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Sample records for helas local helioseismology

  1. Local Helioseismology

    Directory of Open Access Journals (Sweden)

    Gizon Laurent

    2005-11-01

    Full Text Available We review the current status of local helioseismology, covering both theoretical and observational results. After a brief introduction to solar oscillations and wave propagation through inhomogeneous media, we describe the main techniques of local helioseismology: Fourier-Hankel decomposition, ring-diagram analysis, time-distance helioseismology, helioseismic holography, and direct modeling. We discuss local helioseismology of large-scale flows, the solar-cycle dependence of these flows, perturbations associated with regions of magnetic activity, and solar supergranulation.

  2. Helioseismology, Asteroseismology, and MHD Connections

    CERN Document Server

    Gizon, Laurent; Leibacher, John

    2009-01-01

    This volume presents a timely snapshot of the state of helio- and asteroseismology in the era when the SOHO/MDI instrument is about to be replaced by SDO/HMI and the CoRoT space mission is yielding its first long-duration light curves of thousands of stars. The articles and topics in this book are inspired by two seminal conferences, HELAS II and SOHO19/GONG 2007, but contributions from other experts have been commissioned as well. For example, three key papers were invited to describe the current status of asteroseismology, global helioseismology, and local helioseismology. These papers provide a framework for the other contributions and together they form a complete description of our understanding of pressure waves in the Sun and other stars. This volume is aimed at solar physicists and astronomers specializing in helio- and asteroseismology.

  3. Inversion of full acoustic wavefield in local helioseismology: A study with synthetic data

    NARCIS (Netherlands)

    Cobden, L.J.|info:eu-repo/dai/nl/323068758; Tong, C.H.; Warner, M.R.

    We present the first results from the inversion of full acoustic wavefield in the helioseismic context. In contrast to time-distance helioseismology, which involves analyzing the travel times of seismic waves propagating into the solar interior, wavefield tomography models both the travel times and

  4. FOREWORD: HELAS II International Conference

    Science.gov (United States)

    Gizon, Laurent; Roth, Markus

    2008-07-01

    Volume 118 (2008) of Journal of Physics: Conference Series provides a written record of the talks and posters presented at the HELAS II International Conference `Helioseismology, Asteroseismology and MHD Connections'. The conference was held during the week 20-24 August 2007 in Göttingen, Germany, jointly hosted by the Max Planck Institute for Solar System Research and the Faculty of Physics of the University of Göttingen. A total of 140 scientists from all over the world attended. The Scientific Organizing Committee consisted of Conny Aerts, Annie Baglin, Jørgen Christensen-Dalsgaard, Thierry Corbard, Jadwiga Daszyńska-Daszkiewicz, Stefan Dreizler, Yvonne Elsworth, Laurent Gizon (Chairman), Wolfgang Glatzel, Frank Hill, Donald Kurtz, Oskar von der Lühe, Maria Pia Di Mauro, Mário Monteiro, Pere Pallé, Markus Roth, Philip Scherrer, Manfred Schüssler, and Michael Thompson. HELAS stands for the European Helio- and Asteroseismology Network, a Coordination Action supported by the sixth Framework Programme of the European Union. It aims to bring together researchers in the fields of solar and stellar oscillations. This volume consists of 91 articles organized into sections that reflect the scientific programme of the conference: 012001-07 Wave diagnostics in physics, geophysics and astrophysics 012008-09 Perspectives on helio- and asteroseismology 012010-17 Asteroseismology: Observations 012018-25 Asteroseismology: Theory 012026-32 Global helioseismology and solar models 012033-38 Local helioseismology and magnetic activity 012039-44 Future observational projects in helio- and asteroseismology 012045-91 Poster papers. The overwhelming majority of papers discuss the seismology of the Sun and stars. Papers in the first section provide a broader perspective on wave phenomena and techniques for probing other physical systems, from living beings to the universe as a whole. We were extremely fortunate to have particularly distinguished experts to cover these topics

  5. Helioseismology on the moon

    Science.gov (United States)

    Rhodes, E. J., Jr.

    1994-06-01

    The prospect of a future manned lunar base presents an interesting challenge for helioseismology-a field in which observations have been obtained from the Earth's surface, from near-earth satellites, and from interplanetary spacecraft. Similar observations from the Moon would possess several advantages over those from other sites. Advantages over the Earth would include the absence of the terrestrial atmosphere and the lower lunar surface gravity, both of which would allow for larger-aperture telescopes with which the highest-degree solar oscillations could be studied. Compared with the .99 AU halo orbit of the upcoming Solar and Heliospheric Observatory (SOHO) mission a lunar base would have less-restrictive weight, power, and telemetry restrictions. An instrument there could also be repaired in-place and could be operated for at least one solar cycle. Disadvantages compared to SOHO would include interruptions from the lunar night and during lunar eclipses. Temporal sidelobes of frequency spacing comparable to that due to solar surface rotation would be introduced unless a network of lunar stations were operated. FInally, solar Doppler measurements made from the Moon would have to allow for the orbital velocity of the Moon around the Earth, but this should not pose a problem for such measurements.

  6. Helioseismology and the Solar Interior Dynamics

    Indian Academy of Sciences (India)

    On the other hand, the observations of beryllium and helium 3 impose specific constraints on the depth of this mildly mixed zone. Helioseismology also gives information on the internal solar rotation: while differential rotation exists in the convection zone, solid rotation prevails in the radiative zone, and the transition layer ...

  7. Full waveform inversion for time-distance helioseismology

    Energy Technology Data Exchange (ETDEWEB)

    Hanasoge, Shravan M. [Department of Astronomy and Astrophysics, Tata Institute of Fundamental Research, Mumbai 400005 (India); Tromp, Jeroen, E-mail: hanasoge@tifr.res.in [Department of Geosciences, Princeton University, Princeton, NJ 08544 (United States)

    2014-03-20

    Inferring interior properties of the Sun from photospheric measurements of the seismic wavefield constitutes the helioseismic inverse problem. Deviations in seismic measurements (such as wave travel times) from their fiducial values estimated for a given model of the solar interior imply that the model is inaccurate. Contemporary inversions in local helioseismology assume that properties of the solar interior are linearly related to measured travel-time deviations. It is widely known, however, that this assumption is invalid for sunspots and active regions and is likely for supergranular flows. Here, we introduce nonlinear optimization, executed iteratively, as a means of inverting for the subsurface structure of large-amplitude perturbations. Defining the penalty functional as the L {sub 2} norm of wave travel-time deviations, we compute the total misfit gradient of this functional with respect to the relevant model parameters at each iteration around the corresponding model. The model is successively improved using either steepest descent, conjugate gradient, or the quasi-Newton limited-memory Broyden-Fletcher-Goldfarb-Shanno algorithm. Performing nonlinear iterations requires privileging pixels (such as those in the near field of the scatterer), a practice that is not compliant with the standard assumption of translational invariance. Measurements for these inversions, although similar in principle to those used in time-distance helioseismology, require some retooling. For the sake of simplicity in illustrating the method, we consider a two-dimensional inverse problem with only a sound-speed perturbation.

  8. Localization of three human polypeptide GalNAc-transferases in HeLa cells suggests initiation of O-linked glycosylation throughout the Golgi apparatus

    DEFF Research Database (Denmark)

    Röttger, S; White, J; Wandall, H H

    1998-01-01

    O-glycosylation of proteins is initiated by a family of UDP-N-acetylgalactosamine:polypeptide N-acetylgalactos-aminyltransferases (GalNAc-T). In this study, we have localized endogenous and epitope-tagged human GalNAc-T1, -T2 and -T3 to the Golgi apparatus in HeLa cells by subcellular fractionation...... have investigated the possibility of O-glycan initiation in pre-Golgi compartments such as the ER. We could not detect endogenous polypeptide GalNAc-transferase activity in the ER of HeLa cells, neither by subcellular fractionation nor by situ glycosylation of an ER-retained form of CD8 (CD8/E19...

  9. A novel composite hydrogel initiated by Spinacia oleracea L. extract on Hela cells for localized photodynamic therapy.

    Science.gov (United States)

    Pan, Le; Li, Yanjie; Zhu, Lin; Zhang, Buchang; Shen, Yuhua; Xie, Anjian

    2017-06-01

    In this study, the poly (ethylene glycol) double acrylates (PEGDA) was first initiated by Spinacia oleracea L. extract (SOLE) to in situ form a polymer hydrogel shell on Hela cells with near infra-red irradiation. The SOLE was also a natural photosensitizer for photodynamic therapy of tumor. The use of SOLE is simple, eco-friendly, low cost, and convenient. More importantly, the PEGDA/SOLE composite hydrogel shell on Hela cells not merely prevents SOLE from diffusing to normal tissue for reducing the side effects, but also keeps high SOLE concentration on tumor cell for improving the antitumor effect. Such a hydrogel system has good biocompatibility and exhibits high therapeutic efficacy. This study extended the application of the SOLE/PEGDA gel in photodynamic therapy. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Intracellular localization analysis of npAu-PpIX in HeLa cells using specific dyes and confocal microscopy

    Science.gov (United States)

    Roblero-Bartolón, Victoria Gabriela; Maldonado-Alvarado, Elizabeth; Galván-Mendoza, José Iván; Ramón-Gallegos, Eva

    2012-10-01

    Cervical carcinoma (CC) represents the second leading cause of cancer death in Mexican women. No conventional treatments are being developed such as photodynamic therapy (PDT), involving the simultaneous presence of a photosensitizer (Ps), light of a specific wavelength and tissue oxygen. On the other hand, it has seen that the use of gold nanoparticles coupled to protoporphyrin IX increases the effectiveness of PDT. The aim of this study was to determine the site of accumulation of the conjugate npAu-PpIX in cells of cervical cancer by the use of specific dyes and confocal microscopy. The results indicate that the gold nanoparticles coupled to protoporphyrin IX are accumulated in both the cytoplasm and nucleus of HeLa cells.

  11. Full disk helioseismology: repetitive music and the question of gap filling

    Science.gov (United States)

    Fossat, E.; Kholikov, Sh.; Gelly, B.; Schmider, F. X.; Fierry-Fraillon, D.; Grec, G.; Palle, P.; Cacciani, A.; Ehgamberdiev, S.; Hoeksema, J. T.; Lazrek, M.

    1999-03-01

    Helioseismology requires continuous measurements of very long duration, months to years. This paper addresses the specific and limited case of full disk measurements of p-mode oscillations, although it can be generalized, to some extent, to the case of imaged helioseismology. First, a method of mode by mode (or rather pair of modes by pair of modes) interpolation of the signal in gaps is tested, and shown to be efficient for gaps as long as two days, but limited to the frequency range where the signal to noise ratio is good. It is then noted that the autocorrelation function of the full disk signal, after dropping quickly to zero in 20 or 30 minutes, shows secondary quasi periodic bumps, due to the quasi-periodicity of the peak distribution in the Fourier spectrum. The first of these bumps, at 4 hours or so, is higher than 70 percent and climbs to nearly 90 percent in limited frequency ranges. This suggests that an easy gap filling method can be developed, with a confidence of nearly 90 percent across all the frequency range, as long as the gap does not exceed 8 hours, with at least 4 hours of data at both ends. Even a short gap of one or two periods is better filled by the data taken 4 hours earlier or later than by local interpolation. This relaxes quite considerably the requirement of continuity of the observations for the case the full disk p-mode helioseismology. Applied to 7 years of IRIS data, this method permits the detection of all low frequency p-modes already seen by 2 years of the GOLF instrument data, and makes possible the measurement of their frequencies with an accuracy consistent with the partially filled 7 years of statistics.

  12. Computational helioseismology in the frequency domain: acoustic waves in axisymmetric solar models with flows

    Science.gov (United States)

    Gizon, Laurent; Barucq, Hélène; Duruflé, Marc; Hanson, Chris S.; Leguèbe, Michael; Birch, Aaron C.; Chabassier, Juliette; Fournier, Damien; Hohage, Thorsten; Papini, Emanuele

    2017-04-01

    Context. Local helioseismology has so far relied on semi-analytical methods to compute the spatial sensitivity of wave travel times to perturbations in the solar interior. These methods are cumbersome and lack flexibility. Aims: Here we propose a convenient framework for numerically solving the forward problem of time-distance helioseismology in the frequency domain. The fundamental quantity to be computed is the cross-covariance of the seismic wavefield. Methods: We choose sources of wave excitation that enable us to relate the cross-covariance of the oscillations to the Green's function in a straightforward manner. We illustrate the method by considering the 3D acoustic wave equation in an axisymmetric reference solar model, ignoring the effects of gravity on the waves. The symmetry of the background model around the rotation axis implies that the Green's function can be written as a sum of longitudinal Fourier modes, leading to a set of independent 2D problems. We use a high-order finite-element method to solve the 2D wave equation in frequency space. The computation is embarrassingly parallel, with each frequency and each azimuthal order solved independently on a computer cluster. Results: We compute travel-time sensitivity kernels in spherical geometry for flows, sound speed, and density perturbations under the first Born approximation. Convergence tests show that travel times can be computed with a numerical precision better than one millisecond, as required by the most precise travel-time measurements. Conclusions: The method presented here is computationally efficient and will be used to interpret travel-time measurements in order to infer, e.g., the large-scale meridional flow in the solar convection zone. It allows the implementation of (full-waveform) iterative inversions, whereby the axisymmetric background model is updated at each iteration.

  13. Design and fabrication of a microplatform for the proximity effect study of localized ELF-EMF on the growth of in vitro HeLa and PC-12 cells

    Science.gov (United States)

    Chen, Y. C.; Chen, C. C.; Tu, W.; Cheng, Y. T.; Tseng, F. G.

    2010-12-01

    This paper presents a platform technology with experimental results that show the scientists and biologists a way to rapidly investigate and analyze the biological effects of localized extremely low frequency (ELF) electromagnetic field (EMF) on living cells. The proximity effect of the localized ELF-EMF on living cells is revealed using the bio-compatible microplatform on which an on-glass inductive coil array, the source of the localized ELF-EMF in micro scale, is designed, fabricated and operated with a field strength of 1.2 ± 0.1 mT at 60 Hz for cell culturing study. After a 72 h ELF-EMF exposure, HeLa (human cervical cancer) and PC-12 (rat pheochromocytoma) cells exhibit about 18.4% and 12.9% cell proliferation rate reduction, respectively. Furthermore, according to the presented dynamic model, the reduction of the proliferation can be attributed to the interference of signal transduction processes due to the tangential currents induced around the cells.

  14. Verification of the helioseismology travel-time measurement technique and the inversion procedure for sound speed using artificial data

    Energy Technology Data Exchange (ETDEWEB)

    Parchevsky, K. V.; Zhao, J.; Hartlep, T.; Kosovichev, A. G., E-mail: akosovichev@solar.stanford.edu [Stanford University, HEPL, Stanford, CA 94305 (United States)

    2014-04-10

    We performed three-dimensional numerical simulations of the solar surface acoustic wave field for the quiet Sun and for three models with different localized sound-speed perturbations in the interior with deep, shallow, and two-layer structures. We used the simulated data generated by two solar acoustics codes that employ the same standard solar model as a background model, but utilize different integration techniques and different models of stochastic wave excitation. Acoustic travel times were measured using a time-distance helioseismology technique, and compared with predictions from ray theory frequently used for helioseismic travel-time inversions. It is found that the measured travel-time shifts agree well with the helioseismic theory for sound-speed perturbations, and for the measurement procedure with and without phase-speed filtering of the oscillation signals. This testing verifies the whole measuring-filtering-inversion procedure for static sound-speed anomalies with small amplitude inside the Sun outside regions of strong magnetic field. It is shown that the phase-speed filtering, frequently used to extract specific wave packets and improve the signal-to-noise ratio, does not introduce significant systematic errors. Results of the sound-speed inversion procedure show good agreement with the perturbation models in all cases. Due to its smoothing nature, the inversion procedure may overestimate sound-speed variations in regions with sharp gradients of the sound-speed profile.

  15. SENSITIVITY OF HELIOSEISMIC TRAVEL TIMES TO THE IMPOSITION OF A LORENTZ FORCE LIMITER IN COMPUTATIONAL HELIOSEISMOLOGY

    Energy Technology Data Exchange (ETDEWEB)

    Moradi, Hamed; Cally, Paul S., E-mail: hamed.moradi@monash.edu [Monash Centre for Astrophysics, School of Mathematical Sciences, Monash University, Clayton, Victoria 3800 (Australia)

    2014-02-20

    The rapid exponential increase in the Alfvén wave speed with height above the solar surface presents a serious challenge to physical modeling of the effects of magnetic fields on solar oscillations, as it introduces a significant Courant-Friedrichs-Lewy time-step constraint for explicit numerical codes. A common approach adopted in computational helioseismology, where long simulations in excess of 10 hr (hundreds of wave periods) are often required, is to cap the Alfvén wave speed by artificially modifying the momentum equation when the ratio between the Lorentz and hydrodynamic forces becomes too large. However, recent studies have demonstrated that the Alfvén wave speed plays a critical role in the MHD mode conversion process, particularly in determining the reflection height of the upwardly propagating helioseismic fast wave. Using numerical simulations of helioseismic wave propagation in constant inclined (relative to the vertical) magnetic fields we demonstrate that the imposition of such artificial limiters significantly affects time-distance travel times unless the Alfvén wave-speed cap is chosen comfortably in excess of the horizontal phase speeds under investigation.

  16. A New Paradigm for Supergranulation Derived from Large-Distance Time-Distance Helioseismology: Pancakes

    Science.gov (United States)

    Duvall, Thomas L.; Hanasoge, Shravan M.

    2012-01-01

    With large separations (10-24 deg heliocentric), it has proven possible to cleanly separate the horizontal and vertical components of supergranular flow with time-distance helioseismology. These measurements require very broad filters in the k-$\\omega$ power spectrum as apparently supergranulation scatters waves over a large area of the power spectrum. By picking locations of supergranulation as peaks in the horizontal divergence signal derived from f-mode waves, it is possible to simultaneously obtain average properties of supergranules and a high signal/noise ratio by averaging over many cells. By comparing ray-theory forward modeling with HMI measurements, an average supergranule model with a peak upflow of 240 m/s at cell center at a depth of 2.3 Mm and a peak horizontal outflow of 700 m/s at a depth of 1.6 Mm. This upflow is a factor of 20 larger than the measured photospheric upflow. These results may not be consistent with earlier measurements using much shorter separations (<5 deg heliocentric). With a 30 Mm horizontal extent and a few Mm in depth, the cells might be characterized as thick pancakes.

  17. The nonstructural protein NP1 of human bocavirus 1 induces cell cycle arrest and apoptosis in Hela cells

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Bin; Cai, Yingyue; Li, Yongshu [College of Life Science, Central China Normal University, Wuhan 430079, Hubei (China); Li, Jingjing [College of Life Science, Hubei Normal University, Huangshi 435002, Hubei (China); Liu, Kaiyu [College of Life Science, Central China Normal University, Wuhan 430079, Hubei (China); Li, Yi, E-mail: johnli2668@hotmail.com [College of Life Science, Central China Normal University, Wuhan 430079, Hubei (China); Bioengineering Department, Wuhan Bioengineering Institute, Wuhan 430415, Hubei (China); Yang, Yongbo, E-mail: yongboyang@mail.ccnu.edu.cn [College of Life Science, Central China Normal University, Wuhan 430079, Hubei (China)

    2013-05-25

    Human bocavirus type 1 (HBoV1) is a newly identified pathogen associated with human respiratory tract illnesses. Previous studies demonstrated that proteins of HBoV1 failed to cause cell death, which is considered as a possible common feature of bocaviruses. However, our work showed that the NP1 of HBoV1 induced apoptotic cell death in Hela cells in the absence of viral genome replication and expression of other viral proteins. Mitochondria apoptotic pathway was involved in the NP1-induced apoptosis that was confirmed by apoptotic characteristics including morphological changes, DNA fragmentation and caspase activation. We also demonstrated that the cell cycle of NP1-transfected Hela cells was transiently arrested at G2/M phase followed by rapid appearance of apoptosis and that the N terminal domain of NP1 was critical to its nuclear localization and function in apoptosis induction in Hela cells. These findings might provide alternative information for further study of mechanism of HBoV1 pathogenesis. - Highlights: ► NP1 protein of HBoV1 induced apoptosis in Hela cells was first reported. ► NP1 induced-apoptosis followed the cell cycle arrest at G2/M phase. ► The NP1 induced-apoptosis was mediated by mitochondrion apoptotic pathway. ► N terminal of NP1 was critical for apoptosis induction and nuclear localization.

  18. Autophagy Facilitates Salmonella Replication in HeLa Cells

    Science.gov (United States)

    Yu, Hong B.; Croxen, Matthew A.; Marchiando, Amanda M.; Ferreira, Rosana B. R.; Cadwell, Ken; Foster, Leonard J.; Finlay, B. Brett

    2014-01-01

    ABSTRACT Autophagy is a process whereby a double-membrane structure (autophagosome) engulfs unnecessary cytosolic proteins, organelles, and invading pathogens and delivers them to the lysosome for degradation. We examined the fate of cytosolic Salmonella targeted by autophagy and found that autophagy-targeted Salmonella present in the cytosol of HeLa cells correlates with intracellular bacterial replication. Real-time analyses revealed that a subset of cytosolic Salmonella extensively associates with autophagy components p62 and/or LC3 and replicates quickly, whereas intravacuolar Salmonella shows no or very limited association with p62 or LC3 and replicates much more slowly. Replication of cytosolic Salmonella in HeLa cells is significantly decreased when autophagy components are depleted. Eventually, hyperreplication of cytosolic Salmonella potentiates cell detachment, facilitating the dissemination of Salmonella to neighboring cells. We propose that Salmonella benefits from autophagy for its cytosolic replication in HeLa cells. PMID:24618251

  19. Photodynamic Effects of Pterin on HeLa Cells

    DEFF Research Database (Denmark)

    Denofrio, M. Paula; Lorente, Carolina; Breitenbach, Thomas

    2011-01-01

    cells (HeLa) and that these cells die upon UV-A irradiation of Ptr. Cell death was assessed using two tests: (1) the Rhodamine 123 fluorescence assay for mitochondrial viability and (2) the Trypan Blue assay for membrane integrity. The data suggest that, for Ptr-dependent photoinitiated cell death...

  20. Apoptosis of HeLa cells induced by a new targeting photosensitizer-based PDT via a mitochondrial pathway and ER stress.

    Science.gov (United States)

    Li, Donghong; Li, Lei; Li, Pengxi; Li, Yi; Chen, Xiangyun

    2015-01-01

    Photodynamic therapy (PDT) is emerging as a viable treatment for many cancers. To decrease the cutaneous photosensitivity induced by PDT, many attempts have been made to search for a targeting photosensitizer; however, few reports describe the molecular mechanism of PDT mediated by this type of targeting photosensitizer. The present study aimed to investigate the molecular mechanism of PDT induced by a new targeting photosensitizer (PS I), reported previously by us, on HeLa cells. Apoptosis is the primary mode of HeLa cell death in our system, and apoptosis occurs in a manner dependent on concentration, irradiation dose, and drug-light intervals. After endocytosis mediated by the folate receptor, PS I was primarily localized to the mitochondria and the endoplasmic reticulum (ER) of HeLa cells. PS I PDT resulted in rapid increases in intracellular reactive oxygen species (ROS) production and Ca(2+) concentration, both of which reached a peak nearly simultaneously at 15 minutes, followed by the loss of mitochondrial membrane potential at 30 minutes, release of cytochrome c from mitochondria into the cytoplasm, downregulation of Bcl-2 expression, and upregulation of Bax expression. Meanwhile, activation of caspase-3, -9, and -12, as well as induction of C/EBP homologous protein (CHOP) and glucose-regulated protein (GRP78), in HeLa cells after PS I PDT was also detected. These results suggest that apoptosis of HeLa cells induced by PS I PDT is not only triggered by ROS but is also regulated by Ca(2+) overload. Mitochondria and the ER serve as the subcellular targets of PS I PDT, the effective activation of which is responsible for PS I PDT-induced apoptosis in HeLa cells.

  1. Helioseismology H. M. Antia

    Indian Academy of Sciences (India)

    It was soon recognised that in order to obtain high temporal resolution, one needs almost continuous observations covering long periods. This has been possible using a network of ground based observatories, e.g., the Global. Oscillation Network Group (GONG) (Harvey et al. 1996) or from a suitably located satellite such ...

  2. Implementation and Comparison of Acoustic Travel-Time Measurement Procedures for the Helioseismic and Magnetic Imager Time-Distance Helioseismology Pipeline

    Science.gov (United States)

    Couvidat, S.; Zhao, J.; Birch, A. C.; Kosovichev, A. G.; Duvall, T. L., Jr.; Parchevsky, K.; Scherrer, P. H.

    2009-01-01

    The Helioseismic and Magnetic Imager (HMI) instrument on board the Solar Dynamics Observatory (SDO) satellite is designed to produce high-resolution Doppler velocity maps of oscillations at the solar surface with high temporal cadence. To take advantage of these high-quality oscillation data, a time-distance helioseismology pipeline has been implemented at the Joint Science Operations Center (JSOC) at Stanford University. The aim of this pipeline is to generate maps of acoustic travel times from oscillations on the solar surface, and to infer subsurface 3D flow velocities and sound-speed perturbations. The wave travel times are measured from cross covariances of the observed solar oscillation signals. For implementation into the pipeline we have investigated three different travel-time definitions developed in time-distance helioseismology: a Gabor wavelet fitting (Kosovichev and Duvall, 1997), a minimization relative to a reference cross-covariance function (Gizon and Birch, 2002), and a linearized version of the minimization method (Gizon and Birch, 2004). Using Doppler velocity data from the Michelson Doppler Imager (MDI) instrument on board SOHO, we tested and compared these definitions for the mean and difference travel-time perturbations measured from reciprocal signals. Although all three procedures return similar travel times in a quiet Sun region, the method of Gizon and Birch (2004) gives travel times that are significantly different from the others in a magnetic (active) region. Thus, for the pipeline implementation we chose the procedures of Kosovichev and Duvall (1997) and Gizon and Birch (2002). We investigated the relationships among these three travel-time definitions, their sensitivities to fitting parameters, and estimated the random errors they produce

  3. Implementation and Comparison of Acoustic Travel-Time Measurement Procedures for the Solar Dynamics Observatory-Helioseismic and Magnetic Imager Time-Distance Helioseismology Pipeline

    Science.gov (United States)

    Couvidat, S.; Zhao, J.; Birch, A. C.; Kosovichev, A. G.; Duvall, Thomas L., Jr.; Parchevsky, K.; Scherrer, P. H.

    2010-01-01

    The Helioseismic and Magnetic Imager (HMI) instrument onboard the Solar Dynamics Observatory (SDO) satellite is designed to produce high-resolution Doppler-velocity maps of oscillations at the solar surface with high temporal cadence. To take advantage of these high-quality oscillation data, a time - distance helioseismology pipeline (Zhao et al., Solar Phys. submitted, 2010) has been implemented at the Joint Science Operations Center (JSOC) at Stanford University. The aim of this pipeline is to generate maps of acoustic travel times from oscillations on the solar surface, and to infer subsurface 3D flow velocities and sound-speed perturbations. The wave travel times are measured from cross-covariances of the observed solar oscillation signals. For implementation into the pipeline we have investigated three different travel-time definitions developed in time - distance helioseismology: a Gabor-wavelet fitting (Kosovichev and Duvall, SCORE'96: Solar Convection and Oscillations and Their Relationship, ASSL, Dordrecht, 241, 1997), a minimization relative to a reference cross-covariance function (Gizon and Birch, Astrophys. J. 571, 966, 2002), and a linearized version of the minimization method (Gizon and Birch, Astrophys. J. 614, 472, 2004). Using Doppler-velocity data from the Michelson Doppler Imager (MDI) instrument onboard SOHO, we tested and compared these definitions for the mean and difference traveltime perturbations measured from reciprocal signals. Although all three procedures return similar travel times in a quiet-Sun region, the method of Gizon and Birch (Astrophys. J. 614, 472, 2004) gives travel times that are significantly different from the others in a magnetic (active) region. Thus, for the pipeline implementation we chose the procedures of Kosovichev and Duvall (SCORE'96: Solar Convection and Oscillations and Their Relationship, ASSL, Dordrecht, 241, 1997) and Gizon and Birch (Astrophys. J. 571, 966, 2002). We investigated the relationships among

  4. [Prolonged luminescence of HeLa cells in deep hypoxia].

    Science.gov (United States)

    Petukhov, V G; Osin, N S

    1983-03-01

    The technique of registering the afterglow of cells is described. Data are presented on spectral-luminescent characteristics of HeLa cells at room temperature under conditions of deep hypoxia, the residual pressure of oxygen in the medium being under 1 mmHg. The afterglow in the visible part of spectrum is shown to be due to protein tryptophanils with phosphorescence peaks at 415, 445, and 470 nm; metalloporphyrins with delayed fluorescence peaks at 595 and 640 nm, as well as by other cellular components with peaks at about 500 and 540 nm. The latter components are supposed to be NAD, NADH and flavins. The passage of air through the suspension of cells eliminating hypoxia results in practically a complete extinction of the afterglow of cells.

  5. Study of Paclitaxel-Treated HeLa Cells by Differential Electrical Impedance Flow Cytometry

    DEFF Research Database (Denmark)

    Kirkegaard, Julie; Clausen, Casper Hyttel; Rodriguez-Trujíllo, Romén

    2014-01-01

    This work describes the electrical investigation of paclitaxel-treated HeLa cells using a custom-made microfluidic biosensor for whole cell analysis in continuous flow. We apply the method of differential electrical impedance spectroscopy to treated HeLa cells in order to elucidate the changes...

  6. Ultrastructural immunolocalization of polyamines in HeLa cells subjected to fast-freezing fixation and freeze substitution.

    Science.gov (United States)

    Roch, A M; Nicolas, M T; Quash, G

    1997-04-01

    Polyamines have been localized at the ultrastructural level in HeLa cells subjected first to fast-freezing fixation (FFF)-freeze substitution (FS) and then to an immunocytochemical method combining anti-polyamine antibodies and immunogold labelling. Polyamines were found in both the cytoplasm and the nucleus and, in the latter, particularly over the dense chromatin area. To our knowledge, this is the first example of the electron microscopic localization of a hapten after FFF-FS. For the preservation of fine ultrastructural details, this FFF-FS method is not only adequate but also greatly reduces, if not totally eliminates, any leeching-out and redistribution of the polyamines during the preparation of the sample. For the preservation of antigenicity in situ during FS, epoxy resin was more effective than hydrophilic LR white resin, probably due to the solubility of polyamines in the latter.

  7. Cooperative and Stochastic Calcium Releases from Multiple Calcium Puff Sites Generate Calcium Microdomains in Intact HeLa Cells*

    Science.gov (United States)

    Nakamura, Hideki; Bannai, Hiroko; Inoue, Takafumi; Michikawa, Takayuki; Sano, Masaki; Mikoshiba, Katsuhiko

    2012-01-01

    Ca2+ microdomains or locally restricted Ca2+ increases in the cell have recently been reported to regulate many essential physiological events. Ca2+ increases through the inositol 1,4,5-trisphosphate (IP3) receptor (IP3R)/Ca2+ release channels contribute to the formation of a class of such Ca2+ microdomains, which were often observed and referred to as Ca2+ puffs in their isolated states. In this report, we visualized IP3-evoked Ca2+ microdomains in histamine-stimulated intact HeLa cells using a total internal reflection fluorescence microscope, and quantitatively characterized the spatial profile by fitting recorded images to a two-dimensional Gaussian distribution. Ca2+ concentration profiles were marginally spatially anisotropic, with the size increasing linearly even after the amplitude began to decline. We found the event centroid drifted with an apparent diffusion coefficient of 4.20 ± 0.50 μm2/s, which is significantly larger than those estimated for IP3Rs. The sites of maximal Ca2+ increase, rather than initiation or termination sites, were detected repeatedly at the same location. These results indicate that Ca2+ microdomains in intact HeLa cell are generated from spatially distributed multiple IP3R clusters or Ca2+ puff sites, rather than a single IP3R cluster reported in cells loaded with Ca2+ buffers. PMID:22637479

  8. Poetry, science and hope in the Prose des cellules HeLa (prose for the HeLa cells) in Marcel Thiry’s poetic work

    OpenAIRE

    Halen, Pierre

    2014-01-01

    International audience; If the process of natural sciences, so-called “exact sciences”, can be enlightened by theology and philosophy, it can also be seen by a literary gaze, and in this case by a poetic approach. A work published in 1969 by the Belgian poet Marcel Thiry, entitled Prose des cellules HeLa (prose for the HeLa cells), shows the possibility for knowledges to hold a dialogue which brings out their complementary nature. Using resources of various worldviews (myths from Greek Antiqu...

  9. Distribution of Repetitive and Nonrepetitive Sequence Transcripts in HeLa mRNA

    Science.gov (United States)

    Klein, William H.; Murphy, William; Attardi, Giuseppe; Britten, Roy J.; Davidson, Eric H.

    1974-01-01

    Polyadenylated messenger RNA extracted from HeLa cells was hybridized with a mass excess of HeLa DNA. The kinetics of the hybridization reaction demonstrated that most of the messenger RNA is transcribed from nonrepetitive DNA. The amount of messenger RNA hybridized to DNA was measured both with and without prior RNase treatment. Comparison of the results indicates that within the limits of detection, HeLa messenger RNA does not contain repetitive sequence elements covalently linked to nonrepetitive sequence transcripts. However, a small fraction of the HeLa messenger RNA preparation is transcribed entirely from repetitive DNA sequences. This fraction represents about 6% of the total polyadenylated messenger RNA preparation. PMID:4525461

  10. Methanolic extract of Pterocarpus santalinus induces apoptosis in HeLa cells.

    Science.gov (United States)

    Kwon, H J; Hong, Y K; Kim, K H; Han, C H; Cho, S H; Choi, J S; Kim, Byung-Woo

    2006-04-21

    Ptercarpus santalinus (Fabaceae) has been used as a folk remedy in Korea, and it has been shown to exhibit antiinflammations, antiulcers and anticancer effects. In this study, therefore, we report the cytotoxic activity and the mechanism of cell death exhibited by the methanol extract of Ptercarpus santalinus (MEPS) against human cervical adenocarcinoma cell line, HeLa. Treatment of HeLa cells with various concentrations of MEPS resulted in growth inhibition and induction of apoptosis in a dose-dependent manner as determined by cell viability, chromatin condensation, DNA fragmentation and sub-G1 phase accumulation. In Western blot analysis, apoptosis in the HeLa cells was associated with the release of cytochrome C from mitochondria into the cytosol, activation of caspases-3, -8, -9 and proteolytic cleavage of PARP. These results suggest that MEPS exhibits antiproliferative effect on HeLa cells via apoptosis, and it may be a potential candidate in field of anticancer drug discovery.

  11. Comprehensive and definitive molecular cytogenetic characterization of HeLa cells by spectral karyotyping

    National Research Council Canada - National Science Library

    Macville, M; Schröck, E; Padilla-Nash, H; Keck, C; Ghadimi, B M; Zimonjic, D; Popescu, N; Ried, T

    1999-01-01

    .... Twenty clonally abnormal chromosomes were found. Comparison with previously reported HeLa G-banding karyotypes revealed a remarkably stable cytogenetic constitution because 18 of 20 markers that were found were present before...

  12. Mechanisms of hela cell apoptosis induced by abnormal Savda Munziq total phenolics combined with chemotherapeutic agents.

    Science.gov (United States)

    Zhang, Yun-Xia; Abliz, Guzalnur; Ye, Wei-Jun; Mutalipu, Zuohelaguli; Li, Xiao-Wen; Wang, Hai-Qin; Buranjiang, Gulimire; Upur, Halmurat

    2014-01-01

    To investigate the effects of abnormal Savda Munziq (ASMq) total phenolics combined with cisplatin and docetaxel on the Hela cell growth. In vivo cultured Hela cells were treated with cisplatin, docetaxel, total phenolics, cisplatin+total phenolics or docetaxel+total phenolics. MTT was performed to assess inhibition of cell proliferation, flow cytometry to detect apoptosis, and semi-quantitative RT-PCR to test for survivin and Bcl-2 expression. The total phenolics, cisplatin and docetaxel had significant inhibitory and apoptosis-promoting effects on Hela cells (Pphenolics, cisplatin and docetaxel significantly decreased the expression of Bcl-2 and survivin (all Pphenolics, combined with cisplatin and docetaxel, could promote the apoptosis of Hela cells possibly through reducing the expression of Bcl-2 and survivin.

  13. Staphylococcus aureus Lpl Lipoproteins Delay G2/M Phase Transition in HeLa Cells.

    Science.gov (United States)

    Nguyen, Minh-Thu; Deplanche, Martine; Nega, Mulugeta; Le Loir, Yves; Peisl, Loulou; Götz, Friedrich; Berkova, Nadia

    2016-01-01

    The cell cycle is an ordered set of events, leading to cell growth and division into two daughter cells. The eukaryotic cell cycle consists of interphase (G1, S, and G2 phases), followed by the mitotic phase and G0 phase. Many bacterial pathogens secrete cyclomodulins that interfere with the host cell cycle. In Staphylococcus aureus four cyclomodulins have been described so far that all represent toxins and are secreted into the culture supernatant. Here we show that the membrane-anchored lipoprotein-like proteins (Lpl), encoded on a genomic island called νSaα, interact with the cell cycle of HeLa cells. By comparing wild type and lpl deletion mutant it turned out that the lpl cluster is causative for the G2/M phase transition delay and also contributes to increased invasion frequency. The lipoprotein Lpl1, a representative of the lpl cluster, also caused G2/M phase transition delay. Interestingly, the lipid modification, which is essential for TLR2 signaling and activation of the immune system, is not necessary for cyclomodulin activity. Unlike the other staphylococcal cyclomodulins Lpl1 shows no cytotoxicity even at high concentrations. As all Lpl proteins are highly conserved there might be a common function that is accentuated by their multiplicity in a tandem gene cluster. The cell surface localized Lpls' suggests a correlation between G2/M phase transition delay and host cell invasion.

  14. Study of Paclitaxel-Treated HeLa Cells by Differential Electrical Impedance Flow Cytometry

    OpenAIRE

    Kirkegaard, Julie; Clausen, Casper Hyttel; Rodriguez-Trujillo, Romen; Svendsen, Winnie Edith

    2014-01-01

    This work describes the electrical investigation of paclitaxel-treated HeLa cells using a custom-made microfluidic biosensor for whole cell analysis in continuous flow. We apply the method of differential electrical impedance spectroscopy to treated HeLa cells in order to elucidate the changes in electrical properties compared with non-treated cells. We found that our microfluidic system was able to distinguish between treated and non-treated cells. Furthermore, we utilize a model for electr...

  15. Phosphatidylinositol anchor of HeLa cell alkaline phosphatase

    Energy Technology Data Exchange (ETDEWEB)

    Jemmerson, R.; Low, M.G.

    1987-09-08

    Alkaline phosphatase from cancer cells, HeLa TCRC-1, was biosynthetically labeled with either /sup 3/H-fatty acids or (/sup 3/H)ethanolamine as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography of immunoprecipitated material. Phosphatidylinositol-specific phospholipase C (PI-PLC) released a substantial proportion of the /sup 3/H-fatty acid label from immunoaffinity-purified alkaline phosphatase but had no effect on the radioactivity of (/sup 3/H)ethanolamine-labeled material. PI-PLC also liberated catalytically active alkaline phosphatase from viable cells, and this could be selectively blocked by monoclonal antibodies to alkaline phosphatase. However, the alkaline phosphatase released from /sup 3/H-fatty acid labeled cells by PI-PLC was not radioactive. By contrast, treatment with bromelain removed both the /sup 3/H-fatty acid and the (/sup 3/H)ethanolamine label from purified alkaline phosphatase. Subtilisin was also able to remove the (/sup 3/H)ethanolamine label from the purified alkaline phosphatase. The /sup 3/H radioactivity in alkaline phosphatase purified from (/sup 3/H)ethanolamine-labeled cells comigrated with authentic (/sup 3/H)ethanolamine by anion-exchange chromatography after acid hydrolysis. The data suggest that the /sup 3/H-fatty acid and (/sup 3/H)ethanolamine are covalently attached to the carboxyl-terminal segment since bromelain and subtilisin both release alkaline phosphatase from the membrane by cleavage at that end of the polypeptide chain. The data are consistent with findings for other proteins recently shown to be anchored in the membrane through a glycosylphosphatidylinositol structure and indicate that a similar structure contributes to the membrane anchoring of alkaline phosphatase.

  16. Amygdalin induces apoptosis in human cervical cancer cell line HeLa cells.

    Science.gov (United States)

    Chen, Yu; Ma, Jinshu; Wang, Fang; Hu, Jie; Cui, Ai; Wei, Chengguo; Yang, Qing; Li, Fan

    2013-02-01

    Amygdalin, a naturally occurring substance, has been suggested to be efficacious as an anticancer substance. The effect of amygdalin on cervical cancer cells has never been studied. In this study, we found that the viability of human cervical cancer HeLa cell line was significantly inhibited by amygdalin. 4,6-Diamino-2-phenyl indole (DAPI) staining showed that amygdalin-treated HeLa cells developed typical apoptotic changes. The development of apoptosis in the amygdalin-treated HeLa cells were confirmed by double staining of amygdalin-treated HeLa cells with annexin V-FITC and propidium iodide (PI) along with increase in caspase-3 activity in these cells. Further studies indicated that antiapoptotic protein Bcl-2 was downregulated whereas proapoptotic Bax protein was upregulated in the amygdalin-treated HeLa cells implying involvement of the intrinsic pathway of apoptosis. In vivo, amygdalin administration inhibited the growth of HeLa cell xenografts through a mechanism of apoptosis. The results in the present study suggest that amygdalin may offer a new therapeutic option for patients with cervical cancer.

  17. Mycoplasma fermentans binds to and invades HeLa cells: involvement of plasminogen and urokinase.

    Science.gov (United States)

    Yavlovich, Amichai; Katzenell, Avigail; Tarshis, Mark; Higazi, Abd A-R; Rottem, Shlomo

    2004-09-01

    Adherence of Mycoplasma fermentans to HeLa cells followed saturation kinetics, required a divalent cation, and was enhanced by preincubation of the organism at 37 degrees C for 1 h in a low-osmolarity solution. Proteolytic digestion, choline phosphate, or anti-choline phosphate antibodies partially inhibited the adherence, supporting the notion that M. fermentans utilizes at least two surface components for adhesion, a protease-sensitive surface protein and a phosphocholine-containing glycolipid. Plasminogen binding to M. fermentans greatly increased the maximal adherence of the organism to HeLa cells. Anti-plasminogen antibodies and free plasminogen inhibited this increase. These observations suggest that in the presence of plasminogen the organism adheres to novel sites on the HeLa cell surface, which are apparently plasminogen receptors. Plasminogen-bound M. fermentans was detected exclusively on the cell surface of the infected HeLa cells. Nevertheless, plasminogen binding in the presence of the urokinase-type plasminogen activator (uPA) promoted the invasion of HeLa cells by M. fermentans. The latter finding indicates that the invasiveness of M. fermentans does not result from binding plasminogen but from activation of the bound plasminogen to plasmin. Cholesterol depletion and sequestration with beta-cyclodextrin and filipin, respectively, did not affect the capacity of M. fermentans to adhere, but invasion of HeLa cells by uPA-activated plasminogen-bound M. fermentans was impaired, suggesting that lipid rafts are implicated in M. fermentans entry.

  18. Apoptosis of HeLa cells induced by a new targeting photosensitizer-based PDT via a mitochondrial pathway and ER stress

    Directory of Open Access Journals (Sweden)

    Li D

    2015-04-01

    Full Text Available Donghong Li,1 Lei Li,2 Pengxi Li,1 Yi Li,3 Xiangyun Chen1 1State Key Laboratory of Trauma, Burn and Combined Injury, The Second Department of Research Institute of Surgery, 2The First Department of Research Institute of Surgery, 3Cancer Center, Daping Hospital, Third Military Medical University, Chongqing, People’s Republic of China Abstract: Photodynamic therapy (PDT is emerging as a viable treatment for many cancers. To decrease the cutaneous photosensitivity induced by PDT, many attempts have been made to search for a targeting photosensitizer; however, few reports describe the molecular mechanism of PDT mediated by this type of targeting photosensitizer. The present study aimed to investigate the molecular mechanism of PDT induced by a new targeting photosensitizer (PS I, reported previously by us, on HeLa cells. Apoptosis is the primary mode of HeLa cell death in our system, and apoptosis occurs in a manner dependent on concentration, irradiation dose, and drug–light intervals. After endocytosis mediated by the folate receptor, PS I was primarily localized to the mitochondria and the endoplasmic reticulum (ER of HeLa cells. PS I PDT resulted in rapid increases in intracellular reactive oxygen species (ROS production and Ca2+ concentration, both of which reached a peak nearly simultaneously at 15 minutes, followed by the loss of mitochondrial membrane potential at 30 minutes, release of cytochrome c from mitochondria into the cytoplasm, downregulation of Bcl-2 expression, and upregulation of Bax expression. Meanwhile, activation of caspase-3, -9, and -12, as well as induction of C/EBP homologous protein (CHOP and glucose-regulated protein (GRP78, in HeLa cells after PS I PDT was also detected. These results suggest that apoptosis of HeLa cells induced by PS I PDT is not only triggered by ROS but is also regulated by Ca2+ overload. Mitochondria and the ER serve as the subcellular targets of PS I PDT, the effective activation of which

  19. Silencing cytokeratin 18 gene inhibits intracellular replication of Trypanosoma cruzi in HeLa cells but not binding and invasion of trypanosomes

    Directory of Open Access Journals (Sweden)

    de Mello Samanta M

    2008-12-01

    Full Text Available Abstract Background As an obligatory intracellular parasite, Trypanosoma cruzi, the etiological agent of Chagas' disease, must invade and multiply within mammalian cells. Cytokeratin 18 (CK18 is among the host molecules that have been suggested as a mediator of important events during T. cruzi-host cell interaction. Based on that possibility, we addressed whether RNA interference (RNAi-mediated down regulation of the CK18 gene could interfere with the parasite life cycle in vitro. HeLa cells transiently transfected with CK18-RNAi had negligible levels of CK18 transcripts, and significantly reduced levels of CK18 protein expression as determined by immunoblotting or immunofluorescence. Results CK18 negative or positive HeLa cells were invaded equally as well by trypomastigotes of different T. cruzi strains. Also, in CK18 negative or positive cells, parasites recruited host cells lysosomes and escaped from the parasitophorous vacuole equally as well. After that, the growth of amastigotes of the Y or CL-Brener strains, was drastically arrested in CK18 RNAi-treated cells. After 48 hours, the number of amastigotes was several times lower in CK18 RNAi-treated cells when compared to control cells. Simultaneous staining of parasites and CK18 showed that in HeLa cells infected with the Y strain both co-localize. Although the amastigote surface protein-2 contains the domain VTVXNVFLYNR previously described to bind to CK18, in several attempts, we failed to detect binding of a recombinant protein to CK-18. Conclusion The study demonstrates that silencing CK18 by transient RNAi, inhibits intracellular multiplication of the Y and CL strain of T. cruzi in HeLa cells, but not trypanosome binding and invasion.

  20. [Essential oil from Artemisia lavandulaefolia induces apoptosis and necrosis of HeLa cells].

    Science.gov (United States)

    Zhang, Lu-min; Lv, Xue-wei; Shao, Lin-xiang; Ma, Yan-fang; Cheng, Wen-zhao; Gao, Hai-tao

    2013-12-01

    To investigate the effects of Artemisia lavandulaefolia essential oil on apoptosis and necrosis of HeLa cells. Cell viability was assayed using MTT method. The morphological and structure alterations in HeLa cells were observed by microscopy. Furthermore, cell apoptosis was measured by DNA Ladder and flow cytometry. DNA damage was measured by comet assay, and the protein expression was examined by Western blot analysis. MTT assay displayed essential oil from Artemisia lavandulaefolia could inhibit the proliferation of HeLa cells in a dose-dependent manner. After treated with essential oil of Artemisia lavadulaefolia for 24 h, HeLa cells in 100 and 200 microg/mL experiment groups exhibited the typical morphology changes of undergoing apoptosis, such as cell shrinkage and nucleus chromatin condensed. However, the cells in the 400 microg/mL group showed the necrotic morphology changes including cytomembrane rupture and cytoplasm spillover. In addition, DNA Ladder could be demonstrated by DNA electrophoresis in each experiment group. Apoptosis peak was also evident in flow cytometry in each experiment group. After treating the HeLa cells with essential oil of Artemisia lavadulaefolia for 6 h, comet tail was detected by comet assay. Moreover, western blotting analysis showed that caspase-3 was activated and the cleavage of PARP was inactivated. Essential oil from Artemisia lavadulaefolia can inhibit the proliferation of HeLa cells in vitro. Low concentration of essential oil from Artemisia lavadulaefolia can induce apoptosis, whereas high concentration of the compounds result in necrosis of HeLa cells. And,the mechanism may be related to the caspase-3-mediated-PARP apoptotic signal pathway.

  1. Apoptosis and necrosis in vaccinia virus-infected HeLa G and BSC-40 cells.

    Science.gov (United States)

    Liskova, Jana; Knitlova, Jarmila; Honner, Richard; Melkova, Zora

    2011-09-01

    In most cells, vaccinia virus (VACV) infection is considered to cause a lytic cell death, an equivalent of necrosis. However, upon infection of the epithelial cell lines HeLa G and BSC-40 with VACV strain Western Reserve (WR), we have previously observed an increased activation of and activity attributable to caspases, a typical sign of apoptosis. In this paper, we have further analyzed the type of cell death in VACV-infected cells HeLa G and BSC-40. In a cell-based flow cytometric assay, we showed a specific activation of caspase-2 and 4 in HeLa G and BSC-40 cells infected with VACV, strain WR, while we did not find any effects of inhibitors of calpain and cathepsin D and E. The actual activity of the two caspases, but also of caspase-3, was then confirmed in lysates of infected HeLa G, but not in BSC-40 cells. Accordingly, poly(ADP)-ribose polymerase (PARP) cleavage was found increased only in infected HeLa G cells. Consequently, we have determined morphological features of apoptosis and/or activity of the executioner caspase-3 in infected HeLa G cells in situ, while only a background apoptosis was observed in infected BSC-40 cells. Finally, vaccination strains Dryvax and Praha were found to induce apoptosis in both HeLa G and BSC-40 cells, as characterized morphologically and by PARP cleavage. These findings may be important for understanding the differences in VACV-host interactions and post-vaccination complications in different individuals. Copyright © 2011 Elsevier B.V. All rights reserved.

  2. In Vitro Ultramorphological Assessment of Apoptosis Induced by Zerumbone on (HeLa

    Directory of Open Access Journals (Sweden)

    Siddig Ibrahim Abdel Wahab

    2009-01-01

    Full Text Available Zerumbone (ZER, a potential anticancer compound, isolated from the fresh rhizomes of Zingiber zerumbet. In this investigation, the cytotoxic properties of ZER were evaluated, on cancer cells of human cervix (HeLa, breast and ovary, and normal cells of Chinese Hamster ovary, using MTT assay. Apoptogenic effects of ZER on HeLa were studied using fluorescence microscopy (AO/PI double staining, scanning and transmission electron microscopy (SEM and TEM, and colorimetric assay of the apoptosis promoter enzyme, caspase-3. The results of MTT assay showed that ZER has less effect on normal cells compared to cancer cells. The lowest IC50 of ZER was observed on HeLa cells. Cytological observations showed nuclear and chromatin condensation, cell shrinkage, multinucleation, abnormalities of mitochondrial cristae, membrane blebbing, holes, cytoplasmic extrusions and formation of apoptotic bodies as confirmed collectively by double staining of AO/PI, SEM and TEM. Statistical analysis (two-tailed t-test of differential counting of 200 cells under fluorescence microscope revealed significant difference in apoptotic cells populations between treated and untreated HeLa cells. In addition, ZER has increased the cellular level of caspase-3 on the treated HeLa cells. It could be concluded that ZER was able to produce distinctive morphological features of cell death that corresponds to apoptosis.

  3. Adjuvant antiproliferative and cytotoxic effect of aloin in irradiated HeLaS3 cells

    Science.gov (United States)

    Nićiforović, A.; Adžić, M.; Zarić, B.; Radojčić, M. B.

    2007-09-01

    Naturally occurring phytoanthracycline, aloin, was used to radiosensitize HeLaS3 human cervix carcinoma cells. The results indicated that the cytotoxic adjuvant effect of aloin was synergistic with gammaionizing radiation at all drug concentrations and comparable to the cytotoxicity of 5-10 Gy ionizing radiation alone. Radiosensitization of HeLaS3 cells was achieved by 60 μM aloin, which reduced the IC50 dose of ionizing radiation from 3.4 to 2 Gy. Ionizing radiation and aloin alone or in combination are shown to cause perturbation of the HeLaS3 cell-cycle and increase the percentage of cells in the DNA synthesis (S) phase of the cell cycle. While either of the agents applied alone causes programmed cell death by apoptosis, the simultaneous cell damage by both agents through the altered redox balance compromised cell capacity to conduct this program and led to synergic cytotoxic cell death by necrosis.

  4. Selective killing effect of oxytetracycline, propafenone and metamizole on A549 or Hela cells

    Science.gov (United States)

    Feng, Guihua

    2013-01-01

    Objective To determine the selective killing effect of oxytetracycline, propafenone and metamizole on A549 or Hela cells. Methods Proliferation assay, lactate dehydrogenase (LDH) assay, apoptosis detecting, flow cytometry and western blot were performed. Results It was found that treatment with propafenone at the concentration of 0.014 g/L or higher for 48 h could induce apoptosis in Hela cells greatly, while it was not observed in oxytetracycline and metamizole at the concentration of 0.20 g/L for 48 h. Oxytetracycline, propafenone and metamizole all displayed evident inhibitory effects on the proliferation of A549 cells. The results of LDH assay demonstrated that the drugs at the test range of concentration did not cause necrosis in the cells. Propafenone could elevate the protein level of P53 effectively (Pmetamizol (dipyrone) all displayed evident inhibitory effects on the proliferation of A549 cells. Propafenone also displayed evident inhibitory effects on the proliferation of Hela cells. PMID:24385693

  5. Antiproliferative effects of some medicinal plants on HeLa cells

    Directory of Open Access Journals (Sweden)

    Cenić-Milošević Desanka

    2013-01-01

    Full Text Available Medicinal plants maintain the health and vitality of individuals, and also have potential curative effect on various diseases, including cancer. In this study were investigated the antiproliferative effects of water extracts of previously obtained ethanolic dry extracts of three different medicinal plants (Echinacea angustifolia, Salvia officinalis and Melissa officinalis on cell lines derived from human cervix adenocarcinoma (HeLa cells. The best cytotoxic activity (IC50 = 43.52 μg/ml on HeLa cell lines was exhibited by Echinacea angustifolia. The extract of Salvia officinalis also showed a good cytotoxic activity against HeLa cell lines; the IC50 value was 70.41 μg/ml. Melissa officinalis manifested a slightly weaker cytotoxic activity and an IC50 value of 122.22 μg/ml. [Projekat Ministarstva nauke Republike Srbije, br. 34021 i br. 175011

  6. Involvement of S6K1 in mitochondria function and structure in HeLa cells.

    Science.gov (United States)

    Park, Jisoo; Tran, Quangdon; Mun, Kisun; Masuda, Kouhei; Kwon, So Hee; Kim, Seon-Hwan; Kim, Dong-Hoon; Thomas, George; Park, Jongsun

    2016-12-01

    The major biological function of mitochondria is to generate cellular energy through oxidative phosphorylation. Apart from cellular respiration, mitochondria also play a key role in signaling processes, including aging and cancer metabolism. It has been shown that S6K1-knockout mice are resistant to obesity due to enhanced beta-oxidation, with an increased number of large mitochondria. Therefore, in this report, the possible involvement of S6K1 in regulating mitochondria dynamics and function has been investigated in stable lenti-shS6K1-HeLa cells. Interestingly, S6K1-stably depleted HeLa cells showed phenotypical changes in mitochondria morphology. This observation was further confirmed by detailed image analysis of mitochondria shape. Corresponding molecular changes were also observed in these cells, such as the induction of mitochondrial fission proteins (Drp1 and Fis1). Oxygen consumption is elevated in S6K1-depeleted HeLa cells and FL5.12 cells. In addition, S6K1 depletion leads to enhancement of ATP production in cytoplasm and mitochondria. However, the relative ratio of mitochondrial ATP to cytoplasmic ATP is actually decreased in lenti-shS6K1-HeLa cells compared to control cells. Lastly, induction of mitophagy was found in lenti-shS6K1-HeLa cells with corresponding changes of mitochondria shape on electron microscope analysis. Taken together, our results indicate that S6K1 is involved in the regulation of mitochondria morphology and function in HeLa cells. This study will provide novel insights into S6K1 function in mitochondria-mediated cellular signaling. Copyright © 2016 Elsevier Inc. All rights reserved.

  7. Campylobacter jejuni cell lysates differently target mitochondria and lysosomes on HeLa cells.

    Science.gov (United States)

    Canonico, B; Campana, R; Luchetti, F; Arcangeletti, M; Betti, M; Cesarini, E; Ciacci, C; Vittoria, E; Galli, L; Papa, S; Baffone, W

    2014-08-01

    Campylobacter jejuni is the most common cause of bacterial gastroenteritis in humans. The synthesis of cytolethal distending toxin appears essential in the infection process. In this work we evaluated the sequence of lethal events in HeLa cells exposed to cell lysates of two distinct strains, C. jejuni ATCC 33291 and C. jejuni ISS3. C. jejuni cell lysates (CCLys) were added to HeLa cell monolayers which were analysed to detect DNA content, death features, bcl-2 and p53 status, mitochondria/lysosomes network and finally, CD54 and CD59 alterations, compared to cell lysates of C. jejuni 11168H cdtA mutant. We found mitochondria and lysosomes differently targeted by these bacterial lysates. Death, consistent with apoptosis for C. jejuni ATCC 33291 lysate, occurred in a slow way (>48 h); concomitantly HeLa cells increase their endolysosomal compartment, as a consequence of toxin internalization besides a simultaneous and partial lysosomal destabilization. C. jejuni CCLys induces death in HeLa cells mainly via a caspase-dependent mechanism although a p53 lysosomal pathway (also caspase-independent) seems to appear in addition. In C. jejuni ISS3-treated cells, the p53-mediated oxidative degradation of mitochondrial components seems to be lost, inducing the deepest lysosomal alterations. Furthermore, CD59 considerably decreases, suggesting both a degradation or internalisation pathway. CCLys-treated HeLa cells increase CD54 expression on their surface, because of the action of lysate as its double feature of toxin and bacterial peptide. In conclusion, we revealed that C. jejuni CCLys-treated HeLa cells displayed different features, depending on the particular strain.

  8. Evaluation of Antiproliferative Potential of Cerium Oxide Nanoparticles on HeLa Human Cervical Tumor Cell

    Directory of Open Access Journals (Sweden)

    Zoriţa Diaconeasa

    2015-05-01

    Full Text Available Cerium oxide nanoparticles (CeO2 nanoparticles as nanomaterials have promising biomedical applications. In this paper, the cytotoxicity induced by CONPs human cervical tumor cells was investigated. Cerium oxide nanoparticles were synthesized using the precipitation method. The nanoparticles were found to inhibit the proliferation of HeLa human cervical tumor cells in a dose dependent manner but did not showed to be cytotoxic as analyzed by MTT assay. The administrated treatment decreased the HeLa cell viability cells from 100% to 65% at the dose of 100 μg/mL.

  9. Proteomic, cellular, and network analyses reveal new DUSP3 interactions with nucleolar proteins in HeLa cells.

    Science.gov (United States)

    Panico, Karine; Forti, Fabio Luis

    2013-12-06

    DUSP3 (or Vaccinia virus phosphatase VH1-related; VHR) is a small dual-specificity phosphatase known to dephosphorylate c-Jun N-terminal kinases and extracellular signal-regulated kinases. In human cervical cancer cells, DUSP3 is overexpressed, localizes preferentially to the nucleus, and plays a key role in cellular proliferation and senescence triggering. Other DUSP3 functions are still unknown, as illustrated by recent and unpublished results from our group showing that this enzyme mediates DNA damage response or repair processes. In this study, we sought to identify new interactions between DUSP3 and proteins directly or indirectly involved in or correlated with its biological roles in HeLa cells exposed to gamma or UV radiation. By using GST-DUSP as bait, we pulled down interacting proteins and identified them by LC-MS/MS. Of the 46 proteins obtained, six hits were extensively validated by immune techniques; the proteins Nucleophosmin, HnRNP C1/C2, and Nucleolin were the most promising targets found to directly interact with DUSP3. We then analyzed the DUSP3 interactomes using physical protein-protein interaction networks using our hits as the seed list. The validated hits as well as unvalidated hits fluctuated on the DUSP3 interactomes of HeLa cells, independent of the time post radiation, which confirmed our proteomic and experimental data and clearly showed the proximity of DUSP3 to proteins involved in processes intimately related to DNA repair and senescence, such as Ku70 and Tert, via interactions with nucleolar proteins, which were identified in this study, that regulate DNA/RNA structure and functions.

  10. Procyanidins from Pinus koraiensis bark inhibits HeLa cell growth by ...

    African Journals Online (AJOL)

    In this study, the effect of procyanidins from P. koraiensis bark on inhibiting Hela cells growth has been investigated by many approaches which are MTT method, agarose gel electrophoresis and Western blot method in vitro. The results showed that Marron powder were extracted from P. koraiensis bark with the extract ...

  11. Tetrastigma hemsleyanum (Sanyeqing) root tuber extracts induces apoptosis in human cervical carcinoma HeLa cells.

    Science.gov (United States)

    Xiong, Yan; Wu, Xuewen; Rao, Liqun

    2015-05-13

    Tetrastigma hemsleyanum (Sanyeqing) is traditionally used as a folk medicine for the treatment of cancer. However, the underlying mechanisms remain unclear. The purpose of this study was to investigate the possible mechanisms by which petroleum ether fraction (PEF) of Sanyeqing has anti-tumor activity on HeLa cells. The chemical components of PEF were analyzed by gas chromatography-mass spectrometry. The cytotoxicity of PEF on HeLa cells was measured by MTT assay. Apoptosis was evaluated by phosphatidylserine translocation, mitochondrial membrane potential (Δψm) changes and the activation of caspase-3, caspase-8 and caspase-9. The levels in T-SOD, CAT, GSH-PX and MDA were measured. PEF of Sanyeqing inhibited the growth and induced apoptosis of HeLa cells in dose- and time-dependent manner. PEF triggered intrinsic apoptotic pathway indicated by the loss of mitochondrial membrane potential and the activation of caspase-9 and caspase-3. In addition, PEF activated extrinsic apoptotic pathway indicated by the activation of caspase-8. Furthermore, PEF decreased T-SOD, CAT, GSH-PX activities and increased MDA level. Chemical analysis revealed the presence of fatty acids and phytosterol in PEF. PEF of Tetrastigma hemsleyanum Diels et. Gilg (Sanyeqing) exhibits cytotoxic effects, triggers both extrinsic and intrinsic apoptotic pathways, and augments oxidative stress in cervical carcinoma HeLa cells. Sanyeqing has strong potential to be developed as an agent for the treatment of cervical cancer. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  12. Cholesterol esters are detected by Raman microspectroscopy in HeLa cells

    NARCIS (Netherlands)

    van Manen, H.J.; Otto, Cornelis

    2009-01-01

    The detection of trans-unsaturated lipids in single HeLa cells by Raman microspectroscopy was recently reported in this journal by Onogi et al. Based on our previously published Raman microspectroscopy data of individual macrophage foam cells, a detailed comparison between our spectra and spectrum

  13. Visualizing the molecular sociology at the HeLa cell nuclear periphery

    NARCIS (Netherlands)

    Mahamid, Julia; Pfeffer, Stefan; Schaffer, Miroslava; Villa, Elizabeth; Danev, Radostin; Cuellar, Luis Kuhn; Förster, Friedrich|info:eu-repo/dai/nl/412516438; Hyman, Anthony A; Plitzko, Jürgen M; Baumeister, Wolfgang

    2016-01-01

    The molecular organization of eukaryotic nuclear volumes remains largely unexplored. Here we combined recent developments in cryo-electron tomography (cryo-ET) to produce three-dimensional snapshots of the HeLa cell nuclear periphery. Subtomogram averaging and classification of ribosomes revealed

  14. PHA-induced cytotoxicity of human lymphocytes against adherent hela-cells

    NARCIS (Netherlands)

    Huges-Law, G.; de Gast, G. C.; The, T. Hauw

    The conditions for a phytohaemagglutinin(PHA)-induced cytotoxicity test of human peripheral blood lymphocytes were investigated. [3H]thymidine prelabelled HeLa cells were used as target cells. Stimulation with 10 μl PHA/ml during 24 h gave the best measure of lymphocyte cytotoxic capacity.

  15. Effect of Electrode Shape on Impedance of Single HeLa Cell: A COMSOL Simulation.

    Science.gov (United States)

    Wang, Min-Haw; Chang, Wen-Hao

    2015-01-01

    In disease prophylaxis, single cell inspection provides more detailed data compared to conventional examinations. At the individual cell level, the electrical properties of the cell are helpful for understanding the effects of cellular behavior. The electric field distribution affects the results of single cell impedance measurements whereas the electrode geometry affects the electric field distributions. Therefore, this study obtained numerical solutions by using the COMSOL multiphysics package to perform FEM simulations of the effects of electrode geometry on microfluidic devices. An equivalent circuit model incorporating the PBS solution, a pair of electrodes, and a cell is used to obtain the impedance of a single HeLa cell. Simulations indicated that the circle and parallel electrodes provide higher electric field strength compared to cross and standard electrodes at the same operating voltage. Additionally, increasing the operating voltage reduces the impedance magnitude of a single HeLa cell in all electrode shapes. Decreasing impedance magnitude of the single HeLa cell increases measurement sensitivity, but higher operational voltage will damage single HeLa cell.

  16. Single-walled carbon nanotube interactions with HeLa cells

    Directory of Open Access Journals (Sweden)

    Musselman Inga H

    2007-10-01

    Full Text Available Abstract This work concerns exposing cultured human epithelial-like HeLa cells to single-walled carbon nanotubes (SWNTs dispersed in cell culture media supplemented with serum. First, the as-received CoMoCAT SWNT-containing powder was characterized using scanning electron microscopy and thermal gravimetric analyses. Characterizations of the purified dispersions, termed DM-SWNTs, involved atomic force microscopy, inductively coupled plasma – mass spectrometry, and absorption and Raman spectroscopies. Confocal microRaman spectroscopy was used to demonstrate that DM-SWNTs were taken up by HeLa cells in a time- and temperature-dependent fashion. Transmission electron microscopy revealed SWNT-like material in intracellular vacuoles. The morphologies and growth rates of HeLa cells exposed to DM-SWNTs were statistically similar to control cells over the course of 4 d. Finally, flow cytometry was used to show that the fluorescence from MitoSOX™ Red, a selective indicator of superoxide in mitochondria, was statistically similar in both control cells and cells incubated in DM-SWNTs. The combined results indicate that under our sample preparation protocols and assay conditions, CoMoCAT DM-SWNT dispersions are not inherently cytotoxic to HeLa cells. We conclude with recommendations for improving the accuracy and comparability of carbon nanotube (CNT cytotoxicity reports.

  17. Induced senescence in HeLa cervical carcinoma cells containing elevated telomerase activity and extended telomeres.

    Science.gov (United States)

    Goodwin, E C; DiMaio, D

    2001-11-01

    Proliferation of normal somatic human cells in culture is limited by replicative senescence, a growth-arrested state that appears to be triggered by the erosion of telomeres. Tumor cells such as HeLa cervical carcinoma cells, which contain short telomeres, can be induced to undergo senescence by various manipulations including oncogene withdrawal. Repression of the human papillomavirus (HPV) type 18 E6/E7 genes in HeLa cells by the bovine papillomavirus E2 transcriptional regulatory protein results in reactivation of the dormant p53 and p105(Rb) tumor suppressor pathways in these cells, repression of telomerase, and profound growth arrest. Strikingly, the growth-arrested cells rapidly and synchronously acquired numerous characteristics of primary cells undergoing replicative senescence. To explore the role of telomerase and telomere length in induced senescence, we expressed an exogenous hTERT gene, which encodes the catalytic subunit of telomerase, to generate stable HeLa cell clones with elevated telomerase activity and extended telomeres. Expression of the E2 protein in these cells repressed HPV E6/E7 expression, activated tumor suppressor pathways, and induced senescence as assessed by growth arrest, morphological changes, senescence-associated beta-galactosidase expression, and increased autofluorescence. Cells carrying the hTERT gene and control cells displayed identical responses to E2 expression. Therefore, HeLa cell senescence induced by HPV repression is not triggered by short telomeres or low levels of telomerase activity.

  18. Mycoepoxydiene suppresses HeLa cell growth by inhibiting glycolysis and the pentose phosphate pathway.

    Science.gov (United States)

    Jin, Kehua; Li, Li; Sun, Xihuan; Xu, Qingyan; Song, Siyang; Shen, Yuemao; Deng, Xianming

    2017-05-01

    Upregulation of glycolysis and the pentose phosphate pathway (PPP) is a major characteristic of the metabolic reprogramming of cancer and provides cancer cells with energy and vital metabolites to support their rapid proliferation. Targeting glycolysis and the PPP has emerged as a promising antitumor therapeutic strategy. Marine natural products are attractive sources for anticancer therapeutics, as evidenced by the antitumor drug Yondelis. Mycoepoxydiene (MED) is a natural product isolated from a marine fungus that has shown promising inhibitory efficacy against HeLa cells in vitro. We used a proteomic approach with two-dimensional gel electrophoresis (2-DE) coupled with mass spectrometry to explore the cellular targets of MED and to unravel the molecular mechanisms underlying the antitumor activity of MED in HeLa cells. Our proteomic data showed that triosephosphate isomerase (TPI) and 6-phosphogluconolactonase (PGLS), which participate in glycolysis and the PPP, respectively, were significantly downregulated by MED treatment. Functional studies revealed that the expression levels of several other enzymes involved in glycolysis and the PPP, including hexokinase 2 (HK2), phosphofructokinase 1 (PFKM), aldolase A (ALDOA), enolase 1 (ENO1), lactate dehydrogenase A (LDHA), and glucose-6-phosphate dehydrogenase (G6PD), were also reduced in a dose-dependent manner. Moreover, the LDHA and G6PD enzymatic activities in HeLa cells were inhibited by MED, and overexpression of these downregulated enzymes rescued HeLa cells from the growth inhibition induced by MED. Our data suggest that MED suppresses HeLa cell growth by inhibiting glycolysis and the PPP, which provides a mechanistic basis for the development of new therapeutics against cervical cancer.

  19. Growth and apoptosis of HeLa cells induced by intense picosecond pulsed electric field

    Directory of Open Access Journals (Sweden)

    Yuan-yuan HUA

    2011-07-01

    Full Text Available Objective To investigate the growth and apoptosis of HeLa cells induced by intense picosecond pulsed electric field(PEF in vitro.Methods HeLa cells cultured in vitro were divided into experimental group and control group(with or without intense picosecond PEF.With constant pulse width,frequency and voltage,the cells in experimental group were divided into 6 sub-groups according to the number of pulse(100,200,500,1000,1500,2000,the growth inhibition of HeLa cells by PEF and the dose-effect relationship were analyzed by MTT.Caspase 3 protein activity was detected in the cells in 500,1000 and 2000 sub-groups.Mitochondrial transmembrane potential was detected by rhodamine 123 staining with the cells in 2000 sub-groups.Results MTT assay demonstrated that intense picosecond PEF significantly inhibited the proliferation of HeLa cells in dose-dependent manner.The survival rates of cells declined along with the increase in pulse number,and were 96.23%±0.76%,94.11%±2.42%,90.31%±1.77%,64.59%±1.59%,32.95%±0.73%,23.85%±2.38% and 100%,respectively,in 100,200,500,1000,1500,2000 sub-groups and control group(P < 0.01.The Caspase 3 protein activity was significantly enhanced by intense picosecond PEF,and the absorbancy indexes(A were 0.174±0.012,0.232±0.017,0.365±0.016 and 0.122±0.011,respectively,in 500,1000,2000 sub-groups and control group(P < 0.05.The mitochondrial transmembrane potential of HeLa cells was significantly inhibited by intense picosecond PEF,and the fluorescence intensity in 2000 sub-group(76.66±13.38 was much lower than that in control group(155.81±2.33,P < 0.05.Conclusion Intense picosecond PEF may significantly inhibit the growth of HeLa cells,and induce cell apoptosis via mitochondrial pathway.

  20. Curcumin targeting the thioredoxin system elevates oxidative stress in HeLa cells

    Energy Technology Data Exchange (ETDEWEB)

    Cai, Wenqing; Zhang, Baoxin; Duan, Dongzhu [State Key Laboratory of Applied Organic Chemistry, Lanzhou University, Lanzhou, Gansu 730000 (China); Wu, Jincai [College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou, Gansu 730000 (China); Fang, Jianguo, E-mail: fangjg@lzu.edu.cn [State Key Laboratory of Applied Organic Chemistry, Lanzhou University, Lanzhou, Gansu 730000 (China); College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou, Gansu 730000 (China)

    2012-08-01

    The thioredoxin system, composed of thioredoxin reductase (TrxR), thioredoxin (Trx), and NADPH, is ubiquitous in all cells and involved in many redox-dependent signaling pathways. Curcumin, a naturally occurring pigment that gives a specific yellow color in curry food, is consumed in normal diet up to 100 mg per day. This molecule has also been used in traditional medicine for the treatment of a variety of diseases. Curcumin has numerous biological functions, and many of these functions are related to induction of oxidative stress. However, how curcumin elicits oxidative stress in cells is unclear. Our previous work has demonstrated the way by which curcumin interacts with recombinant TrxR1 and alters the antioxidant enzyme into a reactive oxygen species (ROS) generator in vitro. Herein we reported that curcumin can target the cytosolic/nuclear thioredoxin system to eventually elevate oxidative stress in HeLa cells. Curcumin-modified TrxR1 dose-dependently and quantitatively transfers electrons from NADPH to oxygen with the production of ROS. Also, curcumin can drastically down-regulate Trx1 protein level as well as its enzyme activity in HeLa cells, which in turn remarkably decreases intracellular free thiols, shifting the intracellular redox balance to a more oxidative state, and subsequently induces DNA oxidative damage. Furthermore, curcumin-pretreated HeLa cells are more sensitive to oxidative stress. Knockdown of TrxR1 sensitizes HeLa cells to curcumin cytotoxicity, highlighting the physiological significance of targeting TrxR1 by curcumin. Taken together, our data disclose a previously unrecognized prooxidant mechanism of curcumin in cells, and provide a deep insight in understanding how curcumin works in vivo. -- Highlights: ► Curcumin induces oxidative stress by targeting the thioredoxin system. ► Curcumin-modified TrxR quantitatively oxidizes NADPH to generate ROS. ► Knockdown of TrxR1 augments curcumin's cytotoxicity in HeLa cells.

  1. Coxsackievirus B5 induced apoptosis of HeLa cells: effects on p53 and SUMO.

    Science.gov (United States)

    Gomes, Rogério; Guerra-Sá, Renata; Arruda, Eurico

    2010-01-20

    Coxsackievirus B5 (CVB5), a human enterovirus of the family Picornaviridae, is a frequent cause of acute and chronic human diseases. The pathogenesis of enteroviral infections is not completely understood, and the fate of the CVB5-infected cell has a pivotal role in this process. We have investigated the CVB5-induced apoptosis of HeLa cells and found that it happens by the intrinsic pathway by a mechanism dependent on the ubiquitin-proteasome system, associated with nuclear aggregation of p53. Striking redistribution of both SUMO and UBC9 was noted at 4 h post-infection, simultaneously with a reduction in the levels of the ubiquitin-ligase HDM2. Taken together, these results suggest that CVB5 infection of HeLa cells elicit the intrinsic pathway of apoptosis by MDM2 degradation and p53 activation, destabilizing protein sumoylation, by a mechanism that is dependent on a functional ubiquitin-proteasome system.

  2. Study of Paclitaxel-Treated HeLa Cells by Differential Electrical Impedance Flow Cytometry.

    Science.gov (United States)

    Kirkegaard, Julie; Clausen, Casper Hyttel; Rodriguez-Trujillo, Romen; Svendsen, Winnie Edith

    2014-09-01

    This work describes the electrical investigation of paclitaxel-treated HeLa cells using a custom-made microfluidic biosensor for whole cell analysis in continuous flow. We apply the method of differential electrical impedance spectroscopy to treated HeLa cells in order to elucidate the changes in electrical properties compared with non-treated cells. We found that our microfluidic system was able to distinguish between treated and non-treated cells. Furthermore, we utilize a model for electrical impedance spectroscopy in order to perform a theoretical study to clarify our results. This study focuses on investigating the changes in the electrical properties of the cell membrane caused by the effect of paclitaxel. We observe good agreement between the model and the obtained results. This establishes the proof-of-concept for the application in cell drug therapy.

  3. Study of Paclitaxel-Treated HeLa Cells by Differential Electrical Impedance Flow Cytometry

    Directory of Open Access Journals (Sweden)

    Julie Kirkegaard

    2014-08-01

    Full Text Available This work describes the electrical investigation of paclitaxel-treated HeLa cells using a custom-made microfluidic biosensor for whole cell analysis in continuous flow. We apply the method of differential electrical impedance spectroscopy to treated HeLa cells in order to elucidate the changes in electrical properties compared with non-treated cells. We found that our microfluidic system was able to distinguish between treated and non-treated cells. Furthermore, we utilize a model for electrical impedance spectroscopy in order to perform a theoretical study to clarify our results. This study focuses on investigating the changes in the electrical properties of the cell membrane caused by the effect of paclitaxel. We observe good agreement between the model and the obtained results. This establishes the proof-of-concept for the application in cell drug therapy.

  4. Aktivitas Antitumor (Hela dan T47d dan Antioksidan Ekstrak Makroalga Hijau Ulva fasciata

    Directory of Open Access Journals (Sweden)

    Endar Marraskuranto

    2008-12-01

    metode BSLT, uji sitotoksisitas terhadap sel tumor HeLa dan T47D dilakukan dengan metode uji MTT, sedangkan uji antioksidan dilakukan dengan metode DPPH (2,2-diphenyl pikryl hidrazil. Hasil uji toksisitas menunjukkan bahwa fraksi heksan U. fasciata memiliki toksisitas tertinggi dengan LC50 sebesar 19,12 ppm sehingga tergolong sangat toksik. Sementara itu, fraksi heksan makroalga U. fasciata menunjukkan aktivitas sitotoksik yang baik terhadap sel tumor HeLa (IC50 = 25,6 ppm dan terhadap sel tumor T47D (IC50 = 28,7 ppm. Akan tetapi, ekstrak kasar, fraksi metanol dan fraksi heksan makroalga U. fasciata tidak menunjukkan aktivitas antioksidan dengan nilai IC50 yang masih jauh di atas standar vitamin C.

  5. Biocompatibility of various ferrite nanoparticles evaluated by in vitro cytotoxicity assays using HeLa cells

    Science.gov (United States)

    Tomitaka, Asahi; Hirukawa, Atsuo; Yamada, Tsutomu; Morishita, Shin; Takemura, Yasushi

    2009-05-01

    Magnetic nanoparticles for thermotherapy must be biocompatible and possess high thermal efficiency as heating elements. The biocompatibility of Fe 3O 4 (20-30 nm), ZnFe 2O 4 (15-30 nm) and NiFe 2O 4 (20-30 nm) nanoparticles was studied using a cytotoxicity colony formation assay and a cell viability assay. The Fe 3O 4 sample was found to be biocompatible on HeLa cells. While ZnFe 2O 4 and NiFe 2O 4 were non-toxic at low concentrations, HeLa cells exhibited cytotoxic effects when exposed to concentrations of 100 μg/ml nanoparticles.

  6. Biocompatibility of various ferrite nanoparticles evaluated by in vitro cytotoxicity assays using HeLa cells

    Energy Technology Data Exchange (ETDEWEB)

    Tomitaka, Asahi [Department of Electrical and Computer Engineering, Yokohama National University, Tokiwadai 79-5, Yokohama, Kanagawa 240-8501 (Japan)], E-mail: d07gd158@ynu.ac.jp; Hirukawa, Atsuo; Yamada, Tsutomu [Department of Electrical and Computer Engineering, Yokohama National University, Tokiwadai 79-5, Yokohama, Kanagawa 240-8501 (Japan); Morishita, Shin [Department of Mechanical Engineering and Materials Science, Yokohama National University, Tokiwadai 79-5, Yokohama, Kanagawa 240-8501 (Japan); Takemura, Yasushi [Department of Electrical and Computer Engineering, Yokohama National University, Tokiwadai 79-5, Yokohama, Kanagawa 240-8501 (Japan)

    2009-05-15

    Magnetic nanoparticles for thermotherapy must be biocompatible and possess high thermal efficiency as heating elements. The biocompatibility of Fe{sub 3}O{sub 4} (20-30 nm), ZnFe{sub 2}O{sub 4} (15-30 nm) and NiFe{sub 2}O{sub 4} (20-30 nm) nanoparticles was studied using a cytotoxicity colony formation assay and a cell viability assay. The Fe{sub 3}O{sub 4} sample was found to be biocompatible on HeLa cells. While ZnFe{sub 2}O{sub 4} and NiFe{sub 2}O{sub 4} were non-toxic at low concentrations, HeLa cells exhibited cytotoxic effects when exposed to concentrations of 100 {mu}g/ml nanoparticles.

  7. IR-induced autophagy plays a role in survival of HeLa cells

    Energy Technology Data Exchange (ETDEWEB)

    Kang, Mi Young; Jang, Eun Yeong; Ryu, Tae Ho; Chung, Dong Min; Kim, Jin Hong; Kim, Jin Kyu [Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongeup (Korea, Republic of)

    2014-04-15

    Cells respond to stress with repair, or are diverted into irreversible cell cycle exit (senescence) or are eliminated through programmed cell death. There are two major morphologically distinctive forms of programmed cell death, apoptosis and autophagic cell death. Apoptosis contribute to cell death, whereas autophagy can play a dual role in mediating either cell survival or death in response to various stress stimuli. Here we analysed cellular responses induced by IR. The understanding of an appropriate cellular stress response is of crucial importance in foreseeing the cell fate. Apoptotic feagures were not detected in HeLa under our experimental irradiation condition. Autophagic cell death in HeLa may play an important role in cell protection and can result in cell survival.

  8. The comparison of radiation responses in MCF-7 and HeLa cells

    Energy Technology Data Exchange (ETDEWEB)

    Kang, Mi Young; Jang, Eun Yeong; Ryu, Tae Ho; Chung, Dong-Min; Kim, Jin Hong; Kim, Jin Kyu [Advanced Radiation Technology Institute, Jeongeup (Korea, Republic of)

    2014-11-15

    Activation of this pathway temporarily arrests cells at the G1 or G2 checkpoints of cell cycle, or terminates DNA replication and cell division. The present study was carried out to identify the fate of cells to cope with DNA damage stress. Cellular responses following IR treatment were different depending on the characteristics (origin, organism and genes expressed etc.) of cell line used and extent of genomic injury. p53 expression level was increased in a dose-dependent manner in both cells. IR induced a drastic increase in expression of p21 in MCF-7 compared to that in HeLa cells. Cell cycle analysis using flow cytometry showed a significant accumulation in G2/M phase after treatment of MCF-7 with IR. This study identified that IR-induced cell fates were determined through p53-dependent activation of p21, which resulted in senescence of MCF-7 cells and autophagy of HeLa cells.

  9. Bamboo charcoal inhibits growth of HeLa cells in vitro.

    Science.gov (United States)

    Teraoka, Fumio; Hamada, Yoshinosuke; Takahashi, Junzo

    2004-12-01

    The purpose of this work was to investigate the far infrared spectral characteristics of bamboo charcoal powder and its effect on cancer cells for use in the dental field. To analyze the effects of the powder, HeLa and WI-38 cells were used and then assessed by cell adhesion assay and WST-1 assay. The powder emitted far infrared rays at wavelengths between 4 to 16 microm. The multiplication rate of WI-38 cells showed no significant differences between the conventional culture (control group) and culture on the powder (FIR group). However, at six days after incubation, HeLa cells of FIR group had a significantly lower multiplication rate compared with the control group. Based on the far infrared rays emitted in this study, bamboo charcoal powder proved to be a promising dental filler material for cancer prevention.

  10. Hemocyanin from Shrimp Litopenaeus vannamei Has Antiproliferative Effect against HeLa Cell In Vitro.

    Directory of Open Access Journals (Sweden)

    Liyuan Zheng

    Full Text Available Hemocyanin (HMC has been shown to participate in multiple roles of immune defence. In this study, we investigated the antiproliferative effect and underpinning mechanism of HMC from Litopenaeus vannamei in vitro. Sulforhodamine B (SRB assay indicated that HMC could dramatically inhibit the growth of HeLa cells, but not 293T cells under the same conditions. Moreover, typical morphological features of apoptosis in HeLa cells including the formation of apoptotic body-like vesicles, chromatin condensation and margination were observed by using 4, 6-diamidino-2- phenylindole dihydrochloride (DAPI staining and fluorescence analysis. An apoptotic DNA ladder from 180 to 300 bp was also detected. Furthermore, 10 variation proteins associated with apoptosis pathway, viz. G3PDH isoforms 1/2 (G3PDH1/2, aldosereductase, ectodemal dysplasia receptor associated death receptor domain isoform CRA_a (EDARADD, heat shock 60kD protein 1 variant 1 (HSP60, heat shock 70kDa protein 5 precursor (HSP70, heat shock protein 90kDa beta member 1 precursor (HSP90, 14-3-3 protein ζ/δ, Ran and ubiquitin activating enzyme E1(UBE1, were identified from HMC-treated HeLa cells by the proteomic and quantitative real-time RT-PCR strategies. Importantly, the reactive oxygen species (ROS, mitochondrial membrane potential (Δψm and caspase-9/3 activities were changed significantly in HMC-treated HeLa cells. Together, the data suggests that L. vannamei HMC mediates antiproliferative properties through the apoptosis mechanism involving the mitochondria triggered pathway.

  11. Analysis of lysosomal membrane proteins exposed to melanin in HeLa cells

    OpenAIRE

    Bang, Seung Hyuck; Park, Dong Jun; Kim, Yang-Hoon; Min, Jiho

    2016-01-01

    Objectives There have been developed to use targeting ability for antimicrobial, anticancerous, gene therapy and cosmetics through analysis of various membrane proteins isolated from cell organelles. Methods It was examined about the lysosomal membrane protein extracted from lysosome isolated from HeLa cell treated by 100 ppm melanin for 24 hours in order to find associated with targeting ability to melanin using by 2-dimensional electrophoresis. Results The result showed 14 up-regulated (1.5...

  12. Influence of valine deprivation and its reversal on fatty acid metabolism in HeLa cells.

    Science.gov (United States)

    Slayback, J R; Campbell, I M; Vaughan, M H

    1976-05-27

    The effects that amino acid starvation and re-supplementation have on fatty acid metabolism in HeLa cells have been studied using radio gas chromatographic techniques. Deprivation of valine for 13.5 h caused fatty acid de novo biosynthesis, elongation and desaturation to cease. This effect was reversed within 5 h by adding valine back to the culture. During deprivation accumulation of triacylglycerol occurred. The return of valine to the culture caused compositional changes in the triacylglycerols and phosphatidylcholines.

  13. IBTK Differently Modulates Gene Expression and RNA Splicing in HeLa and K562 Cells

    Directory of Open Access Journals (Sweden)

    Giuseppe Fiume

    2016-11-01

    Full Text Available The IBTK gene encodes the major protein isoform IBTKα that was recently characterized as substrate receptor of Cul3-dependent E3 ligase, regulating ubiquitination coupled to proteasomal degradation of Pdcd4, an inhibitor of translation. Due to the presence of Ankyrin-BTB-RCC1 domains that mediate several protein-protein interactions, IBTKα could exert expanded regulatory roles, including interaction with transcription regulators. To verify the effects of IBTKα on gene expression, we analyzed HeLa and K562 cell transcriptomes by RNA-Sequencing before and after IBTK knock-down by shRNA transduction. In HeLa cells, 1285 (2.03% of 63,128 mapped transcripts were differentially expressed in IBTK-shRNA-transduced cells, as compared to cells treated with control-shRNA, with 587 upregulated (45.7% and 698 downregulated (54.3% RNAs. In K562 cells, 1959 (3.1% of 63128 mapped RNAs were differentially expressed in IBTK-shRNA-transduced cells, including 1053 upregulated (53.7% and 906 downregulated (46.3%. Only 137 transcripts (0.22% were commonly deregulated by IBTK silencing in both HeLa and K562 cells, indicating that most IBTKα effects on gene expression are cell type-specific. Based on gene ontology classification, the genes responsive to IBTK are involved in different biological processes, including in particular chromatin and nucleosomal organization, gene expression regulation, and cellular traffic and migration. In addition, IBTK RNA interference affected RNA maturation in both cell lines, as shown by the evidence of alternative 3′- and 5′-splicing, mutually exclusive exons, retained introns, and skipped exons. Altogether, these results indicate that IBTK differently modulates gene expression and RNA splicing in HeLa and K562 cells, demonstrating a novel biological role of this protein.

  14. Taraxerol Induces Cell Apoptosis through A Mitochondria-Mediated Pathway in HeLa Cells.

    Science.gov (United States)

    Yaoi, Xiangyang; Lu, Binyu; Lü, Chaotian; Bai, Qin; Yan, Dazhong; Xu, Hui

    2017-10-01

    Taraxerol acetate has potent anti-cancer effects via the induction of apoptosis, autophagy, cell cycle arrest, and inhibition of cell migration. However, whether taraxerol induced apoptosis and its underlying mechanisms of action is not clear. In the present study, we assess the effects of taraxerol on the mitochondrial apoptotic pathway and determine the release of cytochrome c to the cytosol and activation of caspases. In this experimental study, we mainly investigated the effect of taraxerol on HeLa cells. We tested cell viability by the MTT assay and morphologic changes, analyzed apoptosis by DAPI staining and flow cytometry. We also determined reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) using a Microplate Reader. In addition, the apoptotic proteins were tested by Western blot. Taraxerol enhanced ROS levels and attenuated the MMP (Δψm) in HeLa cells. Taraxerol induced apoptosis mainly via the mitochondrial pathway including the release of cytochrome c to the cytosol and activation of caspases 9 and 3, and anti-poly (ADPribose) polymerase (PARP). Taraxerol could induce the down-regulation of the anti-apoptotic protein Bcl-2 and up-regulation of pro-apoptotic protein Bax. It suppressed the PI3K/ Akt signaling pathway. These results demonstrated that taraxerol induced cell apoptosis through a mitochondria-mediated pathway in HeLa cells. Thus, taraxerol might be a potential anticervical cancer candidate.

  15. Kelussia odoratissima potentiates cytotoxic effects of radiation in HeLa cancer cell line

    Directory of Open Access Journals (Sweden)

    Azar Hosseini

    2017-02-01

    Full Text Available Objective: Cervical cancer is the second most common cause of death from cancer in women throughout the world. The aim of this study was to evaluate the cytotoxic activity of Kelussia odoratissima (K. odoratissima extract associated with radiotherapy in cervical cancer cells (HeLa cell line.Materials and Methods: Different concentration of the extract (25-500µg/ml was tested in HeLa cell lines. Cell cytotoxicity of the extract and the effects of the extract on radiation (2Gy/min-induced damages were assessed by MTT assay. Apoptosis was assessed using flow cytometric analysis.Result: K. odoratissima decreased cell viability in HeLa cell line in a concentration and time-dependent manner. When compared to the control,K. odoratissima induced a sub-G1 peak in the flow cytometry histogram of treated cells, indicating that apoptotic cell death is involved in K. odoratissima-induced toxicity. It was also shown that K. odoratissima sensitizes cells to radiation-induced toxicity.Conclusion: Our result showed the extract increased the radiation effect. This observation may be related to the presence of active compounds such as phthalides and ferulic acid.

  16. A key inactivation factor of HeLa cell viability by a plasma flow

    Energy Technology Data Exchange (ETDEWEB)

    Sato, Takehiko; Yokoyama, Mayo [Institute of Fluid Science, Tohoku University, 2-1-1 Katahira, Aoba-ku, Sendai 980-8577 (Japan); Johkura, Kohei, E-mail: sato@ifs.tohoku.ac.jp [Department of Histology and Embryology, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto 390-8621 (Japan)

    2011-09-21

    Recently, a plasma flow has been applied to medical treatment using effects of various kinds of stimuli such as chemical species, charged particles, heat, light, shock wave and electric fields. Among them, the chemical species are known to cause an inactivation of cell viability. However, the mechanisms and key factors of this event are not yet clear. In this study, we focused on the effect of H{sub 2}O{sub 2} in plasma-treated culture medium because it is generated in the culture medium and it is also chemically stable compared with free radicals generated by the plasma flow. To elucidate the significance of H{sub 2}O{sub 2}, we assessed the differences in the effects of plasma-treated medium and H{sub 2}O{sub 2}-added medium against inactivation of HeLa cell viability. These two media showed comparable effects on HeLa cells in terms of the survival ratios, morphological features of damage processes, permeations of H{sub 2}O{sub 2} into the cells, response to H{sub 2}O{sub 2} decomposition by catalase and comprehensive gene expression. The results supported that among chemical species generated in a plasma-treated culture medium, H{sub 2}O{sub 2} is one of the main factors responsible for inactivation of HeLa cell viability. (fast track communication)

  17. Cytotoxicity and apoptotic effects of nickel oxide nanoparticles in cultured HeLa cells

    Directory of Open Access Journals (Sweden)

    Kezban Ada

    2010-04-01

    Full Text Available The aim of this study was to observe the cytotoxicity and apoptotic effects of nickel oxide nanoparticles on humancervix epithelioid carcinoma cell line (HeLa. Nickel oxide precursors were synthesized by an nickel sulphate-excess ureareaction in boiling aqueous solution. The synthesized NiO nanoparticles (<200 nm were investigated by X-ray diffractionanalysis and transmission electron microscopy techniques. For cytotoxicity experiments, HeLa cells were incubated in50-500 μg/mL NiO for 2, 6, 12 and 16 hours. The viable cells were counted with a haemacytometer using light microscopy.The cytotoxicity was observed low in 50-200 μg/mL concentration for 16 h, but high in 400-500 μg/mL concentration for2-6 h. HeLa cells' cytoplasm membrane was lysed and detached from the well surface in 400 μg/mL concentration NiOnanoparticles. Double staining and M30 immunostaining were performed to quantify the number of apoptotic cells in cultureon the basis of apoptotic cell nuclei scores. The apoptotic effect was observed 20% for 16 h incubation.

  18. ALG-2 knockdown in HeLa cells results in G2/M cell cycle phase accumulation and cell death

    DEFF Research Database (Denmark)

    Høj, Berit Rahbek; la Cour, Peter Jonas Marstrand; Mollerup, Jens

    2009-01-01

    downregulation induces accumulation of HeLa cells in the G2/M cell cycle phase and increases the amount of early apoptotic and dead cells. Caspase inhibition by the pan-caspase inhibitor zVAD-fmk attenuated the increase in the amount of dead cells following ALG-2 downregulation. Thus, our results indicate...... that ALG-2 has an anti-apoptotic function in HeLa cells by facilitating the passage through checkpoints in the G2/M cell cycle phase....

  19. A systematic investigation into the electrical properties of single HeLa cells via impedance measurements and COMSOL simulations.

    Science.gov (United States)

    Wang, Min-Haw; Jang, Ling-Sheng

    2009-05-15

    The electrical properties of single cells provide fundamental insights into their pathological condition and are therefore of immense interest to medical practitioners. Accordingly, this study captures single HeLa cells using a microfluidic device and then measures their impedance properties using a commercial impedance spectroscopy system. The experimental system is modeled by an equivalent electrical circuit and COMSOL simulations are then performed to establish the conductivity, permittivity and impedance of single HeLa cells under various operational frequencies and voltages. At an operational voltage of 0.2 V, the maximum deviation between the experimental and simulation results for the magnitude and phase of the HeLa cell impedance is found to be 9.5% and 4.2%, respectively. In general, both sets of results show that the conductivity and permittivity of single HeLa cells increase with an increasing operational voltage. Moreover, an increasing frequency is found to increase the conductivity of HeLa cells at all values of the operational voltage, but to reduce the permittivity for operational voltages in the range 0.6-1.0 V. Based upon the simulation and experimental results, empirical equations are constructed to predict the conductivity and permittivity of single HeLa cells under specified values of the operational voltage and frequency, respectively. The maximum discrepancy between the predicted results and the simulation results for the permittivity and conductivity of the HeLa cells at an operational voltage of 0.2 V is found to be just 0.5% and 4.5%, respectively.

  20. Presence of autoantibodies against HeLa small nuclear ribonucleoproteins in chagasic and non-chagasic cardiac patients

    Directory of Open Access Journals (Sweden)

    M.C. Bosetto

    2004-01-01

    Full Text Available We detected anti-human small nuclear ribonucleoprotein (snRNP autoantibodies in chagasic patients by different immunological methods using HeLa snRNPs. ELISA with Trypanosoma cruzi total lysate antigen or HeLa human U small nuclear ribonucleoproteins (UsnRNPs followed by incubation with sera from chronic chagasic and non-chagasic cardiac patients was used to screen and compare serum reactivity. Western blot analysis using a T. cruzi total cell extract was also performed in order to select some sera for Western blot and immunoprecipitation assays with HeLa nuclear extract. ELISA showed that 73 and 95% of chronic chagasic sera reacted with HeLa UsnRNPs and T. cruzi antigens, respectively. The Western blot assay demonstrated that non-chagasic cardiac sera reacted with high molecular weight proteins present in T. cruzi total extract, probably explaining the 31% reactivity found by ELISA. However, these sera reacted weakly with HeLa UsnRNPs, in contrast to the chagasic sera, which showed autoantibodies with human Sm (from Stefanie Smith, the first patient in whom this activity was identified proteins (B/B', D1, D2, D3, E, F, and G UsnRNP. Immunoprecipitation reactions using HeLa nuclear extracts confirmed the reactivity of chagasic sera and human UsnRNA/RNPs, while the other sera reacted weakly only with U1snRNP. These findings agree with previously reported data, thus supporting the idea of the presence of autoimmune antibodies in chagasic patients. Interestingly, non-chagasic cardiac sera also showed reactivity with T. cruzi antigen and HeLa UsnRNPs, which suggests that individuals with heart disease of unknown etiology may develop autoimmune antibodies at any time. The detection of UsnRNP autoantibodies in chagasic patients might contribute to our understanding of how they develop upon initial T. cruzi infection.

  1. Photodynamic Effect of Ni Nanotubes on an HeLa Cell Line.

    Directory of Open Access Journals (Sweden)

    Muhammad Hammad Aziz

    Full Text Available Nickel nanomaterials are promising in the biomedical field, especially in cancer diagnostics and targeted therapy, due to their distinctive chemical and physical properties. In this experiment, the toxicity of nickel nanotubes (Ni NTs were tested in an in vitro cervical cancer model (HeLa cell line to optimize the parameters of photodynamic therapy (PDT for their greatest effectiveness. Ni NTs were synthesized by electrodeposition. Morphological analysis and magnetic behavior were examined using a Scanning electron microscope (SEM, an energy dispersive X-ray analysis (EDAX and a vibrating sample magnetometer (VSM analysis. Phototoxic and cytotoxic effects of nanomaterials were studied using the Ni NTs alone as well as in conjugation with aminolevulinic acid (5-ALA; this was performed both in the dark and under laser exposure. Toxic effects on the HeLa cell model were evaluated by a neutral red assay (NRA and by detection of intracellular reactive oxygen species (ROS production. Furthermore, 10-200 nM of Ni NTs was prepared in solution form and applied to HeLa cells in 96-well plates. Maximum toxicity of Ni NTs complexed with 5-ALA was observed at 100 J/cm2 and 200 nM. Up to 65-68% loss in cell viability was observed. Statistical analysis was performed on the experimental results to confirm the worth and clarity of results, with p-values = 0.003 and 0.000, respectively. Current results pave the way for a more rational strategy to overcome the problem of drug bioavailability in nanoparticulate targeted cancer therapy, which plays a dynamic role in clinical practice.

  2. Coxsackievirus B5 induced apoptosis of HeLa cells: effects on p53 and SUMO.

    OpenAIRE

    Gomes,Rogério; Guerra-Sá, Renata; ARRUDA, Eurico

    2010-01-01

    Coxsackievirus B5 (CVB5), a human enterovirus of the family Picornaviridae, is a frequent cause of acute and chronic human diseases. The pathogenesis of enteroviral infections is not completely understood, and the fate of the CVB5-infected cell has a pivotal role in this process. We have investigated the CVB5-induced apoptosis of HeLa cells and found that it happens by the intrinsic pathway by a mechanism dependent on the ubiquitin–proteasome system, associated with nuclear aggregation of ...

  3. Caveolae-mediated endocytosis of biocompatible gold nanoparticles in living Hela cells

    DEFF Research Database (Denmark)

    Hao, Xian; Wu, Jiazhen; Shan, Yuping

    2012-01-01

    the internalization mechanism of small-size AuNPs by living Hela cells. Herein, we found that the caveolae-mediated endocytosis was the dominant pathway for the intracellular delivery of small-size AuNPs. The intracellular delivery was suppressed when we depleted the cholesterol with methyl-β-cyclodextrin (M beta CD......Efficient intracellular delivery of gold nanoparticles (AuNPs) and unraveling the mechanism underlying the intracellular delivery are essential for advancing the applications of AuNPs toward in vivo imaging and therapeutic interventions. We employed fluorescence microscopy to investigate...

  4. The chemosensitizing effect of aqueous extract of sweet fennel on cisplatin treated HeLa cells.

    Science.gov (United States)

    Ramadan, W S; Sait, K H; Anfinan, N M; Sait, H

    2016-01-01

    Cisplatin is an important chemotherapeutic agent that is widely used in treatment of several malignancies, but its side effects on normal tissues and organs limit its use. The aim of this study was to evaluate the effect of aqueous extract of sweet fennel alone and in combination with cisplatin on human cervical cancer adenocarcinoma cell line (HeLa cells) searching for an effective, inexpensive therapy with minimal side effects. HeLa cell line was used to study the cytotoxic effect of different concentrations of the aqueous extract of sweet fennel alone and in combination with 50 μg/ml cisplatin. Quantitative measure of drug interaction was quantified by the combination index. Gas chromatography-mass spectrometry (GC-MS) and high-performance liquid chromatography (HPLC) were used to analyze the sweet fennel decoction. MTT assay was used to examine cell viability percentage. Electron microscopy was applied to study the ultrastructure of the cells. The phenyl propanoids (23%) and phenols (12%) constituted the highest percentage of the aqueous extract. Increasing the concentration of sweet fennel from 50 μg/ml to 80 μg/ml, decreased the percentage of the cell viability of HeLa cells from 86.74% to 78.28%, respectively. Further decrease to 11.31% was demonstrated when 50 μg/ml of fennel was combined with 50 μg/ml cisplatin (additive effect). In addition to the signs of apoptosis observed in HeLa cells at 50 μg/ml of fennel, disruption of both nuclear and cytoplasmic membranes and presence of autophagolysosomes were noticed at a dose of 80 μg/ml. Combination of 50 μg/ml of cisplatin with 60, 70, and 80 μg/ml of sweet fennel revealed no significant difference in comparison to cisplatin alone. The combination with 50 μg/ml of sweet fennel revealed marked vacuolization of the cytoplasm, fragmentation of the nucleus, and complete disruption of nuclear membrane. CONCLUSIOn: Combination of cisplatin and the 50 μg/ml of the fennel could enhance cervical cancer

  5. Surface enhanced Raman spectroscopy analysis of HeLa cells using a multilayer substrate

    Science.gov (United States)

    Aguilar-Hernández, I. A.; Pichardo-Molina, J. L.; Lopez-Luke, T.; Ornelas-Soto, N.

    2017-08-01

    Single cell analysis can provide important information regarding cell composition, and can be used for biomedical applications. In this work, a SERS active substrate formed by 3 layers of gold nanospheres and a final layer of gold nanocubes was used for the label-free SERS analysis of HeLa cells. Nanocubes were selected due to the high electromagnetic enhancement expected in nanoparticles with sharp corners. Significant improvement in the reproducibility and quality of SERS spectra was found when compared to the spectra obtained using a nanosphere-only substrate and normal Raman spectroscopy.

  6. Non-thermal plasma induces mitochondria-mediated apoptotic signaling pathway via ROS generation in HeLa cells.

    Science.gov (United States)

    Li, Wei; Yu, K N; Ma, Jie; Shen, Jie; Cheng, Cheng; Zhou, Fangjian; Cai, Zhiming; Han, Wei

    2017-11-01

    Non-thermal plasma (NTP) has been proposed as a novel therapeutic method for anticancer treatment. Although increasing evidence suggests that NTP selectively induces apoptosis in some types of tumor cells, the molecular mechanisms underlying this phenomenon remain unclear. In this study, we further investigated possible molecular mechanisms for NTP-induced apoptosis of HeLa cells. The results showed that NTP exposure significantly inhibited the growth and viability of HeLa cells. Morphological observation and flow cytometry analysis demonstrated that NTP exposure induced HeLa cell apoptosis. NTP exposure also activated caspase-9 and caspase-3, which subsequently cleaved poly (ADP- ribose) polymerase. Furthermore, NTP exposure suppressed Bcl-2 expression, enhanced Bax expression and translocation to mitochondria, activated mitochondria-mediated apoptotic pathway, followed by the release of cytochrome c. Further studies showed that NTP treatment led to ROS generation, whereas blockade of ROS generation by N-acetyl-l-cysteine (NAC, ROS scavengers) significantly prevented NTP-induced mitochondrial alteration and subsequent apoptosis of HeLa cells via suppressing Bax translocation, cytochrome c and caspase-3 activation. Taken together, our results indicated that NTP exposure induced mitochondria-mediated intrinsic apoptosis of HeLa cells was activated by ROS generation. These findings provide insights to the therapeutic potential and clinical research of NTP as a novel tool in cervical cancer treatment. Copyright © 2017. Published by Elsevier Inc.

  7. Discovery of HeLa Cell Contamination in HES Cells: Call for Cell Line Authentication in Reproductive Biology Research.

    Science.gov (United States)

    Kniss, Douglas A; Summerfield, Taryn L

    2014-08-01

    Continuous cell lines are used frequently in reproductive biology research to study problems in early pregnancy events and parturition. It has been recognized for 50 years that many mammalian cell lines contain inter- or intraspecies contaminations with other cells. However, most investigators do not routinely test their culture systems for cross-contamination. The most frequent contributor to cross-contamination of cell lines is the HeLa cell isolated from an aggressive cervical adenocarcinoma. We report on the discovery of HeLa cell contamination of the human endometrial epithelial cell line HES isolated in our laboratory. Short tandem repeat analysis of 9 unique genetic loci demonstrated molecular identity between HES and HeLa cells. In addition, we verified that WISH cells, isolated originally from human amnion epithelium, were also contaminated with HeLa cells. Inasmuch as our laboratory did not culture HeLa cells at the time of HES cell derivations, the source of contamination was the WISH cell line. These data highlight the need for continued diligence in authenticating cell lines used in reproductive biology research. © The Author(s) 2014.

  8. Combinational light emitting diode-high frequency focused ultrasound treatment for HeLa cell.

    Science.gov (United States)

    Choe, Se-Woon; Park, Kitae; Park, Chulwoo; Ryu, Jaemyung; Choi, Hojong

    2017-12-01

    Light sources such as laser and light emitting diode or ultrasound devices have been widely used for cancer therapy and regenerative medicines, since they are more cost-effective and less harmful than radiation therapy, chemotherapy or magnetic treatment. Compared to laser and low intensity ultrasound techniques, light emitting diode and high frequency focused ultrasound shows enhanced therapeutic effects, especially for small tumors. We propose combinational light emitting diode-high frequency focused ultrasound treatment for human cervical cancer HeLa cells. Individual red, green, and blue light emitting diode light only, high frequency focused ultrasound only, or light emitting diode light combined with high frequency focused ultrasound treatments were applied in order to characterize the responses of HeLa cells. Cell density exposed by blue light emitting diode light combined with high frequency focused ultrasound (2.19 ± 0.58%) was much lower than that of cells exposed by red and green light emitting diode lights (81.71 ± 9.92% and 61.81 ± 4.09%), blue light emitting diode light (11.19 ± 2.51%) or high frequency focused ultrasound only (9.72 ± 1.04%). We believe that the proposed combinational blue light emitting diode-high frequency focused ultrasound treatment could have therapeutic benefits to alleviate cancer cell proliferation.

  9. Evaluation of cytotoxicity of Moringa oleifera Lam. callus and leaf extracts on Hela cells

    Directory of Open Access Journals (Sweden)

    Abbas Jafarain

    2014-01-01

    Full Text Available Background: There are considerable attempts worldwide on herbal and traditional compounds to validate their use as anti-cancer drugs. Plants from Moringaceae family including Moringa oleifera possess several activities such as antitumor effect on tumor cell lines. In this study we sought to determine if callus and leaf extracts of M. oleifera possess any cytotoxicity. Materials and Methods: Ethanol-water (70-30 extracts of callus and leaf of M. oleifera were prepared by maceration method. The amount of phenolic compounds of the extracts was determined by Folin Ciocalteu method. The cytotoxicity of the extracts against Hela tumor cells was carried out using MTT assay. Briefly, cells were seeded in microplates and different concentrations of the extract were added. Cells were incubated for 48 h and their viability was evaluated by addition of tetrazolium salt solution. After 3 h medium was aspirated, dimethyl sulfoxide was added and absorbance was determined at 540 nm with an ELISA plate reader. Cytotoxicity was considered when more than 50% reduction on cell survival was observed. Results: Callus and leaf extracts of M. oleifera significantly decreased the viability of Hela cells in a concentration-dependent manner. However, leaf extract of M. oleifera were more potent than that of callus extract. Conclusion: As the content of phenolic compounds of leaf extract was higher than that of callus extract, it can be concluded that phenolic compounds are involved in the cytotoxicity of M. oleifera.

  10. Evaluation of cytotoxicity of Moringa oleifera Lam. callus and leaf extracts on Hela cells.

    Science.gov (United States)

    Jafarain, Abbas; Asghari, Gholamreza; Ghassami, Erfaneh

    2014-01-01

    There are considerable attempts worldwide on herbal and traditional compounds to validate their use as anti-cancer drugs. Plants from Moringaceae family including Moringa oleifera possess several activities such as antitumor effect on tumor cell lines. In this study we sought to determine if callus and leaf extracts of M. oleifera possess any cytotoxicity. Ethanol-water (70-30) extracts of callus and leaf of M. oleifera were prepared by maceration method. The amount of phenolic compounds of the extracts was determined by Folin Ciocalteu method. The cytotoxicity of the extracts against Hela tumor cells was carried out using MTT assay. Briefly, cells were seeded in microplates and different concentrations of the extract were added. Cells were incubated for 48 h and their viability was evaluated by addition of tetrazolium salt solution. After 3 h medium was aspirated, dimethyl sulfoxide was added and absorbance was determined at 540 nm with an ELISA plate reader. Cytotoxicity was considered when more than 50% reduction on cell survival was observed. Callus and leaf extracts of M. oleifera significantly decreased the viability of Hela cells in a concentration-dependent manner. However, leaf extract of M. oleifera were more potent than that of callus extract. As the content of phenolic compounds of leaf extract was higher than that of callus extract, it can be concluded that phenolic compounds are involved in the cytotoxicity of M. oleifera.

  11. Photoirradiation study of gold nanospheres and rods in Vero and Hela cell lines

    Science.gov (United States)

    Gananathan, Poorani; Aruna, Prakasarao; Ganesan, Singaravelu; Elanchezhiyan, Manickan

    2014-03-01

    Photoirradiation effect of gold nanospheres in conjucation with green light and rods in conjugation with red light corresponds to their absorption wavelength range found to be appreciable. In this present work concentration of nanomaterial and light dose were optimized. Gold nanospheres were synthesized by reduction technique using Sodium Borohydrate as reducing agent and Trisodium Citrate as capping agent. Au nanorods having 680-900nm absorption were synthesized using reduction techniques with CTAB and BDAC polymers. From UV-Vis absorption and Transmission Electron Microscopy the size of nanoparticles were confirmed. 30nm Gold nanospheres and green light source of 530nm wavelength with power 30mW were applied to Vero and Hela cell lines shows higher toxicity for Hela cells. Nanorods were applied and irradiated with 680nm wavelength light source with light intensity 45mW. Post irradiation effect for 24hrs, 48hrs confirms cell proliferation in normal rate in viable cells. The morphological changes in irradiated spot leads to apoptotoic cell death was confirmed with microscopic imaging. The LD50 value was also calculated.

  12. Investigation of role of aspartame on apoptosis process in HeLa cells

    Directory of Open Access Journals (Sweden)

    Muthuraman Pandurangan

    2016-07-01

    Full Text Available Aspartame is an artificial sweetener used as an alternate for sugar in several foods and beverages. The study reports that consumption of aspartame containing product could lead to cancer. However, the effect of aspartame on apoptosis process in cancer is not yet understood clearly. HeLa cells were exposed to different concentrations (0.01–0.05 mg/ml of aspartame for 48 h. Cytotoxicity of aspartame on cancer cells was determined by SRB assay. The result indicates no significant changes on cell viability. Aspartame suppresses apoptosis process in cancer cells by down-regulation of mRNA expression of tumor suppressor gene p53, and pro-apoptotic gene bax. It up-regulates anti-apoptotic gene bcl-2 mRNA expression. In addition, Ki 67 and PCNA mRNA, and protein expressions were determined. Taking all these together, we conclude that aspartame may be a potent substance to slow-down the apoptosis process in HeLa cells. Further works are ongoing to understand the biochemical and molecular mechanism of aspartame in cancer cells.

  13. In vitro oxygen availability modulates the effect of artesunate on HeLa cells.

    Science.gov (United States)

    Murray, James; Gannon, Sandra; Rawe, Sarah; Murphy, James E J

    2014-12-01

    Hypoxia can affect chemotherapeutic drug efficacy in cancer patients, yet related in vitro assays in oxygen-rich environment remain the norm. Such levels are well beyond normoxic/hypoxic levels typically experienced by normal tissues/tumor masses. The present study evaluated how artesunate anti-tumor efficacy is modulated by oxygen availability in HeLa cells and its implications for future in vitro analyses. Real-time cell analysis was employed to evaluate HeLa cell toxicity to artesunate at 21%, 4% or 1% oxygen. Cell count analysis was performed to validate real-time data. An increase in artesunate efficacy was observed when oxygen concentration was reduced from atmospheric levels down to in vivo-relevant levels. Artesunate is more potent than originally reported using standard oxygen conditions during in vitro studies. The inclusion of this long overlooked variable as standard in future in vitro analysis procedures is warranted. Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  14. PVA engineered microcapsules for targeted delivery of camptothecin to HeLa cells

    Energy Technology Data Exchange (ETDEWEB)

    Galbiati, Alice; Rocca, Blasco Morozzo della; Tabolacci, Claudio; Beninati, Simone; Desideri, Alessandro [Dipartimento di Biologia, Universita di Roma Tor Vergata, Via della Ricerca Scientifica, 00133 Rome (Italy); Paradossi, Gaio, E-mail: paradossi@stc.uniroma2.it [Dipartimento di Scienze e Tecnologie Chimiche, Universita di Roma Tor Vergata, Via della Ricerca Scientifica, 00133 Rome (Italy)

    2011-12-01

    Capsular microvectors are an important tool in the recent research field of nanomedicine to address a drug cargo for the therapeutic treatment of several pathologies. In this study we describe how the product of the conjugation of the polysaccharide chitosan with folate can be used as a coating of poly (vinyl alcohol), PVA, based microcapsules for an efficient targeting of HeLa cells. The influence of the coating on the bioadhesive properties of the vector and on its cargo capacity was also considered using camptothecin as an anticancer drug model. The coating strategy was finalized to exploit the good chemical versatility of PVA, used to form the shell of the vector. This study is a follow up of an investigation activity aiming to show the potentialities of PVA-shelled microcapsules or microbubbles as injectable microdevices supporting a theranostic approach for different types of tumour. Highlights: {yields}Coating of PVA-shelled microcapsules with chitosan-folate. {yields} Selective bioadhesion of microcapsules to HeLa Cells. {yields} Effective loading and release of camptothecin. {yields} In vitro anti-proliferative action of camptothecin loaded microcapsules.

  15. Effects of propolis extract on sensitivity to chemotherapeutic agents in HeLa and resistant sublines.

    Science.gov (United States)

    Takara, Kohji; Fujita, Megumi; Matsubara, Mika; Minegaki, Tetsuya; Kitada, Noriaki; Ohnishi, Noriaki; Yokoyama, Teruyoshi

    2007-09-01

    The effects of a propolis extract obtained by supercritical fluid extraction on sensitivity to chemotherapeutic agents were examined in HeLa cells and resistant sublines thereof. In addition, the actions of propolis and caffeic acid phenethyl ester (CAPE), a constituent of propolis, on the multidrug efflux transporter P-glycoprotein/MDR1, were evaluated in paclitaxel-resistant HeLa/TXL cells (MDR1-overexpressing cells). In HeLa cells, the sensitivity to paclitaxel and doxorubicin, substrates of MDR1, was unchanged in the presence of propolis. In HeLa/TXL cells, propolis increased sensitivity to these MDR1 substrates. The accumulation of Rhodamine123, also a substrate for MDR1, by HeLa/TXL cells increased in the presence of 50 microg/mL, but not 10 microg/mL, of the extract. However, the growth inhibition of HeLa/TXL cells by paclitaxel was not changed by CAPE, although the accumulation of Rhodamine123 increased significantly in the presence of 100 microm, but not 1 nM or 1 microm, CAPE. Collectively, the extract was suggested to inhibit the function of MDR1 and to increase the sensitivity to MDR1 substrates in HeLa/TXL cells, effects likely to be caused by constituents other than CAPE.

  16. Electroporation of micro-droplet encapsulated HeLa cells in oil phase

    KAUST Repository

    Xiao, Kang

    2010-08-27

    Electroporation (EP) is a method widely used to introduce foreign genes, drugs or dyes into cells by permeabilizing the plasma membrane with an external electric field. A variety of microfluidic EP devices have been reported so far. However, further integration of prior and posterior EP processes turns out to be very complicated, mainly due to the difficulty of developing an efficient method for precise manipulation of cells in microfluidics. In this study, by means of a T-junction structure within a delicate microfluidic device, we encapsulated HeLa cells in micro-droplet of poration medium in oil phase before EP, which has two advantages: (i) precise control of cell-encapsulating droplets in oil phase is much easier than the control of cell populations or individuals in aqueous buffers; (ii) this can minimize the electrochemical reactions on the electrodes. Finally, we successfully introduced fluorescent dyes into the micro-droplet encapsulated HeLa cells in oil phase. Our results reflected a novel way to realize the integrated biomicrofluidic system for EP. © 2010 Wiley-VCH Verlag GmbH & Co. KGaA.

  17. Uptake and fate of ouabain bound to HeLa cells. [/sup 3/H tracer study

    Energy Technology Data Exchange (ETDEWEB)

    Cook, J. S.; Brake, E. T.

    1977-01-01

    The first step in the pharmacologic interaction of a cardiac glycoside with cells is the binding of the drug to its specific surface receptor, an exteriorly oriented site of the Na/sup +/--K/sup +/ ATPase. The purpose of the study outlined was to follow a glycoside from its initial binding to its ultimate release from a sensitive cell, and in parallel studies to observe the recovery of the cells from glycoside intoxication. The glycoside used is /sup 3/H-ouabain, and the cells are from the S3 clone of HeLa. HeLa cells are used principally because of their intrinsic interest as a human cell type. In addition, having been cloned, they can be grown as a homogeneous population, which greatly simplifies the interpretation of the results. Finally, after pulse treatment with the drug, the cells can be returned to a complete medium with adequate supplies of serum, amino acids, vitamins, and energy sources. In this normal environment, not only can the short term physiologic responses be assessed but, with the growth requirements of the cells fulfilled, any recovery processes dependent on macromolecular-synthesis can also be observed.

  18. Usefulness of HeLa cells to evaluate inverse agonistic activity of antihistamines.

    Science.gov (United States)

    Mizuguchi, Hiroyuki; Ono, Shohei; Hattori, Masashi; Sasaki, Yohei; Fukui, Hiroyuki

    2013-03-01

    Antihistamines are thought to antagonize histamine and prevent it from binding to the histamine H1 receptor (H1R). However, recent studies indicate that antihistamines are classified into two groups, i.e., inverse agonists and neutral antagonists on the basis of their ability to down-regulate the constitutive activity of H1R. As H1R is an allergy-sensitive gene whose expression influences the severity of allergic symptoms, inverse agonists should more potently alleviate allergic symptoms than neutral antagonists by inhibiting H1R constitutive activity. Therefore, it is important to assess inverse agonistic activity of antihistamines. Here we report a novel assay method using HeLa cells expressing H1R endogenously for evaluation of inverse agonistic activity of antihistamines. Pretreatment with inverse agonists down-regulated H1R gene expression below to its basal level. On the other hand, basal H1R mRNA expression was unchanged by neutral antagonist pretreatment. Both inverse agonists and neutral antagonists suppressed histamine-induced H1R mRNA elevation. Classification of antihistamines on the basis of their suppressive activity of basal H1R gene expression was consistent with that of inositol phosphate accumulation in H1R-overexpressed cells. Our data suggest that the assay method using HeLa cells is more convenient and useful than the existing methods and may contribute to develop new antihistamines with inverse agonistic activity. Copyright © 2013 Elsevier B.V. All rights reserved.

  19. Overexpression of atypical protein kinase C in HeLa cells facilitates macropinocytosis via Src activation.

    Science.gov (United States)

    Tisdale, Ellen J; Shisheva, Assia; Artalejo, Cristina R

    2014-06-01

    Atypical protein kinase C (aPKC) is the first recognized kinase oncogene. However, the specific contribution of aPKC to cancer progression is unclear. The pseudosubstrate domain of aPKC is different from the other PKC family members, and therefore a synthetic peptide corresponding to the aPKC pseudosubstrate (aPKC-PS) sequence, which specifically blocks aPKC kinase activity, is a valuable tool to assess the role of aPKC in various cellular processes. Here, we learned that HeLa cells incubated with membrane permeable aPKC-PS peptide displayed dilated heterogeneous vesicles labeled with peptide that were subsequently identified as macropinosomes. A quantitative membrane binding assay revealed that aPKC-PS peptide stimulated aPKC recruitment to membranes and activated Src. Similarly, aPKC overexpression in transfected HeLa cells activated Src and induced macropinosome formation. Src-aPKC interaction was essential; substitution of the proline residues in aPKC that associate with the Src-SH3 binding domain rendered the mutant kinase unable to induce macropinocytosis in transfected cells. We propose that aPKC overexpression is a contributing factor to cell transformation by interacting with and consequently promoting Src activation and constitutive macropinocytosis, which increases uptake of extracellular factors, required for altered cell growth and accelerated cell migration. Copyright © 2014. Published by Elsevier Inc.

  20. Analysis of differentially expressed proteins in Yersinia enterocolitica-infected HeLa cells.

    Science.gov (United States)

    Alugubelly, Navatha; Hercik, Kamil; Kibler, Peter; Nanduri, Bindu; Edelmann, Mariola J

    2016-05-01

    Yersinia enterocolitica is a facultative intracellular pathogen and a causative agent of yersiniosis, which can be contracted by ingestion of contaminated food. Yersinia secretes virulence factors to subvert critical pathways in the host cell. In this study we utilized shotgun label-free proteomics to study differential protein expression in epithelial cells infected with Y.enterocolitica. We identified a total of 551 proteins, amongst which 42 were downregulated (including Prostaglandin E Synthase 3, POH-1 and Karyopherin alpha) and 22 were upregulated (including Rab1 and RhoA) in infected cells. We validated some of these results by western blot analysis of proteins extracted from Caco-2 and HeLa cells. The proteomic dataset was used to identify host canonical pathways and molecular functions modulated by this infection in the host cells. This study constitutes a proteome of Yersinia-infected cells and can support new discoveries in the area of host-pathogen interactions. We describe a proteome of Yersinia enterocolitica-infected HeLa cells, including a description of specific proteins differentially expressed upon infection, molecular functions as well as pathways altered during infection. This proteomic study can lead to a better understanding of Y. enterocolitica pathogenesis in human epithelial cells. Copyright © 2016. Published by Elsevier B.V.

  1. Nuclear proteome analysis of cisplatin-treated HeLa cells

    Energy Technology Data Exchange (ETDEWEB)

    Wu Wei [Department of Toxicology, Zhejiang University School of Public Health, 388 Yu-Hang-Tang Road, Hangzhou, Zhejiang 310058 (China); Institute of Hygiene, Zhejiang Academy of Medical Sciences, Hangzhou, Zhejiang 310013 (China); Yan Chunlan; Gan Tieer; Chen Zhanghui [Department of Toxicology, Zhejiang University School of Public Health, 388 Yu-Hang-Tang Road, Hangzhou, Zhejiang 310058 (China); Lu Xianghong [Department of Pharmacy, Lishui People' s Hospital, Lishui, Zhejiang 323000 (China); Duerksen-Hughes, Penelope J. [Department of Basic Sciences, Division of Biochemistry, Loma Linda University School of Medicine, Loma Linda, CA 92354 (United States); Zhu Xinqiang, E-mail: zhuxq@zju.edu.cn [Department of Toxicology, Zhejiang University School of Public Health, 388 Yu-Hang-Tang Road, Hangzhou, Zhejiang 310058 (China); Yang Jun, E-mail: gastate@zju.edu.cn [Department of Toxicology, Zhejiang University School of Public Health, 388 Yu-Hang-Tang Road, Hangzhou, Zhejiang 310058 (China)

    2010-09-10

    Cisplatin has been widely accepted as one of the most efficient anticancer drugs for decades. However, the mechanisms for the cytotoxic effects of cisplatin are still not fully understood. Cisplatin primarily targets DNA, resulting in the formation of DNA double strand breaks and eventually causing cell death. In this study, we applied two-dimensional electrophoresis coupled with LC-MS/MS to analyze the nuclear proteome of HeLa cells treated with cisplatin, in an effort to uncover new mechanistic clues regarding the cellular response to cisplatin. A total of 19 proteins were successfully identified, and these proteins are involved in a variety of basal metabolic and biological processes in cells, including biosynthesis, cell cycle, glycolysis and apoptosis. Six were related to the regulation of mRNA splicing, and we therefore asked whether the Fas gene might undergo alternative splicing following cisplatin treatment. This proved to be the case, as the splicing forms of Fas were modified in cisplatin-treated HeLa cells. This work provides novel information, from the perspective of the nuclear response, for understanding the cytotoxicity caused by cisplatin-induced DNA damage.

  2. Heterofucan from Sargassum filipendula Induces Apoptosis in HeLa Cells

    Directory of Open Access Journals (Sweden)

    Hugo Alexandre Oliveira Rocha

    2011-04-01

    Full Text Available Fucan is a term used to denominate a family of sulfated polysaccharides rich in sulfated L-fucose. Heterofucan SF-1.5v was extracted from the brown seaweed Sargassum filipendula by proteolytic digestion followed by sequential acetone precipitation. This fucan showed antiproliferative activity on Hela cells and induced apoptosis. However, SF-1.5v was not able to activate caspases. Moreover, SF-1.5v induced glycogen synthase kinase (GSK activation, but this protein is not involved in the heterofucan SF-1.5v induced apoptosis mechanism. In addition, ERK, p38, p53, pAKT and NFκB were not affected by the presence of SF-1.5v. We determined that SF-1.5v induces apoptosis in HeLa mainly by mitochondrial release of apoptosis-inducing factor (AIF into cytosol. In addition, SF-1.5v decreases the expression of anti-apoptotic protein Bcl-2 and increased expression of apoptogenic protein Bax. These results are significant in that they provide a mechanistic framework for further exploring the use of SF-1.5v as a novel chemotherapeutics against human cervical cancer.

  3. 31P nuclear magnetic resonance studies of HeLa cells.

    Science.gov (United States)

    Evans, F E; Kaplan, N O

    1977-11-01

    A survey of phosphorus compounds present in HeLa cells and their acid extracts has been carried out by (31)P nuclear magnetic resonance spectroscopy at 40 MHz. The proton decoupled (31)P spectrum of the neutralized extract had resolution adequate to enable the identification of the main phosphate compounds. The spectral intensities were converted to concentrations. The lower detection limit with extensive signal averaging was 0.02 mumol for the extract. The composition, listed in order of decreasing concentration, was: inorganic phosphate, ATP, phosphorylcholine, creatine phosphate, UTP, NAD(+), glucose 6-phosphate, beta-D-fructose 1,6-bisphosphate, alpha-D-fructose 1,6-bisphosphate, ADP, alpha-glycerophosphorylcholine, and alpha-glycerophosphorylethanolamine. UTP made up (1/5) of the total nucleotide triphosphate content. The composition was compared to the (31)P spectrum of an extract from a human astrocytoma grown in athymic mice. The signal from P-containing macromolecules such as nucleic acids was not detected in the intact HeLa cell spectrum because of broad lines. Effects of the glycolysis inhibitor iodoacetic acid could be clearly shown in spectra of both the intact cell and the extract as buildup of fructose 1,6-bisphosphate at the expense of ATP, UTP, and creatine phosphate.

  4. Exposure to TiO2 nanoparticles increases Staphylococcus aureus infection of HeLa cells.

    Science.gov (United States)

    Xu, Yan; Wei, Ming-Tzo; Ou-Yang, H Daniel; Walker, Stephen G; Wang, Hong Zhan; Gordon, Chris R; Guterman, Shoshana; Zawacki, Emma; Applebaum, Eliana; Brink, Peter R; Rafailovich, Miriam; Mironava, Tatsiana

    2016-04-22

    Titanium dioxide (TiO2) is one of the most common nanoparticles found in industry ranging from food additives to energy generation. Approximately four million tons of TiO2 particles are produced worldwide each year with approximately 3000 tons being produced in nanoparticulate form, hence exposure to these particles is almost certain. Even though TiO2 is also used as an anti-bacterial agent in combination with UV, we have found that, in the absence of UV, exposure of HeLa cells to TiO2 nanoparticles significantly increased their risk of bacterial invasion. HeLa cells cultured with 0.1 mg/ml rutile and anatase TiO2 nanoparticles for 24 h prior to exposure to bacteria had 350 and 250 % respectively more bacteria per cell. The increase was attributed to bacterial polysaccharides absorption on TiO2 NPs, increased extracellular LDH, and changes in the mechanical response of the cell membrane. On the other hand, macrophages exposed to TiO2 particles ingested 40 % fewer bacteria, further increasing the risk of infection. In combination, these two factors raise serious concerns regarding the impact of exposure to TiO2 nanoparticles on the ability of organisms to resist bacterial infection.

  5. Regulation of Chemokine Production via Oxidative Pathway in HeLa Cells

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    Shinichiro Kina

    2009-01-01

    Full Text Available Inflammation is associated with disease progression and, by largely unknown mechanisms, has been said to drive oncogenesis. At inflamed sites, neutrophils deploy a potent antimicrobial arsenal that includes proteinases, antimicrobial peptides, and ROS. Reactive oxygen species (ROSs induce chemokines. In the present study, the concentrations of IL-8 in culture supernatants of HeLa cells treated with ROS were determined by enzyme-linked immunosorbent assay. We used -phenanthroline to deplete Fe2+ in order to investigate the mechanisms through which ROSs induce IL-8 secretion in our system. The iron chelator -phenanthroline effectively inhibited H2O2-induced ERK2 activation. Enzyme-linked immunosorbent assays showed that IL-8 protein secretion was elevated in ROS-treated HeLa cells. When Fe2+ was removed from these cells, IL-8 secretion was inhibited. Collectively, these results indicate that Fe2+-mediated Erk pathway activation is an important signal transduction pathway in ROS-induced IL-8 secretion in epithelial cells.

  6. Metabolism of HeLa cells revealed through autofluorescence lifetime upon infection with enterohemorrhagic Escherichia coli

    Science.gov (United States)

    Buryakina, Tatyana Yu.; Su, Pin-Tzu; Syu, Wan-Jr; Allen Chang, C.; Fan, Hsiu-Fang; Kao, Fu-Jen

    2012-10-01

    Fluorescence lifetime imaging microscopy (FLIM) is a sensitive technique in monitoring functional and conformational states of nicotinamide adenine dinucleotide reduced (NADH) and flavin adenine dinucleotide (FAD),main compounds participating in oxidative phosphorylation in cells. In this study, we have applied FLIM to characterize the metabolic changes in HeLa cells upon bacterial infection and made comparison with the results from the cells treated with staurosporine (STS), a well-known apoptosis inducer. The evolving of NADH's average autofluorescence lifetime during the 3 h after infection with enterohemorragic Escherichia coli (EHEC) or STS treatment has been observed. The ratio of the short and the long lifetime components' relative contributions of NADH increases with time, a fact indicating cellular metabolic activity, such as a decrease of oxidative phosphorylation over the course of infection, while opposite dynamics is observed in FAD. Being associated with mitochondria, FAD lifetimes and redox ratio could indicate heterogeneous mitochondrial function, microenvironment with bacterial infection, and further pathway to cell death. The redox ratios for both EHEC-infected and STS-treated HeLa cells have been observed and these observations also indicate possible apoptosis induced by bacterial infection.

  7. Growth inhibition of HeLa cell by internalization of Mycobacterium bovis Bacillus Calmette-Guérin (BCG Tokyo

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    Asahina Izumi

    2009-12-01

    Full Text Available Abstract Background Intravesical BCG immunotherapy is effective for preventing recurrence and progression in none muscle-invasive bladder cancer but the dosing schedule and duration of treatment remain empirical. The mechanisms by which intravesical BCG treatment mediates antitumor activity are currently poorly understood. Results HeLa cell infected with Mycobacterium bovis Bacillus Calmette-Guérin(BCG Tokyo which were different multiplicity of infection(MOI. Proliferation of HeLa cell reduced in a dose-dependent manner by live BCG. The cytoplasm of the HeLa cell showed variety lysosomal stages by internalized and interacted BCG. Conclusion Proliferated Live BCG secreted the protein and depressed the growth of tumor. The possibility for clinical introduction of BCG therapy for carcinoma reported with review of literature.

  8. Primordial oscillations in life: Direct observation of glycolytic oscillations in individual HeLa cervical cancer cells

    Science.gov (United States)

    Amemiya, Takashi; Shibata, Kenichi; Itoh, Yoshihiro; Itoh, Kiminori; Watanabe, Masatoshi; Yamaguchi, Tomohiko

    2017-10-01

    We report the first direct observation of glycolytic oscillations in HeLa cervical cancer cells, which we regard as primordial oscillations preserved in living cells. HeLa cells starved of glucose or both glucose and serum exhibited glycolytic oscillations in nicotinamide adenine dinucleotide (NADH), exhibiting asynchronous intercellular behaviors. Also found were spatially homogeneous and inhomogeneous intracellular NADH oscillations in the individual cells. Our results demonstrate that starved HeLa cells may be induced to exhibit glycolytic oscillations by either high-uptake of glucose or the enhancement of a glycolytic pathway (Crabtree effect or the Warburg effect), or both. Their asynchronous collective behaviors in the oscillations were probably due to a weak intercellular coupling. Elucidation of the relationship between the mechanism of glycolytic dynamics in cancer cells and their pathophysiological characteristics remains a challenge in future.

  9. γ-Tocotrienol Inhibits Proliferation and Induces Apoptosis via the Mitochondrial Pathway in Human Cervical Cancer HeLa Cells

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    Weili Xu

    2017-08-01

    Full Text Available γ-Tocotrienol, a kind of isoprenoid phytochemical, has antitumor activity. However, there is limited evidence that it has an effect on cervical cancer. In this study, the capacity to inhibit proliferation and induce apoptosis in human cervical cancer HeLa cells and the mechanism underlying these effects were examined. The results indicated that a γ-tocotrienol concentration over 30 μM inhibited the growth of HeLa cells with a 50% inhibitory concentration (IC50 of 46.90 ± 3.50 μM at 24 h, and significantly down-regulated the expression of proliferative cell nuclear antigen (PCNA and Ki-67. DNA flow cytometric analysis indicated that γ-tocotrienol arrested the cell cycle at G0/G1 phase and reduced the S phase in HeLa cells. γ-tocotrienol induced apoptosis of HeLa cells in a time- and dose-dependent manner. γ-tocotrienol-induced apoptosis in HeLa cells was accompanied by down-regulation of Bcl-2, up-regulation of Bax, release of cytochrome from mitochondria, activation of caspase-9 and caspase-3, and subsequent poly (ADP-ribose polymerase (PARP cleavage. These results suggested that γ-tocotrienol could significantly inhibit cell proliferation through G0/G1 cell cycle arrest, and induce apoptosis via the mitochondrial apoptotic pathway in human cervical cancer HeLa cells. Thus, our findings revealed that γ-tocotrienol may be considered as a potential agent for cervical cancer therapy.

  10. Hesperidin inhibits HeLa cell proliferation through apoptosis mediated by endoplasmic reticulum stress pathways and cell cycle arrest.

    Science.gov (United States)

    Wang, Yaoxian; Yu, Hui; Zhang, Jin; Gao, Jing; Ge, Xin; Lou, Ge

    2015-10-12

    Hesperidin (30, 5, 9-dihydroxy-40-methoxy-7-orutinosyl flavanone) is a flavanone that is found mainly in citrus fruits and has been shown to have some anti-neoplastic effects. The aim of the present study was to investigate the effect of hesperidin on apoptosis in human cervical cancer HeLa cells and to identify the mechanism involved. Cells were treated with hesperidin (0, 20, 40, 60, 80, and 100 μM) for 24, 48, or 72 h and relative cell viability was assessed using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Hesperidin inhibited the proliferation of HeLa cells in a concentration- and time-dependent manner. Hesperidin-induced apoptosis in HeLa cells was characterized by increased nuclear condensation and DNA fragmentation. Furthermore, increased levels of GADD153/CHOP and GRP78 indicated hesperidin-induced apoptosis in HeLa cells involved a caspase-dependent pathway, presumably downstream of the endoplasmic reticulum stress pathway. Both of these proteins are hallmarks of endoplasmic reticulum stress. Hesperidin also promoted the formation of reactive oxygen species, mobilization of intracellular Ca(2+), loss of mitochondrial membrane potential (ΔΨm), increased release of cytochrome c and apoptosis-inducing factor from mitochondria, and promoted capase-3 activation. It also arrested HeLa cells in the G0/G1 phase in the cell cycle by downregulating the expression of cyclinD1, cyclinE1, and cyclin-dependent kinase 2 at the protein level. The effect of hesperidin was also verified on the human colon cancer cell HT-29 cells. We concluded that hesperidin inhibited HeLa cell proliferation through apoptosis involving endoplasmic reticulum stress pathways and cell cycle arrest.

  11. Nicotinamide induces mitochondrial-mediated apoptosis through oxidative stress in human cervical cancer HeLa cells.

    Science.gov (United States)

    Feng, Yi; Wang, Yonghua; Jiang, Chengrui; Fang, Zishui; Zhang, Zhiqiang; Lin, Xiaoying; Sun, Liwei; Jiang, Weiying

    2017-07-15

    Nicotinamide participates in energy metabolism and influences cellular redox status and modulates multiple pathways related with both cellular survival and death. Recent studies have shown that it induced proliferation inhibition and apoptosis in many cancer cells. However, little is known about the effects of nicotinamide on human cervical cancer cells. We aimed to evaluate the effects of the indicated concentrations nicotinamide on cell proliferation, apoptosis and redox-related parameters in HeLa cells and investigated the apoptotic mechanism. After the treatment of the indicated concentrations nicotinamide, HeLa cell proliferation was evaluated by the CCK-8 assay and the production of ROS (reactive oxygen species) was measured using 2',7'-Dichlorofluorescin diacetate. The apoptotic effect was confirmed by observing the cellular and nuclear morphologies with fluorescence microscope and apoptotic rate of HeLa cell apoptosis was measured by flow cytometry using Annexin-V method. Moreover, we examined the mitochondrial membrane potential by JC-1 method and measured the expression of apoptosis related genes using qRT-PCR and immunoblotting. Nicotinamide restrained the HeLa cell proliferation and significantly increased the accumulation of ROS and depletion of GSH at relatively high concentrations. Furthermore, nicotinamide promoted HeLa cell apoptosis via the intrinsic mitochondrial apoptotic pathway. Our study revealed that nicotinamide induced the apoptosis through oxidative stress and intrinsic mitochondrial apoptotic pathways in HeLa cell. The results emerge that nicotinamide may be an inexpensive, safe and promising therapeutic agent or a neoadjuvant chemotherapy for cervical cancer patients, as well useful to find new drugs for cervical cancer therapy. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. Poly(3-hydroxybutyrate)/caffeic acid electrospun fibrous materials coated with polyelectrolyte complex and their antibacterial activity and in vitro antitumor effect against HeLa cells

    Energy Technology Data Exchange (ETDEWEB)

    Ignatova, Milena G. [Laboratory of Bioactive Polymers, Institute of Polymers, Bulgarian Academy of Sciences, Acad. G. Bonchev St, Bl. 103A, BG-1113 Sofia (Bulgaria); Manolova, Nevena E., E-mail: manolova@polymer.bas.bg [Laboratory of Bioactive Polymers, Institute of Polymers, Bulgarian Academy of Sciences, Acad. G. Bonchev St, Bl. 103A, BG-1113 Sofia (Bulgaria); Rashkov, Iliya B. [Laboratory of Bioactive Polymers, Institute of Polymers, Bulgarian Academy of Sciences, Acad. G. Bonchev St, Bl. 103A, BG-1113 Sofia (Bulgaria); Markova, Nadya D. [Institute of Microbiology, Bulgarian Academy of Sciences, Acad. G. Bonchev Bl. 26, BG-1113 Sofia (Bulgaria); Toshkova, Reneta A.; Georgieva, Ani K.; Nikolova, Elena B. [Institute of Experimental Morphology, Pathology and Anthropology with Museum, Bulgarian Academy of Sciences, Acad. G. Bonchev St, bl. 25, BG-1113 Sofia (Bulgaria)

    2016-08-01

    The purpose of this work was to investigate the possibility for the preparation of new poly(3-hydroxybutyrate) (PHB)/poly(ethylene glycol) (PEG)-based fibrous materials containing natural phenolic compound caffeic acid (CA) of diverse architectures, as well as to study the impact of the fiber composition on the in vitro CA release profile and on the biological properties of the fibrous materials. The application of the one-pot electrospinning enabled the fabrication of nanofibrous materials from PHB and PEG loaded with the CA. Materials with targeted design were obtained by coating with polyelectrolyte complex of alginate (Alg) and N,N,N-trimethylchitosan (TMCh). Three different processing paths were used to obtain coated mats: (i) with CA incorporated in the PHB/PEG core; (ii) with CA embedded in the Alg layer; and (iii) with CA included in the TMCh layer. The in vitro release of CA was modulated by controlling the composition and the architecture of the nanofibrous mats. The performed microbiological screening and MTT cell viability studies revealed that in contrast to the bare mats, the CA-containing nanofibrous materials were effective in suppressing the growth of the Gram-positive bacteria Staphylococcus aureus and the Gram-negative bacteria Escherichia coli and displayed good cytotoxicity against human cervical HeLa tumor cells. In addition, the proliferation of murine spleen lymphocytes and peritoneal macrophages was increased by the prepared CA-containing nanofibrous materials. The obtained materials are promising for antibacterial wound dressing applications as well as for application in local treatment of cervical tumors. - Highlights: • New caffeic acid-loaded materials from PHB and PEG were prepared by electrospinning. • Different design is achieved by coating and formation of polyelectrolyte complexes. • The control on the architecture of the mats enables modulating caffeic acid release. • The caffeic acid-loaded mats suppress the growth of

  13. Mobility of tethering factor EEA1 on endosomes is decreased upon stimulation of EGF receptor endocytosis in HeLa cells

    Energy Technology Data Exchange (ETDEWEB)

    Kosheverova, Vera V., E-mail: kosheverova_vera@incras.ru [Institute of Cytology of RAS, 4, Tikhoretsky Ave, St. Petersburg, 194064 (Russian Federation); Kamentseva, Rimma S., E-mail: rkamentseva@yandex.ru [Institute of Cytology of RAS, 4, Tikhoretsky Ave, St. Petersburg, 194064 (Russian Federation); St. Petersburg State University, 7-9, Universitetskaya nab, St. Petersburg, 199034 (Russian Federation); Gonchar, Ilya V., E-mail: ample@mail.ru [Institute of Cytology of RAS, 4, Tikhoretsky Ave, St. Petersburg, 194064 (Russian Federation); Kharchenko, Marianna V., E-mail: mariannakharchenko@gmail.com [Institute of Cytology of RAS, 4, Tikhoretsky Ave, St. Petersburg, 194064 (Russian Federation); Kornilova, Elena S., E-mail: lenkor@mail.cytspb.rssi.ru [Institute of Cytology of RAS, 4, Tikhoretsky Ave, St. Petersburg, 194064 (Russian Federation); St. Petersburg State University, 7-9, Universitetskaya nab, St. Petersburg, 199034 (Russian Federation); Department of Medical Physics, Peter the Great St. Petersburg Polytechnic University, 29, Polytechnicheskaya, St.Petersburg, 195251 (Russian Federation)

    2016-04-22

    Tethering factor EEA1, mediating homotypic fusion of early endosomes, was shown to be localized in membrane-bound state both in serum-deprived and stimulated for EGF receptor endocytosis cells. However, it is not known whether dynamics behavior of EEA1 is affected by EGF stimulation. We investigated EEA1 cytosol-to-membrane exchange rate in interphase HeLa cells by FRAP analysis. The data obtained fitted two-states binding model, with the bulk of membrane-associated EEA1 protein represented by the mobile fraction both in serum-starved and EGF-stimulated cells. Fast recovery state had similar half-times in the two cases: about 1.6 s and 2.8 s, respectively. However, the recovery half-time of slowly cycled EEA1 fraction significantly increased in EGF-stimulated comparing to serum-starved cells (from 21 to 99 s). We suppose that the retardation of EEA1 fluorescence recovery upon EGF-stimulation may be due to the increase of activated Rab5 on endosomal membranes, the growth of the number of tethering events between EEA1-positive vesicles and their clustering. - Highlights: • EEA1 mobility was compared in serum-starved and EGF-stimulated interphase HeLa cells. • FRAP analysis revealed fast and slow components of EEA1 recovery in both cases. • Stimulation of EGFR endocytosis did not affect fast EEA1 turnover. • EGF stimulation significantly increased half-time of slowly exchanged EEA1 fraction.

  14. Mechanistic aspects of fluorescent gold nanocluster internalization by live HeLa cells

    Science.gov (United States)

    Yang, Linxiao; Shang, Li; Nienhaus, G. Ulrich

    2013-01-01

    We have studied cellular uptake of ultrasmall fluorescent gold nanoclusters (AuNCs) by HeLa cells by confocal fluorescence microscopy in combination with quantitative image analysis. Water solubilized, lipoic acid-protected AuNCs, which had an overall hydrodynamic diameter of 3.3 nm and emitted fluorescence in the near-infrared region at ~700 nm, were observed to accumulate on the cell membrane prior to internalization. The internalization mechanisms were analyzed using inhibitors known to interfere with specific pathways. Cellular uptake of AuNCs is energy-dependent and involves multiple mechanisms: clathrin-mediated endocytosis and macropinocytosis appear to play a significant role, whereas the caveolin-mediated pathway contributes only to a lesser extent. Co-labeling of different cell organelles showed that intracellular trafficking of AuNCs mainly follows through endosomal pathways. The AuNCs were ultimately transferred to lysosomes; they were completely excluded from the nucleus even after 24 h.We have studied cellular uptake of ultrasmall fluorescent gold nanoclusters (AuNCs) by HeLa cells by confocal fluorescence microscopy in combination with quantitative image analysis. Water solubilized, lipoic acid-protected AuNCs, which had an overall hydrodynamic diameter of 3.3 nm and emitted fluorescence in the near-infrared region at ~700 nm, were observed to accumulate on the cell membrane prior to internalization. The internalization mechanisms were analyzed using inhibitors known to interfere with specific pathways. Cellular uptake of AuNCs is energy-dependent and involves multiple mechanisms: clathrin-mediated endocytosis and macropinocytosis appear to play a significant role, whereas the caveolin-mediated pathway contributes only to a lesser extent. Co-labeling of different cell organelles showed that intracellular trafficking of AuNCs mainly follows through endosomal pathways. The AuNCs were ultimately transferred to lysosomes; they were completely excluded

  15. Monitoring the elasticity changes of HeLa cells during mitosis by atomic force microscopy

    Science.gov (United States)

    Jiang, Ningcheng; Wang, Yuhua; Zeng, Jinshu; Ding, Xuemei; Xie, Shusen; Yang, Hongqin

    2016-10-01

    Cell mitosis plays a crucial role in cell life activity, which is one of the important phases in cell division cycle. During the mitosis, the cytoskeleton micro-structure of the cell changed and the biomechanical properties of the cell may vary depending upon different mitosis stages. In this study, the elasticity property of HeLa cells during mitosis was monitored by atomic force microscopy. Also, the actin filaments in different mitosis stages of the cells were observed by confocal imaging. Our results show that the cell in anaphase is stiffer than that in metaphase and telophase. Furthermore, lots of actin filaments gathered in cells' center area in anaphase, which contributes to the rigidity of the cell in this phase. Our findings demonstrate that the nano-biomechanics of living cells could provide a new index for characterizing cell physiological states.

  16. Primary cilia found on HeLa and other cancer cells.

    Science.gov (United States)

    Kowal, Tia J; Falk, Matthias M

    2015-11-01

    For many years now, researchers have known of a sensory appendage on the surface of most differentiated cell types called primary cilium. Primary cilia are both chemo- and mechano-sensory in function and have an obvious role in cell cycle control. Because of this, it has been thought that primary cilia are not found on rapidly proliferating cells, for example, cancer cells. Here we report using immunofluorescent staining for the ciliary protein Arl13b that primary cilia are frequently found on HeLa (human epithelial adenocarcinoma) and other cancer cell lines such as MG63 (human osteosarcoma) commonly used for cell culture studies and that the ciliated population is significantly higher (ave. 28.6% and 46.5%, respectively in starved and 15.7-18.6% in un-starved cells) than previously anticipated. Our finding impacts the current perception of primary cilia formed in highly proliferative cells. © 2015 International Federation for Cell Biology.

  17. Evaluation of the effects of paederus beetle extract and gamma irradiation on HeLa cells

    Directory of Open Access Journals (Sweden)

    Fariba Samani

    2014-04-01

    Full Text Available Objective(s:Cervical cancer is a malignancy that is the second most common cause of death from cancer in women throughout the world. Paederus beetle (Paederus fuscipes extract (PBE, contains bioactive compounds such as pederine which has cytotoxic properties and blocks DNA and protein synthesis at very low concentrations. In this investigation we tried to determine the effects co-treatment with PBE and gamma irradiation on HeLa cells. Materials and Methods: The viability of the cells was measured by two methods: MTT and Colony assay. Results: We found that supplementing gamma irradiation therapy with PBE does not increase cell death and it might even interfere with its cytotoxicty at the concentrations below 0.1 ng/ml and the viability for irradiation vs irradiation + PBE was 37%: 60%.   Conclusion: This finding might be due to radioprotective effects of the very low doses of PBE against gamma radiation.

  18. The pro-survival function of p53 in HeLa cells

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Jin Kyu; Kang, Mi Young; Jang, Eun Yeong; Kim, Jin Hong [Korea Atomic Energy Research Institute, Advanced Radiation Technology Institute, Jeongeup (Korea, Republic of)

    2014-11-15

    The rate of apoptosis and autophagy was variable with different p53 status after IR treatment of cells. The influence of p53 status on cell fate suggests a role of p53 in two fundamentally important cell biological pathways: autophagy and apoptosis. p53 coordinates cell cycle arrest and apoptosis to govern cell fate. This study was done to identify p53-mediated regulation of cell's fate. Autophagy induced by IR may prevent cells from undergoing apoptosis, implying an interlink modulation between autophagy and apoptosis. The rate of apoptosis and autophagy was determined with different p53 status after IR treatment of HeLa cells in this study. Our research on IR-induced cellular responses may provide new information about fate decision between the processes of apoptosis and autophagy.

  19. How valine deprivation and its reversal affect fatty acid metabolism in HeLa cells.

    Science.gov (United States)

    Slayback, J R; Campbell, I M

    1976-10-21

    A protocol based on radio gas chromatography demonstrates that when HeLa cells are deprived of valine for short periods of time (6-7.5 h), their overall fatty acid biosynthetic activity is depressed after a latency of a few hours. The transfer of newly synthesized fatty acyl units to phospholipids is curtailed much faster than their transfer to triacyglycerols. Despite the cut-back in fatty acid biosynthesis, valine deprivation causes a lipid accumulation in the cells. Valine deprivation appears to affect de novo synthesis of fatty acid units from acetate more rapidly than desaturation and elongation. When valine is returned to the valine-deprived culture, overall fatty acid biosynthesis is resumed well within 2 h. Newly synthesized fatty acyl units are transferred to both the phospholipids and the 1,2-diacylglycerols of the cells but not initially to the triacylglycerols.

  20. Branched dimerization of Tat peptide improves permeability to HeLa and hippocampal neuronal cells.

    Science.gov (United States)

    Monreal, I Abrrey; Liu, Qian; Tyson, Katherine; Bland, Tyler; Dalisay, Doralyn S; Adams, Erin V; Wayman, Gary A; Aguilar, Hector C; Saludes, Jonel P

    2015-03-28

    A dimeric branched peptide TATp-D designed as an analogue of the HIV-Tat protein transduction domain (TATp), a prototypical cell penetrating peptide (CPP), demonstrates significantly enhanced cell uptake at 0.25 to 2.5 μM. Live cell confocal laser scanning microscopy revealed that multivalency dramatically improved the permeation potency of TATp-D to HeLa and primary hippocampal neuronal cells. The observed enhanced ability of TATp-D to translocate through the membrane is highlighted by a non-linear dependence on concentration, exhibiting the greatest uptake at sub-micromolar concentrations as compared to TATp. Multimerization via bis-Fmoc Lysine offered a synthetically straightforward method to investigate the effects of multivalent CPPs while offering orthogonal handles for cargo attachment, increasing the utility of CPPs at significantly lower concentrations.

  1. Short-term desensitization of the histamine H1 receptor in human HeLa cells : involvement of protein kinase C dependent and independent pathways

    NARCIS (Netherlands)

    Smit, M J; Bloemers, S M; Leurs, R; Tertoolen, L G; Bast, A; de Laat, S W; Timmerman, H

    1992-01-01

    1. In this study we have investigated the effects of short-term exposure of cells to histamine on the subsequent H1 receptor responsiveness in HeLa cells, using Ca2+ fluorescence microscopy and video digital imaging. 2. In HeLa cells, histamine (100 microM) induces an immediate H1 receptor-mediated

  2. Acid stress suggests different determinants for polystyrene and HeLa cell adhesion in Lactobacillus casei.

    Science.gov (United States)

    Haddaji, N; Khouadja, S; Fdhila, K; Krifi, B; Ben Ismail, M; Lagha, R; Bakir, K; Bakhrouf, A

    2015-07-01

    Adhesion has been regarded as one of the basic features of probiotics. The aim of this study was to investigate the influence of acid stress on the functional properties, such as hydrophobicity, adhesion to HeLa cells, and composition of membrane fatty acids, of Lactobacillus probiotics strains. Two strains of Lactobacillus casei were used. Adhesion on polystyrene, hydrophobicity, epithelial cells adhesion, and fatty acids analysis were evaluated. Our results showed that the membrane properties such as hydrophobicity and fatty acid composition of stressed strains were significantly changed with different pH values. However, we found that acid stress caused a change in the proportions of unsaturated and saturated fatty acid. The ratio of saturated fatty acid to unsaturated fatty acids observed in acid-stressed Lactobacillus casei cells was significantly higher than the ration in control cells. In addition, we observed a significant decrease in the adhesion ability of these strains to HeLa cells and to a polystyrene surface at low pH. The present finding could first add new insight about the acid stress adaptation and, thus, enable new strategies to be developed aimed at improving the industrial performance of this species under acid stress. Second, no relationship was observed between changes in membrane composition and fluidity induced by acid treatment and adhesion to biotic and abiotic surfaces. In fact, the decrease of cell surface hydrophobicity and the adhesion ability to abiotic surface and the increase of the capacity of adhesion to biotic surface demonstrate that adhesive characteristics will have little relevance in probiotic strain-screening procedures. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  3. Yeast CUP1 protects HeLa cells against copper-induced stress

    Energy Technology Data Exchange (ETDEWEB)

    Xie, X.X. [Department of Animal Sciences, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai (China); Shanghai Key Laboratory of Veterinary Biotechnology, Shanghai (China); College of Biological and Environmental Engineering, Zhejiang University of Technology, Hangzhou (China); Ma, Y.F.; Wang, Q.S.; Chen, Z.L.; Liao, R.R.; Pan, Y.C. [Department of Animal Sciences, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai (China); Shanghai Key Laboratory of Veterinary Biotechnology, Shanghai (China)

    2015-06-12

    As an essential trace element, copper can be toxic in mammalian cells when present in excess. Metallothioneins (MTs) are small, cysteine-rich proteins that avidly bind copper and thus play an important role in detoxification. YeastCUP1 is a member of the MT gene family. The aim of this study was to determine whether yeast CUP1 could bind copper effectively and protect cells against copper stress. In this study,CUP1 expression was determined by quantitative real-time PCR, and copper content was detected by inductively coupled plasma mass spectrometry. Production of intracellular reactive oxygen species (ROS) was evaluated using the 2',7'-dichlorofluorescein-diacetate (DCFH-DA) assay. Cellular viability was detected using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and the cell cycle distribution of CUP1 was analyzed by fluorescence-activated cell sorting. The data indicated that overexpression of yeast CUP1 in HeLa cells played a protective role against copper-induced stress, leading to increased cellular viability (P<0.05) and decreased ROS production (P<0.05). It was also observed that overexpression of yeast CUP1 reduced the percentage of G1 cells and increased the percentage of S cells, which suggested that it contributed to cell viability. We found that overexpression of yeast CUP1 protected HeLa cells against copper stress. These results offer useful data to elucidate the mechanism of the MT gene on copper metabolism in mammalian cells.

  4. Deformation-based nuclear morphometry: capturing nuclear shape variation in HeLa cells.

    Science.gov (United States)

    Rohde, Gustavo K; Ribeiro, Alexandre J S; Dahl, Kris N; Murphy, Robert F

    2008-04-01

    The empirical characterization of nuclear shape distributions is an important unsolved problem with many applications in biology and medicine. Numerous genetic diseases and cancers have alterations in nuclear morphology, and methods for characterization of morphology could aid in both diagnoses and fundamental understanding of these disorders. Automated approaches have been used to measure features related to the size and shape of the cell nucleus, and statistical analysis of these features has often been performed assuming an underlying Euclidean (linear) vector space. We discuss the difficulties associated with the analysis of nuclear shape in light of the fact that shape spaces are nonlinear, and demonstrate methods for characterizing nuclear shapes and shape distributions based on spatial transformations that map one nucleus to another. By combining large deformation metric mapping with multidimensional scaling we offer a flexible approach for elucidating the intrinsic nonlinear degrees of freedom of a distribution of nuclear shapes. More specifically, we demonstrate approaches for nuclear shape interpolation and computation of mean nuclear shape. We also provide a method for estimating the number of free parameters that contribute to shape as well as an approach for visualizing most representative shape variations within a distribution of nuclei. The proposed methodology can be completely automated, is independent of the dimensionality of the images, and can handle complex shapes. Results obtained by analyzing two sets of images of HeLa cells are shown. In addition to identifying the modes of variation in normal HeLa nuclei, the effects of lamin A/C on nuclear morphology are quantitatively described. (c) 2007 International Society for Analytical Cytology.

  5. Magnetic capture of polydopamine-encapsulated Hela cells for the analysis of cell surface proteins.

    Science.gov (United States)

    Liu, Yiying; Yan, Guoquan; Gao, Mingxia; Zhang, Xiangmin

    2017-10-16

    A novel method to characterize cell surface proteins and complexes has been developed. Polydopamine (PDA)-encapsulated Hela cells were prepared for plasma membrane proteome research. Since the PDA protection, the encapsulated cells could be maintained for more than two weeks. Amino groups functionalized magnetic nanoparticles were also used for cell capture by the reaction with the PDA coatings. Plasma membrane fragments were isolated and enriched with assistance of an external magnetic field after disruption of the coated cells by ultrasonic treatment. Plasma membrane proteins (PMPs) and complexes were well preserved on the fragments and identified by shot-gun proteomic analytical strategy. 385 PMPs and 1411 non-PMPs were identified using the method. 85.2% of these PMPs were lipid-raft associated proteins. Ingenuity Pathway Analysis was employed for bio-information extraction from the identified proteins. It was found that 653 non-PMPs had interactions with 140 PMPs. Among them, epidermal growth factor receptor and its complexes, and a series of important pathways including STAT3 pathway were observed. All these results demonstrated that the new approach is of great importance in applying to the research of physiological function and mechanism of the plasma membrane proteins. This work developed a novel strategy for the proteomic analysis of cell surface proteins. According to the results, 73.3% of total identified proteins were lipid-raft associated proteins, which imply that the proposed method is of great potential in the identification of lipid-raft associated proteins. In addition, a series of protein-protein interactions and pathways related to Hela cells were pointed out. All these results demonstrated that our proposed approach is of great importance and could well be applied to the physiological function and mechanism research of plasma membrane proteins. Copyright © 2017. Published by Elsevier B.V.

  6. Polyketide Derivatives from Annona muricata Linn Leaves as Potencial Anticancer Material by Combination Treatment With Doxorubicin on Hela Cell Line

    Science.gov (United States)

    Artanti, A. N.; Astirin, O. P.; Prayito, A.; Widiyaningsih, R. F.; Prihapsara, F.

    2017-02-01

    One of the compounds found effication as an anticancer agent on cervical cancer is acetogenin, a polyketide compound that is abundant in Annona muricata L. leaves. This study has been done to examine polyketide derivatives was isolated from Annona muricata L. which has potency to induce apoptosis by p53 expression on hela cell line. An approach recently develop to overcome side effect of chemoterapeutic agent is used of combined chemoterapeutic agent, i.e doxorubicin. The determination of cytotoxic combination activity from polyketide derivative and doxorubicin was evaluated using MTT assay to obtain the value of CI (combination index). The expression of p53 profile was evaluated by immunohistochemistry on hela cell line. Data analysis showed that combination of polyketide derivative from Annona muricata L. (38,5 µg/ml) and doxorubicin with all of concentration performed synergistic effect on hela cell line with CI value from 0,33 - 0,65. The analysis on immucytochemistry showed that polyketide derivative from Annona muricata L. leaves could enhance p53 pathway significantly on hela cell line.

  7. Intense picosecond pulsed electric fields induce apoptosis through a mitochondrial-mediated pathway in HeLa cells

    Science.gov (United States)

    HUA, YUAN-YUAN; WANG, XIAO-SHU; ZHANG, YU; YAO, CHEN-GUO; ZHANG, XI-MING; XIONG, ZHENG-AI

    2012-01-01

    The application of pulsed electric fields (PEF) is emerging as a new technique for tumor therapy. Picosecond pulsed electric fields (psPEF) can be transferred to target deep tissue non-invasively and precisely, but the research of the biological effects of psPEF on cells is limited. Electric theory predicts that intense psPEF will target mitochondria and lead to changes in transmembrane potential, therefore, it is hypothesized that it can induce mitochondrial-mediated apoptosis. HeLa cells were exposed to psPEF in this study to investigate this hypothesis. MTT assay demonstrated that intense psPEF significantly inhibited the proliferation of HeLa cells in a dose-dependent manner. Typical characteristics of apoptosis in HeLa cells were observed, using transmission electron microscopy. Loss of mitochondrial transmembrane potential was explored using laser scanning confocal microscopy with Rhodamine-123 (Rh123) staining. Furthermore, the mitochondrial apoptotic events were also confirmed by western blot analysis for the release of cytochrome C and apoptosis-inducing factor from mitochondria into the cytosol. In addition, activation of caspase-3, caspase-9, upregulation of Bax, p53 and downregulation of Bcl-2 were observed in HeLa cells also indicating apoptosis. Taken together, these results demonstrate that intense psPEF induce cell apoptosis through a mitochondrial-mediated pathway. PMID:22307872

  8. Anticancer Activity of a Hexapeptide from Skate (Raja porosa) Cartilage Protein Hydrolysate in HeLa Cells.

    Science.gov (United States)

    Pan, Xin; Zhao, Yu-Qin; Hu, Fa-Yuan; Chi, Chang-Feng; Wang, Bin

    2016-08-16

    In this study, the hexapeptide Phe-Ile-Met-Gly-Pro-Tyr (FIMGPY), which has a molecular weight of 726.9 Da, was separated from skate (Raja porosa) cartilage protein hydrolysate using ultrafiltration and chromatographic methods, and its anticancer activity was evaluated in HeLa cells. Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay indicated that FIMGPY exhibited high, dose-dependent anti-proliferation activities in HeLa cells with an IC50 of 4.81 mg/mL. Acridine orange/ethidium bromide (AO/EB) fluorescence staining and flow cytometry methods confirmed that FIMGPY could inhibit HeLa cell proliferation by inducing apoptosis. Western blot assay revealed that the Bax/Bcl-2 ratio and relative intensity of caspase-3 in HeLa cells treated with 7-mg/mL FIMGPY were 2.63 and 1.83, respectively, significantly higher than those of the blank control (p skate cartilage protein hydrolysate may have applications as functional foods and nutraceuticals for the treatment and prevention of cancer.

  9. Anticancer Activity of a Hexapeptide from Skate (Raja porosa Cartilage Protein Hydrolysate in HeLa Cells

    Directory of Open Access Journals (Sweden)

    Xin Pan

    2016-08-01

    Full Text Available In this study, the hexapeptide Phe-Ile-Met-Gly-Pro-Tyr (FIMGPY, which has a molecular weight of 726.9 Da, was separated from skate (Raja porosa cartilage protein hydrolysate using ultrafiltration and chromatographic methods, and its anticancer activity was evaluated in HeLa cells. Methylthiazolyldiphenyl-tetrazolium bromide (MTT assay indicated that FIMGPY exhibited high, dose-dependent anti-proliferation activities in HeLa cells with an IC50 of 4.81 mg/mL. Acridine orange/ethidium bromide (AO/EB fluorescence staining and flow cytometry methods confirmed that FIMGPY could inhibit HeLa cell proliferation by inducing apoptosis. Western blot assay revealed that the Bax/Bcl-2 ratio and relative intensity of caspase-3 in HeLa cells treated with 7-mg/mL FIMGPY were 2.63 and 1.83, respectively, significantly higher than those of the blank control (p < 0.01. Thus, FIMGPY could induce apoptosis by upregulating the Bax/Bcl-2 ratio and caspase-3 activation. Using a DNA ladder method further confirmed that the anti-proliferation activity of FIMGPY was attributable to its role in inducing apoptosis. These results suggest that FIMGPY from skate cartilage protein hydrolysate may have applications as functional foods and nutraceuticals for the treatment and prevention of cancer.

  10. Extracellular ATP elevates cytoplasmatic free Ca2+ in HeLa cells by the interaction with a 5'-nucleotide receptor

    NARCIS (Netherlands)

    Smit, M J; Leurs, R; Bloemers, S M; Tertoolen, L G; Bast, A; De Laat, S W; Timmerman, H

    1993-01-01

    In the present study we have characterized the effects of ATP and several other nucleotides on the intracellular Ca2+ levels of HeLa cells. Using fura-2 microscopy fluorescence measurements, the ATP-mediated increase in intracellular Ca2+ was shown to consist of a rapid rise which decreased after a

  11. Subnanomolar antisense activity of phosphonate-peptide nucleic acid (PNA) conjugates delivered by cationic lipids to HeLa cells

    DEFF Research Database (Denmark)

    Shiraishi, Takehiko; Hamzavi, Ramin; Nielsen, Peter E

    2008-01-01

    oligomer. This modification of the PNA does not interfere with the nucleic acid target binding affinity based on thermal stability of the PNA/RNA duplexes. When delivered to cultured HeLa pLuc705 cells by Lipofectamine, the PNAs showed dose-dependent nuclear antisense activity in the nanomolar range...

  12. 1.5-NM PROJECTION STRUCTURE OF HELA-CELL PROSOME-MCP (PROTEASOME) PROVIDED BY 2-DIMENSIONAL CRYSTALS

    NARCIS (Netherlands)

    PERKINS, GA; BERGSMASCHUTTER, W; KEEGSTRA, W; ARNBERG, AC; COUX, O; SCHERRER, K

    1994-01-01

    We grew two-dimensional crystals of HeLa cell prosomes, also called multicatalytic proteinases (MCP) and proteasomes, for a structure determination by electron microscopy. The molecules were arranged in side views in these crystals. The crystals have p21 plane group symmetry with one particle per

  13. Effects of Agaricus blazei Murill extract on sensitivity to chemotherapeutic agents in HeLa cells and its resistant sublines.

    Science.gov (United States)

    Takara, Kohji; Shin, Yasunori; Obata, Yukihisa; Kitada, Noriaki; Ohnishi, Noriaki; Yokoyama, Teruyoshi

    2008-01-01

    Agaricus blazei Murill (ABM; Japanese name: Kawahiratake or Agarikusutake) extract is a widely used dietary supplement. However, limited information is available on the effects of the extract on the effectiveness of the chemotherapeutic agents. In this study, we examined the effects of ABM extract (Kyowa Wellness Co., Ltd.) on sensitivity to chemotherapeutic agents, paclitaxel and doxorubicin as MDR1/P-glycoprotein substrates, and cisplatin and 5-fluorouracil as non-substrates, in human cervical carcinoma HeLa cells, and paclitaxel-resistant and cisplatin-resistant derivatives (HeLa/TXL and HeLa/CDDP, respectively). The extract had no growth inhibitory effects on HeLa and the resistant cells at concentrations ranging from 7.6 × 10(-4) μ g/ml to 8.0 × 10(2)μ g/ml, indicating no remarkable cytotoxic activity in vitro. In the presence of 0.1, 0.5, and 1 μ g/ml of ABM extract, sensitivity to paclitaxel, cisplatin and 5-fluorouracil did not change in HeLa, HeLa/TXL and HeLa/CDDP cells. However, the extract reduced sensitivity to doxorubicin in HeLa/TXL and HeLa/CDDP cells in a concentration-dependent manner. In conclusion, the concomitant use of ABM extract minimally affected sensitivity to various chemotherapeutic agents in HeLa cells and resistant sublines in vitro.

  14. HEAT-INDUCED PROTEIN DENATURATION IN THE PARTICULATE FRACTION OF HELA S3 CELLS - EFFECT OF THERMOTOLERANCE

    NARCIS (Netherlands)

    BURGMAN, PWJJ; KONINGS, AWT

    1992-01-01

    In this study we investigated the effect of heat on the proteins of the particulate fraction (PF) of HeLa S3 cells using electron spin resonance (ESR) and thermal gel analysis (TGA). ESR detects overall conformational changes in proteins, while TGA detects denaturation (aggregation due to formation

  15. Cytotoxic Effects of Native and Recombinant Frutalin, a Plant Galactose-Binding Lectin, on HeLa Cervical Cancer Cells

    Directory of Open Access Journals (Sweden)

    Carla Oliveira

    2011-01-01

    Full Text Available Frutalin is the α-D-galactose-binding lectin isolated from breadfruit seeds. Frutalin was obtained from two different sources: native frutalin was purified from its natural origin, and recombinant frutalin was produced and purified from Pichia pastoris. This work aimed to study and compare the effect of native and recombinant frutalin on HeLa cervical cancer cells proliferation and apoptosis. Furthermore, the interaction between frutalin and the HeLa cells was investigated by confocal microscopy. Despite having different carbohydrate-binding affinities, native and recombinant frutalin showed an identical magnitude of cytotoxicity on HeLa cells growth (IC50~100 μg/mL and equally induced cell apoptosis. The interaction studies showed that both lectins were rapidly internalised and targeted to HeLa cell's nucleus. Altogether, these results indicate that frutalin action is not dependent on its sugar-binding properties. This study provides important information about the bioactivity of frutalin and contributes to the understanding of the plant lectins cytotoxic activity.

  16. [Extraction and analysis of the essential oil in Pogostemon cablin by enzymatic hydrolysis and inhibitory activity against Hela cell proliferation].

    Science.gov (United States)

    Yu, Jing; Qi, Yue; Luo, Gang; Duan, Hong-quan; Zhou, Jing

    2012-05-01

    To optimize the extraction method of essential oil in Pogostemon cablin and analyze its inhibitory activity against Hela cell proliferation. The Pogostemon cablin was treated by hemicellulase before steam distillation. The enzyme dosage, treatment time, treatment temperature, pH were optimized through orthogonal experimental design. The components of essential oil were identified by gas chromatography-mass spectrometry (GC-MS). Inhibitory activity of patchouli oil against Hela cell proliferation was determined by MTP method. The optimum extraction process was as follows: pH 4.5, temperature 45 degrees C, the ratio of hemicellulase to Pogostemon cablin was 1% and enzymatic hydrolysis for 1.0 hour. Extraction ratio of the patchouli oil in steam distillation and hemicellulase extraction method was 2.2220 mg/g, 3.1360 mg/g respectively. Patchouli oil could inhibit Hela cell proliferation. IC50 of the patchouli oil in steam distillation and hemicellulase extraction method was 12.2 +/- 0.46 microg/mL and 0.36 +/- 0.03 microg/mL respectively. In comparison with steam distillation method, extraction ratios of essential oil and the inhibitory activity against Hela cell proliferation can be increased by the hemicellulase extraction method.

  17. On Biophysical Properties and Sensitivity to Gap Junction Blockers of Connexin 39 Hemichannels Expressed in HeLa Cells

    Science.gov (United States)

    Vargas, Anibal A.; Cisterna, Bruno A.; Saavedra-Leiva, Fujiko; Urrutia, Carolina; Cea, Luis A.; Vielma, Alex H.; Gutierrez-Maldonado, Sebastian E.; Martin, Alberto J. M.; Pareja-Barrueto, Claudia; Escalona, Yerko; Schmachtenberg, Oliver; Lagos, Carlos F.; Perez-Acle, Tomas; Sáez, Juan C.

    2017-01-01

    Although connexins (Cxs) are broadly expressed by cells of mammalian organisms, Cx39 has a very restricted pattern of expression and the biophysical properties of Cx39-based channels [hemichannels (HCs) and gap junction channels (GJCs)] remain largely unknown. Here, we used HeLa cells transfected with Cx39 (HeLa-Cx39 cells) in which intercellular electrical coupling was not detected, indicating the absence of GJCs. However, functional HCs were found on the surface of cells exposed to conditions known to increase the open probability of other Cx HCs (e.g., extracellular divalent cationic-free solution (DCFS), extracellular alkaline pH, mechanical stimulus and depolarization to positive membrane potentials). Cx39 HCs were blocked by some traditional Cx HC blockers, but not by others or a pannexin1 channel blocker. HeLa-Cx39 cells showed similar resting membrane potentials (RMPs) to those of parental cells, and exposure to DCFS reduced RMPs in Cx39 transfectants, but not in parental cells. Under these conditions, unitary events of ~75 pS were frequent in HeLa-Cx39 cells and absent in parental cells. Real-time cellular uptake experiments of dyes with different physicochemical features, as well as the application of a machine-learning approach revealed that Cx39 HCs are preferentially permeable to molecules characterized by six categories of descriptors, namely: (1) electronegativity, (2) ionization potential, (3) polarizability, (4) size and geometry, (5) topological flexibility and (6) valence. However, Cx39 HCs opened by mechanical stimulation or alkaline pH were impermeable to Ca2+. Molecular modeling of Cx39-based channels suggest that a constriction present at the intracellular portion of the para helix region co-localizes with an electronegative patch, imposing an energetic and steric barrier, which in the case of GJCs may hinder channel function. Results reported here demonstrate that Cx39 form HCs and add to our understanding of the functional roles of Cx39 HCs

  18. Local Foods, Local Places

    Science.gov (United States)

    The Local Foods, Local Places technical assistance program protects human health and the environment, spurs revitalization, increases access to healthy foods, and creates economic opportunities by promoting local foods.

  19. Effects of natural flavones on membrane properties and citotoxicity of HeLa cells

    Directory of Open Access Journals (Sweden)

    Tatiana Herrerias

    Full Text Available The aim of this study was to determine whether eupafolin and hispidulin, flavones extracted from Eupatorium littorale Cabrera, Asteraceae, have the ability to change properties of biological membranes and promote cytotoxic effects. Eupafolin (50-200 µM decreased approximately 30% the rate and total amplitude of valinomycin induced swelling and 60-100% the energy-dependent mitochondrial swelling. Moreover, eupafolin (200 µM reduced 35% the mitochondrial permeability transition, and hispidulin did not change this parameter in any of the doses tested. The evaluation of phase transition of DMPC liposomes with the probe DPH demonstrated that hispidulin and eupafolin affect gel and fluid phase. With mitochondrial membrane as model, hispidulin increased the polarization of fluorescence when used DPH-PA probe. Eupafolin and hispidulin (100 µM promoted a reduction of 40% in cellular viability of HeLa cells in 24 h. Our results suggest that eupafolin and hispidulin have cytotoxic effects that can be explained, in part, by alterations promoted on biological membranes properties and mitochondrial bioenergetics.

  20. Serine integrase chimeras with activity in E. coli and HeLa cells

    Directory of Open Access Journals (Sweden)

    Alfonso P. Farruggio

    2014-09-01

    Full Text Available In recent years, application of serine integrases for genomic engineering has increased in popularity. The factor-independence and unidirectionality of these large serine recombinases makes them well suited for reactions such as site-directed vector integration and cassette exchange in a wide variety of organisms. In order to generate information that might be useful for altering the specificity of serine integrases and to improve their efficiency, we tested a hybridization strategy that has been successful with several small serine recombinases. We created chimeras derived from three characterized members of the serine integrase family, phiC31, phiBT1, and TG1 integrases, by joining their amino- and carboxy-terminal portions. We found that several phiBT1-phiC31 (BC and phiC31-TG1 (CT hybrid integrases are active in E. coli. BC chimeras function on native att-sites and on att-sites that are hybrids between those of the two donor enzymes, while CT chimeras only act on the latter att-sites. A BC hybrid, BC{−1}, was also active in human HeLa cells. Our work is the first to demonstrate chimeric serine integrase activity. This analysis sheds light on integrase structure and function, and establishes a potentially tractable means to probe the specificity of the thousands of putative large serine recombinases that have been revealed by bioinformatics studies.

  1. Effects of Circular DNA Length on Transfection Efficiency by Electroporation into HeLa Cells.

    Science.gov (United States)

    Hornstein, Benjamin D; Roman, Dany; Arévalo-Soliz, Lirio M; Engevik, Melinda A; Zechiedrich, Lynn

    2016-01-01

    The ability to produce extremely small and circular supercoiled vectors has opened new territory for improving non-viral gene therapy vectors. In this work, we compared transfection of supercoiled DNA vectors ranging from 383 to 4,548 bp, each encoding shRNA against GFP under control of the H1 promoter. We assessed knockdown of GFP by electroporation into HeLa cells. All of our vectors entered cells in comparable numbers when electroporated with equal moles of DNA. Despite similar cell entry, we found length-dependent differences in how efficiently the vectors knocked down GFP. As vector length increased up to 1,869 bp, GFP knockdown efficiency per mole of transfected DNA increased. From 1,869 to 4,257 bp, GFP knockdown efficiency per mole was steady, then decreased with increasing vector length. In comparing GFP knockdown with equal masses of vectors, we found that the shorter vectors transfect more efficiently per nanogram of DNA transfected. Our results rule out cell entry and DNA mass as determining factors for gene knockdown efficiency via electroporation. The length-dependent effects we have uncovered are likely explained by differences in nuclear translocation or transcription. These data add an important step towards clinical applications of non-viral vector delivery.

  2. Calculation of HELAS amplitudes for QCD processes using graphics processing unit (GPU)

    Science.gov (United States)

    Hagiwara, K.; Kanzaki, J.; Okamura, N.; Rainwater, D.; Stelzer, T.

    2010-11-01

    We use a graphics processing unit (GPU) for fast calculations of helicity amplitudes of quark and gluon scattering processes in massless QCD. New HEGET ( HELAS Evaluation with GPU Enhanced Technology) codes for gluon self-interactions are introduced, and a C++ program to convert the MadGraph generated FORTRAN codes into HEGET codes in CUDA (a C-platform for general purpose computing on GPU) is created. Because of the proliferation of the number of Feynman diagrams and the number of independent color amplitudes, the maximum number of final state jets we can evaluate on a GPU is limited to 4 for pure gluon processes ( gg→4 g), or 5 for processes with one or more quark lines such as qoverline{q}→ 5g and qq→ qq+3 g. Compared with the usual CPU-based programs, we obtain 60-100 times better performance on the GPU, except for 5-jet production processes and the gg→4 g processes for which the GPU gain over the CPU is about 20.

  3. Cell Cycle Synchronization of HeLa Cells to Assay EGFR Pathway Activation.

    Science.gov (United States)

    Wee, Ping; Wang, Zhixiang

    2017-01-01

    Progression through the cell cycle causes changes in the cell's signaling pathways that can alter EGFR signal transduction. Here, we describe drug-derived protocols to synchronize HeLa cells in various phases of the cell cycle, including G1 phase, S phase, G2 phase, and mitosis, specifically in the mitotic stages of prometaphase, metaphase, and anaphase/telophase. The synchronization procedures are designed to allow synchronized cells to be treated for EGF and collected for the purpose of Western blotting for EGFR signal transduction components.S phase synchronization is performed by thymidine block, G2 phase with roscovitine, prometaphase with nocodazole, metaphase with MG132, and anaphase/telophase with blebbistatin. G1 phase synchronization is performed by culturing synchronized mitotic cells obtained by mitotic shake-off. We also provide methods to validate the synchronization methods. For validation by Western blotting, we provide the temporal expression of various cell cycle markers that are used to check the quality of the synchronization. For validation of mitotic synchronization by microscopy, we provide a guide that describes the physical properties of each mitotic stage, using their cellular morphology and DNA appearance. For validation by flow cytometry, we describe the use of imaging flow cytometry to distinguish between the phases of the cell cycle, including between each stage of mitosis.

  4. Human cytosolic thymidine kinase: purification and physical characterization of the enzyme from HeLa cells

    Energy Technology Data Exchange (ETDEWEB)

    Sherley, J.L.; Kelly, T.J.

    1988-01-05

    The mammalian cytosolic thymidine kinase is one of a number of enzymes involved in DNA replication whose activities increase dramatically during S phase of the cell cycle. As a first step in defining the mechanisms that control the S phase induction of thymidine kinase activity, the authors have purified the human enzyme from HeLa cells and raised a specific immune serum against the purified protein. The enzyme was isolated from cells arrested in S phase by treatment with methotrexate and purified to near homogeneity by ion-exchange and affinity chromatography. Stabilization of the purified enzyme was achieved by the addition of digitonin. An electrophoretic R/sub m/ of 0.2 in nondenaturing gels characterizes the purified enzyme activity as cytosolic thymidine kinase. The enzyme has a Stoke's radius of 40 A determined by gel filtration and a sedimentation coefficient of 5.5 S determined by glycerol gradient sedimentation. Based on these hydrodynamic values, a native molecular weight of 96,000 was calculated for the purified enzyme. When electrophoresed in denaturing sodium dodecyl sulfate-polyacrylamide gels under reducing conditions, the most purified enzyme fraction was found to contain one predominant polypeptide of M/sub r/ = 24,000. Several lines of evidence indicate that this polypeptide is responsible for thymidine kinase enzymatic activity.

  5. Quantitative Proteomic and Interaction Network Analysis of Cisplatin Resistance in HeLa Cells

    Science.gov (United States)

    Chavez, Juan D.; Hoopmann, Michael R.; Weisbrod, Chad R.; Takara, Kohji; Bruce, James E.

    2011-01-01

    Cisplatin along with other platinum based drugs are some of the most widely used chemotherapeutic agents. However drug resistance is a major problem for the successful chemotherapeutic treatment of cancer. Current evidence suggests that drug resistance is a multifactorial problem due to changes in the expression levels and activity of a wide number of proteins. A majority of the studies to date have quantified mRNA levels between drug resistant and drug sensitive cell lines. Unfortunately mRNA levels do not always correlate with protein expression levels due to post-transcriptional changes in protein abundance. Therefore global quantitative proteomics screens are needed to identify the protein targets that are differentially expressed in drug resistant cell lines. Here we employ a quantitative proteomics technique using stable isotope labeling with amino acids in cell culture (SILAC) coupled with mass spectrometry to quantify changes in protein levels between cisplatin resistant (HeLa/CDDP) and sensitive HeLa cells in an unbiased fashion. A total of 856 proteins were identified and quantified, with 374 displaying significantly altered expression levels between the cell lines. Expression level data was then integrated with a network of protein-protein interactions, and biological pathways to obtain a systems level view of proteome changes which occur with cisplatin resistance. Several of these proteins have been previously implicated in resistance towards platinum-based and other drugs, while many represent new potential markers or therapeutic targets. PMID:21637840

  6. Quantitative proteomic and interaction network analysis of cisplatin resistance in HeLa cells.

    Directory of Open Access Journals (Sweden)

    Juan D Chavez

    Full Text Available Cisplatin along with other platinum based drugs are some of the most widely used chemotherapeutic agents. However drug resistance is a major problem for the successful chemotherapeutic treatment of cancer. Current evidence suggests that drug resistance is a multifactorial problem due to changes in the expression levels and activity of a wide number of proteins. A majority of the studies to date have quantified mRNA levels between drug resistant and drug sensitive cell lines. Unfortunately mRNA levels do not always correlate with protein expression levels due to post-transcriptional changes in protein abundance. Therefore global quantitative proteomics screens are needed to identify the protein targets that are differentially expressed in drug resistant cell lines. Here we employ a quantitative proteomics technique using stable isotope labeling with amino acids in cell culture (SILAC coupled with mass spectrometry to quantify changes in protein levels between cisplatin resistant (HeLa/CDDP and sensitive HeLa cells in an unbiased fashion. A total of 856 proteins were identified and quantified, with 374 displaying significantly altered expression levels between the cell lines. Expression level data was then integrated with a network of protein-protein interactions, and biological pathways to obtain a systems level view of proteome changes which occur with cisplatin resistance. Several of these proteins have been previously implicated in resistance towards platinum-based and other drugs, while many represent new potential markers or therapeutic targets.

  7. Characterization of HeLa 5'-nucleotidase: a stable plasma membrane marker

    Energy Technology Data Exchange (ETDEWEB)

    Brake, E.T. (Oak Ridge National Laboratory, TN); Will, P.C.; Cook, J.S.

    1977-11-22

    The enzyme 5'-nucleotidase, assayed as 5'-AMPase, has been extensively characterized and established as a stable, quantitative plasma membrane marker in HeLa S3 cells. The 5'-AMPase has a K/sub m/ of 7.0 ..mu..m. There are activity optima at pH7 and 10; the latter is Mg/sup 2 +/-dependent. The membrane preparations have a small amount of acid phosphatase activity that is distinct from 5'-AMPase activity but no alkaline phosphatase. ADP, ATP and ..cap alpha.., ..beta..-methylene adenosine-5'-diphosphate are strongly inhibitory. Mg/sup 2 +/, Ca/sup 2 +/, or Co/sup 2 +/ do not affect the pH 7.0 activity; Mn/sup 2 +/ activates slightly, whereas Zn/sup 2 +/, Cu/sup 2 +/, and Ni/sup 2 +/ are inhibitory. EDTA slowly inactivates, but removal of the EDTA without the addition of divalent cations restores activity. The inactivation is also substantially reversed by Co/sup 2 +/ or Mn/sup 2 +/. ConA strongly inhibits, and ..cap alpha..-methyl-D-mannoside or glucose relieves the inhibition, indicating that the 5'-AMPase is a glycoprotein. Histidine is also inhibitory. Ouabain, phloretin, cytochalasin B, cysteine, phenylalanine, N-ethylmaleimide, and iodoacetic acid are without effect.

  8. Variation of stemline karyotype in a HeLa cell line.

    Science.gov (United States)

    Ghosh, S; Ghosh, I

    1975-10-27

    100 karyotypes of a HeLa cell line (modal number 69) were studied in details. It was observed that the stemline cells of this "triploid" somatic cell population showed high degree of chromosomal polymorphism. It has been discussed that the stemline cells have number of extra chromosomes (or parts of chromosomes) which are not essential for the genetic integrity of this cell population. A loss of some of these chromosomes (or their parts) does not induce any appreciable change in the genetic make-up of these cells. The broken chromosomes lose their identity and are grouped together with the non-homologues, when the Denver system of classification is followed. However, it has been argued that in absence of any other acceptable system for classification, the Denver system can still be employed for analyzing human chromosomes both at diploid and abnormal heteroploid level. It has been held in contrast to the pseudo-stemline concept that these cells in general, have the essential genes in common and are responsible for the genetic make-up of this cell line and constitute together the stemline of this somatic cell population.

  9. Simultaneous imaging of two initiator caspases during cisplatin-induced HeLa apoptosis

    Science.gov (United States)

    Chu, Jun; Wang, Liang; Luo, Qingming; Zhang, Zhihong

    2008-02-01

    Caspase-2 is an initiating caspase required for stress-induced apoptosis in various human cancer cells. Activation of caspase-9, a key event in stress-mediated apoptosis, also has been shown to be an initiator caspase. However, the timing or activation sequence of these initiator caspases, which trigger apoptotic pathway, is unclear. Here we report caspase-2 and caspase-9 dynamics during cisplatin-induced HeLa apoptosis using Double Fluorescence Resonance Energy Transfer (FRET) technique. Two FRET probes were constructed that each encoded a CRS (caspase-2 or caspase-9 recognition Site) fused with a cyan/yellow fluorescent protein (CFP/YFP) and a red fluorescent protein (DsRed) (CFP/YFP-CRS-DsRed). By using two probes, CFP-C2-DsRed and YFP-C9-DsRed, we carried out simultaneous double-FRET analysis and revealed that activation of caspase-2 had the same time course with caspase-9. These data suggest parallel activation of initiator caspase-2 and caspase-9 in cisplatin-induced cell death.

  10. Targeting Thioredoxin Reductase by Parthenolide Contributes to Inducing Apoptosis of HeLa Cells*

    Science.gov (United States)

    Duan, Dongzhu; Zhang, Junmin; Yao, Juan; Liu, Yaping; Fang, Jianguo

    2016-01-01

    Parthenolide (PTL), a major active sesquiterpene lactone from the herbal plant Tanacetum parthenium, has been applied in traditional Chinese medicine for centuries. Although PTL demonstrates potent anticancer efficacy in numerous types of malignant cells, the cellular targets of PTL have not been well defined. We reported here that PTL interacts with both cytosolic thioredoxin reductase (TrxR1) and mitochondrial thioredoxin reductase (TrxR2), two ubiquitous selenocysteine-containing antioxidant enzymes, to elicit reactive oxygen species-mediated apoptosis in HeLa cells. PTL selectively targets the selenocysteine residue in TrxR1 to inhibit the enzyme function, and further shifts the enzyme to an NADPH oxidase to generate superoxide anions, leading to reactive oxygen species accumulation and oxidized thioredoxin. Under the conditions of inhibition of TrxRs in cells, PTL does not cause significant alteration of cellular thiol homeostasis, supporting selective target of TrxRs by PTL. Importantly, overexpression of functional TrxR1 or Trx1 confers protection, whereas knockdown of the enzymes sensitizes cells to PTL treatment. Targeting TrxRs by PTL thus discloses an unprecedented mechanism underlying the biological activity of PTL, and provides deep insights to understand the action of PTL in treatment of cancer. PMID:27002142

  11. Comparison of the HeLa DNA-synthesis inhibition test and the Ames test for screening of mutagenic carcinogens

    Energy Technology Data Exchange (ETDEWEB)

    Painter, R.B.; Howard, R.

    1978-01-01

    The action of most mutagens is mediated by damage to DNA, which causes at least a temporary inhibition of DNA syntesis in mammalian cells. Assays for mammalian DNA-synthesis inhibition, both in vivo (mouse testes) and in vitro (HeLa cells), have been proposed as possible screening tests for mutagenic carcinogens. The mouse system has recently been chekced with 100 chemicals; of 88 known carcinogens and/or mutagens in this group, 76 were positive. The most generally used non-animal screening procedure is the Ames test, which uses auxotrophic strains of Salmonella typhimurium to measure mutagenesis. In this communication we summarize our results with 19 chemicals tested in HeLa cells and show that they correlate very well with the results obtained in the Ames test. Most of these chemicals act by alkylation, but an intercalator (adriamycin) is included among them as well as aflatoxin B/sub 1/, whose action is not established.

  12. Opposed to the being of Henrietta: bioslavery, pop culture and the third life of HeLa cells.

    Science.gov (United States)

    Moore, Marlon Rachquel

    2017-03-01

    Operating at the intersection of thanatopolitics and African-American cultural studies, this essay argues that the commercial sale of HeLa-themed art and other bioproducts perpetuates the bioslavery of HeLa cells, a circumstance created by legal and medical discourses tracing back to US racial slavery. Racial slavery normalised economic, social and legal inequities that the nation continues to struggle with and, the article posits, laid foundation for the dynamics that currently exist between Henrietta Lacks' genealogical family, the HeLa cell line, and the medical-pharmaceutical establishment. The author turns to fashion ethics discourse and trademark law as potential sites for reparations. 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/.

  13. Quantitative and real-time effects of carbon quantum dots on single living HeLa cell membrane permeability.

    Science.gov (United States)

    Kong, Weiqian; Liu, Juan; Liu, Ruihua; Li, Hao; Liu, Yang; Huang, Hui; Li, Kunyang; Liu, Jian; Lee, Shuit-Tong; Kang, Zhenhui

    2014-05-21

    The interaction between carbon quantum dots (CQDs) and a single living cell was explored in real time. Here, we provide the quantitative data on the permeability of the HeLa cell membrane in the presence of CQDs with different surface functional groups (CQDs terminated with -OH/-COOH (CQD-OH), -PEG (CQD-PEG), and -NH2 (CQD-NH2)). Although these CQDs have very low toxicity towards HeLa cells, they still increase the cell membrane permeability by 8%, 13%, and 19% for CQD-PEG, CQD-OH, and CQD-NH2, respectively, and this kind of permeability was irreversible. These observations are valuable for promoting the bio-applications of carbon nanostructures in living systems.

  14. Anticancer Activity of Certain Herbs and Spices on the Cervical Epithelial Carcinoma (HeLa) Cell Line

    OpenAIRE

    Danielle Berrington; Namrita Lall

    2012-01-01

    Acetone extracts of selected plant species were evaluated for their in vitro cytotoxicity against a noncancerous African green monkey kidney (Vero) cell line and an adenocarcinoma cervical cancer (HeLa) cell line. The plants studied were Origanum vulgare L. (Oregano), Rosmarinus officinalis L. (Upright and ground cove rosemary), Lavandula spica L. (Lavender), Laurus nobilis L. (Bay leaf), Thymus vulgaris L. (Thyme), Lavandula x intermedia L. (Margaret Roberts Lavender), Petroselinum crispum M...

  15. Curcumin causes DNA damage and affects associated protein expression in HeLa human cervical cancer cells.

    Science.gov (United States)

    Shang, Hung-Sheng; Chang, Chuan-Hsun; Chou, Yu-Ru; Yeh, Ming-Yang; Au, Man-Kuan; Lu, Hsu-Feng; Chu, Yung-Lin; Chou, Hsiao-Min; Chou, Hsiu-Chen; Shih, Yung-Luen; Chung, Jing-Gung

    2016-10-01

    Cervical cancer is one of the most common cancers in women worldwide and it is a prominent cause of cancer mortality. Curcumin is one of the major compounds from Turmeric and has been shown to induce cytotoxic cell death in human cervical cancer cells. However, there is no study to show curcumin induced DNA damage action via the effect on the DNA damage and repair protein in cervical cancer cells in detail. In this study, we investigated whether or not curcumin induced cell death via DNA damage, chromatin condensation in human cervical cancer HeLa cells by using comet assay and DAPI staining, respectively, we found that curcumin induced cell death through the induction of DNA damage, and chromatin condensation. Western blotting and confocal laser microscopy examination were used to examine the effects of curcumin on protein expression associated with DNA damage, repair and translocation of proteins. We found that curcumin at 13 µM increased the protein levels associated with DNA damage and repair, such as O6-methylguanine-DNA methyltransferase, early-onset breast cancer 1 (BRCA1), mediator of DNA damage checkpoint 1, p-p53 and p-H2A.XSer140 in HeLa cells. Results from confocal laser systems microscopy indicated that curcumin increased the translocation of p-p53 and p-H2A.XSer140 from cytosol to nuclei in HeLa cells. In conclusion, curcumin induced cell death in HeLa cells via induction of DNA damage, and chromatin condensation in vitro.

  16. Introduction of disease-related mitochondrial DNA deletions into HeLa cells lacking mitochondrial DNA results in mitochondrial dysfunction.

    OpenAIRE

    Hayashi, J; Ohta, S.; Kikuchi, A; Takemitsu, M; Goto, Y.; Nonaka, I

    1991-01-01

    Mutant mitochondrial DNA with large-scale deletions (delta-mtDNA) has been frequently observed in patients with chronic progressive external ophthalmoplegia (CPEO), a subgroup of the mitochondrial encephalomyopathies. To exclude involvement of the nuclear genome in expression of the mitochondrial dysfunction characteristic of CPEO, we introduced the mtDNA of a CPEO patient into clonal mtDNA-less HeLa cells and isolated cybrid clones. Quantitation of delta-mtDNA in the cybrids revealed that de...

  17. Molecular changes to HeLa cells on continuous exposure to SN-38, an active metabolite of irinotecan hydrochloride.

    Science.gov (United States)

    Takara, Kohji; Kitada, Noriaki; Yoshikawa, Eri; Yamamoto, Kazuhiro; Horibe, Sayo; Sakaeda, Toshiyuki; Nishiguchi, Kohshi; Ohnishi, Noriaki; Yokoyama, Teruyoshi

    2009-06-08

    It is important to clarify the molecular characteristics of tumor cells showing multidrug resistance (MDR) and to identify the novel targets or biomarkers for chemotherapy. The aim of this study is to establish resistant HeLa sublines through exposure to SN-38, an active metabolite of irinotecan hydrochloride, and to investigate their molecular changes. HeLa cells were exposed to SN-38 at 1, 10, or 100 nM, and resistant clones were isolated and named HeLa/SN1, HeLa/SN10, and HeLa/SN100, respectively. Their cellular changes were examined based on growth inhibition assays, the function of ABCG2/BCRP, and a RT-PCR analysis of MDR-related protein. The sublines showed a decrease in sensitivity to not only SN-38 but also other chemotherapeutic agents as compared with HeLa cells. mRNA and protein levels of ABCG2/BCRP were increased, and the transport activity of ABCG2/BCRP was enhanced, in the resistant cells. In addition, the expression levels of ABCC1/MRP1, ABCC3/MRP3, and ABCC5/MRP5 were higher than in HeLa cells. The mRNA levels of GGT1 encoding a gamma-glutamyl transferase, but not GCS encoding a gamma-glutamyl cysteine synthetase, were also higher. Other factors examined, i.e., topoisomerase, SLCO1B1, and apoptosis-regulating factors, were comparable among the cells. The overexpression of ABCG2/BCRP was involved in the mechanism of resistance in SN-38-tolerant cells, and ABCC1/MRP1, ABCC3/MRP3, ABCC5/MRP5, and GGT1 may also have participated.

  18. The Cytotoxicity of Dextran-coated Iron Oxide Nanoparticles on Hela and MCF-7 Cancerous Cell Lines

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    Masoud Rezaei

    2017-09-01

    Full Text Available Background: Recently, iron oxide nanoparticles have attracted attention in various diagnosis and treatment fields. The aim of the present study was to investigate the cytotoxicity of various concentrations and incubation times of dextran-coated iron oxide nanoparticles (DIONPs on HeLa and MCF-7 cancerous cell lines. Methods: This in-vitro study was conducted at Pharmaceutical Sciences Research Center, Kermanshah University of Medical Sciences, Kermanshah, Iran in 2016. The dextran-coated iron oxide nanoparticles (DIONPs uptake and cytotoxicity at different concentrations (10, 40 and 80 µg/ml and different incubation times (6, 12 and 24 h were assessed on HeLa and MCF-7 cell lines. The viability of the cells was measured by MTT assay. Results: DIONPs entered into the HeLa and MCF-7 cells. After 6, 12 and 24 h incubation times and in all concentrations, the viability of HeLa cells was more than 94%. For MCF-7 cell line, increasing incubation time from 6 to 24 h at a concentration of 10 μg/ml decreased the cells viability from 98% to 95%. When the cells were exposed to concentrations of 40 and 80 μg/ml of the nanoparticles, significant reductions in the cells viability was observed from 98% to 91.6% and from 95% to 88%, respectively. Conclusion: DIONPs cytotoxicity increased by increasing the incubation time from 6 to 24 h and also increased with increasing the nanoparticles concentration from 0 to 80 μg/ml. In general, DIONPs did not cause considerable toxicity in both cell lines especially at lower concentrations. Therefore, these nanoparticles are good candidates for use in biomedical and cancer research studies.

  19. Effects of serum amyloid A protein on lymphocytes, HeLa, and MRC5 cells in culture.

    Science.gov (United States)

    Peristeris, P; Gaspar, A; Gros, P; Laurent, P; Bernon, H; Bienvenu, J

    1989-07-01

    A major human acute phase protein, the serum amyloid A protein, has been tested in vitro for its effect on lymphocyte proliferation, the formation of E-stable rosettes, as well as the growth of HeLa and MRC5 cell cultures. Serum amyloid A protein has been found to be markedly inhibitory at 30, 100, 200, and 300 micrograms/mL, and is a very potent inhibitor of in vitro biological functions.

  20. Rapid Biosynthesis of Silver Nanoparticles Using Pepino (Solanum muricatum Leaf Extract and Their Cytotoxicity on HeLa Cells

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    Mónica Gorbe

    2016-04-01

    Full Text Available Within nanotechnology, gold and silver nanostructures have unique physical, chemical, and electronic properties [1,2], which make them suitable for a number of applications. Moreover, biosynthetic methods are considered to be a safer alternative to conventional physicochemical procedures for both the environmental and biomedical applications, due to their eco-friendly nature and the avoidance of toxic chemicals in the synthesis. For this reason, employing bio routes in the synthesis of functionalized silver nanoparticles (FAgNP have gained importance recently in this field. In the present study, we report the rapid synthesis of FAgNP through the extract of pepino (Solanum muricatum leaves and employing microwave oven irradiation. The core-shell globular morphology and characterization of the different shaped and sized FAgNP, with a core of 20–50 nm of diameter is established using the UV-Visible spectroscopy (UV-vis, field emission scanning electron microscopy (FESEM, transmission electron microscopy (TEM and Zeta potential and dynamic light scanning (DLS studies. Moreover, cytotoxic studies employing HeLa (human cervix carcinoma cells were undertaken to understand FAgNP interactions with cells. HeLa cells showed significant dose dependent antiproliferative activity in the presence of FAgNP at relatively low concentrations. The calculated IC50 value was 37.5 µg/mL, similar to others obtained for FAgNPs against HeLa cells.

  1. Induction of apoptosis in HeLa cells by chloroform fraction of seed extracts of Nigella sativa

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    Alshatwi Ali A

    2009-11-01

    Full Text Available Abstract Background Cancer remains one of the most dreaded diseases causing an astonishingly high death rate, second only to cardiac arrest. The fact that conventional and newly emerging treatment procedures like chemotherapy, catalytic therapy, photodynamic therapy and radiotherapy have not succeeded in reverting the outcome of the disease to any drastic extent, has made researchers investigate alternative treatment options. The extensive repertoire of traditional medicinal knowledge systems from various parts of the world are being re-investigated for their healing properties. This study progresses in the direction of identifying component(s from Nigella sativa with anti cancer acitivity. In the present study we investigated the efficacy of Organic extracts of Nigella sativa seed powder for its clonogenic inhibition and induction of apoptosis in HeLa cancer cell. Results Methanolic, n-Hexane and chloroform extracts of Nigella sativa seedz effectively killed HeLa cells. The IC50 values of methanolic, n-hexane, and chloroform extracts of Nigella sativa were 2.28 μg/ml, 2.20 μg/ml and 0.41 ng/ml, respectively. All three extracts induced apoptosis in HeLa cells. Apoptosis was confirmed by DNA fragmentation, western blot and terminal transferase-mediated dUTP-digoxigenin-end labeling (TUNEL assay. Conclusion Western Blot and TUNEL results suggested that Nigella sativa seed extracts regulated the expression of pro- and anti- apoptotic genes, indicating its possible development as a potential therapeutic agent for cervical cancer upon further investigation.

  2. CRISPR-Cas9 Mediated NOX4 Knockout Inhibits Cell Proliferation and Invasion in HeLa Cells.

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    Naser Jafari

    Full Text Available Increased expression of NOX4 protein is associated with cancer progression and metastasis but the role of NOX4 in cell proliferation and invasion is not fully understood. We generated NOX4 knockout HeLa cell lines using the CRISPR-Cas9 gene editing system to explore the cellular functions of NOX4. After transfection of CRISPR-Cas9 construct, we performed T7 endonuclease 1 assays and DNA sequencing to generate and identify insertion and deletion of the NOX4 locus. We confirmed the knockout of NOX4 by Western blotting. NOX4 knockout cell lines showed reduced cell proliferation with an increase of sub-G1 cell population and the decrease of S/G2/M population. Moreover, NOX4 deficiency resulted in a dramatic decrease in invadopodium formation and the invasive activity. In addition, NOX4 deficiency also caused a decrease in focal adhesions and cell migration in HeLa cells. These results suggest that NOX4 is required for both efficient proliferation and invasion of HeLa cells.

  3. Methanolic Extracts from Brown Seaweeds Dictyota cilliolata and Dictyota menstrualis Induce Apoptosis in Human Cervical Adenocarcinoma HeLa Cells

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    Dayanne Lopes Gomes

    2015-04-01

    Full Text Available Carcinoma of the uterine cervix is the second most common female tumor worldwide, surpassed only by breast cancer. Natural products from seaweeds evidencing apoptotic activity have attracted a great deal of attention as new leads for alternative and complementary preventive or therapeutic anticancer agents. Here, methanol extracts from 13 species of tropical seaweeds (Rhodophytas, Phaeophyta and Chlorophyta collected from the Northeast of Brazil were assessed as apoptosis-inducing agents on human cervical adenocarcinoma (HeLa. All extracts showed different levels of cytotoxicity against HeLa cells; the most potent were obtained from the brown alga Dictyota cilliolata (MEDC and Dictyota menstrualis (MEDM. In addition, MEDC and MEDM also inhibits SiHa (cervix carcinoma cell proliferation. Studies with these two extracts using flow cytometry and fluorescence microscopy showed that HeLa cells exposed to MEDM and MEDC exhibit morphological and biochemical changes that characterize apoptosis as shown by loss of cell viability, chromatin condensation, phosphatidylserine externalization, and sub-G1 cell cycle phase accumulation, also MEDC induces cell cycle arrest in cell cycle phase S. Moreover, the activation of caspases 3 and 9 by these extracts suggests a mitochondria-dependent apoptosis route. However, other routes cannot be ruled out. Together, these results point out the methanol extracts of the brown algae D. mentrualis and D. cilliolata as potential sources of molecules with antitumor activity.

  4. Stable gene amplification and overexpression of sodium- and potassium-activated ATPase in HeLa cells.

    Science.gov (United States)

    Pauw, P G; Johnson, M D; Moore, P; Morgan, M; Fineman, R M; Kalka, T; Ash, J F

    1986-01-01

    Cell lines stably resistant to ouabain were isolated from an unstably resistant HeLa line after growth in nonselective medium. Stable resistant lines bound ouabain at levels 10-fold higher than did HeLa cells and at similar levels to those bound by the unstable C+ line previously described (J. F. Ash, R. M. Fineman, T. Kalka, M. Morgan, and B. Wire, J. Cell Biol. 99: 971-983). Expression and synthesis of the Na+, K+ -ATPase alpha chain showed a similar amplification over that for HeLa cells by Western blots and [35S]methionine pulse-labeling. In addition, a glycoprotein labeled with [3H]fucose and comigrating with the Na+, K+ -ATPase beta chain was eight- to ninefold amplified in stably resistant lines. Dot blots with a cDNA clone specific for Na+, K+ -ATPase alpha chain gene sequences confirmed the amplification of this gene. Karyotyping suggested that the amplification is associated with an expanded, abnormal banded region on the long (q) arm of one chromosome 17. Images PMID:3023874

  5. Identification of a cancer stem cell-like side population in the HeLa human cervical carcinoma cell line.

    Science.gov (United States)

    Wang, Kefang; Zeng, Jianfang; Luo, Lijing; Yang, Jiaxin; Chen, Jie; Li, Bin; Shen, Keng

    2013-12-01

    The present study aimed to identify the stem cell characteristics of side population (SP) cells sorted from the widely-used HeLa human cervical carcinoma cell line. The SP cells were sorted from the HeLa cell line using fluorescence-activating cell sorting (FACS). Stem cell characteristics of the SP cells, including proliferation, self-renewal, differentiation and the ability to form xenografts, were investigated in vitro and in vivo . The SP cells demonstrated strong tumorigenesis following in vivo transplantation into five to six-week-old female Balb/c mice. The SP cells were observed to be more resistant to chemotherapy and radiotherapy compared with non-side population (NSP) cells. A higher expression of CD133 was observed in the SP cells compared with the NSP cells following FACS. The results demonstrated that the SP cells from the HeLa human cervical carcinoma cell line exhibit stem cell characteristics in vitro and also have a strong ability to form tumors in vivo . The cell surface marker CD133 may serve as a potential molecular marker for the identification of cervical cancer stem cells (CSCs).

  6. Aktivitas Sitotoksik, Induksi Apoptosis dan Ekspresi Gen P53 Fraksi Metanol Spons Petrosia cf nigricans Terhadap Sel Tumor Hela

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    Muhammad Nursid

    2007-12-01

    Full Text Available Spons merupakan hewan invertebrata laut yang kaya akan kandungan senyawa bioaktif. Senyawa bioaktif dari spons banyak dieksplorasi sebagai bahan obat antitumor. Penelitian ini bertujuan untuk mengetahui aktivitas sitotoksik, induksi apoptosis dan ekspresi gen p53 fraksi metanol spons Petrosia cf. nigricans terhadap sel tumor HeLa. Uji aktivitas pendahuluan dan sitotoksik masing‑masing dilakukan dengan metode Brine Shrimp Lethality Test (BSLT dan MTT ([3‑(4,5‑dimethylthiazol‑2yl‑2,5‑diphenyltetrazolium bromide] assay. Induksi apoptosis dilakukan dengan metode pengecatan menggunakan akridin oranye dan etidium bromida. Analisis PCR menggunakan primer p53 dilakukan untuk mengetahui ekspresi gen p53. Hasil uji BSLT memperlihatkan bahwa ekstrak kasar P. cf nigricans memiliki aktivitas tahap awal yang sangat baik dengan LC50 = 23,4 µg/mL. Hasil uji MTT menunjukkan bahwa fraksi metanol memiliki aktivitas sitotoksik terhadap sel HeLa dengan nilai LC50 sebesar 11,9 µg/mL. Berdasarkan uji induksi apoptosis metode pengecatan dapat dinyatakan bahwa fraksi metanol mampu menginduksi peristiwa apoptosis pada sel HeLa. Fraksi metanol juga mampu meningkatkan ekspresi gen p53.

  7. Sechium edule (Jacq. Swartz, a New Cultivar with Antiproliferative Potential in a Human Cervical Cancer HeLa Cell Line

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    Sandra Salazar-Aguilar

    2017-07-01

    Full Text Available The Sechium edule Perla Negra cultivar is a recently-obtained biological material whose progenitors are S. edule var. nigrum minor and S. edule var. amarus silvestrys, the latter of which has been reported to have antiproliferative activity against the HeLa P-388 and L-929 cancer cell lines. The present study aimed to determine if the methanolic extract of the fruit of the Perla Negra cultivar had the same biological activity. The methanolic extract was phytochemically characterized by thin layer chromatography (TLC and column chromatography (CC, identifying the terpenes and flavonoids. The compounds identified via high performance liquid chromatography (HPLC were Cucurbitacins B, D, E, and I for the terpene fractions, and Rutin, Phlorizidin, Myricetin, Quercetin, Naringenin, Phloretin, Apigenin, and Galangin for the flavonoid fractions. Biological activity was evaluated with different concentrations of the methanolic extract in the HeLa cell line and normal lymphocytes. The methanolic extract inhibited the proliferation of HeLa cells (IC50 1.85 µg·mL−1, but the lymphocytes were affected by the extract (IC50 30.04 µg·mL−1. Some fractions, and the pool of all of them, showed inhibition higher than 80% at a concentration of 2.11 µg·mL−1. Therefore, the biological effect shown by the methanolic extract of the Perla Negra has some specificity in inhibiting tumor cells and not normal cells; an unusual feature among molecules investigated as potential biomedical agents.

  8. Reovirus type 3 synthesizes proteins in interferon-treated HeLa cells without reversing the antiviral state.

    Science.gov (United States)

    Feduchi, E; Esteban, M; Carrasco, L

    1988-06-01

    Treatment of HeLa cells with human lymphoblastoid interferon (IFN-alpha) does not inhibit reovirus type 3 protein synthesis during virus infection. In contrast, reovirus translation is blocked by treatment of L cells with mouse IFN-alpha. The (2'-5')A synthetase activity is induced in HeLa cells by IFN-alpha treatment and is activated after reovirus infection, since cell lysates from these cells synthesize in vitro (2'-5')A oligonucleotides. The IFN-induced protein kinase activity is also triggered in those lysates upon dsRNA addition. Thus, contrary to DNA-containing viruses, such as vaccinia virus or adenovirus, reovirus infection does not destroy or reverse the IFN-induced antiviral state. In support of this conclusion, superinfection with poliovirus or vesicular stomatitis virus of reovirus-infected HeLa cells treated with IFN leads only to a blockade of translation of the former viruses. These results provide a remarkable example where in the same cells doubly infected with two different viruses, the antiviral state induced by IFN-alpha is manifested by selectively inhibiting translation of one kind of virus (poliovirus or vesicular stomatitis virus) without affecting the translation of reovirus type 3. In addition, these results indicate that the resistance of reovirus translation to inhibition by IFN is different from the mechanism of resistance induced by DNA-containing viruses.

  9. Antioxidant potential of Ulva rigida extracts: protection of HeLa cells against H₂O₂ cytotoxicity.

    Science.gov (United States)

    Mezghani, Sana; Bourguiba, Ines; Hfaiedh, Imen; Amri, Mohamed

    2013-09-01

    The rising demand for natural antioxidants instead of synthetic materials, especially in biomedical applications, has led to increased interest in the search for bioactive compounds with potent antioxidant activity. In the present study, we tested the antioxidant effect of both a crude extract and an ethanol precipitate of Ulva rigida in HeLa cells exposed to hydrogen peroxide (H₂O₂). HeLa cells treated with H₂O₂ (1 mmol l⁻¹ for 3 h) exhibited significant damage to their morphology, a significant decrease in cell survival, and a remarkable leakage of lactate dehydrogenase (LDH). However, the co-exposure of cells to H₂O₂ and the crude extract or ethanol precipitate of U. rigida induced fewer morphological cytotoxic effects, a significant increase in cell viability, and a significant decrease in LDH release. Biochemical studies have demonstrated that U. rigida extracts have a strong radicalscavenging activity and contain protein, sugar, and phenolic content. The overall results suggest that U. rigida extracts protect HeLa cells from death induced by oxidative stress, and it is likely that these effects are related to the phenolic, protein, and polysaccharide compounds contained in this alga. Hence, U. rigida can be used to treat diseases ascribed to oxidative disorders.

  10. Cytotoxic activity of proteins isolated from extracts of Corydalis cava tubers in human cervical carcinoma HeLa cells

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    Balcerkiewicz Stanislaw

    2010-12-01

    Full Text Available Abstract Background Corydalis cava Schweigg. & Koerte, the plant of numerous pharmacological activities, together with the studied earlier by our group Chelidonium majus L. (Greater Celandine, belong to the family Papaveraceae. The plant grows in Central and South Europe and produces the sizeable subterraneous tubers, empty inside, which are extremely resistant to various pathogen attacks. The Corydalis sp. tubers are a rich source of many biologically active substances, with the extensive use in European and Asian folk medicine. They have analgetic, sedating, narcotic, anti-inflammatory, anti-allergic and anti-tumour activities. On the other hand, there is no information about possible biological activities of proteins contained in Corydalis cava tubers. Methods Nucleolytic proteins were isolated from the tubers of C. cava by separation on a heparin column and tested for DNase activity. Protein fractions showing nucleolytic activity were tested for cytotoxic activity in human cervical carcinoma HeLa cells. Cultures of HeLa cells were conducted in the presence of three protein concentrations: 42, 83 and 167 ng/ml during 48 h. Viability of cell cultures was appraised using XTT colorimetric test. Protein fractions were separated and protein bands were excised and sent for identification by mass spectrometry (LC-ESI-MS/MS. Results The studied protein fractions showed an inhibiting effect on mitochondrial activity of HeLa cells, depending on the administered dose of proteins. The most pronounced effect was obtained with the highest concentration of the protein (167 ng/ml - 43.45 ± 3% mitochondrial activity of HeLa cells were inhibited. Mass spectrometry results for the proteins of applied fractions showed that they contained plant defense- and pathogenesis-related (PR proteins. Conclusions The cytotoxic effect of studied proteins toward HeLa cell line cells has been evident and dependent on increasing dose of the protein. The present study, most

  11. Impact of Exopolysaccharides (EPSs) of Lactobacillus gasseri strains isolated from human vagina on cervical tumor cells (HeLa).

    Science.gov (United States)

    Sungur, Tolga; Aslim, Belma; Karaaslan, Cagtay; Aktas, Busra

    2017-10-01

    Lactobacilli, commonly used as probiotics, have been shown to maintain vaginal health and contribute to host microbiota interaction. Exopolysaccharides (EPSs) produced by lactobacillus have been found to have an important role in probiotic activity; however, there is limited knowledge concerning their impact on cervical cancer and urogenital health. The objective of this study is to investigate and compare EPSs of L. gasseri strains (G10 and H15), isolated from a healthy human vagina, for their capability to inhibit cervical cancer cell (HeLa) growth and modulate immune response. HeLa cells were treated with live culture at ∼108 CFU/ml or increasing concentration of lyophilized EPS (L-EPS) (100, 200, or 400 μg/ml) of L. gasseri strains and their ability to adhere to host cells, inhibit proliferation, and modulate immune response were evaluated. Additionally, monosaccharide composition of the L-EPSs produced by L. gasseri strains was determined by HPLC. The sugar component was the same; however, relative proportions of the individual monosaccharides except mannose were different. Although they both produce similar amount of EPS, the most adhesive strain was G10. Both live and L-EPS of L. gasseri strains were capable of inhibiting the cell proliferation of HeLa cells with the impact of L-EPS being strain specific. L-EPSs of L. gasseri strains induced apoptosis in HeLa cells in a strain dependent manner. The ability to induce apoptosis by G10 associated with an upregulation of Bax and Caspase 3. L. gasseri strains showed an anti-inflammatory impact on HeLa cells by decreasing the production of TNF-α and increasing the IL-10 production. In conclusion, diversity in sugar composition of EPS might contribute to adhesion and proliferation properties. Although our results suggest a relationship between the ability of a strain to induce apoptosis and its sugar composition of EPS, further research is required to determine the probiotic mechanisms of action by

  12. Doxorubicin-induced cell death requires cathepsin B in HeLa cells.

    Science.gov (United States)

    Bien, S; Rimmbach, C; Neumann, H; Niessen, J; Reimer, E; Ritter, C A; Rosskopf, D; Cinatl, J; Michaelis, M; Schroeder, H W S; Kroemer, H K

    2010-11-15

    The cysteine protease cathepsin B acts as a key player in apoptosis. Cathepsin B-mediated cell death is induced by various stimuli such as ischemia, bile acids or TNFα. Whether cathepsin B can be influenced by anticancer drugs, however, has not been studied in detail. Here, we describe the modulation of doxorubicin-induced cell death by silencing of cathepsin B expression. Previously, it was shown that doxorubicin, in contrast to other drugs, selectively regulates expression and activity of cathepsin B. Selective silencing of cathepsin B by siRNA or the cathepsin B specific inhibitor CA074Me modified doxorubicin-mediated cell death in Hela tumor cells. Both Caspase 3 activation and PARP cleavage were significantly reduced in cells lacking cathepsin B. Moreover, mitochondrial membrane permeabilization as well as the release of cytochrome C and AIF from mitochondria into cytosol induced by doxorubicin were significantly diminished in cathepsin B suppressed cells. In addition, doxorubicin associated down-regulation of XIAP was not observed in cathepsin B silenced cells. Lack of cathepsin B significantly modified cell cycle regulatory proteins such as cdk1, Wee1 and p21 without significant changes in G(1), S or G(2)M cell cycle phases maybe indicating further cell cycle independent actions of these proteins. Consequently, cell viability following doxorubicin was significantly elevated in cells with cathepsin B silencing. In summary, our data strongly suggest a role of cathepsin B in doxorubicin-induced cell death. Therefore, increased expression of cathepsin B in various types of cancer can modify susceptibility towards doxorubicin. Copyright © 2010 Elsevier Inc. All rights reserved.

  13. Combined antitumor activity of the nitroreductase/CB1954 suicide gene system and γ-rays in HeLa cells in vitro.

    Science.gov (United States)

    Teng, Geling; Ju, Yuanrong; Yang, Yepeng; Hua, Hu; Chi, Jingyu; Mu, Xiuan

    2016-12-01

    Escherichia coli nitroreductase (NTR) may convert the prodrug CB1954 (5-(aziridin-1-yl)-2,4-dinitrobenzamide) into a bifunctional alkylating agent, which may lead to DNA crosslinks and the apoptosis of cancer cells. NTR/CB1954 has been demonstrated to be an effective gene therapy in cancer cells. The present study examined whether the NTR/CB1954 suicide gene system had cytotoxic effects on HeLa cells and may improve the radiosensitivity of HeLa cells to γ‑rays. It was observed that the NTR/CB1954 suicide gene system exerted marked cytotoxic effects on HeLa cells. The combined therapeutic effects of NTR/CB1954 and γ‑rays on HeLa cells demonstrated a synergistic effect. CB1954 at concentrations of 12.5 and 25 µmol/l increased the sensitization enhancement ratio of HeLa cells to 1.54 and 1.66, respectively. Therefore, when compared with monotherapy, the combined therapy of NTR/CB1954 and γ‑rays may increase the apoptotic rate and enhance the radiosensitivity of HeLa cells. The combined therapy of γ‑ray radiation and the NTR/CB1954 suicide gene system may be a novel and potent therapeutic method for the treatment of cervical carcinoma.

  14. Subcellular localization of the five members of the human steroid 5α-reductase family

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    Antonella Scaglione

    2017-06-01

    We report the cloning and transient expression in HeLa cells of the five members of the human steroid 5α-reductase family as both N- and C-terminus green fluorescent protein tagged protein constructs. Following the intrinsic fluorescence of the tag, we have determined that the subcellular localization of these enzymes is in the endoplasmic reticulum, upon expression in HeLa cells. The presence of the tag at either end of the polypeptide chain can affect protein expression and, in the case of trans enoyl-CoA reductase, it induces the formation of protein aggregates.

  15. Proteasome Inhibition Contributed to the Cytotoxicity of Arenobufagin after Its Binding with Na, K-ATPase in Human Cervical Carcinoma HeLa Cells.

    Directory of Open Access Journals (Sweden)

    Qingxi Yue

    Full Text Available Although the possibility of developing cardiac steroids/cardiac glycosides as novel cancer therapeutic agents has been recognized, the mechanism of their anticancer activity is still not clear enough. Toad venom extract containing bufadienolides, which belong to cardiac steroids, has actually long been used as traditional Chinese medicine in clinic for cancer therapy in China. The cytotoxicity of arenobufagin, a bufadienolide isolated from toad venom, on human cervical carcinoma HeLa cells was checked. And, the protein expression profile of control HeLa cells and HeLa cells treated with arenobufagin for 48 h was analyzed using two-dimensional electrophoresis, respectively. Differently expressed proteins in HeLa cells treated with arenobufagin were identified and the pathways related to these proteins were mapped from KEGG database. Computational molecular docking was performed to verify the binding of arenobufagin and Na, K-ATPase. The effects of arenobufagin on Na, K-ATPase activity and proteasome activity of HeLa cells were checked. The protein-protein interaction network between Na, K-ATPase and proteasome was constructed and the expression of possible intermediate proteins ataxin-1 and translationally-controlled tumor protein in HeLa cells treated with arenobufagin was then checked. Arenobufagin induced apoptosis and G2/M cell cycle arrest in HeLa cells. The cytotoxic effect of arenobufagin was associated with 25 differently expressed proteins including proteasome-related proteins, calcium ion binding-related proteins, oxidative stress-related proteins, metabolism-related enzymes and others. The results of computational molecular docking revealed that arenobufagin was bound in the cavity formed by the transmembrane alpha subunits of Na, K-ATPase, which blocked the pathway of extracellular Na+/K+ cation exchange and inhibited the function of ion exchange. Arenobufagin inhibited the activity of Na, K-ATPase and proteasome, decreased the

  16. Helioseismology and the Solar Interior Dynamics

    Indian Academy of Sciences (India)

    tribpo

    well as several instruments aboard the space mission SOHO, continuously observe the solar oscillations. Millions of solar p-modes have been detected. The inversion of the measured frequencies yields accurate and detailed information about the sound velocity in the Sun's interior, which in turn leads to constraints on the ...

  17. Waveform tomography in geophysics and helioseismology

    NARCIS (Netherlands)

    Cobden, L.J.; Fichtner, A.; Tong, Vincent

    2015-01-01

    Seismic tomography – in which we construct images of a body's interior using seismic waves – is an inverse problem; that is, our goal is to find a model that fits a set of existing data observations. This is much less straightforward than the reverse, forward problem (i.e., generating synthetic data

  18. Anticancer Activity of Certain Herbs and Spices on the Cervical Epithelial Carcinoma (HeLa Cell Line

    Directory of Open Access Journals (Sweden)

    Danielle Berrington

    2012-01-01

    Full Text Available Acetone extracts of selected plant species were evaluated for their in vitro cytotoxicity against a noncancerous African green monkey kidney (Vero cell line and an adenocarcinoma cervical cancer (HeLa cell line. The plants studied were Origanum vulgare L. (Oregano, Rosmarinus officinalis L. (Upright and ground cove rosemary, Lavandula spica L. (Lavender, Laurus nobilis L. (Bay leaf, Thymus vulgaris L. (Thyme, Lavandula x intermedia L. (Margaret Roberts Lavender, Petroselinum crispum Mill. (Curly leaved parsley, Foeniculum vulgare Mill. (Fennel, and Capsicum annuum L. (Paprika. Antioxidant activity was determined using a quantitative DPPH (1,1-diphenyl-2-picryl hydrazyl assay. The rosemary species exhibited effective radical scavenging capacity with 50% inhibitory concentration (IC50 of 3.48±0.218 μg/mL and 10.84±0.125 μg/mL and vitamin C equivalents of 0.351 g and 1.09 g for McConnell’s Blue and Tuscan Blue, respectively. Cytotoxicity was measured using XTT (Sodium 3′-[1-(phenyl amino-carbonyl-3,4-tetrazolium]-bis-[4-methoxy-6-nitro] benzene sulfonic acid hydrate colorimetric assay. Only L. nobilis and O. vulgare exhibited pronounced effects on the HeLa cell line. Dose-dependent studies revealed IC50 of 34.46±0.48 μg/mL and 126.3±1.00 μg/mL on the HeLa cells and on the Vero cells 124.1 μg/mL ± 18.26 and 163.8 μg/mL ± 2.95 for L. nobilis and O. vulgare, respectively. Light (eosin and haematoxylin staining and confocal microscopy (Hoechst 33342, acridine orange, and propidium iodide staining were used to evaluate the cytotoxic mechanism of action for L. nobilis and O. vulgare.

  19. Anticancer Activity of Certain Herbs and Spices on the Cervical Epithelial Carcinoma (HeLa) Cell Line.

    Science.gov (United States)

    Berrington, Danielle; Lall, Namrita

    2012-01-01

    Acetone extracts of selected plant species were evaluated for their in vitro cytotoxicity against a noncancerous African green monkey kidney (Vero) cell line and an adenocarcinoma cervical cancer (HeLa) cell line. The plants studied were Origanum vulgare L. (Oregano), Rosmarinus officinalis L. (Upright and ground cove rosemary), Lavandula spica L. (Lavender), Laurus nobilis L. (Bay leaf), Thymus vulgaris L. (Thyme), Lavandula x intermedia L. (Margaret Roberts Lavender), Petroselinum crispum Mill. (Curly leaved parsley), Foeniculum vulgare Mill. (Fennel), and Capsicum annuum L. (Paprika). Antioxidant activity was determined using a quantitative DPPH (1,1-diphenyl-2-picryl hydrazyl) assay. The rosemary species exhibited effective radical scavenging capacity with 50% inhibitory concentration (IC(50)) of 3.48 ± 0.218 μg/mL and 10.84 ± 0.125 μg/mL and vitamin C equivalents of 0.351 g and 1.09 g for McConnell's Blue and Tuscan Blue, respectively. Cytotoxicity was measured using XTT (Sodium 3'-[1-(phenyl amino-carbonyl)-3,4-tetrazolium]-bis-[4-methoxy-6-nitro] benzene sulfonic acid hydrate) colorimetric assay. Only L. nobilis and O. vulgare exhibited pronounced effects on the HeLa cell line. Dose-dependent studies revealed IC(50) of 34.46 ± 0.48 μg/mL and 126.3 ± 1.00 μg/mL on the HeLa cells and on the Vero cells 124.1 μg/mL ± 18.26 and 163.8 μg/mL ± 2.95 for L. nobilis and O. vulgare, respectively. Light (eosin and haematoxylin staining) and confocal microscopy (Hoechst 33342, acridine orange, and propidium iodide staining) were used to evaluate the cytotoxic mechanism of action for L. nobilis and O. vulgare.

  20. In vitro anticancer and cytotoxic activities of some plant extracts on HeLa and Vero cell lines.

    Science.gov (United States)

    Tugba Artun, Fulya; Karagoz, Ali; Ozcan, Gul; Melikoglu, Gulay; Anil, Sezin; Kultur, Sukran; Sutlupinar, Nurhayat

    2016-01-01

    The aim of our study was to evaluate the effect of in vitro anticancer and cytotoxic activity of the methanolic extracts of 14 medicinal plants, 8 of which are endemic species in Anatolia, against the human HeLa cervical cancer cell line and to compare to the normal African green monkey kidney epithelial cell line (Vero) using the MTT colorimetric assay. Values for cytotoxicity measured by MTT assay were expressed as the concentration that causes 50% decrease in cell viability (IC50, μg/mL). The degree of selectivity of the compounds can be expressed by its selectivity index (SI) value. High SI value (>2) of a compound gives the selective toxicity against cancer cells (SI = IC50 normal cell/IC50 cancer cell). Dose-dependent studies revealed IC50 of 293 mg/mL and >1000 mg/mL for Cotinus coggygria Scop., IC50 of 265 μg/mL and >1000 mg/mL for Rosa damascena Miller, IC50 of 2 μg/mL and 454 mg/mL for Colchicum sanguicolle K.M. Perss, IC50 of 427 μg/mL and >1000 μg/mL for Centaurea antiochia Boiss. var. praealta (Boiss & Bal) Wagenitz on the HeLa cells and the Vero cells, respectively. Four plants showed significant SI values which were 227 for Colchicum sanguicolle K.M. Perss (endemic species), >3.8 for Rosa damascena Miller, >3.4 for Cotinus coggygria Scop. and >2.3 for Centaurea antiochia Boiss. var. praealta (Boiss & Bal)Wagenitz (endemic species). According to our study, 4 methanolic extracts of 14 tested plants exhibit greater activity on the HeLa cell line and little activity on the Vero cell line, meaning that these plants can be evaluated for potential promising anticancer activity.

  1. Synergistic combination of fluoro chalcone and doxorubicin on HeLa cervical cancer cells by inducing apoptosis

    Science.gov (United States)

    Arianingrum, Retno; Arty, Indyah Sulistyo; Atun, Sri

    2017-03-01

    Doxorubicin (Dox), a primary chemotherapeutic agent used for cancer treatment is known to have various side effect included multidrug resistance (MDR) phenomenon. Combination chemotherapy is one of some approaches to reduce Dox side effect. Chalcones have been reported to reduce the proliferation of many cancer cells. The research were conducted to investigate the cytotoxic activity and apoptosis induction of a chalcone derivate which is containing fluoro substituent [1 - (4" - fluorophenyl) -3 - (4' - hydroxy - 3' - methoxyphenyl) - 2 - propene - 1 -on] (FHM) and its combination with Dox on HeLa cells line. The observation of the cytotoxic activity was conducted using MTT [3 - (4, 5 - dimethyl thiazol - 2 - y1) - 2.5 - diphenyltetrazolium bromide] assay. Apoptosis induction was determined by flow cytometric. The changes of cell morphology were observed using phase contrast microscopy. The combination index (CI) was used to determine the effect of the combination. The study showed that FHM inhibited the HeLa cell growth with IC50 of 34 μM, while the IC50 of Dox was 1 μM. The combination had a higher inhibitory effect on cell growth compare to the single treatment of FHM and Dox. All of the combination doses under IC50 of FHM and Dox gave synergistic (CI: - 0.7) up to strong synergistic effect (CI: 0.l - 0.3). The synergistic effects of the combination were due to their ability to induce apoptosis in the HeLa cells. According to the result, FHM was potential to be developed as a co-chemotherapeutic agent with Dox for cervical cancer.

  2. In vitro studies of the toxic effects of silver nanoparticles on HeLa and U937 cells

    Directory of Open Access Journals (Sweden)

    Kaba SI

    2015-03-01

    Full Text Available Said I Kaba, Elena M Egorova Institute of General Pathology and Pathophysiology, Moscow, Russia Abstract: In the last decade, much attention has been paid to studies of the effect of silver nanoparticles (Ag NPs on tumor cells. Apart from elucidation of the mechanism of NPs’ interaction with mammalian cells, these studies are aimed at discovering new effective antitumor drugs. In this work, we report about the toxic effects of Ag NPs observed on two types of tumor cells: HeLa (adhesive cells and U937 (suspension cells. The Ag NPs were obtained by an original method of biochemical synthesis. Particle size was 13.2±4.72 nm, and zeta potential was -61.9±3.2 mV. The toxicity of Ag NPs in the concentration range 0.5–8.0 µg Ag/mL was determined by means of 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide assay and cytofluorometry after 4 and 24 hours' incubation. It was found that Ag NPs had high toxicity toward both cell types. The minimal concentrations where a toxicity effect was registered (toxicity thresholds lied in the range 0.5–2.0 µg Ag/mL. In parallel with the Ag NP solution, cells were incubated with water solutions of the NP stabilizer (aerosol-OT and Ag+ ions (as silver nitrate. It was shown that aerosol-OT had no effect on the viability on HeLa cells, but was moderately toxic toward U937, though less dangerous for these cells than Ag NPs. With Ag+ ions, for HeLa no toxic effect was observed, while for U937 they were as toxic as the Ag NPs. The data obtained indicate that Ag NPs as used in this study may prove to be useful for the creation of medicines for cancer therapy. Keywords: silver nanoparticles, cell viability, apoptosis, tumor cells

  3. Off-target responses in the HeLa proteome subsequent to transient plasmid-mediated transfection.

    Science.gov (United States)

    Hagen, Lars; Sharma, Animesh; Aas, Per Arne; Slupphaug, Geir

    2015-01-01

    Transient transfection of mammalian cells with plasmid expression vectors and chemical transfection reagents is widely used to study protein transport and dynamics as well as phenotypic alterations mediated by the overexpressed protein. Despite the undisputed impact of this technique, surprisingly little is known about the cellular effects mediated by the transfection process per se. Conceivably, off-target effects could have implications upon proteins or processes being studied and understanding the molecular pathways affected would add value to the interpretation of experimental observations subsequent to cell transfection. Here we have used a SILAC-based proteomic approach to study differentially expressed proteins after transfection of HeLa cells with ECFP vector using a commonly employed non-liposome based transfection reagent, Fugene®HD. Whereas the transfection reagent itself mediated minimal effects upon protein expression, 11 proteins were found to be significantly upregulated after transfection, all of which were associated with an interferon type I/II response. The upregulated proteins might potentially inflict major cellular processes such as RNA splicing, chromatin remodeling, post-translational protein modification and cell cycle control. The results were validated by western analysis as well as quantitative RT-PCR and this demonstrated that an essentially identical response was induced in HeLa by transfection using an empty pUC18 vector, which does not contain a mammalian virus promoter, as well as a liposome-based transfection reagent, Lipofectamine(TM)2000. Notably, no induction of the interferon response was observed in HEK293 cells, suggesting that these cells might be preferable to HeLa to avoid undesired off-target effects in transfection studies encompassing interferon-signaling and antiviral responses. Copyright © 2014 Elsevier B.V. All rights reserved.

  4. Ctotoxic and apoptogenic effects of Perovskia abrotanoides flower extract on MCF-7 and HeLa cell lines

    Directory of Open Access Journals (Sweden)

    Mohamad Ali Geryani

    2016-06-01

    Full Text Available Objective: Perovskia abrotanoides Karel, belongs to the family Lamiaceae and grows wild alongside the mountainous roads inarid and cold climate of Northern Iran. The anti-tumor activity of P. abrotanoides root extract has been shown previously. This study was designed to examine in vitro anti-proliferative and pro-apoptotic effects of flower extract of P. abrotanoides on MCF-7 and Hela cell lines. Materials and Methods: Cells were cultured in DMEM medium with 10% fetal bovine serum, 100 units/ml penicillin and 100 µg/ml streptomycin and incubated with different concentrations of plant extracts. Cell viability was quantified by MTT assay. Apoptotic cells were determined using propidium iodide (PI staining of DNA fragmentation by flow cytometry (sub-G1 peak. Results: P. abrotanoides extract inhibited the growth of malignant cells in a time and dose-dependent manner and 1000 µg/ml of extract following 48h of incubation was the most cytotoxic dose against Hela cell in comparison with other doses; however, in MCF-7 cells,1000 and 500 µg/ml PA induced toxicity at all time points but with different features.. Analysis of flowcytometry histogram of treated cells compared with control cells indicated that the cytotoxic effect is partly due toapoptosis induction. Conclusion: Hydro-alcoholic extract of P. abrotanoides flowers inhibits the growth of MCF-7 and HeLa cell lines, partly via inducing apoptosis. Their inhibitory effect was increased in a time and dose-dependent manner, especially in MCF7 cells. However, further studies are needed to reveal the mechanisms of P. abrotanoides extract-induced cell death.

  5. Ethanol metabolism by HeLa cells transduced with human alcohol dehydrogenase isoenzymes: control of the pathway by acetaldehyde concentration.

    Science.gov (United States)

    Matsumoto, Michinaga; Cyganek, Izabela; Sanghani, Paresh C; Cho, Won Kyoo; Liangpunsakul, Suthat; Crabb, David W

    2011-01-01

    Human class I alcohol dehydrogenase 2 isoenzymes (encoded by the ADH1B locus) have large differences in kinetic properties; however, individuals inheriting the alleles for the different isoenzymes exhibit only small differences in alcohol elimination rates. This suggests that other cellular factors must regulate the activity of the isoenzymes. The activity of the isoenzymes expressed from ADH1B*1, ADH1B*2, and ADH1B*3 cDNAs was examined in stably transduced HeLa cell lines, including lines which expressed human low K(m) aldehyde dehydrogenase (ALDH2). The ability of the cells to metabolize ethanol was compared with that of HeLa cells expressing rat class I alcohol dehydrogenase (ADH) (HeLa-rat ADH cells), rat hepatoma (H4IIEC3) cells, and rat hepatocytes. The isoenzymes had similar protein half-lives in the HeLa cells. Rat hepatocytes, H4IIEC3 cells, and HeLa-rat ADH cells oxidized ethanol much faster than the cells expressing the ADH1B isoenzymes. This was not explained by high cellular NADH levels or endogenous inhibitors; but rather because the activity of the β1 and β2 ADHs was constrained by the accumulation of acetaldehyde, as shown by the increased rate of ethanol oxidation by cell lines expressing β2 ADH plus ALDH2. The activity of the human β2 ADH isoenzyme is sensitive to inhibition by acetaldehyde, which likely limits its activity in vivo. This study emphasizes the importance of maintaining a low steady-state acetaldehyde concentration in hepatocytes during ethanol metabolism. Copyright © 2010 by the Research Society on Alcoholism.

  6. Development of electrochemical reporter assay using HeLa cells transfected with vector plasmids encoding various responsive elements

    Energy Technology Data Exchange (ETDEWEB)

    Shiku, Hitoshi, E-mail: shiku@bioinfo.che.tohoku.ac.jp [Graduate School of Environmental Studies, Tohoku University, 6-6-11-604 Aramaki-Aoba, Sendai 980-8579 (Japan); Takeda, Michiaki; Murata, Tatsuya [Graduate School of Environmental Studies, Tohoku University, 6-6-11-604 Aramaki-Aoba, Sendai 980-8579 (Japan); Akiba, Uichi; Hamada, Fumio [Graduate School of Engineering and Resource Science, Akita University, 1-1 Tegata gakuen-machi, Akita 010-8502 (Japan); Matsue, Tomokazu, E-mail: matsue@bioinfo.che.tohoku.ac.jp [Graduate School of Environmental Studies, Tohoku University, 6-6-11-604 Aramaki-Aoba, Sendai 980-8579 (Japan)

    2009-04-27

    Electrochemical assay using HeLa cell lines transfected with various plasmid vectors encoding SEAP (secreted alkaline phosphatase) as the reporter has been performed by using SECM (scanning electrochemical microscopy). The plasmid vector contains different responsive elements that include GRE (glucocorticoid response elements), CRE (cAMP responsive elements), or {kappa}B (binding site for NF{kappa}B (nuclear factor kappa B)) upstream of the SEAP sequence. The transfected HeLa cells were patterned on a culture dish in a 4 x 4 array of circles of diameter 300 {mu}m by using the PDMS (poly(dimethylsiloxane)) stencil technique. The cellular array was first exposed to 100 ng mL{sup -1} dexamethasone, 10 ng mL{sup -1} forskolin, or 100 ng mL{sup -1} TNF-{alpha} (tumor necrosis factor {alpha}) after which it was further cultured in an RPMI culture medium for 6 h. After incubation, the cellular array was soaked in a measuring solution containing 4.7 mM PAPP (p-aminophenylphosphate) at pH 9.5, following which electrochemical measurements were performed immediately within 40 min. The SECM method allows parallel evaluation of different cell lines transfected with pGRE-SEAP, pCRE-SEAP, and pNF{kappa}B-SEAP patterned on the same solid support for detection of the oxidation current of PAP (p-aminophenol) flux produced from only 300 HeLa cells in each stencil pattern. The results of the SECM method were highly sensitive as compared to those obtained from the conventional CL (chemiluminescence) protocol with at least 5 x 10{sup 4} cells per well.

  7. Caveolin-1 and CDC42 mediated endocytosis of silica-coated iron oxide nanoparticles in HeLa cells

    Directory of Open Access Journals (Sweden)

    Nils Bohmer

    2015-01-01

    Full Text Available Nanomedicine is a rapidly growing field in nanotechnology, which has great potential in the development of new therapies for numerous diseases. For example iron oxide nanoparticles are in clinical use already in the thermotherapy of brain cancer. Although it has been shown, that tumor cells take up these particles in vitro, little is known about the internalization routes. Understanding of the underlying uptake mechanisms would be very useful for faster and precise development of nanoparticles for clinical applications. This study aims at the identification of key proteins, which are crucial for the active uptake of iron oxide nanoparticles by HeLa cells (human cervical cancer as a model cell line. Cells were transfected with specific siRNAs against Caveolin-1, Dynamin 2, Flotillin-1, Clathrin, PIP5Kα and CDC42. Knockdown of Caveolin-1 reduces endocytosis of superparamagnetic iron oxide nanoparticles (SPIONs and silica-coated iron oxide nanoparticles (SCIONs between 23 and 41%, depending on the surface characteristics of the nanoparticles and the experimental design. Knockdown of CDC42 showed a 46% decrease of the internalization of PEGylated SPIONs within 24 h incubation time. Knockdown of Dynamin 2, Flotillin-1, Clathrin and PIP5Kα caused no or only minor effects. Hence endocytosis in HeLa cells of iron oxide nanoparticles, used in this study, is mainly mediated by Caveolin-1 and CDC42. It is shown here for the first time, which proteins of the endocytotic pathway mediate the endocytosis of silica-coated iron oxide nanoparticles in HeLa cells in vitro. In future studies more experiments should be carried out with different cell lines and other well-defined nanoparticle species to elucidate possible general principles.

  8. Bid and calpains cooperate to trigger oxaliplatin-induced apoptosis of cervical carcinoma HeLa cells.

    Science.gov (United States)

    Anguissola, Sergio; Köhler, Barbara; O'Byrne, Robert; Düssmann, Heiko; Cannon, Mary D; Murray, Frank E; Concannon, Caoimhin G; Rehm, Markus; Kögel, Donat; Prehn, Jochen H M

    2009-11-01

    The Bcl-2 homology 3-only protein Bid is an important mediator of death receptor-induced apoptosis. Recent reports and this study suggest that Bid may also mediate genotoxic drug-induced apoptosis of various human cancer cells. Here, we characterized the role of Bid and the mechanism of Bid activation during oxaliplatin-induced apoptosis of HeLa cervical cancer cells. Small hairpin RNA-mediated silencing of Bid protected HeLa cells against both death receptor- and oxaliplatin-induced apoptosis. Expression of a Bid mutant in which caspase-8 cleavage site was mutated (D59A) reactivated oxaliplatin-induced apoptosis in Bid-deficient cells but failed to reactivate death receptor-induced apoptosis, suggesting that caspase-8-mediated Bid cleavage did not contribute to oxaliplatin-induced apoptosis. Overexpression of bcl-2 or treatment with the pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-dl-Asp-fluoromethylketone abolished caspase-2, -8, -9, and -3 activation as well as Bid cleavage in response to oxaliplatin, suggesting that Bid cleavage occurred downstream of mitochondrial permeabilization and was predominantly mediated by caspases. We also detected an early activation of calpains in response to oxaliplatin. Calpain inhibition reduced Bid cleavage, mitochondrial depolarization, and activation of caspase-9, -3, -2, and -8 in response to oxaliplatin. Further experiments, however, suggested that Bid cleavage by calpains was not a prerequisite for oxaliplatin-induced apoptosis: single-cell imaging experiments using a yellow fluorescent protein-Bid-cyan fluorescent protein probe demonstrated translocation of full-length Bid to mitochondria that was insensitive to calpain or caspase inhibition. Moreover, calpain inhibition showed a potent protective effect in Bid-silenced cells. In conclusion, our data suggest that calpains and Bid act in a cooperative, but mutually independent, manner to mediate oxaliplatin-induced apoptosis of HeLa cells.

  9. Pravastatin and simvastatin inhibit the adhesion, replication and proliferation of Toxoplasma gondii (RH strain) in HeLa cells.

    Science.gov (United States)

    Sanfelice, Raquel Arruda; da Silva, Suelen Santos; Bosqui, Larissa Rodrigues; Miranda-Sapla, Milena Menegazzo; Barbosa, Bellisa Freitas; Silva, Rafaela José; Ferro, Eloísa A Vieira; Panagio, Luciano Aparecido; Navarro, Italmar Teodorico; Bordignon, Juliano; Conchon-Costa, Ivete; Pavanelli, Wander Rogerio; Almeida, Ricardo Sergio; Costa, Idessania Nazareth

    2017-03-01

    The conventional treatment for toxoplasmosis with pyrimethamine and sulfadiazine shows toxic effects to the host, and it is therefore necessary to search for new drugs. Some studies suggest the use of statins, which inhibit cholesterol synthesis in humans and also the initial processes of isoprenoid biosynthesis in the parasite. Thus, the objective of this study was to evaluate the activity of the statins pravastatin and simvastatin in HeLa cells infected in vitro with the RH strain of T. gondii. HeLa cells (1×10(5)) were infected with T. gondii tachyzoites (5×10(5)) following two different treatment protocols. In the first protocol, T. gondii tachyzoites were pretreated with pravastatin (50 and 100μg/mL) and simvastatin (1.56 and 3.125μg/mL) for 30min prior to infection. In the second, HeLa cells were first infected (5×10(5)) with tachyzoites and subsequently treated with pravastatin and simvastatin for 24h at the concentrations noted above. Initially, we evaluated the cytotoxicity of drugs by the MTT assay, number of tachyzoites adhered to cells, number of infected cells, and viability of tachyzoites by trypan blue exclusion. The supernatant of the cell cultures was collected post-treatment for determination of the pattern of Th1/Th2/Th17 cytokines by cytometric bead array. There was no cytotoxicity to HeLa cells with 50 and 100μg/mL pravastatin and 1.56 and 3.125μg/mL simvastatin. There was no change in the viability of tachyzoites that received pretreatment. Regarding the pre- and post-treatment of the cells with pravastatin and simvastatin alone, there was a reduction in adhesion, invasion and proliferation of cells to T. gondii. As for the production of cytokines, we found that IL-6 and IL-17 were significantly reduced in cells infected with T. gondii and treated with pravastatin and simvastatin, when compared to control. Based on these results, we can infer that pravastatin and simvastatin alone possess antiproliferative effects on tachyzoites forms

  10. Sorting and identification of side population cells in the human cervical cancer cell line HeLa.

    Science.gov (United States)

    Qi, Wenjuan; Zhao, Chao; Zhao, Lijun; Liu, Ning; Li, Xiaoping; Yu, Weidong; Wei, Lihui

    2014-01-13

    Several reports have revealed that cancer stem cells (CSCs) exist in many types of solid tumors. Some studies have demonstrated that side population (SP) cells isolated from diverse cancer lines harbor cancer stem-like properties, but there are few reports examining the characteristic of SP cells in human cervical cancer. The aim of this study is 1) to find out a feasible way to detect the tumor stem-like cells in cervical cancer, and 2) to analyze the properties of the SP cells being sorted. Isolated SP and non-SP cells from human cervical cancer cell line Hela by Hoechst 33342 dying method and flow cytometry analysis. Observing morphology of SP and non-SP cells. The expression of various biomarkers putatively related to cancer stem cells were investigated by immucytochemistry of SP and non-SP cells. We also analyzed cell cycle and cell apoptosis for sorted cells. The oncogenicity of the SP and non-SP cells were analyzed by tumor formation in nonobesediabeti- c/severe combined immune- deficient (NOD/SCID) mice. The drug-resistant and radiation-resistant index between SP, non-SP and Hela cells was estimated by MTS assay. The fraction of SP cells in Hela was approximately 1.07 ± 0.32%. SP cells were smaller and rounder in shape than non-SP cells, and mostly showed colony-like growth. Immunocytochemistry showed that stem cell makers (Oct3/4, CD133, BCRP) were highly expressed in SP cells. Moreover, the number of apoptotic cells among non-SP cells (17.6 ± 3.7%) was significantly higher compared with that among SP cells (4.4 ± 1.2%). The HE staining of in vivo grown tumors result from SP cells showed more poor differentiation, though no significant differences were shown between SP and non-SP cells in NOD/SCID mice tumorigenicity. Furthermore, SP cells demonstrated a higher degree of drug resistance against trichostatin A (TSA) compared with that of non-SP and Hela cells. SP cells were also found to be more resistant against radiotherapy. SP cells

  11. Transport of NaYF4:Er3+, Yb3+ up-converting nanoparticles into HeLa cells

    Science.gov (United States)

    Sikora, Bożena; Fronc, Krzysztof; Kamińska, Izabela; Koper, Kamil; Szewczyk, Sebastian; Paterczyk, Bohdan; Wojciechowski, Tomasz; Sobczak, Kamil; Minikayev, Roman; Paszkowicz, Wojciech; Stępień, Piotr; Elbaum, Danek

    2013-06-01

    An effective, simple and practically useful method to incorporate fluorescent nanoparticles inside live biological cells was developed. The internalization time and concentration dependence of a frequently used liposomal transfection factor (Lipofectamine 2000) was studied. A user friendly, one-step technique to obtain water and organic solvent soluble Er3+ and Yb3+ doped NaYF4 nanoparticles coated with polyvinylpyrrolidone was obtained. Structural analysis of the nanoparticles confirmed the formation of nanocrystals of the desired sizes and spectral properties. The internalization of NaYF4 nanoparticles in HeLa cervical cancer cells was determined at different nanoparticle concentrations and for incubation periods from 3 to 24 h. The images revealed a redistribution of nanoparticles inside the cell, which increases with incubation time and concentration levels, and depends on the presence of the transfection factor. The study identifies, for the first time, factors responsible for an effective endocytosis of the up-converting nanoparticles to HeLa cells. Thus, the method could be applied to investigate a wide range of future ‘smart’ theranostic agents. Nanoparticles incorporated into the liposomes appear to be very promising fluorescent probes for imaging real-time cellular dynamics.

  12. Multiple origins of spontaneously arising micronuclei in HeLa cells: Direct evidence from long-term live cell imaging

    Energy Technology Data Exchange (ETDEWEB)

    Rao Xiaotang; Zhang Yingyin; Yi Qiyi; Hou Heli; Xu Bo; Chu Liang; Huang Yun; Zhang Wenrui [Laboratory of Molecular and Cell Genetics, Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027 (China); Fenech, Michael [CSIRO Human Nutrition, PO Box 10041, Adelaide BC, Adelaide, SA 5000 (Australia); Shi Qinghua [Laboratory of Molecular and Cell Genetics, Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027 (China)], E-mail: qshi@ustc.edu.cn

    2008-11-10

    Although micronuclei (MNi) are extensively used to evaluate genotoxic effects and chromosome instability, the most basic issue regarding their origins has not been completely addressed due to limitations of traditional methods. Recently, long-term live cell imaging was developed to monitor the dynamics of single cell in a real-time and high-throughput manner. In the present study, this state-of-the-art technique was employed to examine spontaneous micronucleus (MN) formation in untreated HeLa cells. We demonstrate that spontaneous MNi are derived from incorrectly aligned chromosomes in metaphase (displaced chromosomes, DCs), lagging chromosomes (LCs) and broken chromosome bridges (CBs) in later mitotic stages, but not nuclear buds in S phase. However, most of bipolar mitoses with DCs (91.29%), LCs (73.11%) and broken CBs (88.93%) did not give rise to MNi. Our data also show directly, for the first time, that MNi could originate spontaneously from (1) MNi already presented in the mother cells; (2) nuclear fragments that appeared during mitosis with CB; and (3) chromosomes being extruded into a minicell which fused with one of the daughter cells later. Quantitatively, most of MNi originated from LCs (63.66%), DCs (10.97%) and broken CBs (9.25%). Taken together, these direct evidences show that there are multiple origins for spontaneously arising MNi in HeLa cells and each mechanism contributes to overall MN formation to different extents.

  13. Caspase-3-dependent apoptosis of citreamicin ε-induced heLa iells Is associated with reactive oxygen species generation

    KAUST Repository

    Liu, Lingli

    2013-07-15

    Citreamicins, members of the polycyclic xanthone family, are promising antitumor agents that are produced by Streptomyces species. Two diastereomers, citreamicin ε A (1) and B (2), were isolated from a marine-derived Streptomyces species. The relative configurations of these two diastereomers were determined using NMR spectroscopy and successful crystallization of citreamicin ε A (1). Both diastereomers showed potent cytotoxic activity against HeLa (cervical cancer) and HepG2 (hepatic carcinoma) cells with IC 50 values ranging from 30 to 100 nM. The terminal deoxynucleotidyl transferase dUTP nick-end labeling assay confirmed that citreamicin ε A (1) induced cellular apoptosis, and Western blot analysis showed that apoptosis occurred via activation of caspase-3. The 2,7-dichlorofluorescein diacetate assay indicated that citreamicin ε substantially increased the intracellular concentration of reactive oxygen species (ROS). To confirm the hypothesis that citreamicin ε induced apoptosis through an increase in the intracellular ROS concentration, the oxidized products, oxicitreamicin ε A (3) and B (4), were obtained from a one-step reaction catalyzed by Ag 2O. These products, with a reduced capacity to increase the intracellular ROS concentration, exhibited a significantly weakened cytotoxicity in both HeLa and HepG2 cells compared with that of citreamicin ε A (1) and B (2). © 2013 American Chemical Society.

  14. A new paradigm for MAPK: structural interactions of hERK1 with mitochondria in HeLa cells.

    Directory of Open Access Journals (Sweden)

    Soledad Galli

    Full Text Available Extracellular signal-regulated protein kinase 1 and 2 (ERK1/2 are members of the MAPK family and participate in the transduction of stimuli in cellular responses. Their long-term actions are accomplished by promoting the expression of specific genes whereas faster responses are achieved by direct phosphorylation of downstream effectors located throughout the cell. In this study we determined that hERK1 translocates to the mitochondria of HeLa cells upon a proliferative stimulus. In the mitochondrial environment, hERK1 physically associates with (i at least 5 mitochondrial proteins with functions related to transport (i.e. VDAC1, signalling, and metabolism; (ii histones H2A and H4; and (iii other cytosolic proteins. This work indicates for the first time the presence of diverse ERK-complexes in mitochondria and thus provides a new perspective for assessing the functions of ERK1 in the regulation of cellular signalling and trafficking in HeLa cells.

  15. tRNA modifying enzymes, NSUN2 and METTL1, determine sensitivity to 5-fluorouracil in HeLa cells.

    Directory of Open Access Journals (Sweden)

    Mayumi Okamoto

    2014-09-01

    Full Text Available Nonessential tRNA modifications by methyltransferases are evolutionarily conserved and have been reported to stabilize mature tRNA molecules and prevent rapid tRNA decay (RTD. The tRNA modifying enzymes, NSUN2 and METTL1, are mammalian orthologs of yeast Trm4 and Trm8, which are required for protecting tRNA against RTD. A simultaneous overexpression of NSUN2 and METTL1 is widely observed among human cancers suggesting that targeting of both proteins provides a novel powerful strategy for cancer chemotherapy. Here, we show that combined knockdown of NSUN2 and METTL1 in HeLa cells drastically potentiate sensitivity of cells to 5-fluorouracil (5-FU whereas heat stress of cells revealed no effects. Since NSUN2 and METTL1 are phosphorylated by Aurora-B and Akt, respectively, and their tRNA modifying activities are suppressed by phosphorylation, overexpression of constitutively dephosphorylated forms of both methyltransferases is able to suppress 5-FU sensitivity. Thus, NSUN2 and METTL1 are implicated in 5-FU sensitivity in HeLa cells. Interfering with methylation of tRNAs might provide a promising rationale to improve 5-FU chemotherapy of cancer.

  16. EFEK SITOTOKSIK DAN PEMACUAN APOPTOSIS FRAKSI PETROLEUM ETER EKSTRAK ETANOL DAUN TAPAK LIMAN (Elephantopus scaber Linn TERHADAP SEL HELA

    Directory of Open Access Journals (Sweden)

    Yeni Listyowati

    2013-11-01

    Full Text Available Elephantopus scaber Linn. has been reported to have cytotoxic effects on breast cancer cells and the potential to be developed as anticancer agent. This study aims was to determine the cytotoxic activity and apoptosis induction effect of petroleum ether fractions of ethanolic extract of (Elephantopus scaber Linn leaves against cervical cancer cells (HeLa. Petroleum ether fraction was obtained by dissolving eyhanolic extract in petroleum ether, and the soluble fraction was as petroleum ether fraction. The method used for cytotoxic activity test was MTT test. The concentration series used were 2000; 1500; 1000; 800; 400; 200; 100; 50; 25; 12.5; 6.25 and 3.125 mg/ml. The IC50 used as cytotoxic parameters. The apoptotic observations was conducted using acridine orange and ethidium bromide. The study showed the IC50 of petroleum ether fraction of (Elephantopus scaber Linn ethanolic extract was 185 ug/ml. The study also showed the potency to stimulate apoptosis in HeLa cells.

  17. Blue light induced reactive oxygen species from flavin mononucleotide and flavin adenine dinucleotide on lethality of HeLa cells.

    Science.gov (United States)

    Yang, Ming-Yeh; Chang, Chih-Jui; Chen, Liang-Yü

    2017-08-01

    Photodynamic therapy (PDT) is a safe and non-invasive treatment for cancers and microbial infections. Various photosensitizers and light sources have been developed for clinical cancer therapies. Flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) are the cofactor of enzymes and are used as photosensitizers in this study. Targeting hypoxia and light-triggering reactive oxygen species (ROS) are experimental strategies for poisoning tumor cells in vitro. HeLa cells are committed to apoptosis when treated with FMN or FAD and exposed to visible blue light (the maximum emitted wavelength of blue light is 462nm). Under blue light irradiation at 3.744J/cm2 (=0.52mW/cm2 irradiated for 2h), the minimal lethal dose is 3.125μM and the median lethal doses (LD50) for FMN and FAD are 6.5μM and 7.2μM, respectively. Individual exposure to visible blue light irradiation or riboflavin photosensitizers does not produce cytotoxicity and no side effects are observed in this study. The western blotting results also show that an intrinsic apoptosis pathway is activated by the ROS during photolysis of riboflavin analogues. Blue light triggers the cytotoxicity of riboflavins on HeLa cells in vitro. Based on these results, this is a feasible and efficient of PDT with an intrinsic photosensitizer for cancer research. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. The study of single anticancer peptides interacting with HeLa cell membranes by single molecule force spectroscopy

    Science.gov (United States)

    Shan, Yuping; Huang, Jinfeng; Tan, Juanjuan; Gao, Gui; Liu, Shuheng; Wang, Hongda; Chen, Yuxin

    2012-02-01

    To determine the effects of biophysical parameters (e.g. charge, hydrophobicity, helicity) of peptides on the mechanism of anticancer activity, we applied a single molecule technique--force spectroscopy based on atomic force microscope (AFM)--to study the interaction force at the single molecule level. The activity of the peptide and analogs against HeLa cells exhibited a strong correlation with the hydrophobicity of peptides. Our results indicated that the action mode between α-helical peptides and cancer cells was largely hydrophobicity-dependent.To determine the effects of biophysical parameters (e.g. charge, hydrophobicity, helicity) of peptides on the mechanism of anticancer activity, we applied a single molecule technique--force spectroscopy based on atomic force microscope (AFM)--to study the interaction force at the single molecule level. The activity of the peptide and analogs against HeLa cells exhibited a strong correlation with the hydrophobicity of peptides. Our results indicated that the action mode between α-helical peptides and cancer cells was largely hydrophobicity-dependent. Electronic supplementary information (ESI) available: Peptide design, biophysical properties, biological activities and experimental section. See DOI: 10.1039/c2nr11541g

  19. On-Line Monitoring the Growth of E. coli or HeLa Cells Using an Annular Microelectrode Piezoelectric Biosensor.

    Science.gov (United States)

    Tong, Feifei; Lian, Yan; Han, Junliang

    2016-12-18

    Biological information is obtained from the interaction between the series detection electrode and the organism or the physical field of biological cultures in the non-mass responsive piezoelectric biosensor. Therefore, electric parameter of the electrode will affect the biosensor signal. The electric field distribution of the microelectrode used in this study was simulated using the COMSOL Multiphysics analytical tool. This process showed that the electric field spatial distribution is affected by the width of the electrode finger or the space between the electrodes. In addition, the characteristic response of the piezoelectric sensor constructed serially with an annular microelectrode was tested and applied for the continuous detection of Escherichia coli culture or HeLa cell culture. Results indicated that the piezoelectric biosensor with an annular microelectrode meets the requirements for the real-time detection of E. coli or HeLa cells in culture. Moreover, this kind of piezoelectric biosensor is more sensitive than the sensor with an interdigital microelectrode. Thus, the piezoelectric biosensor acts as an effective analysis tool for acquiring online cell or microbial culture information.

  20. Ectopic overexpression of LAPTM5 results in lysosomal targeting and induces Mcl-1 down-regulation, Bak activation, and mitochondria-dependent apoptosis in human HeLa cells.

    Directory of Open Access Journals (Sweden)

    Do Youn Jun

    Full Text Available Human lysosomal-associated protein multispanning membrane 5 (LAPTM5 was identified by an ordered differential display-polymerase chain reaction (ODD-PCR as an up-regulated cDNA fragment during 12-O-tetradecanoylphorbol 13-acetate (TPA-induced differentiation of U937 cells into monocytes/macrophages. After TPA-treatment, the levels of LAPTM5 mRNA and protein increased and reached a maximum at 18-36 h. In healthy human tissues, LAPTM5 mRNA was expressed at high levels in hematopoietic cells and tissues, at low levels in the lung and fetal liver, and was not detected in other non-hematopoietic tissues. LAPTM5 mRNA was detected in immature malignant cells of myeloid lineage, such as K562, HL-60, U937, and THP-1 cells, and in unstimulated peripheral T cells, but was absent or barely detectable in lymphoid malignant or non-hematopoietic malignant cells. The LAPTM5 level in HL-60 cells increased more significantly during TPA-induced monocyte/macrophage differentiation than during DMSO-induced granulocyte differentiation. Ectopic expression of GFP-LAPTM5 or LAPTM5 in HeLa cells exhibited the localization of LAPTM5 to the lysosome. In HeLa cells overexpressing LAPTM5, the Mcl-1 and Bid levels declined markedly and apoptosis was induced via Bak activation, Δψm loss, activation of caspase-9, -8 and -3, and PARP degradation without accompanying necrosis. However, these LAPTM5-induced apoptotic events except for the decline of Bid level were completely abrogated by concomitant overexpression of Mcl-1. The pan-caspase inhibitor (z-VAD-fmk could suppress the LAPTM5-induced apoptotic sub-G1 peak by ~40% but failed to block the induced Δψm loss, whereas the broad-range inhibitor of cathepsins (Cathepsin Inhibitor I could suppress the LAPTM5-induced apoptotic sub-G1 peak and Δψm loss, by ~22% and ~23%, respectively, suggesting that the LAPTM5-mediated Δψm loss was exerted at least in part in a cathepsin-dependent manner. Together, these results

  1. Characterisation of ribosomal proteins from HeLa and Krebs II mouse ascites tumor cells by different two-dimensional polyacrylamide gel electrophoresis techniques

    DEFF Research Database (Denmark)

    Issinger, O G; Beier, H

    1978-01-01

    Electrophoresis of ribosomal proteins according to Kaltschmidt and Wittmann, 1970a, b (pH 8.6/pH 4.5 urea system) yielded 29 proteins for the small subunits and 35 and 37 proteins for the large subunits of Krebs II ascites and HeLa ribosomes, respectively. Analysis of the proteins according...... to a modified technique by Mets and Bogorad (1974) (pH 4.5/pH 8.6 SDS system) revealed 28 and 29 proteins in the small subunits and 37 and 38 proteins in the large subunits of Krebs II ascites and HeLa ribosomes. The molecular weights of the individual proteins were determined by: 1. "three-dimensional" gel...... using the pH 4.5/pH 8.6 SDS system. The molecular weights Krebs II ascites and HeLa ribosomal proteins are compared with those obtained by other authors for different mammalian species....

  2. Differential effects of calcium antagonists on ABCG2/BCRP-mediated drug resistance and transport in SN-38-resistant HeLa cells.

    Science.gov (United States)

    Takara, Kohji; Matsubara, Mika; Yamamoto, Kazuhiro; Minegaki, Tetsuya; Takegami, Shigehiko; Takahashi, Minoru; Yokoyama, Teruyoshi; Okumura, Katsuhiko

    2012-03-01

    The effects of 9 calcium antagonists on ABCG2/BCRP-mediated resistance and transport were examined in HeLa and SN-38-resistant HeLa (HeLa/SN100) cells, overexpressing ABCG2/BCRP. Sensitivity to mitoxantrone, an ABCG2/BCRP substrate, in HeLa/SN100 cells was significantly reversed by the coexistence of the calcium antagonists, except for diltiazem and verapamil. The accelerated transport activity of Hoechst33342, an ABCG2/BCRP substrate, in HeLa/SN100 cells was significantly decreased by the presence of the calcium antagonists, except for diltiazem, nifedipine or verapamil, returning to the level of HeLa cells. The present study classifies the calcium antagonists into 3 categories: strong (benidipine, felodipine, nicardipine, nisoldipine and nitrendipine), moderate (amlodipine and nifedipine) and weak (diltiazem and verapamil) inhibitors of ABCG2/BCRP.

  3. Cervical Cancer HeLa Cell Autocrine Apoptosis Induced by Co-immobilized TNF-α plus IFN-γ Biomaterials.

    Science.gov (United States)

    Li, Jian; Zhang, Yuxiao; Chen, Liyi; Lu, Xinhua; Li, Zhibin; Xue, Yongyong; Guan, Yan-Qing

    2018-02-13

    Based on our earlier work with co-immobilized TNF-α plus IFN-γ induction of HeLa cell death, we used FRET and XRD techniques to examine the differences in the structure of co-immobilized TNF-α plus IFN-γ and free TNF-α plus IFN-γ. The expressions of both TNF-α and IFN-α increased significantly, as determined by gene microarray analysis, however, in the presence of TNF-α plus IFN-α inhibitors, TNF-α and IFN-α did not increase in HeLa cells induced by co-immobilized TNF-γ plus IFN-α. This suggests that the TNF-α and IFN-α are the results of autocrine signaling in HeLa cells, and that cell death is caused by these substances. The mortality was then measured by flow cytometry and the results demonstrate that co-immobilized TNF-α plus IFN-γ induced death is a type of autocrine-induced apoptosis in HeLa cells. In addition, we performed Elisa, RT-PCR, flow cytometry, and western blot analyses, as well as a series of analytical tests at the animal level. We demonstrated that at both the cellular and animal levels, HeLa cells induced by co-immobilized IFN-γ plus TNF-α secrete much more IFN-α and TNF-α, than these two cytokines could exert on HeLa cells alone, and that this leads to cancer cell death.

  4. Interaction of C60 fullerene complexed to doxorubicin with model bilipid membranes and its uptake by HeLa cells.

    Science.gov (United States)

    Prylutskyy, Yu; Bychko, A; Sokolova, V; Prylutska, S; Evstigneev, M; Rybalchenko, V; Epple, M; Scharff, P

    2016-02-01

    With an aim to elucidate the effects of C60 fullerene complexed with antibiotic doxorubicin (Dox) on model bilipid membranes (BLM), the investigation of the electrical properties of BLM under the action of Dox and C60 fullerene, and of their complex, C60+Dox,was performed. The complex as well as its components exert a clearly detectable influence on BLM, which is concentration-dependent and also depends on phospholipid composition. The mechanism of this effect originates either from intermolecular interaction of the drug with fatty-acid residues of phospholipids, or from membranotropic effects of the drug-induced lipid peroxidation, or from the sum of these two effects. By fluorescence microscopy the entering of C60 + Dox complex into HeLa cells was directly shown.

  5. Labeling of HeLa cells using ZrO2:Yb3+-Er3+ nanoparticles with upconversion emission

    Science.gov (United States)

    Ceja-Fdez, Andrea; López-Luke, Tzarara; Oliva, Jorge; Vivero-Escoto, Juan; Gonzalez-Yebra, Ana Lilia; Rojas, Ruben A. Rodriguez; Martínez-Pérez, Andrea; de la Rosa, Elder

    2015-04-01

    This work reports the synthesis, structural characterization, and optical properties of ZrO2:Yb3+-Er3+ (2-1 mol%) nanocrystals. The nanoparticles were coated with 3-aminopropyl triethoxysilane (APTES) and further modified with biomolecules, such as Biotin-Anti-rabbit (mouse IgG) and rabbit antibody-AntiKi-67, through a conjugation method. The conjugation was successfully confirmed by Fourier transform infrared, zeta potential, and dynamic light scattering. The internalization of the conjugated nanoparticles in human cervical cancer (HeLa) cells was followed by two-photon confocal microscopy. The ZrO2:Yb3+-Er3+ nanocrystals exhibited strong red emission under 970-nm excitation. Moreover, the luminescence change due to the addition of APTES molecules and biomolecules on the nanocrystals was also studied. These results demonstrate that ZrO2:Yb3+-Er3+ nanocrystals can be successfully functionalized with biomolecules to develop platforms for biolabeling and bioimaging.

  6. Haemagglutinins and adherence properties to HeLa and intestine 407 cells of Klebsiella pneumoniae and Klebsiella oxytoca isolates.

    Science.gov (United States)

    Podschun, R; Heineken, P; Sonntag, H G

    1987-03-01

    The occurrence of haemagglutination (HA) and adherence properties were examined in 50 strains of K. pneumoniae and 17 K. oxytoca strains isolated from humans. All isolates except three exhibited HA activity. Mannose-sensitive haemagglutinins (MSHA) were expressed by the majority of K. pneumoniae strains, but only by one K. oxytoca isolate. Mannose-resistant haemagglutination (MRHA) to human or guinea pig erythrocytes could not be detected; haemagglutinins of the MR/K type were found in both species with similar frequencies. Adhesive properties could be demonstrated in K. pneumoniae as often as in K. oxytoca: About half of the strains adhered to two human cell lines: HeLa and Intestine 407. The incidence of HA activity was similar in adhering and nonadhering strains. A correlation between MSHA, MR/K-HA and adherence to tissue-cultured cells could not be detected.

  7. Sulfated fucan from marine alga inhibits HeLa cells infection by HTLV-1 free particles: semi-quantitative analysis

    Directory of Open Access Journals (Sweden)

    Maria T. V. Romanos

    2011-04-01

    Full Text Available A sulfated fucan from Laminaria abyssalis marine alga prevented the interaction of HTLV-1 particles, purified from the MT-2 cell line, with HeLa cells. The infection obtained using a concentrated virus suspension was detected only by amplification of the newly synthesized HTLV-1 proviral cDNA by the nested-polymerase chain reaction (PCR. The sulfated polysaccharide was not toxic to the cells at a concentration of 100 µg/mL and prevented infection by the viral particles when added to the cell monolayers. The proviral cDNA was only detected when the sulfated polysaccharide was added to the cells three hours post-infection, indicating that the inhibitory activity occurred in the initial stages of virus-cell interaction. Our results demonstrate, for the first time, the ability of a sulfated fucan from marine algae to inhibit virus transmission through free virus particles.

  8. Gene expression responses of HeLa cells to chemical species generated by an atmospheric plasma flow

    Energy Technology Data Exchange (ETDEWEB)

    Yokoyama, Mayo, E-mail: yokoyama@plasma.ifs.tohoku.ac.jp [Institute of Fluid Science, Tohoku University, 2-1-1 Katahira, Aoba-ku, Sendai 980-8577 (Japan); Johkura, Kohei, E-mail: kohei@shinshu-u.ac.jp [Department of Histology and Embryology, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto 390-8621 (Japan); Sato, Takehiko, E-mail: sato@ifs.tohoku.ac.jp [Institute of Fluid Science, Tohoku University, 2-1-1 Katahira, Aoba-ku, Sendai 980-8577 (Japan)

    2014-08-08

    Highlights: • Response of HeLa cells to a plasma-irradiated medium was revealed by DNA microarray. • Gene expression pattern was basically different from that in a H{sub 2}O{sub 2}-added medium. • Prominently up-/down-regulated genes were partly shared by the two media. • Gene ontology analysis showed both similar and different responses in the two media. • Candidate genes involved in response to ROS were detected in each medium. - Abstract: Plasma irradiation generates many factors able to affect the cellular condition, and this feature has been studied for its application in the field of medicine. We previously reported that hydrogen peroxide (H{sub 2}O{sub 2}) was the major cause of HeLa cell death among the chemical species generated by high level irradiation of a culture medium by atmospheric plasma. To assess the effect of plasma-induced factors on the response of live cells, HeLa cells were exposed to a medium irradiated by a non-lethal plasma flow level, and their gene expression was broadly analyzed by DNA microarray in comparison with that in a corresponding concentration of 51 μM H{sub 2}O{sub 2}. As a result, though the cell viability was sufficiently maintained at more than 90% in both cases, the plasma-medium had a greater impact on it than the H{sub 2}O{sub 2}-medium. Hierarchical clustering analysis revealed fundamentally different cellular responses between these two media. A larger population of genes was upregulated in the plasma-medium, whereas genes were downregulated in the H{sub 2}O{sub 2}-medium. However, a part of the genes that showed prominent differential expression was shared by them, including an immediate early gene ID2. In gene ontology analysis of upregulated genes, the plasma-medium showed more diverse ontologies than the H{sub 2}O{sub 2}-medium, whereas ontologies such as “response to stimulus” were common, and several genes corresponded to “response to reactive oxygen species.” Genes of AP-1 proteins, e.g., JUN

  9. Red-luminescent europium (III) doped silica nanoshells: synthesis, characterization, and their interaction with HeLa cells

    Science.gov (United States)

    Yang, Jian; Sandoval, Sergio; Alfaro, Jesus G.; Aschemeyer, Sharraya; Liberman, Alex; Martin, David T.; Makale, Milan; Kummel, Andrew C.; Trogler, William C.

    2011-01-01

    A simple method to fabricate Eu3+ doped silica nanoshells particles with 100 and 200 nm diameters is reported. Amino polystyrene beads were used as templates, and an 8 to 10 nm thick silica gel coating was formed by the sol-gel reaction. After removing the template by calcination, porous dehydrated silica gel nanoshells of uniform size were obtained. The Eu3+ doped silica nanoshells exhibited a red emission at 615 nm on UV excitation. The porous structure of the silica shell wall was characterized by transmission electron microscopy measurements, while particle size and zeta potentials of the particles suspended in aqueous solution were characterized by dynamic light scattering. Two-photon microscopy was used to image the nanoshells after assimilation by HeLa cancer cells. PMID:21721813

  10. Cytotoxic evaluation of volatile oil from Descurainia sophia seeds on MCF-7 and HeLa cell lines.

    Science.gov (United States)

    Khodarahmi, E; Asghari, G H; Hassanzadeh, F; Mirian, M; Khodarahmi, G A

    2015-01-01

    Descurainia sophia is a plant widely distributed and used as folk medicine throughout the world. Different extracts of aerial parts and seeds of this plant have been shown to inhibit the growth of different cancer cell lines in vitro. In this study, cytotoxic activity of D. sophia seed volatile oil was evaluated. D. sophia seed powder was mixed with distilled water and left at 25 °C for 17 h (E1), 23 h (E2) and 28 h (E3) to autolyse. Then, the volatile fractions of E1, E2, and E3 were collected after steam distillation for 3 h. Cytotoxic effects of the volatile oils alone or in combination with doxorubicin (mixture of E1 or E2 at 50 μg/ml or E1 at 100 μg/ml with doxorubicin at 0.1, 1, 10 μM) against MCF-7 cell line were determined using MTT assay. Cytotoxic effect of E1 volatile oil was also determined on HeLa cell line. The results indicated that 1-buten-4-isothiocyanate was the major isothiocyanate found in the volatile oils. The results of cytotoxic evaluations showed that volatile constituents were more toxic on MCF-7 cells with IC50 100 μg/ml. No significant differences were observed between cytotoxic activities of E1, E2 and E3 on MCF-7 cell line. Concomitant use of E1 and E2 (50 μg/ml) with doxurubicin (1 μM) significantly reduced the viability of MCF-7 cells compared to the negative control, doxorubicin alone, or each volatile fraction. The same result was obtained on HeLa cells, when E1 (100 μg/ml) was concurrently used with doxorubicin (1 μM).

  11. Inducible HSP70 antagonizes IL-1β cytocidal effects through inhibiting NF-kB activation via destabilizing TAK1 in HeLa cells.

    Directory of Open Access Journals (Sweden)

    Xiang Cao

    Full Text Available BACKGROUND: Despite several reports describing the HSP70-mediated cytoprotection against IL-1, the precise mechanism for this phenomenon remains to be determined. METHODS/PRINCIPAL FINDINGS: Here we used HeLa cells, a human epithelial carcinoma cell line, to evaluate the role of inducible HSP70 in response of IL-1β stimulation. We found that inducible HSP70 antagonized the cytotoxicity of IL-1β and improved the survival of HeLa cells. Further investigation demonstrated that increased expression level of inducible HSP70 reduced the complex of TAK1 and HSP90, and promoted the degradation of TAK1 protein via proteasome pathway. By overexpression and RNAi knockdown, we showed that inducible HSP70 modulated the NF-kB but not MAPKs signalings through influencing the stability of TAK1 protein in HeLa cells. Moreover, overexpression of HSP70 attenuated the production of iNOS upon IL-1β stimulation, validating that inducible HSP70 serves as a cytopretective factor to antagonize the cytocidal effects of IL-1β in HeLa cells. CONCLUSIONS/SIGNIFICANCE: Our observations provide evidence for a novel signaling mechanism involving HSP70, TAK1, and NF-κB in the response of IL-1β cytocidal effects. This research also provides insight into mechanisms by which HSP70 exerts its cytoprotective action upon toxic stimuli in tumor cells.

  12. Dynamically-enhanced retention of gold nanoclusters in HeLa cells following X-rays exposure: A cell cycle phase-dependent targeting approach.

    Science.gov (United States)

    Liu, Yan; Chen, Weiqiang; Zhang, Pengcheng; Jin, Xiaodong; Liu, Xinguo; Li, Ping; Li, Feifei; Zhang, Hongpeng; Zou, Guozhang; Li, Qiang

    2016-06-01

    Cell cycle phase could affect the cellular uptake of nanoparticles. Based on the fact that ionizing radiation exposure can delay cell cycle progression including inducing G2/M phase arrest, we propose that ionizing radiation exposure is a cell cycle phase-dependent targeting approach for intracellular delivery of nano-agents in tumor cells. We synthesized luminescent gold nanoclusters (AuNCs) using a one-pot green synthetic method. Subsequently, we used the as-prepared AuNCs as both "nano-agents" and fluorescent trafficking probes for our study using human cervical carcinoma HeLa cells. Estimating the cellular uptake of AuNCs and cell cycle analysis were performed following X-rays irradiation and cell synchronization. Our work showed that X-rays irradiation could delay the division of HeLa cells and thereby enhance the retention of AuNCs in HeLa cells, which is a reverse strategy compared with other studies on synergistic nano-radiotherapy. Our results demonstrated that the cell cycle synchronization influenced the cellular uptake processes of AuNCs, suggesting that dynamic cell cycle progression could affect the cellular uptake kinetics of AuNCs. We consider that the radiation-induced cell division delay might provide a possible mechanism underlying the enhanced effect for the cellular uptake of AuNCs in irradiated HeLa cells. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  13. Inhibition of nucleoside diphosphate kinase activity by in vitro phosphorylation by protein kinase CK2. Differential phosphorylation of NDP kinases in HeLa cells in culture

    DEFF Research Database (Denmark)

    Biondi, R M; Engel, M; Sauane, M

    1996-01-01

    that in vitro protein kinase CK2 catalyzed phosphorylation of human NDPK A inhibits its enzymatic activity by inhibiting the first step of its ping-pong mechanism of catalysis: its autophosphorylation. Upon in vivo 32P labeling of HeLa cells, we observed that both human NDPKs, A and B, were autophosphorylated...

  14. Oxidative stress-mediated cytotoxicity and apoptosis induction by TiO2 nanofibers in HeLa cells

    DEFF Research Database (Denmark)

    Ramkumar, Kunga Mohan; Manjula, Chinnasamy; GnanaKumar, Georgepeter

    2012-01-01

    attracted a lot of attention due to their possible negative health effects as suggested by their morphological similarities with asbestos. In the present study, therefore, toxicity of TiO2NFs was evaluated in human cervical adenocarcinoma HeLa cells. The TEM and XRD analyses showed that TiO2NFs used...

  15. Metallothionein isoform 2A expression is inducible and protects against ROS-mediated cell death in rotenone-treated HeLa cells.

    NARCIS (Netherlands)

    Reinecke, F.; Levanets, O.; Olivier, Y.; Louw, R.; Semete, B.; Grobler, A.; Hidalgo, J.; Smeitink, J.A.M.; Olckers, A.; Westhuizen, F.H. van der

    2006-01-01

    The role of MT (metallothionein) gene expression was investigated in rotenone-treated HeLa cells to induce a deficiency of NADH:ubiquinone oxidoreductase (complex I). Complex I deficiency leads to a diversity of cellular consequences, including production of ROS (reactive oxygen species) and

  16. Uptake of Shiga‐toxigenic Escherichia coli SubAB by HeLa cells requires an actin‐ and lipid raft‐dependent pathway

    National Research Council Canada - National Science Library

    Nagasawa, Sayaka; Ogura, Kohei; Tsutsuki, Hiroyasu; Saitoh, Hisako; Moss, Joel; Iwase, Hirotaro; Noda, Masatoshi; Yahiro, Kinnosuke

    2014-01-01

    ...‐dependent cell death. However, the SubAB uptake mechanism in HeLa cells is unknown. In this study, a variety of inhibitors and siRNAs were employed to characterize the SubAB uptake process. SubAB...

  17. Inhibitors of cysteine cathepsin and calpain do not prevent ultraviolet-B-induced apoptosis in human keratinocytes and HeLa cells

    DEFF Research Database (Denmark)

    Bang, Bo; Baadsgaard, Ole; Skov, Lone

    2004-01-01

    been demonstrated to play a role in the execution of programmed cell death induced by other stimuli, e.g. TNF-alpha. The purpose of the present study was therefore to investigate whether inhibitors of cysteine cathepsins and calpains could prevent UVB-induced apoptosis in HeLa cells and keratinocytes....... This was done by investigating the effect of the irreversible cysteine protease inhibitor zFA-fmk, the cathepsin B inhibitor CA-074-Me and the calpain inhibitor ALLN on the viability of UVB-irradiated human keratinocytes and HeLa cells. At concentrations of 10 microM and above zVAD-fmk conferred partial dose......-dependent protection against UVB-induced apoptosis in HeLa cells and keratinocytes. Moreover, caspase-3 activity was completely blocked at zVAD-fmk concentrations of 1 microM in HeLa cells. This indicates that caspase-independent mechanisms could be involved in UVB-induced apoptosis. However, the protease inhibitors z...

  18. Unique characteristics of AAV1, 2, and 5 viral entry, intracellular trafficking, and nuclear import define transduction efficiency in HeLa cells.

    Science.gov (United States)

    Keiser, Nicholas W; Yan, Ziying; Zhang, Yulong; Lei-Butters, Diana C M; Engelhardt, John F

    2011-11-01

    Biological differences between recombinant adeno-associated virus (rAAV) serotypes define their efficiencies in expressing a transgene in a particular target cell. Few studies have directly compared how differences in viral entry, intracellular trafficking, and nuclear import of rAAV serotypes influence the effectiveness of transduction in the same cell type. We evaluated these characteristics for three rAAV serotypes in HeLa cells, using biochemical techniques and fluorescence-based detection of multiple serotypes in the same cell. Although rAAV2 exhibited the slowest entry, intracellular trafficking, and nuclear import among the three serotypes, it elicited the highest levels of transduction. Conversely, rAAV1 exhibited more rapid entry and nuclear import than the other serotypes, yet was ineffective at transducing HeLa cells due to impaired capsid disassembly in the nucleus. rAAV5, which entered the cell less rapidly than rAAV1, was imported efficiently into the nucleus, but then rapidly degraded, resulting in poor transduction of HeLa cells. We conclude that rAAV1, 2, and 5 utilize distinct mechanisms for intracellular trafficking, and that post-nuclear events play an important role in determining the efficiency of HeLa cell transduction by these serotypes. Thus, overcoming post-nuclear barriers that limit uncoating and/or promote virion degradation may enhance the efficiency of certain AAV serotypes.

  19. Mutations in VP1 and 3A proteins improve binding and replication of rhinovirus C15 in HeLa-E8 cells.

    Science.gov (United States)

    Bochkov, Yury A; Watters, Kelly; Basnet, Sarmila; Sijapati, Shakher; Hill, Marchel; Palmenberg, Ann C; Gern, James E

    2016-12-01

    Viruses in the rhinovirus C species (RV-C) can cause severe respiratory illnesses in children including pneumonia and asthma exacerbations. A transduced cell line (HeLa-E8) stably expressing the CDHR3-Y529 receptor variant, supports propagation of RV-C after infection. C15 clinical or recombinant isolates replicate in HeLa-E8, however progeny yields are lower than those of related strains of RV-A and RV-B. Serial passaging of C15 in HeLa-E8 resulted in stronger cytopathic effects and increased (≥10-fold) virus binding to cells and progeny yields. The adaptation was acquired by two mutations which increased binding (VP1 T125K) and replication (3A E41K), respectively. A similar 3A mutation engineered into C2 and C41 cDNAs also improved viral replication (2-8 fold) in HeLa but the heparan sulfate mediated cell-binding enhancement by the VP1 change was C15-specific. The findings now enable large-scale cost-effective C15 production by infection and the testing of RV-C infectivity by plaque assay. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. [Comparison of the indirect immunofluorescence assay performance of Bartonella henselae antigens obtained by co-cultivation in Vero and HeLa cells].

    Science.gov (United States)

    Ergin, Cağrı; Akkaya, Yüksel; Kiriş Satılmış, Ozgün; Yılmaz, Cansev

    2011-07-01

    The laboratory diagnosis of Bartonella henselae infection is mainly based on serological testing by indirect immunofluorescence assay (IFA). Cell line co-cultivation with B.henselae and agar derivated antigens are the two major procedures used for evaluation of anti-Bartonella antibodies. Vero and Hep-2 cell lines are the most commonly used media for co-cultivation both in-house and commercial diagnostic kits production. However, HeLa cells which are easily supplied and grown, also can easily be infected by B.henselae. The aim of this study was to compare the performances of antigens obtained by co-cultivation of B.henselae ATCC 49882 (Houston-1) in Vero and HeLa Cells in IFA serology. Out of 381 sera samples, 127 (33.3%) were found positive and 195 (51.2%) were found negative by IFA performed by both cell line co-cultivations. The total agreement between the methods were found as 84.5% (322/381), and Cohen kappa value was calculated as 0.68 (strong, coherent). As a result, He-La cells were found to be useful for the preparation of B.henselae antigens to be used in IFA for the serologic diagnosis of B.henselae infections. However different genotype strains and cross reactions with other infectious agents should be investigated by further studies before routine applications of HeLa cell co-cultivations procedure is established.

  1. Polypeptide Fraction from Arca subcrenata Induces Apoptosis and G2/M Phase Arrest in HeLa Cells via ROS-Mediated MAPKs Pathways

    Directory of Open Access Journals (Sweden)

    Xianjing Hu

    2015-01-01

    Full Text Available Arca subcrenata is documented in the literature of marine Traditional Chinese Medicine. Polypeptide fraction from A. subcrenata, coded as P2, was demonstrated to possess significant antitumor activity in our previous study. However, the underlying mechanism remains undefined. The present study was carried out to investigate the underlying antitumor mechanism of P2 in human cervical cancer HeLa cells by MTT, FCM, LSCM, and western blot assays. The results revealed that P2 significantly induced apoptosis of HeLa cells in a concentration- and time-dependent manner. High level of ROS was provoked by P2, which was in turn responsible for induction of apoptosis through activation of intrinsic mitochondrial pathway and JNK1/2, p38 MAPK pathways, as well as inhibition of ERK1/2 pathway, as evidenced by the abrogation of P2’s effect on HeLa cells preincubated with the ROS scavenger NAC. P2 also was observed to display significant effect on G2/M phase arrest by downregulating the expression of cyclin B1/cdc2 complex and upregulating the expression of p21. These findings demonstrate that P2 induces apoptosis and G2/M phase arrest in HeLa cells through ROS-mediated MAPKs pathways, suggesting that P2 would be worth investigating as a promising agent within the scope of marine drugs for treatment of cervical cancer.

  2. Activation of the endogenous p53 growth inhibitory pathway in HeLa cervical carcinoma cells by expression of the bovine papillomavirus E2 gene.

    Science.gov (United States)

    Hwang, E S; Naeger, L K; DiMaio, D

    1996-02-15

    We previously showed that expression of the bovine papillomavirus (BPV) E2 gene results in a dramatic inhibition of the proliferation of several human cervical carcinoma cell lines, including HeLa cells which contain human papillomavirus (HPV) type 18 DNA. We have assessed the status of endogenous G1 cell cycle regulatory proteins, including the tumor suppressor proteins, p53 and p105Rb, in order to investigate growth regulatory pathways in HeLa cells following E2 expression. The p53 tumor suppressor protein is stabilized following the introduction of the E2 gene into HeLa cells. This results in the induction of the p53-responsive gene encoding the cyclin dependent kinase (cdk) inhibitor, p21/WAF1, complex formation between p21/WAF1 and cdk2 and reduction of in vitro cdk2/cyclin E kinase activity. The reduced cdk kinase activity is accompanied by the accumulation of the growth inhibitory hypophosphorylated form of the tumor suppressor protein, p105Rb. The level of the p105Rb-regulated transcription factor, E2F1, is reduced, as is transcription of a variety of E2F1-regulated genes, including B-myb. Thus, the p53 growth inhibitory pathway has evidently not accumulated mutations in HeLa cells but rather appears intact. However, this pathway remains dormant, until it is mobilized by appropriate manipulations, such as the expression of the BPV E2 protein.

  3. SPONTANEOUS AND MNNG-INDUCED REVERSION OF AN EGFP CONSTRUCT IN HELA CELLS: AN ASSAY FOR OBSERVING MUTATIONS IN LIVING CELLS BY FLUORESCENT MICROSCOPY

    Science.gov (United States)

    A HeLa cell line stably expressing the Enhanced Green Fluorescence Protein (EGFP) gene, interrupted by the IVS2-654 intron, was studied without treatment and after treatment with a single standard dose of 15 ?M of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). This assay was done ...

  4. Rose Bengal acetate photodynamic therapy (RBAc-PDT) induces exposure and release of Damage-Associated Molecular Patterns (DAMPs) in human HeLa cells.

    Science.gov (United States)

    Panzarini, Elisa; Inguscio, Valentina; Fimia, Gian Maria; Dini, Luciana

    2014-01-01

    The new concept of Immunogenic Cell Death (ICD), associated with Damage Associated Molecular Patterns (DAMPs) exposure and/or release, is recently becoming very appealing in cancer treatment. In this context, PhotoDynamic Therapy (PDT) can give rise to ICD and to immune response upon dead cells removal. The list of PhotoSensitizers (PSs) able to induce ICD is still short and includes Photofrin, Hypericin, Foscan and 5-ALA. The goal of the present work was to investigate if Rose Bengal Acetate (RBAc), a powerful PS able to trigger apoptosis and autophagy, enables photosensitized HeLa cells to expose and/or release pivotal DAMPs, i.e. ATP, HSP70, HSP90, HMGB1, and calreticulin (CRT), that characterize ICD. We found that apoptotic HeLa cells after RBAc-PDT exposed and released, early after the treatment, high amount of ATP, HSP70, HSP90 and CRT; the latter was distributed on the cell surface as uneven patches and co-exposed with ERp57. Conversely, autophagic HeLa cells after RBAc-PDT exposed and released HSP70, HSP90 but not CRT and ATP. Exposure and release of HSP70 and HSP90 were always higher on apoptotic than on autophagic cells. HMGB1 was released concomitantly to secondary necrosis (24 h after RBAc-PDT). Phagocytosis assay suggests that CRT is involved in removal of RBAc-PDT generated apoptotic HeLa cells. Altogether, our data suggest that RBAc has all the prerequisites (i.e. exposure and/or release of ATP, CRT, HSP70 and HSP90), that must be verified in future vaccination experiments, to be considered a good PS candidate to ignite ICD. We also showed tha CRT is involved in the clearance of RBAc photokilled HeLa cells. Interestingly, RBAc-PDT is the first cancer PDT protocol able to induce the translocation of HSP90 and plasma membrane co-exposure of CRT with ERp57.

  5. Molecular mechanism of inhibitory effects of C-phycocyanin combined with all-trans-retinoic acid on the growth of HeLa cells in vitro.

    Science.gov (United States)

    Yang, Fan; Li, Bing; Chu, Xian-Ming; Lv, Cong-Yi; Xu, Ying-Jie; Yang, Peng

    2014-06-01

    We studied the effects of all-trans-retinoic acid (ATRA), C-phycocyanin (C-PC), or ATRA+C-PC on the growth of cervical cells (HeLa cells), cell cycle distribution, and apoptosis. The anticancer mechanism of the drug combination was revealed. MTT assay was adopted to determine the effects of C-PC and ATRA on the growth of HeLa cells. The expression quantities of cyclin-dependent kinase (CDK) 4, cyclin D1, Bcl-2, caspase-3, and CD59 were determined by in situ hybridization, immunofluorescence, immunohistochemistry staining, Western blot, and RT-PCR. TUNEL assay was adopted to determine the cellular apoptosis levels. Both C-PC and ATRA could inhibit the growth of HeLa cells, and the combination of ATRA+C-PC functioned cooperatively to induce apoptosis in HeLa cells. The dosage of ATRA was reduced when it cooperated with C-PC to reduce the toxicity. ATRA treated with C-PC could induce more cell cycle arrests than the single drug used by decrease in cyclin D1 and CDK4 expression. The combination of the two drugs could upregulate caspase-3 and downregulate the Bcl-2 gene and induce cell apoptosis. Moreover, the combination therapy has an important immunological significance in decreased expression of the CD59 protein. Singly, C-PC or ATRA could inhibit the growth of HeLa cells, and the effects of treatment were further enhanced in the combination group. In combination with C-PC, the dosage of ATRA was effectively reduced. The C-PC + ATRA combination might take effect by inhibiting the progress of the cell cycle, inducing cell apoptosis and promoting complement-mediated cytolysis.

  6. Systems biology approach to transplant tolerance: proof of concept experiments using RNA interference (RNAi) to knock down hub genes in Jurkat and HeLa cells in vitro.

    Science.gov (United States)

    Lwin, Wint Wah; Park, Ken; Wauson, Matthew; Gao, Qin; Finn, Patricia W; Perkins, David; Khanna, Ajai

    2012-07-01

    Systems biology is gaining importance in studying complex systems such as the functional interconnections of human genes [1]. To investigate the molecular interactions involved in T cell immune responses, we used databases of physical gene-gene interactions to constructed molecular interaction networks (interconnections) with R language algorithms. This helped to identify highly interconnected "hub" genes AT(1)P5C1, IL6ST, PRKCZ, MYC, FOS, JUN, and MAPK1. We hypothesized that suppression of these hub genes in the gene network would result in significant phenotypic effects on T cells and examined this in vitro. The molecular interaction networks were then analyzed and visualized with Cytoscape. Jurkat and HeLa cells were transfected with siRNA for the selected hub genes. Cell proliferation was measured using ATP luminescence and BrdU labeling, which were measured 36, 72, and 96 h after activation. Following T cell stimulation, we found a significant decrease in ATP production (P cells. However, HeLa cells showed a significant (P cell proliferation when the genes MAPK1, IL6ST, ATP5C1, JUN, and FOS were knocked down. In both Jurkat and HeLa cells, targeted gene knockdown using siRNA showed decreased cell proliferation and ATP production in both Jurkat and HeLa cells. However, Jurkat T cells and HELA cells use different hub genes to regulate activation responses. This experiment provides proof of principle of applying siRNA knockdown of T cell hub genes to evaluate their proliferative capacity and ATP production. This novel concept outlines a systems biology approach to identify hub genes for targeted therapeutics. Published by Elsevier Inc.

  7. A systems biology analysis of the changes in gene expression via silencing of HPV-18 E1 expression in HeLa cells.

    Science.gov (United States)

    Castillo, Andres; Wang, Lu; Koriyama, Chihaya; Eizuru, Yoshito; Jordan, King; Akiba, Suminori

    2014-10-01

    Previous studies have reported the detection of a truncated E1 mRNA generated from HPV-18 in HeLa cells. Although it is unclear whether a truncated E1 protein could function as a replicative helicase for viral replication, it would still retain binding sites for potential interactions with different host cell proteins. Furthermore, in this study, we found evidence in support of expression of full-length HPV-18 E1 mRNA in HeLa cells. To determine whether interactions between E1 and cellular proteins play an important role in cellular processes other than viral replication, genome-wide expression profiles of HPV-18 positive HeLa cells were compared before and after the siRNA knockdown of E1 expression. Differential expression and gene set enrichment analysis uncovered four functionally related sets of genes implicated in host defence mechanisms against viral infection. These included the toll-like receptor, interferon and apoptosis pathways, along with the antiviral interferon-stimulated gene set. In addition, we found that the transcriptional coactivator E1A-binding protein p300 (EP300) was downregulated, which is interesting given that EP300 is thought to be required for the transcription of HPV-18 genes in HeLa cells. The observed changes in gene expression produced via the silencing of HPV-18 E1 expression in HeLa cells indicate that in addition to its well-known role in viral replication, the E1 protein may also play an important role in mitigating the host's ability to defend against viral infection.

  8. Confident site localization using a simulated phosphopeptide spectral library.

    Science.gov (United States)

    Suni, Veronika; Imanishi, Susumu Y; Maiolica, Alessio; Aebersold, Ruedi; Corthals, Garry L

    2015-05-01

    We have investigated if phosphopeptide identification and simultaneous site localization can be achieved by spectral library searching. This allows taking advantage of comparison of specific spectral features, which would lead to improved discrimination of differential localizations. For building a library, we propose a spectral simulation strategy where all possible single phosphorylations can be simply and accurately (re)constructed on enzymatically dephosphorylated peptides, by predicting the diagnostic fragmentation events produced in beam-type CID. To demonstrate the performance of our approach, enriched HeLa phosphopeptides were dephosphorylated with alkaline phosphatase and analyzed with higher energy collisional dissociation (HCD), which were then used for creating a spectral library of simulated phosphopeptides. Spectral library searching using SpectraST was performed on data sets of synthetic phosphopeptides and the HeLa phosphopeptides, and subsequently compared to Mascot and Sequest database searching followed by phosphoRS and Ascore afforded localization, respectively. Our approach successfully led to accurate localization, and it outperformed other methods, when phosphopeptides were covered by the library. These results suggest that the searching with simulated spectral libraries serves as a crucial approach for both supplementing and validating the phosphorylation sites obtained by database searching and localization tools. For future development, simulation of multiply phosphorylated peptides remains to be implemented.

  9. Intracellular trafficking mechanism of cationic phospholipids including cationic liposomes in HeLa cells.

    Science.gov (United States)

    Un, K; Sakai-Kato, K; Goda, Y

    2014-07-01

    The development of gene delivery methods is essential for the achievement of effective gene therapy. Elucidation of the intracellular transfer mechanism for cationic carriers is in progress, but there are few reports regarding the intracellular trafficking processes of the cationic phospholipids taken up into cells. In the present work, the trafficking processes of a cationic phospholipid (1,2-dioleoyl-3-trimethylammonium-propane, DOTAP) were investigated from intracellular uptake to extracellular efflux using cationic liposomes in vitro. Following intracellular transport of liposomes via endocytosis, DOTAP was localized in the endoplasmic reticulum, Golgi apparatus, and mitochondria. Moreover, the proteins involved in DOTAP intracellular trafficking and extracellular efflux were identified. In addition, helper lipids of cationic liposomes were found to partially affect this intracellulartrafficking. These findings might provide valuable information for designing cationic carriers and avoiding unexpected toxic side effects derived from cationic liposomal components.

  10. Acyl-CoA-binding protein (ACBP) localizes to the endoplasmic reticulum and Golgi in a ligand-dependent manner in mammalian cells

    DEFF Research Database (Denmark)

    Hansen, Jesper S; Færgeman, Nils J; Kragelund, Birthe B

    2008-01-01

    In the present study, we microinjected fluorescently labelled liver bovine ACBP (acyl-CoA-binding protein) [FACI-50 (fluorescent acyl-CoA indicator-50)] into HeLa and BMGE (bovine mammary gland epithelial) cell lines to characterize the localization and dynamics of ACBP in living cells. Results s...

  11. Local stimulation of cultured myocyte cells by femtosecond laser-induced stress wave

    Science.gov (United States)

    Kuo, Yung-En; Wu, Cheng-Chi; Hosokawa, Yoichiroh; Maezawa, Yasuyo; Okano, Kazunori; Masuhara, Hiroshi; Kao, Fu-Jen

    2010-12-01

    When an 800 nm femtosecond laser is tightly focused into cell culture medium a stress wave is generated at the laser focal point. Since the stress wave localizes in a few tens of μm, it is possible to locally stimulate single cells in vitro. In this work, several kinds of cultured mammalian cells, HeLa, PC12, P19CL6, and C2C12, were stimulated by the stress wave and the cell growth after the stress loading with the laser irradiation was investigated. In comparison with the control conditions, cell growth after the laser irradiation was enhanced for the cells of C2C12 and P19CL6, which can differentiate into myocytes, and suppressed for PC12 and HeLa cell lines. These results suggest a possibility of cell growth enhancement due to myogenic cells response to the femtosecond laser-induced stress.

  12. The hereditary hemochromatosis protein, HFE, specifically regulates transferrin-mediated iron uptake in HeLa cells.

    Science.gov (United States)

    Roy, C N; Penny, D M; Feder, J N; Enns, C A

    1999-03-26

    HFE is the protein product of the gene mutated in the autosomal recessive disease hereditary hemochromatosis (Feder, J. N., Gnirke, A., Thomas, W., Tsuchihashi, Z., Ruddy, D. A., Basava, A., Dormishian, F., Domingo, R. J., Ellis, M. C., Fullan, A., Hinton, L. M., Jones, N. L., Kimmel, B. E., Kronmal, G. S., Lauer, P., Lee, V. K., Loeb, D. B., Mapa, F. A., McClelland, E., Meyer, N. C., Mintier, G. A., Moeller, N., Moore, T., Morikang, E., Prasss, C. E., Quintana, L., Starnes, S. M., Schatzman, R. C., Brunke, K. J., Drayna, D. T., Risch, N. J., Bacon, B. R., and Wolff, R. R. (1996) Nat. Genet. 13, 399-408). At the cell surface, HFE complexes with transferrin receptor (TfR), increasing the dissociation constant of transferrin (Tf) for its receptor 10-fold (Gross, C. N., Irrinki, A., Feder, J. N., and Enns, C. A. (1998) J. Biol. Chem. 273, 22068-22074; Feder, J. N., Penny, D. M., Irrinki, A., Lee, V. K., Lebron, J. A., Watson, N. , Tsuchihashi, Z., Sigal, E., Bjorkman, P. J., and Schatzman, R. C. (1998) Proc. Natl. Acad. Sci. U S A 95, 1472-1477). HFE does not remain at the cell surface, but traffics with TfR to Tf-positive internal compartments (Gross et al., 1998). Using a HeLa cell line in which the expression of HFE is controlled by tetracycline, we show that the expression of HFE reduces 55Fe uptake from Tf by 33% but does not affect the endocytic or exocytic rates of TfR cycling. Therefore, HFE appears to reduce cellular acquisition of iron from Tf within endocytic compartments. HFE specifically reduces iron uptake from Tf, as non-Tf-mediated iron uptake from Fe-nitrilotriacetic acid is not altered. These results explain the decreased ferritin levels seen in our HeLa cell system and demonstrate the specific control of HFE over the Tf-mediated pathway of iron uptake. These results also have implications for the understanding of cellular iron homeostasis in organs such as the liver, pancreas, heart, and spleen that are iron loaded in hereditary hemochromatotic

  13. Fluid shear stress induction of the transcriptional activator c-fos in human and bovine endothelial cells, HeLa, and Chinese hamster ovary cells.

    Science.gov (United States)

    Ranjan, V; Waterbury, R; Xiao, Z; Diamond, S L

    1996-02-20

    The c-fos protein belongs to a family of transcriptional cofactors that can complex with proteins of the Jun family and activate mRNA transcription from gene promoters containing an activator protein 1 (AP-1) binding element. The shear stress inducibility of the c-fos protein was studied in human and animal cell lines of vastly different origins. Primary human umbilical vein endothelial cells (HUVEC), bovine aortic endothelial cells (BAEC, passage 2-14), HeLa cells, and Chinese hamster ovary (CHO) cells were subjected to steady laminar shear stress using a parallel plate flow apparatus. After 1 h of flow exposure at 25 dyn/cm(2), the c-fos levels in nuclei of shear stress HUVEC, BAEC, HeLa, and CHO were 5.4 +/- 2.0 (n = 3), 2.25 +/- 1.38 (n = 6), 2.14 +/- 0.07 (n = 8), 1.92 +/- 0.58 (n = 2) times higher, respectively, than in matched stationary controls. Flow exposure at 4 dyn/cm(2) caused no enhancement of c-fos levels in any of the cell lines tested, but caused significant reduction in c-fos expression in the HeLa cells. The c-fos induction by shear stress could be blocked by pharmacological agents. For example, the flow induction of the c-fos protein levels was blocked by 50% with the preincubation of HUVEC with a protein kinase C inhibitor, H7 (10 muM) and blocked completely in HeLa cells preincubated with the phospholipase C inhibitor, neomycin (5 mM). The minimum time of shear stress exposure required to induce the c-fos protein expression in HeLa cells was found to be as low as 1 min. By Northern analysis, the c-fos mRNA levels were found to be elevated in BAEC, CHO, and HeLa cells exposed to 25 dyn/cm(2) for 30 min. These studies indicate that c-fos induction is a consistent genetic response in a variety of mammalian cells that may alter cellular phenotype in mechanical environments. (c) 1996 John Wiley & Sons, Inc.

  14. Local food:

    DEFF Research Database (Denmark)

    Sundbo, Donna Isabella Caroline

    2013-01-01

    Recently there has been more focus on food in general and local food in particular. But what is local food? And what are the perceptions of this concept according to theory and to providers and consumers of local food? This article first summarises and compares three different theoretical perspec...... the providers and consumers studied on what local food is. However, this may change in time.......Recently there has been more focus on food in general and local food in particular. But what is local food? And what are the perceptions of this concept according to theory and to providers and consumers of local food? This article first summarises and compares three different theoretical...... perspectives on local food, namely experience economy, local food systems and what is termed pro-industrialism. These have differing and sometimes opposite conceptualisations and aims for the concept of local food. Using the perspective of experience economy as theoretical background, the concept of local food...

  15. A new carbamidemethyl-linked lanthanoid chelating tag for PCS NMR spectroscopy of proteins in living HeLa cells

    Energy Technology Data Exchange (ETDEWEB)

    Hikone, Yuya [Tokyo Metropolitan University, Department of Chemistry, Graduate School of Science and Engineering (Japan); Hirai, Go [RIKEN, Synthetic Organic Chemistry Laboratory (Japan); Mishima, Masaki [Tokyo Metropolitan University, Department of Chemistry, Graduate School of Science and Engineering (Japan); Inomata, Kohsuke [RIKEN, Quantitative Biology Center (Japan); Ikeya, Teppei; Arai, Souichiro [Tokyo Metropolitan University, Department of Chemistry, Graduate School of Science and Engineering (Japan); Shirakawa, Masahiro [Japan Agency for Medical Research and Development, AMED-CREST (Japan); Sodeoka, Mikiko [RIKEN, Synthetic Organic Chemistry Laboratory (Japan); Ito, Yutaka, E-mail: ito-yutaka@tmu.ac.jp [Tokyo Metropolitan University, Department of Chemistry, Graduate School of Science and Engineering (Japan)

    2016-10-15

    Structural analyses of proteins under macromolecular crowding inside human cultured cells by in-cell NMR spectroscopy are crucial not only for explicit understanding of their cellular functions but also for applications in medical and pharmaceutical sciences. In-cell NMR experiments using human cultured cells however suffer from low sensitivity, thus pseudocontact shifts from protein-tagged paramagnetic lanthanoid ions, analysed using sensitive heteronuclear two-dimensional correlation NMR spectra, offer huge potential advantage in obtaining structural information over conventional NOE-based approaches. We synthesised a new lanthanoid-chelating tag (M8-CAM-I), in which the eight-fold, stereospecifically methylated DOTA (M8) scaffold was retained, while a stable carbamidemethyl (CAM) group was introduced as the functional group connecting to proteins. M8-CAM-I successfully fulfilled the requirements for in-cell NMR: high-affinity to lanthanoid, low cytotoxicity and the stability under reducing condition inside cells. Large PCSs for backbone N–H resonances observed for M8-CAM-tagged human ubiquitin mutant proteins, which were introduced into HeLa cells by electroporation, demonstrated that this approach readily provides the useful information enabling the determination of protein structures, relative orientations of domains and protein complexes within human cultured cells.

  16. A novel and simple fluorescence probe for detecting main group magnesium ion in HeLa cells and Arabidopsis.

    Science.gov (United States)

    Yu, Tingting; Sun, Ping; Hu, Yijie; Ji, Yinggang; Zhou, Hongping; Zhang, Baowei; Tian, Yupeng; Wu, Jieying

    2016-12-15

    A simple-molecule fluorescence probe L has been designed, synthesized and characterized, which shows high selectivity and sensitivity for the main group magnesium ion through fluorescence "turn-on" response in ethanol solution, and no interference from calcium ion in particular. Detection limit of probe L is 1.47×10(-6) M and the rapid response could reach about 15-20s. The recognition mechanism has been established by fluorescence spectra, (1)H NMR study. Moreover, probe L presents a great photostability, low toxicity and cellular permeability, then we have carried out fluorescent bio-imaging of the probe L for magnesium ions in HeLa cells, which showed that probe L could be utilized to detect the intracellular magnesium ion. Furthermore, it is successfully used as a magnesium ion developer in plant tissues, which shows that it not only can be well tracking the transport of magnesium ion but also make a corresponding fluorescence response to different concentrations magnesium ion. These results would make this probe a great potential application for detecting Mg(2+) in biological system. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. Properties of Surfactin C-15 Nanopeptide and Its Cytotoxic Effect on Human Cervix Cancer (HeLa Cell Line

    Directory of Open Access Journals (Sweden)

    Zahra Nozhat

    2012-01-01

    Full Text Available Surfactin is one of the most powerful biosurfactants that has been known so far. It is an acidic cyclic nonribosomal lipoheptapeptide that is produced by Bacillus subtilis. In this presentation we investigated different properties of surfactin C-15. The nanomicelle forming ability of surfactin C-15 in different aqueous environments with various ionic strengths was studied by scanning electron microscope. Surfactin second structure was investigated by Far-UV CD spectrum. Its hemolytic activity and cytotoxicity were measured by hemolysis and MTT assays, respectively. Surfactin formed spherical nanomicelles in distilled water and amorphous nanomicelles in PBS buffer . The hemolysis assay results indicated that HC50 of surfactin was 47 μM. Surfactin C-15 arrested growth of human cervix cancer HeLa cell line in a time- and dosage-dependent method, so that its IC50 at 16, 24, and 48h were 86.9, 73.1, and 50.2 μM, respectively.

  18. Effects of Tatariside G Isolated from Fagopyrum tataricum Roots on Apoptosis in Human Cervical Cancer HeLa Cells

    Directory of Open Access Journals (Sweden)

    Yuan Li

    2014-07-01

    Full Text Available Cervical cancer is the second most common female carcinoma. Current therapies are often unsatisfactory, especially for advanced stage patients. The aim of this study was to explore the effects of tatariside G (TG on apoptosis in human cervical cancer HeLa cells and the possible mechanism of action involved. An MTT assay was employed to evaluate cell viability. Hoechst 33258 staining and flow cytometry (FCM assays were used to detect cell apoptosis. The protein expression of phosphorylated JNK, P38, ERK and Akt and cleaved caspase-3 and caspase-9 was evaluated by western blot analysis. Additionally, the mRNA expression of caspase-3 and caspase-9 was measured by fluorescent quantitative reverse transcription-PCR (FQ-RT-PCR. TG notably inhibited cell viability, enhanced the percentage of apoptotic cells, facilitated the phosphorylation of p38 MAPK and JNK proteins and caspase-3 and caspase-9 cracking, downregulated the phosphorylation level of Akt, and increased the loss of MMP and the mRNA expression of caspase-3 and caspase-9. TG-induced apoptosis is associated with activation of the mitochondrial death pathway. TG may be an effective candidate for chemotherapy against cervical cancer.

  19. Novel localisation and possible function of LIN7 and IRSp53 in mitochondria of HeLa cells.

    Science.gov (United States)

    Ferrari, Ilaria; Crespi, Arianna; Fornasari, Diego; Pietrini, Grazia

    2016-08-01

    By means of immunofluorescence and subcellular fractionation experiments, we here demonstrate mitochondrial distribution of LIN7 and IRSp53 in HeLa cells. These peripheral proteins displayed a tight association with mitochondria and coimmunoprecipitated from mitochondrial fractions. In line with a role for LIN7 in the regulation of IRSp53 activity on actin dynamics, the morphology of mitochondria was similarly altered by changing the expression levels of either each protein or both, whereas mitochondrial morphology was preserved in cells overexpressing IRSp53 deleted of its binding domains for LIN7 (IRSp53Δ5) or for actin polymerisation modulators (IRSp53ΔSH3). In particular, the overexpression of full length LIN7 and/or IRSp53 increased the percentage of cells with short mitochondria, while downregulation of the endogenous proteins by shRNAs increased the amount of cells with elongated and perinuclear clustered mitochondria. These mitochondria were only partially resistant to fragmentation induced by dissipation of the mitochondrial membrane potential (i.e. treatment with sodium azide), whereas mitochondria were fully protected by the fission defective mutant Drp1 K38A. Overexpression of LIN7 or IRSp53 did not prevent the formation of hyperfused mitochondria in cells coexpressing the Drp1 K38A mutant, thus suggesting that LIN7-IRSp53 complex requires functional Drp1 to regulate mitochondrial morphology. Copyright © 2016 Elsevier GmbH. All rights reserved.

  20. Prx II and CKBB proteins interaction under physiological and thermal stress conditions in A549 and HeLa cells

    Directory of Open Access Journals (Sweden)

    A. D. Rakhmetov

    2016-02-01

    Full Text Available Peroxiredoxins (Prxs are versatile enzymes that demonstrate various cell functions as peroxidases, protein chaperones, functions of signal modulators and binding partners. It is well established that Prxs can interact with multiple proteins in cells, such as ASK1, Cdk5-p35, JNK, MIF, PDGF, TKR4 and others. In this study, we attempted to evaluate a possible association between ubiquitous Prx II and ATP/ADP buffering enzyme – brain-type creatine kinase (CKBB. Our co-immunoprecipitation (Co-IP results from the A549 and HeLa cell lysates with overexpressed HA-Prx II and Flag-CKBB have demonstrated strong association between two proteins under non-stressed conditions. This protein interaction was enhanced by the heat treatment with further HA-Prx II precipitation to the immobilized Flag-CKBB depending on the temperature increase. Temperature induced oligomerization of Prx II may contribute to the formation of Prx II conglomera­tes, which in turn, can associate with CKBB and increase signal intensities on the blotted membranes. Thus, such association and oligomerization of Prx II could take part in recovery and protection of the CKBB enzyme activity from inactivation during heat-induced stress.

  1. Uracil DNa-glycosylase from HeLa cells: general properties, substrate specificity and effect of uracil analogs.

    Science.gov (United States)

    Krokan, H; Wittwer, C U

    1981-06-11

    Uracil-DNA glycosylase was partially purified from HeLa cells. Various substrates containing [3H]dUMP residues were prepared by nick-translation of calf thymus DNA. The standard substrate was double-stranded DNA with [3H]dUMP located internally in the chain. Compared to the release of uracil from this substrate, a 3-fold increase in the rate was seen with single-stranded DNA, and a 20-fold reduction in the rate was observed when the [3H]dUMP-residue was located at the 3'end. The rate of [3H]uracil release decreased progressively when one, two or three of the dNMP residues were replaced by the corresponding rNMP; in the extreme case when the substrate contained [3H]dUMP in addition to rCMP, rGMP, and rAMP, the rate of [3H]uracil release was less than 3% of that of the control. The enzyme was inhibited to the same extent by uracil and the uracil analogs 6-aminouracil and 5-azauracil, but very weakly, or not at all, by 5 other analogs. Our results suggest strongly that uracil-DNA glycosylase has a high degree of selectivity for uracil in dUMP residues located internally in DNA chains and that the recognition of the correct substrate also depends on the residues flanking dUMP being deoxyribonucleotides.

  2. [EFFECT OF FUCOIDANS ISOLATED FROM SEAWEEDS LAMINARIA DIGITATA AND FUCUS VESICULOSUS ON CELL LINES HELA G-63, ECV 304 AND PC 12].

    Science.gov (United States)

    Zhurishkina, E V; Lapina, I M; Ivanen, D R; Stepanov, S I; Shvetsova, S V; Shavarda, A L; Giliano, N Ya; Kulminskaya, A A

    2015-01-01

    The aim of the research was to investigate cytotoxicity of fucoidans on mammals cells. Three different samples of fucoidans were isolated from mechanically grounded brown algae Laminaria digitata and Fucus ve- siculosus. The sample F2 that differed from the others by higher sulfatation level and suppression of HeLa G-63 line culture growth was taken for further study in cell lines HeLa G-63, ECV 304 and PC 12. We have shown that fucoidan preparation F2 inhibits proliferation and induces cell death in a dose- and time-dependent manner for all investigated cell lines. Neuroendocrine tumor rat cell line PC 12 appeared to be the most sensitive to fucoidan treatment whereas endothelial human cells ECV 304 were the least sensitive.

  3. Inhibition of clathrin by pitstop 2 activates the spindle assembly checkpoint and induces cell death in dividing HeLa cancer cells

    Directory of Open Access Journals (Sweden)

    Smith Charlotte M

    2013-01-01

    Full Text Available Abstract Background During metaphase clathrin stabilises the mitotic spindle kinetochore(K-fibres. Many anti-mitotic compounds target microtubule dynamics. Pitstop 2™ is the first small molecule inhibitor of clathrin terminal domain and inhibits clathrin-mediated endocytosis. We investigated its effects on a second function for clathrin in mitosis. Results Pitstop 2 did not impair clathrin recruitment to the spindle but disrupted its function once stationed there. Pitstop 2 trapped HeLa cells in metaphase through loss of mitotic spindle integrity and activation of the spindle assembly checkpoint, phenocopying clathrin depletion and aurora A kinase inhibition. Conclusions Pitstop 2 is therefore a new tool for investigating clathrin spindle dynamics. Pitstop 2 reduced viability in dividing HeLa cells, without affecting dividing non-cancerous NIH3T3 cells, suggesting that clathrin is a possible novel anti-mitotic drug target.

  4. Low-level laser therapy: Effects on human face aged skin and cell viability of HeLa cells exposed to UV radiation

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    Mezghani Sana

    2015-01-01

    Full Text Available Chronic and excessive exposure to UV radiation leads to photoaging and photocarcinogenesis. Adequate protection of the skin against the deleterious effects of UV irradiation is essential. Low-level laser therapy (LLLT is a light source in the red to near-infrared range that has been accepted in a variety of medical applications. In this study, we explored the effect of LLLT in human face aged skin and the cell viability of HeLa cells exposed to UV radiation. We found that LLLT significantly reduced visible wrinkles and the loss of firmness of facial skin in aging subjects. Additionally, treatment of cultured HeLa cells with LLLT prior to or post UVA or UVB exposure significantly protected cells from UV-mediated cell death. All results showed the beneficial effects of LLLT on relieving signs of skin aging and its prevention and protection of the cell viability against UV-induced damage.

  5. Biochemical features of the adhesion G protein-coupled receptor CD97 related to its auto-proteolysis and HeLa cell attachment activities.

    Science.gov (United States)

    Yang, Li-Yun; Liu, Xiao-Fang; Yang, Yang; Yang, Lin-Lin; Liu, Kai-Wen; Tang, Yu-Bo; Zhang, Min; Tan, Min-Jia; Cheng, Shan-Mei; Xu, Ye-Chun; Yang, Huai-Yu; Liu, Zhi-Jie; Song, Gao-Jie; Huang, Wei

    2017-01-01

    CD97 belongs to the adhesion GPCR family characterized by a long ECD linked to the 7TM via a GPCR proteolytic site (GPS) and plays important roles in modulating cell migration and invasion. CD97 (EGF1-5) is a splicing variant of CD97 that recognizes a specific ligand chondroitin sulfate on cell membranes and the extracellular matrix. The aim of this study was to elucidate the extracellular molecular basis of the CD97 EGF1-5 isoform in protein expression, auto-proteolysis and cell adhesion, including epidermal growth factor (EGF)-like domain, GPCR autoproteolysis-inducing (GAIN) domain, as well as GPS mutagenesis and N-glycosylation. Both wild-type (WT) CD97-ECD and its truncated, GPS mutated, PNGase F-deglycosylated, and N-glycosylation site mutated forms were expressed and purified. The auto-proteolysis of the proteins was analyzed with Western blotting and SDS-PAGE. Small angle X-ray scattering (SAXS) and molecular modeling were used to determine a structural profile of the properly expressed receptor. Potential N-glycosylation sites were identified using MS and were modulated with PNGase F digestion and glyco-site mutations. A flow cytometry-based HeLa cell attachment assay was used for all aforementioned CD97 variants to elucidate the molecular basis of CD97-HeLa interactions. A unique concentration-dependent GPS auto-proteolysis was observed in CD97 EGF1-5 isoform with the highest concentration (4 mg/mL) per sample was self-cleaved much faster than the lower concentration (0.1 mg/mL), supporting an intermolecular mechanism of auto-proteolysis that is distinct to the reported intramolecular mechanism for other CD97 isoforms. N-glycosylation affected the auto-proteolysis of CD97 EGF1-5 isoform in a similar way as the other previously reported CD97 isoforms. SAXS data for WT and deglycosylated CD97ECD revealed a spatula-like shape with GAIN and EGF domains constituting the body and handle, respectively. Structural modeling indicated a potential interaction

  6. MicroRNA-21 promotes cell proliferation and down-regulates the expression of programmed cell death 4 (PDCD4) in HeLa cervical carcinoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Yao, Qing; Xu, Hui; Zhang, Qian-Qian; Zhou, Hui [Key Laboratory of Gene Engineering of the Ministry of Education, State Key Laboratory for Biocontrol, Sun Yat-Sen University, Guangzhou 510275 (China); Qu, Liang-Hu, E-mail: lssqlh@mail.sysu.edu.cn [Key Laboratory of Gene Engineering of the Ministry of Education, State Key Laboratory for Biocontrol, Sun Yat-Sen University, Guangzhou 510275 (China)

    2009-10-23

    MicroRNAs are involved in cancer-related processes. The microRNA-21(miR-21) has been identified as the only miRNA over-expressed in a wide variety of cancers, including cervical cancer. However, the function of miR-21 is unknown in cervical carcinomas. In this study, we found that the inhibition of miR-21 in HeLa cervical cancer cells caused profound suppression of cell proliferation, and up-regulated the expression of the tumor suppressor gene PDCD4. We also provide direct evidence that PDCD4-3'UTR is a functional target of miR-21 and that the 18 bp putative target site can function as the sole regulatory element in HeLa cells. These results suggest that miR-21 may play an oncogenic role in the cellular processes of cervical cancer and may serve as a target for effective therapies.

  7. Binding of host extracellular matrix proteins to Mycoplasma fermentans and its effect on adherence to, and invasion of HeLa cells.

    Science.gov (United States)

    Yavlovich, Amichai; Rottem, Shlomo

    2007-01-01

    In the present study, we show that intact Mycoplasma fermentans cells have a wealth of adhesive interactions with components of the extracellular matrix. Mycoplasma fermentans intensively bind plasminogen, and to a lesser extent, fibronectin, heparin, and laminin. The binding of collagen type III, IV, or V was low. The binding of plasminogen, collagen type III, or collagen type V markedly enhanced the adherence of M. fermentans to HeLa cells, whereas the binding of fibronectin, heparin, laminin, or collagen IV induced only a small effect on mycoplasma adherence. Utilizing plasminogen-treated M. fermentans preparations, we detected microorganisms within host HeLa cells by the gentamicin protection assay or by confocal laser scanning microscopy of immunofluorescent preparations. However, no intracellular M. fermentans was detected when M. fermentans preparations treated with fibronectin, heparin, laminin, or collagen type III, IV, or V were utilized.

  8. Persistently Elevated Level of IL-8 in Chlamydia trachomatis Infected HeLa 229 Cells is Dependent on Intracellular Available Iron

    Directory of Open Access Journals (Sweden)

    Harsh Vardhan

    2009-01-01

    Full Text Available Chlamydia trachomatis is a leading cause of sexually transmitted infection worldwide and responsible for myriad of immunopathological changes associated with reproductive health. Delayed secretion of proinflammatory chemokine interleukin (IL-8 is a hallmark of chlamydial infection and is dependent on chlamydial growth. We examined the effect of iron chelators on IL-8 production in HeLa 229 (cervix epitheloid cell, CCL2 cells infected with C. trachomatis. IL-8 production was induced by Iron chelator DFO and Mimosine, however, synergy with chlamydial infection was obtained with DFO only. Temporal expression of proinflammatory secreted cytokines IL-1beta, TNF-alpha, and IL-8 did not show synchrony in Chlamydia trachomatis infected cells. Secretion of IL-8 from Hela cells infected with C. trachomatis was not dependent on IL-1 beta and TNF- alpha induction. These results indicate towards involvement of iron in chlamydia induced IL-8 production.

  9. Cell death in HeLa mediated by thermoplastic polyurethane with co-immobilized IFN-γ plus TNF-α.

    Science.gov (United States)

    Guan, Yan-Qing; Zheng, Zhe; Li, Zhibin; Liu, Jun-Ming

    2012-03-01

    In order to prohibit the toxicity of free IFN-γ plus TNF-α in treating human cervical cancer HeLa cells, two kinds of thermoplastic polyurethane (polyester/polyether) biomaterials with co-immobilized IFN-γ plus TNF-α on the surfaces are prepared. The programmed cell death of HeLa induced by these biomaterials is investigated. The surface modification of these biomaterials with co-immobilized IFN-γ plus TNF-α is performed by the photo-immobilization method, and the surface structures are characterized by various techniques. The cell morphology, cell mortality, cell cycle arrest, and functional status of caspases, upon the treatment by these biomaterials, are characterized. The results show that the as-prepared biomaterials have high inhibition activity against the growth of HeLa cells. The HeLa cells mediated by the two kinds of biomaterials are mainly arrested in the G(1) phase, while those cells mediated directly by free IFN-γ plus TNF-α are mainly arrested in the S phase. It is suggested that the programmed cell death mechanism induced by these two kinds of biomaterials is both caspase-dependent and caspase-independent. Our data provide the knowledge of microscopic surface structures and cell biology basis for synthesizing the thermoplastic polyurethane biomaterials with co-immobilized IFN-γ plus TNF-α, which are promising for novel therapeutics (e.g. drug cup) design for cervical cancer patients. Copyright © 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  10. Unique Characteristics of AAV1, 2, and 5 Viral Entry, Intracellular Trafficking, and Nuclear Import Define Transduction Efficiency in HeLa Cells

    OpenAIRE

    Keiser, Nicholas W; Yan, Ziying; Zhang, Yulong; Diana C M Lei-Butters; Engelhardt, John F.

    2011-01-01

    Biological differences between recombinant adeno-associated virus (rAAV) serotypes define their efficiencies in expressing a transgene in a particular target cell. Few studies have directly compared how differences in viral entry, intracellular trafficking, and nuclear import of rAAV serotypes influence the effectiveness of transduction in the same cell type. We evaluated these characteristics for three rAAV serotypes in HeLa cells, using biochemical techniques and fluorescence-based detectio...

  11. Taxifolin synergizes Andrographolide-induced cell death by attenuation of autophagy and augmentation of caspase dependent and independent cell death in HeLa cells.

    Directory of Open Access Journals (Sweden)

    Mazen Alzaharna

    Full Text Available Andrographolide (Andro has emerged recently as a potential and effective anticancer agent with induction of apoptosis in some cancer cell lines while induction of G2/M arrest with weak apoptosis in others. Few studies have proved that Andro is also effective in combination therapy. The flavonoid Taxifolin (Taxi has showed anti-oxidant and antiproliferative effects against different cancer cells. Therefore, the present study investigated the cytotoxic effects of Andro alone or in combination with Taxi on HeLa cells. The combination of Andro with Taxi was synergistic at all tested concentrations and combination ratios. Andro alone induced caspase-dependent apoptosis which was enhanced by the combination with Taxi and attenuated partly by using Z-Vad-Fmk. Andro induced a protective reactive oxygen species (ROS-dependent autophagy which was attenuated by Taxi. The activation of p53 was involved in Andro-induced autophagy where the use of Taxi or pifithrin-α (PFT-α decreased it while the activation of JNK was involved in the cell death of HeLa cells but not in the induction of autophagy. The mitochondrial outer-membrane permeabilization (MOMP plays an important role in Andro-induced cell death in HeLa cells. Andro alone increased the MOMP which was further increased in the case of combination. This led to the increase in AIF and cytochrome c release from mitochondria which consequently increased caspase-dependent and independent cell death. In conclusion, Andro induced a protective autophagy in HeLa cells which was reduced by Taxi and the cell death was increased by increasing the MOMP and subsequently the caspase-dependent and independent cell death.

  12. Human Papillomavirus E6 Knockdown Restores Adenovirus Mediated-estrogen Response Element Linked p53 Gene Transfer in HeLa Cells.

    Science.gov (United States)

    Kajitani, Koji; Honda, Ken-Ichi; Terada, Hiroyuki; Yasui, Tomoyo; Sumi, Toshiyuki; Koyama, Masayasu; Ishiko, Osamu

    2015-01-01

    The p53 gene is inactivated by the human papillomavirus (HPV) E6 protein in the majority of cervical cancers. Treatment of HeLa S3 cells with siRNA for HPV E6 permitted adenovirus-mediated transduction of a p53 gene linked to an upstream estrogen response element (ERE). Our previous study in non-siRNA treated HHUA cells, which are derived from an endometrial cancer and express estrogen receptor β, showed enhancing effects of an upstream ERE on adenovirus-mediated p53 gene transduction. In HeLa S3 cells treated with siRNA for HPV E6, adenovirus-mediated transduction was enhanced by an upstream ERE linked to a p53 gene carrying a proline variant at codon 72, but not for a p53 gene with arginine variant at codon 72. Expression levels of p53 mRNA and Coxsackie/adenovirus receptor (CAR) mRNA after adenovirus-mediated transfer of an ERE-linked p53 gene (proline variant at codon 72) were higher compared with those after non-ERE-linked p53 gene transfer in siRNA-treated HeLa S3 cells. Western blot analysis showed lower β-tubulin levels and comparatively higher p53/β-tubulin or CAR /β-tubulin ratios in siRNA-treated HeLa S3 cells after adenovirus-mediated ERE-linked p53 gene (proline variant at codon 72) transfer compared with those in non-siRNA-treated cells. Apoptosis, as measured by annexin V binding, was higher after adenovirus-mediated ERE-linked p53 gene (proline variant at codon 72) transfer compared with that after non-ERE-linked p53 gene transfer in siRNA-treated cells.

  13. Repression of the human papillomavirus E6 gene initiates p53-dependent, telomerase-independent senescence and apoptosis in HeLa cervical carcinoma cells.

    Science.gov (United States)

    Horner, Stacy M; DeFilippis, Rosa Anna; Manuelidis, Laertes; DiMaio, Daniel

    2004-04-01

    Cervical cancer cells express high-risk human papillomavirus (HPV) E6 and E7 proteins. When both HPV oncogenes are repressed in HeLa cervical carcinoma cells, the dormant p53 and retinoblastoma (Rb) tumor suppressor pathways are activated, and the cells undergo senescence in the absence of apoptosis. When the E6 gene is repressed in cells that continue to express an E7 gene, the p53 pathway, but not the Rb pathway, is activated, and both senescence and apoptosis are triggered. To determine the role of p53 signaling in senescence or apoptosis after repression of HPV oncogenes, we introduced a dominant-negative allele of p53 into HeLa cells. Dominant-negative p53 prevented senescence and apoptosis when E6 alone was repressed but did not inhibit senescence when both E6 and E7 were repressed. To determine whether reduced telomerase activity was involved in senescence or apoptosis after E6 repression, we generated HeLa cells stably expressing an exogenous hTERT gene, which encodes the catalytic subunit of telomerase. Although these cells contained markedly elevated telomerase activity and elongated telomeres, hTERT expression did not prevent senescence and apoptosis when E6 alone was repressed. These results demonstrate that when the Rb tumor suppressor pathway is inactivated by the E7 protein, E6 repression activates p53 signaling, which in turn is required for growth inhibition, senescence, and apoptosis. Thus, sustained inactivation of the p53 pathway by the E6 protein is required for maintenance of the proliferative phenotype of HeLa cervical carcinoma cells.

  14. Anti-Proliferative Activity and Apoptosis Induction of an Ethanolic Extract of Boesenbergia pandurata (Roxb.) Schlecht. against HeLa and Vero Cell Lines.

    Science.gov (United States)

    Listyawati, Shanti; Sismindari; Mubarika, Sofia; Murti, Yosi Bayu; Ikawati, Muthi

    2016-01-01

    Rhizomes of Boesenbergia pandurata (Roxb.) Schlecht have been reported to contain active compounds with anticancer properties. This research was carried out to examine anti-proliferative and apoptotic induction against HeLa and Vero cells-line. Dried powder of B. pandurata rhizomes was extracted by a maceration method using 90% ethanol. Cytotoxic assays to determine IC50 and anti-proliferative effects were carried out by MTT methods. Observation of apoptosis was achieved with double staining using acridine orange and ethidium bromide. The results showed that ethanolic extract of B. pandurata was more cytotoxic against HeLa cells (IC50 of 60 μg/ mL) than Vero cells (IC50 of 125 μg/mL). The extract had higher anti-proliferative activity as well as apoptotic induction in HeLa than Vero cells. Therefore, it was concluded that the ethanolic extract of B. pandurata had anti-proliferative as well as apoptosis induction activity dependent on the cell type.

  15. Anticancer activity of novel oleanolic acid methyl ester derivative in HeLa cervical cancer cells is mediated through apoptosis induction and reactive oxygen species production

    Directory of Open Access Journals (Sweden)

    Xin Song

    2015-12-01

    Full Text Available The objective of this study was to determine the anticancer and apoptotic effects of a novel oleanolic acid methyl vanillate derivative in human cervical cancer cells (HeLa. We also evaluated effect of the compound on reactive oxygen species (ROS generation. MTT and LDH assay were used to evaluate the effect of this compound on HeLa cancer cell cytotoxicity. Confocal microscopy and flow cytometry evaluated the effect on apoptosis induction, while as ROS production was detected by using fluorescent CM-DCFH2-DA. The compound induced potent cytotoxic effects in HeLa cells in a dose-dependent as well as time-dependent manner. The compound also induced characteristic morphological changes indicative of apoptosis. The compound induced both early and late apoptosis in a concentration-dependent and time-dependent manner. The compound led to a significant and concentration dependent increase in the ROS production as the concentration of oleanolic acid methyl vanillate derivative was increased from 0 to 15, 60 and 120 μM.

  16. Nanosecond pulsed electric fields induce poly(ADP-ribose) formation and non-apoptotic cell death in HeLa S3 cells.

    Science.gov (United States)

    Morotomi-Yano, Keiko; Akiyama, Hidenori; Yano, Ken-ichi

    2013-08-30

    Nanosecond pulsed electric fields (nsPEFs) have recently gained attention as effective cancer therapy owing to their potency for cell death induction. Previous studies have shown that apoptosis is a predominant mode of nsPEF-induced cell death in several cell lines, such as Jurkat cells. In this study, we analyzed molecular mechanisms for cell death induced by nsPEFs. When nsPEFs were applied to Jurkat cells, apoptosis was readily induced. Next, we used HeLa S3 cells and analyzed apoptotic events. Contrary to our expectation, nsPEF-exposed HeLa S3 cells exhibited no molecular signs of apoptosis execution. Instead, nsPEFs induced the formation of poly(ADP-ribose) (PAR), a hallmark of necrosis. PAR formation occurred concurrently with a decrease in cell viability, supporting implications of nsPEF-induced PAR formation for cell death. Necrotic PAR formation is known to be catalyzed by poly(ADP-ribose) polymerase-1 (PARP-1), and PARP-1 in apoptotic cells is inactivated by caspase-mediated proteolysis. Consistently, we observed intact and cleaved forms of PARP-1 in nsPEF-exposed and UV-irradiated cells, respectively. Taken together, nsPEFs induce two distinct modes of cell death in a cell type-specific manner, and HeLa S3 cells show PAR-associated non-apoptotic cell death in response to nsPEFs. Copyright © 2013 Elsevier Inc. All rights reserved.

  17. Assessment of Cytotoxic Activity of Rosemary (Rosmarinus officinalis L.), Turmeric (Curcuma longa L.), and Ginger (Zingiber officinale R.) Essential Oils in Cervical Cancer Cells (HeLa).

    Science.gov (United States)

    Santos, P A S R; Avanço, G B; Nerilo, S B; Marcelino, R I A; Janeiro, V; Valadares, M C; Machinski, Miguel

    2016-01-01

    The objective of this study was to evaluate the cytotoxic activity of rosemary (REO, Rosmarinus officinalis L.), turmeric (CEO, Curcuma longa L.), and ginger (GEO, Zingiber officinale R.) essential oils in HeLa cells. Cytotoxicity tests were performed in vitro, using tetrazolium (MTT) and neutral red assays for evaluation of antiproliferative activity by different mechanisms, trypan blue assay to assess cell viability and evaluation of cell morphology for Giemsa to observe the cell damage, and Annexin V to evaluate cell death by apoptosis. CEO and GEO exhibited potent cytotoxic activity against HeLa cells. IC50 obtained was 36.6 μg/mL for CEO and 129.9 μg/mL for GEO. The morphology of HeLa cells showed condensation of chromatin, loss of cell membrane integrity with protrusions (blebs), and cell content leakage for cells treated with CEO and GEO, from the lowest concentrations studied, 32.81 μg/mL of CEO and 32.12 μg/mL of GEO. The Annexin V assay revealed a profile of cell death by apoptosis for both CEO and GEO. The results indicate cytotoxic activity in vitro for CEO and GEO, suggesting potential use as anticancer agents for cervical cancer cells.

  18. Effect of Static Magnetic Field on the Rate of Proliferation and Viability in HeLa Cancer Cells and Normal Fibroblasts

    Directory of Open Access Journals (Sweden)

    E. Shams

    2017-01-01

    Full Text Available Aims: The increasing use of the electromagnetic devices in daily life leads to higher electromagnetic filed effects. The effects on the organic systems are contradictory and controversial. The aim of this study was to investigate the effects of different intensities and durations of the static magnetic fields on the living cells and their proliferation rate. Materials & Methods: In the applied study, two HeLa cancer cell lines and human fibroblast natural cells were studied. At first, the cells were cultured on DMEN medium. Three magnetic intensities (7, 14, and 21T and two durations (24 and 48h were used, and the cells were treated by static magnetic field. The living cell percentage and cell proliferation rate were assessed by MTT method. Trypan blue was used in staining. And an optical microscope was used in enumeration. Data was analyzed by Graphpad Prism 5 using one-way ANOVA. Findings: The higher the static magnetic field and the more the duration were, the lesser the percentage of living cells and cell proliferation, showing a significant reduction in the HeLa cancer cells, while it was insignificant in the fibroblast natural cells. The highest reduction in the living cell percentage and cell proliferation rate was in 48-hour 21T (p<0.05. Conclusion: The static magnetic field affects the HeLa cancer cells more than the fibroblast cells. The higher the field intensity and the more the duration are, the lesser the alive cell percentage and cell proliferation rate.

  19. Enhanced cellular uptake of albumin-based lyophilisomes when functionalized with cell-penetrating peptide TAT in HeLa cells.

    Directory of Open Access Journals (Sweden)

    Etienne van Bracht

    Full Text Available Lyophilisomes are a novel class of biodegradable proteinaceous nano/micrometer capsules with potential use as drug delivery carrier. Cell-penetrating peptides (CPPs including the TAT peptide have been successfully implemented for intracellular delivery of a broad variety of cargos including various nanoparticulate pharmaceutical carriers. In the present study, lyophilisomes were modified using CPPs in order to achieve enhanced cellular uptake. Lyophilisomes were prepared by a freezing, annealing, and lyophilization method and a cystein-elongated TAT peptide was conjugated to the lyophilisomes using a heterobifunctional linker. Fluorescent-activated cell sorting (FACS was utilized to acquire a lyophilisome population with a particle diameter smaller than 1000 nm. Cultured HeLa, OVCAR-3, Caco-2 and SKOV-3 cells were exposed to unmodified lyophilisomes and TAT-conjugated lyophilisomes and examined with FACS. HeLa cells were investigated in more detail using a trypan blue quenching assay, confocal microscopy, and transmission electron microscopy. TAT-conjugation strongly increased binding and cellular uptake of lyophilisomes in a time-dependent manner in vitro, as assessed by FACS. These results were confirmed by confocal microscopy. Transmission electron microscopy indicated rapid cellular uptake of TAT-conjugated lyophilisomes via phagocytosis and/or macropinocytosis. In conclusion, TAT-peptides conjugated to albumin-based lyophilisomes are able to enhance cellular uptake of lyophilisomes in HeLa cells.

  20. Identification of HPV integration and gene mutation in HeLa cell line by integrated analysis of RNA-Seq and MS/MS data.

    Science.gov (United States)

    Sun, Han; Chen, Chen; Lian, Baofeng; Zhang, Menghuan; Wang, Xiaojing; Zhang, Bing; Li, Yixue; Yang, Pengyuan; Xie, Lu

    2015-04-03

    HeLa cell line, which was derived from cervical carcinoma, provides an idea platform to study both the integration of human papillomavirus and the massive mutations occurring on the cancer cell genome. Proteogenomics is a field with the intersection of proteomics and genomics to perform gene annotation and identify gene mutation. In this work, we first identified the SNV/INDEL, structural variation (SV), and virus infection/integration events from RNA-Seq data of HeLa cell line; then, by applying proteogenomics strategy, we were able to detect some of the genomic events with the tandem mass spectrometry (MS/MS) data from the same sample. Furthermore, some of the mutated peptides were experimentally validated using multiple reaction monitoring technology. The integrated analysis of the RNA-Seq and MS/MS data not only renders the discovery of HeLa cell genome variations more credible but also illustrates a practical workflow for protein-coding mutation discovery in cancer-related studies.

  1. Dioscin Induces Apoptosis in Human Cervical Carcinoma HeLa and SiHa Cells through ROS-Mediated DNA Damage and the Mitochondrial Signaling Pathway.

    Science.gov (United States)

    Zhao, Xinwei; Tao, Xufeng; Xu, Lina; Yin, Lianhong; Qi, Yan; Xu, Youwei; Han, Xu; Peng, Jinyong

    2016-06-04

    Dioscin, a natural product, has activity against glioblastoma multiforme, lung cancer and colon cancer. In this study, the effects of dioscin against human cervical carcinoma HeLa and SiHa cells were further confirmed, and the possible mechanism(s) were investigated. A transmission electron microscopy (TEM) assay and DAPI staining were used to detect the cellular morphology. Flow cytometry was used to assay cell apoptosis, ROS and Ca(2+) levels. Single cell gel electrophoresis and immunofluorescence assays were used to test DNA damage and cytochrome C release. The results showed that dioscin significantly inhibited cell proliferation and caused DNA damage in HeLa and SiHa cells. The mechanistic investigation showed that dioscin caused the release of cytochrome C from mitochondria into the cytosol. In addition, dioscin significantly up-regulated the protein levels of Bak, Bax, Bid, p53, caspase-3, caspase-9, and down-regulated the protein levels of Bcl-2 and Bcl-xl. Our work thus demonstrated that dioscin notably induces apoptosis in HeLa and SiHa cells through adjusting ROS-mediated DNA damage and the mitochondrial signaling pathway.

  2. Assessment of Cytotoxic Activity of Rosemary (Rosmarinus officinalis L., Turmeric (Curcuma longa L., and Ginger (Zingiber officinale R. Essential Oils in Cervical Cancer Cells (HeLa

    Directory of Open Access Journals (Sweden)

    P. A. S. R. Santos

    2016-01-01

    Full Text Available The objective of this study was to evaluate the cytotoxic activity of rosemary (REO, Rosmarinus officinalis L., turmeric (CEO, Curcuma longa L., and ginger (GEO, Zingiber officinale R. essential oils in HeLa cells. Cytotoxicity tests were performed in vitro, using tetrazolium (MTT and neutral red assays for evaluation of antiproliferative activity by different mechanisms, trypan blue assay to assess cell viability and evaluation of cell morphology for Giemsa to observe the cell damage, and Annexin V to evaluate cell death by apoptosis. CEO and GEO exhibited potent cytotoxic activity against HeLa cells. IC50 obtained was 36.6 μg/mL for CEO and 129.9 μg/mL for GEO. The morphology of HeLa cells showed condensation of chromatin, loss of cell membrane integrity with protrusions (blebs, and cell content leakage for cells treated with CEO and GEO, from the lowest concentrations studied, 32.81 μg/mL of CEO and 32.12 μg/mL of GEO. The Annexin V assay revealed a profile of cell death by apoptosis for both CEO and GEO. The results indicate cytotoxic activity in vitro for CEO and GEO, suggesting potential use as anticancer agents for cervical cancer cells.

  3. The Cytotoxicity Mechanism of 6-Shogaol-Treated HeLa Human Cervical Cancer Cells Revealed by Label-Free Shotgun Proteomics and Bioinformatics Analysis

    Directory of Open Access Journals (Sweden)

    Qun Liu

    2012-01-01

    Full Text Available Cervical cancer is one of the most common cancers among women in the world. 6-Shogaol is a natural compound isolated from the rhizome of ginger (Zingiber officinale. In this paper, we demonstrated that 6-shogaol induced apoptosis and G2/M phase arrest in human cervical cancer HeLa cells. Endoplasmic reticulum stress and mitochondrial pathway were involved in 6-shogaol-mediated apoptosis. Proteomic analysis based on label-free strategy by liquid chromatography chip quadrupole time-of-flight mass spectrometry was subsequently proposed to identify, in a non-target-biased manner, the molecular changes in cellular proteins in response to 6-shogaol treatment. A total of 287 proteins were differentially expressed in response to 24 h treatment with 15 μM 6-shogaol in HeLa cells. Significantly changed proteins were subjected to functional pathway analysis by multiple analyzing software. Ingenuity pathway analysis (IPA suggested that 14-3-3 signaling is a predominant canonical pathway involved in networks which may be significantly associated with the process of apoptosis and G2/M cell cycle arrest induced by 6-shogaol. In conclusion, this work developed an unbiased protein analysis strategy by shotgun proteomics and bioinformatics analysis. Data observed provide a comprehensive analysis of the 6-shogaol-treated HeLa cell proteome and reveal protein alterations that are associated with its anticancer mechanism.

  4. Assessment of Cytotoxic Activity of Rosemary (Rosmarinus officinalis L.), Turmeric (Curcuma longa L.), and Ginger (Zingiber officinale R.) Essential Oils in Cervical Cancer Cells (HeLa)

    Science.gov (United States)

    Santos, P. A. S. R.; Avanço, G. B.; Nerilo, S. B.; Marcelino, R. I. A.; Janeiro, V.; Valadares, M. C.

    2016-01-01

    The objective of this study was to evaluate the cytotoxic activity of rosemary (REO, Rosmarinus officinalis L.), turmeric (CEO, Curcuma longa L.), and ginger (GEO, Zingiber officinale R.) essential oils in HeLa cells. Cytotoxicity tests were performed in vitro, using tetrazolium (MTT) and neutral red assays for evaluation of antiproliferative activity by different mechanisms, trypan blue assay to assess cell viability and evaluation of cell morphology for Giemsa to observe the cell damage, and Annexin V to evaluate cell death by apoptosis. CEO and GEO exhibited potent cytotoxic activity against HeLa cells. IC50 obtained was 36.6 μg/mL for CEO and 129.9 μg/mL for GEO. The morphology of HeLa cells showed condensation of chromatin, loss of cell membrane integrity with protrusions (blebs), and cell content leakage for cells treated with CEO and GEO, from the lowest concentrations studied, 32.81 μg/mL of CEO and 32.12 μg/mL of GEO. The Annexin V assay revealed a profile of cell death by apoptosis for both CEO and GEO. The results indicate cytotoxic activity in vitro for CEO and GEO, suggesting potential use as anticancer agents for cervical cancer cells. PMID:28042599

  5. Local Content

    CSIR Research Space (South Africa)

    Gibberd, Jeremy

    2016-10-01

    Full Text Available is also delineated in order to demonstrate the implications of local content on building design, construction and operation. The advantages and disadvantages of local content approaches are discussed and illustrated through examples. Finally, broad...

  6. Functional analysis of a recombinant PIII-SVMP, GST-acocostatin; an apoptotic inducer of HUVEC and HeLa, but not SK-Mel-28 cells

    Science.gov (United States)

    Teklemariam, Takele; Seoane, Agustin I.; Ramos, Carla J.; Sanchez, Elda E.; Lucena, Sara E.; Perez, John C.; Mandal, Stephanie A.; Soto, Julio G.

    2011-01-01

    Disintegrins and disintegrin-like peptides interact with integrins and interfere with cell-cell and cell-matrix interactions. A disintegrin-like snake venom gene, Acocostatin was cloned from the venom gland mRNA of Agkistrodon contortrix contortrix. Acocostatin belongs to the PIII-SVMP subfamily of disintegrin-like peptides. The recombinant acocostatin peptide was produced and purified as GST-fusion. The GST-acocostatin peptide, at 44 μg/mL, inhibited platelet aggregation by 30% in PRP and 18% in whole blood. In addition GST-acocostatin, at 220μg/mL, inhibited SK-Mel-28 cell migration by 48%, but did not inhibit T24 cell migration. The GST-acocostatin peptide ability to induce apoptosis on HUVEC, HeLa, and SK-Mel-28 cells was determined using Annexin-V-FITC and chromatin fragmentation assays after 24 h of treatment. At 5 μM GST-acocostatin peptide, 19.68% +/− 3.09 of treated HUVEC, and 35.86% +/− 2.05 of treated HeLa cells were in early apoptosis. The GST-acocostatin peptide also caused chromatin fragmentation of HUVEC and HeLa cells as determined by fluorescent microscopy and Hoechst staining. The GST-acocostatin peptide failed to induce apoptosis of SK-Mel-28 cells. We characterized the HUVEC, HeLa, and T24 integrin expression by flow cytometry, as the first step in determining GST-acocostatin binding specificity. Our results indicate that HUVEC express αv, αvβ3, αvβ5, α6, β1, and β3 integrin receptors. HeLa cells express α1, α2, α6, αv, αvβ5, and β1 integrin receptors. T24 cells express α1, α3, α6, αv, αvβ3, αvβ5, β1, β3, and β6 integrin receptors. PMID:21255601

  7. Local complications

    NARCIS (Netherlands)

    van den Akker, H.P.; Baart, J.A.; Baart, J.A.; Brand, H.S.

    2017-01-01

    Local anaesthesia is frequently used in dentistry and seldom leads to serious local complications. Nevertheless, it is of great importance to be aware of the causes of each local complication and – if necessary – implement correct treatment. The patient must be informed extensively and, if

  8. Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells.

    Science.gov (United States)

    Casanova, Alain; Low, Shyan H; Emmenlauer, Mario; Conde-Alvarez, Raquel; Salcedo, Suzana P; Gorvel, Jean-Pierre; Dehio, Christoph

    2016-08-05

    Brucella species are facultative intracellular pathogens that infect animals as their natural hosts. Transmission to humans is most commonly caused by direct contact with infected animals or by ingestion of contaminated food and can lead to severe chronic infections. Brucella can invade professional and non-professional phagocytic cells and replicates within endoplasmic reticulum (ER)-derived vacuoles. The host factors required for Brucella entry into host cells, avoidance of lysosomal degradation, and replication in the ER-like compartment remain largely unknown. Here we describe two assays to identify host factors involved in Brucella entry and replication in HeLa cells. The protocols describe the use of RNA interference, while alternative screening methods could be applied. The assays are based on the detection of fluorescently labeled bacteria in fluorescently labeled host cells using automated wide-field microscopy. The fluorescent images are analyzed using a standardized image analysis pipeline in CellProfiler which allows single cell-based infection scoring. In the endpoint assay, intracellular replication is measured two days after infection. This allows bacteria to traffic to their replicative niche where proliferation is initiated around 12 hr after bacterial entry. Brucella which have successfully established an intracellular niche will thus have strongly proliferated inside host cells. Since intracellular bacteria will greatly outnumber individual extracellular or intracellular non-replicative bacteria, a strain constitutively expressing GFP can be used. The strong GFP signal is then used to identify infected cells. In contrast, for the entry assay it is essential to differentiate between intracellular and extracellular bacteria. Here, a strain encoding for a tetracycline-inducible GFP is used. Induction of GFP with simultaneous inactivation of extracellular bacteria by gentamicin enables the differentiation between intracellular and extracellular

  9. Development of kinetochores during early mitosis in HeLa cells and the stability of a trilaminar kinetochore structure

    Energy Technology Data Exchange (ETDEWEB)

    Schroeter, D.; Paweletz, N.; Finze, E.M.; Kiesewetter, U.L. [German Cancer Research Center, Heidelberg (Germany)

    1993-12-31

    The ultrastructure of the kinetochore varies during the course of mitosis. This variation is considered to be a progressive maturation process that may correlate with kinetochore function. We examined the development of kinetochore structures on condensing prophase chromosomes in HeLa cells. Usually, one accepts the three laminar structure of the kinetochore of a condensed metaphase chromosome to reflect the general state and to be a prerequisite for microtubule attachment. The study of developing kinetochores in mitotic cells revealed unquestionable exceptions from this dogma. At the onset of chromatin condensation, indicating the beginning of prophase, the nuclear envelope and the nucleoli are still intact. First indications for kinetochore formation, documented as clouds of a fibrous material or as the {open_quotes}kinetochore ball structure{close_quotes} can be identified along or close to distinct heterochromatic, condensed chromatin elements. These fibrils extend from or interdigitate with the folding chromatin. Enhanced density of the ball structure represents a condensation process. Concomitantly a segregation of a plate structure is detected, occuring at a distal zone but obviously not at the end of the fibrils. This differentiation into kinetochore plate and corona more or less coincides with two other important prophase processes: the first tangential contacts to single microtubules and the mitotic breakdown of the nuclear envelope. But evidence will be discussed that these early prophase features not necessarily depend on each other and that the formation of a characteristic, trilaminar kinetochore structure does not guarantee its function. Conversely, structurally incomplete kinetochores can establish microtubular contacts for the attachment of chromosomes into the spindle.

  10. Extracellular gentamicin reduces the activity of connexin hemichannels and interferes with purinergic Ca2+ signaling in HeLa cells

    Directory of Open Access Journals (Sweden)

    Vania A Figueroa

    2014-09-01

    Full Text Available Gap junction channels (GJCs and hemichannels (HCs are composed of protein subunits termed connexins (Cxs and are permeable to ions and small molecules. In most organs, GJCs communicate the cytoplasm of adjacent cells, while HCs communicate the intra and extracellular compartments. In this way, both channel types coordinate physiological responses of cell communities. Cx mutations explain several genetic diseases, including about 50% of autosomal recessive nonsyndromic hearing loss. However, the possible involvement of Cxs in the etiology of acquired hearing loss remains virtually unknown. Factors that induce post-lingual hearing loss are diverse, exposure to gentamicin an aminoglycoside antibiotic, being the most common. Gentamicin has been proposed to block GJCs, but its effect on HCs remains unknown. In this work, the effect of gentamicin on the functional state of HCs was studied and its effect on GJCs was reevaluated in HeLa cells stably transfected with Cxs. We focused on Cx26 because it is the main Cx expressed in the cochlea of mammals where it participates in purinergic signaling pathways. We found that gentamicin applied extracellularly reduces the activity of HCs, while dye transfer across GJCs was not affected. HCs were also blocked by streptomycin, another aminoglycoside antibiotic. Gentamicin also reduced the ATP release and the HC-dependent oscillations of cytosolic free-Ca2+ signal. Moreover, gentamicin drastically reduced the Cx26 HC-mediated membrane currents in Xenopus laevis oocytes. Therefore, the extracellular gentamicin-induced inhibition of Cx HCs may adversely affect autocrine and paracrine signaling, including the purinergic one, which might partially explain its ototoxic effects.

  11. A Stable HeLa Cell Line That Inducibly Expresses Poliovirus 2Apro: Effects on Cellular and Viral Gene Expression

    Science.gov (United States)

    Barco, Angel; Feduchi, Elena; Carrasco, Luis

    2000-01-01

    A HeLa cell clone (2A7d) that inducibly expresses the gene for poliovirus protease 2A (2Apro) under the control of tetracycline has been obtained. Synthesis of 2Apro induces severe morphological changes in 2A7d cells. One day after tetracycline removal, cells round up and a few hours later die. Poliovirus 2Apro cleaves both forms of initiation factor eIF4G, causing extensive inhibition of capped-mRNA translation a few hours after protease induction. Methoxysuccinyl-Ala-Ala-Pro-Val-chloromethylketone, a selective inhibitor of 2Apro, prevents both eIF4G cleavage and inhibition of translation but not cellular death. Expression of 2Apro still allows both the replication of poliovirus and the translation of mRNAs containing a picornavirus leader sequence, while vaccinia virus replication is drastically inhibited. Translation of transfected capped mRNA is blocked in 2A7d-On cells, while luciferase synthesis from a mRNA bearing a picornavirus internal ribosome entry site (IRES) sequence is enhanced by the presence of 2Apro. Moreover, synthesis of 2Apro in 2A7d cells complements the translational defect of a poliovirus 2Apro-defective variant. These results show that poliovirus 2Apro expression mimics some phenotypical characteristics of poliovirus-infected cells, such as cell rounding, inhibition of protein synthesis and enhancement of IRES-driven translation. This cell line constitutes a useful tool to further analyze 2Apro functions, to complement poliovirus 2Apro mutants, and to test antiviral compounds. PMID:10666269

  12. [Studies of the origin of malignant rhabdoid tumor(MRT)--experimental researches on the MRT evolving in nude mice inoculated with violently variable HeLa cells].

    Science.gov (United States)

    Zhang, D L; Huang, G S; Li, L J; He, X Y; Xia, G T; Gao, B X; Bai, X H; Liu, S G

    2000-01-01

    Under the prerequisite that the incidence of cancer or tumor in negatively-controlled nude mice inoculated subcutaneously with feline or canine kidney cell cultures purified in vitro at passage 3 or higher (the modal chromosome number of FKC on passage 3 was 38 of diploid at the rate of 80%) was 0%(0/22) and 0%(0/10) respectively, and the incidence of progressively negative growing tumor in controlled nude mice inoculated subcutaneously with repeatedly-frozen- and thawed-HeLa cell cultures of X strain was 20%(1/5), the negative growing malignant tumor (MT) was found in half of the nude mice inoculated subcutaneously with HeLa cell cultures of H strain(with modal chromosome number of 78 +/- 2 of sub-tetraploid at the rate of 40%), the progressively-growing malignant tumor was found in all the other 40 nude mice inoculated subcutaneously with HeLa cell cultures of other strains, with the incidence of MT in nude mice with KB strain (with modal chromosome number of 60 +/- 3 of hyperdiploid at the rate of 72%-76%) 10/10, the incidence of poorly-differentiated MT originated from epithelia in nude mice with X strain (with modal chromosomal number of 62 +/- 3 of hyperdiploid at the rate of 69%) 25/25, and the incidence of MRT in nude mice with in vitro cultured tumor cell NM20/X strain (with modal chromosome number of 68 +/- 3 of both hyperdiploid and subtetraploid at the rate of 52%) 5/5. After being continuously cultivated for 20 passages in vitro, HeLa cell of X strain was subcutaneously inoculated into nude mice and cultivated for 1 passage in vivo within 15 days, and then the developed growing MT was collected as HeLa cell of NM20/X strain on passage 0 and continuously cultivated for 11 passages to prepare for transplanting into nude mice again. Therefore, the highly variable strain of HeLa cells can be successfully selected by alternate cultivation in vitro and in vivo. Occasionally in another experiment, the progressively-growing MRT was found in all the 4 nude mice

  13. [Expression, subcellular localization and nuclear translocation of transcription factor up stream stimulatory factor-1 in odontoblasts].

    Science.gov (United States)

    Wu, Li-An; Wen, Ling-Ying; Yang, Fu-Sheng; Wang, Xiao-Jing; Fang, Jun

    2007-09-01

    To examine the expression and subcellular localization of transcription factor USF1 in odontoblasts and investigate whether nuclear translocation occurs under stimuli. Odontoblasts MDPC-23 were cultured on coverslips and divided into 2 groups. Group 1 received no stimuli, and group 2 was stimulated by nicotine with various concentrations respectively for 1h. Then the mountings of odontoblasts were prepared and immunocytochemical staining was performed with specific USF1 antibody via SABC method. Hela cells were used as positive control. The staining was positive in the cytoplasm of odontoblasts in group 1, but in the nuclei of Hela cells and in 100 mg/L nicotine-stimulated odontoblasts in group 2. There exists USF1 protein in odontoblasts, which locates in the cytoplasm and could translocate into nuclei under the stimulation of nicotine.

  14. Biocompatible chitosan based hydrogels for potential application in local tumour therapy.

    Science.gov (United States)

    Olaru, Anda-Mihaela; Marin, Luminita; Morariu, Simona; Pricope, Gabriela; Pinteala, Mariana; Tartau-Mititelu, Liliana

    2018-01-01

    A series of hydrogels based on chitosan polyamine and nitrosalicylaldehyde were prepared via dynamic covalent chemistry (DCC), by imination and transimination reactions towards ordered clusters which play the role of crosslinking nodes of the chitosan network. The hydrogelation mechanism has been proved through NMR and FTIR spectroscopy, X-ray diffraction and polarized light microscopy. The successful preparation of the hydrogels and their mechanical properties were further investigated using rheological measurements. By electron scanning microscopy, the hydrogels exhibited a channels microstructure morphology which critically influenced their fast swelling by capillarity. The hydrogels cytotoxicity was explored in vitro on HeLa cancer cells and their biocompatibility was monitored in vivo by subcutaneous implantation on rats. The novel hydrogels proved good in vitro cytotoxicity on the HeLa cells and also in vivo biocompatibility in rats. Thus, these novel biomaterials promise to be suitable for local cancer therapy. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. [miR-143 inhibits cell proliferation through targeted regulating the expression of K-ras gene in HeLa cells].

    Science.gov (United States)

    Qin, H X; Cui, H K; Pan, Y; Hu, R L; Zhu, L H; Wang, S J

    2016-12-23

    Objective: To explore the effect of microRNA miR-143 on the proliferation of cervical cancer HeLa cells through targeted regulating the expression of K-ras gene. Methods: The luciferase report carrier containing wild type 3'-UTR of K-ras gene (K-ras-wt) or mutated 3'-UTR of the K-ras (K-ras-mut) were co-transfected with iR-143 mimic into the HeLa cells respectively, and the targeting effect of miR-143 in the transfectants was verified by the dual luciferase report system. HeLa cells were also transfected with miR-143 mimic (miR-143 mimic group), mimic control (negative control group), and miR-143 mimic plus K-ras gene (miR-143 mimic+ K-ras group), respectively. The expression of miR-143 in the transfected HeLa cells was detected by real-time PCR (RT-PCR), and the expression of K-ras protein was detected by Western blot. The cell proliferation activity of each group was examined by MTT assay. In addition, human cervical cancer tissue samples (n=5) and cervical intraepithelial neoplasia tissue samples (n=5) were also examined for the expression of miR-143 and K-ras protein by RT-PCR and Western blot, respectively. Results: The luciferase report assay showed that co-transfection with miR-143 mimic decreased the luciferase activity of the K-ras-wt significantly, but did not inhibit the luciferase activity of the K-ras-mut. The expression of miR-143 in the HeLa cells transfected with miR-143 mimic was significantly higher than that in the HeLa cells transfected with the mimic control (3.31±0.45 vs 0.97±0.22, Pras group was also significantly lower than the control group (Pras protein in the miR-143 mimic group, the negative control group and the miR-143 mimic+ K-ras group were lowest, moderate, and highest, respectively (115.27±34.08, 521.36±41.89, and 706.52±89.44, all Pras protein expression in the cervical cancer group was significantly higher than that in the cervical intraepithelial neoplasia group (584.39±72.34 vs. 114.23±25.82, Pras gene. In human cervical

  16. Localized Scleroderma

    Science.gov (United States)

    ... scleroderma, or who are experts at examining the skin, can arrive at the diagnosis without much difficulty after a careful examination. In ... especially in patients who continue to have active skin problems or develop new ... Diagnosis of Localized Scleroderma? The diagnosis of localized scleroderma ...

  17. Net Locality

    DEFF Research Database (Denmark)

    de Souza e Silva, Adriana Araujo; Gordon, Eric

    Provides an introduction to the new theory of Net Locality and the profound effect on individuals and societies when everything is located or locatable. Describes net locality as an emerging form of location awareness central to all aspects of digital media, from mobile phones, to Google Maps...... of emerging technologies, from GeoCities to GPS, Wi-Fi, Wiki Me, and Google Android....

  18. Radiation dose rate affects the radiosensitization of MCF-7 and HeLa cell lines to X-rays induced by dextran-coated iron oxide nanoparticles.

    Science.gov (United States)

    Khoshgard, Karim; Kiani, Parvaneh; Haghparast, Abbas; Hosseinzadeh, Leila; Eivazi, Mohammad Taghi

    2017-08-01

    The aim of radiotherapy is to deliver lethal damage to cancerous tissue while preserving adjacent normal tissues. Radiation absorbed dose of the tumoral cells can increase when high atomic nanoparticles are present in them during irradiation. Also, the dose rate is an important aspect in radiation effects that determines the biological results of a given dose. This in vitro study investigated the dose-rate effect on the induced radiosensitivity by dextran-coated iron oxide in cancer cells. HeLa and MCF-7 cells were cultured in vitro and incubated with different concentrations of dextran-coated iron oxide nanoparticles. They were then irradiated with 6 MV photons at dose rates of 43, 185 and 370 cGy/min. The MTT test was used to obtain the cells' survival after 48 h of irradiations. Incubating the cells with the nanoparticles at concentrations of 10, 40 and 80 μg/ml showed no significant cytotoxicity effect. Dextran-coated iron oxide nanoparticles showed more radiosensitivity effect by increasing the dose rate and nanoparticles concentration. Radiosensitization enhancement factors of MCF-7 and HeLa cells at a dose-rate of 370 cGy/min and nanoparticles' concentration of 80 μg/ml were 1.21 ± 0.06 and 1.19 ± 0.04, respectively. Increasing the dose rate of 6 MV photons irradiation in MCF-7 and HeLa cells increases the radiosensitization induced by the dextran-coated iron nanoparticles in these cells.

  19. Isolation of Melittin from Iranian Honey Bee Venom and Investigation of Its Effect on Proliferation of Cervical Cancer- HeLa Cell Line

    Directory of Open Access Journals (Sweden)

    K Pooshang Bagheri

    2013-06-01

    Full Text Available Introduction: Cervical cancer is the second prevalent cancer in developing countries and the sixth prevalent cancer in USA. Since conventional treatment methods are associated with detrimental side effects, searching for new drugs using natural ingredients is very important. Previous studies have shown that melittin (main component of honey bee venom has anticancer properties along with the effect on cell membrane and activation of apoptosis. In this study, inhibitory effects of melittin on the viability and proliferation of cervical cancer cell line (HeLa was investigated. Methods: Melittin was purified from honeybee venom using reversed-phase HPLC method. Then, biological activity of melittin was examined by hemolytic activity analysis on the red blood cells. In order to investigate whether melittin inhibits proliferation of HeLa cell, MTT assay was performed. HeLa cells were plated in a 96-well plate and treated with serially diluted concentrations of melittin for 12 and 24 hours. The viability of the cells was measured via MTT assay at 540nm. Results: Melittin showed a strong hemolytic activity (HD50=0.5 µg/ml which can be reduced by FBS(HD50=2 µg/ml. Results of MTT assay indicated that melittin shows cytotoxic effect on cervical cancer cells with IC50 = 1.2 ug/ml at 12h incubation period. Conclusion: In this study, biological activity of melittin and inhibitory effect of FBS on hemolysis were determined via hemolytic activity analysis. MTT assay indicated that melittin induced cytotoxic effects in a dose dependent manner on cervical cancer cells and it also revealed dependence on incubation time as well.

  20. 1-(2-Hydroxy-5-methylphenyl-3-phenyl-1,3-propanedione Induces G1 Cell Cycle Arrest and Autophagy in HeLa Cervical Cancer Cells

    Directory of Open Access Journals (Sweden)

    Jie-Heng Tsai

    2016-08-01

    Full Text Available The natural agent, 1-(2-hydroxy-5-methylphenyl-3-phenyl-1,3-propanedione (HMDB, has been reported to have growth inhibitory effects on several human cancer cells. However, the role of HMDB in cervical cancer remains unclear. Herein, we found that HMDB dose- and time-dependently inhibited growth of HeLa cervical cancer cells, accompanied with G1 cell cycle arrest. HMDB decreased protein expression of cyclins D1/D3/E and cyclin-dependent kinases (CDKs 2/4/6 and reciprocally increased mRNA and protein levels of CDK inhibitors (p15, p16, p21, and p27, thereby leading to the accumulation of hypophosphorylated retinoblastoma (Rb protein. HMDB also triggered the accumulation of acidic vesicles and formation of microtubule-associated protein-light chain 3 (LC3, followed by increased expression of LC3 and Beclin-1 and decreased expression of p62, suggesting that HMDB triggered autophagy in HeLa cells. Meanwhile, suppression of the expression of survivin and Bcl-2 implied that HMDB-induced autophagy is tightly linked to apoptosis. Exploring the action mechanism, HMDB induced autophagy via the modulation of AMP-activated protein kinase (AMPK and mTOR signaling pathway rather than the class III phosphatidylinositol 3-kinase pathway. These results suggest that HMDB inhibits HeLa cell growth by eliciting a G1 arrest through modulation of G1 cell cycle regulators and by concomitantly inducing autophagy through the mediation of AMPK-mTOR and Akt-mTOR pathways, and may be a promising antitumor agent against cervical cancer.

  1. Human papillomavirus type 16 and 18 L1 protein peptide binding to VERO and HeLa cells inhibits their VLPs binding.

    Science.gov (United States)

    Vera-Bravo, Ricardo; Ocampo, Marisol; Urquiza, Mauricio; García, Javier E; Rodríguez, Luis E; Puentes, Alvaro; López, Ramses; Curtidor, Hernando; Suárez, Jorge E; Torres, Elizabeth; Guzmán, Fanny; Díaz, Diana; Cortes, Jimena; Bravo, María M; Cómbita, Alba L; Orozco, Oscar; Patarroyo, Manuel E

    2003-11-10

    Human papillomaviruses (HPVs) are the cause of epithelial lesions, HPV type 16 and type 18 being associated with the development of anogenital cancer. The L1 Major Capsid Protein (L1) represents about 90% of total HPV protein and is involved in virus-host cell interaction, but little is known about this binding process. L1 sequences from HPV types 16 and 18 were synthesized in 56 20-mer peptides, covering the entire protein, HPLC-purified, (125)I-radiolabeled and tested in VERO and HeLa cell-binding assays to identify those peptides with high specific binding activity. Peptides 18283 (residues 54-77) and 18294 (274-308) from HPV16 L1, as well as 18312 (59-78) and 18322 (259-278) from HPV18 L1, presented high specific target cell binding activity. Peptide 18283 and 18294 affinity constants were 300 and 600 nM, respectively. Enzyme cell treatment before binding assay indicated that VERO and HeLa cell peptide receptor is a surface-exposed protein. There was a 60% reduction in peptide 18283 binding to heparin lyase-treated cells. Cross-linking assays showed that these proteins molecular weights were around 69 and 54 kDa. Peptides 18283 and 18294 specifically inhibited HPV-16 VLP binding to HeLa cells. According to the L1- and VLP-reported structure, both peptides are close on the VLP-surface, belonging to the outer surface broad pockets suggested as being potential receptor sites. Furthermore, it has been reported that a conserved motif from peptide 18294 is the target for neutralizing antibodies. These results suggest that such binding sequences are used by the virus as cell-binding regions. Copyright 2003 Wiley-Liss, Inc.

  2. Using HeLa cell stress response to introduce first year students to the scientific method, laboratory techniques, primary literature, and scientific writing.

    Science.gov (United States)

    Resendes, Karen K

    2015-01-01

    Incorporating scientific literacy into inquiry driven research is one of the most effective mechanisms for developing an undergraduate student's strength in writing. Additionally, discovery-based laboratories help develop students who approach science as critical thinkers. Thus, a three-week laboratory module for an introductory cell and molecular biology course that couples inquiry-based experimental design with extensive scientific writing was designed at Westminster College to expose first year students to these concepts early in their undergraduate career. In the module students used scientific literature to design and then implement an experiment on the effect of cellular stress on protein expression in HeLa cells. In parallel the students developed a research paper in the style of the undergraduate journal BIOS to report their results. HeLa cells were used to integrate the research experience with the Westminster College "Next Chapter" first year program, in which the students explored the historical relevance of HeLa cells from a sociological perspective through reading The Immortal Life of Henrietta Lacks by Rebecca Skloot. In this report I detail the design, delivery, student learning outcomes, and assessment of this module, and while this exercise was designed for an introductory course at a small primarily undergraduate institution, suggestions for modifications at larger universities or for upper division courses are included. Finally, based on student outcomes suggestions are provided for improving the module to enhance the link between teaching students skills in experimental design and execution with developing student skills in information literacy and writing. © 2015 The International Union of Biochemistry and Molecular Biology.

  3. Do altered activities of superoxide dismutases and the level of NF-kB modulate the effects of gamma radiation in HeLaS3 cells?

    Directory of Open Access Journals (Sweden)

    ANA NICIFOROVIC

    2007-10-01

    Full Text Available Most experimental models, including cell culture studies, have demon­strated that over-expression of manganese superoxide dismutase (MnSOD in cells bearing a carcinoma phenotype has anti-proliferative and tumour suppression chara­cteristics. In contrast, when cervical carcinoma biopsies express MnSOD, there is a poor prognosis and resistance to radiation therapy. The results herein indicate that human cervical adenocarcinoma (HeLaS3 cells have increased MnSOD activity (up to 50 % of the total SOD activity due to low expression of its repressor p53 and a high level of oxidative stress arising from the cell culture conditions. High MnSOD activity may be related to HeLaS3 cell radioresistance, illustrated by a high IC50 of 3.4 Gy and by a relatively high level of cell viability after gamma irradiation. In contrast to MnSOD activity, cytosolic CuZnSOD activity decreased after ionising radiation. The catalase (Cat activity was unchanged. IR also increa­sed the nitric oxide synthase (NOS activity. Such conditions lead to increased con­centrations of the superoxide radical, hydrogen peroxide and NO., which together may be responsible for the decreased expression of NF-kB and unaltered Cat ac­tivity. Therefore, the disturbed redox balance within HeLaS3 cells may be respon­sible for the cytotoxicity observed at higher irradiation doses. It could be concluded that inhibition of the CuZnSOD activity may be an important target for the selective killing of radioresistant cancer cells.

  4. Quantitative and qualitative effect of gH625 on the nanoliposome-mediated delivery of mitoxantrone anticancer drug to HeLa cells.

    Science.gov (United States)

    Perillo, Emiliana; Allard-Vannier, Emilie; Falanga, Annarita; Stiuso, Paola; Vitiello, Maria Teresa; Galdiero, Massimiliano; Galdiero, Stefania; Chourpa, Igor

    2015-07-05

    The present work investigates in vitro the delivery of the anticancer drug mitoxantrone (MTX) to HeLa cancer cells by means of polyethylene glycol (PEG) liposomes functionalized with the novel cell penetrating peptide gH625. This hydrophobic peptide enhances the delivery of doxorubicin (Doxo) to the cytoplasm of cancer cells, while the mechanism of this enhancement has not yet been understood. Here, in order to get a better insight into the role of gH625 on the mechanism of liposome-mediated drug delivery, we treated HeLa cells with liposomes functionalized with gH625 and loaded with MTX; functionalized and not liposome were characterized in terms of their physico-chemical properties and drug release kinetics. To quantify the MTX uptake and to study the subcellular drug distribution and interaction, we took advantage of the intrinsic fluorescence of MTX and of the fluorescence-based techniques like fluorescence-activated cell sorting (FACS) and confocal spectral imaging (CSI). FACS data confirmed that gH625 increases the total intracellular MTX content. CSI data indicated that when liposomes are decorated with gH625 an enhanced staining of the internalized drug is observed mainly in hydrophobic regions of the cytoplasm, where the increased presence of an oxidative metabolite of the drug is observed. The cytotoxicity on HeLa cell line was higher for functionalized liposomes within 4-6h of treatment. To summarise, the MTX delivery with gH625-decorated nanoliposomes enhances the quantity of both the intracellular drug and of its oxidative metabolite and contributes to higher anticancer efficacy of the drug at the delay of 4-6h. Copyright © 2015 Elsevier B.V. All rights reserved.

  5. The E8 repression domain can replace the E2 transactivation domain for growth inhibition of HeLa cells by papillomavirus E2 proteins.

    Science.gov (United States)

    Stubenrauch, Frank; Straub, Elke; Fertey, Jasmin; Iftner, Thomas

    2007-11-15

    Continuous expression of the human papillomavirus (HPV) oncoproteins E6 and E7 is required for the growth of cervical cancer cell lines. So far, only the overexpression of the wild type papillomavirus E2 protein has been shown to induce growth arrest in HPV18-positive HeLa cells by repressing E6/E7 transcription. Growth arrest by E2 requires the aminoterminal transcription activation domain in addition to the carboxyterminal DNA-binding domain. Several papillomaviruses such as the carcinogenic HPV31 express in addition to E2 an E8(wedge)E2C fusion protein in which the E8 domain, which is required for repression of replication and transcription, replaces the E2 activation domain. In this report, we demonstrate that the HPV31 E8(wedge)E2C protein is able to inhibit the growth of HeLa cells but not of HPV-negative C33A cervical cancer cells. Growth repression by E8(wedge)E2C correlates with repression of the endogenous HPV18 E6/E7 promoter and the reappearance of E6- and E7-regulated p53, pRb and p21 proteins, suggesting that E8(wedge)E2C inhibits growth by reactivating dormant tumor suppressor pathways. Growth inhibition requires an intact E8 repression domain in addition to the carboxyterminal E2C DNA binding domain. Chromatin immunoprecipitation experiments suggest that the E8 repression domain enhances binding to the HPV18 promoter sequence in vivo. In summary, our results demonstrate that the small E8 repression domain can functionally replace the large E2 transactivation domain for growth inhibition of HeLa cervical cancer cells. (c) 2007 Wiley-Liss, Inc.

  6. Low angle x-ray diffraction studies of HeLa metaphase chromosomes: effects of histone phosphorylation and chromosome isolation procedure

    OpenAIRE

    1983-01-01

    To test whether gross changes in chromatin structure occur during the cell cycle, we compared HeLa mitotic metaphase chromosomes and interphase nuclei by low angle x-ray diffraction. Interphase nuclei and metaphase chromosomes differ only in the 30-40-nm packing reflection, but not in the higher angle part of the x-ray diffraction pattern. Our interpretation of these results is that the transition to metaphase affects only the packing of chromatin fibers and not, to the resolution of our meth...

  7. Mapping and identification of interferon gamma-regulated HeLa cell proteins separated by immobilized pH gradient two-dimensional gel electrophoresis

    DEFF Research Database (Denmark)

    Shaw, AC; Rossel Larsen, M; Roepstorff, P

    1999-01-01

    . A semiconfluent layer of HeLa cells was grown on tissue culture plates, and changes in protein expression due to 100 U/mL IFN-gamma were investigated at different periods after treatment, using pulse labeling with [35S]methionine/cysteine in combination with 2-D PAGE (IPG). The identity of eight protein spots...... was elucidated by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS), and several variants of the IFN-gamma-inducible tryptophanyl-tRNA synthetase (hWRS) were detected by immunoblotting....

  8. Cytotoxicity Studies of the Extracts, Fractions, and Isolated Compound of Pseudocedrela kotschyi on Cervical Cancer (HeLa), Breast Cancer (MCF-7) and Skeletal Muscle Cancer (RD) Cells.

    Science.gov (United States)

    Elufioye, Taiwo O; Abdul, Abolaji A; Moody, Jone O

    2017-01-01

    This study determined the cytotoxic effects of root and stem bark extracts, fractions, and isolated compounds derived from Pseudocedrela kotschyi on HeLa, MCF-7, and RD cells. The cytotoxic activity was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetric assay against three cell lines (RD, HeLa, and MCF 7) at concentrations ranging from 0.01 to 1000 μg/mL. Isolation of crude saponin was done from the most active ethyl acetate fraction and further purified using vacuum liquid chromatography and preparative thin layer chromatographic techniques. The cytotoxicity assay revealed that the methanol extract from the root bark and the ethyl acetate fraction from the stem bark exhibited marked anticancer activity with IC50 of 87.36 μg/ml and 21.53 μg/ml, respectively, on HeLa cancer cell line and 101.51 μg/mL and 38.46 μg/mL, respectively, on RD cell line. These values are comparable with that obtained from vinblastine and methotrexate used as standard drugs (IC50 values of 0.01 μg/mL and 0.05 μg/mL, respectively). The isolated crude saponins also gave IC50 values of 5.28 μg/mL and 81.52 μg/mL against the RD cell lines and IC50values of 1.05 μg/mL and 86.8 μg/mL for the MCF 7 cancer cell lines. PTLC led to the isolation of a compound from the crude saponin which was identified as 7-deacetoxy-7-oxogedunin through spectroscopic analysis and comparison with literature data. P. kotschyi could be considered as a potential source of chemotherapeutic agent. However, further research to determine the exact mechanism of action needs to be carried out. Pseudocedrela kotschyi methanol extract from the root bark and the ethyl acetate fraction from the stem bark exhibited marked anticancer activity on HeLa, MCF-7, and RD cell lines7-deacetoxy-7-oxogedunin isolated as a white crystalline substance from the most active ethyl acetate fraction contributed to the observed activity. Abbreviations Used: MTT: 3-(4,5-dimethylthiazol-2-yl)-2

  9. Repression of the Human Papillomavirus E6 Gene Initiates p53-Dependent, Telomerase-Independent Senescence and Apoptosis in HeLa Cervical Carcinoma Cells

    OpenAIRE

    Horner, Stacy M.; DeFilippis, Rosa Anna; Manuelidis, Laertes; DiMaio, Daniel

    2004-01-01

    Cervical cancer cells express high-risk human papillomavirus (HPV) E6 and E7 proteins. When both HPV oncogenes are repressed in HeLa cervical carcinoma cells, the dormant p53 and retinoblastoma (Rb) tumor suppressor pathways are activated, and the cells undergo senescence in the absence of apoptosis. When the E6 gene is repressed in cells that continue to express an E7 gene, the p53 pathway, but not the Rb pathway, is activated, and both senescence and apoptosis are triggered. To determine th...

  10. Repression of human papillomavirus oncogenes in HeLa cervical carcinoma cells causes the orderly reactivation of dormant tumor suppressor pathways

    OpenAIRE

    Goodwin, Edward C.; DiMaio, Daniel

    2000-01-01

    Most cervical carcinomas express high-risk human papillomaviruses (HPVs) E6 and E7 proteins, which neutralize cellular tumor suppressor function. To determine the consequences of removing the E6 and E7 proteins from cervical cancer cells, we infected HeLa cells, a cervical carcinoma cell line that contains HPV18 DNA, with a recombinant virus that expresses the bovine papillomavirus E2 protein. Expression of the E2 protein resulted in rapid repression of HPV E6 and E7 e...

  11. Microarray dataset of transient and permanent DNA methylation changes in HeLa cells undergoing inorganic arsenic-mediated epithelial-to-mesenchymal transition

    OpenAIRE

    Eckstein, Meredith; Rea, Matthew; Fondufe-Mittendorf, Yvonne N.

    2017-01-01

    The novel dataset presented here represents the results of the changing pattern of DNA methylation profiles in HeLa cells exposed to chronic low dose (0.5 µM) sodium arsenite, resulting in epithelial-to-mesenchymal transition, as well as DNA methylation patterns in cells where inorganic arsenic has been removed. Inorganic arsenic is a known carcinogen, though not mutagenic. Several mechanisms have been proposed as to how inorganic arsenic drives carcinogenesis such as regulation of the cell׳s...

  12. The C proteins of HeLa 40S nuclear ribonucleoprotein particles exist as anisotropic tetramers of (C1)3 C2.

    OpenAIRE

    Barnett, S F; Friedman, D L; LeStourgeon, W M

    1989-01-01

    The C proteins (C1 and C2) of HeLa 40S heterogeneous nuclear ribonucleoprotein particles copurify under native conditions as a stable complex with a fixed molar protein ratio (S.F. Barnett, W.M. LeStourgeon, and D.L. Friedman, J. Biochem. Biophys. Methods 16:87-97, 1988). Gel filtration chromatography and velocity sedimentation analyses of these complexes revealed a large Stokes radius (6.2 nm) and a sedimentation coefficient of 5.8S. On the basis of these values and a partial specific volume...

  13. High Cytotoxic Activity of Phosphonium Salts and Their Complementary Selectivity towards HeLa and K562 Cancer Cells: Identification of Tri-n-butyl-n-hexadecylphosphonium bromide as a Highly Potent Anti-HeLa Phosphonium Salt.

    Science.gov (United States)

    Bachowska, Barbara; Kazmierczak-Baranska, Julia; Cieslak, Marcin; Nawrot, Barbara; Szczęsna, Dorota; Skalik, Joanna; Bałczewski, Piotr

    2012-02-01

    Quaternary ammonium and phosphonium salts have been screened for their toxic effect on HeLa and K562 cancer cell lines, as well as on normal HUVEC cells. Tri-n-butyl-n-hexadecylphosphonium bromide, the first phosphonium salt with a halogen anion tested against HeLa cells, was 12 times more potent (IC50 phosphonium salt to be evaluated in HeLa cells. However, it was inactive against K562 cells (24 and 48 h). According to a caspase-3/7 assay, its toxicity has not been connected with the induction of apoptosis. In contrast, triphenylalkylphosphonium iodides with shorter C1-5 alkyl chains were inactive against HeLa cells but very active against K562 cells (IC50=6-10 μm after 48 h). Phosphonium cations with halide counterions proved to be more potent than those with (CF3SO2)2N(-) as the anion, as in the anticancer agent NSC 747251, or other anions in molecules with similar alkyl chain lengths. On the other hand, a series of ammonium salts containing a short methylthiomethyl or methoxymethyl side chain revealed low cytotoxicity (IC50 >500 μm after 24 and 48 h) against both HeLa and K562 cancer cell lines as well as normal HUVEC cells, showing that the nontoxic N(+)CH2YMe (Y=S, O) structural motif in ammonium salts could be suitable for further optimization and development, especially in transfection experiments.

  14. Mapping and identification of HeLa cell proteins separated by immobilized pH-gradient two-dimensional gel electrophoresis and construction of a two-dimensional polyacrylamide gel electrophoresis database

    DEFF Research Database (Denmark)

    Shaw, AC; Rossel Larsen, M; Roepstorff, P

    1999-01-01

    The HeLa cell line, a human adenocarcinoma, is used in many research fields, since it can be infected with a wide range of viruses and intracellular bacteria. Therefore, the mapping of HeLa cell proteins is useful for the investigation of parasite host cell interactions. Because of the recent imp...... these and future data accessible for interlaboratory comparison, we constructed a 2-D PAGE database on the World Wide Web.......The HeLa cell line, a human adenocarcinoma, is used in many research fields, since it can be infected with a wide range of viruses and intracellular bacteria. Therefore, the mapping of HeLa cell proteins is useful for the investigation of parasite host cell interactions. Because of the recent...... the mapping of [35S]methionine/cysteine-labeled HeLa cell proteins with the 2-D PAGE (IPG)-system, using matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) and N-terminal sequencing for protein identification. To date 21 proteins have been identified and mapped. In order to make...

  15. The epimer of kaurenoic acid from Croton antisyphiliticus is cytotoxic toward B-16 and HeLa tumor cells through apoptosis induction.

    Science.gov (United States)

    Fernandes, V C; Pereira, S I V; Coppede, J; Martins, J S; Rizo, W F; Beleboni, R O; Marins, M; Pereira, P S; Pereira, A M S; Fachin, A L

    2013-04-02

    Cancer has become the leading cause of death in developing countries due to increased life expectancy of the population and changes in lifestyle. Studies on active principles of plant have motivated researchers to develop new antitumor agents that are specific and effective for treatment of neoplasms. Kaurane diterpenes are considered important compounds in the development of new and highly effective anticancer chemotherapeutic agents due to their cytotoxic properties in the induction of apoptosis. We evaluated the cytotoxic and apoptotic activity of the epimer of kaurenoic acid (EKA) isolated from the medicinal plant Croton antisyphiliticus (Euphorbiaceae) toward tumor cell lines HeLa and B-16 and normal fibroblasts 3T3. Based on analyses with the MTT test, EKA showed cytotoxic activity, with half maximal inhibitory concentration values of 59.41, 68.18 and 60.30 µg/mL for the B-16, HeLa and 3T3 cell lines, respectively. The assay for necrotic or apoptotic cells by differential staining showed induction of apoptosis in all three cell lines. We conclude that EKA is not selective between tumor and normal cell lines; the mechanism of action of EKA is induction of apoptosis, which is part of the innate mechanism of cell defense against neoplasia.

  16. Carboxymethyl chitosan nanoparticles coupled with CD59-specific ligand peptide for targeted delivery of C-phycocyanin to HeLa cells.

    Science.gov (United States)

    Yang, Peng; Li, Bing; Yin, Qi-Feng; Wang, Yu-Juan

    2017-03-01

    The combination of nanotechnology and medicine will be the next generation of vehicles for targeted drug delivery. Carboxymethyl chitosan loaded with the anticancer drug C-phycocyanin and the CD59-specific ligand peptide for cancer cell targeting were used to create C-phycocyanin/carboxymethyl chitosan-CD59-specific ligand peptide nanoparticles using the ionic-gelation method. Optimal synthesis conditions, selected by response surface methodology, comprised the ratio carboxymethyl chitosan:C-phycocyanin = 3:1, and carboxymethyl chitosan and CaCl2 concentrations of 2.0 and 1.0 mg/mL, respectively. The resulting nanoparticles were spherical, with diameters of approximately 200 nm; the entrapment efficient was about 65%; and the drug loading was about 20%. The release of C-phycocyanin from C-phycocyanin/carboxymethyl chitosan nanoparticles was pH sensitive and had a sustainable effect in vitro. Guided by the CD59-specific ligand peptide, the nanoparticles efficiently targeted the surface of HeLa cells and had an obvious inhibitory effect on HeLa cell proliferation as determined by methyl thiazolyl tetrazolium assays. The nanoparticles were hemocompatible and induced apoptosis by upregulation of cleaved caspase-3 and cleaved polyADP-ribose polymerase proteins, and downregulation of Bcl-2 proteins. Our study provides a novel approach to the research and development of marine drugs, and support for targeted therapy using anticancer drugs.

  17. Coating lanthanide nanoparticles with carbohydrate ligands elicits affinity for HeLa and RAW264.7 cells, enhancing their photodamaging effect.

    Science.gov (United States)

    Kanamori, Takashi; Sawamura, Takashi; Tanaka, Tatsumi; Sotokawa, Izumi; Mori, Ryota; Inada, Kotaro; Ohkubo, Akihiro; Ogura, Shun-Ichiro; Murayama, Yasutoshi; Otsuji, Eigo; Yuasa, Hideya

    2017-01-15

    Lanthanide nanoparticles (LNPs) conjugated with monosaccharides were synthesized as a photon energy-upconverting nanodevice with affinity to cancer cells. The conjugates were designed to selectively damage the cancer cells containing protoporphyrin IX, a photosensitizer endogenously synthesized from priorly administrated 5-aminolevlunic acid (ALA), by a highly tissue-penetrative near-infrared (NIR) irradiation. First of all, the affinities of monosaccharides toward cells (HeLa, RAW264.7, and MKN45) were assessed by a novel cell aggregation assay with trivalent monosaccharide-citric acid conjugates. As a result, HeLa exhibited high affinity for glucose, while RAW264.7 for glucose, galactose, mannose, and fucose. A similar cell-monosaccharide affinity was microscopically observed when the cells were mixed with monosaccharide-LNP conjugates and rinsed, in which the high affinity LNP probes luminesced on the cells. The high affinity monosaccharide-LNPs showed greater photodamaging effects than the unmodified LNP toward the corresponding cells, when the cells were pretreated with ALA and irradiated by NIR. This study demonstrates that carbohydrates can be used as selective ligands for cancer cells in a photodynamic therapy with LNP. Copyright © 2016 Elsevier Ltd. All rights reserved.

  18. The neem limonoids azadirachtin and nimbolide induce cell cycle arrest and mitochondria-mediated apoptosis in human cervical cancer (HeLa) cells.

    Science.gov (United States)

    Priyadarsini, R Vidya; Murugan, R Senthil; Sripriya, P; Karunagaran, D; Nagini, S

    2010-06-01

    Limonoids from the neem tree (Azadirachta indica) have attracted considerable research attention in recent years owing to their potent antioxidant and anti-proliferative effects. The present study was designed to investigate the cellular and molecular mechanisms by which azadirachtin and nimbolide exert cytotoxic effects in the human cervical cancer (HeLa) cell line. Both azadirachtin and nimbolide significantly suppressed the viability of HeLa cells in a dose-dependent manner by inducing cell cycle arrest at G0/G1 phase accompanied by p53-dependent p21 accumulation and down-regulation of the cell cycle regulatory proteins cyclin B, cyclin D1 and PCNA. Characteristic changes in nuclear morphology, presence of a subdiploid peak and annexin-V staining pointed to apoptosis as the mode of cell death. Increased generation of reactive oxygen species with decline in the mitochondrial transmembrane potential and release of cytochrome c confirmed that the neem limonoids transduced the apoptotic signal via the mitochondrial pathway. Altered expression of the Bcl-2 family of proteins, inhibition of NF-kappaB activation and over-expression of caspases and survivin provide compelling evidence that azadirachtin and nimbolide induce a shift of balance toward a pro-apoptotic phenotype. Antioxidants such as azadirachtin and nimbolide that can simultaneously arrest the cell cycle and target multiple molecules involved in mitochondrial apoptosis offer immense potential as anti-cancer therapeutic drugs.

  19. Visualisation of cell cycle modifications by X-ray irradiation of single HeLa cells using fluorescent ubiquitination-based cell cycle indicators.

    Science.gov (United States)

    Kaminaga, K; Noguchi, M; Narita, A; Sakamoto, Y; Kanari, Y; Yokoya, A

    2015-09-01

    To explore the effects of X-ray irradiation on mammalian cell cycle dynamics, single cells using the fluorescent ubiquitination-based cell cycle indicator (Fucci) technique were tracked. HeLa cells expressing Fucci were used to visualise cell cycle modifications induced by irradiation. After cultured HeLa-Fucci cells were exposed to 5 Gy X-rays, fluorescent cell images were captured every 20 min for 48 h using a fluorescent microscope. Time dependence of the fluorescence intensity of S/G2 cells was analysed to examine the cell cycle dynamics of irradiated and non-irradiated control cells. The results showed that irradiated cells could be divided into two populations: one with similar cell cycle dynamics to that of non-irradiated cells, and another displaying a prolonged G2 phase. Based on these findings, it is proposed in this article that an underlying switch mechanism is involved in cell cycle regulation and the G2/M checkpoint of HeLa cells. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  20. Ethanolic Neem (Azadirachta indica Leaf Extract Prevents Growth of MCF-7 and HeLa Cells and Potentiates the Therapeutic Index of Cisplatin

    Directory of Open Access Journals (Sweden)

    Chhavi Sharma

    2014-01-01

    Full Text Available The present study was designed to gain insight into the antiproliferative activity of ethanolic neem leaves extract (ENLE alone or in combination with cisplatin by cell viability assay on human breast (MCF-7 and cervical (HeLa cancer cells. Nuclear morphological examination and cell cycle analysis were performed to determine the mode of cell death. Further, to identify its molecular targets, the expression of genes involved in apoptosis, cell cycle progression, and drug metabolism was analyzed by RT-PCR. Treatment of MCF-7, HeLa, and normal cells with ENLE differentially suppressed the growth of cancer cells in a dose- and time-dependent manner through apoptosis. Additionally, lower dose combinations of ENLE with cisplatin resulted in synergistic growth inhibition of these cells compared to the individual drugs (combination index <1. ENLE significantly modulated the expression of bax, cyclin D1, and cytochrome P450 monooxygenases (CYP 1A1 and CYP 1A2 in a time-dependent manner in these cells. Conclusively, these results emphasize the chemopreventive ability of neem alone or in combination with chemotherapeutic treatment to reduce the cytotoxic effects on normal cells, while potentiating their efficacy at lower doses. Thus, neem may be a prospective therapeutic agent to combat gynecological cancers.

  1. Cytotoxicity of different extracts of arial parts of Ziziphus spina-christi on Hela and MDA-MB-468 tumor cells

    Science.gov (United States)

    Jafarian, Abbas; Zolfaghari, Behzad; Shirani, Kobra

    2014-01-01

    Background: It has been shown that plants from the family Rhamnaceae possess anticancer activity. In this study, we sought to determine if Ziziphus spina-christi, a species from this family, has cytotoxic effect on cancer cell lines. Materials and Methods: Using maceration method, different extracts of leaves of Z. spina-christi were prepared. Hexane, chloroform, chloroform-methanol (9:1), methanol-water (7:1) methanol, butanol and water were used for extraction, after preliminary phytochemical analyses were done. The cytotoxic activity of the extracts against Hela and MDA-MB-468 tumor cells was evaluated by MTT assay. Briefly, cells were seeded in microplates and different concentrations of extracts were added. After incubation of cells for 72 h, their viability was evaluated by addition of tetrazolium salt solution. After 3 h medium was aspirated, dimethyl sulfoxide was added and absorbance was determined at 540 nm with an ELISA plate reader. Extracts were considered cytotoxic when more than 50% reduction on cell survival was observed. Results: Hexane, chloroform, chloroform-methanol, butanol, methanol-water and aqueous extracts of Z. spina-christi significantly and concentration-dependently reduced viability of Hela and MAD-MB-468 cells. In the both cell lines, chloroform-methanol extract of Z. spina-christi was more potent than the other extracts. Results: From the finding of this study it can be concluded that Z. spina-christi is a good candidate for further study for new cytotoxic agents. PMID:24627846

  2. Induction of Apoptotic Effects of Antiproliferative Protein from the Seeds of Borreria hispida on Lung Cancer (A549 and Cervical Cancer (HeLa Cell Lines

    Directory of Open Access Journals (Sweden)

    S. Rupachandra

    2014-01-01

    Full Text Available A 35 KDa protein referred to as F3 was purified from the seeds of Borreria hispida by precipitation with 80% ammonium sulphate and gel filtration on Sephadex G-100 column. RP-HPLC analysis of protein fraction (F3 on an analytical C-18 column produced a single peak, detected at 220 nm. F3 showed an apparent molecular weight of 35 KDa by SDS PAGE and MALDI-TOF-MS analyses. Peptide mass fingerprinting analysis of F3 showed the closest homology with the sequence of 1-aminocyclopropane-1-carboxylate deaminase of Pyrococcus horikoshii. The protein (F3 exhibited significant cytotoxic activity against lung (A549 and cervical (HeLa cancer cells in a dose-dependent manner at concentrations ranging from 10 µg to 1000 µg/mL, as revealed by the MTT assay. Cell cycle analysis revealed the increased growth of sub-G0 population in both cell lines exposed to a concentration of 1000 µg/mL of protein fraction F3 as examined from flow cytometry. This is the first report of a protein from the seeds of Borreria hispida with antiproliferative and apoptotic activity in lung (A549 and cervical (HeLa cancer cells.

  3. SYNTHESIS, IDENTIFICATION AND IN VITRO ANTITUMOUR PRESCREENING TEST OF TRIPHENYLTIN BENZOATE TOWARDS A HUMAN CERVICAL CARCINOMA CELL LINE, HeLa

    Directory of Open Access Journals (Sweden)

    Ida Bagus Putra Manuaba

    2010-06-01

    Full Text Available In this study, triphenyltin benzoate was synthesized first, and followed by antitumor prescreening test of the compound towards a human cervical carcinoma cell line, HeLa. Three reaction steps were employed to obtain the compound needed, i.e. 1 synthesizing of tetraphenyltin compound via insitu phenilmagnesiumbromide Grignard reaction to tin(IVchloride, 2 synthesizing triphenyltin chloride via redistribution reaction of tetraphenyltin to tin(IV chloride without any solvent, the reaction completed depends on the temperature, in this case a good results was achieved at temperature 220 °C for 6 h, 3 finally, triphenyltin benzoate was produced through a methathetical reaction of triphenyltin chloride to an excess of sodium benzoate in ethanol. In vitro prescreening antitumour activity of the compound towards a human cervical tumour cell line, HeLa was carried out following an enzyme linked immunosorbent assays (ELISA. By this method, the test ended with good promising results. This indicates by the IC50 of 170 nM which is compared well to cisplatinum with IC50 950 nM.   Keywords: redistribution reaction, methathetical reaction, cell line, in vitro, antitumour

  4. Core/shell Fe3O4 @SiO2 nanoparticles modified with PAH as a vector for EGFP plasmid DNA delivery into HeLa cells.

    Science.gov (United States)

    Shi, Mengran; Liu, Yiyao; Xu, Mingming; Yang, Hong; Wu, Chunhui; Miyoshi, Hirokazu

    2011-11-10

    Novel stable core/shell Fe(3)O(4)@SiO(2)/PAH nanoparticles are synthesized using 15 nm Fe(3)O(4) as the template that is modified with PAH. The resulting nanoparticles can absorb plasmid DNA to mediate gene transfer in cultured HeLa cells. An electrophoretic assay suggests that the Fe(3)O(4)@SiO(2)/PAH nanoparticles protect the plasmid DNA from serum and DNase I degradation. A cell viability assay shows that the Fe(3)O(4)@SiO(2)/PAH nanoparticles exhibit a low cytotoxicity toward endothelial cells. Qualitative analysis of transfection in HeLa cells by nanoparticles carrying a plasmid DNA encoding EGFP demonstrates a fairly high expression level, even in the presence of serum. Thus, Fe(3)O(4)@SiO(2)/PAH nanoparticles are biocompatible and suitable for nonviral delivery, and may find applications in cancer therapy. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. A class of DNA-binding peptides from wheat bud causes growth inhibition, G2 cell cycle arrest and apoptosis induction in HeLa cells

    Directory of Open Access Journals (Sweden)

    Elgjo Kjell

    2009-07-01

    Full Text Available Abstract Background Deproteinized DNA from eukaryotic and prokaryotic cells still contains a low-molecular weight peptidic fraction which can be dissociated by alkalinization of the medium. This fraction inhibits RNA transcription and tumor cell growth. Removal from DNA of normal cells causes amplification of DNA template activity. This effect is lower or absent in several cancer cell lines. Likewise, the amount of active peptides in cancer cell DNA extracts is lower than in DNA preparation of the corresponding normal cells. Such evidence, and their ubiquitous presence, suggests that they are a regulatory, conserved factor involved in the control of normal cell growth and gene expression. Results We report that peptides extracted from wheat bud chromatin induce growth inhibition, G2 arrest and caspase-dependent apoptosis in HeLa cells. The growth rate is decreased in cells treated during the S phase only and it is accompanied by DNA damage and DNA synthesis inhibition. In G2 cells, this treatment induces inactivation of the CDK1-cyclin B1 complex and an increase of active chk1 kinase expression. Conclusion The data indicate that the chromatin peptidic pool inhibits HeLa cell growth by causing defective DNA replication which, in turn, arrests cell cycle progression to mitosis via G2 checkpoint pathway activation.

  6. Myricetin and methyl eugenol combination enhances the anticancer activity, cell cycle arrest and apoptosis induction of cis-platin against HeLa cervical cancer cell lines.

    Science.gov (United States)

    Yi, Jin-Ling; Shi, Song; Shen, Yan-Li; Wang, Ling; Chen, Hai-Yan; Zhu, Jun; Ding, Yan

    2015-01-01

    Drug combination therapies are common practice in the treatment of cancer. In this study, we evaluated the anticancer effects of myricetin (MYR), methyl eugenol (MEG) and cisplatin (CP) both separately as well as in combination against cervical cancer (HeLa) cells. To demonstrate whether MYR and MEG enhance the anticancer activity of CP against cervical cancer cells, we treated HeLa cells with MYR and MEG alone or in combination with cisplatin and evaluated cell growth and apoptosis using MTT (3 (4, 5 dimethyl thiazol 2yl) 2, 5 diphenyltetrazolium bromide) assay, LDH release assay, flow cytometry and fluorescence microscopy. The results revealed that, as compared to single drug treatment, the combination of MYR or MEG with CP resulted in greater effect in inhibiting cancer cell growth and inducing apoptosis. Cell apoptosis induction, Caspase-3 activity, cell cycle arrest and mitochondrial membrane potential loss were systematically studied to reveal the mechanisms of synergy between MYR, MEG and CP. Combination of MYR or MEG with CP resulted in more potent apoptosis induction as revealed by fluorescence microscopy using Hoechst 33258 and AO-ETBR staining. The combination treatment also increased the number of cells in G0/G1 phase dramatically as compared to single drug treatment. Mitochondrial membrane potential loss (ΛΨm) as well as Caspase-3 activity was much higher in combination treatment as compared to single drug treatment. Findings of this investigation suggest that MYR and MEG combined with cisplatin is a potential clinical chemotherapeutic approach in human cervical cancer.

  7. Ezrin-Radixin-Moesin Binding Phosphoprotein 50 (EBP50) Suppresses the Metastasis of Breast Cancer and HeLa Cells by Inhibiting Matrix Metalloproteinase-2 Activity.

    Science.gov (United States)

    Wang, Lei; Qi, Yijun; Xiong, Ying; Peng, Zhiqiang; Ma, Qiang; Zhang, Yue; Song, Jian; Zheng, Junfang

    2017-08-01

    Expression of ezrin-radixin-moesin-binding phosphoprotein-50 (EBP50) is correlated with human breast and cervical cancer development, but its effects on the metastasis of breast and cervical cancer and the underlying mechanism are not fully understood. In this study, EBP50 was overexpressed in MDA-MB-231 breast cancer and HeLa cervical cancer cells; moreover, EBP50 was knocked-down in MCF-7 breast cancer cells and HeLa cells. Metastasis-related ability and matrix metalloproteinase-2 (MMP-2) activity of these cells were investigated. Cell adhesion, wound-healing and invasion were significantly suppressed in EBP50-overexpressing cells. Contrarily, EBP50-knockdown promoted cell adhesion, wound healing and invasion. EBP50 overexpression inhibited MMP-2 activity, and the knockdown of EBP50 promoted the activity of MMP-2, suggesting that EBP50 inhibited cell metastasis via suppression of MMP-2 activity. Our work reveals the anti-metastatic effect and a new mechanism of EBP50 action in breast and cervical cancer cells. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  8. Fluorescence quenching of free and bound NADH in HeLa cells determined by hyperspectral imaging and unmixing of cell autofluorescence.

    Science.gov (United States)

    Rehman, Aziz Ul; Anwer, Ayad G; Gosnell, Martin E; Mahbub, Saabah B; Liu, Guozhen; Goldys, Ewa M

    2017-03-01

    Carbonyl cyanide-p-trifluoro methoxyphenylhydrazone (FCCP) is a well-known mitochondrial uncoupling agent. We examined FCCP-induced fluorescence quenching of reduced nicotinamide adenine dinucleotide / nicotinamide adenine dinucleotide phosphate (NAD(P)H) in solution and in cultured HeLa cells in a wide range of FCCP concentrations from 50 to 1000µM. A non-invasive label-free method of hyperspectral imaging of cell autofluorescence combined with unsupervised unmixing was used to separately isolate the emissions of free and bound NAD(P)H from cell autofluorescence. Hyperspectral image analysis of FCCP-treated HeLa cells confirms that this agent selectively quenches fluorescence of free and bound NAD(P)H in a broad range of concentrations. This is confirmed by the measurements of average NAD/NADH and NADP/NADPH content in cells. FCCP quenching of free NAD(P)H in cells and in solution is found to be similar, but quenching of bound NAD(P)H in cells is attenuated compared to solution quenching possibly due to a contribution from the metabolic and/or antioxidant response in cells. Chemical quenching of NAD(P)H fluorescence by FCCP validates the results of unsupervised unmixing of cell autofluorescence.

  9. Antitumor effects of a natural anthracycline analog (Aloin) involve altered activity of antioxidant enzymes in HeLaS3 cells.

    Science.gov (United States)

    Nićiforović, Ana; Adzić, Miroslav; Spasić, Snezana D; Radojcić, Marija B

    2007-08-01

    The antiproliferative and cytotoxic potential of the natural anthracycline aloin from Aloe vera was tested on human uterine carcinoma HeLaS3 cells. Aloin showed a pronounced antiproliferative effect at physiological concentration (IC50 = 97 microM), caused cell cycle arrest in the S phase and markedly increased HeLaS3 cell apoptosis (to 24%). In the concentration range of 20-100 microM, its action was accompanied by remarkable changes in the activity of almost all antioxidant enzymes: MnSOD activity was increased many fold, while CuZnSOD and iNOS activities were inhibited. Moreover, inhibition of CuZnSOD was shown to occur by direct aloin interaction with the enzyme. As catalase activity was not changed, it is suggested that such conditions were responsible for antiproliferative and cytotoxic effects owing to accumulation of H2O2. Aloin alone was a more potent proapoptotic agent than a 2 Gy fractional dose of ionizing radiation or a combination of the two. Compared to other currently used therapeutics, aloin, due to its less undesirable side effects and antimetastatic potential, may prove to be the agent of choice on which clinical protocols for the treatment of human cervical carcinoma should rely in future.

  10. The role of hyaluronic acid capsular material of Streptococcus equi subsp. zooepidemicus in mediating adherence to HeLa cells and in resisting phagocytosis.

    Science.gov (United States)

    Wibawan, I W; Pasaribu, F H; Utama, I H; Abdulmawjood, A; Lämmler, C

    1999-10-01

    Hyaluronic acid is thought to be one of the critical virulence factors of Streptococcus equi subsp. zooepidemicus. The present study was designed to study the role of hyaluronic acid capsular material in mediating adherence and to resist the phagocytosis of the host's immune defence. The studies were performed with two encapsulated S. equi subsp. zooepidemicus and two unencapsulated phase variants. The bacteria had been previously isolated from diseased pigs and monkeys in Indonesia. The presence of capsular material was determined using the hyaluronic acid decapsulation test and by electron microscopic studies. Both encapsulated bacteria showed mucoid colonies after cultivation on blood agar, grew with diffuse colonies in soft agar media and reacted negatively in the salt aggregation test. The unencapsulated bacteria grew with small colonies on blood agar, formed compact colonies in soft agar media and reacted positively in the salt aggregation test. Adherence and phagocytosis studies revealed that the encapsulated bacteria adhered significantly more to HeLa cells and were less phagocytosed by murine macrophages compared to unencapsulated bacteria. Pretreatment of the HeLa cells using hyaluronic acid or pretreatment of the bacteria by hyaluronidase decreased the adherence value of encapsulated bacteria. Pretreatment of bacteria with pronase had no effect. The presented results strongly indicate that the hyaluronic acid capsular material contributes to adherence properties of S. equi subsp. zooepidemicus and might help the bacteria to resist phagocytosis by macrophages. Copyright 1999 Harcourt Publishers Ltd.

  11. Investigation of siRNA Nanoparticle Formation Using Mono-Cationic Detergents and Its Use in Gene Silencing in Human HeLa Cells

    Energy Technology Data Exchange (ETDEWEB)

    Yamada, Yuma; Suzuki, Ryosuke; Harashima, Hideyoshi, E-mail: harashima@pharm.hokudai.ac.jp [Laboratory for Molecular Design of Pharmaceutics, Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812 (Japan)

    2013-11-01

    The focus of recent research has been on the development of siRNA vectors to achieve an innovative gene therapy. Most of the conventional vectors are siRNA nanoparticles complexed with cationic polymers and liposomes, making it difficult to release siRNA. In this study, we report on the use of MCD, a quaternary ammonium salt detergent containing a long aliphatic chain (L-chain) as an siRNA complexation agent using human HeLa cells (a model cancer cell). We prepared siRNA nanoparticles using various MCDs, and measured the diameters and zeta-potentials of the particles. The use of an MCD with a long L-chain resulted in the formation of a positively charged nanoparticle. In contrast, a negatively charged nanoparticle was formed when a MCD with a short L-chain was used. We next evaluated the gene silencing efficiency of the nanoparticles using HeLa cells expressing the luciferase protein. The results showed that the siRNA/MCD nanoparticles showed a higher gene silencing efficiency than Lipofectamine 2000. We also found that the efficiency of gene silencing is a function of the length of the alkyl chain in MCD and zeta-potential of the siRNA/MCD nanoparticles. Such information provides another viewpoint for designing siRNA vectors.

  12. Marine Streptomyces sp. derived antimycin analogues suppress HeLa cells via depletion HPV E6/E7 mediated by ROS-dependent ubiquitin–proteasome system

    Science.gov (United States)

    Zhang, Weiyi; Che, Qian; Tan, Hongsheng; Qi, Xin; Li, Jing; Li, Dehai; Gu, Qianqun; Zhu, Tianjiao; Liu, Ming

    2017-01-01

    Four new antimycin alkaloids (1–4) and six related known analogs (5–10) were isolated from the culture of a marine derived Streptomyces sp. THS-55, and their structures were elucidated by extensive spectroscopic analysis. All of the compounds exhibited potent cytotoxicity in vitro against HPV-transformed HeLa cell line. Among them, compounds 6–7 were derived as natural products for the first time, and compound 5 (NADA) showed the highest potency. NADA inhibited the proliferation, arrested cell cycle distribution, and triggered apoptosis in HeLa cancer cells. Our molecular mechanic studies revealed NADA degraded the levels of E6/E7 oncoproteins through ROS-mediated ubiquitin-dependent proteasome system activation. This is the first report that demonstrates antimycin alkaloids analogue induces the degradation of high-risk HPV E6/E7 oncoproteins and finally induces apoptosis in cervical cancer cells. The present work suggested that these analogues could serve as lead compounds for the development of HPV-infected cervical cancer therapeutic agents, as well as research tools for the study of E6/E7 functions. PMID:28176847

  13. Comparative proteomic analyses demonstrate enhanced Interferon and STAT-1 activation in reovirus T3D-infected HeLa cells

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    Peyman eEzzati

    2015-04-01

    Full Text Available As obligate intracellular parasites, viruses are exclusively and intimately dependent upon their host cells for replication. During replication viruses induce profound changes within cells, including: induction of signaling pathways, morphological changes, and cell death. Many such cellular perturbations have been analyzed at the transcriptomic level by gene arrays and recent efforts have begun to analyze cellular proteomic responses. We recently described comparative stable isotopic (SILAC analyses of reovirus, strain type 3 Dearing (T3D-infected HeLa cells. For the present study we employed the complementary labeling strategy of iTRAQ (isobaric tags for relative and absolute quantitation to examine HeLa cell changes induced by T3D, another reovirus strain, type 1 Lang, and UV-inactivated T3D (UV-T3D. Triplicate replicates of cytosolic and nuclear fractions identified a total of 2375 proteins, of which 50, 57, and 46 were significantly up-regulated, and 37, 26 and 44 were significantly down-regulated by T1L, T3D and UV-T3D, respectively. Several pathways, most notably the Interferon signaling pathway and the EIF2 and ILK signaling pathways, were induced by virus infection. Western blots confirmed that cells were more strongly activated by live T3D as demonstrated by elevated levels of key proteins like STAT-1, ISG-15, IFIT-1, IFIT-3 and Mx1. This study expands our understanding of reovirus-induced host responses.

  14. Cytotoxic Effect of Ethanolic Extract of Sarang Semut (Myrmecodia pendens on HeLa Cervix Cancer Cell Line In Vitro Experimental Study

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    Dina Fatmawati

    2011-12-01

    Design and Method: The method was quasi experimental with post test only non equivalent control group design. HeLa cell was divided into two groups. The first group as positive control with doxorubicin, second group as treatment with ethanolic extract of sarang semut at various concentrations. Ethanolic extract of sarang semut concentrations used were 3,91 μg/ml; 7,81 μg/ml; 15,63 μg/ml; 31,25 μg/ml; 62,50 μg/ml; 125 μg/ml; 250 μg/ml; 500 μg/ml; 1000 μg/ml. Cytotoxic effect was evaluated by direct counting method with tryphan blue dye then using probit regression analysis to find IC50 value. Result: Inhibitory concentration 50 (IC50 value ethanol extract of sarang semut was 33,28 μg/ml. Ethanol extract of sarang semut had a cytotoxicity effect categorized as the moderately active (20 ìg/ml< IC50< 100ìg/ ml. Inhibitory concentration 50 (IC50 value doxorubicin was 5,56 μg/ml. Cytotoxicity effect of doxorubisin higher than cytotoxicity effect of ethanolic extract of sarang semut. Conclusion: Ethanolic extract of sarang semut (Myrmecodia pendens had a cytotoxic effect categorized as the moderately active on HeLa cell (Sains Medika, 3(2:112-120.

  15. Local Foods, Local Places Toolkit

    Science.gov (United States)

    Toolkit to help communities that want to use local foods to spur revitalization. The toolkit gives step-by-step instructions to help communities plan and host a workshop and create an action plan to implement.

  16. Locals Collection

    Directory of Open Access Journals (Sweden)

    Stephen Hastings-King

    2010-03-01

    Full Text Available A locals collection is a set of parameters that are used to delimit data-mining operations. This piece uses a collection of locals from around Essex Massachusetts to shape and delimit an interrogation of post-reality in contemporary America. It explores the notion of crisis, the possibility of a crisis of empire that may or may not emerge in a media-space that does not allow crisis of empire to be mentioned and relations this maybe-crisis to the various levels of economic dysfunction that have become evident since late 2008. But mostly this piece explores ways in which particular stories about particular people do and do not link/link to these larger-scale narratives. This is the first of a potential series of locals collections that will mine the American post-real.

  17. Desarrollo local

    Directory of Open Access Journals (Sweden)

    Luciano Palmitesta

    2001-12-01

    Full Text Available Tras explicar la génesis del concepto de desarrollo y a quien se le aplica la necesidad de desarrollarse, el autor de este ensayo hace un recorrido por las escuelas más representativas de pensamiento sobre el tema. A continuación, entra a reflexionar el concepto de lo local y de sociedad local y sus elementos. En la segunda parte, se analiza la Economía Local y se hace un intento de aplicar esta teoría a la realidad nicaragüense. En esta parte se examinan las diferentes formas empresariales más comunes en Nicaragua. Igualmente, se estudian las dificultades y ventajas de este tipo de empresas en un mundo globalizado y se plantean algunos posibles mecanismos para afrontar los problemas. El trabajo cierra con algunas breves conclusiones.

  18. The Roles of Platelet GPIIb/IIIa and αvβ3 Integrins during HeLa Cells Adhesion, Migration, and Invasion to Monolayer Endothelium under Static and Dynamic Shear Flow

    Directory of Open Access Journals (Sweden)

    Yiyao Liu

    2009-01-01

    Full Text Available During their passage through the circulatory system, tumor cells undergo extensive interactions with various host cells including endothelial cells and platelets. Mechanisms mediating tumor cell adhesion, migration, and metastasis to vessel wall under flow condition are largely unknown. The aim of this study was to investigate the potential roles of GPIIb/IIIa and αvβ3 integrins underlying the HeLa-endothelium interaction in static and dynamic flow conditions. HeLa cell migration and invasion were studied by using Millicell cell culture insert system. The numbers of transmigrated or invaded HeLa cells significantly increased by thrombin-activated platelets and reduced by eptifibatide, a platelet inhibitor. Meanwhile, RGDWE peptides, a specific inhibitor of αvβ3 integrin, also inhibited HeLa cell transmigration. Interestingly, the presence of endothelial cells had significant effect on HeLa cell migration regardless of static or cocultured flow condition. The adhesion capability of HeLa cells to endothelial monolayer was also significantly affected by GPIIb/IIIa and αvβ3 integrins. The arrested HeLa cells increased nearly 5-fold in the presence of thrombin-activated platelets at shear stress condition (1.84 dyn/cm2 exposure for 1 hour than the control (static. Our findings showed that GPIIb/IIIa and αvβ3 integrins are important mediators in the pathology of cervical cancer and provide a molecular basis for the future therapy, and the efficient antitumor benefit should target multiple receptors on tumor cells and platelets.

  19. DNA Methylation Changes in Valproic Acid-Treated HeLa Cells as Assessed by Image Analysis, Immunofluorescence and Vibrational Microspectroscopy.

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    Giovana M B Veronezi

    Full Text Available Valproic acid (VPA, a well-known histone deacetylase inhibitor, has been reported to affect the DNA methylation status in addition to inducing histone hyperacetylation in several cell types. In HeLa cells, VPA promotes histone acetylation and chromatin remodeling. However, DNA demethylation was not checked in this cell model for standing effects longer than those provided by histone acetylation, which is a rapid and transient phenomenon. Demonstration of VPA-induced DNA demethylation in HeLa cells would contribute to understanding the effect of VPA on an aggressive tumor cell line. In the present work, DNA demethylation in VPA-treated HeLa cells was assessed by image analysis of chromatin texture, the abundance of 5-methylcytosine (5mC immunofluorescence signals and Fourier transform-infrared (FT-IR microspectroscopy centered on spectral regions related to the vibration of-CH3 groups. Image analysis indicated that increased chromatin unpacking promoted by a 4-h-treatment with 1.0 mM VPA persisted for 24 h in the absence of the drug, suggesting the occurrence of DNA demethylation that was confirmed by decreased 5mC immunofluorescence signals. FT-IR spectra of DNA samples from 1 mM or 20 mM VPA-treated cells subjected to a peak fitting analysis of the spectral window for-CH3 stretching vibrations showed decreased vibrations and energy of these groups as a function of the decreased abundance of 5mC induced by increased VPA concentrations. Only the 20 mM-VPA treatment caused an increase in the ratio of -CH3 bending vibrations evaluated at 1375 cm-1 in relation to in-plane vibrations of overall cytosines evaluated at 1492 cm-1. CH3 stretching vibrations showed to be more sensitive than-CH3 bending vibrations, as detected with FT-IR microspectroscopy, for studies aiming to associate vibrational spectroscopy and changes in DNA 5mC abundance.

  20. Cell-type specific DNA-protein interactions at the tissue-type plasminogen activator promoter in human endothelial and HeLa cells in vivo and in vitro.

    Science.gov (United States)

    Arts, J; Herr, I; Lansink, M; Angel, P; Kooistra, T

    1997-01-15

    Tissue-type plasminogen activator (t-PA) gene expression in human endothelial cells and HeLa cells is stimulated by the protein kinase C activator phorbol 12-myristate 13-acetate (PMA) at the level of transcription. To study the mechanism of transcriptional regulation, we have characterized a segment of the t-PA gene extending from -135 to +100 by in vivo footprinting analysis [dimethyl sulphate (DMS) method] and gel mobility shift assay. In vivo footprinting analysis revealed changes in cleavage pattern in five distinct promoter elements in both endothelial cells and HeLa cells, including a PMA-responsive element (TRE), a CTF/NF-1 binding site and three GC-boxes, and an altered cleavage pattern of the TRE and CTF/NF-1 element after PMA treatment of HeLa cells. Although endothelial cells and HeLa cells differed in the exact G residues protected by nuclear proteins,in vitro bandshift analysis showed that nuclear protein binding to the t-PA promoter was qualitatively and quantitatively very similar in both cell types, except for the TRE. Protein binding to the TRE under non- stimulated conditions was much higher in human endothelial cells than in HeLa cells, and this TRE-bound protein showed a lower dissociation rate in the endothelial cells than in HeLa cells. In endothelial cells, the proteins bound to the TRE consisted mainly of the AP-1 family members JunD and Fra-2, while in HeLa cells predominantly JunD, FosB and Fra-2 were bound. The proteins bound to the other protected promoter elements were identified as SP-1 (GC-box II and III) and CTF/NF-1 (CTF/NF-1 binding site). After PMA treatment of the cells, AP-1 and SP-1 binding was increased two-fold in endothelial cell nuclear extracts and >20-fold in HeLa nuclear extracts. In the endothelial cells, all Jun and Fos forms (c-Jun, JunB, JunD, c-Fos, FosB, Fra-1 and Fra-2) were part of the AP-1 complex after PMA induction. In HeLa cells, the complex consisted predominantly of c-Jun and the Fos family members Fos

  1. Localized shocks

    Energy Technology Data Exchange (ETDEWEB)

    Roberts, Daniel A. [Center for Theoretical Physics and Department of Physics,Massachusetts Institute of Technology, Cambridge, MA (United States); Stanford, Douglas [Stanford Institute for Theoretical Physics and Department of Physics,Stanford University, Stanford, CA (United States); School of Natural Sciences, Institute for Advanced Study,Princeton, NJ (United States); Susskind, Leonard [Stanford Institute for Theoretical Physics and Department of Physics,Stanford University, Stanford, CA (United States)

    2015-03-10

    We study products of precursors of spatially local operators, W{sub x{sub n}}(t{sub n})…W{sub x{sub 1}}(t{sub 1}), where W{sub x}(t)=e{sup −iHt}W{sub x}e{sup iHt}. Using chaotic spin-chain numerics and gauge/gravity duality, we show that a single precursor fills a spatial region that grows linearly in t. In a lattice system, products of such operators can be represented using tensor networks. In gauge/gravity duality, they are related to Einstein-Rosen bridges supported by localized shock waves. We find a geometrical correspondence between these two descriptions, generalizing earlier work in the spatially homogeneous case.

  2. Effect of EBI3 on radiation-induced immunosuppression of cervical cancer HeLa cells by regulating Treg cells through PD-1/PD-L1 pathway.

    Science.gov (United States)

    Zhang, Song-An; Niyazi, Hu-Er-Xi-Dan; Hong, Wen; Tuluwengjiang, Gu-Li-Xian; Zhang, Lei; Zhang, Yang; Su, Wei-Peng; Bao, Yong-Xing

    2017-03-01

    This study aimed to investigate the effect of EBI3 on radiation-induced immunosuppression of cervical cancer HeLa cells by regulating Treg cells through PD-1/PD-L1 signaling pathway. A total of 43 adult female Wistar rats were selected and injected with HeLa cells in the caudal vein to construct a rat model of cervical cancer. All model rats were randomly divided into the radiotherapy group ( n = 31) and the control group ( n = 12). The immunophenotype of Treg cells was detected by the flow cytometry. The protein expressions of EBI3, PD-1, and PD-L1 in cervical cancer tissues were tested by the streptavidin-peroxidase method. HeLa cells in the logarithmic growth phase were divided into four groups: the blank, the negative control group, the EBI3 mimics group, and the EBI3 inhibitors group. Western blotting was used to detect PD-1 and PD-L1 protein expressions. MTT assay was performed to measure the proliferation of Treg cells. Flow cytometry was used to detect cell cycle and apoptosis, and CD4(+)/CD8(+) T cell ratio in each group. Compared with before and 1 week after radiotherapy, the percentages of CD4(+)T cells and CD8(+)T cells were significantly decreased in the radiotherapy group at 1 month after radiotherapy. Furthermore, down-regulation of EBI3 and up-regulation of PD-1 and PD-L1 were observed in cervical cancer tissues at 1 month after radiotherapy. In comparison to the blank and negative control groups, increased expression of EBI3 and decreased expressions of PD-1 and PD-L1 were found in the EBI3 mimics group. However, the EBI3 inhibitors group had a lower expression of EBI3 and higher expressions of PD-1 and PD-L1 than those in the blank and negative control groups. The EBI3 mimics group showed an increase in the optical density value (0.43 ± 0.05), while a decrease in the optical density value (0.31 ± 0.02) was found in the EBI3 inhibitors group. Moreover, compared with the blank and negative control groups, the apoptosis rates

  3. An evidence on G2/M arrest, DNA damage and caspase mediated apoptotic effect of biosynthesized gold nanoparticles on human cervical carcinoma cells (HeLa)

    Energy Technology Data Exchange (ETDEWEB)

    Jeyaraj, M. [Department of Biotechnology and Genetic Engineering, School of Biotechnology, Bharathidasan University, Tiruchirappalli 620024, Tamil Nadu (India); Arun, R. [Department of Biomedical Sciences, Bharathidasan University, Tiruchirappalli 620024 (India); Sathishkumar, G. [Department of Biotechnology and Genetic Engineering, School of Biotechnology, Bharathidasan University, Tiruchirappalli 620024, Tamil Nadu (India); MubarakAli, D. [Central Inter-Disciplinary Research Facility, Mahatma Gandhi Medical College and Research Institute Campus, Pondicherry 607402 (India); Rajesh, M.; Sivanandhan, G.; Kapildev, G.; Manickavasagam, M. [Department of Biotechnology and Genetic Engineering, School of Biotechnology, Bharathidasan University, Tiruchirappalli 620024, Tamil Nadu (India); Thajuddin, N. [Department of Microbiology, Bharathidasan University, Tiruchirappalli 620024 (India); Ganapathi, A., E-mail: aganapathi2007@gmail.com [Department of Biotechnology and Genetic Engineering, School of Biotechnology, Bharathidasan University, Tiruchirappalli 620024, Tamil Nadu (India)

    2014-04-01

    Highlights: • Gold nanoparticles (AuNPs) have been synthesized using Podophyllum hexandrum L. • AuNPs induces the oxidative stress to cell death in human cervical carcinoma cells. • It activates the caspase-cascade to cellular death. • It is actively blocks G2/M phase of cell cycle. - Abstract: Current prospect of nanobiotechnology involves in the greener synthesis of nanostructured materials particularly noble metal nanoparticles for various biomedical applications. In this study, biologically (Podophyllum hexandrum L.) synthesized crystalline gold nanoparticles (AuNPs) with the size range between 5 and 35 nm were screened for its anticancereous potential against human cervical carcinoma cells (HeLa). Stoichiometric proportion of the reaction mixture and conditions were optimized to attain stable nanoparticles with narrow size range. Different high throughput techniques like transmission electron microscope (TEM), X-ray diffraction (XRD) and UV–vis spectroscopy were adopted for the physio-chemical characterization of AuNPs. Additionally, Fourier transform infrared spectroscopy (FTIR) study revealed that the water soluble fractions present in the plant extract solely influences the reduction of AuNPs. Sublimely, synthesized AuNPs exhibits an effective in vitro anticancer activity against HeLa cells via induction of cell cycle arrest and DNA damage. Furthermore, it was evidenced that AuNPs treated cells are undergone apoptosis through the activation of caspase cascade which subsequently leads to mitochondrial dysfunction. Thereby, this study proves that biogenic colloidal AuNPs can be developed as a promising drug candidature for human cervical cancer therapy.

  4. CXCL12 and [N33A]CXCL12 in 5637 and HeLa cells: regulating HER1 phosphorylation via calmodulin/calcineurin.

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    Antonella Rigo

    Full Text Available In the human neoplastic cell lines 5637 and HeLa, recombinant CXCL12 elicited, as expected, downstream signals via both G-protein-dependent and β-arrestin-dependent pathways responsible for inducing a rapid and a late wave, respectively, of ERK1/2 phosphorylation. In contrast, the structural variant [N33A]CXCL12 triggered no β-arrestin-dependent phosphorylation of ERK1/2, and signaled via G protein-dependent pathways alone. Both CXCL12 and [N33A]CXCL12, however, generated signals that transinhibited HER1 phosphorylation via intracellular pathways. 1 Prestimulation of CXCR4/HER1-positive 5637 or HeLa cells with CXCL12 modified the HB-EGF-dependent activation of HER1 by delaying the peak phosphorylation of tyrosine 1068 or 1173. 2 Prestimulation with the synthetic variant [N33A]CXCL12, while preserving CXCR4-related chemotaxis and CXCR4 internalization, abolished HER1 phosphorylation. 3 In cells knockdown of β-arrestin 2, CXCL12 induced a full inhibition of HER1 like [N33A]CXCL12 in non-silenced cells. 4 HER1 phosphorylation was restored as usual by inhibiting PCK, calmodulin or calcineurin, whereas the inhibition of CaMKII had no discernable effect. We conclude that both recombinant CXCL12 and its structural variant [N33A]CXCL12 may transinhibit HER1 via G-proteins/calmodulin/calcineurin, but [N33A]CXCL12 does not activate β-arrestin-dependent ERK1/2 phosphorylation and retains a stronger inhibitory effect. Therefore, we demonstrated that CXCL12 may influence the magnitude and the persistence of signaling downstream of HER1 in turn involved in the proliferative potential of numerous epithelial cancer. In addition, we recognized that [N33A]CXCL12 activates preferentially G-protein-dependent pathways and is an inhibitor of HER1.

  5. The acquired radioresistance in HeLa cells under conditions mimicking hypoxia was attenuated by a decreased expression of HIF subunit genes induced by RNA interference

    Energy Technology Data Exchange (ETDEWEB)

    Doi, Nobutaka [Department of Radiological Sciences, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama 930-0194 (Japan); New Products Research & Development, Gene Engineering Division, NIPPON GENE Co., Ltd. (Japan); Ogawa, Ryohei, E-mail: ogawa@med.u-toyama.ac.jp [Department of Radiological Sciences, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama 930-0194 (Japan); Cui, Zheng-Guo [Department of Public Health, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama (Japan); Morii, Akihiro; Watanabe, Akihiko [Department of Urology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama (Japan); Kanayama, Shinji; Yoneda, Yuko [New Products Research & Development, Gene Engineering Division, NIPPON GENE Co., Ltd. (Japan); Kondo, Takashi [Department of Radiological Sciences, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama 930-0194 (Japan)

    2015-05-01

    The cancer cells residing in the hypoxic layer are resistant to radiation and these are ones responsible for cancer recurrence after radiation therapy. One of the reasons why hypoxic cancer cells acquire radioresistance may be attributable to changes in the gene expression profile by the activation of hypoxia inducible factors (HIFs). However, the details underlying this process remain unknown. In this study, we investigated the effects of knockdown of HIF subunit genes to elucidate how HIF subunit genes may be involved in the radioresistance acquired by HeLa cells following exposure to a hypoxia mimic. Interestingly, HIF-1α and HIF-2α seemed mutually complementary for each other when either of them was suppressed. We thus suppressed the expression of both genes simultaneously. To do this, we developed a short hairpin RNA (shRNA) targeting a high homology region between HIF-1α and HIF-2α. It was shown that the expression of the shRNA effectively suppressed the acquisition of radioresistance following the hypoxia mimic. Moreover, it was confirmed that suppression of both subunits resulted in the downregulation of stem cell markers and the suppression of spheroid formation during the hypoxia mimicking-conditions. This shRNA-mediated knockdown method targeting a common region shared by a family of genes may offer a new candidate cancer treatment. - Highlights: • Incubation with CoCl{sub 2} confers radioresistance to HeLa cells. • Both HIF-1α and HIF-2α are involved in the acquisition of radioresistance. • An shRNA to a homology region of HIF-1α and HIF-2α suppressed the radioresistance. • The shRNA decreased cells with stem cell markers and a stem cell phenotype.

  6. Association of cap-binding protein with eucaryotic initiation factor 3 in initiation factor preparations from uninfected and poliovirus-infected HeLa cells.

    Science.gov (United States)

    Hansen, J; Etchison, D; Hershey, J W; Ehrenfeld, E

    1982-04-01

    Extracts from poliovirus-infected HeLa cells are unable to translate vesicular stomatitis virus or cellular mRNAs in vitro, probably reflecting the poliovirus-induced inhibition of host cell protein synthesis which occurs in vivo. Crude initiation factors from uninfected HeLa cells are able to restore translation of vesicular stomatitis virus mRNA in infected cell lysates. This restoring activity separates into the 0 to 40% ammonium sulfate fractional precipitate of ribosomal salt wash. Restoring activity is completely lacking in the analogous fractions prepared from poliovirus-infected cells. The 0 to 40% ammonium sulfate precipitates from both uninfected and infected cells contain eucaryotic initiation factor 3 (eIF-3), eIf-4B, and the cap-binding protein (CBP), which is detected by means of a cross-linking assay, as well as other proteins. The association of eIF-3 and cap binding protein was examined. The 0 to 40% ammonium sulfate precipitate of ribosomal salt wash from uninfected and infected cells was sedimented in sucrose gradients. Each fraction was examined for the presence of eIF-3 antigens by an antibody blot technique and for the presence of the CBP by cross-linking to cap-labeled mRNAs. From uninfected cells, a major proportion of the CBP cosedimented with eIF-3; however, none of the CBP from infected cells sedimented with eIF-3. The results suggest that the association of the CBP with eIF-3 into a functional complex may have been disrupted during the course of poliovirus infection.

  7. CRISPR/Cas9-mediated knockout of c-REL in HeLa cells results in profound defects of the cell cycle

    Science.gov (United States)

    Ruiz-Perera, Lucia M.; Kadhim, Hussamadin M.; Tertel, Tobias; Henkel, Elena; Hübner, Wolfgang; Huser, Thomas; Kaltschmidt, Barbara; Kaltschmidt, Christian

    2017-01-01

    Cervical cancer is the fourth common cancer in women resulting worldwide in 266,000 deaths per year. Belonging to the carcinomas, new insights into cervical cancer biology may also have great implications for finding new treatment strategies for other kinds of epithelial cancers. Although the transcription factor NF-κB is known as a key player in tumor formation, the relevance of its particular subunits is still underestimated. Here, we applied CRISPR/Cas9n-mediated genome editing to successfully knockout the NF-κB subunit c-REL in HeLa Kyoto cells as a model system for cervical cancers. We successfully generated a homozygous deletion in the c-REL gene, which we validated using sequencing, qPCR, immunocytochemistry, western blot analysis, EMSA and analysis of off-target effects. On the functional level, we observed the deletion of c-REL to result in a significantly decreased cell proliferation in comparison to wildtype (wt) without affecting apoptosis. The impaired proliferative behavior of c-REL-/- cells was accompanied by a strongly decreased amount of the H2B protein as well as a significant delay in the prometaphase of mitosis compared to c-REL+/+ HeLa Kyoto cells. c-REL-/- cells further showed significantly decreased expression levels of c-REL target genes in comparison to wt. In accordance to our proliferation data, we observed the c-REL knockout to result in a significantly increased resistance against the chemotherapeutic agents 5-Fluoro-2’-deoxyuridine (5-FUDR) and cisplatin. In summary, our findings emphasize the importance of c-REL signaling in a cellular model of cervical cancer with direct clinical implications for the development of new treatment strategies. PMID:28767691

  8. Effect of Tetrodotoxin from Crude Puffer Fish (Tetraodon fluviatilis Liver Extract on Intracellular Calcium Level and Apoptosis of HeLa Cell Culture

    Directory of Open Access Journals (Sweden)

    Natanael Untario

    2017-01-01

    Full Text Available Cervical cancer is the third most commonly diagnosed cancer and fourth leading cause of women death with 8% of total death caused by cancer in women in 2008. Tetrodotoxin (TTX is a potent neurotoxin found in inner organs puffer fish, with the specific mechanism of sodium channel blocking, and widely used for research purposes. Previous reports claimed that TTX has the capability of inhibiting the metastatic process of cancer and apoptotic effect. Studies also show that apoptosis is a process involving the increase of intracellular calcium level, yet the connection between TTX and increase of intracellular calcium level, therefore triggering apoptosis, has not been established. This is an experimental study with post test only control group design, carried out by exposing HeLa cell culture to a crude liver extract of a puffer fish species, Tetraodon fluviatilis. Crude puffer fish liver extract is administered into HeLa cell culture well in different concentrations 10-4, 10-2, and 10-1. Intracellular calcium level and apoptosis were then measured after 18 hours of incubation. Measurements of intracellular calcium level were done by using CLSM with Fura-2AM staining, and apoptosis by using flowcytometry with Annexin V/PI.  The result shows that there is a significant difference between samples both in intracellular calcium (p < 0.05 and apoptosis (p < 0,05. Both intracellular calcium and apoptosis levels are proportional to liver fish extract concentration. Pearson’s correlation test shows correlation between treatment and intracellular calcium levels (p = 0.000, between treatment and apoptosis (p = 0.002, but not between intracellular calcium and apoptosis (p = 0.05. These results suggest that TTX induces an increase in intracellular calcium level and apoptosis, but calcium pathway is not the sole cause of the apoptosis.

  9. Application of a FRET probe for Caspase-3 activation in living HeLa cells by sequentially treated cisplatin and TRAIL

    Science.gov (United States)

    Lin, Juqiang; Zhang, Zhihong; Yi, Qiushi; Zeng, Shaoqun; Luo, Qingming

    2006-02-01

    Caspase-3 is a kind of cysteine proteases that plays an important role in cell apoptosis. We have constructed a FRET (fluorescence resonance energy transfer) probe fused with ECFP (enhanced cyan fluorescence protein) and DsRed (Discosoma red fluorescent protein) with a linker containing a caspase-3 cleavage sequence (CCS, DEVD).It could be observed much change in fluorescence emission ratio when the probe was cleaved by caspase-3. Therefore, application of this probe we can real-time detected the activation of caspase-3. It was already confirmed that caspase-3 was activated in HeLa cells treated by cisplatin or TRAIL (Tumor necrosis factor (TNF)-related apoptosis-inducing ligand). In the present study, we detected the activation of caspase-3 during cisplatin or TRAIL induced apoptosis in living HeLa cells, and also observed the activation of caspase-3 caused by both cisplatin and TRAIL combined treatment. Our results demonstrated a synergistic effect between cisplatin and TRAIL. Cisplatin is one of the most broadly used drugs in the Clinical applications of cancer chemotherapy, and TRAIL, which belongs to the TNF family proteins, can selectively induce apoptosis in many transformed cells but not in normal cells. Therefore, TRAIL is a very valuably prospective utility as its potential tumor-specific cancer therapeutic. Most of anticancer drugs can induce apoptosis which mediated by the activation of caspase pathway. We can select the best synergistic effect group by our FRET probe. This finding would be useful in the design of treatment modalities for patients.

  10. Non-radioactive 2-deoxy-2-fluoro-D-glucose inhibits glucose uptake in xenograft tumours and sensitizes HeLa cells to doxorubicin in vitro.

    Science.gov (United States)

    Niccoli, Sarah; Boreham, Douglas R; Phenix, Christopher P; Lees, Simon J

    2017-01-01

    A glucose analog called 2-deoxy-D-glucose (2DG) has been successfully used to sensitize cancer cells to ROS-inducing cancer treatments such as ionizing radiation, through the inhibition of glycolysis. However, the use of 2DG can be limited by several factors such as availability, non-specific cytotoxicity, and chemoresistance under hypoxic conditions. The purpose of this study was to investigate the use of non-radioactive 2-deoxy-2-fluoro-D-glucose (19FDG), a drug that potentially addresses current limitations of 2DG. The effectiveness of using either 2DG or 19FDG in combination with doxorubicin (Dox) in HeLa cells was determined in both normoxia and hypoxia. We have also shown that under both oxygen conditions, 19FDG-treated cells produce less lactate than 2DG-treated cells, an important finding that suggests improved inhibition of glycolysis, the preferential pathway for cancerous cells. When used in combination with Dox, we have demonstrated a significant decrease in the number of viable cells, with the effect of 19FDG remaining stable across both normoxic and hypoxic conditions. Moreover, the assessment of apoptosis and necrosis revealed that 19FDG maintained its ability to sensitize HeLa cells to Dox in hypoxia, but 2DG was only effective under normoxic conditions. The retained effectiveness of 19FDG in combination with Dox under hypoxic conditions, suggests that 19FDG may be efficacious for sensitizing hypoxic regions of solid tumour masses. Importantly, the ability of 19FDG to inhibit glucose uptake in vivo was also confirmed using positron emission tomography (PET) of xenograft tumours. The results displayed here suggest 19FDG is a promising combination therapy, which may lead to decreased ROS scavenging via glycolysis, and enhanced treatment success.

  11. Analysis and prediction of pathways in HeLa cells by integrating biological levels of organization with systems-biology approaches.

    Science.gov (United States)

    Higareda-Almaraz, Juan Carlos; Valtierra-Gutiérrez, Ilse A; Hernandez-Ortiz, Magdalena; Contreras, Sandra; Hernandez, Erika; Encarnación-Guevara, Sergio; Encarnacion, Sergio

    2013-01-01

    It has recently begun to be considered that cancer is a systemic disease and that it must be studied at every level of complexity using many of the currently available approaches, including high-throughput technologies and bioinformatics. To achieve such understanding in cervical cancer, we collected information on gene, protein and phosphoprotein expression of the HeLa cell line and performed a comprehensive analysis of the different signaling pathways, transcription networks and metabolic events in which they participate. A total expression analysis by RNA-Seq of the HeLa cell line showed that 19,974 genes were transcribed. Of these, 3,360 were over-expressed, and 2,129 under-expressed when compared to the NHEK cell line. A protein-protein interaction network was derived from the over-expressed genes and used to identify central elements and, together with the analysis of over-represented transcription factor motifs, to predict active signaling and regulatory pathways. This was further validated by Metal-Oxide Affinity Chromatography (MOAC) and Tandem Mass Spectrometry (MS/MS) assays which retrieved phosphorylated proteins. The 14-3-3 family members emerge as important regulators in carcinogenesis and as possible clinical targets. We observed that the different over- and under-regulated pathways in cervical cancer could be interrelated through elements that participate in crosstalks, therefore belong to what we term "meta-pathways". Additionally, we highlighted the relations of each one of the differentially represented pathways to one or more of the ten hallmarks of cancer. These features could be maintained in many other types of cancer, regardless of mutations or genomic rearrangements, and favor their robustness, adaptations and the evasion of tissue control. Probably, this could explain why cancer cells are not eliminated by selective pressure and why therapy trials directed against molecular targets are not as effective as expected.

  12. Functional interaction between hMYH and hTRADD in the TNF-α-mediated survival and death pathways of HeLa cells

    Energy Technology Data Exchange (ETDEWEB)

    Vy Tran, An Hue; Hahm, Soo-Hyun; Han, Se Hee [Department of Advanced Technology Fusion, Konkuk University, Hwayang-dong, Gwangjin-gu, Seoul 143-701 (Korea, Republic of); Chung, Ji Hyung [Department of Applied Bioscience, College of Life Science, CHA University, Gyeonggi-do 463-836 (Korea, Republic of); Park, Geon Tae [Cornell University, Ithaca, NY 14850 (United States); Han, Ye Sun, E-mail: yshan@konkuk.ac.kr [College of Global Integrated Studies, Division of Interdisciplinary Studies, Konkuk University, Hwayang-dong, Gwangjin-gu, Seoul 143-701 (Korea, Republic of)

    2015-07-15

    Highlights: • We determine the interaction between hMYH and hTRADD. • We examine changes in the level of hMYH–hTRADD interaction under TNF-α treatment. • hTRADD–hMYH association is involved in the nuclear translocation of NFκB. • hTRADD–hMYH complex influences the TNFR1–TRADD association. - Abstract: The tumor necrosis factor (TNF) signaling pathway is a classical immune system pathway that plays a key role in regulating cell survival and apoptosis. The TNF receptor-associated death domain (TRADD) protein is recruited to the death domain of TNF receptor 1 (TNFR1), where it interacts with TNF receptor-associated factor 2 (TRAF2) and receptor-interacting protein (RIP) for the induction of apoptosis, necrosis, nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB), and mitogen-activated protein (MAP) kinase activation. In this study, we found that the human MutY homolog (hMYH) interacted with human TRADD (hTRADD) via the C-terminal domain of hMYH. Moreover, under conditions promoting TNF-α-induced cell death or survival in HeLa cells, this interaction was weakened or enhanced, respectively. The interaction between hMYH and hTRADD was important for signaling pathways mediated by TNF-α. Our results also suggested that the hTRADD–hMYH association was involved in the nuclear translocation of NFκB and formation of the TNFR1–TRADD complex. Thus, this study identified a novel mechanism through which the hMYH–hTRADD interaction may affect the TNF-α signaling pathway. Implications: In HeLa cells, the hTRADD–hMYH interaction functioned in both cell survival and apoptosis pathways following TNF-α stimulation.

  13. Inhibition of autophagic flux by ROS promotes apoptosis during DTT-induced ER/oxidative stress in HeLa cells.

    Science.gov (United States)

    Xiang, Xi-Yan; Yang, Xiao-Chun; Su, Jin; Kang, Jing-Song; Wu, Yao; Xue, Ya-Nan; Dong, Yu-Tong; Sun, Lian-Kun

    2016-06-01

    As targets for cancer therapy, endoplasmic reticulum (ER) stress and autophagy are closely linked. However, the signaling pathways responsible for induction of autophagy in response to ER stress and its cellular consequences appear to vary with cell type and stimulus. In the present study, we showed that dithiothreitol (DTT) induced ER stress in HeLa cells in a time- and dose-dependent fashion. With increased ER stress, reactive oxygen species (ROS) production increased and autophagy flux, assessed by intracellular accumulation of LC3B-II and p62, was inhibited. N-acetyl-L-cysteine (NAC), a classic antioxidant, exacerbated cell death induced by 3.2 mM of DTT, but attenuated that induced by 6.4 mM DTT. Low cytotoxic doses of DTT transiently activated c-JNU N-terminal kinase (JNK) and p38, whereas high dose of DTT persistently activated JNK and p38 and simultaneously reduced extracellular signal-regulated kinase (ERK) activity. Combined treatment with DTT and U0126, an inhibitor of ERK upstream activators mitogen-activated protein kinase (MAPK) kinase 1 and 2 (MEK1/2), blocked autophagy flux in HeLa cells. This effect was similar to that caused by a combination of DTT and chloroquine (CQ). These data suggested that insufficient autophagy was accompanied by increased ROS production during DTT-induced ER stress. ROS appeared to regulate MAPK signaling, switching from a pro-survival to a pro-apoptotic signal as ER stress increased. ERK inhibition by ROS during severe ER stress blocked autophagic flux. Impaired autophagic flux, in turn, aggravated ER stress, ultimately leading to cell death. Taken together, our data provide the first reported evidence that ROS may control cell fate through regulating the MAPK pathways and autophagic flux during DTT-induced ER/oxidative stress.

  14. Repression of human papillomavirus oncogenes in HeLa cervical carcinoma cells causes the orderly reactivation of dormant tumor suppressor pathways.

    Science.gov (United States)

    Goodwin, E C; DiMaio, D

    2000-11-07

    Most cervical carcinomas express high-risk human papillomaviruses (HPVs) E6 and E7 proteins, which neutralize cellular tumor suppressor function. To determine the consequences of removing the E6 and E7 proteins from cervical cancer cells, we infected HeLa cells, a cervical carcinoma cell line that contains HPV18 DNA, with a recombinant virus that expresses the bovine papillomavirus E2 protein. Expression of the E2 protein resulted in rapid repression of HPV E6 and E7 expression, followed approximately 12 h later by profound inhibition of cellular DNA synthesis. Shortly after E6/E7 repression, there was dramatic posttranscriptional induction of p53. Two p53-responsive genes, mdm2 and p21, were induced with slightly slower kinetics than p53 and appeared to be functional, as assessed by inhibition of cyclin-dependent kinase activity and p53 destabilization. There was also dramatic posttranscriptional induction of p105(Rb) and p107 after E6/E7 repression, followed shortly thereafter by induction of p130. By 24 h after infection, only hypophosphorylated p105(Rb) was detectable and transcription of several Rb/E2F-regulated genes was dramatically repressed. Constitutive expression of the HPV16 E6/E7 genes alleviated E2-induced growth inhibition and impaired activation of the Rb pathway and repression of E2F-responsive genes. This dynamic response strongly suggests that the p53 and Rb tumor suppressor pathways are intact in HeLa cells and that repression of HPV E6 and E7 mobilizes these pathways in an orderly fashion to deliver growth inhibitory signals to the cells. Strikingly, the major alterations in the cell cycle machinery underlying cervical carcinogenesis can be reversed by repression of the endogenous HPV oncogenes.

  15. The potentized homeopathic drug, Lycopodium clavatum (5C and 15C) has anti-cancer effect on hela cells in vitro.

    Science.gov (United States)

    Samadder, Asmita; Das, Sreemanti; Das, Jayeeta; Paul, Avijit; Boujedaini, Naoual; Khuda-Bukhsh, Anisur Rahman

    2013-08-01

    Cancer is a disease that needs a multi-faceted approach from different systems of medicine. The purpose of this study was to evaluate whether homeopathically-potentized ultra-high dilutions of Lycopodium Clavatum (LC-5C and LC-15C, respectively) have any anti-cancer effects on HeLa cells. Cells were exposed to either LC-5C (diluted below Avogadro's limit, i.e., 10(-10)) or LC-15C (diluted beyond Avogadro's limit, i.e., 10(-30)) (drug-treated) or to 30% succussed ethanol ("vehicle" of the drug). The drug-induced modulation in the percent cell viability, the onset of apoptosis, and changes in the expressions of Bax, Bcl2, caspase 3, and Apaf proteins in inter-nucleosomal DNA, in mitochondrial membrane potentials and in the release of cytochrome-c were analyzed by utilizing different experimental protocols. Results revealed that administration of LC-5C and LC-15C had little or no cytotoxic effect in normal peripheral blood mononuclear cells, but caused considerable cell death through apoptosis in cancer (HeLa) cells, which was evident from the induction of DNA fragmentation, the increases in the expressions of protein and mRNA of caspase 3 and Bax, and the decreases in the expressions of Bcl2 and Apaf and in the release of cytochrome-c. Thus, the highly-diluted, dynamized homeopathic remedies LC-5C and LC-15C demonstrated their capabilities to induce apoptosis in cancer cells, signifying their possible use as supportive medicines in cancer therapy. Copyright © 2013. Published by Elsevier B.V.

  16. Bid participates in genotoxic drug-induced apoptosis of HeLa cells and is essential for death receptor ligands' apoptotic and synergistic effects.

    Science.gov (United States)

    Köhler, Barbara; Anguissola, Sergio; Concannon, Caoimhin G; Rehm, Markus; Kögel, Donat; Prehn, Jochen H M

    2008-07-30

    The BH3-only protein Bid is an important component of death receptor-mediated caspase activation. Bid is cleaved by caspase-8 or -10 into t-Bid, which translocates to mitochondria and triggers the release of caspase-activating factors. Bid has also been reported to be cleaved by other proteases. To test the hypothesis that Bid is a central mediator of stress-induced apoptosis, we investigated the effects of a small molecule Bid inhibitor on stress-induced apoptosis, and generated HeLa cells deficient for Bid. Stable knockdown of bid lead to a pronounced resistance to Fas/CD95- and TRAIL-induced caspase activation and apoptosis, and significantly increased clonogenic survival. While Bid-deficient cells were equally sensitive to ER stress-induced apoptosis, they showed moderate, but significantly reduced levels of apoptosis, as well as increased clonogenic survival in response to the genotoxic drugs Etoposide, Oxaliplatin, and Doxorubicin. Similar effects were observed using the Bid inhibitor BI6C9. Interestingly, Bid-deficient cells were dramatically protected from apoptosis when subtoxic concentrations of ER stressors, Etoposide or Oxaliplatin were combined with subtoxic TRAIL concentrations. Our data demonstrate that Bid is central for death receptor-induced cell death and participates in anti-cancer drug-induced apoptosis in human cervical cancer HeLa cells. They also show that the synergistic effects of TRAIL in combination with either ER stressors or genotoxic anti-cancer drugs are nearly exclusively mediated via an increased activation of Bid-induced apoptosis signalling.

  17. Comparison of phosphorylation of ribosomal proteins from HeLa and Krebs II ascites-tumour cells by cyclic AMP-dependent and cyclic GMP-dependent protein kinases

    DEFF Research Database (Denmark)

    Issinger, O G; Beier, H; Speichermann, N

    1980-01-01

    identified by two-dimensional gel electrophoresis. Almost identical results were obtained when ribosomal subunits from HeLa or ascites-tumour cells were used. About 50-60% of the total radioactive phosphate incorporated into small-subunit ribosomal proteins by either kinase was associated with protein S6...

  18. Cell-type specific DNA-protein interactions at the tissue-type plasminogen activator promoter in human endothelial and HeLa cells in vivo and in vitro

    NARCIS (Netherlands)

    Arts, J.; Herr, I.; Lansink, M.; Angel, P.; Kooistra, T.

    1997-01-01

    Tissue-type plasminogen activator (t-PA) gene expression in human endothelial cells and HeLa cells is stimulated by the protein kinase C activator phorbol 12-myristate 13-acetate (PMA) at the level of transcription. To study the mechanism of transcriptional regulation, we have characterized a

  19. Localized shocks

    OpenAIRE

    Stanford, Douglas; Susskind, Leonard; Roberts, Daniel Adam

    2014-01-01

    We study products of precursors of spatially local operators, W[subscript xn](tn)⋅⋅⋅W[subscript x1](t[subscript 1]), where W [subscript x] (t) = e [superscript − iHt] W [subscript x] e [superscript iHt]. Using chaotic spin-chain numerics and gauge/gravity duality, we show that a single precursor fills a spatial region that grows linearly in t. In a lattice system, products of such operators can be represented using tensor networks. In gauge/gravity duality, they are related to Einstein-Rosen ...

  20. Efecto citotóxico del extracto metanólico de tres ecotipos de Lepidium peruvianum Chacón sobre líneas celulares HeLa y HT-29

    Directory of Open Access Journals (Sweden)

    Libertad Alzamora

    2013-05-01

    Full Text Available La búsqueda de compuestos naturales con actividad citotóxica y antitumoral es una de las prioridades actuales de la lucha contra el cáncer; motivo por el cual el objetivo del presente trabajo fue evaluar la actividad citotóxica de los extractos metanólicos (EM de los ecotipos negro, morado y amarillo de Lepidium peruvianum, Chacón (conocida también como Lepidium meyenii Walp. (maca sobre las líneas celulares HeLa (Human Epithelial Carcinoma y HT-29 (Human Colon Adenocarcinoma. Se determinó que la concentración inhibitoria del 50% del crecimiento celular (IC50 para la línea celular HT-29, con los ecotipos negro, morado y amarillo fue de 8,32 mg/ml, 9,28 mg/ml y 0,487 mg/ml respectivamente, mientras que para la línea celular HeLa fue de 2,4 mg/ml, 1,93 mg/ml y 0,66 mg/ml respectivamente. Adicionalmente, se evaluó un EM del ecotipo amarillo con dos años de almacenamiento (10 ºC determinándose como IC50 4,29 mg/ml para HT-29 y 4,17 mg/ml para HeLa. Se concluye que el efecto citotóxico del ecotipo amarillo sobre HT-29 y HeLa fue superior al mostrado por los ecotipos negro y morado; que la línea celular más sensible a los ecotipos amarillo, negro y morado es HeLa, y que el EM del ecotipo amarillo conservó sus propiedades citotóxicas pese al tiempo de almacenamiento, aunque éstas disminuyeron.

  1. Long-distance interaction of the integrated HPV fragment with MYC gene and 8q24.22 region upregulating the allele-specific MYC expression in HeLa cells.

    Science.gov (United States)

    Shen, Congle; Liu, Yongzhen; Shi, Shu; Zhang, Ruiyang; Zhang, Ting; Xu, Qiang; Zhu, Pengfei; Chen, Xiangmei; Lu, Fengmin

    2017-08-01

    Human papillomavirus (HPV) infection is the most important risk factor for cervical cancer development. In HeLa cell line, the HPV viral genome is integrated at 8q24 in one allele of chromosome 8. It has been reported that the HPV fragment integrated in HeLa genome can cis-activate the expression of proto-oncogene MYC, which is located at 500 kb downstream of the integrated site. However, the underlying molecular mechanism of this regulation is unknown. A recent study reported that MYC was highly expressed exclusively from the HPV-integrated haplotype, and a long-range chromatin interaction between the integrated HPV fragment and MYC gene has been hypothesized. In this study, we provided the experimental evidences supporting this long-range chromatin interaction in HeLa cells by using Chromosome Conformation Capture (3C) method. We found that the integrated HPV fragment, MYC and 8q24.22 was close to each other and might form a trimer in spatial location. When knocking out the integrated HPV fragment or 8q24.22 region from chromosome 8 by CRISPR/Cas9 system, the expression of MYC reduced dramatically in HeLa cells. Interestingly, decreased expression was only observed in three from eight cell clones, when only one 8q24.22 allele was knocked out. Functionally, HPV knockout caused senescence-associated acidic β-gal activity in HeLa cells. These data indicate a long-distance interaction of the integrated HPV fragment with MYC gene and 8q24.22 region, providing an alternative mechanism relevant to the carcinogenicity of HPV integration. © 2017 The Authors International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.

  2. Helioseismology with long-range dark matter-baryon interactions

    DEFF Research Database (Denmark)

    Lopes, I.; Panci, Paolo; Silk, J.

    2014-01-01

    the agreement between the best solar model and the helioseismic data without being excluded by direct detection experiments. In particular, the LUX detector will soon be able to either constrain or confirm our best-fit solar model in the presence of a dark sector with long-range interactions that reconcile......Assuming the existence of a primordial asymmetry in the dark sector, we study how long-range dark matter (DM)-baryon interactions, induced by the kinetic mixing of a new U(1) gauge boson and a photon, affect the evolution of the Sun and, in turn, the sound speed the profile obtained from...

  3. Helioseismology and the Solar Cycle: Past, Present and Future ...

    Indian Academy of Sciences (India)

    similar to methods used in terrestrial seismology. Acoustic waves emitted at the surface travel downward into the solar interior, are refracted upwards and reappear some time later and some distance away from the source. The time and distance is influenced by the conditions of the plasma that the wave propagates through.

  4. Helioseismology and the Solar Cycle: Past, Present and Future

    Indian Academy of Sciences (India)

    Since January 2016, the Journal of Astrophysics and Astronomy has moved to Continuous Article Publishing (CAP) mode. This means that each accepted article is being published immediately online with DOI and article citation ID with starting page number 1. Articles are also visible in Web of Science immediately.

  5. Study of HeLa cells clone survival after X-ray irradiation in the presence of cisplatin

    Science.gov (United States)

    Baulin, A. A.; Sukhikh, E. S.; Vasilyev, S. A.; Sukhikh, L. G.; Sheino, I. N.

    2017-09-01

    Radiation therapy in the presence of heavy elements nuclei (Z > 53) is widely developed these days. The presence of such nuclei in cancer cells results in the local increase of energy release from primary photon beam thus increasing relative biological efficiency. In this paper we present the preliminary results of the cell survival study while irradiating cells by X-Ray photon beam in the presence of cisplatin (Pt, Z = 78). The preliminary results show the decrease of the cell survival in the presence of both radiation and cisplatin.

  6. Biofabrication of Ag nanoparticles using Sterculia foetida L. seed extract and their toxic potential against mosquito vectors and HeLa cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Rajasekharreddy, Pala; Rani, Pathipati Usha, E-mail: usharani65@yahoo.com

    2014-06-01

    A one-step and eco-friendly process for the synthesis of silver-(protein-lipid) nanoparticles (Ag-PL NPs) (core–shell) has been developed using the seed extract from wild Indian Almond tree, Sterculia foetida (L.) (Sterculiaceae). The reaction temperature played a major role in controlling the size and shell formation of NPs. The amount of NPs synthesized and qualitative characterization was done by UV–vis spectroscopy and transmission electron microscopy (TEM), respectively. TEM studies exhibited controlled dispersity of spherical shaped NPs with an average size of 6.9 ± 0.2 nm. Selected area electron diffraction (SAED) and X-ray diffraction (XRD) revealed ‘fcc’ phase and crystallinity of the particles. X-ray photoelectron spectroscopy (XPS) was used to identify the protein–lipid (PL) bilayer that appears as a shell around the Ag core particles. The thermal stability of the Ag-PL NPs was examined using thermogravimetric analysis (TGA). Further analysis was carried out by using Fourier transform infrared spectroscopy (FTIR), where the spectra provided evidence for the presence of proteins and lipid moieties ((2n-octylcycloprop-1-enyl)-octanoic acid (I)), and their role in synthesis and stabilization of Ag NPs. This is the first report of plant seed assisted synthesis of PL conjugated Ag NPs. These formed Ag-PL NPs showed potential mosquito larvicidal activity against Aedes aegypti (L.), Anopheles stephensi Liston and Culex quinquefasciatus Say. These Ag-PL NPs can also act as promising agents in cancer therapy. They exhibited anti-proliferative activity against HeLa cancer cell lines and a promising toxicity was observed in a dose dependent manner. Toxicity studies were further supported by the cellular DNA fragmentation in the Ag-PL NPs treated HeLa cells. - Highlights: • Green synthesis of protein-lipid conjugated Ag NPs using S. foetida L. seed extract. • S. foetida seed extract acted as good reducing and stabilizing agent for Ag NPs. • XPS and

  7. Analysis of gene expression profiles in HeLa cells in response to overexpression or siRNA-mediated depletion of NASP

    Directory of Open Access Journals (Sweden)

    Alekseev Oleg

    2009-05-01

    Full Text Available Abstract Background NASP (Nuclear Autoantigenic Sperm Protein is a linker histone chaperone required for normal cell division. Changes in NASP expression significantly affect cell growth and development; loss of gene function results in embryonic lethality. However, the mechanism by which NASP exerts its effects in the cell cycle is not understood. To understand the pathways and networks that may involve NASP function, we evaluated gene expression in HeLa cells in which NASP was either overexpressed or depleted by siRNA. Methods Total RNA from HeLa cells overexpressing NASP or depleted of NASP by siRNA treatment was converted to cRNA with incorporation of Cy5-CTP (experimental samples, or Cy3-CTP (control samples. The labeled cRNA samples were hybridized to whole human genome microarrays (Agilent Technologies, Wilmington, Delaware, USA. Various gene expression analysis techniques were employed: Significance Analysis of Microarrays (SAM, Expression Analysis Systematic Explorer (EASE, and Ingenuity Pathways Analysis (IPA. Results From approximately 36 thousand genes present in a total human genome microarray, we identified a set of 47 up-regulated and 7 down-regulated genes as a result of NASP overexpression. Similarly we identified a set of 56 up-regulated and 71 down-regulated genes as a result of NASP siRNA treatment. Gene ontology, molecular network and canonical pathway analysis of NASP overexpression demonstrated that the most significant changes were in proteins participating in organismal injury, immune response, and cellular growth and cancer pathways (major "hubs": TNF, FOS, EGR1, NFκB, IRF7, STAT1, IL6. Depletion of NASP elicited the changed expression of proteins involved in DNA replication, repair and development, followed by reproductive system disease, and cancer and cell cycle pathways (major "hubs": E2F8, TP53, FGF, FSH, FST, hCG, NFκB, TRAF6. Conclusion This study has demonstrated that NASP belongs to a network of genes and

  8. Quantum Locality?

    Energy Technology Data Exchange (ETDEWEB)

    Stapp, Henry

    2011-11-10

    vagaries that he cites do not upset the proof in question. It is show here in detail why the precise statement of this theorem justifies the specified application of CQT. It is also shown, in response to his challenge, why a putative proof of locality that he has proposed is not valid.

  9. [6]-Gingerol induces caspase 3 dependent apoptosis and autophagy in cancer cells: drug-DNA interaction and expression of certain signal genes in HeLa cells.

    Science.gov (United States)

    Chakraborty, Debrup; Bishayee, Kausik; Ghosh, Samrat; Biswas, Raktim; Mandal, Sushil Kumar; Khuda-Bukhsh, Anisur Rahman

    2012-11-05

    [6]-Gingerol, a pharmacologically important bioactive component of ginger, has been reported to have anti-hyperglycemic, anti-cancer and anti-oxidative properties, but mechanisms through which these are achieved are largely unclear. The present study focuses on apoptosis and autophagy, two key events of anti-cancer activity, in HeLa cells treated with [6]-gingerol. The treated cells showed several morphological changes, including externalization of phosphatidyl serine, degradation of DNA and increase in TUNEL positivity. Furthermore, there was depolarization of mitochondrial membrane potential, providing evidence of mitochondria mediated apoptosis. The expression of caspase 3 and PARP was increased in cells exposed to [6]-gingerol. Circular dichroism study for testing drug-DNA interaction with both calf thymus and nuclear DNA as target revealed that the drug had potential to bind with the nuclear DNA and induce conformational changes of DNA. The over-expression of NFkβ, AKT and Bcl2 genes in cancer cells was down-regulated by [6]-gingerol treatment. On the other hand the expression levels of TNFα, Bax and cytochrome c were enhanced in [6]-gingerol treated cells. Thus, overall results suggest that [6]-gingerol has potential to bind with DNA and induce cell death by autophagy and caspase 3 mediated apoptosis. Copyright © 2012 Elsevier B.V. All rights reserved.

  10. Thymosin beta4 inhibits ADF/cofilin stimulated F-actin cycling and hela cell migration: reversal by active Arp2/3 complex.

    Science.gov (United States)

    Al Haj, Abdulatif; Mazur, Antonina Joanna; Buchmeier, Sabine; App, Christine; Theiss, Carsten; Silvan, Unai; Schoenenberger, Cora-Ann; Jockusch, Brigitte M; Hannappel, Ewald; Weeds, Alan G; Mannherz, Hans Georg

    2014-02-01

    F-actin treadmilling plays a key part in cell locomotion. Because immunofluorescence showed colocalisation of thymosin beta4 (Tβ4) with cofilin-1 and Arp2/3 complex in lamellipodia, we analyzed combinations of these proteins on F-actin-adenosine triphosphate (ATP)-hydrolysis, which provides a measure of actin treadmilling. Actin depolymerising factor (ADF)/cofilin stimulated treadmilling, while Tβ4 decreased treadmilling, presumably by sequestering monomers. Tβ4 added together with ADF/cofilin also inhibited the treadmilling, relative to cofilin alone, but both the rate and extent of depolymerization were markedly enhanced in the presence of both these proteins. Arp2/3 complex reversed the sequestering activity of Tβ4 when equimolar to actin, but not in the additional presence of cofilin-1 or ADF. Transfection experiments to explore the effects of changing the intracellular concentration of Tβ4 in HeLa cells showed that an increase in Tβ4 resulted in reduced actin filaments bundles and narrower lamellipodia, and a conspicuous decrease of cell migration as seen by two different assays. In contrast, cells transfected with a vector leading to Tβ4 knockdown by small interfering RNA (siRNA) displayed prominent actin filament networks within the lamellipodia and the leading lamella and enhanced migration. The experiments reported here demonstrate the importance of the interplay of these different classes of actin-binding proteins on cell behaviour. Copyright © 2013 Wiley Periodicals, Inc.

  11. Individual expression features of GPX2, NQO1 and SQSTM1 transcript variants induced by hydrogen peroxide treatment in HeLa cells

    Directory of Open Access Journals (Sweden)

    Anna A. Belanova

    2017-05-01

    Full Text Available Abstract Pathway activity assessment-based approaches are becoming highly influential in various fields of biology and medicine. However, these approaches mostly rely on analysis of mRNA expression, and total mRNA from a given locus is measured in the majority of cases. Notably, a significant portion of protein-coding genes produces more than one transcript. This biological fact is responsible for significant noise when changes in total mRNA transcription of a single gene are analyzed. The NFE2L2/AP-1 pathway is an attractive target for biomedical applications. To date, there is a lack of data regarding the agreement in expression of even classical target genes of this pathway. In the present paper we analyzed whether transcript variants of GPX2, NQO1 and SQSTM1 were characterized by individual features of expression when HeLa cells were exposed to pro-oxidative stimulation with hydrogen peroxide. We found that all the transcripts (10 in total appeared to be significantly individually regulated under the conditions tested. We conclude that individual transcripts, rather than total mRNA, are best markers of pathway activation. We also discuss here some biological roles of individual transcript regulation.

  12. The Effect of Coatings on the Affinity of Lanthanide Nanoparticles to MKN45 and HeLa Cancer Cells and Improvement in Photodynamic Therapy Efficiency

    Science.gov (United States)

    Sawamura, Takashi; Tanaka, Tatsumi; Ishige, Hiroyuki; Iizuka, Masayuki; Murayama, Yasutoshi; Otsuji, Eigo; Ohkubo, Akihiro; Ogura, Shun-Ichiro; Yuasa, Hideya

    2015-01-01

    An improvement in photodynamic therapy (PDT) efficiency against a human gastric cancer cell line (MKN45) with 5-aminolevulinic acid (ALA) and lanthanide nanoparticles (LNPs) is described. An endogenous photosensitizer, protoporphyrin IX, biosynthesized from ALA and selectively accumulated in cancer cells, is sensitizable by the visible lights emitted from up-conversion LNPs, which can be excited by a near-infrared light. Ten kinds of surface modifications were performed on LNPs, NaYF4(Sc/Yb/Er) and NaYF4(Yb/Tm), in an aim to distribute these irradiation light sources near cancer cells. Among these LNPs, only the amino-functionalized LNPs showed affinity to MKN45 and HeLa cancer cells. A PDT assay with MKN45 demonstrated that amino-modified NaYF4(Sc/Yb/Er) gave rise to a dramatically enhanced PDT effect, reaching almost perfect lethality, whereas NaYF4(Yb/Tm)-based systems caused little improvement in PDT efficiency. The improvement of PDT effect with the amino-modified NaYF4(Sc/Yb/Er) is promising for a practical PDT against deep cancer cells that are reachable only by near-infrared lights. PMID:26389895

  13. Visualizing cell-cycle kinetics after hypoxia/reoxygenation in HeLa cells expressing fluorescent ubiquitination-based cell cycle indicator (Fucci).

    Science.gov (United States)

    Goto, Tatsuaki; Kaida, Atsushi; Miura, Masahiko

    2015-12-10

    Hypoxia induces G1 arrest in many cancer cell types. Tumor cells are often exposed to hypoxia/reoxygenation, especially under acute hypoxic conditions in vivo. In this study, we investigated cell-cycle kinetics and clonogenic survival after hypoxia/reoxygenation in HeLa cells expressing fluorescent ubiquitination-based cell cycle indicator (Fucci). Hypoxic treatment halted cell-cycle progression during mid-S to G2 phase, as determined by the cell cycle-regulated E3 ligase activities of SCF(Skp2) and APC/C(Cdh1), which are regulators of the Fucci probes; however, the DNA content of the arrested cells was equivalent to that in G1 phase. After reoxygenation, time-lapse imaging and DNA content analysis revealed that all cells reached G2 phase, and that Fucci fluorescence was distinctly separated into two fractions 24h after reoxygenation: red cells that released from G2 arrest after repairing DNA double-strand breaks (DSBs) exhibited higher clonogenic survival, whereas most cells that stayed green contained many DSBs and exhibited lower survival. We conclude that hypoxia disrupts coordination of DNA synthesis and E3 ligase activities associated with cell-cycle progression, and that DSB repair could greatly influence cell-cycle kinetics and clonogenic survival after hypoxia/reoxygenation. Copyright © 2015 Elsevier Inc. All rights reserved.

  14. Visualizing the effect of tumor microenvironments on radiation-induced cell kinetics in multicellular spheroids consisting of HeLa cells

    Energy Technology Data Exchange (ETDEWEB)

    Kaida, Atsushi; Miura, Masahiko, E-mail: masa.mdth@tmd.ac.jp

    2013-10-04

    Highlights: •We visualized radiation-induced cell kinetics in spheroids. •HeLa-Fucci cells were used for detection of cell-cycle changes. •Radiation-induced G2 arrest was prolonged in the spheroid. •The inner and outer cell fractions behaved differently. -- Abstract: In this study, we visualized the effect of tumor microenvironments on radiation-induced tumor cell kinetics. For this purpose, we utilized a multicellular spheroid model, with a diameter of ∼500 μm, consisting of HeLa cells expressing the fluorescent ubiquitination-based cell-cycle indicator (Fucci). In live spheroids, a confocal laser scanning microscope allowed us to clearly monitor cell kinetics at depths of up to 60 μm. Surprisingly, a remarkable prolongation of G2 arrest was observed in the outer region of the spheroid relative to monolayer-cultured cells. Scale, an aqueous reagent that renders tissues optically transparent, allowed visualization deeper inside spheroids. About 16 h after irradiation, a red fluorescent cell fraction, presumably a quiescent G0 cell fraction, became distinct from the outer fraction consisting of proliferating cells, most of which exhibited green fluorescence indicative of G2 arrest. Thereafter, the red cell fraction began to emit green fluorescence and remained in prolonged G2 arrest. Thus, for the first time, we visualized the prolongation of radiation-induced G2 arrest in spheroids and the differences in cell kinetics between the outer and inner fractions.

  15. A stable human progesterone receptor expressing HeLa reporter cell line as a tool in chemical evaluation at the different cell-cycle phases.

    Science.gov (United States)

    Mori, Tetsushi; Murata, Mai; Yoshino, Tomoko; Nakasono, Satoshi; Saito, Fumiyo; Takeyama, Haruko; Matsunaga, Tadashi

    2009-04-25

    Specific molecular events, characteristic of each cell-cycle phase may have direct effect to the functionality of nuclear receptors. Based on this understanding, the evaluation of lipophilic chemicals at the different cell-cycle phases is significant and should be considered. In order to achieve the aim of performing large-scale dose-response analysis on the effects of lipophilic chemicals at the different cell-cycle phases, a stable, sensitive and highly selective human progesterone receptor (hPR) expressing HeLa reporter cell line, hPRLuc-20, was established. Upon the establishment of the hPRLuc-20 cells, they were synchronized to the G(1), S and G(2) phases and treated with progesterone (PROG) and promegestone (R5020). The cells successfully showed that at the different cell-cycle phase, both agonists resulted in different cellular responses. The differences in response supports that hPR expressed within the hPRLuc-20 cells do respond in a cell-cycle dependent manner, thus showing the cells' compatibility in large-scale dose-response analyses of chemicals. It is hopeful that the advanced application of the hPRLuc-20 cells could contribute to provide fundamental hints to further understand the function of hPR, and provide key observations to elucidate the nature of these chemicals with hPR, its corresponding co-regulators and transcription factors.

  16. Demonstration in vitro that eucaryotic initiation factor 3 is active but that a cap-binding protein complex is inactive in poliovirus-infected HeLa cells.

    Science.gov (United States)

    Etchison, D; Hansen, J; Ehrenfeld, E; Edery, I; Sonenberg, N; Milburn, S; Hershey, J W

    1984-01-01

    Protein synthesis initiation factor preparations from poliovirus-infected HeLa cells have reduced ability to initiate translation on capped mRNA. The defect in initiation factors has been variously attributed to inactivation of eucaryotic initiation factor 3 (eIF3), eIF4B, or a cap-binding protein (CBP) complex. We have developed a series of in vitro protein synthesis assays to show that eIF3 is active but a CBP complex activity is inactivated after poliovirus infection. eIF3 activity, when determined in the presence of purified CBP complex, is present in sucrose gradients of factors from both infected and uninfected cells. CBP complex activity, determined in the presence of eIF3 from poliovirus-infected cells, is present in uninfected cells only and comigrates on sucrose gradient with an activity which restores the ability of crude initiation factors from infected cells to translate capped globin mRNA. This is the first demonstration by a fractionated translation system that an activity which is attributable to CBP complex is inactivated in poliovirus-infected cells. The results also indicate that eIF3 is undetectable or has greatly reduced activity in the absence of CBP complex. Images PMID:6088805

  17. Production of a Novel Fucoidanase for the Green Synthesis of Gold Nanoparticles by Streptomyces sp. and Its Cytotoxic Effect on HeLa Cells.

    Science.gov (United States)

    Manivasagan, Panchanathan; Oh, Junghwan

    2015-11-12

    Marine actinobacteria-produced fucoidanases have received considerable attention as one of the major research topics in recent years, particularly for the medical exploitation of fucoidans and their degradation products. The present study describes the optimization and production of a novel fucoidanase for the green synthesis of gold nanoparticles and its biological applications. The production of fucoidanase was optimized using Streptomyces sp. The medium components were selected in accordance with the Plackett-Burman design and were further optimized via response surface methodology. The fucoidanase was statistically optimized with the most significant factors, namely wheat bran 3.3441 g/L, kelp powder 0.7041 g/L, and NaCl 0.8807 g/L, respectively. The biosynthesized gold nanoparticles were determined by UV-vis spectroscopy and were further characterized by X-ray diffraction analysis, Fourier transform infrared spectroscopy, field emission scanning electron microscopy, energy dispersive X-ray analysis, and high-resolution transmission electron microscopy. Furthermore, the biosynthesized gold nanoparticles exhibited a dose-dependent cytotoxicity against HeLa cells and the inhibitory concentration (IC50) was found to be 350 µg/mL at 24 h and 250 µg/mL at 48 h. Therefore, the production of novel fucoidanase for the green synthesis of gold nanoparticles has comparatively rapid, less expensive and wide application to anticancer therapy in modern medicine.

  18. SIRT6 Is a Positive Regulator of Aldose Reductase Expression in U937 and HeLa cells under Osmotic Stress: In Vitro and In Silico Insights.

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    Ahmet Can Timucin

    Full Text Available SIRT6 is a protein deacetylase, involved in various intracellular processes including suppression of glycolysis and DNA repair. Aldose Reductase (AR, first enzyme of polyol pathway, was proposed to be indirectly associated to these SIRT6 linked processes. Despite these associations, presence of SIRT6 based regulation of AR still remains ambiguous. Thus, regulation of AR expression by SIRT6 was investigated under hyperosmotic stress. A unique model of osmotic stress in U937 cells was used to demonstrate the presence of a potential link between SIRT6 and AR expression. By overexpressing SIRT6 in HeLa cells under hyperosmotic stress, its role on upregulation of AR was revealed. In parallel, increased SIRT6 activity was shown to upregulate AR in U937 cells under hyperosmotic milieu by using pharmacological modulators. Since these modulators also target SIRT1, binding of the inhibitor, Ex-527, specifically to SIRT6 was analyzed in silico. Computational observations indicated that Ex-527 may also target SIRT6 active site residues under high salt concentration, thus, validating in vitro findings. Based on these evidences, a novel regulatory step by SIRT6, modifying AR expression under hyperosmotic stress was presented and its possible interactions with intracellular machinery was discussed.

  19. Microarray dataset of transient and permanent DNA methylation changes in HeLa cells undergoing inorganic arsenic-mediated epithelial-to-mesenchymal transition.

    Science.gov (United States)

    Eckstein, Meredith; Rea, Matthew; Fondufe-Mittendorf, Yvonne N

    2017-08-01

    The novel dataset presented here represents the results of the changing pattern of DNA methylation profiles in HeLa cells exposed to chronic low dose (0.5 µM) sodium arsenite, resulting in epithelial-to-mesenchymal transition, as well as DNA methylation patterns in cells where inorganic arsenic has been removed. Inorganic arsenic is a known carcinogen, though not mutagenic. Several mechanisms have been proposed as to how inorganic arsenic drives carcinogenesis such as regulation of the cell׳s redox potential and/or epigenetics. In fact, there are gene specific studies and limited genome-wide studies that have implicated epigenetic factors such as DNA methylation in inorganic arsenic-mediated epithelial-to-mesenchymal transition (EMT). However, genome-wide studies about the impact of 1) chronic, low-dose inorganic arsenic exposure on DNA methylation patterns during inorganic arsenic-induced epithelial-to-mesenchymal transition, and 2) the removal inorganic arsenic (reversal) on DNA methylation patterns, is lacking. For this dataset, two replicates were performed with each of the samples - non-treated, inorganic arsenic-treated, and reverse-treated cells. We provide normalized and processed data, and log2 fold change in DNA methylation. The raw microarray data are available through NCBI GEO, accession number GSE95232 and a related research paper has been accepted for published in Toxicology and Applied Pharmacology (Eckstein et al., 2017) [1].

  20. The dynamin chemical inhibitor dynasore impairs cholesterol trafficking and sterol-sensitive genes transcription in human HeLa cells and macrophages.

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    Emmanuelle Girard

    Full Text Available Intracellular transport of cholesterol contributes to the regulation of cellular cholesterol homeostasis by mechanisms that are yet poorly defined. In this study, we characterized the impact of dynasore, a recently described drug that specifically inhibits the enzymatic activity of dynamin, a GTPase regulating receptor endocytosis and cholesterol trafficking. Dynasore strongly inhibited the uptake of low-density lipoprotein (LDL in HeLa cells, and to a lower extent in human macrophages. In both cell types, dynasore treatment led to the abnormal accumulation of LDL and free cholesterol (FC within the endolysosomal network. The measure of cholesterol esters (CE further showed that the delivery of regulatory cholesterol to the endoplasmic reticulum (ER was deficient. This resulted in the inhibition of the transcriptional control of the three major sterol-sensitive genes, sterol-regulatory element binding protein 2 (SREBP-2, 3-hydroxy-3-methyl-coenzymeA reductase (HMGCoAR, and low-density lipoprotein receptor (LDLR. The sequestration of cholesterol in the endolysosomal compartment impaired both the active and passive cholesterol efflux in HMDM. Our data further illustrate the importance of membrane trafficking in cholesterol homeostasis and validate dynasore as a new pharmacological tool to study the intracellular transport of cholesterol.

  1. Expression of aggregative adherence to hela cells by Escherichia coli strains isolated from sick horses Expressão de aderência agregativa em células HeLa por amostras de E. coli isoladas de eqüinos doentes

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    Ana Maria Alvim Liberatore

    2007-03-01

    Full Text Available The virulence attributes of 56 Escherichia coli strains isolated from sick horses (secretions of uterine cervices; gastrointestinal and lung fragments of necropsy; diarrheic feces, and tracheal washings was examined by determining their adherence pattern to HeLa cells and searching for the presence of virulence genes of the various E. coli pathotypes. Two non-adherent strains presented astA, which encodes the enteroaggregative E. coli heat-stable toxin. Twenty-seven strains (48.2% adhered to HeLa cells, 21 (77.8% of which presented the aggregative adherence pattern (AA that characterize the Enteroaggregative E. coli pathotype (EAEC. Nine of the strains presenting AA were isolated from secretions of uterine cervix, including one carrying virulence genes of the EAEC pathotype (aggR,aap,irp2, and pic. This is the first description of the AA phenotype amongst E. coli strains from sick horses. Such strains should be further evaluated regarding their potential role in the pathogenesis of diverse equine diseases and as reservoirs of human infections.Características de virulência de 56 amostras de Escherichia coli isoladas de eqüinos doentes (secreção de colo uterino, fragmentos de necrópsia do trato gastrointestinal e de pulmões, fezes diarréicas e lavado traqueal foram examinadas para determinar o padrão de aderência em células HeLa e pesquisar a presença de genes de virulência de vários patotipos de E. coli. Duas amostras não aderentes apresentaram astA, gene que codifica a toxina termo-estável de E. coli enteroagregativa. Das vinte e sete amostras (48,2% que aderiram a células HeLa, 21 (77,8% apresentaram o padrão de aderência agregativa (AA que caracteriza o patotipo de E. coli Enteroagregativa (EAEC. Nove destas amostras que apresentaram AA foram isoladas de secreção de colo uterino, incluindo uma que apresentava genes de virulência de patotipos de EAEC (aggR,aap,irp2 e pic. Esta é a primeira descrição do fenótipo AA em

  2. Detecção da citotoxicidade de materiais biocompatíveis nas linhagens celulares MRC-5, HeLa e RC-IAL MRC-5, HeLa and RC-IAL cell lines sensitivity for detection of cytotoxicity of biocompatible materials

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    Aurea S. Cruz

    1992-04-01

    Full Text Available A sensibilidade de uma linhagem celular diplóide e duas heteroplóides, para a detecção de citotoxicidade através do método de difusão em camada de ágar sobre culturas celulares, foi avaliada experimentalmente com solução de ácido ascórbico em diferentes concentrações e, na prática, frente a 562 amostras de 21 diferentes materiais industriais enviados para análise na Seção de Culturas Celulares do Instituto Adolfo Lutz. A linhagem celular heteroplóide designada RC-IAL apresentou, em relação às linhagens MRC-5 e HeLa, maior sensibilidade porque revelou a presença de efeito citotóxico nas menores concentrações utilizadas (10 e 25 ug/ml do ácido ascórbico e apresentou maior diâmetro do halo citotóxico em 15 amostras e igual diâmetro em 16 das 43 amostras (7,6% que resultaram positivas. Nas 43 amostras positivas, a linhagem MRC-5 não revelou citotoxicidade em 3 amostras de espuma e 1 de resina acrílica. O polivinilcloreto (PVC e o polietileno, raramente revelaram positividade, enquanto plástico, algodão e resinas acrílicas revelaram citotoxicidade ao redor de 5%. Em vista dos resultados é discutida a proposta da utilização da linhagem RC-IAL e HeLa para a continuidade das futuras análises solicitadas ao Instituto Adolfo LutzThe sensitivity of diploid and heteroploid cell lines for detection of cytotoxicity using the agar diffusion method on cell culture, was tested with ascorbic acid solution of different concentrations. A total of 562 samples of 21 various materials were tested. The heteroploid cell line, RC-IAL, showed in relation to the MRC-5 and HeLa cell lines, greater sensitivity because it showed the presence of cytotoxic effect with the lowest concentration used (10 and 25ug/ml of ascorbic acid and showed greater diameter of cytotoxic halo in 15 samples and equal diameter in 16 of the 43 positive samples (7.6%. Out of 43 positive samples, the MRC-5 line did not show cytotoxicity in 3 sponge samples and

  3. Phospholipase C-β1 and β4 Contribute to Non-Genetic Cell-to-Cell Variability in Histamine-Induced Calcium Signals in HeLa Cells

    Science.gov (United States)

    Ishida, Sachiko; Matsu-ura, Toru; Fukami, Kiyoko; Michikawa, Takayuki; Mikoshiba, Katsuhiko

    2014-01-01

    A uniform extracellular stimulus triggers cell-specific patterns of Ca2+ signals, even in genetically identical cell populations. However, the underlying mechanism that generates the cell-to-cell variability remains unknown. We monitored cytosolic inositol 1,4,5-trisphosphate (IP3) concentration changes using a fluorescent IP3 sensor in single HeLa cells showing different patterns of histamine-induced Ca2+ oscillations in terms of the time constant of Ca2+ spike amplitude decay and the Ca2+ oscillation frequency. HeLa cells stimulated with histamine exhibited a considerable variation in the temporal pattern of Ca2+ signals and we found that there were cell-specific IP3 dynamics depending on the patterns of Ca2+ signals. RT-PCR and western blot analyses showed that phospholipase C (PLC)-β1, -β3, -β4, -γ1, -δ3 and -ε were expressed at relatively high levels in HeLa cells. Small interfering RNA-mediated silencing of PLC isozymes revealed that PLC-β1 and PLC-β4 were specifically involved in the histamine-induced IP3 increases in HeLa cells. Modulation of IP3 dynamics by knockdown or overexpression of the isozymes PLC-β1 and PLC-β4 resulted in specific changes in the characteristics of Ca2+ oscillations, such as the time constant of the temporal changes in the Ca2+ spike amplitude and the Ca2+ oscillation frequency, within the range of the cell-to-cell variability found in wild-type cell populations. These findings indicate that the heterogeneity in the process of IP3 production, rather than IP3-induced Ca2+ release, can cause cell-to-cell variability in the patterns of Ca2+ signals and that PLC-β1 and PLC-β4 contribute to generate cell-specific Ca2+ signals evoked by G protein-coupled receptor stimulation. PMID:24475116

  4. Phospholipase C-β1 and β4 contribute to non-genetic cell-to-cell variability in histamine-induced calcium signals in HeLa cells.

    Directory of Open Access Journals (Sweden)

    Sachiko Ishida

    Full Text Available A uniform extracellular stimulus triggers cell-specific patterns of Ca(2+ signals, even in genetically identical cell populations. However, the underlying mechanism that generates the cell-to-cell variability remains unknown. We monitored cytosolic inositol 1,4,5-trisphosphate (IP3 concentration changes using a fluorescent IP3 sensor in single HeLa cells showing different patterns of histamine-induced Ca(2+ oscillations in terms of the time constant of Ca(2+ spike amplitude decay and the Ca(2+ oscillation frequency. HeLa cells stimulated with histamine exhibited a considerable variation in the temporal pattern of Ca(2+ signals and we found that there were cell-specific IP3 dynamics depending on the patterns of Ca(2+ signals. RT-PCR and western blot analyses showed that phospholipase C (PLC-β1, -β3, -β4, -γ1, -δ3 and -ε were expressed at relatively high levels in HeLa cells. Small interfering RNA-mediated silencing of PLC isozymes revealed that PLC-β1 and PLC-β4 were specifically involved in the histamine-induced IP3 increases in HeLa cells. Modulation of IP3 dynamics by knockdown or overexpression of the isozymes PLC-β1 and PLC-β4 resulted in specific changes in the characteristics of Ca(2+ oscillations, such as the time constant of the temporal changes in the Ca(2+ spike amplitude and the Ca(2+ oscillation frequency, within the range of the cell-to-cell variability found in wild-type cell populations. These findings indicate that the heterogeneity in the process of IP3 production, rather than IP3-induced Ca(2+ release, can cause cell-to-cell variability in the patterns of Ca(2+ signals and that PLC-β1 and PLC-β4 contribute to generate cell-specific Ca(2+ signals evoked by G protein-coupled receptor stimulation.

  5. The aqueous extract of Ficus religiosa induces cell cycle arrest in human cervical cancer cell lines SiHa (HPV-16 Positive and apoptosis in HeLa (HPV-18 positive.

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    Amit S Choudhari

    Full Text Available Natural products are being extensively explored for their potential to prevent as well as treat cancer due to their ability to target multiple molecular pathways. Ficus religiosa has been shown to exert diverse biological activities including apoptosis in breast cancer cell lines. In the present study, we report the anti-neoplastic potential of aqueous extract of F. religiosa (FRaq bark in human cervical cancer cell lines, SiHa and HeLa. FRaq altered the growth kinetics of SiHa (HPV-16 positive and HeLa (HPV-18 positive cells in a dose-dependent manner. It blocked the cell cycle progression at G1/S phase in SiHa that was characterized by an increase in the expression of p53, p21 and pRb proteins with a simultaneous decrease in the expression of phospho Rb (ppRb protein. On the other hand, in HeLa, FRaq induced apoptosis through an increase in intracellular Ca(2+ leading to loss of mitochondrial membrane potential, release of cytochrome-c and increase in the expression of caspase-3. Moreover, FRaq reduced the migration as well as invasion capability of both the cervical cancer cell lines accompanied with downregulation of MMP-2 and Her-2 expression. Interestingly, FRaq reduced the expression of viral oncoproteins E6 and E7 in both the cervical cancer cell lines. All these data suggest that F. religiosa could be explored for its chemopreventive potential in cervical cancer.

  6. Using multi-channel level sets to measure the cytoplasmic localization of HCMV pUL97 in GFP-B-gal fusion constructs.

    Science.gov (United States)

    Held, Christian; Webel, Rike; Palmisano, Ralf; Hutterer, Corina; Marschall, Manfred; Wittenberg, Thomas

    2014-04-01

    Human cytomegalovirus UL97-encoded protein kinase (pUL97) phosphorylates cellular and viral proteins and is critical for viral replication. To quantify the efficiency of nuclear translocation and to elucidate the role of putative nuclear localization signal (NLS) elements, immunofluorescence analysis of different pUL97 expression constructs was performed. Since manual quantitation of respective expression levels lacks objectivity and reproducibility, and is time-consuming as well, a computer-based model is established. This model enables objective quantitation of the degree of cytoplasmic localization λ. To determine the degree of cytoplasmic localization of different pUL97-GFP-β-gal fusion proteins automatically, a multi-channel segmentation of the nucleus and cytoplasm of transfected HeLa cells is performed in DAPI and GFP micrographs. A watershed transform-based segmentation scheme is used for the segmentation of the cell nuclei. Subsequently, the cytoplasm is segmented using a fast marching level set method. Based on the segmentation of cell nuclei and cytoplasm, λ can be determined for each HeLa cell by quantitation of the ratio of average signal intensity outside and inside the nucleus. The degree of cytoplasmic localization of an individual construct is then determined by evaluating the average and standard deviation of λ for the corresponding HeLa cells. Evaluation demonstrates that nuclear transport of pUL97 is a multilayered mechanism resulting in different efficiencies of nuclear translocation between a small and a large isoform and objective quantitation of the cytoplasmic localization is possible with a high accuracy (96.7% and 94.3%). Copyright © 2013 Elsevier B.V. All rights reserved.

  7. Synthesis of 7-hydroxy-4'-methoxyflavanone and 7-hydroxy-4'-methoxyflavone as a candidate anticancer against cervical (HeLa) cancer cell and colon (WiDr) cancer cell by in vitro

    Science.gov (United States)

    Matsjeh, Sabirin; Anwar, Chairil; Solikhah, Eti Nurwening; Farah, Harra Ismi; Nurfitria, Kurnia

    2017-03-01

    The compound 7-hydroxy-4'-methoxyflavanone and 7-hydroxy-4'-methoxyflavone have been synthesized through cyclization reaction of 2 ', 4'-dihydroxy-4-methoxychalcone (1,3-diphenyl-2-propene-1-one). The 2 ', 4'-dihydroxy-4-methoxychalcone were synthesized through Claisen-Schmidt condensation from 2,4-dihydroxyacetophenone and 4-methoxybenzaldehyde (anisaldehyde) in aqueous KOH as a catalyst in ethanol. The 7-hydroxy-4'-methoxyflavanone has been synthesized through cyclization reaction of 2 ', 4'-dihydroxy-4-methoxychalcone by Oxa-Michael addition reaction with sulfuric acid as a catalyst in ethanol. The 7-hydroxy-4'-methoxyflavone has been synthesized through oxidative cyclization reaction of 2 ', 4'-dihydroxy-4-methoxychalcone using I2 in DMSO as a catalyst with a mole ratio (1: 1) mol. All these producets were characterized by FT-IR, GC-MS, and 1H-NMR and 13C-NMR spectrometer. Both of these compounds were tested citotoxycity activity as an anticancer against cervical and colon cancer cells (HeLa and WiDr cell lines) using MTT assay in vitro. Dose series given test solution concentration on HeLa and WiDr cells starting from 0,78; 1,56; 3,12; 6,25; 12,50; 25; 50 and 100 µg/mL with a long incubation treatment for 24 hours. The results study showed that the 7-hydroxy-4'-methoxyflavanone as bright yellow crystals with a melting point 172-174 ° C and a yield of 56.67% and the 7-hydroxy-4'-methoxyflavone as bright yellow crystals with a yield of 88, 31%, and a melting point of 263-265 ° C. The test results cytotoxic 7-hydroxy-4-methoxyflavone showed active against HeLa cells with IC50 value of 25.73 µg/mL and was quite active in the WiDr cells with IC50 value of 83.75 µg/mL. The result of the activity of 7-hydroxy-4-methoxyflavanone show active cytotoxic activity against HeLa and WiDr cell growth with IC50 value of 40.13 µg/mL and 37.85 µg/mL. IC50 value indicated that 7-hydroxy-4'-methoxyflavone and 7-hydroxy-4'-methoxyflavanone potential as inhibitors in HeLa and

  8. Synthesis of 2',4-dihydroxy-3-methoxychalcone and 2',4',4-trihydroxy-3-methoxychalcone as a candidate anticancer against cervical (WiDr), colon (HeLa), and breast (T47d) cancer cell lines in vitro

    Science.gov (United States)

    Matsjeh, Sabirin; Swasono, Respati Tri; Anwar, Chairil; Solikhah, Eti Nurwening; Lestari, Endang

    2017-03-01

    The compound 2',4-dihydroxy-3-methoxychalcone and 2',4',4-trihydroxy-3-methoxychalcone have been synthesized through Claisen-Schmidt reaction from 2-hydroxyacetophenone and 2,4-dihydroxyacetophenone with 4-hydroxy-3-methoxy benzaldehida (vanillin) in aqueous KOH 40% and KSF montmorillonite as catalyst in methanol. All these products were characterized by FT-IR, TLC Scanner, GC-MS, MS-Direct, and 1H-NMR and 13C-NMR spectrometer. Both of these compounds were tested citotoxycity activity as an anticancer against cervical, colon, and breast cancer cells (Hela, WiDr, and T47D cell lines) using MTT assay in vitro. Dose series given test solution concentration on Hela, WiDr, and T47D cells started from 6,25; 25; 50 and 100 µg/mL with incubation treatment for 24 hours. The result of study showed that the 2',4-dihydroxy-3-methoxychalcone as bright yellow crystal with the melting point of 114-115 °C and the yield of 13.77% and the 2',4',4-trihydroxy-3-methoxychalcone as bright yellow crystals with the melting point of 195-197 °C and the yield of 6%. Other 2',4-dihydroxy-3-methoxychalcone and 2',4',4-trihydroxy-3-methoxychalcone also exhibited cytotoxic activity against the cancer cell lines, with the 2',4',4-trihydroxy-3-methoxychalcone showed greater activities than the 2',4-dihydroxy-3-methoxychalcone in WiDr cell lines. The 2',4-dihydroxy-3-methoxychalcone and 2',4',4-trihydroxy-3-methoxychalcone exhibited strong anticancer activities with IC50 value below 20 µg/mL. The activity of 2',4',4-trihydroxy-3-methoxychalcone showed the most active against Hela and WiDr cell lines with IC50 value 8.53 and 2.66 µg/mL respectively, than T47D cell lines with IC50 value 24.61 µg/mL. The test results cytotoxic of 2',4-dihydroxy-3-methoxychalcone showed the most active against Hela and WiDr cell lines with IC50 value 12.80, 19.57 µg/mL than T47D cell lines with IC50 value of 20.73 µg/mL. IC50 value indicated that 2',4-dihydroxy-3-methoxychalcone and 2',4',4-trihydroxy-3

  9. Carbon ion beam triggers both caspase-dependent and caspase-independent pathway of apoptosis in HeLa and status of PARP-1 controls intensity of apoptosis.

    Science.gov (United States)

    Ghorai, Atanu; Sarma, Asitikantha; Bhattacharyya, Nitai P; Ghosh, Utpal

    2015-04-01

    High linear energy transfer (LET) carbon ion beam (CIB) is becoming very promising tool for various cancer treatments and is more efficient than conventional low LET gamma or X-rays to kill malignant or radio-resistant cells, although detailed mechanism of cell death is still unknown. Poly (ADP-ribose) polymerase-1 (PARP-1) is a key player in DNA repair and its inhibitors are well-known as radio-sensitizer for low LET radiation. The objective of our study was to find mechanism(s) of induction of apoptosis by CIB and role of PARP-1 in CIB-induced apoptosis. We observed overall higher apoptosis in PARP-1 knocked down HeLa cells (HsiI) compared with negative control H-vector cells after irradiation with CIB (0-4 Gy). CIB activated both intrinsic and extrinsic pathways of apoptosis via caspase-9 and caspase-8 activation respectively, followed by caspase-3 activation, apoptotic body, nucleosomal ladder formation and sub-G1 accumulation. Apoptosis inducing factor translocation into nucleus in H-vector but not in HsiI cells after CIB irradiation contributed caspase-independent apoptosis. Higher p53 expression was observed in HsiI cells compared with H-vector after exposure with CIB. Notably, we observed about 37 % fall of mitochondrial membrane potential, activation of caspase-9 and caspase-3 and mild activation of caspase-8 without any detectable apoptotic body formation in un-irradiated HsiI cells. We conclude that reduction of PARP-1 expression activates apoptotic signals via intrinsic and extrinsic pathways in un-irradiated cells. CIB irradiation further intensified both intrinsic and extrinsic pathways of apoptosis synergistically along with up-regulation of p53 in HsiI cells resulting overall higher apoptosis in HsiI than H-vector.

  10. Cytotoxic evaluation of medicinal smoke \\"Anbar Nasara\\" against cancer cells (Hela & KB and a normal cell line (L929, using MTT assay

    Directory of Open Access Journals (Sweden)

    hojat Sadeghi-aliabadi

    2013-09-01

    Full Text Available Background: Medicinal smoke has been in natural health products by many nations all over the worlds. Dung's smoke called "Anbar Nasara" is one of the most used medicinal smokes in Iranian traditional medicine. Anber Nasara has been administered as inhalation in the treatment of viral or bacterial infections as well as for tumors and cysts in vaginal diseases in women. Its cytotoxic effects have been proposed in these studies. Materials and Methods: Dung's of female donkey have been collected from Shahreza, Isfahan province in summer 2011. The samples were burned in a specially designed apparatus and its smoke was collected in n-hexane solvent over ice-cold water. Solvent was evaporated in the air and dried residue was dissolved in DMSO and diluted in RPMI So that the final concentrations of 0.4, 0.3, 0.25, 0.2, 0.125, 0.1, 0.0625, 0.0312 mg/ml were obtained for MTT assay as a cytotoxic evaluation method against two cancer cells (KB and Hela and a normal one (L929. After 48h incubation percent cell survival and IC50 values were obtained using ELISA plate reader at 540nm. Results: Results showed that dung's smoke were cytotoxic in tested concentrations against cancer cell lines in a dose dependent manner. The IC50 was calculated ≥ 0.2 mg/ml. Conclusion: Dung's smoke consists of too many constituents and its cytotoxic effect may belong to one or complexes of constituents. More researches are under way to clarify these aspects of the study.

  11. Impact of capsaicin, an active component of chili pepper, on pathogenic chlamydial growth (Chlamydia trachomatis and Chlamydia pneumoniae) in immortal human epithelial HeLa cells.

    Science.gov (United States)

    Yamakawa, Kazuya; Matsuo, Junji; Okubo, Torahiko; Nakamura, Shinji; Yamaguchi, Hiroyuki

    2018-02-01

    Chlamydia trachomatis is the leading cause of sexually transmitted infections worldwide. Capsaicin, a component of chili pepper, which can stimulate actin remodeling via capsaicin receptor TRPV1 (transient receptor potential vanilloid 1) and anti-inflammatory effects via PPARγ (peroxisome proliferator-activated receptor-γ) and LXRα (liver X receptor α), is a potential candidate to control chlamydial growth in host cells. We examined whether capsaicin could inhibit C. trachomatis growth in immortal human epithelial HeLa cells. Inclusion forming unit and quantitative PCR assays showed that capsaicin significantly inhibited bacterial growth in cells in a dose-dependent manner, even in the presence of cycloheximide, a eukaryotic protein synthesis inhibitor. Confocal microscopic and transmission electron microscopic observations revealed an obvious decrease in bacterial numbers to inclusions bodies formed in the cells. Although capsaicin can stimulate the apoptosis of cells, no increase in cleaved PARP (poly (ADP-ribose) polymerase), an apoptotic indicator, was observed at a working concentration. All of the drugs tested (capsazepine, a TRPV1 antagonist; 5CPPSS-50, an LXRα inhibitor; and T0070907, a PPARγ inhibitor) had no effect on chlamydial inhibition in the presence of capsaicin. In addition, we also confirmed that capsaicin inhibited Chlamydia pneumoniae growth, indicating a phenomena not specific to C. trachomatis. Thus, we conclude that capsaicin can block chlamydial growth without the requirement of host cell protein synthesis, but by another, yet to be defined, mechanism. Copyright © 2017 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

  12. CRISPR-Cas9 induced mutations along de novo purine synthesis in HeLa cells result in accumulation of individual enzyme substrates and affect purinosome formation.

    Science.gov (United States)

    Baresova, Veronika; Krijt, Matyas; Skopova, Vaclava; Souckova, Olga; Kmoch, Stanislav; Zikanova, Marie

    2016-11-01

    Purines are essential molecules for nucleic acid synthesis and are the most common carriers of chemical energy in all living organisms. The cellular pool of purines is maintained by the balance between their de novo synthesis (DNPS), recycling and degradation. DNPS includes ten reactions catalysed by six enzymes. To date, two genetically determined disorders of DNPS enzymes have been described, and the existence of other defects manifested by neurological symptoms and the accumulation of DNPS intermediates in bodily fluids is highly presumable. In the current study, we prepared specific recombinant DNPS enzymes and used them for the biochemical preparation of their commercially unavailable substrates. These compounds were used as standards for the development and validation of quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS). To simulate manifestations of known and putative defects of DNPS we prepared CRISPR-Cas9 genome-edited HeLa cells deficient for the individual steps of DNPS (CR-cells), assessed the substrates accumulation in cell lysates and growth media and tested how the mutations affect assembly of the purinosome, the multi-enzyme complex of DNPS enzymes. In all model cell lines with the exception of one, an accumulation of the substrate(s) for the knocked out enzyme was identified. The ability to form the purinosome was reduced. We conclude that LC-MS/MS analysis of the dephosphorylated substrates of DNPS enzymes in bodily fluids is applicable in the selective screening of the known and putative DNPS disorders. This approach should be considered in affected individuals with neurological and neuromuscular manifestations of unknown aetiology. Prepared in vitro human model systems can serve in various studies that aim to provide a better characterization and understanding of physiology and pathology of DNPS, to study the role of each DNPS protein in the purinosome formation and represent an interesting way for the screening of potential

  13. Validation of a novel Mho microarray for a comprehensive characterisation of the Mycoplasma hominis action in HeLa cell infection.

    Directory of Open Access Journals (Sweden)

    Birgit Henrich

    Full Text Available Mycoplasma hominis is the second smallest facultative pathogen of the human urogenital tract. With less than 600 protein-encoding genes, it represents an ideal model organism for the study of host-pathogen interactions. For a comprehensive characterisation of the M. hominis action in infection a customized Mho microarray, which was based on two genome sequences (PG21 and LBD-4, was designed to analyze the dynamics of the mycoplasma transcriptome during infection and validated for M. hominis strain FBG. RNA preparation was evaluated and adapted to ensure the highest recovery of mycoplasmal mRNAs from in vitro HeLa cell infection assays. Following cRNA hybridization, the read-out strategy of the hybridization results was optimized and confirmed by RT-PCR. A statistically robust infection assay with M. hominis strain FBG enabled the identification of differentially regulated key effector molecules such as critical cytoadhesins (4 h post infection (pI, invasins (48 h pI and proteins associated with establishing chronic infection of the host (336 h pI. Of the 294 differentially regulated genes (>2-fold 128 (43.5% encoded hypothetical proteins, including lipoproteins that seem to play a central role as virulence factors at each stage of infection: P75 as a novel cytoadhesin candidate, which is also differentially upregulated in chronic infection; the MHO_2100 protein, a postulated invasin and the MHO_730-protein, a novel ecto-nuclease and domain of an ABC transporter, the function of which in chronic infection has still to be elucidated. Implementation of the M. hominis microarray strategy led to a comprehensive identification of to date unknown candidates for virulence factors at relevant stages of host cell infection.

  14. Non-biased enrichment does not improve quantitative proteomic delineation of reovirus T3D-infected HeLa cell protein alterations

    Directory of Open Access Journals (Sweden)

    Jieyuan eJiang

    2012-09-01

    Full Text Available Mass spectrometry-based methods have allowed elucidation of alterations in complex proteomes, such as eukaryotic cells. Such studies have identified and measured relative abundances of thousands of host proteins after cells are infected with a virus. One of the potential limitations in such studies is that generally only the most abundant proteins are identified, leaving the deep richness of the cellular proteome largely unexplored. We differentially labeled HeLa cells with light and heavy stable isotopic forms of lysine and arginine (SILAC and infected cells with reovirus strain T3D. Cells were harvested at 24 hours post-infection. Heavy-labeled infected and light-labeled mock-infected cells were mixed together 1:1. Cells were then divided into cytosol and nuclear fractions and each fraction analyzed, both by standard 2D-HPLC/MS, and also after each fraction had been reacted with a random hexapeptide library (Proteominer® beads to attempt to enrich for low-abundance cellular proteins. A total of 2736 proteins were identified by 2 or more peptides at >99% confidence, of which 66 were significantly up-regulated and 67 were significantly down-regulated. Up-regulated proteins included those involved in antimicrobial and antiviral responses, GTPase activity, nucleotide binding, interferon signaling, and enzymes associated with energy generation. Down-regulated proteins included those involved in cell and biological adhesion, regulation of cell proliferation, structural molecule activity, and numerous molecular binding activities. Comparisons of the r2 correlations, degree of dataset overlap, and numbers of peptides detected suggest that non-biased enrichment approaches may not provide additional data to allow deeper quantitative and comparative mining of complex proteomes.

  15. A stable HeLa cell line that inducibly expresses poliovirus 2A(pro): effects on cellular and viral gene expression.

    Science.gov (United States)

    Barco, A; Feduchi, E; Carrasco, L

    2000-03-01

    A HeLa cell clone (2A7d) that inducibly expresses the gene for poliovirus protease 2A (2A(pro)) under the control of tetracycline has been obtained. Synthesis of 2A(pro) induces severe morphological changes in 2A7d cells. One day after tetracycline removal, cells round up and a few hours later die. Poliovirus 2A(pro) cleaves both forms of initiation factor eIF4G, causing extensive inhibition of capped-mRNA translation a few hours after protease induction. Methoxysuccinyl-Ala-Ala-Pro-Val-chloromethylketone, a selective inhibitor of 2A(pro), prevents both eIF4G cleavage and inhibition of translation but not cellular death. Expression of 2A(pro) still allows both the replication of poliovirus and the translation of mRNAs containing a picornavirus leader sequence, while vaccinia virus replication is drastically inhibited. Translation of transfected capped mRNA is blocked in 2A7d-On cells, while luciferase synthesis from a mRNA bearing a picornavirus internal ribosome entry site (IRES) sequence is enhanced by the presence of 2A(pro). Moreover, synthesis of 2A(pro) in 2A7d cells complements the translational defect of a poliovirus 2A(pro)-defective variant. These results show that poliovirus 2A(pro) expression mimics some phenotypical characteristics of poliovirus-infected cells, such as cell rounding, inhibition of protein synthesis and enhancement of IRES-driven translation. This cell line constitutes a useful tool to further analyze 2A(pro) functions, to complement poliovirus 2A(pro) mutants, and to test antiviral compounds.

  16. Magnetic luminescent porous silicon microparticles for localized delivery of molecular drug payloads.

    Science.gov (United States)

    Gu, Luo; Park, Ji-Ho; Duong, Kim H; Ruoslahti, Erkki; Sailor, Michael J

    2010-11-22

    Magnetic manipulation, fluorescent tracking, and localized delivery of a drug payload to cancer cells in vitro is demonstrated, using nanostructured porous silicon microparticles as a carrier. The multifunctional microparticles are prepared by electrochemical porosification of a silicon wafer in a hydrofluoric acid-containing electrolyte, followed by removal and fracture of the porous layer into particles using ultrasound. The intrinsically luminescent particles are loaded with superparamagnetic iron oxide nanoparticles and the anti-cancer drug doxorubicin. The drug-containing particles are delivered to human cervical cancer (HeLa) cells in vitro, under the guidance of a magnetic field. The high concentration of particles in the proximity of the magnetic field results in a high concentration of drug being released in that region of the Petri dish, and localized cell death is confirmed by cellular viability assay (Calcein AM).

  17. Local duality for 2-dimensional local ring

    Indian Academy of Sciences (India)

    We prove a local duality for some schemes associated to a 2-dimensional complete local ring whose residue field is an -dimensional local field in the sense of Kato–Parshin. Our results generalize the Saito works in the case =0 and are applied to study the Bloch–Ogus complex for such rings in various cases.

  18. Adherence to HeLa cells, typing by killer toxins and susceptibility to antifungal agents of Candida dubliniensis strains Adesão a células HeLa, tipagem pelas toxinas "killer" e sensibilidade a antifúngicos de cepas de Candida dubliniensis

    Directory of Open Access Journals (Sweden)

    Gismari Miranda da Silva

    2007-03-01

    Full Text Available The aim of this study was to evaluate the adherence capability to HeLa cells, the susceptibility to killer toxins and the in vitro susceptibility to antifungal agents (eTest? method - AB Biodisk, Solna, Sweden of 9 Candida dubliniensis isolates recovered from HIV+ and AIDS patients. The adherence test was strongly positive for strain ATCC 777 and positive for all other strains. Typing by killer toxins revealed two different biotypes among the 9 isolates studied: 888 and 688. Only biotype 688 (ATCC 777 was susceptible to the K2 toxin. There was a significant inverse correlation between adherence and killer toxin susceptibility (r = -0.8525 - p = 0.0035. No strains presented resistance to fluconazole, itraconazole, ketoconazole, voriconazole, flucytosine or amphotericin-B. With the exception of ATCC 777, all the other isolates presented similar behavior.O objetivo do presente trabalho foi avaliar o comportamento de cepas de Candida dubliniensis recuperadas de pacientes HIV+ e com AIDS por meio da pesquisa de capacidade de adesão a células HeLa, susceptibilidade a toxinas "Killer" e resistência in vitro a antifúngicos (eTest® AB Biodisk, Solna, Suécia. O ensaio de adesão foi fortemente aderente para a amostra padrão ATCC 777, e aderente para os demais isolados. Os testes de tipagem das amostras frente às cepas-padr��o produtoras de toxinas "Killer" mostraram dois biótipos diferentes dos 9 isolados estudados: 888 e 688. Somente o biótipo 688 (ATCC 777 de C. dubliniensis foi sensível à toxina K2. Houve correlação inversa significativa entre adesão e sensibilidade a toxinas "killer" (r = -0,8525 - p = 0,0035. Em relação à pesquisa de resistência a antifúngicos, as amostras de C. dubliniensis foram sensíveis ao fluconazol, itraconazol, cetoconazol, voriconazol, à flucitosina e anfotericina B. Com exceção da amostra ATCC 777, todas as demais mostraram comportamento similar.

  19. ASPM and citron kinase co-localize to the midbody ring during cytokinesis.

    Science.gov (United States)

    Paramasivam, Murugan; Chang, Yoon Jeung; LoTurco, Joseph J

    2007-07-01

    Mutations in ASPM (abnormal spindle-like microcephaly associated) and citron kinase (CITK) cause primary microcephaly in humans and rodents, respectively. Both proteins are expressed during neurogenesis and play important roles in neuronal progenitor cell division. ASPM is localized to the spindle pole, and is essential for maintaining proliferative cell division. CITK is present at the cytokinesis furrow and midbody ring, and it is essential for cellular abscission. We report here that ASPM also localizes to the midbody ring in mammalian cells. ASPM co-localizes with CITK at the midbody ring and coimmunoprecipitates with CITK in lysates prepared from HeLa cells and embryonic neuroepithelium. Furthermore, a GFP-tagged fragment of the N-terminus of ASPM localizes to centrosomes and spindle poles, while a GFP-tagged fragment of the C-terminus localizes to midbodies. All reported ASPM mutations that cause microcephaly involve a truncation or mutation of the C-terminus. In addition, at least two other microcephaly-related proteins, CENPJ and CDK5RAP2, previously localized to spindle poles, also localize to midbodies. Together our observations support a model of neurogenesis in which spindle dynamics and cellular abscission are coordinated.

  20. The effect of lance geometry and carbon coating of silicon lances on propidium iodide uptake in lance array nanoinjection of HeLa 229 cells

    Science.gov (United States)

    Sessions, John W.; Lindstrom, Dallin L.; Hanks, Brad W.; Hope, Sandra; Jensen, Brian D.

    2016-04-01

    Connecting technology to biologic discovery is a core focus of non-viral gene therapy biotechnologies. One approach that leverages both the physical and electrical function of microelectromechanical systems (MEMS) in cellular engineering is a technology previously described as lance array nanoinjection (LAN). In brief, LAN consists of a silicon chip measuring 2 cm by 2 cm that has been etched to contain an array of 10 μm tall, solid lances that are spaced every 10 μm in a grid pattern. This array of lances is used to physically penetrate hundreds of thousands of cells simultaneously and to then electrically deliver molecular loads into cells. In this present work, two variables related to the microfabrication of the silicon lances, namely lance geometry and coating, are investigated. The purpose of both experimental variables is to assess these parameters’ effect on propidium iodide (PI), a cell membrane impermeable dye, uptake to injected HeLa 229 cells. For the lance geometry experimentation, three different microfabricated lance geometries were used which include a flat/narrow (FN, 1 μm diameter), flat/wide (FW, 2-2.5 μm diameter), and pointed (P, 1 μm diameter) lance geometries. From these tests, it was shown that the FN lances had a slightly better cell viability rate of 91.73% and that the P lances had the best PI uptake rate of 75.08%. For the lance coating experimentation, two different lances were fabricated, both silicon etched lances with some being carbon coated (CC) in a  <100 nm layer of carbon and the other lances being non-coated (Si). Results from this experiment showed no significant difference between lance types at three different nanoinjection protocols (0V, +1.5V DC, and  +5V Pulsed) for both cell viability and PI uptake rates. One exception to this is the comparison of CC/5V Pul and Si/5V Pul samples, where the CC/5V Pul samples had a cell viability rate 5% higher. Both outcomes were unexpected and reveal how to better

  1. Temporo-spatial cell-cycle kinetics in HeLa cells irradiated by Ir-192 high dose-rate remote afterloading system (HDR-RALS).

    Science.gov (United States)

    Asahina, Taito; Kaida, Atsushi; Goto, Tatsuaki; Yoshimura, Ryo-Ichi; Sasai, Keisuke; Miura, Masahiko

    2016-07-29

    Intracavitary irradiation plays a pivotal role in definitive radiotherapy for cervical cancer, and the Ir-192 high dose-rate remote afterloading system (HDR-RALS) is often used for this purpose. Under this condition, tumor tissues receive remarkably different absorption doses, with a steep gradient, depending on distance from the radiation source. To obtain temporo-spatial information regarding cell-cycle kinetics in cervical cancer following irradiation by Ir-192 HDR-RALS, we examined HeLa cells expressing the fluorescence ubiquitination-based cell cycle indicator (Fucci), which allowed us to visualize cell-cycle progression. HeLa-Fucci cells, which emit red and green fluorescence in G1 and S/G2/M phases, respectively, were grown on 35-mm dishes and irradiated by Ir-192 HDR-RALS under normoxic and hypoxic conditions. A 6 French (Fr) catheter was used as an applicator. A radiation dose of 6 Gy was prescribed at hypothetical treatment point A, located 20 mm from the radiation source. Changes in Fucci fluorescence after irradiation were visualized for cells from 5 to 20 mm from the Ir-192 source. Several indices, including first green phase duration after irradiation (FGPD), were measured by analysis of time-lapse images. Cells located 5 to 20 mm from the Ir-192 source became green, reflecting arrest in G2, in a similar manner up to 12 h after irradiation; at more distant positions, however, cells were gradually released from the G2 arrest and became red. This could be explained by the observation that the FGPD was longer for cells closer to the radiation source. Detailed observation revealed that FGPD was significantly longer in cells irradiated in the green phase than in the red phase at positions closer to the Ir-192 source. Unexpectedly, the FGPD was significantly longer after irradiation under hypoxia than normoxia, due in large part to the elongation of FGPD in cells irradiated in the red phase. Using HeLa-Fucci cells, we obtained the first temporo

  2. Two zinc-aminoclays' in-vitro cytotoxicity assessment in HeLa cells and in-vivo embryotoxicity assay in zebrafish.

    Science.gov (United States)

    Chun, Hang-Suk; Park, Duckshin; Eun Lim, Song; Jeong, Kwang-Hun; Park, Ji-Seon; Park, Han-Jin; Kang, Shinyoung; Kang, Kyoung Suk; Park, Hyun Gyu; An, Ha-Rim; Huh, Yun Suk; Lee, Young-Chul

    2017-03-01

    Two zinc-aminoclays [ZnACs] with functionalized primary amines [(-CH2)3NH2] were prepared by a simple sol-gel reaction using cationic metal precursors of ZnCl2 and Zn(NO3)2 with 3-aminopropyl triethoxysilane [APTES] under ambient conditions. Due to the facile interaction of heavy metals with primary amine sites and Zn-related intrinsic antimicrobial activity, toxicity assays of ZnACs nanoparticles (NPs) prior to their environmental and human-health applications are essential. However, such reports remain rare. Thus, in the present study, a cell viability assay of in-vitro HeLa cells comparing ZnCl2, Zn(NO3)2 salts, and ZnO (~50nm average diameter) NPs was performed. Interestingly, compared with the ZnCl2, and Zn(NO3)2 salts, and ZnO NPs (18.73/18.12/51.49µg/mL and 18.12/15.19/46.10µg/mL of IC50 values for 24 and 48h), the two ZnACs NPs exhibited the highest toxicity (IC50 values of 21.18/18.36µg/mL and 18.37/17.09µg/mL for 24 and 48h, respectively), whose concentrations were calculated on Zn elemental composition. This might be due to the enhanced bioavailability and uptake into cells of ZnAC NPs themselves and their positively charged hydrophilicity by reactive oxygen species (ROS) generation, particularly as ZnACs exist in cationic NP's form, not in released Zn(2+) ionic form (i.e., dissolved nanometal). However, in an in-vivo embryotoxicity assay in zebrafish, ZnACs and ZnO NPs showed toxic effects at 50-100µg/mL (corresponding to 37.88-75.76 of Zn wt% µg/mL). The hatching rate (%) of zebrafish was lowest for the ZnO NPs, particularly where ZnAC-[(NO3)2] is slightly more toxic than ZnAC-[Cl2]. These results are all very pertinent to the issue of ZnACs' potential applications in the environmental and biomedical fields. Copyright © 2016 Elsevier Inc. All rights reserved.

  3. VT Certified Local Governments

    Data.gov (United States)

    Vermont Center for Geographic Information — Vermont established its Certified Local Government (CLG) program in 1985 to better help local governments integrate historic preservation concerns with planning and...

  4. In vitro Evaluation of Cytotoxic Activities of Essential Oil from Moringa oleifera Seeds on HeLa, HepG2, MCF-7, CACO-2 and L929 Cell Lines.

    Science.gov (United States)

    Elsayed, Elsayed Ahmed; Sharaf-Eldin, Mahmoud A; Wadaan, Mohammad

    2015-01-01

    Moringa oleifera Lam. (Moringaceae) is widely consumed in tropical and subtropical regions for their valuable nutritional and medicinal characteristics. Recently, extensive research has been conducted on leaf extracts of M. oleifera to evaluate their potential cytotoxic effects. However, with the exception of antimicrobial and antioxidant activities, little information is present on the cytotoxic activity of the essential oil obtained from M. oleifera seeds. Therefore, the present investigation was designed to investigate the potential cytotoxic activity of seed essential oil obtained from M. oleifera on HeLa, HepG2, MCF-7, CACO-2 and L929 cell lines. The different cell lines were subjected to increasing oil concentrations ranging from 0.15 to 1 mg/mL for 24h, and the cytotoxicity was assessed using MTT assay. All treated cell lines showed a significant reduction in cell viability in response to the increasing oil concentration. Moreover, the reduction depended on the cell line as well as the oil concentration applied. Additionally, HeLa cells were the most affected cells followed by HepG2, MCF-7, L929 and CACO-2, where the percentages of cell toxicity recorded were 76.1, 65.1, 59.5, 57.0 and 49.7%, respectively. Furthermore, the IC50 values obtained for MCF-7, HeLa and HepG2 cells were 226.1, 422.8 and 751.9 μg/mL, respectively. Conclusively, the present investigation provides preliminary results which suggest that seed essential oil from M. oleifera has potent cytotoxic activities against cancer cell lines.

  5. Efflux Transport Characterization of Resveratrol Glucuronides in UDP-Glucuronosyltransferase 1A1 Transfected HeLa Cells: Application of a Cellular Pharmacokinetic Model to Decipher the Contribution of Multidrug Resistance-Associated Protein 4.

    Science.gov (United States)

    Wang, Shuai; Li, Feng; Quan, Enxi; Dong, Dong; Wu, Baojian

    2016-04-01

    Resveratrol undergoes extensive metabolism to form biologically active glucuronides in humans. However, the transport mechanisms for resveratrol glucuronides are not fully established. Here, we aimed to characterize the efflux transport of resveratrol glucuronides using UGT1A1-overexpressing HeLa cells (HeLa1A1 cells), and to determine the contribution of multidrug resistance-associated protein (MRP) 4 to cellular excretion of the glucuronides. Two glucuronide isomers [i.e., resveratrol 3-O-glucuronide (R3G) and resveratrol 4'-O-glucuronide (R4'G)] were excreted into the extracellular compartment after incubation of resveratrol (1-100 μM) with HeLa1A1 cells. The excretion rate was linearly related to the level of intracellular glucuronide, indicating that glucuronide efflux was a nonsaturable process. MK-571 (a dual inhibitor of UGT1A1 and MRPs) significantly decreased the excretion rates of R3G and R4'G while increasing their intracellular levels. Likewise, short-hairpin RNA (shRNA)-mediated silencing of MRP4 caused a significant reduction in glucuronide excretion but an elevation in glucuronide accumulation. Furthermore, β-glucuronidase expressed in the cells catalyzed the hydrolysis of the glucuronides back to the parent compound. A cellular pharmacokinetic model integrating resveratrol transport/metabolism with glucuronide hydrolysis/excretion was well fitted to the experimental data, allowing derivation of the efflux rate constant values in the absence or presence of shRNA targeting MRP4. It was found that a large percentage of glucuronide excretion (43%-46%) was attributed to MRP4. In conclusion, MRP4 participated in cellular excretion of R3G and R4'G. Integration of mechanistic pharmacokinetic modeling with transporter knockdown was a useful method to derive the contribution percentage of an exporter to overall glucuronide excretion. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

  6. Sniffing for gene-silencing efficiency of siRNAs in HeLa cells in comparison with that in HEK293T cells: correlation between knockdown efficiency and sustainability of sirnas revealed by FRET-based probing.

    Science.gov (United States)

    Shin, Seonmi; Kim, Yea Seul; Kim, Jisu; Kwon, Hyun-Mi; Kim, Dong-Eun; Hah, Sang Soo

    2013-04-01

    Investigation of the intracellular fate of small interfering RNAs (siRNAs) following their delivery into cells is of great importance to elucidate their dynamics in cytoplasm. Here we describe the use of an advanced fluorescence-based method to probe the dissociation and/or degradation of double-labeled siRNAs in HeLa cells in comparison with that in human embryonic kidney 293T (HEK293T) cells. This work was performed with three siRNAs labeled with fluorescence resonance energy transfer (FRET) dyes, allowing a non-destructive and non-invasive assessment of the dissociation and degradation state of siRNAs in cultured cells. Our FRET analysis not only shows the asymmetric degradation as well as the time-dependent dissociation of each siRNA strand during the measured time period, underlining the high intrinsic nuclease resistance of duplex siRNAs, but also reveals the longer sustainability of siRNAs in HeLa cells compared with that in HEK293T cells, explaining the gene silencing in HeLa cells is more efficient than that in HEK293T cells. In addition, our single-molecule FRET assays demonstrate the potential of the delineated fluorescence-based technique for future research on biological behavior of siRNAs even at the single-molecule level. The fluorescence-based method is a straightforward technique to gain direct information on siRNA integrity inside living cells, which can provide a detection tool for dynamics of biological molecules.

  7. Local nanomechanical properties of HeLa-cell surfaces

    Science.gov (United States)

    Gigler, A.; Holzwarth, M.; Marti, O.

    2007-04-01

    Using the Digital Pulsed Force Mode (DPFM) approach, the local mechanical properties of living HeLa cells have been examined. The cells were attached to a favorable glass-like substrate. The AFM used for the experiments was a commercial AFM setup by WITec. At every point of the image, approach and retract curves have been performed. The repetition rate of the cycle was 175 Hz. A total of about 500,000 curves has been recorded and completely evaluated for each experiment. The substrate served as an online reference material for calibration purposes. First, the force trajectories were corrected for the viscous drag force in the liquid environment. Second, the curves within the region of the substrate were phase corrected to compensate for the time lag of the signal in the setup assuming a purely elastic response of the reference material. Finally, all the force traces have been corrected by using this information and evaluated according to common continuum-elastic models. The resulting images allow the assignment of values of Young's modulus, local adhesion, hysteretic behavior, etc. at a high lateral resolution all over the cell body. In this paper, we describe the procedure of our measurement and the corresponding signal correction strategy of our automated data evaluation.

  8. Correcting chromatic offset in multicolor super-resolution localization microscopy.

    Science.gov (United States)

    Erdelyi, Miklos; Rees, Eric; Metcalf, Daniel; Schierle, Gabriele S Kaminski; Dudas, Laszlo; Sinko, Jozsef; Knight, Alex E; Kaminski, Clemens F

    2013-05-06

    Localization based super-resolution microscopy techniques require precise drift correction methods because the achieved spatial resolution is close to both the mechanical and optical performance limits of modern light microscopes. Multi-color imaging methods require corrections in addition to those dealing with drift due to the static, but spatially-dependent, chromatic offset between images. We present computer simulations to quantify this effect, which is primarily caused by the high-NA objectives used in super-resolution microscopy. Although the chromatic offset in well corrected systems is only a fraction of an optical wavelength in magnitude (super-resolution methods is impossible without appropriate image correction. The simulated data are in excellent agreement with experiments using fluorescent beads excited and localized at multiple wavelengths. Finally we present a rigorous and practical calibration protocol to correct for chromatic optical offset, and demonstrate its efficacy for the imaging of transferrin receptor protein colocalization in HeLa cells using two-color direct stochastic optical reconstruction microscopy (dSTORM).

  9. Local food and tourism

    DEFF Research Database (Denmark)

    Boesen, Morten; Sundbo, Donna; Sundbo, Jon

    2017-01-01

    is on the actions and attitude logics of local food networks and tourism actors, and whether their respective logics fit as a factor to explain why or why not development of local food concepts lead to increased local tourism. Six local food networks and their collaboration with local tourism actors are studied......This article investigates the question: Why local food networks succeed or fail in collaborating with local tourism actors to create more tourism based on local food? The article focuses on entrepreneurial local food networks and their collaboration with local tourism actors. Emphasis......, which emphasises the community aspect (i.e. that both parties want to support the local community and brand it). The non-fitting logics that mostly hinder this are dominant market and industry logic, which emphasise egoistic economic gain....

  10. Proliferative activity of a blend of Echinacea angustifolia and Echinacea purpurea root extracts in human vein epithelial, HeLa, and QBC-939 cell lines, but not in Beas-2b cell lines

    Directory of Open Access Journals (Sweden)

    Simon Angelo Cichello

    2016-04-01

    Full Text Available Echinacea is used for its immunostimulating properties and may have a role in modulating adverse immune effects of chemotherapy (i.e., use of 5-fluorouracil (5-FU; fluorouracil and its immunosuppressive effect. Patients may seek herbal remedies such as Echinacea (Echinacea angustifolia and Echinacea purpurea for immune stimulation. Echinacea extracts have been prescribed to supplement cancer chemotherapy for their immune-supportive effects; however, the extracts may also influence tumourgenesis. Our study aimed to determine the proliferative effect of the ethanolic blend of E. angustifolia and E. purpurea on various cancer cervical and bile duct cell lines, including HELA and QBC-939. Various cancer cells (HeLa and QBC-939 and human vein epithelial cells (HUVEC were treated with the Echinacea blend sample that was evaporated and reconstituted in Dimethyl sulfoxide (DMSO. As the extract concentration of Echinacea was increased from 12.5 μg/mL to 25 μg/mL, there was an increase in cell inhibition up to 100%, which then reduced to 90% over the next three concentrations, 50 μg/mL, 100 μg/mL, and 200 μg/mL, in HeLa cells; further inhibitory effects were observed in QBC-939 cells, from 9% inhibition at a concentration of 25 μg/mL up to 37.96% inhibition at 100 μg/mL concentration. Moreover, this is the first study to report the growth-promoting effects of this Echinacea blend in HUVEC, up to 800% at a dose concentration of 200 μg/mL. Previous studies have suggested that chicoric acid of Echinacea spp. is responsible for the increased cell growth. The results of this study show that the hydroethanolic extract of Echinacea herbal medicine promotes the growth of HeLa cells and QBC-939 cancer cell proliferation, and may interfere with cancer treatment (i.e., chemotherapy drugs such as 5-fluorouracil and Cisplatin (DDP. However, the Echinacea blend shows potential in neurodegenerative diseases with growth-promoting effects in HUVEC

  11. Proliferative activity of a blend of Echinacea angustifolia and Echinacea purpurea root extracts in human vein epithelial, HeLa, and QBC-939 cell lines, but not in Beas-2b cell lines.

    Science.gov (United States)

    Cichello, Simon Angelo; Yao, Qian; He, Xiao Qiong

    2016-04-01

    Echinacea is used for its immunostimulating properties and may have a role in modulating adverse immune effects of chemotherapy (i.e., use of 5-fluorouracil (5-FU); fluorouracil and its immunosuppressive effect). Patients may seek herbal remedies such as Echinacea (Echinacea angustifolia and Echinacea purpurea) for immune stimulation. Echinacea extracts have been prescribed to supplement cancer chemotherapy for their immune-supportive effects; however, the extracts may also influence tumourgenesis. Our study aimed to determine the proliferative effect of the ethanolic blend of E. angustifolia and E. purpurea on various cancer cervical and bile duct cell lines, including HELA and QBC-939. Various cancer cells (HeLa and QBC-939) and human vein epithelial cells (HUVEC) were treated with the Echinacea blend sample that was evaporated and reconstituted in Dimethyl sulfoxide (DMSO). As the extract concentration of Echinacea was increased from 12.5 μg/mL to 25 μg/mL, there was an increase in cell inhibition up to 100%, which then reduced to 90% over the next three concentrations, 50 μg/mL, 100 μg/mL, and 200 μg/mL, in HeLa cells; further inhibitory effects were observed in QBC-939 cells, from 9% inhibition at a concentration of 25 μg/mL up to 37.96% inhibition at 100 μg/mL concentration. Moreover, this is the first study to report the growth-promoting effects of this Echinacea blend in HUVEC, up to 800% at a dose concentration of 200 μg/mL. Previous studies have suggested that chicoric acid of Echinacea spp. is responsible for the increased cell growth. The results of this study show that the hydroethanolic extract of Echinacea herbal medicine promotes the growth of HeLa cells and QBC-939 cancer cell proliferation, and may interfere with cancer treatment (i.e., chemotherapy drugs such as 5-fluorouracil and Cisplatin (DDP)). However, the Echinacea blend shows potential in neurodegenerative diseases with growth-promoting effects in HUVEC. Further animal

  12. Locally orderless registration

    DEFF Research Database (Denmark)

    Darkner, Sune; Sporring, Jon

    2013-01-01

    approach is based on local intensity histograms and built upon the technique of Locally Orderless Images. Histograms by Locally Orderless Images are well posed and offer explicit control over the 3 inherent and unavoidable scales: the spatial resolution, intensity levels, and spatial extent of local...

  13. Local duality for 2-dimensional local ring

    Indian Academy of Sciences (India)

    dimensional complete local ring whose residue field is an n-dimensional local field in the sense of. Kato–Parshin. Our results generalize the Saito works in the case n = 0 and are applied to study the Bloch–Ogus complex for such rings in various cases.

  14. carcinomes nasopharynges localement avances locally advanced ...

    African Journals Online (AJOL)

    East Asia and North Africa. The prognosis remains poor for locally advanced stages (IIB -IVB), more than one third of cases presented locally recurrent and ..... Lee A.W.M, Poon Y.F, Foo W et al. Retrospective analysis of 5 037 patients with nasopharyngeal carcinoma treated during 1976-85: ove- rall survival and patterns of ...

  15. Defining local food

    DEFF Research Database (Denmark)

    Eriksen, Safania Normann

    2013-01-01

    Despite evolving local food research, there is no consistent definition of “local food.” Various understandings are utilized, which have resulted in a diverse landscape of meaning. The main purpose of this paper is to examine how researchers within the local food systems literature define local...... food, and how these definitions can be used as a starting point to identify a new taxonomy of local food based on three domains of proximity....

  16. Analysis of the subcellular localization of the human histone methyltransferase SETDB1

    Energy Technology Data Exchange (ETDEWEB)

    Tachibana, Keisuke, E-mail: nya@phs.osaka-u.ac.jp [Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan); Gotoh, Eiko; Kawamata, Natsuko [Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan); Ishimoto, Kenji [Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan); Laboratory for System Biology and Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro, Tokyo 153-8904 (Japan); Uchihara, Yoshie [Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan); Iwanari, Hiroko [Department of Quantitative Biology and Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro, Tokyo 153-8904 (Japan); Sugiyama, Akira; Kawamura, Takeshi [Radioisotope Center, The University of Tokyo, 2-11-16 Yayoi, Bunkyo, Tokyo 113-0032 (Japan); Mochizuki, Yasuhiro [Department of Quantitative Biology and Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro, Tokyo 153-8904 (Japan); Tanaka, Toshiya [Laboratory for System Biology and Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro, Tokyo 153-8904 (Japan); Sakai, Juro [Division of Metabolic Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro, Tokyo 153-8904 (Japan); Hamakubo, Takao [Department of Quantitative Biology and Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro, Tokyo 153-8904 (Japan); Kodama, Tatsuhiko [Laboratory for System Biology and Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro, Tokyo 153-8904 (Japan); and others

    2015-10-02

    SET domain, bifurcated 1 (SETDB1) is a histone methyltransferase that methylates lysine 9 on histone H3. Although it is important to know the localization of proteins to elucidate their physiological function, little is known of the subcellular localization of human SETDB1. In the present study, to investigate the subcellular localization of hSETDB1, we established a human cell line constitutively expressing enhanced green fluorescent protein fused to hSETDB1. We then generated a monoclonal antibody against the hSETDB1 protein. Expression of both exogenous and endogenous hSETDB1 was observed mainly in the cytoplasm of various human cell lines. Combined treatment with the nuclear export inhibitor leptomycin B and the proteasome inhibitor MG132 led to the accumulation of hSETDB1 in the nucleus. These findings suggest that hSETDB1, localized in the nucleus, might undergo degradation by the proteasome and be exported to the cytosol, resulting in its detection mainly in the cytosol. - Highlights: • Endogenous human SETDB1 was localized mainly in the cytoplasm. • Combined treatment with LMB and MG132 led to accumulation of human SETDB1 in the nucleus. • HeLa cells expressing EFGP-hSETDB1 are useful for subcellular localization analyses.

  17. Investigating the consequences of eIF4E2 (4EHP interaction with 4E-transporter on its cellular distribution in HeLa cells.

    Directory of Open Access Journals (Sweden)

    Dorota Kubacka

    Full Text Available In addition to the canonical eIF4E cap-binding protein, eukaryotes have evolved sequence-related variants with distinct features, some of which have been shown to negatively regulate translation of particular mRNAs, but which remain poorly characterised. Mammalian eIF4E proteins have been divided into three classes, with class I representing the canonical cap-binding protein eIF4E1. eIF4E1 binds eIF4G to initiate translation, and other eIF4E-binding proteins such as 4E-BPs and 4E-T prevent this interaction by binding eIF4E1 with the same consensus sequence YX 4Lϕ. We investigate here the interaction of human eIF4E2 (4EHP, a class II eIF4E protein, which binds the cap weakly, with eIF4E-transporter protein, 4E-T. We first show that ratios of eIF4E1:4E-T range from 50:1 to 15:1 in HeLa and HEK293 cells respectively, while those of eIF4E2:4E-T vary from 6:1 to 3:1. We next provide evidence that eIF4E2 binds 4E-T in the yeast two hybrid assay, as well as in pull-down assays and by recruitment to P-bodies in mammalian cells. We also show that while both eIF4E1 and eIF4E2 bind 4E-T via the canonical YX 4Lϕ sequence, nearby downstream sequences also influence eIF4E:4E-T interactions. Indirect immunofluorescence was used to demonstrate that eIF4E2, normally homogeneously localised in the cytoplasm, does not redistribute to stress granules in arsenite-treated cells, nor to P-bodies in Actinomycin D-treated cells, in contrast to eIF4E1. Moreover, eIF4E2 shuttles through nuclei in a Crm1-dependent manner, but in an 4E-T-independent manner, also unlike eIF4E1. Altogether we conclude that while both cap-binding proteins interact with 4E-T, and can be recruited by 4E-T to P-bodies, eIF4E2 functions are likely to be distinct from those of eIF4E1, both in the cytoplasm and nucleus, further extending our understanding of mammalian class I and II cap-binding proteins.

  18. Activation of double-stranded RNA-activated protein kinase in HeLa cells after poliovirus infection does not result in increased phosphorylation of eucaryotic initiation factor-2.

    Science.gov (United States)

    Ransone, L J; Dasgupta, A

    1987-01-01

    Protein kinase activity in general is stimulated at least 5- to 10-fold in ribosomal salt wash preparations from poliovirus-infected HeLa cells compared with those from mock-infected cells. The stimulation of kinase activity is manifested by increased phosphorylation of ribosome-associated polypeptides having approximate molecular weights of 135,000, 120,000, 85,000, 68,000, 65,000, 40,000, 28,000, 25,000, and 21,000. The Mr 68,000 phosphoprotein is structurally identical to the interferon-induced, double-stranded RNA-activated protein kinase (P1) which phosphorylates the alpha subunit of eucaryotic initiation factor-2 (eIF-2). A similar protein of Mr 68,000 is more phosphorylated in poliovirus-infected cells than in mock-infected cells. Increased phosphorylation of P1 protein in poliovirus-infected cells, however, does not result in an increased phosphorylation of the alpha subunit of endogenous or exogenously added eIF-2, both in vitro and in vivo. These results suggest that a mechanism must exist in poliovirus-infected HeLa cells which prevents further phosphorylation of eIF-2 by the activated kinase. Images PMID:3033310

  19. Locally analytic vectors in representations of locally

    CERN Document Server

    Emerton, Matthew J

    2017-01-01

    The goal of this memoir is to provide the foundations for the locally analytic representation theory that is required in three of the author's other papers on this topic. In the course of writing those papers the author found it useful to adopt a particular point of view on locally analytic representation theory: namely, regarding a locally analytic representation as being the inductive limit of its subspaces of analytic vectors (of various "radii of analyticity"). The author uses the analysis of these subspaces as one of the basic tools in his study of such representations. Thus in this memoir he presents a development of locally analytic representation theory built around this point of view. The author has made a deliberate effort to keep the exposition reasonably self-contained and hopes that this will be of some benefit to the reader.

  20. How local is the local field potential?

    Science.gov (United States)

    Kajikawa, Yoshinao; Schroeder, Charles E

    2011-12-08

    Local field potentials (LFPs) are of growing importance in neurophysiological investigations. LFPs supplement action potential recordings by indexing activity relevant to EEG, magnetoencephalographic, and hemodynamic (fMRI) signals. Recent reports suggest that LFPs reflect activity within very small domains of several hundred micrometers. We examined this conclusion by comparing LFP, current source density (CSD), and multiunit activity (MUA) signals in macaque auditory cortex. Estimated by frequency tuning bandwidths, these signals' "listening areas" differ systematically with an order of MUA local contributions. Direct measurements indicate passive spread of LFPs to sites more than a centimeter from their origins. These findings appear to be independent of the frequency content of the LFP. Our results challenge the idea that LFP recordings typically integrate over extremely circumscribed local domains. Rather, LFPs appear as a mixture of local potentials with "volume conducted" potentials from distant sites. Copyright © 2011 Elsevier Inc. All rights reserved.

  1. Local electroporation of a single cell using a scanning ion conductance microscope

    Science.gov (United States)

    Iwata, Futoshi; Yamazaki, Koji; Ishizaki, Kimihiro; Ushiki, Tatuo

    2014-03-01

    We developed a novel electroporation technique for molecular delivery into a single cell. A nanopipette, a thermally pulled glass capillary, is prepared as to act as a pair of tiny electrodes for single-cell electroporation. An Ag/AgCl wire is inserted into the nanopipette, and the outside edge of the nanopipette is coated by Ag sputtering. Electric pulses are applied between the outside and inside electrodes to form a local electric field at the edge of the nanopipette. To position the pipette edge in the vicinity of the cell membrane, we control the probe-surface distance using a scanning ion conductance microscope (SICM). The SICM technique achieves non-contact approach of the nanopipette edge on the cell membrane, which allows low-invasive electroporation of a single cell. As a demonstration of this technique, a fluorescent molecule of propidium iodide was successfully delivered into a single HeLa cell.

  2. Nuclear localization of Rad51B is independent of BRCA2

    Energy Technology Data Exchange (ETDEWEB)

    Miller, K A; Hinz, J M; Yamada, A; Thompson, L H; Albala, J S

    2005-06-28

    Human Rad51 is critical for the maintenance of genome stability through its role in the repair of DNA double-strand breaks. Rad51B (Rad51L1/hRec2) is one of the five known paralogs of human Rad51 found in a multi-protein complex with three other Rad51 paralogs, Rad51C, Rad51D and Xrcc2. Examination of EGFP-Rad51B fusion protein in HeLa S3 cells and immunofluorescence in several human cell lines confirms the nuclear localization of Rad51B. This is the first report to detail putative interactions of a Rad51 paralog protein with BRCA2. Utilization of a BRCA2 mutant cell line, CAPAN-1 suggests that Rad51B localizes to the nucleus independent of BRCA2. Although both Rad51B and BRCA2 are clearly involved in the homologous recombinational repair pathway, Rad51B and BRCA2 do not appear to associate directly. Furthermore, mutations in the KKLK motif of Rad51B, amino acid residues 4-7, mislocalizes Rad51B to the cytoplasm suggesting that this is the nuclear localization signal for the Rad51B protein. Examination of wild-type EGFP-Rad51B fusion protein in mammalian cells deficient in Rad51C showed that Rad51B localizes to the nucleus independent of Rad51C; further suggesting that Rad51B, like Rad51C, contains its own nuclear localization signal.

  3. Clarifying local integration

    Directory of Open Access Journals (Sweden)

    Sarah Meyer

    2006-08-01

    Full Text Available Ana Low’s article in FMR 251 highlights the need to reexamineand re-invigorate debate on local integration as adurable solution for refugees. However, the Self-RelianceStrategy (SRS in Uganda which she describes does notprovide an adequate model of local integration as adurable solution – in fact, local integration is not its aim.

  4. Importin β1 protein-mediated nuclear localization of death receptor 5 (DR5) limits DR5/tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-induced cell death of human tumor cells.

    Science.gov (United States)

    Kojima, Yuko; Nakayama, Masafumi; Nishina, Takashi; Nakano, Hiroyasu; Koyanagi, Makoto; Takeda, Kazuyoshi; Okumura, Ko; Yagita, Hideo

    2011-12-16

    Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)/death receptor 5 (DR5)-mediated cell death plays an important role in the elimination of tumor cells and transformed cells. Recently, recombinant TRAIL and agonistic anti-DR5 monoclonal antibodies have been developed and applied to cancer therapy. However, depending on the type of cancer, the sensitivity to TRAIL has been reportedly different, and some tumor cells are resistant to TRAIL-mediated apoptosis. Using confocal microscopy, we found that large amounts of DR5 were localized in the nucleus in HeLa and HepG2 cells. Moreover, these tumor cells were resistant to TRAIL, whereas DU145 cells, which do not have nuclear DR5, were highly sensitive to TRAIL. By means of immunoprecipitation and Western blot analysis, we found that DR5 and importin β1 were physically associated, suggesting that the nuclear DR5 was transported through the nuclear import pathway mediated by importin β1. Two functional nuclear localization signals were identified in DR5, the mutation of which abrogated the nuclear localization of DR5 in HeLa cells. Moreover, the nuclear transport of DR5 was also prevented by the knockdown of importin β1 using siRNA, resulting in the up-regulation of DR5 expression on the cell surface and an increased sensitivity of HeLa and HepG2 cells to TRAIL. Taken together, our findings suggest that the importin β1-mediated nuclear localization of DR5 limits the DR5/TRAIL-induced cell death of human tumor cells and thus can be a novel target to improve cancer therapy with recombinant TRAIL and anti-DR5 antibodies.

  5. Disorder-Free Localization

    Science.gov (United States)

    Smith, A.; Knolle, J.; Kovrizhin, D. L.; Moessner, R.

    2017-06-01

    The venerable phenomena of Anderson localization, along with the much more recent many-body localization, both depend crucially on the presence of disorder. The latter enters either in the form of quenched disorder in the parameters of the Hamiltonian, or through a special choice of a disordered initial state. Here, we present a model with localization arising in a very simple, completely translationally invariant quantum model, with only local interactions between spins and fermions. By identifying an extensive set of conserved quantities, we show that the system generates purely dynamically its own disorder, which gives rise to localization of fermionic degrees of freedom. Our work gives an answer to a decades old question whether quenched disorder is a necessary condition for localization. It also offers new insights into the physics of many-body localization, lattice gauge theories, and quantum disentangled liquids.

  6. Superior antibacterial activity of zinc oxide/graphene oxide composites originating from high zinc concentration localized around bacteria.

    Science.gov (United States)

    Wang, Yan-Wen; Cao, Aoneng; Jiang, Yu; Zhang, Xin; Liu, Jia-Hui; Liu, Yuanfang; Wang, Haifang

    2014-02-26

    New materials with good antibacterial activity and less toxicity to other species attract numerous research interest. Taking advantage of zinc oxide (ZnO) and graphene oxide (GO), the ZnO/GO composites were prepared by a facile one-pot reaction to achieve superior antibacterial properties without damaging other species. In the composites, ZnO nanoparticles (NPs), with a size of about 4 nm, homogeneously anchored onto GO sheets. The typical bacterium Escherichia coli and HeLa cell were used to evaluate the antibacterial activity and cytotoxicity of the ZnO/GO composites, respectively. The synergistic effects of GO and ZnO NPs led to the superior antibacterial activity of the composites. GO helped the dispersion of ZnO NPs, slowed the dissolution of ZnO, acted as the storage site for the dissolved zinc ions, and enabled the intimate contact of E. coli with ZnO NPs and zinc ions as well. The close contact enhanced the local zinc concentration pitting on the bacterial membrane and the permeability of the bacterial membrane and thus induced bacterial death. In addition, the ZnO/GO composites were found to be much less toxic to HeLa cells, compared to the equivalent concentration of ZnO NPs in the composites. The results indicate that the ZnO/GO composites are promising disinfection materials to be used in surface coatings on various substrates to effectively inhibit bacterial growth, propagation, and survival in medical devices.

  7. Aging in Sweden: local variation, local control.

    Science.gov (United States)

    Davey, Adam; Malmberg, Bo; Sundström, Gerdt

    2014-08-01

    Aging in Sweden has been uniquely shaped by its history-most notably the long tradition of locally controlled services for older adults. We considered how local variations and local control shape the experience of aging in Sweden and organized the paper into 3 sections. First, we examine aging in Sweden along demography, economy, and housing. Next, we trace the origins and development of the Swedish welfare state to consider formal supports (service provision) and informal supports (caregiving and receipt of care). Finally, we direct researchers to additional data resources for understanding aging in Sweden in greater depth. Sweden was one of the first countries to experience rapid population aging. Quality of life for a majority of older Swedes is high. Local control permits a flexible and adaptive set of services and programs, where emphasis is placed on improving the quality and targeting of services that have already reached a plateau as a function of population and expenditures. © The Author 2013. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  8. HTP Nutraceutical Screening for Histone Deacetylase Inhibitors and Effects of HDACis on Tumor-suppressing miRNAs by Trichostatin A and Grapeseed (Vitis vinifera) in HeLa cells.

    Science.gov (United States)

    Mazzio, Elizabeth A; Soliman, Karam F A

    2017-01-02

    Aggressive tumor malignancies are a consequence of delayed diagnosis, epigenetic/phenotype changes and chemo-radiation resistance. Histone deacetylases (HDACs) are a major epigenetic regulator of transcriptional repression, which are highly overexpressed in advanced malignancy. While original chemotherapy drugs were modeled after phytochemicals elucidated by botanical screenings, HDAC inhibitors (HDACi) such as apicidin, trichostatin A (TSA) and butyrate were discovered as products of fungus and microbes, in particular, gut microbiota. Therefore, a persistent question remains as to the inherent existence of HDACis in raw undigested dietary plant material. In this study, we conduct a high-throughput (HTP) screening of ~1,600 non-fermented commonly used nutraceuticals (spices, herbs, teas, vegetables, fruits, seeds, rinds etc.) at (screening was then conducted, followed by a study comparing biological effects of HDACis in HeLa cells, including analysis of whole-transcriptome non-coding RNAs using Affymetrix miRNA 4.1-panel arrays. The HTP screening results confirmed 44/1600 as potential HDACis to which 31 were further eliminated as false-positives. Methodological challenges/concerns are addressed regarding plant product false-positives that arise from the signal reduction of commercial lysine development reagents. Only 13 HDACis were found having an IC50 under <200 μg/ml: Grapeseed extract (Vitis vinifera), Great burnet root (Sanguisorba Officinalis), Babul (Acacia arabica), Chinese gallnut (Melaphis chinensis), Konaberry extract (Coffea arabica), Uva Ursi (Arctostaphylos uva ursi), Green tea (Camellia sinensis), Meadowsweet (Filipendula ulmaria), Sassafras (Sassafras officinale), Turkey rhubarb (Rheum palmatum), epigallocatechin gallate (EGCG), gossypol and gallic acid. Next, we investigate the biological consequence of HDACi panel drugs in HeLa cells, where the data suggest predominant effects are anti-mitotic rather than cytotoxic. Lastly, differential effects of

  9. Ca2+-mediated potentiation of the swelling-induced taurine efflux from HeLa cells: On the role of calmodulin and novel protein kinase C isoforms

    DEFF Research Database (Denmark)

    Falktoft, Birgitte; Lambert, Ian H.

    2004-01-01

    The present work sets out to investigate how Ca2+ regulates the volume-sensitive taurine-release pathway in HeLa cells. Addition of Ca2+-mobilizing agonists at the time of exposure to hypotonic NaCl medium augments the swelling-induced taurine release and subsequently accelerates the inactivation...... of the release pathway. The accelerated inactivation is not observed in hypotonic Ca2+-free or high-K+ media. Addition of Ca2+-mobilizing agonists also accelerates the regulatory volume decrease, which probably reflects activation of Ca2+-activated K+ channels. The taurine release from control cells and cells...... exposed to Ca2+ agonists is equally affected by changes in cell volume, application of DIDS and arachidonic acid, indicating that the volume-sensitive taurine leak pathway mediates the Ca2+-augmented taurine release. Exposure to Ca2+-mobilizing agonists prior to a hypotonic challenge also augments...

  10. [Local anaesthesia in ruminants].

    Science.gov (United States)

    Nuss, Karl; Schwarz, Andrea; Ringer, Simone

    2017-06-20

    The use of local anaesthesia in ruminants allows many surgical procedures to be conducted free of pain, efficiently and inexpensively in the field. Local anaesthesia combined with sedation and immobilisation of the animal can replace general anaesthesia for many procedures (e. g. castration, claw amputation). The level of difficulty differs among various local anaesthetic techniques: local infiltration of tissue or anaesthesia of the cornual nerve are easily performed, whereas local anaesthesia of the eye, regional anaesthesia in limbs or anaesthesia for umbilical surgery are more difficult to carry out. This article presents an illustrated overview of the most common local anaesthetic procedures in cattle as well as in small ruminants and serves as a practical guide for veterinarians in the field. In principle, these techniques can likewise be applied in other ruminants or artiodactyls.

  11. Local Worlds of Activation

    DEFF Research Database (Denmark)

    Jacobsson, Kerstin; Hollertz, Katarina; Garsten, Christina

    2017-01-01

    . This article studies local activation policy and practice in three Swedish municipalities, representing three distinct ‘local worlds of activation’. The analysis shows that policy orientations in the municipalities studied ranged from ‘work-first’ to ‘life-first’ approaches to activation. Governance...... of Coordination Unions, as multi-party collaborate organisational structures established for activation policy implementation for certain target groups. Thus, activation must be approached not as a fixed and universal policy for social inclusion, but as susceptible to local practice and hence open to influence...... from local politics, established local traditions, patterns of networking and modes of collaborating, as the notion of ‘local words of activation’ intends to capture....

  12. Local Social Networks

    OpenAIRE

    Sapuppo, Antonio; Sørensen, Lene Tolstrup

    2011-01-01

    Online social networks have become essential for many users in their daily communication. Through a combination of the online social networks with opportunistic networks, a new concept arises: Local Social Networks. The target of local social networks is to promote social networking benefits in physical environment in order to leverage personal affinities in the users' surroundings. The purpose of this paper is to present and discuss the concept of local social networks as a new social commun...

  13. Condurango (Gonolobus condurango Extract Activates Fas Receptor and Depolarizes Mitochondrial Membrane Potential to Induce ROS-dependent Apoptosis in Cancer Cells in vitro CE-treatment on HeLa: a ROS-dependent mechanism

    Directory of Open Access Journals (Sweden)

    Kausik Bishayee

    2015-09-01

    Full Text Available Objectives: Condurango (Gonolobus condurango extract is used by complementary and alternative medicine (CAM practitioners as a traditional medicine, including homeopathy, mainly for the treatment of syphilis. Condurango bark extract is also known to reduce tumor volume, but the underlying molecular mechanisms still remain unclear. Methods: Using a cervical cancer cell line (HeLa as our model, the molecular events behind condurango extract’s (CE’s anticancer effect were investigated by using flow cytometry, immunoblotting and reverse transcriptase-polymerase chain reaction (RT-PCR. Other included cell types were prostate cancer cells (PC3, transformed liver cells (WRL-68, and peripheral blood mononuclear cells (PBMCs. Results: Condurango extract (CE was found to be cytotoxic against target cells, and this was significantly deactivated in the presence of N-acetyl cysteine (NAC, a scavenger of reactive oxygen species (ROS, suggesting that its action could be mediated through ROS generation. CE caused an increase in the HeLa cell population containing deoxyribonucleic acid (DNA damage at the G zero/Growth 1 (G0/G1 stage. Further, CE increased the tumor necrosis factor alpha (TNF-α and the fas receptor (FasR levels both at the ribonucleic acid (RNA and the protein levels, indicating that CE might have a cytotoxic mechanism of action. CE also triggered a sharp decrease in the expression of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB both at the RNA and the protein levels, a possible route to attenuation of B-cell lymphoma 2 (Bcl-2, and caused an opening of the mitochondrial membrane’s permeability transition (MPT pores, thus enhancing caspase activities. Conclusion: Overall, our results suggest possible pathways for CE mediated cytotoxicity in model cancer cells.

  14. Connexin30.2: in vitro interaction with connexin36 in HeLa cells and expression in AII amacrine cells and intrinsically photosensitive ganglion cells in the mouse retina

    Directory of Open Access Journals (Sweden)

    Arndt eMeyer

    2016-05-01

    Full Text Available Electrical coupling via gap junctions is an abundant phenomenon in the mammalian retina and occurs in all major cell types. Gap junction channels are assembled from different connexin subunits, and the connexin composition of the channel confers specific properties to the electrical synapse. In the mouse retina, gap junctions were demonstrated between intrinsically photosensitive ganglion cells and displaced amacrine cells but the underlying connexin remained undetermined. In the primary rod pathway, gap junctions play a crucial role, coupling AII amacrine cells among each other and to ON cone bipolar cells. Although it has long been known that connexin36 and connexin45 are necessary for the proper functioning of this most sensitive rod pathway, differences between homocellular AII/AII gap junctions and AII/ON bipolar cell gap junctions suggested the presence of an additional connexin in AII amacrine cells. Here, we used a connexin30.2-lacZ mouse line to study the expression of connexin30.2 in the retina. We show that connexin30.2 is expressed in intrinsically photosensitive ganglion cells and AII amacrine cells. Moreover, we tested whether connexin30.2 and connexin36 – both expressed in AII amacrine cells – are able to interact with each other and are deposited in the same gap junctional plaques. Using newly generated anti-connexin30.2 antibodies, we show in HeLa cells that both connexins are indeed able to interact and may form heteromeric channels: both connexins were co-immunoprecipitated from transiently transfected HeLa cells and connexin30.2 gap junction plaques became significantly larger when co-expressed with connexin36. These data suggest that connexin36 is able to form heteromeric gap junctions with another connexin. We hypothesize that co-expression of connexin30.2 and connexin36 may endow AII amacrine cells with the means to differentially regulate its electrical coupling to different synaptic partners.

  15. Connexin30.2: In Vitro Interaction with Connexin36 in HeLa Cells and Expression in AII Amacrine Cells and Intrinsically Photosensitive Ganglion Cells in the Mouse Retina.

    Science.gov (United States)

    Meyer, Arndt; Tetenborg, Stephan; Greb, Helena; Segelken, Jasmin; Dorgau, Birthe; Weiler, Reto; Hormuzdi, Sheriar G; Janssen-Bienhold, Ulrike; Dedek, Karin

    2016-01-01

    Electrical coupling via gap junctions is an abundant phenomenon in the mammalian retina and occurs in all major cell types. Gap junction channels are assembled from different connexin subunits, and the connexin composition of the channel confers specific properties to the electrical synapse. In the mouse retina, gap junctions were demonstrated between intrinsically photosensitive ganglion cells and displaced amacrine cells but the underlying connexin remained undetermined. In the primary rod pathway, gap junctions play a crucial role, coupling AII amacrine cells among each other and to ON cone bipolar cells. Although it has long been known that connexin36 and connexin45 are necessary for the proper functioning of this most sensitive rod pathway, differences between homocellular AII/AII gap junctions and AII/ON bipolar cell gap junctions suggested the presence of an additional connexin in AII amacrine cells. Here, we used a connexin30.2-lacZ mouse line to study the expression of connexin30.2 in the retina. We show that connexin30.2 is expressed in intrinsically photosensitive ganglion cells and AII amacrine cells. Moreover, we tested whether connexin30.2 and connexin36-both expressed in AII amacrine cells-are able to interact with each other and are deposited in the same gap junctional plaques. Using newly generated anti-connexin30.2 antibodies, we show in HeLa cells that both connexins are indeed able to interact and may form heteromeric channels: both connexins were co-immunoprecipitated from transiently transfected HeLa cells and connexin30.2 gap junction plaques became significantly larger when co-expressed with connexin36. These data suggest that connexin36 is able to form heteromeric gap junctions with another connexin. We hypothesize that co-expression of connexin30.2 and connexin36 may endow AII amacrine cells with the means to differentially regulate its electrical coupling to different synaptic partners.

  16. Unusual expression of red fluorescence at M phase induced by anti-microtubule agents in HeLa cells expressing the fluorescent ubiquitination-based cell cycle indicator (Fucci)

    Energy Technology Data Exchange (ETDEWEB)

    Honda-Uezono, Asumi [Section of Oral Radiation Oncology, Department of Oral Health Sciences, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549 (Japan); Section of Maxillofacial Surgery, Department of Maxillofacial and Neck Reconstruction, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549 (Japan); Kaida, Atsushi [Section of Oral Radiation Oncology, Department of Oral Health Sciences, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549 (Japan); Michi, Yasuyuki; Harada, Kiyoshi [Section of Maxillofacial Surgery, Department of Maxillofacial and Neck Reconstruction, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549 (Japan); Hayashi, Yoshiki; Hayashi, Yoshio [Department of Medicinal Chemistry, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo 192-0392 (Japan); Miura, Masahiko, E-mail: masa.mdth@tmd.ac.jp [Section of Oral Radiation Oncology, Department of Oral Health Sciences, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549 (Japan)

    2012-11-16

    Highlights: Black-Right-Pointing-Pointer Fucci visualizes cell cycle by green and red fluorescence. Black-Right-Pointing-Pointer Plinabulin, induced unusual red fluorescence at M-phase in HeLa-Fucci cells. Black-Right-Pointing-Pointer The unusual pattern was followed by mitotic catastrophe. Black-Right-Pointing-Pointer The unusual pattern may be an early indicator of cell death in HeLa cells. -- Abstract: Plinabulin (NPI-2358) is a novel microtubule-depolymerizing agent. In HeLa cells, plinabulin arrests the cell-cycle at M phase and subsequently induces mitotic catastrophe. To better understand the effects on this compound on the cell-cycle, we used the fluorescent ubiquitination-based cell cycle indicator (Fucci), which normally enables G1 and S/G2/M cells to emit red and green fluorescence, respectively. When HeLa-Fucci cells were treated with 50 nM plinabulin, cells began to fluoresce both green and red in an unusual pattern; most cells exhibited the new pattern after 24 h of treatment. X-irradiation efficiently induced G2 arrest in plinabulin-treated cells and significantly retarded the emergence of the unusual pattern, suggesting that entering M phase is essential for induction of the pattern. By simultaneously visualizing chromosomes with GFP-histone H2B, we established that the pattern emerges after nuclear envelope breakdown but before metaphase. Pedigree assay revealed a significant relationship between the unusual expression and mitotic catastrophe. Nocodazole, KPU-133 (a more potent derivative of plinabulin), and paclitaxel also exerted similar effects. From these data, we conclude that the unusual pattern may be associated with dysregulation of late M phase-specific E3 ligase activity and mitotic catastrophe following treatment with anti-microtubule agents.

  17. Local Social Networks

    DEFF Research Database (Denmark)

    Sapuppo, Antonio; Sørensen, Lene Tolstrup

    2011-01-01

    in physical environment in order to leverage personal affinities in the users' surroundings. The purpose of this paper is to present and discuss the concept of local social networks as a new social communication system. Particularly, the preliminary architecture and the prototype of local social networks......Online social networks have become essential for many users in their daily communication. Through a combination of the online social networks with opportunistic networks, a new concept arises: Local Social Networks. The target of local social networks is to promote social networking benefits...

  18. Local Democracy in Myanmar

    DEFF Research Database (Denmark)

    Kyed, Helene Maria; Harrisson, Annika Pohl; McCarthy, Gerard

    local elections of village tract and urban ward administrators and reflects on the future of local democracy and decentralization in Myanmar. Local administrators are the primary point of contact between the state and citizens. How they are elected and how they govern in the everyday are essential....... However, often overlooked in this larger picture of transition is the state of local democracy, including village level governance and everyday state-citizen engagements. Political changes at this level are equally crucial for the wider democratization process. This roundtable summary discusses the 2016...

  19. Localization of magnetic pills

    Science.gov (United States)

    Laulicht, Bryan; Gidmark, Nicholas J.; Tripathi, Anubhav; Mathiowitz, Edith

    2011-01-01

    Numerous therapeutics demonstrate optimal absorption or activity at specific sites in the gastrointestinal (GI) tract. Yet, safe, effective pill retention within a desired region of the GI remains an elusive goal. We report a safe, effective method for localizing magnetic pills. To ensure safety and efficacy, we monitor and regulate attractive forces between a magnetic pill and an external magnet, while visualizing internal dose motion in real time using biplanar videofluoroscopy. Real-time monitoring yields direct visual confirmation of localization completely noninvasively, providing a platform for investigating the therapeutic benefits imparted by localized oral delivery of new and existing drugs. Additionally, we report the in vitro measurements and calculations that enabled prediction of successful magnetic localization in the rat small intestines for 12 h. The designed system for predicting and achieving successful magnetic localization can readily be applied to any area of the GI tract within any species, including humans. The described system represents a significant step forward in the ability to localize magnetic pills safely and effectively anywhere within the GI tract. What our magnetic pill localization strategy adds to the state of the art, if used as an oral drug delivery system, is the ability to monitor the force exerted by the pill on the tissue and to locate the magnetic pill within the test subject all in real time. This advance ensures both safety and efficacy of magnetic localization during the potential oral administration of any magnetic pill-based delivery system. PMID:21257903

  20. Localization of Interference Fringes.

    Science.gov (United States)

    Simon, J. M.; Comastri, Silvia A.

    1980-01-01

    Discusses a proof for determining the localized fringes position arrived at when one considers the interference of two extended sources when one is able to observe fringes only at certain points in space. Shows how the localized fringes may be found in a device used to observe Newton's rings. (Author/CS)

  1. Local Exhaust Ventilation

    DEFF Research Database (Denmark)

    Madsen, Ulla; Breum, N. O.; Nielsen, Peter V.

    Capture efficiency of a local exhaust system, e.g. a kitchen hood, should include only contaminants being direct captured. In this study basic concepts of local exhaust capture efficiency are given, based on the idea of a control box. A validated numerical model is used for estimation...

  2. Local anesthesia in dentistry

    National Research Council Canada - National Science Library

    Bahl, Ritu

    2004-01-01

    ... upon achieving adequate local anesthesia. Research has shown that the fear of pain associated with dentistry is closely associated with the most common method for blocking pain during dental procedures-intraoral administration of local anesthetics. This is considered aversive due to the pain associated with the injection and the perceived threat ...

  3. Locally orderless registration code

    DEFF Research Database (Denmark)

    2012-01-01

    This is code for the TPAMI paper "Locally Orderless Registration". The code requires intel threadding building blocks installed and is provided for 64 bit on mac, linux and windows.......This is code for the TPAMI paper "Locally Orderless Registration". The code requires intel threadding building blocks installed and is provided for 64 bit on mac, linux and windows....

  4. Time to Go Local!

    Science.gov (United States)

    ... example, a person who has found information about breast cancer on MedlinePlus would be able to click on GoLocal and be instantly connected to information about local screening services, cancer care facilities, support groups, exercise and yoga classes, providers of wigs ...

  5. Clustering by Local Gravitation.

    Science.gov (United States)

    Wang, Zhiqiang; Yu, Zhiwen; Chen, C L Philip; You, Jane; Gu, Tianlong; Wong, Hau-San; Zhang, Jun

    2017-05-02

    The objective of cluster analysis is to partition a set of data points into several groups based on a suitable distance measure. We first propose a model called local gravitation among data points. In this model, each data point is viewed as an object with mass, and associated with a local resultant force (LRF) generated by its neighbors. The motivation of this paper is that there exist distinct differences between the LRFs (including magnitudes and directions) of the data points close to the cluster centers and at the boundary of the clusters. To capture these differences efficiently, two new local measures named centrality and coordination are further investigated. Based on empirical observations, two new clustering methods called local gravitation clustering and communication with local agents are designed, and several test cases are conducted to verify their effectiveness. The experiments on synthetic data sets and real-world data sets indicate that both clustering approaches achieve good performance on most of the data sets.

  6. Local sleep and learning.

    Science.gov (United States)

    Huber, Reto; Ghilardi, M Felice; Massimini, Marcello; Tononi, Giulio

    2004-07-01

    Human sleep is a global state whose functions remain unclear. During much of sleep, cortical neurons undergo slow oscillations in membrane potential, which appear in electroencephalograms as slow wave activity (SWA) of sleep. It has been suggested that SWA homeostasis may reflect synaptic changes underlying a cellular need for sleep. If this were so, inducing local synaptic changes should induce local SWA changes, and these should benefit neural function. Here we show that sleep homeostasis indeed has a local component, which can be triggered by a learning task involving specific brain regions. Furthermore, we show that the local increase in SWA after learning correlates with improved performance of the task after sleep. Thus, sleep homeostasis can be induced on a local level and can benefit performance.

  7. Explosion localization via infrasound.

    Science.gov (United States)

    Szuberla, Curt A L; Olson, John V; Arnoult, Kenneth M

    2009-11-01

    Two acoustic source localization techniques were applied to infrasonic data and their relative performance was assessed. The standard approach for low-frequency localization uses an ensemble of small arrays to separately estimate far-field source bearings, resulting in a solution from the various back azimuths. This method was compared to one developed by the authors that treats the smaller subarrays as a single, meta-array. In numerical simulation and a field experiment, the latter technique was found to provide improved localization precision everywhere in the vicinity of a 3-km-aperture meta-array, often by an order of magnitude.

  8. Local Anesthetic Microencapsulation.

    Science.gov (United States)

    1983-11-04

    local anesthetic by Freud and Koller in 1884 (Ritchie and Grune 1980), local anes- thetics have played an important role in clinical medicine. Local...J * ana , NCO C L) ..0C%j U C - r_ c 00 4- C) LU- V~~ 0 )a IrL LUI ii;:7-7 73 -04 &001 EE 4-- 4)4 m’ CJ ou u o 0>’. U L o_ 0 0 -l -L 0 4) U U 0 0 4-L

  9. A neural network classifier capable of recognizing the patterns of all major subcellular structures in fluorescence microscope images of HeLa cells.

    Science.gov (United States)

    Boland, M V; Murphy, R F

    2001-12-01

    Assessment of protein subcellular location is crucial to proteomics efforts since localization information provides a context for a protein's sequence, structure, and function. The work described below is the first to address the subcellular localization of proteins in a quantitative, comprehensive manner. Images for ten different subcellular patterns (including all major organelles) were collected using fluorescence microscopy. The patterns were described using a variety of numeric features, including Zernike moments, Haralick texture features, and a set of new features developed specifically for this purpose. To test the usefulness of these features, they were used to train a neural network classifier. The classifier was able to correctly recognize an average of 83% of previously unseen cells showing one of the ten patterns. The same classifier was then used to recognize previously unseen sets of homogeneously prepared cells with 98% accuracy. Algorithms were implemented using the commercial products Matlab, S-Plus, and SAS, as well as some functions written in C. The scripts and source code generated for this work are available at http://murphylab.web.cmu.edu/software. murphy@cmu.edu

  10. Focus on Local Reaction.

    Science.gov (United States)

    Howe, Rod

    1999-01-01

    Discusses how the student newspaper "The Lance" (at Westside High School in Omaha, Nebraska) covered the shootings at Columbine High School. Notes that the staff localized the event and brought the student body into the story. (RS)

  11. Stereotype locally convex spaces

    Energy Technology Data Exchange (ETDEWEB)

    Akbarov, S S

    2000-08-31

    We give complete proofs of some previously announced results in the theory of stereotype (that is, reflexive in the sense of Pontryagin duality) locally convex spaces. These spaces have important applications in topological algebra and functional analysis.

  12. Stereotype locally convex spaces

    Science.gov (United States)

    Akbarov, S. S.

    2000-08-01

    We give complete proofs of some previously announced results in the theory of stereotype (that is, reflexive in the sense of Pontryagin duality) locally convex spaces. These spaces have important applications in topological algebra and functional analysis.

  13. rural y conocimiento local

    Directory of Open Access Journals (Sweden)

    Teresa Acampora

    2007-01-01

    Full Text Available En el contexto de la crisis de los modelos alimentarios en el siglo XXI, las producciones agroalimentarias están en un período de redescubrimiento. Las autoras analizan las principales estrategias que tienen los actores locales para perseguir la valorización de los recursos locales con identidad territorial; para luego ahondar en las políticas de diferenciación de los productos con marcas colectivas certifi-cadas. Finalmente, se ponen en evidencia las relaciones entre productos típicos, conservación de la agrobiodiversidad y conocimientos locales. Aquí se articula el debate sobre valorización de los productos típicos con el debate internacional sobre el conocimiento tradicional y el folclor, que se ha generado a partir de la preocupación internacional sobre la erosión de la biodiversidad y los conocimientos locales.

  14. Characterizing local EMS systems.

    Science.gov (United States)

    2013-08-01

    Emergency medical services (EMS) systems are configured differently depending on several factors, including the size, demographics, geography, and politics of the local communities they serve. Although some information exists about the organization, ...

  15. nuevo gobierno local

    Directory of Open Access Journals (Sweden)

    José Vargas Hernández

    2005-01-01

    Full Text Available Este trabajo tiene como objetivo analizar las funciones y estructura de ingeniería social del nuevo gobierno local para dar respuesta a las demandas sociales, promover el desarrollo y regular los nuevos servicios públicos. A partir de una delimitación de los conceptos de soberanía y autonomía del nuevo gobierno local, se reconfigura una nueva estructura de gobernabilidad de los gobiernos locales que implica una mayor participación ciudadana en los procesos de toma de decisiones y la formulación de las políticas públicas, mediante la facilitación de procesos de descentralización de funciones y mecanismos coordinadores de servicios públicos. Finalmente se analizan las implicaciones en la gobernabilidad de la ingeniería social del nuevo gobierno local.

  16. Local Authority Compensatory Policy

    NARCIS (Netherlands)

    M.C.M. Turkenburg

    1999-01-01

    Original title: Gemeentelijke onderwijsachterstandenbeleid. The introduction in the Netherlands of the Municipal Compensatory Policy (Education) Act on 1 August 1998 made the former educational priority policy a responsibility of local authorities. The intention is that the educational

  17. RNA Localization in Astrocytes

    DEFF Research Database (Denmark)

    Thomsen, Rune

    2012-01-01

    Messenger RNA (mRNA) localization is a mechanism by which polarized cells can regulate protein synthesis to specific subcellular compartments in a spatial and temporal manner, and plays a pivotal role in multiple physiological processes from embryonic development to cell differentiation......, regulation of the blood brain barrier and glial scar tissue formation. Despite the involvement in various CNS functions only a limited number of studies have addressed mRNA localization in astrocytes. This PhD project was initially focused on developing and implementing methods that could be used to asses mRNA...... localization in astrocyte protrusions, and following look into the subcellular localization pattern of specific mRNA species of both primary astrocytes isolated from cortical hemispheres of newborn mice, and the mouse astrocyte cell line, C8S. The Boyden chamber cell fractionation assay was optimized, in a way...

  18. Mining local process models

    OpenAIRE

    Tax, Niek; Sidorova, Natalia; Haakma, Reinder; van der Aalst, Wil M.P.

    2016-01-01

    In this paper we describe a method to discover frequent behavioral patterns in event logs. We express these patterns as \\emph{local process models}. Local process model mining can be positioned in-between process discovery and episode / sequential pattern mining. The technique presented in this paper is able to learn behavioral patterns involving sequential composition, concurrency, choice and loop, like in process mining. However, we do not look at start-to-end models, which distinguishes ou...

  19. On Minds' Localization

    OpenAIRE

    Crocco, Prof M

    2004-01-01

    A confluence of clues from a range of academic topics suggests that minds localization in nature consists of relativistically moving microphysical particles, whose motion is physiologically modulated. Here those clues are shown to imply that the localization of the operations of observers (minds or existentialities) in nature are the actions carriers of a force field, which action carriers are slightly slowed from near-c speed motion by electroneurobiological variations in brain physiology – ...

  20. Local Authority Compensatory Policy

    OpenAIRE

    M.C.M. Turkenburg

    1999-01-01

    Original title: Gemeentelijke onderwijsachterstandenbeleid. The introduction in the Netherlands of the Municipal Compensatory Policy (Education) Act on 1 August 1998 made the former educational priority policy a responsibility of local authorities. The intention is that the educational compensatory policy should be developed and implemented at local level in the coming years in collaboration with school boards. The predominant argument for the decentralisation of the educational compensatory ...

  1. Desempleo y justicia local

    Directory of Open Access Journals (Sweden)

    Elster, Jon

    2003-04-01

    Full Text Available In this paper Jon Elster presents a set of criteria of local justice that regulate dismissals. In situations where work is limited and companies adopt means of employment redundancy, the decision of dismissing certain workers —when this one tries not to be arbitrary— can be based on local criteria of fairness like merit, efficiency, age or other analysed in this work. So, after defining the concept of local justice and presenting some examples, the paper focuses on the importance of the work to organize and structure the life of people. Unemployment has serious consequences for the persons who suffer it and the author advises that it is necessary to subordinate dismissals to criteria of local justice.

    En este artículo se presentan una serie de criterios de justicia local para regular los despidos. En situaciones en las que el trabajo es escaso y las empresas adoptan medidas de regulación de empleo, la decisión de despedir a unos trabajadores o a otros se puede basar, cuando se intenta que no sea arbitraria, en criterios locales de equidad como el mérito, la eficacia, la edad u otros que se analizan en este trabajo. Así, tras definir el concepto de justicia local y presentar algunos ejemplos, el artículo se centra en la importancia del trabajo para organizar y estructurar la vida de la gente. Las graves consecuencias que tiene, por ello, el desempleo para quien lo sufre, aconseja que los despidos se sometan a criterios de justicia local.

  2. Locally Weighted Naive Bayes

    OpenAIRE

    Frank, Eibe; Hall, Mark; Pfahringer, Bernhard

    2012-01-01

    Despite its simplicity, the naive Bayes classifier has surprised machine learning researchers by exhibiting good performance on a variety of learning problems. Encouraged by these results, researchers have looked to overcome naive Bayes primary weakness - attribute independence - and improve the performance of the algorithm. This paper presents a locally weighted version of naive Bayes that relaxes the independence assumption by learning local models at prediction time. Experimental results s...

  3. TOURISM AND LOCAL DEVELOPMENT

    Directory of Open Access Journals (Sweden)

    BORMA AFRODITA

    2015-04-01

    Full Text Available The paper presents a theoretical approach of the relation between tourism and local development. The research method used is based on documenting and summarizing aspects related to the importance of tourism on local development. According to data provided by the World Tourism Organization (WTO, 2014, compared to 2010, a 91% increase of the number of international tourist arrivals is forecasted in 2030, from 948 million arrivals (2010 to 1,810 million arrivals (2030. Therefore, it is no surprise that tourism is viewed as a development strategy in many national economies. Tourism literature captures a series of arguments that emphasize the contribution of tourism to local development by: creating new jobs, revitalizing cultural traditions, improving infrastructure, environmental protection, etc. For Romania, as well, it may be noted that tourism strategies included in the 2013-2016 Government Programme are oriented towards local tourism by: decentralizing the authorization and approval activity, developing an integrated tourism by capitalizing all the local resources (cultural, agricultural, handicraft, etc., connecting Romanian tourism to international trends, etc. In order to achieve the aforementioned objectives, the 2013-2016 Government Program provides the clarification on the issue of decentralization, since the current government believes that a balanced development of Romania can only be achieved through transfer of authority to the local, county and regional levels.

  4. Local Lorentz covariance in finite-dimensional local quantum physics

    Science.gov (United States)

    Raasakka, Matti

    2017-10-01

    We show that local Lorentz covariance arises canonically as the group of transformations between local thermal states in the framework of local quantum physics, given the following three postulates: (i) Local observable algebras are finite-dimensional. (ii) Minimal local observable algebras are isomorphic to M2(C ) , the observable algebra of a single qubit. (iii) The vacuum restricted to any minimal local observable algebra is a nonmaximally mixed thermal state. The derivation reveals a new and surprising relation between spacetime structure and local quantum states. In particular, we show how local restrictions of the vacuum can determine the connection between different local inertial reference frames.

  5. A novel human TPIP splice-variant (TPIP-C2 mRNA, expressed in human and mouse tissues, strongly inhibits cell growth in HeLa cells.

    Directory of Open Access Journals (Sweden)

    Rasmi Rekha Mishra

    Full Text Available Alternative splicing of mRNAs is known to involve a major regulation of gene expression at RNA level in mammalian cells. The PTEN (Phosphatase and TENsin homologue deleted from the human chromosome 10, TPTE (Transmembrane Phosphatase with TEnsin homology and TPIP (TPTE and PTEN homologous Inositol lipid Phosphatase belong to a family of dual-specific lipid and protein phosphatases. PTEN is a well characterized tumor suppressor, which plays crucial role in cell survival, cell cycle regulation, cell proliferation as well as adhesion, motility and migration of cells. The C2-domain of PTEN is essential for PTEN-functions. We have isolated a novel 1019 bp human TPIP cDNA (TPIP-C2 from a human testis cDNA library. In silico analysis of the cDNA revealed that it is produced from the TPIP-locus on the human chromosome 13 by alternative RNA-splicing. It has a unique 5'-Alu sequence, a LINE sequence followed by a 582 bp Open Reading Frame (ORF encoding a 193 aa polypeptide with a partial phosphatase domain and a C2-domain. TPIP-C2 mRNA is expressed in human testis and in mouse tissues. Mouse testis and brain showed higher levels of TPIP-C2 mRNA in comparison to the heart, liver and kidney under normal physiological conditions. TPIP-C2 mRNAs from human and mouse testes show extensive sequence identity. Over-expression of TPIP-C2 cDNA in HeLa cells strongly (up to 85% inhibited cell growth/proliferation and caused apoptosis in a caspase 3-dependent manner. These findings suggest for the first time that a TPIP splice-variant mRNA with a partial phosphatase domain and a C2-domain is expressed in cells and tissues of human and murine origins under normal physiological conditions. Inhibition of cell growth/proliferation and induction of apoptosis by overexpression of TPIP-C2 mRNA in HeLa cells suggest that it may be involved in negative regulation of cell growth/proliferation.

  6. Bypass of a site-specific cis-Syn thymine dimer in an SV40 vector during in vitro replication by HeLa and XPV cell-free extracts.

    Science.gov (United States)

    Ensch-Simon, I; Burgers, P M; Taylor, J S

    1998-06-02

    The key step in skin cancer induction by UV light is thought to be the mutagenic DNA synthesis past a DNA photoproduct in a proto-oncogene or tumor suppressor gene. To investigate this critical step, we have constructed an SV40 vector containing a cis-syn thymine dimer, the major DNA photoproduct induced by UVB light, within an AseI site at a location that would initially be replicated by leading strand synthesis. When the dimer-containing SV40 vector was incubated with cell-free HeLa extracts in the presence of TAg, and then digested with AseI, a 2325 bp fragment corresponding to inhibition of cleavage at the dimer site was observed, suggesting that the dimer had terminated synthesis and/or had been bypassed. When the reaction was limited to one round of replication and the products of restriction enzyme digestion were examined by denaturing gel electrophoresis, bands corresponding to both termination and bypass were observed in roughly a one-to-one ratio. Whereas increasing the dNTP concentration from 10 microM to 1 mM increased the ratio of bypass to termination from 0.6 to 2.6, it had no effect on the site of termination, which occurred exclusively one nucleotide before the dimer. Experiments in which dGTP was held constant at 25 microM and various combinations of the remaining nucleotides were raised from 25 microM to 1 mM showed substantial increases in the bypass-to-termination ratio, with the greatest effect seen for raising all three nucleotides to 1 mM. Replication by primary fibroblast XPV extracts was also investigated and found to be greatly stimulated by rhRPA, whereas the stimulatory effect for HeLa cell extracts was variable. In the presence of rhRPA, the XPV extracts were also found to bypass the cis-syn dimer, which contrasts with a recent report that could not detect dimer bypass in SV40 transformed XPV extracts in the absence of added replication factors [Cordeiro-Stone, M., et al. (1997) J. Biol. Chem. 272, 13945-13954].

  7. Beyond local climate

    DEFF Research Database (Denmark)

    D'haen, Sarah Ann Lise; Nielsen, Jonas Østergaard; Lambin, Eric F.

    2014-01-01

    as a safety net against adverse local rainfall events. Results show that household decisions to participate in the nonfarm economy could not be directly linked with local rainfall events during the study period in the mid-2000s. However, household participation was determined by adverse rainfall conditions......At the household level, nonfarm activities are thought to help rural poor households buffer against agricultural risks related to local climate variability by providing them with cash to buy food in the case of harvest shortfalls. Over the recent decades, households in rural Sub-Sahara have been...... found less dependent on land and subsistence agriculture and an increasing number of households here derive their income from nonfarm activities. This study tests the hypothesis that rural households in Burkina Faso have diversified to the extent that they no longer rely on nonfarm activities...

  8. Tackling Health Inequalities Locally

    DEFF Research Database (Denmark)

    Diderichsen, Finn; Scheele, Christian Elling; Little, Ingvild Gundersen

    equality and growth (64). Health equity has been anexplicit political goal in Scandinavia for decades. Nevertheless, in the health domain, average improvement has not been followed by reduced inequality – at least not between socioeconomic groups. It has in other words turned out to be a challenge...... of translating small inequalities in wealth into small inequalities in health. Denmark, Norway and Sweden all have legislation that indifferent ways offers local governments key roles in public health. This is partly due to local governments’ responsibility for many policy areas of great relevance to health...... and health equity. National governments have thus largely made the WHO and EU recommendation of ‘Health in All Policies’a local responsibility. In his analysis for the Nordic Council of Ministers, former Swedish Minister of Health Bo Könberg identifies tackling health inequalities as one of 14 prioritised...

  9. Development of Local Chicken Production Based on Local Feed Ingredients

    Directory of Open Access Journals (Sweden)

    Cecep Hidayat

    2012-06-01

    Full Text Available Development of local chicken production based on local feed ingredient is in line with the vision of Indonesian goverment to fulfill meat and egg national requirement based on local resources. There are two big problem which become stumblingblock in developing local chicken production. The first problem is the difficulty to get day old chick of local chicken. This problem can be solved by integrating breeder institutions belong to goverment with research institution and with local chicken producer association. The second problem is the low performance of local chicken. To improve local chicken performance, it can be done by improving the breed, feed and management. Several research results show that good performance of local chicken were obtained by inclusion of local feed ingredients in the ration. Therefore, development of local chicken production based an local feed resources can be applied.

  10. Frequently Asked Questions (FAQs) About Local Foods, Local Places

    Science.gov (United States)

    Answers to frequently asked questions about EPA's Local Foods, Local Places planning assistance program to help communities revitalize downtowns, create economic opportunities, and improve access to healthy food by promoting local foods.

  11. Photon Localization Revisited

    Directory of Open Access Journals (Sweden)

    Izumi Ojima

    2015-09-01

    Full Text Available In the light of the Newton–Wigner–Wightman theorem of localizability question, we have proposed before a typical generation mechanism of effective mass for photons to be localized in the form of polaritons owing to photon-media interactions. In this paper, the general essence of this example model is extracted in such a form as quantum field ontology associated with the eventualization principle, which enables us to explain the mutual relations, back and forth, between quantum fields and various forms of particles in the localized form of the former.

  12. Local versus national

    DEFF Research Database (Denmark)

    Milan, Christian; Nielsen, Mads Pagh; Bojesen, Carsten

    2014-01-01

    of individual energy supply systems based on on-site weather and building conditions, as well as considering the expected energy consumption profile. However, local planning processes are problematic if they do not take regional or national impacts into account. Given the grid connection, the local building...... is to adapt the earlier proposed methodology by integrating flexible national electricity prices and thus taking account for the aforementioned effects. The methodology is applied in a case study for a single family house under Danish conditions. The results show that the system configuration might...

  13. Passive versus active local microrheology in mammalian cells and amoebae

    Science.gov (United States)

    Riviere, C.; Gazeau, F.; Marion, S.; Bacri, J.-C.; Wilhelm, C.

    2004-12-01

    We compare in this paper the rotational magnetic microrheology detailed by Marion et al [18] and Wilhelm et al [19] to the passive tracking microrheology. The rotational microrheology has been designed to explore, using magnetic rotating probes, the local intracellular microenvironment of living cells in terms of viscoelasticity. Passive microrheology techniques is based on the analysis of spontaneous diffusive motions of Brownian probes. The dependence of mean square displacement (MSD) with the time then directly reflects the type of movement (sub-, hyper- or diffusive motions). Using the same intracellular probes, we performed two types of measurements (active and passive). Based on the fluctuation-dissipation theorem, one should obtain the same information from the both techniques in a thermally equilibrium system. Interestingly, our measurements differ, and the discordances directly inform on active biological processes, which add to thermally activated fluctuations in our out-of equilibrium systems. In both cell models used, mammalian Hela cells and amoebae Entamoeba Histolytica, a hyper-diffusive regime at a short time is observed, which highlights the presence of an active non-thermal driving force, acting on the probe. However, the nature of this active force in mammalian cells and amoebae is different, according to their different phenotypes. In mammalian cells active processes are governed by the transport, via molecular motors, on the microtubule network. In amoebae, which are highly motile cells free of microtubule network, the active processes are dominated by strong fluxes of cytoplasm driven by extension of pseudopodia, in random directions, leading to an amplitude of motion one order of magnitude higher than for mammalian cells. Figs 7, Refs 32.

  14. Rev1, Rev3, or Rev7 siRNA Abolishes Ultraviolet Light-Induced Translesion Replication in HeLa Cells: A Comprehensive Study Using Alkaline Sucrose Density Gradient Sedimentation

    Science.gov (United States)

    Takezawa, Jun; Ishimi, Yukio; Aiba, Naomi; Yamada, Kouichi

    2010-01-01

    When a replicative DNA polymerase stalls upon encountering a lesion on the template strand, it is relieved by other low-processivity polymerase(s), which insert nucleotide(s) opposite the lesion, extend by a few nucleotides, and dissociate from the 3′-OH. The replicative polymerase then resumes DNA synthesis. This process, termed translesion replication (TLS) or replicative bypass, may involve at least five different polymerases in mammals, although the participating polymerases and their roles have not been entirely characterized. Using siRNAs originally designed and an alkaline sucrose density gradient sedimentation technique, we verified the involvement of several polymerases in ultraviolet (UV) light-induced TLS in HeLa cells. First, siRNAs to Rev3 or Rev7 largely abolished UV-TLS, suggesting that these 2 gene products, which comprise Polζ, play a main role in mutagenic TLS. Second, Rev1-targeted siRNA also abrogated UV-TLS, indicating that Rev1 is also indispensable to mutagenic TLS. Third, Polη-targeted siRNA also prevented TLS to a greater extent than our expectations. Forth, although siRNA to Polι had no detectable effect, that to Polκ delayed UV-TLS. To our knowledge, this is the first study reporting apparent evidence for the participation of Polκ in UV-TLS. PMID:21151666

  15. New Sesquiterpene Lactone Dimer, Uvedafolin, Extracted from Eight Yacon Leaf Varieties (Smallanthus sonchifolius): Cytotoxicity in HeLa, HL-60, and Murine B16-F10 Melanoma Cell Lines.

    Science.gov (United States)

    Kitai, Yurika; Hayashi, Kana; Otsuka, Moe; Nishiwaki, Hisashi; Senoo, Tatsuya; Ishii, Tomohiko; Sakane, Genta; Sugiura, Makoto; Tamura, Hirotoshi

    2015-12-23

    Uvedafolin, 1, a new sesquiterpene lactone dimer, was isolated from the leaves of Smallanthus sonchifolius with five related compounds, 2-6, and their cytotoxicity was assessed against three tumor cell lines (HeLa, HL-60, B16-F10 melanoma). The stereostructure of 1 was newly elucidated by ESI-TOF-MS, 1D/2D NMR, and single-crystal X-ray diffraction. Dimers 1 and 2 had the most effective IC50 values, 0.2-1.9 μM, against the three tumor cell lines when compared with monomers 3-6 (IC50 values 0.7-9.9 μM) and etoposide (IC50 values 0.8-114 μM). The ester linkages of two sets of monomers, uvedalin, 5, and sonchifolin, 6, for 1, and enhydrin, 4, and sonchifolin, 6, for 2, as well as the acetyl group at the C-9 position, were essential for the high cytotoxicity. Dimers 1 and 2 would have potential as anticancer agents.

  16. Traditional Chinese medicine herbal mixture LQ arrests FUCCI-expressing HeLa cells in G₀/G₁ phase in 2D plastic, 2.5D Matrigel, and 3D Gelfoam culture visualized with FUCCI imaging.

    Science.gov (United States)

    Zhang, Lei; Wu, Chengyu; Bouvet, Michael; Yano, Shuya; Hoffman, Robert M

    2015-03-10

    We used the fluorescence ubiquitination-based cell cycle indicator (FUCCI) to monitor cell cycle arrest after treatment of FUCCI-expressing HeLa cells (FUCCI-HeLa) with a traditional Chinese medicine (TCM) herbal mixture LQ, previously shown to have anti-tumor and anti-metastatic activity in mouse models. Paclitaxel was used as the positive control. In 2D monolayer culture, the untreated control had approximately 45% of the cells in S/G₂/M phase. In contrast, the LQ-treated cells (9 mg/ml) were mostly in the G₀/G₁ (>90%) after 72 hours. After treatment with paclitaxel (0.01 μm), for 72 hours, 95% of the cells were in S/G₂/M. In 2.5D Matrigel culture, the colonies in the untreated control group had 40% of the cells in S/G₂/M. LQ arrested the cells in G₀/G₁ after 72 hours. Paclitaxel arrested almost all the cells in S/G₂/M after 72 hours. In 3D Gelfoam culture, the untreated control culture had approximately 45% of cells in G₂/M. In contrast, the LQ-treated cells were mostly in G₀/G₁ phase (>80%) after 72 hours treatment. Paclitaxel resulted in 90% of the cells arrested in S/G₂/M after 72 hours. The present report suggests the non-toxic LQ has potential to maintain cancers in a quiescent state for long periods of time.

  17. Coexpression of PPE 34.9 Antigen of Mycobacterium avium subsp. Paratuberculosis with Murine Interferon Gamma in HeLa Cell Line and Study of Their Immunogenicity in Murine Model

    Directory of Open Access Journals (Sweden)

    Rajib Deb

    2011-01-01

    Full Text Available Mycobacterium avium subsp. paratuberculosis (Map is the causative agent of johne's disease whose immunopathology mainly depends on cell mediated immuneresponse. Genome sequencing revealed various PPE (Proline-Proline-Glutamic acid protein family of Map which are immunologically importance candidate genes In present study we have developed a bicistrionic construct pIR PPE/IFN containing a 34.9 kDa PPE protein (PPE 34.9 of Map along with a cytokine gene encoding murine gamma Interferon gene (IFNγ and a monocistrionic construct pIR PPE using a mammalian vector system pIRES 6.1. The construct were transfected in HeLa cell line and expression were studied by Western blot as well as Immunefluroscent assay using recombinant sera. Further we have compared the immunereactivity of these two constructs in murine model by means of DTH study, LTT, NO assay and ELISA. DTH response was higher in pIR PPE/IFN than pIR PPE group of mice, similar finding also observed in case of LTT and NO production assay . ELISA titer of the pIR PPE/IFN was less than that with PPE only. These preliminary finding can revealed a CMI response of this PPE protein of Map and IFNγ having synergistic effect on this PPE protein to elicit a T cell based immunity in mice.

  18. In vitro excision of adeno-associated virus DNA from recombinant plasmids: Isolation of an enzyme fraction from HeLa cells that cleaves DNA at poly(G) sequences

    Energy Technology Data Exchange (ETDEWEB)

    Gottlieb, J.; Muzyczka, N.

    1988-06-01

    When circular recombinant plasmids containing adeno-associated virus (AAV) DNA sequences are transfected into human cells, the AAV provirus is rescued. Using these circular AAV plasmids as substrates, the authors isolated an enzyme fraction from HeLa cell nuclear extracts that excises intact AAV DNA in vitro from vector DNA and produces linear DNA products. The recognition signal for the enzyme is a polypurine-polypyrimidine sequence which is at least 9 residues long and rich in G . C base pairs. Such sequences are present in AAV recombinant plasmids as part of the first 15 base pairs of the AAV terminal repeat and in some cases as the result of cloning the AAV genome by G . C tailing. The isolated enzyme fraction does not have significant endonucleolytic activity on single-stranded or double-stranded DNA. Plasmid DNA that is transfected into tissue culture cells is cleaved in vivo to produce a pattern of DNA fragments similar to that seen with purified enzyme in vitro. The activity has been called endo R for rescue, and its behavior suggests that it may have a role in recombination of cellular chromosomes.

  19. Rev1, Rev3, or Rev7 siRNA Abolishes Ultraviolet Light-Induced Translesion Replication in HeLa Cells: A Comprehensive Study Using Alkaline Sucrose Density Gradient Sedimentation

    Directory of Open Access Journals (Sweden)

    Jun Takezawa

    2010-01-01

    Full Text Available When a replicative DNA polymerase stalls upon encountering a lesion on the template strand, it is relieved by other low-processivity polymerase(s, which insert nucleotide(s opposite the lesion, extend by a few nucleotides, and dissociate from the 3′-OH. The replicative polymerase then resumes DNA synthesis. This process, termed translesion replication (TLS or replicative bypass, may involve at least five different polymerases in mammals, although the participating polymerases and their roles have not been entirely characterized. Using siRNAs originally designed and an alkaline sucrose density gradient sedimentation technique, we verified the involvement of several polymerases in ultraviolet (UV light-induced TLS in HeLa cells. First, siRNAs to Rev3 or Rev7 largely abolished UV-TLS, suggesting that these 2 gene products, which comprise Polζ, play a main role in mutagenic TLS. Second, Rev1-targeted siRNA also abrogated UV-TLS, indicating that Rev1 is also indispensable to mutagenic TLS. Third, Polη-targeted siRNA also prevented TLS to a greater extent than our expectations. Forth, although siRNA to Polι had no detectable effect, that to Polκ delayed UV-TLS. To our knowledge, this is the first study reporting apparent evidence for the participation of Polκ in UV-TLS.

  20. Global and Local Textuality.

    Science.gov (United States)

    Campbell, B. G.

    Coherence and cohesion are fundamental considerations of the composing process that help to define the global and local components of texuality. Global text coherence centers on those aspects of the familiar rhetorical situation. Coherence operates at the paragraph and essay levels, answering questions about focus, tone, mode, topic, and thesis.…

  1. Local Color Correction

    Directory of Open Access Journals (Sweden)

    Juan Gabriel Gomila Salas

    2011-09-01

    Full Text Available In this paper we present a local algorithm for contrast enhancement developed by N. Moroney at Hewlett-Packard Laboratories and presented at the IS&T/SID Eight Color Imaging Conference, in 2000. The algorithm uses a non-linear masking, is fast and does not require any manual parameter adjustments.

  2. Local anaesthetic toxicity

    African Journals Online (AJOL)

    The Austrian pharmacologist Karl Damian Ritter von Schorff was the first to think of cocaine as a narcotic. He noticed skin insensibility after the application of cocaine and postulated a central nervous system effect as mechanism of action. In 1884,. Sigmund Freud proposed the use of cocaine as local anaesthetic to his friend ...

  3. Robust and Secure Localization

    Science.gov (United States)

    2009-06-03

    attacker-chosen location to the nodes as they are deployed.  establish wormhole links and tunnel legitimate messages to distant locations in the...network which are beyond the radio range of the source nodes. The wormhole attack can potentially introduce significant localization error of any node

  4. Local government in Ethiopia

    African Journals Online (AJOL)

    LAW

    farmers would find themselves being rounded up at gun point by the local defence squad, and forcibly dispatched...”69. The involvement of UDAs and PAs in the forceful conscription of young people into the government's army was another control oriented action. Derg was forcefully conscripting young Ethiopians for the ...

  5. Stroke? Localized, otogenic meningitis!

    DEFF Research Database (Denmark)

    Ingolfsdottir, Harpa Maria; Thomasen, Per Caye

    2011-01-01

    We report the case of a patient admitted with aphasia, treated for a stroke. Subsequently, it was revealed that the symptoms were caused by complicated otitis media with localized meningitis. This case draws attention to the possible intracranial spread of infection when neurological symptoms occur...

  6. HOW PARTICIPATION SHAPES LOCAL DEVELOPMENT

    Directory of Open Access Journals (Sweden)

    Muhadjir Darwin

    2016-12-01

    Full Text Available This article elaborates citizens’ participation in shaping local development in the Indonesian context. The main question is how to make local development more participatory as a guarantee that citizens’ rights are fully realized. In that respects, social accountability is a key that should be improved from supply and demand sides simultaneously. From the supply side, there is a need to reform local governance, in order to improve the quality of local regulations, local planning, local budgeting, and local public service provision. While from the demand side, there is a need to empower local citizens so that they are engaged in five forms or local development efforts: local regulation formulation, local development planning, local budgeting, community driven development, and public service provision.

  7. Electron localization functions and local measures of the covariance

    Indian Academy of Sciences (India)

    The electron localization measure proposed by Becke and Edgecombe is shown to be related to the covariance of the electron pair distribution. Just as with the electron localization function, the local covariance does not seem to be, in and of itself, a useful quantity for elucidating shell structure. A function of the local ...

  8. Brane-Localized Gravity

    CERN Document Server

    Mannheim, Philip D

    2005-01-01

    This timely and valuable book provides a detailed pedagogical introduction and treatment of the brane-localized gravity program of Randall and Sundrum, in which gravitational signals are able to localize around our four-dimensional world in the event that it is a brane embedded in an infinitely-sized, higher dimensional anti-de Sitter bulk space. A completely self-contained development of the material needed for brane-world studies is provided for both students and workers in the field, with a significant amount of the material being previously unpublished. Particular attention is given to issues not ordinarily treated in the brane-world literature, such as the completeness of tensor gravitational fluctuation modes, the causality of brane-world propagators, and the status of the massless graviton fluctuation mode in brane worlds in which it is not normalizable.

  9. Photonic local oscillator development

    Science.gov (United States)

    Kimberk, Robert; Tong, Edward; Hunter, Todd R.; Christensen, Robert; Blundell, Ray

    2006-12-01

    In the receiver lab, we have developed a 200 GHz to 230 GHz local oscillator constructed from mostly commercially available 1550 nm laser communication components. Theoretical and experimental work show that the laser adds negligible phase noise to this photonic local oscillator system and that spectral purity and phase stability are similar to Gunn oscillator based local oscillator output. The optical path consists of a single 1550 nm diode laser, a lithium niobate optical phase modulator, a Mach Zehnder interferometer (MZI) with a free spectral range of 75 GHz, and a 160 GHz to 260 GHz photomixer whose output is connected to a horn antenna. All of the optical devices and connections are polarization maintaining, and the photomixer was designed and fabricated at the CCLRC Rutherford Appleton Laboratory. The electrical path consists of a YIG synthesizer, operating in the frequency range 14-20 GHz, a frequency doubler, and a power amplifier connected to the RF port of the phase modulator. At the SMA on Mauna Kea, we incorporated the photonic LO into one element (Antenna 6) of a five antenna array for test observations of CO J=2-1 made towards the ultracompact HII region G138.295+1.555. Spectral features of comparable width occur on baselines with and without antenna 6, and noise increases with baseline length independent of antenna number. Continuum observations were also made toward the quasar 3c454.3 for a period of about one hour. In summary, the SMA has proven that the photonic local oscillator operates with adequate phase and frequency stability for radio-interferometry.

  10. Rationalising localism and brokerage

    DEFF Research Database (Denmark)

    Kusche, Isabel

    2017-01-01

    . The article employs the notion of global scripts and the concept of domestication of global trends in order to analyse how the question of the appropriate relationship between deputies and voters is informed both by a general trend towards the professionalisation of politicians and a domestic tradition...... of the domestic setting on the other. In this way the article contributes to a better understanding of the entanglement of the global and the local....

  11. Local Rotational Symmetries.

    Science.gov (United States)

    1985-08-01

    way to choose among them. Spirals can occur in natural figures, e.g. a spiralled tail or a coil of rope or vine tendril, and in line drawings. Since...generated and removes it and all regions similar to it from the list of regions. The end result is a pruned list of distinct optimal regions. 4.7...that, at least to a first approximation, the potential symmetry regions pruned by the locality restriction are not perceptually salient. For example

  12. Local Noncollinear Spin Analysis.

    Science.gov (United States)

    Abate, Bayileyegn A; Joshi, Rajendra P; Peralta, Juan E

    2017-12-12

    In this work, we generalize the local spin analysis of Clark and Davidson [J. Chem. Phys. 2001 115 (16), 7382] for the partitioning of the expectation value of the molecular spin square operator, ⟨Ŝ 2 ⟩, into atomic contributions, ⟨Ŝ A ·Ŝ B ⟩, to the noncollinear spin case in the framework of density functional theory (DFT). We derive the working equations, and we show applications to the analysis of the noncollinear spin solutions of typical spin-frustrated systems and to the calculation of magnetic exchange couplings. In the former case, we employ the triangular H 3 He 3 test molecule and a Mn 3 complex to show that the local spin analysis provides additional information that complements the standard one-particle spin population analysis. For the calculation of magnetic exchange couplings, J AB , we employ the local spin partitioning to extract ⟨Ŝ A ·Ŝ B ⟩ as a function of the interatomic spin orientation given by the angle θ. This, combined with the dependence of the electronic energy with θ, provides a methodology to extract J AB from DFT calculations that, in contrast to conventional energy differences based methods, does not require the use of ad hoc S A and S B values.

  13. Many-box locality

    Science.gov (United States)

    Zhou, Yuqian; Cai, Yu; Bancal, Jean-Daniel; Gao, Fei; Scarani, Valerio

    2017-11-01

    There is an ongoing search for a physical or operational definition for quantum mechanics. Several informational principles have been proposed which are satisfied by a theory less restrictive than quantum mechanics. Here, we introduce the principle of "many-box locality," which is a refined version of the previously proposed "macroscopic locality." These principles are based on coarse graining the statistics of several copies of a given box. The set of behaviors satisfying many-box locality for N boxes is denoted LNM B. We study these sets in the bipartite scenario with two binary measurements, in relation with the sets Q and Q1 +A B of quantum and "almost quantum" correlations, respectively. We find that the LNM B sets are, in general, not convex. For unbiased marginals, by working in the Fourier space we can prove analytically that LNM B⊈Q for any finite N , while L∞M B=Q . Then, with suitably developed numerical tools, we find an example of a point that belongs to L16M B but not to Q1 +A B. Among the problems that remain open is whether Q ⊂L∞M B .

  14. Local dependency in networks

    Directory of Open Access Journals (Sweden)

    Kudĕlka Miloš

    2015-06-01

    Full Text Available Many real world data and processes have a network structure and can usefully be represented as graphs. Network analysis focuses on the relations among the nodes exploring the properties of each network. We introduce a method for measuring the strength of the relationship between two nodes of a network and for their ranking. This method is applicable to all kinds of networks, including directed and weighted networks. The approach extracts dependency relations among the network’s nodes from the structure in local surroundings of individual nodes. For the tasks we deal with in this article, the key technical parameter is locality. Since only the surroundings of the examined nodes are used in computations, there is no need to analyze the entire network. This allows the application of our approach in the area of large-scale networks. We present several experiments using small networks as well as large-scale artificial and real world networks. The results of the experiments show high effectiveness due to the locality of our approach and also high quality node ranking comparable to PageRank.

  15. New Communication Technologies, Local Journalism and the Perception of Locality

    Directory of Open Access Journals (Sweden)

    Ali ÖZCAN

    2016-12-01

    Full Text Available Studies on local journalism as an important element of participatory democracy generally focus on the history of the local press, the financial insufficiencies of local newspapers as well as technical/ technological incompetency that occurs as a result of these factors. This research analyzes local newspapers with a new scientific point of view by focusing on the perception of locality as a mental projection of geographical constraint. Perspectives regarding the transformations on perceptions of locality as a result of the disappearance of time and space constraints due to new communication technologies, and the opportunities that new communication environments offer to local newspapers will be provided. Moreover, the ways in which local newspapers reflect on locality with their printed versions under time and space constraints will be analyzed. The research scope of the study has been limited to 15 newspapers in Konya. Structured interviews and content analysis methods were used for data collection and analysis.

  16. Localized defects in ophthalmic lenses.

    Science.gov (United States)

    Stroud, J S

    1989-03-01

    Ophthalmic lenses made from glass, hard resin, and polycarbonate are inspected with a shadowgraph to determine the nature, frequency, and severity of localized defects, including power aberrations. The most common localized defects are scratches and pits left by incomplete polishing and localized power aberrations caused by variations in the thickness of scratch-resistant coatings. Localized defects are more likely in polycarbonate lenses than in glass or hard resin lenses. The compliance of lenses with standards that limit localized defects is discussed.

  17. Synthesis, characterisation and in vitro investigation of photodynamic activity of 5-(4-octadecanamidophenyl)-10,15,20-tris(N-methylpyridinium-3-yl)porphyrin trichloride on HeLa cells using low light fluence rate.

    Science.gov (United States)

    Malatesti, Nela; Harej, Anja; Kraljević Pavelić, Sandra; Lončarić, Martin; Zorc, Hrvoje; Wittine, Karlo; Andjelkovic, Uros; Josic, Djuro

    2016-09-01

    Photodynamic therapy (PDT) is a treatment that aims to kill cancer cells by reactive oxygen species, mainly singlet oxygen, produced through light activation of a photosensitiser (PS). Amongst photosensitisers that attracted the most attention in the last decade are cationic and amphiphilic molecules based on porphyrin, chlorin and phthalocyanine structures. Our aim was to join this search for more optimal balance of the lipophilic and hydrophilic moieties in a PS. A new amphiphilic porphyrin, 5-(4-octadecanamidophenyl)-10,15,20-tris(N-methylpyridinium-3-yl)porphyrin trichloride (5) was synthesised and characterised by (1)H NMR, UV-vis and fluorescence spectroscopy, and by MALDI-TOF/TOF spectrometry. In vitro photodynamic activity of 5 was evaluated on HeLa cell lines and compared to the activity of the hydrophilic 5-(4-acetamidophenyl)-10,15,20-tris(N-methylpyridinium-3-yl)porphyrin trichloride (7). Low fluence rate (2mWcm(-2)) of red light (643nm) was used for the activation, and both porphyrins showed a drug dose-response as well as a light dose-response relationship, but the amphiphilic porphyrin was presented with significantly lower IC50 values. The obtained IC50 values for 5 were 1.4μM at 15min irradiation time and 0.7μM when the time of irradiation was 30min, while for 7 these values were 37 and 6 times higher, respectively. These results confirm the importance of the lipophilic component in a PS and show a potential for 5 to be used as a PS in PDT applications. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Terbium doped SnO2 nanoparticles as white emitters and SnO2:5Tb/Fe3O4 magnetic luminescent nanohybrids for hyperthermia application and biocompatibility with HeLa cancer cells.

    Science.gov (United States)

    Singh, Laishram Priyobarta; Singh, Ningthoujam Premananda; Srivastava, Sri Krishna

    2015-04-14

    SnO2:5Tb (SnO2 doped with 5 at% Tb(3+)) nanoparticles were synthesised by a polyol method and their luminescence properties at different annealing temperatures were studied. Characterization of nanomaterials was done by X-ray diffraction (XRD), Fourier transformation infrared spectroscopy (FTIR), transmission electron microscopy (TEM) and vibrating sample magnetometry (VSM). XRD studies indicate that the prepared nanoparticles were of tetragonal structures. Upon Tb(3+) ion incorporation into SnO2, Sn(4+) changes to Sn(2+) and, on annealing again at higher temperature, Sn(2+) changes to Sn(4+). The prepared nanoparticles were spherical in shape. Sn-O vibrations were found from the FTIR studies. In photoluminescence studies, the intensity of the emission peaks of Tb(3+) ions increases with the increase of annealing temperature, and emission spectra lie in the region of white emission in the CIE diagram. CCT calculations show that the SnO2:5Tb emission lies in cold white emission. Quantum yields up to 38% can be obtained for 900 °C annealed samples. SnO2:5Tb nanoparticles were well incorporated into the PVA polymer and such a material incorporated into the polymer can be used for display devices. The SnO2:5Tb/Fe3O4 nanohybrid was prepared and investigated for hyperthermia applications at different concentrations of the nanohybrid. This achieves a hyperthermia temperature (42 °C) under an AC magnetic field. The hybrid nanomaterial SnO2:5Tb/Fe3O4 was found to exhibit biocompatibility with HeLa cells (human cervical cancer cells) at concentrations up to 74% for 100 μg L(-1). Also, this nanohybrid shows green emission and thus it will be helpful in tracing magnetic nanoparticles through optical imaging in vivo and in vitro application.

  19. Localization of CANDU technology

    Energy Technology Data Exchange (ETDEWEB)

    Alizadeh, Ala

    2010-09-15

    The CANDU pressurized heavy water reactor's principal design features suit it particularly well for technology transfer and localization. When the first commercial CANDU reactors of 540 MWe entered service in 1971, Canada's population of less than 24 million supported a 'medium' level of industrial development, lacking the heavy industrial capabilities of larger countries like the USA, Japan and Europe. A key motivation for Canada in developing the CANDU design was to ensure that Canada would have the autonomous capacity to build and operate nuclear power reactors without depending on foreign sources for key components or enriched fuel.

  20. Local analytic geometry

    CERN Document Server

    Abhyankar, Shreeram Shankar

    1964-01-01

    This book provides, for use in a graduate course or for self-study by graduate students, a well-motivated treatment of several topics, especially the following: (1) algebraic treatment of several complex variables; (2) geometric approach to algebraic geometry via analytic sets; (3) survey of local algebra; (4) survey of sheaf theory. The book has been written in the spirit of Weierstrass. Power series play the dominant role. The treatment, being algebraic, is not restricted to complex numbers, but remains valid over any complete-valued field. This makes it applicable to situations arising from

  1. Localized Acoustic Surface Modes

    KAUST Repository

    Farhat, Mohamed

    2015-08-04

    We introduce the concept of localized acoustic surface modes (ASMs). We demonstrate that they are induced on a two-dimensional cylindrical rigid surface with subwavelength corrugations under excitation by an incident acoustic plane wave. Our results show that the corrugated rigid surface is acoustically equivalent to a cylindrical scatterer with uniform mass density that can be represented using a Drude-like model. This, indeed, suggests that plasmonic-like acoustic materials can be engineered with potential applications in various areas including sensing, imaging, and cloaking.

  2. Spatially localized magnetoconvection

    Energy Technology Data Exchange (ETDEWEB)

    Lo Jacono, D; Bergeon, A [Universite de Toulouse (France); INPT, UPS, Institut de Mecanique des Fluides de Toulouse (IMFT), Allee Camille Soula, F-31400 Toulouse (France); Knobloch, E, E-mail: lojacono@imft.fr, E-mail: bergeon@imft.fr, E-mail: knobloch@berkeley.edu [Department of Physics, University of California, Berkeley, CA 94720 (United States)

    2012-06-01

    Numerical continuation is used to compute branches of time-independent, spatially localized convectons in an imposed vertical magnetic field focusing on values of the Chandrasekhar number Q in the range 10 < Q < 10{sup 3}. The calculations reveal that convectons initially grow by nucleating additional cells on either side, but with the build-up of field outside owing to flux expulsion, the convectons are able to transport more heat only by expanding the constituent cells. Thus, at large Q and large Rayleigh numbers, convectons consist of a small number of broad cells. (paper)

  3. 'Joined Up' Local Governments?

    DEFF Research Database (Denmark)

    Bjørnå, Hilde; Casale, Donatella; Hajnal, Gyorgy

    of such studies and comparative studies are lacking. With this, we ask how can local JUG initiatives be explained and what lessons can be drawn from a comparative perspective? Drawing on some components from the framework by Pollitt and Bouckaert (2011) for public management reforms, we analyse JUG reforms in six....... forthc. 2015, Lægreid et al. 2014, Pollitt 2003). A growing body of research analyses JUG initiatives in several countries, but often in the form of single-case studies with central government focus (see e.g. Talbot 2011 or Lægreid et al. 2014). On municipal level, however, there is only a scarce number...

  4. The Local Fractional Bootstrap

    DEFF Research Database (Denmark)

    Bennedsen, Mikkel; Hounyo, Ulrich; Lunde, Asger

    new resampling method, the local fractional bootstrap, relies on simulating an auxiliary fractional Brownian motion that mimics the fine properties of high frequency differences of the Brownian semistationary process under the null hypothesis. We prove the first order validity of the bootstrap method...... and in simulations we observe that the bootstrap-based hypothesis test provides considerable finite-sample improvements over an existing test that is based on a central limit theorem. This is important when studying the roughness properties of time series data; we illustrate this by applying the bootstrap method...

  5. Changing local land systems

    DEFF Research Database (Denmark)

    Friis, Cecilie; Reenberg, Anette; Heinimann, Andreas

    2016-01-01

    This paper investigates the direct and cascading land system consequences of a Chinese company's land acquisition for rubber cultivation in northern Laos. Transnational land acquisitions are increasingly acknowledged as an important driver of direct land use conversion with implications for local...... land-based livelihoods. The paper presents an empirical case study of the village of Na Nhang Neua in Nambak District, Luang Prabang Province, using a mixed methods approach to investigate the positive and negative implications for household agricultural strategies, income generation and food security...

  6. Local Laplacian Coding From Theoretical Analysis of Local Coding Schemes for Locally Linear Classification.

    Science.gov (United States)

    Pang, Junbiao; Qin, Lei; Zhang, Chunjie; Zhang, Weigang; Huang, Qingming; Yin, Baocai

    2015-12-01

    Local coordinate coding (LCC) is a framework to approximate a Lipschitz smooth function by combining linear functions into a nonlinear one. For locally linear classification, LCC requires a coding scheme that heavily determines the nonlinear approximation ability, posing two main challenges: 1) the locality making faraway anchors have smaller influences on current data and 2) the flexibility balancing well between the reconstruction of current data and the locality. In this paper, we address the problem from the theoretical analysis of the simplest local coding schemes, i.e., local Gaussian coding and local student coding, and propose local Laplacian coding (LPC) to achieve the locality and the flexibility. We apply LPC into locally linear classifiers to solve diverse classification tasks. The comparable or exceeded performances of state-of-the-art methods demonstrate the effectiveness of the proposed method.

  7. Locality and reality

    Energy Technology Data Exchange (ETDEWEB)

    Stapp, Henry P.

    1980-10-01

    Einstein's principle that no signal travels faster than suggests that observations in one spacetime region should not depend on whether or not a radioactive decay is detected in a spacelike separated region. This locality property is incompatible with the predictions of quantum theory, and this incompatibility holds independently of the questions of realism, objective reality, and hidden variables. It holds both in the pragmatic quantum theory of Bohr and in realistic frameworks. It is shown here to hold in a completed realistic quantum theory that reconciles Einstein's demand for a description of reality itself with Bohr's contention that quantum theory is complete. This completed realistic quantum theory has no hidden variables, and no objective reality in which observable attributes can become definite independently of observers. The, theory is described in some detail, with particular attention to those aspects related to the question of locality. This completed realistic quantum theory is in principle more comprehensive than Bohr.' s pragmatic quantum theory because it is not limited in principle by the requirement that the observed system be physically separated from the observing one. Applications are discussed.

  8. Pattern Driven Stress Localization

    Science.gov (United States)

    Croll, Andrew; Crosby, Alfred

    2010-03-01

    The self-assembly of patterns from isotropic initial states is a major driver of modern soft-matter research. This avenue of study is directed by the desire to understand the complex physics of the varied structures found in Nature, and by technological interest in functional materials that may be derived through biomimicry. In this work we show how a simple striped phase can respond with significant complexity to an appropriately chosen perturbation. In particular, we show how a buckled elastic plate transitions into a state of stress localization using a simple, self-assembled variation in surface topography. The collection of topographic boundaries act in concert to change the state from isotropic sinusoidal wrinkles, to sharp folds or creases separated by relatively flat regions. By varying the size of the imposed topographic pattern or the wavelength of the wrinkles, we construct a state diagram of the system. The localized state has implications for both biological systems, and for the control of non-linear pattern formation.

  9. Local moduli and singularities

    CERN Document Server

    Laudal, Olav Arnfinn

    1988-01-01

    This research monograph sets out to study the notion of a local moduli suite of algebraic objects like e.g. schemes, singularities or Lie algebras and provides a framework for this. The basic idea is to work with the action of the kernel of the Kodaira-Spencer map, on the base space of a versal family. The main results are the existence, in a general context, of a local moduli suite in the category of algebraic spaces, and the proof that, generically, this moduli suite is the quotient of a canonical filtration of the base space of the versal family by the action of the Kodaira-Spencer kernel. Applied to the special case of quasihomogenous hypersurfaces, these ideas provide the framework for the proof of the existence of a coarse moduli scheme for plane curve singularities with fixed semigroup and minimal Tjurina number . An example shows that for arbitrary the corresponding moduli space is not, in general, a scheme. The book addresses mathematicians working on problems of moduli, in algebraic or in complex an...

  10. A digital local studies collection

    Directory of Open Access Journals (Sweden)

    Simona Resman

    2005-01-01

    Full Text Available Local studies and local studies departments reflect the entire spectrum of knowledge within the local community. Transition to the new digital environment means just continuing the basic functions of local studies collections: acquisition, cataloguing and preservation of materials in different formats on different media. The background of each digital library is the technical architecture of system that enables interaction between the user and the library, saving digital material and building a platform for searching and indexing digital objects. Using advanced ICT requests from librarians, designers of digital local studies collections a reflection about traditional roles. A digital local studies collection with new technology enlarges local boarders, local contents become more and more interesting for a wider sphere of people. In collecting of local materials a collaboration with archives and museums, with academic community, with community groups and with individuals comes in forefront. Digital local studies collections with their contents support local diversity, lifelong learning and social inclusion. The crucial elements of a digital local studies collection are attractive local contents with fast and simple access from one place – a portal. In the digital age public libraries become managers of knowledge also by establishing digital local studies collections.

  11. Local government reform: problematic aspects

    Directory of Open Access Journals (Sweden)

    Y. P. Sharov

    2014-06-01

    Full Text Available In the article the actual problems aspects of realization of reform of local self-government are worked up, namely: reformation of institute of local state administrations, creation of executive branches of local Rada, «technology» of realization of administrative-territorial device, place and role of strategic and project approaches, statutory and regulatory support of integrative forms of territorial communities activity, modern forms of professional communication of local government’s personal, using the potential of associations of local government’s practitioners; directions of development of human factor of reform and administrative system are analyzed in local self-government on principles of management achievements.

  12. Natural resources management by local associations in Ifedore local ...

    African Journals Online (AJOL)

    Management of natural resource by local associations not only in its socio ecological but also in its socio economic context will go a long way in reducing environmental degradation in some local communities. This study examined the operational capacity for natural resource management by local associations in Ifedore ...

  13. Local Agenda 21 - from global idea to local action

    DEFF Research Database (Denmark)

    Hoffmann, Birgitte

    1999-01-01

    The article give a status of the Danish works with Local Agenda 21 and discusses Local Agenda 21 as a planning tool. It describes the idea of Local Agenda 21 as a large meeting, which everybody attends. This picture is elaborated and discussed form different angles: the items on the agenda...

  14. Finance reform under local integration

    Directory of Open Access Journals (Sweden)

    S. S. Seryohin

    2016-06-01

    Full Text Available Abstract Revealed the essence of local finance manifested in economic relations, and their structure is a combination of local budgets, special purpose budget funds; budget funds of local governments; public funds of funds banks if their share capital formed at the expense of local governments; fund municipal enterprises, institutions and organizations. The analysis on fundamental scientific research on local budgets.Defined-purpose local governments.Systematized theoretical definition of municipal finances. Formed impact of reform of local finance to ensure the improvement of socio-economic status of the region to the European Union for more confident of Ukraine’s integration into Europe. Found that implementing fiscal policy in terms of spending, local authorities are guided by the priorities of socio-economic development of the region or administrative unit in line with national principles defined government policy document. Discovered topical issues of local budgets as the existence of significant differences in the regulatory and legislative framework - the excessive centralization of local budgets unstable sources of local revenues inadequacy of intergovernmental relations. It was found that to radically change the existing system of functioning of local budgets, as required considerable time gap and improving the theoretical and methodological and regulatory framework of the local budgets.

  15. Headphone localization of speech

    Science.gov (United States)

    Begault, Durand R.; Wenzel, Elizabeth M.

    1993-01-01

    Three-dimensional acoustic display systems have recently been developed that synthesize virtual sound sources over headphones based on filtering by head-related transfer functions (HRTFs), the direction-dependent spectral changes caused primarily by the pinnae. In this study, 11 inexperienced subjects judged the apparent spatial location of headphone-presented speech stimuli filtered with nonindividualized HRTFs. About half of the subjects 'pulled' their judgments toward either the median or the lateral-vertical planes, and estimates were almost always elevated. Individual differences were pronounced for the distance judgments; 15 to 46 percent of stimuli were heard inside the head, with the shortest estimates near the median plane. The results suggest that most listeners can obtain useful azimuth information from speech stimuli filtered by nonindividualized HRTFs. Measurements of localization error and reversal rates are comparable with a previous study that used broadband noise stimuli.

  16. Local Stellarator Equilibrium Model

    Science.gov (United States)

    Hudson, Stuart R.; Hegna, Chris C.; Lewandowski, Jerome W.

    2000-10-01

    Extensive calculations of ballooning and drift waves spectrums in asymmetric toroidal configurations (e.g. stellarators) to appreciate the role of magnetic geometry and profile variations are usually are usually prohibitive as the evaluation of the magneto-hydrodynamic (MHD) equilibrium is in itself a non-trivial problem. Although simple analytical MHD model equilibria do exist for tokamak configurations, their stellarator counterparts are usually crude or very approximate. In order to make more extensive stability calculations (of both ideal ballooning and drift-type modes), a technique for generating three-dimensional magneto-static equilibria, localized to a magnetic surface, has been developed. The technique allows one to easily manipulate various 3-D shaping and profile effects on a magnetic surface avoiding the need to recompute an entire three dimensional solution of the equilibrium. The model equilibrium has been implemented into existing ideal MHD ballooning and drift wave numerical codes. Marginal ballooning stability diagrams and drift wave calculations will be reported.

  17. Support your local species

    DEFF Research Database (Denmark)

    Stärk, Johanna

    Nearly a quarter of all animal species within the European Union are threatened with extinction. Protecting many of these species will require the full spectrum of conservation actions from in-situ to ex-situ management. Holding an estimated 44% of EU Red Listed terrestrial vertebrates, zoos hereby...... play an important role in protecting local species. However, outcomes of conservation actions are often highly uncertain and if European zoos want to support the conservation of threatened species, they are faced with the question of which species to target first and which conservation strategy...... to choose. Current decision-making in resource allocation and conservation planning is often, as in many other disciplines, based on little scientific ground. Here, we propose a Decision Analysis framework to support the Ex-situ guidelines of the Species Survival Commission of the IUCN. In which we assessed...

  18. Narcotics and local anesthetics.

    Science.gov (United States)

    Kamerling, S G

    1993-12-01

    The recognition and alleviation of animal pain is a growing veterinary and public concern. Pain can be of an acute or chronic nature with different behavioral manifestations. Physiologically, pain is a dynamic and complex phenomenon that produces changes in the central and autonomic nervous systems as well as in the endocrine system. Horses and other animals appear to possess an endogenous pain-suppressing system involving the brainstem and spinal cord. This system can modulate pain perception and the responses to it. The recently discovered endogenous opioid peptides (endorphins and enkephalins) appear to play a role in this system, which is activated by stress. Opioids (narcotic analgesics) act to selectively depress pain-sensitive cells. Opioid analgesics may act via multiple opioid receptors. Each subclass of opioid receptor has a different pharmacologic profile. Classical opioids that act at mu (morphine) receptors typically produce analgesia, increased locomotor activity, cardiorespiratory stimulation, and a decrease in intestinal peristalsis in the horse. Opioids that act at kappa receptors produce analgesia, sedation, ataxia, and minimal autonomic effects in the horse. Owing to their lack of excitatory actions, the kappa opioids represent a potentially useful class of analgesics for use in equine species. Local anesthetics depress all excitable cells and can diminish sensory, motor, and muscular function. They do not act selectively on pain fibers, although pain is among the first sensations lost following a nerve block. Local anesthetic activity is enhanced by increased extraneuronal pH, nerve cooling, increased nervous activity, coadministration of a vasoconstrictor or hyaluronidase, delayed systemic absorption, prolonged drug metabolism, and by using agents with high lipid solubility. Procaine, lidocaine, and mepivacaine are among the most widely used and studied agents in horses. These agents and/or their metabolites can be readily detected in urine; in

  19. Local communities as meso structures.

    Directory of Open Access Journals (Sweden)

    Ferrand, Alexis

    2002-09-01

    Full Text Available In between micro and macro levels: meso. If macro is defined by national, state or market regulation, and micro by relational interpersonnal regulation, a major agency of intermediate regulation has been defined as "local communities" (Wellmann, Fischer. Apart the organizational (local organizations, clubs, shops... and institutional (local power, parties,... dimensions of localities, it is possible to define some pertinent structural properties of systems of private interpersonal relations : one is the degree of local closure (Barnes : local vs transversal networks, or the "duality" of these systems. Grounded on the idea that the efficiency of "bridges" rest upon the kind of cliques they connect (Lin, it is possible to define various types of micro-structures, the distributions of which in different localities are proxy indicators of the different meso structures of relational systems as combinations of "local" and "unlocal" networks.

  20. Local Climatological Data ACSII Format

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — Comma-delimited text files used to create the Local Climatological Data PDF files found in the Local Climatological Data library. Period of record begins in 1998,...