to manage thousands of clones simultaneously, ACE maintains a relational database to store information about clones at various completion stages, project processing parameters and acceptance criteria. In a direct comparison, the automated analysis by ACE took less time and was more accurate than a manual analysis of a 93 gene clone set. Conclusion ACE was designed to facilitate high throughput clone sequence

A novel nucleo-cytoplasmic hybrid clone formed via androgenesis in polyploid gibel carp

Directory of Open Access Journals (Sweden)

Zhou Li

2011-03-01

Full Text Available Abstract Background Unisexual vertebrates have been demonstrated to reproduce by gynogenesis, hybridogenesis, parthenogenesis, or kleptogenesis, however, it is uncertain how the reproduction mode contributes to the clonal diversity. Recently, polyploid gibel carp has been revealed to possess coexisting dual modes of unisexual gynogenesis and sexual reproduction and to have numerous various clones. Using sexual reproduction mating between clone D female and clone A male and subsequent 7 generation multiplying of unisexual gynogenesis, we have created a novel clone strain with more than several hundred millions of individuals. Here, we attempt to identify genetic background of the novel clone and to explore the significant implication for clonal diversity contribution. Methods Several nuclear genome markers and one cytoplasmic marker, the mitochondrial genome sequence, were used to identify the genetic organization of the randomly sampled individuals from different generations of the novel clone. Results Chromosome number, Cot-1 repetitive DNA banded karyotype, microsatellite patterns, AFLP profiles and transferrin alleles uniformly indicated that nuclear genome of the novel clone is identical to that of clone A, and significantly different from that of clone D. However, the cytoplasmic marker, its complete mtDNA genome sequence, is same to that of clone D, and different from that of clone A. Conclusions The present data indicate that the novel clone is a nucleo-cytoplasmic hybrid between the known clones A and D, because it originates from the offspring of gonochoristic sexual reproduction mating between clone D female and clone A male, and contains an entire nuclear genome from the paternal clone A and a mtDNA genome (cytoplasm from the maternal clone D. It is suggested to arise via androgenesis by a mechanism of ploidy doubling of clone A sperm in clone D ooplasm through inhibiting the first mitotic division. Significantly, the selected nucleo

A novel nucleo-cytoplasmic hybrid clone formed via androgenesis in polyploid gibel carp

Science.gov (United States)

2011-01-01

Background Unisexual vertebrates have been demonstrated to reproduce by gynogenesis, hybridogenesis, parthenogenesis, or kleptogenesis, however, it is uncertain how the reproduction mode contributes to the clonal diversity. Recently, polyploid gibel carp has been revealed to possess coexisting dual modes of unisexual gynogenesis and sexual reproduction and to have numerous various clones. Using sexual reproduction mating between clone D female and clone A male and subsequent 7 generation multiplying of unisexual gynogenesis, we have created a novel clone strain with more than several hundred millions of individuals. Here, we attempt to identify genetic background of the novel clone and to explore the significant implication for clonal diversity contribution. Methods Several nuclear genome markers and one cytoplasmic marker, the mitochondrial genome sequence, were used to identify the genetic organization of the randomly sampled individuals from different generations of the novel clone. Results Chromosome number, Cot-1 repetitive DNA banded karyotype, microsatellite patterns, AFLP profiles and transferrin alleles uniformly indicated that nuclear genome of the novel clone is identical to that of clone A, and significantly different from that of clone D. However, the cytoplasmic marker, its complete mtDNA genome sequence, is same to that of clone D, and different from that of clone A. Conclusions The present data indicate that the novel clone is a nucleo-cytoplasmic hybrid between the known clones A and D, because it originates from the offspring of gonochoristic sexual reproduction mating between clone D female and clone A male, and contains an entire nuclear genome from the paternal clone A and a mtDNA genome (cytoplasm) from the maternal clone D. It is suggested to arise via androgenesis by a mechanism of ploidy doubling of clone A sperm in clone D ooplasm through inhibiting the first mitotic division. Significantly, the selected nucleo-cytoplasmic hybrid female

Maternal endometrial oedema may increase perinatal mortality of cloned and transgenic piglets

DEFF Research Database (Denmark)

Schmidt, Mette; Winter, K.D.; Dantzer, Vibeke

2011-01-01

The perinatal mortality of cloned animals is a well-known problem. In the present retrospective study, we report on mortality of cloned transgenic or non-transgenic piglets produced as part of several investigations. Large White (LW) sows (n = 105) received hand-made cloned LW or minipig...... endometrial oedema in sows pregnant with cloned and transgenic piglets, as well as in empty recipients, at term. The growth of certain organs in some of the cloned piglets was reduced and the rate of stillborn piglets was greater in cloned and transgenic piglets delivered vaginally, possibly because of oedema...

TAWS: TABLE ASSISTED WALK STRATEGY IN CLONE ATTACK DETECTION

Directory of Open Access Journals (Sweden)

J Sybi Cynthia

2016-12-01

Full Text Available Wireless Sensor Networks (WSNs deployed in the destructive atmosphere are susceptible to clone attacks. Clone attack in wireless sensor network is a complicated problem because it deployed in hostile environments, and also the nodes could be physically compromised by an adversary. For valuable clone attack detection, the selection criteria play an important role in the proposed work. In this paper, it has been classified the existing detection schemes regarding device type, detection methodologies, deployment strategies and detection ranges and far explore various proposals in deployment based selection criteria category. And also this paper provides a review of detection methodology based on various clone attack detection techniques. It is also widely agreed that clones should be detected quickly as possible with the best optional. Our work is exploratory in that the proposed algorithm concern with table assisted random walk with horizontal and vertical line, frequent level key change and revokes the duplicate node. Our simulation results show that it is more efficient than the detection criteria in terms of security feature, and in detection rate with high resiliency. Specifically, it concentrates on deployment strategy which includes grid based deployment technique. These all come under the selection criteria for better security performance. Our protocol analytically provides effective and clone attack detection capability of robustness.

ReMixT: clone-specific genomic structure estimation in cancer.

Science.gov (United States)

McPherson, Andrew W; Roth, Andrew; Ha, Gavin; Chauve, Cedric; Steif, Adi; de Souza, Camila P E; Eirew, Peter; Bouchard-Côté, Alexandre; Aparicio, Sam; Sahinalp, S Cenk; Shah, Sohrab P

2017-07-27

Somatic evolution of malignant cells produces tumors composed of multiple clonal populations, distinguished in part by rearrangements and copy number changes affecting chromosomal segments. Whole genome sequencing mixes the signals of sampled populations, diluting the signals of clone-specific aberrations, and complicating estimation of clone-specific genotypes. We introduce ReMixT, a method to unmix tumor and contaminating normal signals and jointly predict mixture proportions, clone-specific segment copy number, and clone specificity of breakpoints. ReMixT is free, open-source software and is available at http://bitbucket.org/dranew/remixt .

High-throughput cloning and expression in recalcitrant bacteria

NARCIS (Netherlands)

Geertsma, Eric R.; Poolman, Bert

We developed a generic method for high-throughput cloning in bacteria that are less amenable to conventional DNA manipulations. The method involves ligation-independent cloning in an intermediary Escherichia coli vector, which is rapidly converted via vector-backbone exchange (VBEx) into an

MEANS AND METHODS FOR CLONING NUCLEIC ACID SEQUENCES

NARCIS (Netherlands)

Geertsma, Eric Robin; Poolman, Berend

2008-01-01

The invention provides means and methods for efficiently cloning nucleic acid sequences of interest in micro-organisms that are less amenable to conventional nucleic acid manipulations, as compared to, for instance, E.coli. The present invention enables high-throughput cloning (and, preferably,

Myths about Cloning

Science.gov (United States)

... aging normally. In fact, the first cattle clones ever produced are alive, healthy, and are 10 years old as of January 2008. Back to the ... until we finish assessing their safety. To the best of our knowledge, they have done so. After years of detailed study and analysis, FDA has concluded ...

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

Science.gov (United States)

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.

Japan. Human cloning ban allows some research.

Science.gov (United States)

Normile, D

2000-12-08

TOKYO--Japanese legislators last week approved a ban on human cloning that leaves room for the use of certain techniques in basic research. The action comes at the same time officials in two other countries--China and France--aired similar proposals that would prohibit so-called reproductive cloning while recognizing the possible importance of the technology in combating disease and improving human health.

Potencial forrageiro de novos clones de capim-elefante

Directory of Open Access Journals (Sweden)

Botrel Milton de Andrade

2000-01-01

Full Text Available O objetivo deste trabalho foi avaliar o comportamento de novos clones selecionados de capim-elefante. O experimento foi realizado na Embrapa Gado de Leite, em Coronel Pacheco -- MG, por um período de dois anos. Foi avaliado o potencial forrageiro de 20 clones de capim-elefante, obtidos pelo programa de melhoramento, e mais duas cultivares tradicionais (Cameroon e Taiwan A-146 usadas como testemunhas. O delineamento experimental foi o de blocos ao acaso com quatro repetições. As adubações para estabelecimento e manutenção foram realizadas de acordo com a análise do solo, visando suprir as exigências nutricionais do capim-elefante. Observaram-se diferenças significativas entre os clones, quanto ao potencial para produção de forragem, à relação folha/colmo e ao perfilhamento aéreo e basal. A maioria dos clones avaliados apresentou maior produção de matéria seca que as cultivares tradicionais, Cameroon e Taiwan A-146, durante o período seco e chuvoso. Não houve diferença significativa no teor de proteína bruta da matéria seca das cultivares controles (Cameroon e Taiwan A-146 e dos clones avaliados, em ambas as estações (águas e seca. O clone F 27-01, lançado pela Embrapa Gado de Leite com o nome de cultivar Pioneiro, destacou-se para quase todas as características agronômicas estudadas.

Cloning transformations in spin networks without external control

International Nuclear Information System (INIS)

De Chiara, Gabriele; Fazio, Rosario; Montangero, Simone; Macchiavello, Chiara; Palma, G. Massimo

2005-01-01

In this paper we present an approach to quantum cloning with unmodulated spin networks. The cloner is realized by a proper design of the network and a choice of the coupling between the qubits. We show that in the case of phase covariant cloner the XY coupling gives the best results. In the 1→2 cloning we find that the value for the fidelity of the optimal cloner is achieved, and values comparable to the optimal ones in the general N→M case can be attained. If a suitable set of network symmetries are satisfied, the output fidelity of the clones does not depend on the specific choice of the graph. We show that spin network cloning is robust against the presence of static imperfections. Moreover, in the presence of noise, it outperforms the conventional approach. In this case the fidelity exceeds the corresponding value obtained by quantum gates even for a very small amount of noise. Furthermore, we show how to use this method to clone qutrits and qudits. By means of the Heisenberg coupling it is also possible to implement the universal cloner although in this case the fidelity is 10% off that of the optimal cloner

Peripheral blood and intrathyroidal T cell clones from patients with thyroid autoimmune diseases.

Science.gov (United States)

Massart, C; Caroff, G; Maugendre, D; Genetet, N; Gibassier, J

1999-01-01

For a better understanding of the pathogenesis of thyroid autoimmune diseases, we have studied morphological and functional properties of T clones from peripheral blood lymphocytes (PBL) and from intrathyroidal lymphocytes (ITL) obtained from 3 patients with Graves' disease or 1 Hashimoto's thyroiditis. Investigations were carried out on clones cultured alone or cocultured with autologous thyrocytes. Clonage efficiency ranged from 30% to 33% for PBL and 10% to 36% for ITL. A predominance of CD4-positive clones was observed whatever the origin of the lymphocytes or the autoimmune pathology. Gamma interferon (IFN-gamma) was detected in the majority (17/19) of the clones tested. Intracytoplasmic interleukin (IL-4) was secreted in 7/19 clones and both cytokines were produced in 5/19 clones. In coculture a proliferative response and tumour necrosis factor (TNF-alpha) production were observed with 6 clones (4 from Graves thyrocytes and 2 from thyroiditis). No cytotoxic clone was derived from Graves or thyroiditis tissues. These data demonstrate that the large majority of T clones are principally CD4-T cells; all the clones secreted TNF-alpha and a large majority produced IFN-gamma. Only a few clones produced IL-4 alone or associated with IFN-gamma. Six T clones induced proliferative response and of TNF-alpha secretion in coculture. Further investigations must be performed on these antigen-reactive T clones to analyse their role in the pathogenesis of the human thyroid autoimmune diseases.

Evaluation of Quantitative and Qualitative Traits of 18 Potato Clones

Directory of Open Access Journals (Sweden)

A. R Bolandi

2016-10-01

Full Text Available Introduction Introducing potato cultivars with high yield, early maturing and desirable quality have a key role in food security, decreasing the fluctuation of the price and the store costs and also providing fresh crops throughout the year. Potato (Solanum tuberosum L. plant is one of leading agricultural products in the world with 365 million ton glands in year stands in fourth place after wheat, rice and corn. The main objective of the breeding program is yield. Increase in plant yield in the past due to the gradual elimination of defects visible by experts and today the new criteria for selection are based on principles of morphological and functional characteristics associated with the plant. Variety is one of the effective factors on plant growth and development on potato that yields components of potato is heavily dependent on it. Yield increasing in each variety affect the genetic and natural structure of variety. Nine clones of Solanum tuberosum L. cv. Kennebec from sources in Victoria, South Australia and Tasmania, and the commercially grown clone, clone 1, which was imported from Vancouver, were multiplied from pathogen-tested seed and compared in 3 Victorian potato districts during 2 seasons. The results showed that differences exist in total and size grade yield and tuber number and appearance between clones of a cultivar. They further highlight the importance of selection work to maintain desirable characteristics of established cultivars and to remove mutants with undesirable characteristics. The results of the study, Hassanpanah and Hassanabadi (2012 showed that the clones 397003-7, 396151-27, 397045-100 and Savalan (check produced higher total and marketable tuber yield, tuber number and weight per plant, plant height, main stem number per plant, tuber size average and stable tuber yield. These clones produced high and mid-uniform tuber, tuber inner crack and tuber inner ring, mid-late maturity and mid and high dry in comparison

DNA damage in Populus tremuloides clones exposed to elevated O3

International Nuclear Information System (INIS)

Tai, Helen H.; Percy, Kevin E.; Karnosky, David F.

2010-01-01

The effects of elevated concentrations of atmospheric tropospheric ozone (O 3 ) on DNA damage in five trembling aspen (Populus tremuloides Michx.) clones growing in a free-air enrichment experiment in the presence and absence of elevated concentrations of carbon dioxide (CO 2 ) were examined. Growing season mean hourly O 3 concentrations were 36.3 and 47.3 ppb for ambient and elevated O 3 plots, respectively. The 4th highest daily maximum 8-h ambient and elevated O 3 concentrations were 79 and 89 ppb, respectively. Elevated CO 2 averaged 524 ppm (+150 ppm) over the growing season. Exposure to O 3 and CO 2 in combination with O 3 increased DNA damage levels above background as measured by the comet assay. Ozone-tolerant clones 271 and 8L showed the highest levels of DNA damage under elevated O 3 compared with ambient air; whereas less tolerant clone 216 and sensitive clones 42E and 259 had comparably lower levels of DNA damage with no significant differences between elevated O 3 and ambient air. Clone 8L was demonstrated to have the highest level of excision DNA repair. In addition, clone 271 had the highest level of oxidative damage as measured by lipid peroxidation. The results suggest that variation in cellular responses to DNA damage between aspen clones may contribute to O 3 tolerance or sensitivity. - Ozone tolerant clones and sensitive Populus tremuloides clones show differences in DNA damage and repair.

Cloning of T lymphocytes from bronchoalveolar lavage fluid

NARCIS (Netherlands)

Hol, B. E.; Krouwels, F. H.; Bruinier, B.; Reijneke, R. M.; Mengelers, H. J.; Koenderman, L.; Jansen, H. M.; Out, T. A.

1992-01-01

We have prepared T-cell clones from bronchoalveolar lavage fluid (BALF) from four healthy, nonsmoking persons and from four patients with allergic asthma. T cells were cloned by direct limiting dilution and with the use of a fluorescent activated cell sorter with an automated cell deposition unit.

Optimal cloning of qubits given by an arbitrary axisymmetric distribution on the Bloch sphere

International Nuclear Information System (INIS)

Bartkiewicz, Karol; Miranowicz, Adam

2010-01-01

We find an optimal quantum cloning machine, which clones qubits of arbitrary symmetrical distribution around the Bloch vector with the highest fidelity. The process is referred to as phase-independent cloning in contrast to the standard phase-covariant cloning for which an input qubit state is a priori better known. We assume that the information about the input state is encoded in an arbitrary axisymmetric distribution (phase function) on the Bloch sphere of the cloned qubits. We find analytical expressions describing the optimal cloning transformation and fidelity of the clones. As an illustration, we analyze cloning of qubit state described by the von Mises-Fisher and Brosseau distributions. Moreover, we show that the optimal phase-independent cloning machine can be implemented by modifying the mirror phase-covariant cloning machine for which quantum circuits are known.

Sex-reversed somatic cell cloning in the mouse.

Science.gov (United States)

Inoue, Kimiko; Ogonuki, Narumi; Mekada, Kazuyuki; Yoshiki, Atsushi; Sado, Takashi; Ogura, Atsuo

2009-10-01

Somatic cell nuclear transfer has many potential applications in the fields of basic and applied sciences. However, it has a disadvantage that can never be overcome technically-the inflexibility of the sex of the offspring. Here, we report an accidental birth of a female mouse following nuclear transfer using an immature Sertoli cell. We produced a batch of 27 clones in a nuclear transfer experiment using Sertoli cells collected from neonatal male mice. Among them, one pup was female. This "male-derived female" clone grew into a normal adult and produced offspring by natural mating with a littermate. Chromosomal analysis revealed that the female clone had a 39,X karyotype, indicating that the Y chromosome had been deleted in the donor cell or at some early step during nuclear transfer. This finding suggests the possibility of resuming sexual reproduction after a single male is cloned, which should be especially useful for reviving extinct or endangered species.

The origin and evolution of the term "clone".

Science.gov (United States)

Steensma, David P

2017-06-01

In biology, the term "clone" is most widely used to designate genetically identical cells or organisms that are asexually descended from a common progenitor. The concept of clonality in hematology-oncology has received much attention in recent years, as the advent of next-generation sequencing platforms has provided new tools for detection of clonal populations in patients, and experiments on primary cells have provided fascinating new insights into the clonal architecture of human malignancies. The term "clone" is used more loosely by the general public to mean any close or identical copy. Cloning of humans has been a staple of science fiction films and dystopian novels since Aldous Huxley's Brave New World was published in 1932. Here I trace the origin and evolution of the word clone, from its first use as an agricultural and botanical term in 1903, to its widespread adoption in biology, adaptation by artists, and contemporary use in hematology-oncology. Copyright © 2017 Elsevier Ltd. All rights reserved.

Babesia bovis clones: biochemical and enzymatic characterization

International Nuclear Information System (INIS)

Rodriguez Camarillo, S.D.

1985-01-01

Studies were undertaken to generate additional knowledge of the biochemistry of Babesia bovis. A modified in vitro culture technique used for cloning B. bovis. This technique included a low oxygen concentration atmosphere (2%, O 2 , 5% CO 2 , 93% N 2 ) and 4 mm fluid level. Cultures initiated with one infected erythrocyte were maintained until parasitemias of positive wells reached 2% parasitemia. Primary clones were obtained and from these, nine clones were recloned twice and used for subsequent studies. A procedure was developed to concentrate and separate B. bovis merozoites and infected erythrocytes by Percoll density gradients. Merozoites separated at 1.087 g/ml specific density, whereas infected erythrocytes separated at 1.121 g/ml. Viability of purified parasites was not affected. Agarose gel electrophoresis was used to identify metabolic enzyme in B. bovis and B. bigemina. The enzymes LDH, GDH, GPI and HK were detected in both species. Molecular analysis by one and two-dimensional gel electrophoresis of proteins metabolically labeled with 35 S-methionine indicated that two clones, derived from the same field strain, were similar but not identical to the parent. Fewer proteins were observed in the parental strain. Growth of two 60-Co irradiated B. bovis clones indicated a dose-effect relationship. Growth of parasites exposed for the longest period was initially retarded but returned to normal growth after two or three subcultures. Cultures exposed for shorter periods were unaffected with respect to the rate of growth. Analysis of electrophoretic mobility of metabolic enzyme showed a change in migration pattern

Cosmological constant, inflation and no-cloning theorem

Energy Technology Data Exchange (ETDEWEB)

Huang Qingguo, E-mail: huangqg@itp.ac.cn [State Key Laboratory of Theoretical Physics, Institute of Theoretical Physics, Chinese Academy of Science, Beijing 100190 (China); Lin Fengli, E-mail: linfengli@phy.ntnu.edu.tw [Department of Physics, Massachusetts Institute of Technology, Cambridge, MA 02139 (United States); Department of Physics, National Taiwan Normal University, Taipei, 116, Taiwan (China)

2012-05-30

From the viewpoint of no-cloning theorem we postulate a relation between the current accelerated expansion of our universe and the inflationary expansion in the very early universe. It implies that the fate of our universe should be in a state with accelerated expansion. Quantitatively we find that the no-cloning theorem leads to a lower bound on the cosmological constant which is compatible with observations.

Transplantation and differentiation of donor cells in the cloned pigs

International Nuclear Information System (INIS)

Shimada, Arata; Tomii, Ryo; Kano, Koichiro; Nagashima, Hiroshi

2006-01-01

The application of nuclear transfer technology is an interesting approach to investigate stem and progenitor cell transplantation therapy. If stem cells are used as a nuclear donor, donor cells can engraft into cloned animals without histocompatible problems. However, it is still uncertain whether donor cells can engraft to cloned animal and differentiate in vivo. To address this problem, we transplanted donor cells to dermal tissues of cloned pigs developed by using preadipocytes as donor cells. Preadipocytes are adipocytic progenitor which can differentiate to mature adipocytes in vitro. We showed that the donor preadipocytes were successfully transplanted into the cloned pigs without immune rejection and they differentiated into mature adipocytes in vivo 3 weeks after transplantation. In contrast, allogenic control preadipocytes, which can differentiate in vitro, did not differentiate in vivo. These results indicate that donor progenitor cells can differentiate in cloned animal

Somatic cell nuclear transfer cloning: practical applications and current legislation.

Science.gov (United States)

Niemann, H; Lucas-Hahn, A

2012-08-01

Somatic cloning is emerging as a new biotechnology by which the opportunities arising from the advances in molecular genetics and genome analysis can be implemented in animal breeding. Significant improvements have been made in SCNT protocols in the past years which now allow to embarking on practical applications. The main areas of application of SCNT are: Reproductive cloning, therapeutic cloning and basic research. A great application potential of SCNT based cloning is the production of genetically modified (transgenic) animals. Somatic cell nuclear transfer based transgenic animal production has significant advances over the previously employed microinjection of foreign DNA into pronuclei of zygotes. This cell based transgenesis is compatible with gene targeting and allows both, the addition of a specific gene and the deletion of an endogenous gene. Efficient transgenic animal production provides numerous opportunities for agriculture and biomedicine. Regulatory agencies around the world have agreed that food derived from cloned animals and their offspring is safe and there is no scientific basis for questioning this. Commercial application of somatic cloning within the EU is via the Novel Food regulation EC No. 258/97. Somatic cloning raises novel questions regarding the ethical and moral status of animals and their welfare which has prompted a controversial discussion in Europe which has not yet been resolved. © 2012 Blackwell Verlag GmbH.

Do Managers Clone Themselves?

Science.gov (United States)

Baron, Alma S.

1981-01-01

A recent questionnaire survey provides statistics on male managers' views of female managers. The author recommends that male managers break out of their cloning behavior and that the goal ought to be a plurality in management. (Author/WD)

BASIC DENSITY AND RETRACTIBILITY OF WOOD CLONES OF THREE Eucalyptus SPECIES

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Djeison Cesar Batista

2010-12-01

Full Text Available Among the planted forests that supply the national wood industry, the genus Eucalyptus has become the most important, due to its fast growth, ease of large scale planting and variability of wood use. The generation of new hybrids and clones is a reality in the national practice of silviculture, and there is great interest currently in finding genetic improvements, mainly for higher volumetric gains and resistance in rough conditions of planting, such as pest attacks, periods of drought, low soil fertility, etc. The basic density is one of the most important physical properties of wood because it relates directly to other properties, including the anisotropic shrinkage. Such properties indicate the rational use of a species in a certain wood product. The aim of this work was to determine the basic density and the anisotropic shrinkage of five wood clones for each one of the following species: Eucalyptus saligna, Eucalyptus grandis and Eucalyptus dunnii. Clone 5 of Eucalyptus saligna presented the highest basic density (0.56 g/cm³ and was the most dimensionally instable. Of all the species, there was only a direct relation among basic density, maximum volumetric shrinkage and maximum volumetric shrinkage coefficient in this clone. Considering maximum volumetric shrinkage as the criterion, clone 3 was the most dimensionally stable. Clones 2 and 3 of Eucalyptus grandis presented the least and the highest basic density, respectively, with 0.40 and 0.49 g/cm³. It was not possible to distinguish among clones 1, 3 and 4 in terms of dimensional stability, and considering maximum volumetric shrinkage coefficient as the criterion, clone 5 was the most dimensionally instable. For Eucalyptus saligna and Eucalyptus dunnii it was not possible to distinguish which clone presented the least basic density. Clone 3 of Eucalyptus dunnii presented the highest basic density (0.65 g/cm³ and considering maximum volumetric shrinkage coefficient as the criterion, it

Molecular cloning, expression analysis and sequence prediction of ...

African Journals Online (AJOL)

CCAAT/enhancer-binding protein beta as an essential transcriptional factor, regulates the differentiation of adipocytes and the deposition of fat. Herein, we cloned the whole open reading frame (ORF) of bovine C/EBPβ gene and analyzed its putative protein structures via DNA cloning and sequence analysis. Then, the ...

Evaluation of flooring produced from small diameters logs of Eucalyptus sp. clones

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José Reinaldo Moreira da Silva

2010-12-01

Full Text Available This study evaluated two Eucalyptus clones, MN 249 and MN 89, for the flooring production using small diameters logs. It was considered the wood physical properties - NBR 7190/97 (ABNT, 1997 and simulation of the product in service (ASTM D 2394/83 with two thicknesses, 8 and 14 mm. The basic density of the clone 89 NM was the highest one (0,615 g/cm3. The contractions were more pronounced in clone NM 249, however, the anisotropy coefficient of this clone was small. In the simulation tests, the floor produced by clone MN 249 presented the lowest deformation rate. The floor of 8 mm, in addition to the differences between clones, there was significant interaction between the positions for the indentation test caused by loads applied in small areas. The deformations obtained for the floor with 14 mm thickness, produced with the MN clone 89, were higher than those found in the literature for the indentation load applied on a small area test. The clone MN 249 presented the best results in both thicknesses.

Probabilistic cloning of coherent states without a phase reference

DEFF Research Database (Denmark)

Müller, Christian R.; Wittmann, Christoffer; Marek, Petr

2012-01-01

We present a probabilistic cloning scheme operating independently of any phase reference. The scheme is based solely on a phase-randomized displacement and photon counting, omitting the need for nonclassical resources and nonlinear materials. In an experimental implementation, we employ the scheme...... to clone coherent states from a phase covariant alphabet and demonstrate that the cloner is capable of outperforming the hitherto best-performing deterministic scheme. An analysis of the covariances between the output states shows that uncorrelated clones can be approached asymptotically...

Entamoeba Clone-Recognition Experiments: Morphometrics, Aggregative Behavior, and Cell-Signaling Characterization.

Science.gov (United States)

Espinosa, Avelina; Paz-Y-Miño-C, Guillermo; Hackey, Meagan; Rutherford, Scott

2016-05-01

Studies on clone- and kin-discrimination in protists have proliferated during the past decade. We report clone-recognition experiments in seven Entamoeba lineages (E. invadens IP-1, E. invadens VK-1:NS, E. terrapinae, E. moshkovskii Laredo, E. moshkovskii Snake, E. histolytica HM-1:IMSS and E. dispar). First, we characterized morphometrically each clone (length, width, and cell-surface area) and documented how they differed statistically from one another (as per single-variable or canonical-discriminant analyses). Second, we demonstrated that amebas themselves could discriminate self (clone) from different (themselves vs. other clones). In mix-cell-line cultures between closely-related (E. invadens IP-1 vs. E. invadens VK-1:NS) or distant-phylogenetic clones (E. terrapinae vs. E. moshkovskii Laredo), amebas consistently aggregated with same-clone members. Third, we identified six putative cell-signals secreted by the amebas (RasGap/Ankyrin, coronin-WD40, actin, protein kinases, heat shock 70, and ubiquitin) and which known functions in Entamoeba spp. included: cell proliferation, cell adhesion, cell movement, and stress-induced encystation. To our knowledge, this is the first multi-clone characterization of Entamoeba spp. morphometrics, aggregative behavior, and cell-signaling secretion in the context of clone-recognition. Protists allow us to study cell-cell recognition from ecological and evolutionary perspectives. Modern protistan lineages can be central to studies about the origins and evolution of multicellularity. © 2016 The Author(s) Journal of Eukaryotic Microbiology © 2016 International Society of Protistologists.

Recent advancements in cloning by somatic cell nuclear transfer.

Science.gov (United States)

Ogura, Atsuo; Inoue, Kimiko; Wakayama, Teruhiko

2013-01-05

Somatic cell nuclear transfer (SCNT) cloning is the sole reproductive engineering technology that endows the somatic cell genome with totipotency. Since the first report on the birth of a cloned sheep from adult somatic cells in 1997, many technical improvements in SCNT have been made by using different epigenetic approaches, including enhancement of the levels of histone acetylation in the chromatin of the reconstructed embryos. Although it will take a considerable time before we fully understand the nature of genomic programming and totipotency, we may expect that somatic cell cloning technology will soon become broadly applicable to practical purposes, including medicine, pharmaceutical manufacturing and agriculture. Here we review recent progress in somatic cell cloning, with a special emphasis on epigenetic studies using the laboratory mouse as a model.

Recent advancements in cloning by somatic cell nuclear transfer

Science.gov (United States)

Ogura, Atsuo; Inoue, Kimiko; Wakayama, Teruhiko

2013-01-01

Somatic cell nuclear transfer (SCNT) cloning is the sole reproductive engineering technology that endows the somatic cell genome with totipotency. Since the first report on the birth of a cloned sheep from adult somatic cells in 1997, many technical improvements in SCNT have been made by using different epigenetic approaches, including enhancement of the levels of histone acetylation in the chromatin of the reconstructed embryos. Although it will take a considerable time before we fully understand the nature of genomic programming and totipotency, we may expect that somatic cell cloning technology will soon become broadly applicable to practical purposes, including medicine, pharmaceutical manufacturing and agriculture. Here we review recent progress in somatic cell cloning, with a special emphasis on epigenetic studies using the laboratory mouse as a model. PMID:23166393

Clone-specific differences in Pragmites australis: Effects of ploidy level and geographic origin

DEFF Research Database (Denmark)

Hansen, D.; Lambertini, Carla; Jampeetong, Arunothai

2007-01-01

by the geographic origin, the euploidy level (4x, 6x, 8x and 12x), and to assess differences between native and introduced clones in North America. Growth, morphology, photosynthetic characteristics, photosynthetic pigments and enzymes were measured on 11 geographically distinct clones propagated in a common...... result in an increase in plant size, probably because the number of cell divisions during development is reduced. Four North American clones were included in the study. The clone from the Atlantic Coast and the supposed invasive European clone resembled each other. The Gulf Coast clone differed from...

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.

Directory of Open Access Journals (Sweden)

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.

[TSA improve transgenic porcine cloned embryo development and transgene expression].

Science.gov (United States)

Kong, Qing-Ran; Zhu, Jiang; Huang, Bo; Huan, Yan-Jun; Wang, Feng; Shi, Yong-Qian; Liu, Zhong-Feng; Wu, Mei-Ling; Liu, Zhong-Hua

2011-07-01

Uncompleted epigenetic reprogramming is attributed to the low efficiency of producing transgenic cloned animals. Histone modification associated with epigenetics can directly influence the embryo development and transgene expression. Trichostatin A (TSA), as an inhibitor of histone deacetylase, can change the status of histone acetylation, improve somatic cell reprogramming, and enhance cloning efficiency. TSA prevents the chromatin structure from being condensed, so that transcription factor could binds to DNA sequence easily and enhance transgene expression. Our study established the optimal TSA treatment on porcine donor cells and cloned embryos, 250 nmol/L, 24 h and 40 nmol/L, 24 h, respectively. Furthermore, we found that both the cloned embryo and the donor cell treated by TSA resulted in the highest development efficiency. Meanwhile, TSA can improve transgene expression in donor cell and cloned embryo. In summary, TSA can significantly improve porcine reconstructed embryo development and transgene expression.

Cloning arbuscule-related genes from mycorrhizas

DEFF Research Database (Denmark)

Burleigh, Stephen

2000-01-01

Until recently little was known about the identity of the genes expressed in the arbuscules of mycorrhizas, due in part to problems associated with cloning genes from the tissues of an obligate symbiont. However, the combination of advanced molecular techniques, innovative use of the materials...... available and fortuitous cloning has resulted in the recent identification of a number of arbuscule-related genes. This article provides a brief summary of the genes involved in arbuscule development, function and regulation, and the techniques used to study them. Molecular techniques include differential...

Cloned cattle derived from a novel zona-free embryo reconstruction system

NARCIS (Netherlands)

Oback, B; Wiersema, AT; Gaynor, P; Laible, G; Tucker, FC; Oliver, JE; Miller, AL; Troskie, HE; Wilson, KL; Forsyth, JT; Berg, MC; Cockrem, K; Mcmillan, [No Value; Tervit, HR; Wells, DN

2003-01-01

As the demand for cloned embryos and offspring increases, the need arises for the development of nuclear transfer procedures that are improved in both efficiency and ease of operation. Here, we describe a novel zona-free cloning method that doubles the throughput in cloned bovine embryo production

Performance of quantum cloning and deleting machines over coherence

Science.gov (United States)

Karmakar, Sumana; Sen, Ajoy; Sarkar, Debasis

2017-10-01

Coherence, being at the heart of interference phenomena, is found to be an useful resource in quantum information theory. Here we want to understand quantum coherence under the combination of two fundamentally dual processes, viz., cloning and deleting. We found the role of quantum cloning and deletion machines with the consumption and generation of quantum coherence. We establish cloning as a cohering process and deletion as a decohering process. Fidelity of the process will be shown to have connection with coherence generation and consumption of the processes.

Putative porcine embryonic stem cell lines derived from aggregated four-celled cloned embryos produced by oocyte bisection cloning.

Science.gov (United States)

Siriboon, Chawalit; Lin, Yu-Hsuan; Kere, Michel; Chen, Chun-Da; Chen, Lih-Ren; Chen, Chien-Hong; Tu, Ching-Fu; Lo, Neng-Wen; Ju, Jyh-Cherng

2015-01-01

We attempted to isolate ES cell lines using inner cell masses from high-quality cloned porcine blastocysts. After being seeded onto feeders, embryos had better (P cloned embryos (62.8, 42.6 and 12.8% vs. 76.2, 55.2 and 26.2%, respectively) compared to the non-aggregated group (41.6, 23.4 and 3.9%). Effects of feeder types (STO vs. MEF) and serum sources (FBS vs. KSR) on extraction of cloned embryo-derived porcine ES cells were examined. More (17.1%) ntES cell lines over Passage 3 were generated in the MEF/KSR group. However, ntES cells cultured in KSR-supplemented medium had a low proliferation rate with defective morphology, and eventually underwent differentiation or apoptosis subsequently. Approximately 26.1, 22.7 and 35.7% of primary colonies were formed after plating embryos in DMEM, DMEM/F12 and α-MEM media, respectively. Survival rates of ntES cells cultured in α-MEM, DMEM and DMEM/F12 were 16.7, 4.3 and 6.8%, respectively (P > 0.05). We further examined the beneficial effect of TSA treatment of 3× aggregated cloned embryos on establishment of ntES cell lines. Primary colony numbers and survival rates of ntES cells beyond passage 3 were higher (P cells, remaining undifferentiated over 25 passages, had alkaline phosphatase activity and expressed ES specific markers Oct4, Nanog, Sox2, and Rex01. Moreover, these ntES cells successfully differentiated into embryoid bodies (EBs) that expressed specific genes of all three germ layers after being cultured in LIF-free medium. In conclusion, we have successfully derived putative porcine ntES cells with high efficiency from quality cloned embryos produced by embryo aggregation, and optimized the ES cell culture system suitable for establishing and maintaining ntES cell lines in undifferentiated state.

Cloning, recombinant expression and characterization of a new ...

African Journals Online (AJOL)

A new amylase gene APGA1 was cloned from Aureobasidium pullulans NRRL 12974 and expressed in Pichia pastoris. This is the first report on cloning and expression of amylolytic gene from the industrially important microorganism A. pullulans. The purified recombinant protein with MW of 66 kDa and specific activity of ...

Effect of the I(to) activator NS5806 on cloned K(V)4 channels depends on the accessory protein KChIP2

DEFF Research Database (Denmark)

Lundby, Alicia; Jespersen, Thomas; Schmitt, Nicole

2010-01-01

The compound NS5806 increases the transient outward current (I(to)) in canine ventricular cardiomyocytes and slows current decay. In human and canine ventricle, I(to) is thought to be mediated by K(V)4.3 and various ancillary proteins, yet, the exact subunit composition of I(to) channels is still...... debated. Here we characterize the effect of NS5806 on heterologously expressed putative I(to) channel subunits and other potassium channels....

Demographic profile of states with human cloning laws: morality policy meets political economy.

Science.gov (United States)

Stabile, Bonnie

2007-03-01

This analysis seeks to identify factors that may shape the policy stance - whether restrictive or permissive - that each state in the United States with a human cloning law in place takes toward human therapeutic cloning. The investigation also considers if cloning policy is more the product of morality politics or political economy. Results show that among states with human cloning policies in place, those with a greater biotechnological capacity, more permissive abortion laws, fewer Evangelical Protestants, and higher political liberalism rankings are more likely to have permissive cloning laws. A higher Roman Catholic population is strongly associated with permissive cloning laws, rather than restrictive cloning laws as originally supposed. Factors with morality policy and economic bases were both found to be associated with cloning policy outcomes. Results suggest that morality policies, though distinct in some ways, do share determinants with public policies based on political economy.

Histone Deacetylase Inhibitors Prolong Cardiac Repolarization through Transcriptional Mechanisms.

Science.gov (United States)

Spence, Stan; Deurinck, Mark; Ju, Haisong; Traebert, Martin; McLean, LeeAnne; Marlowe, Jennifer; Emotte, Corinne; Tritto, Elaine; Tseng, Min; Shultz, Michael; Friedrichs, Gregory S

2016-09-01

Histone deacetylase (HDAC) inhibitors are an emerging class of anticancer agents that modify gene expression by altering the acetylation status of lysine residues of histone proteins, thereby inducing transcription, cell cycle arrest, differentiation, and cell death or apoptosis of cancer cells. In the clinical setting, treatment with HDAC inhibitors has been associated with delayed cardiac repolarization and in rare instances a lethal ventricular tachyarrhythmia known as torsades de pointes. The mechanism(s) of HDAC inhibitor-induced effects on cardiac repolarization is unknown. We demonstrate that administration of structurally diverse HDAC inhibitors to dogs causes delayed but persistent increases in the heart rate corrected QT interval (QTc), an in vivo measure of cardiac repolarization, at timepoints far removed from the Tmax for parent drug and metabolites. Transcriptional profiling of ventricular myocardium from dogs treated with various HDAC inhibitors demonstrated effects on genes involved in protein trafficking, scaffolding and insertion of various ion channels into the cell membrane as well as genes for specific ion channel subunits involved in cardiac repolarization. Extensive in vitro ion channel profiling of various structural classes of HDAC inhibitors (and their major metabolites) by binding and acute patch clamp assays failed to show any consistent correlations with direct ion channel blockade. Drug-induced rescue of an intracellular trafficking-deficient mutant potassium ion channel, hERG (G601S), and decreased maturation (glycosylation) of wild-type hERG expressed by CHO cells in vitro correlated with prolongation of QTc intervals observed in vivo The results suggest that HDAC inhibitor-induced prolongation of cardiac repolarization may be mediated in part by transcriptional changes of genes required for ion channel trafficking and localization to the sarcolemma. These data have broad implications for the development of these drug classes and

Dual entanglement measures based on no local cloning and no local deleting

International Nuclear Information System (INIS)

Horodecki, Michal; Sen, Aditi; Sen, Ujjwal

2004-01-01

The impossibility of cloning and deleting of unknown states constitute important restrictions on processing of information in the quantum world. On the other hand, a known quantum state can always be cloned or deleted. However, if we restrict the class of allowed operations, there will arise restrictions on the ability of cloning and deleting machines. We have shown that cloning and deleting of known states is in general not possible by local operations. This impossibility hints at quantum correlation in the state. We propose dual measures of quantum correlation based on the dual restrictions of no local cloning and no local deleting. The measures are relative entropy distances of the desired states in a (generally impossible) perfect local cloning or local deleting process from the best approximate state that is actually obtained by imperfect local cloning or deleting machines. Just like the dual measures of entanglement cost and distillable entanglement, the proposed measures are based on important processes in quantum information. We discuss their properties. For the case of pure states, estimations of these two measures are also provided. Interestingly, the entanglement of cloning for a maximally entangled state of two two-level systems is not unity

DIFFERENTIAL RESPONSE OF CLONES OF EUCALYPT TO GLYPHOSATE1

Directory of Open Access Journals (Sweden)

Leonardo Bianco de Carvalho

2015-02-01

Full Text Available Weed control is commonly performed by the inter-row mechanical weeding associated to intrarow glyphosate directed spraying, causing a risk for drift or accidental herbicide application, that can affect the crop of interest. The objective was to evaluate the response of clones C219, GG100, I144, and I224 of eucalypt (Eucalyptus grandis x E. urophylla to glyphosate doses of 0, 18, 36, 72, 180, 360, and 720 g of acid equivalent per hectare. The clones showed different growth patterns with regard to height, leaf number, stem dry weight, relative growth rate, net assimilation rate, and relative leaf growth rate. The clones I144 and GG100 were more susceptible to glyphosate, showing the doses required to reduce dry weight by 50% of 113.4 and 119.6 g acid equivalent per hectare, respectively. The clones C219 and I224 were less susceptible to glyphosate, showing the doses required to reduce dry weight by 50% of 237.5 and 313.5 g acid equivalent per hectare, respectively. Eucalyptus clones respond differently to glyphosate exposure, so that among I224, C219, GG100, and I144, the susceptibility to the herbicide is increasing.

The global governance of human cloning: the case of UNESCO.

Science.gov (United States)

Langlois, Adèle

2017-03-21

Since Dolly the Sheep was cloned in 1996, the question of whether human reproductive cloning should be banned or pursued has been the subject of international debate. Feelings run strong on both sides. In 2005, the United Nations adopted its Declaration on Human Cloning to try to deal with the issue. The declaration is ambiguously worded, prohibiting "all forms of human cloning inasmuch as they are incompatible with human dignity and the protection of human life". It received only ambivalent support from UN member states. Given this unsatisfactory outcome, in 2008 UNESCO (the United Nations Educational, Scientific and Cultural Organization) set up a Working Group to investigate the possibility of a legally binding convention to ban human reproductive cloning. The Working Group was made up of members of the International Bioethics Committee, established in 1993 as part of UNESCO's Bioethics Programme. It found that the lack of clarity in international law is unhelpful for those states yet to formulate national regulations or policies on human cloning. Despite this, member states of UNESCO resisted the idea of a convention for several years. This changed in 2015, but there has been no practical progress on the issue. Drawing on official records and first-hand observations at bioethics meetings, this article examines the human cloning debate at UNESCO from 2008 onwards, thus building on and advancing current scholarship by applying recent ideas on global governance to an empirical case. It concludes that, although human reproductive cloning is a challenging subject, establishing a robust global governance framework in this area may be possible via an alternative deliberative format, based on knowledge sharing and feasibility testing rather than the interest-based bargaining that is common to intergovernmental organizations and involving a wide range of stakeholders. This article is published as part of a collection on global governance.

Realizing directional cloning using sticky ends produced by 3′-5 ...

Indian Academy of Sciences (India)

http://www.ias.ac.in/jbiosci. J. Biosci. 38(5), December 2013, 857–866, © Indian Academy of Sciences. Supplementary material. Supplementary figure 1. Sequencing of PCR positive clones. (A) Forward insertion of non-directional cloning. (B) Reverse insertion of non-directional cloning. (C) Forward insertion of directional ...

Oncogenesis of melanoma B16 cell clones mutagenized by space environment

International Nuclear Information System (INIS)

Guo Yupeng; Yang Hongsheng; Tang Jingtian; Xu Mei; Geng Chuanying; Fang Qing; Xu Bo; Li Hongyan; Xiang Xing; Pan Lin

2005-01-01

Objective: To explore the oncogenesis of the melanoma B16 cell clones mutagenized by space environment, and find the B16 cell clones with remarkably mutated immunogenicity. Methods: B16 cells were carried by the Chinese 20th recoverable satellite to the outer space, and were harvested after 18 days' spaceflight and then monocloned. Four cell clones, which were randomly selected from the total 110 clones obtained , and the control clone were routinely cultured. The cultured cells were injected to 10 groups of C57BL/6J mice, 82.1 mice in each group. Five groups of mice received hypodermic injection and another 5 groups of mice received abdominal injection. The survival time was observed in abdominal injection groups. The mice in hypodermic injection groups were sacrificed after 14 days, the tumor, spleen and thymus were weighted, and the serum IL-2 concentration was determined. Moreover, the melanoma tumor tissues were examined histopathologically. Results: An experiment program suitable to screening space mutagenesis of B16 tumor cell clones in vivo and the observation indices were basically established. One clone was found out which was remarkably different from the control clone in latent period of tumor formation, tumor weight, survival time of the tumor-bearing mice and the expression of IL-2. Conclusions: Cultured melanoma B16 cells could be mutated by outer space environment. The further study will be focused on the influence of space environment on immunogenicity of mutagenized B16 cells. (authors)