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Sample records for show prolonged intracellular

  1. Prolonged Intracellular Na+ Dynamics Govern Electrical Activity in Accessory Olfactory Bulb Mitral Cells.

    PubMed

    Zylbertal, Asaph; Kahan, Anat; Ben-Shaul, Yoram; Yarom, Yosef; Wagner, Shlomo

    2015-12-01

    Persistent activity has been reported in many brain areas and is hypothesized to mediate working memory and emotional brain states and to rely upon network or biophysical feedback. Here, we demonstrate a novel mechanism by which persistent neuronal activity can be generated without feedback, relying instead on the slow removal of Na+ from neurons following bursts of activity. We show that mitral cells in the accessory olfactory bulb (AOB), which plays a major role in mammalian social behavior, may respond to a brief sensory stimulation with persistent firing. By combining electrical recordings, Ca2+ and Na+ imaging, and realistic computational modeling, we explored the mechanisms underlying the persistent activity in AOB mitral cells. We found that the exceptionally slow inward current that underlies this activity is governed by prolonged dynamics of intracellular Na+ ([Na+]i), which affects neuronal electrical activity via several pathways. Specifically, elevated dendritic [Na+]i reverses the Na+-Ca2+ exchanger activity, thus modifying the [Ca2+]i set-point. This process, which relies on ubiquitous membrane mechanisms, is likely to play a role in other neuronal types in various brain regions. PMID:26674618

  2. Prolonged Intracellular Na+ Dynamics Govern Electrical Activity in Accessory Olfactory Bulb Mitral Cells

    PubMed Central

    Zylbertal, Asaph; Kahan, Anat; Ben-Shaul, Yoram; Yarom, Yosef; Wagner, Shlomo

    2015-01-01

    Persistent activity has been reported in many brain areas and is hypothesized to mediate working memory and emotional brain states and to rely upon network or biophysical feedback. Here, we demonstrate a novel mechanism by which persistent neuronal activity can be generated without feedback, relying instead on the slow removal of Na+ from neurons following bursts of activity. We show that mitral cells in the accessory olfactory bulb (AOB), which plays a major role in mammalian social behavior, may respond to a brief sensory stimulation with persistent firing. By combining electrical recordings, Ca2+ and Na+ imaging, and realistic computational modeling, we explored the mechanisms underlying the persistent activity in AOB mitral cells. We found that the exceptionally slow inward current that underlies this activity is governed by prolonged dynamics of intracellular Na+ ([Na+]i), which affects neuronal electrical activity via several pathways. Specifically, elevated dendritic [Na+]i reverses the Na+-Ca2+ exchanger activity, thus modifying the [Ca2+]i set-point. This process, which relies on ubiquitous membrane mechanisms, is likely to play a role in other neuronal types in various brain regions. PMID:26674618

  3. Intracellular Potassium Activity in Guinea Pig Papillary Muscle during Prolonged Hypoxia

    PubMed Central

    Guarnieri, Thomas; Strauss, Harold C.

    1982-01-01

    During prolonged hypoxia, intracellular potassium concentration, [K]i has been reported to fall by 70% with a concomitant decrease in the calculated potassium equilibrium potential, EK. Nevertheless, resting membrane potential, Vm, declined only slightly. Because Vm depolarized very little in relation to the calculated EK, it was hypothesized that electrogenic Na-K pumping contributed up to 40 mV to Vm during prolonged hypoxia. To further test this hypothesis we studied what changes prolonged hypoxia makes in the thermodynamically active fraction of cellular potassium, intracellular potassium activity, αKi, and how change in αKi affects the relationship between Vm, EK and, by inference, the Na-K pump. Using double-barrel K-selective electrodes, Vm and αKi were measured in quiescent guinea pig right ventricular papillary muscles superfused for 8 h with hypoxic Tyrode's solution. Over the 8-h period both Vm and αKi decreased. However, the decline in Vm was paralleled by a decrease in the EK calculated from αKi. At no time was there hyperpolarization of Vm beyond EK. After 8 h the Na-K pump was inhibited by exposing the muscles to 0.1 mM ouabain. The onset of an increase in extracellular potassium activity, measured with a double-barrel electrode, was used to mark the amount of depolarization of Vm due solely to pump inhibition. After hypoxia, Vm depolarized 8.4±4.4 mV before extracellular potassium activity (αKe) increased. Thus, the decrease in αKi during hypoxia is much less than that reported for [K]i. The parallel decline in Vm and EK and the small depolarization of Vm with ouabain suggest that after prolonged hypoxia the Na-K pump continues to contribute to Vm, but the amount of this contribution is substantially less than previously reported. PMID:6276442

  4. Iridium Oxide Nanotube Electrodes for Highly Sensitive and Prolonged Intracellular Measurement of Action Potentials

    PubMed Central

    Lin, Ziliang Carter; Xie, Chong; Osakada, Yasuko; Cui, Yi; Cui, Bianxiao

    2014-01-01

    Intracellular recording of action potentials is important to understand electrically-excitable cells. Recently, vertical nanoelectrodes have been developed to achieve highly sensitive, minimally invasive, and large scale intracellular recording. It has been demonstrated that the vertical geometry is crucial for the enhanced signal detection. Here we develop nanoelectrodes made up of nanotubes of iridium oxide. When cardiomyocytes are cultured upon those nanotubes, the cell membrane not only wraps around the vertical tubes but also protrudes deep into the hollow center. We show that this geometry enhances cell-electrode coupling and results in measuring much larger intracellular action potentials. The nanotube electrodes afford much longer intracellular access and are minimally invasive, making it possible to achieve stable recording up to an hour in a single session and more than 8 days of consecutive daily recording. This study suggests that the electrode performance can be significantly improved by optimizing the electrode geometry. PMID:24487777

  5. Effect of prolonged depolarizations on twitch tension and intracellular sodium activity in sheep cardiac Purkinje fibres.

    PubMed Central

    Brill, D M; Fozzard, H A; Makielski, J C; Wasserstrom, J A

    1987-01-01

    1. Twitch tension and intracellular Na+ activity (aiNa) were measured in voltage-clamped sheep cardiac Purkinje fibres. aiNa was measured using Na+-sensitive micro-electrodes filled with the liquid ion exchange resin. ETH 227. The stimulus for contraction was a constant 200 ms depolarizing pulse to 0 mV from a holding potential of -80 mV delivered at 0.25 Hz. Prolonged test pulses for 1.8 s (post-pulses) were applied at the end of the stimulus pulse. The effects of post-pulses on twitch tension and aiNa were examined. 2. Post-pulses in the range of -40 mV reduced twitch tension below control force produced without post-pulse. Progressively more positive post-pulses to levels above 0 mV profoundly increased twitch tension, with a greater than 400% rise in tension at +50 to +60 mV compared to control tension. aiNa declined at positive post-pulse potentials by more than 2 mM at +30 to +40 mV. 3. Tetrodotoxin (100 microM) did not affect the post-pulse voltage-tension or voltage-aiNa relation. Ca2+ channel modulation with nitrendipine (1 microM) similarly did not alter the post-pulse voltage-tension relation. 4. Removal of extracellular Na+ eliminated the nadir in tension at post-pulses to -40 mV and the augmentation of tension at post-pulses above 0 mV. 5. We interpret these findings as evidence of voltage-sensitive Na-Ca exchange promoting net Ca2+ influx and net Na+ efflux during positive post-pulses. The unusual shape of the post-pulse voltage-tension relation curve can be accounted for by a charged-carrier model of electrogenic Na-Ca exchange. The inverse relation between aiNa and twitch tension probably reflects the combined effects of reduced aiNa leak and changes in Na+ and Ca2+ flux via voltage-sensitive Na-Ca exchange. PMID:2443661

  6. Prolonged Oxaliplatin Exposure Alters Intracellular Calcium Signaling: A New Mechanism To Explain Oxaliplatin-Associated Peripheral Neuropathy

    PubMed Central

    Schulze, Christin; McGowan, Margit; Jordt, Sven; Ehrlich, Barbara E

    2012-01-01

    Oxaliplatin is a platinum based cytotoxic agent commonly used to treat colorectal cancers. Despite its effectiveness, oxaliplatin administration is associated with the development of cold-induced peripheral neuropathy. This potentially permanent side effect is provoked by cold exposure and can range from mild and self limited to severe and debilitating. Even with tumor shrinkage, these painful side effects can force dose-reduction or discontinuation of treatment. Neither the mechanism of action of oxaliplatin nor that of cold-induced neuropathy is understood. Paclitaxel, an entirely different chemotherapeutic agent used to treat a variety of malignancies, also is associated with the development of peripheral neuropathy. Unlike oxaliplatin, neurotoxicity arising from paclitaxel treatment is better understood and was found to have profound effects on intracellular calcium signaling (1,2). In this study we examined the effects of oxaliplatin on calcium signaling pathways and found that acute exposure of either a neuroblastoma cell line or primary neurons with therapeutic concentrations of oxaliplatin had no effect on intracellular calcium signaling. We also found that cellular temperature sensors (TRP channels) were also not activated by oxaliplatin. Interestingly, prolonged exposure of oxaliplatin sensitized cells to subsequent stimuli and enhanced the magnitude of intracellular calcium responses. Taken together, our results suggest that acute oxaliplatin exposure will not induce abnormal calcium signaling but oxaliplatin-primed cells do exhibit enhanced sensitivity. These findings provide new insight to the mechanism behind oxaliplatin-induced neuropathy. PMID:21859566

  7. Ecological changes in Miocene mammalian record show impact of prolonged climatic forcing.

    PubMed

    Badgley, Catherine; Barry, John C; Morgan, Michèle E; Nelson, Sherry V; Behrensmeyer, Anna K; Cerling, Thure E; Pilbeam, David

    2008-08-26

    Geohistorical records reveal the long-term impacts of climate change on ecosystem structure. A 5-myr record of mammalian faunas from floodplain ecosystems of South Asia shows substantial change in species richness and ecological structure in relation to vegetation change as documented by stable isotopes of C and O from paleosols. Between 8.5 and 6.0 Ma, C(4) savannah replaced C(3) forest and woodland. Isotopic historical trends for 27 mammalian herbivore species, in combination with ecomorphological data from teeth, show three patterns of response. Most forest frugivores and browsers maintained their dietary habits and disappeared. Other herbivores altered their dietary habits to include increasing amounts of C(4) plants and persisted for >1 myr during the vegetation transition. The few lineages that persisted through the vegetation transition show isotopic enrichment of delta(13)C values over time. These results are evidence for long-term climatic forcing of vegetation structure and mammalian ecological diversity at the subcontinental scale. PMID:18711123

  8. Ecological changes in Miocene mammalian record show impact of prolonged climatic forcing

    PubMed Central

    Badgley, Catherine; Barry, John C.; Morgan, Michèle E.; Nelson, Sherry V.; Behrensmeyer, Anna K.; Cerling, Thure E.; Pilbeam, David

    2008-01-01

    Geohistorical records reveal the long-term impacts of climate change on ecosystem structure. A 5-myr record of mammalian faunas from floodplain ecosystems of South Asia shows substantial change in species richness and ecological structure in relation to vegetation change as documented by stable isotopes of C and O from paleosols. Between 8.5 and 6.0 Ma, C4 savannah replaced C3 forest and woodland. Isotopic historical trends for 27 mammalian herbivore species, in combination with ecomorphological data from teeth, show three patterns of response. Most forest frugivores and browsers maintained their dietary habits and disappeared. Other herbivores altered their dietary habits to include increasing amounts of C4 plants and persisted for >1 myr during the vegetation transition. The few lineages that persisted through the vegetation transition show isotopic enrichment of δ13C values over time. These results are evidence for long-term climatic forcing of vegetation structure and mammalian ecological diversity at the subcontinental scale. PMID:18711123

  9. Prolonged Hypoxia Increases Survival Even in Zebrafish (Danio rerio) Showing Cardiac Arrhythmia

    PubMed Central

    Kopp, Renate; Bauer, Ines; Ramalingam, Anil; Egg, Margit; Schwerte, Thorsten

    2014-01-01

    Tolerance towards hypoxia is highly pronounced in zebrafish. In this study even beneficial effects of hypoxia, specifically enhanced survival of zebrafish larvae, could be demonstrated. This effect was actually more pronounced in breakdance mutants, which phenotypically show cardiac arrhythmia. Breakdance mutants (bre) are characterized by chronically reduced cardiac output. Despite an about 50% heart rate reduction, they become adults, but survival rate significantly drops to 40%. Normoxic bre animals demonstrate increased hypoxia inducible factor 1 a (Hif-1α) expression, which indicates an activated hypoxic signaling pathway. Consequently, cardiovascular acclimation, like cardiac hypertrophy and increased erythrocyte concentration, occurs. Thus, it was hypothesized, that under hypoxic conditions survival might be even more reduced. When bre mutants were exposed to hypoxic conditions, they surprisingly showed higher survival rates than under normoxic conditions and even reached wildtype values. In hypoxic wildtype zebrafish, survival yet exceeded normoxic control values. To specify physiological acclimation, cardiovascular and metabolic parameters were measured before hypoxia started (3 dpf), when the first differences in survival rate occurred (7 dpf) and when survival rate plateaued (15 dpf). Hypoxic animals expectedly demonstrated Hif-1α accumulation and consequently enhanced convective oxygen carrying capacity. Moreover, bre animals showed a significantly enhanced heart rate under hypoxic conditions, which reached normoxic wildtype values. This improvement in convective oxygen transport ensured a sufficient oxygen and nutrient supply and was also reflected in the significantly higher mitochondrial activity. The highly optimized energy metabolism observed in hypoxic zebrafish larvae might be decisive for periods of higher energy demand due to organ development, growth and increased activity. However, hypoxia increased survival only during a short period of

  10. Randomized, Controlled, Thorough QT/QTc Study Shows Absence of QT Prolongation with Luseogliflozin in Healthy Japanese Subjects

    PubMed Central

    Kumagai, Yuji; Hasunuma, Tomoko; Sakai, Soichi; Ochiai, Hidekazu; Samukawa, Yoshishige

    2015-01-01

    Luseogliflozin is a selective sodium glucose co-transporter 2 (SGLT2) inhibitor. To evaluate the cardiac safety of luseogliflozin, a thorough QT/QTc study was conducted in healthy Japanese subjects. The effects of moxifloxacin on QT prolongation in Japanese subjects were also evaluated. In this double-blind, placebo- and open-label positive-controlled, 4-way crossover study, 28 male and 28 female subjects received a single dose of luseogliflozin 5 mg (therapeutic dose), luseogliflozin 20 mg (supratherapeutic dose), placebo, and moxifloxacin 400 mg. Serial triplicate digital 12-lead electrocardiograms (ECGs) were recorded before and after dosing, and results were analyzed using the Fridericia correction (QTcF) method. Serial blood sampling was performed for pharmacokinetic analyses of luseogliflozin and moxifloxacin to analyze the relationship between QTcF interval and plasma concentration. The upper limits of the two-sided 90% confidence intervals (CIs) for baseline and placebo-adjusted QTcF intervals (ΔΔQTcF) in the 5 mg and 20 mg luseogliflozin groups were less than 10 ms at all time points. No correlation between plasma luseogliflozin concentrations and ΔΔQTcF was observed. In the moxifloxacin group, the lower limits of the two-sided 90% CIs for ΔΔQTcF were greater than 5 ms at all time points. A positive relationship was observed between plasma moxifloxacin concentration and change in ΔΔQTcF. Luseogliflozin was well tolerated at both dose levels. The majority of adverse events were mild in severity, and no serious or life-threatening adverse events occurred. Neither therapeutic (5 mg) nor supratherapeutic (20 mg) doses of luseogliflozin affected QT prolongation in healthy Japanese subjects. PMID:26444986

  11. Neutrophils from patients with SAPHO syndrome show no signs of aberrant NADPH oxidase-dependent production of intracellular reactive oxygen species

    PubMed Central

    Wekell, Per; Björnsdottir, Halla; Björkman, Lena; Sundqvist, Martina; Christenson, Karin; Osla, Veronica; Berg, Stefan; Fasth, Anders; Welin, Amanda; Bylund, Johan

    2016-01-01

    Objective. We aimed to investigate if aberrant intracellular production of NADPH oxidase-derived reactive oxygen species (ROS) in neutrophils is a disease mechanism in the autoinflammatory disease SAPHO syndrome, characterized by synovitis, acne, pustulosis, hyperostosis and osteitis, as has previously been suggested based on a family with SAPHO syndrome-like disease. Methods. Neutrophil function was explored in a cohort of four patients with SAPHO syndrome, two of whom were sampled during both inflammatory and non-inflammatory phase. Intracellular neutrophil ROS production was determined by luminol-amplified chemiluminescence in response to phorbol myristate acetate. Results. Cells from all patients produced normal amounts of ROS, both intra- and extracellularly, when compared with internal controls as well as with a large collection of healthy controls assayed in the laboratory over time (showing an extensive inter-personal variability in a normal population). Further, intracellular production of ROS increased during the inflammatory phase. Neutrophil activation markers were comparable between patients and controls. Conclusion. Dysfunctional generation of intracellular ROS in neutrophils is not a generalizable feature in SAPHO syndrome. Secondly, serum amyloid A appears to be a more sensitive inflammatory marker than CRP during improvement and relapses in SAPHO syndrome. PMID:27121779

  12. Why is intracellular ice lethal? A microscopical study showing evidence of programmed cell death in cryo-exposed embryonic axes of recalcitrant seeds of Acer saccharinum

    PubMed Central

    Wesley-Smith, James; Walters, Christina; Pammenter, N. W.

    2015-01-01

    Background and Aims Conservation of the genetic diversity afforded by recalcitrant seeds is achieved by cryopreservation, in which excised embryonic axes (or, where possible, embryos) are treated and stored at temperatures lower than −180 °C using liquid nitrogen. It has previously been shown that intracellular ice forms in rapidly cooled embryonic axes of Acer saccharinum (silver maple) but this is not necessarily lethal when ice crystals are small. This study seeks to understand the nature and extent of damage from intracellular ice, and the course of recovery and regrowth in surviving tissues. Methods Embryonic axes of A. saccharinum, not subjected to dehydration or cryoprotection treatments (water content was 1·9 g H2O g−1 dry mass), were cooled to liquid nitrogen temperatures using two methods: plunging into nitrogen slush to achieve a cooling rate of 97 °C s−1 or programmed cooling at 3·3 °C s−1. Samples were thawed rapidly (177 °C s−1) and cell structure was examined microscopically immediately, and at intervals up to 72 h in vitro. Survival was assessed after 4 weeks in vitro. Axes were processed conventionally for optical microscopy and ultrastructural examination. Key Results Immediately following thaw after cryogenic exposure, cells from axes did not show signs of damage at an ultrastructural level. Signs that cells had been damaged were apparent after several hours of in vitro culture and appeared as autophagic decomposition. In surviving tissues, dead cells were sloughed off and pockets of living cells were the origin of regrowth. In roots, regrowth occurred from the ground meristem and procambium, not the distal meristem, which became lethally damaged. Regrowth of shoots occurred from isolated pockets of surviving cells of peripheral and pith meristems. The size of these pockets may determine the possibility for, the extent of and the vigour of regrowth. Conclusions Autophagic degradation and ultimately autolysis of cells following

  13. Why is intracellular ice lethal? A microscopical study showing evidence of programmed cell death in cryo-exposed embryonic axes of recalcitrant seeds of Acer saccharinum

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Intracellular ice formed in rapidly cooled embryonic axes of Acer saccharinum and was not necessarily lethal when ice crystals were small. This study seeks to understand the nature and extent of damage from intracellular ice, and the course of recovery and regrowth in surviving tissues. Embryonic a...

  14. Polymeric micelles with α-glutamyl-terminated PEG shells show low non-specific protein adsorption and a prolonged in vivo circulation time.

    PubMed

    Wang, Xiaoju; Yang, Cuiping; Wang, Chenhong; Guo, Leijia; Zhang, Tianhong; Zhang, Zhenqing; Yan, Husheng; Liu, Keliang

    2016-02-01

    Although PEG remains the gold standard for stealth functionalization in drug delivery field up to date, complete inhibition of protein corona formation on PEG-coated nanoparticles remains a challenge. To improve the stealth property of PEG, herein an α-glutamyl group was conjugated to the end of PEG and polymeric micelles with α-glutamyl-terminated PEG shells were prepared. After incubation with bovine serum albumin or in fetal calf serum, the size of the micelles changed slightly, while the size of the micelles of similar diblock copolymer but without α-glutamyl group increased markedly. These results indicated that the micelles with α-glutamyl-terminated PEG shells showed low non-specific protein adsorption. In vivo blood clearance kinetics assay showed that the micelles with α-glutamyl-terminated PEG shells exhibited a longer in vivo blood circulation time compared with similar micelles but without α-glutamyl groups. The better stealth property of the micelles with α-glutamyl-terminated PEG shells was presumably attributed to the zwitterionic property of the α-glutamyl groups. PMID:26652431

  15. Prolonged pregnancy.

    PubMed

    Hollis, Brian

    2002-04-01

    Prolonged pregnancy is defined as any pregnancy that lasts 294 days or more. It is now well recognized that prolonged pregnancy is associated with an increased risk of perinatal mortality and morbidity. It is these complications of pregnancy that have led obstetricians to adopt a policy of induction of labour before the onset of the post-term period. The induction of labour between 41 and 42 weeks is, however, a very crude strategy for reducing term and post-term stillbirth rates. Although the risk of fetal death is increased after 42 weeks, many more fetuses die in utero between 37 and 42 weeks than die in the post-term period. It appears that smaller term fetuses run a greater risk than their larger counterparts, and that current methods of antepartum assessment of the term fetus are still inadequate. It behoves us as obstetricians to improve our capabilities in identifying the compromised fetus at term. This review puts into perspective the most recent publications and highlights areas requiring further study. PMID:11914699

  16. Prolonged contraction duration in aged myocardium.

    PubMed

    Lakatta, E G; Gerstenblith, G; Angell, C S; Shock, N W; Weisfeldt, M L

    1975-01-01

    Isometric performance at 29degreesC was measured in left ventricular trabeculae carneae from young adult (6-mo) and aged (25-mo) rats (n equals 18 in each group). Active tension and maximal rate of tension development did not differ with age, but contraction duration was 255plus or minus6 ms in the young adult and 283plus or minus6 ms in the aged group (P less than0.001). Although catecholamine content per gram heart weight was less in the aged myocardium, additional experiments showed that neither 1 times 10-6 M propranolol nor pretreatment with 6-hydroxydopamine eliminated the age difference in contraction duration. To determine if this age difference resulted from a prolonged active state, electromechanical dissociation and the overshoot of contraction duration during recovery from hypoxia were measured. During paired stimulation greater mechanical refractoriness was found in aged muscles (P less than0.01), but intracellular action potential recordings showed no age difference in the electrical refractory period. On recovery from hypoxia, contraction duration overshoot was 117plus or minus 4percent of control in the young and 138plus or minus 4percent of control in the aged muscles (P less than0.01). The greater electromechanical dissociation and greater overshoot in contraction duration following hypoxia in aged myocardium suggests that prolonged contraction duration in aged myocardium results from a prolonged active state rather than changes in passive properties or myocardial catecholamine content. PMID:1109181

  17. Intracellular proteoglycans.

    PubMed Central

    Kolset, Svein Olav; Prydz, Kristian; Pejler, Gunnar

    2004-01-01

    Proteoglycans (PGs) are proteins with glycosaminoglycan chains, are ubiquitously expressed and have a wide range of functions. PGs in the extracellular matrix and on the cell surface have been the subject of extensive structural and functional studies. Less attention has so far been given to PGs located in intracellular compartments, although several reports suggest that these have biological functions in storage granules, the nucleus and other intracellular organelles. The purpose of this review is, therefore, to present some of these studies and to discuss possible functions linked to PGs located in different intracellular compartments. Reference will be made to publications relevant for the topics we present. It is beyond the scope of this review to cover all publications on PGs in intracellular locations. PMID:14759226

  18. Correlative Light-Electron Microscopy Shows RGD-Targeted ZnO Nanoparticles Dissolve in the Intracellular Environment of Triple Negative Breast Cancer Cells and Cause Apoptosis with Intratumor Heterogeneity.

    PubMed

    Othman, Basmah A; Greenwood, Christina; Abuelela, Ayman F; Bharath, Anil A; Chen, Shu; Theodorou, Ioannis; Douglas, Trevor; Uchida, Maskai; Ryan, Mary; Merzaban, Jasmeen S; Porter, Alexandra E

    2016-06-01

    ZnO nanoparticles (NPs) are reported to show a high degree of cancer cell selectivity with potential use in cancer imaging and therapy. Questions remain about the mode by which the ZnO NPs cause cell death, whether they exert an intra- or extracellular effect, and the resistance among different cancer cell types to ZnO NP exposure. The present study quantifies the variability between the cellular toxicity, dynamics of cellular uptake, and dissolution of bare and RGD (Arg-Gly-Asp)-targeted ZnO NPs by MDA-MB-231 cells. Compared to bare ZnO NPs, RGD-targeting of the ZnO NPs to integrin αvβ3 receptors expressed on MDA-MB-231 cells appears to increase the toxicity of the ZnO NPs to breast cancer cells at lower doses. Confocal microscopy of live MDA-MB-231 cells confirms uptake of both classes of ZnO NPs with a commensurate rise in intracellular Zn(2+) concentration prior to cell death. The response of the cells within the population to intracellular Zn(2+) is highly heterogeneous. In addition, the results emphasize the utility of dynamic and quantitative imaging in understanding cell uptake and processing of targeted therapeutic ZnO NPs at the cellular level by heterogeneous cancer cell populations, which can be crucial for the development of optimized treatment strategies. PMID:27111660

  19. Intracellular microlasers

    NASA Astrophysics Data System (ADS)

    Humar, Matjaž; Hyun Yun, Seok

    2015-09-01

    Optical microresonators, which confine light within a small cavity, are widely exploited for various applications ranging from the realization of lasers and nonlinear devices to biochemical and optomechanical sensing. Here we use microresonators and suitable optical gain materials inside biological cells to demonstrate various optical functions in vitro including lasing. We explore two distinct types of microresonator—soft and hard—that support whispering-gallery modes. Soft droplets formed by injecting oil or using natural lipid droplets support intracellular laser action. The laser spectra from oil-droplet microlasers can chart cytoplasmic internal stress (˜500 pN μm-2) and its dynamic fluctuations at a sensitivity of 20 pN μm-2 (20 Pa). In a second form, whispering-gallery modes within phagocytized polystyrene beads of different sizes enable individual tagging of thousands of cells easily and, in principle, a much larger number by multiplexing with different dyes.

  20. Intracellular microlasers

    PubMed Central

    Humar, Matjaž; Yun, Seok Hyun

    2015-01-01

    Optical microresonators1 which confine light within a small cavity are widely exploited for various applications ranging from the realization of lasers2 and nonlinear devices3, 4, 5 to biochemical and optomechanical sensing6, 7, 8, 9, 10, 11. Here we employ microresonators and suitable optical gain materials inside biological cells to demonstrate various optical functions in vitro including lasing. We explored two distinct types of microresonators: soft and hard, that support whispering-gallery modes (WGM). Soft droplets formed by injecting oil or using natural lipid droplets support intracellular laser action. The laser spectra from oil-droplet microlasers can chart cytoplasmic internal stress (~500 pN/μm2) and its dynamic fluctuations at a sensitivity of 20 pN/μm2 (20 Pa). In a second form, WGMs within phagocytized polystyrene beads of different sizes enable individual tagging of thousands of cells easily and, in principle, a much larger number by multiplexing with different dyes. PMID:26417383

  1. Managing intracellular transport

    PubMed Central

    Chua, John J.E.; Jahn, Reinhard; Klopfenstein, Dieter R.

    2013-01-01

    Formation and normal function of neuronal synapses are intimately dependent on the delivery to and removal of biological materials from synapses by the intracellular transport machinery. Indeed, defects in intracellular transport contribute to the development and aggravation of neurodegenerative disorders. Despite its importance, regulatory mechanisms underlying this machinery remain poorly defined. We recently uncovered a phosphorylation-regulated mechanism that controls FEZ1-mediated Kinesin-1-based delivery of Stx1 into neuronal axons. Using C. elegans as a model organism to investigate transport defects, we show that FEZ1 mutations resulted in abnormal Stx1 aggregation in neuronal cell bodies and axons. This phenomenon closely resembles transport defects observed in neurodegenerative disorders. Importantly, diminished transport due to mutations of FEZ1 and Kinesin-1 were concomitant with increased accumulation of autophagosomes. Here, we discuss the significance of our findings in a broader context in relation to regulation of Kinesin-mediated transport and neurodegenerative disorders. PMID:24058857

  2. Prolonged daily light exposure increases body fat mass through attenuation of brown adipose tissue activity

    PubMed Central

    Kooijman, Sander; van den Berg, Rosa; Ramkisoensing, Ashna; Boon, Mariëtte R.; Kuipers, Eline N.; Loef, Marieke; Zonneveld, Tom C. M.; Lucassen, Eliane A.; Sips, Hetty C. M.; Chatzispyrou, Iliana A.; Houtkooper, Riekelt H.; Meijer, Johanna H.; Coomans, Claudia P.; Biermasz, Nienke R.; Rensen, Patrick C. N.

    2015-01-01

    Disruption of circadian rhythmicity is associated with obesity and related disorders, including type 2 diabetes and cardiovascular disease. Specifically, prolonged artificial light exposure associates with obesity in humans, although the underlying mechanism is unclear. Here, we report that increasing the daily hours of light exposure increases body adiposity through attenuation of brown adipose tissue (BAT) activity, a major contributor of energy expenditure. Mice exposed to a prolonged day length of 16- and 24-h light, compared with regular 12-h light, showed increased adiposity without affecting food intake or locomotor activity. Mechanistically, we demonstrated that prolonged day length decreases sympathetic input into BAT and reduces β3-adrenergic intracellular signaling. Concomitantly, prolonging day length decreased the uptake of fatty acids from triglyceride-rich lipoproteins, as well as of glucose from plasma selectively by BAT. We conclude that impaired BAT activity is an important mediator in the association between disturbed circadian rhythm and adiposity, and anticipate that activation of BAT may overcome the adverse metabolic consequences of disturbed circadian rhythmicity. PMID:25964318

  3. Targeted Cancer Therapy: Correlative Light-Electron Microscopy Shows RGD-Targeted ZnO Nanoparticles Dissolve in the Intracellular Environment of Triple Negative Breast Cancer Cells and Cause Apoptosis with Intratumor Heterogeneity (Adv. Healthcare Mater. 11/2016).

    PubMed

    Othman, Basmah A; Greenwood, Christina; Abuelela, Ayman F; Bharath, Anil A; Chen, Shu; Theodorou, Ioannis; Douglas, Trevor; Uchida, Maskai; Ryan, Mary; Merzaban, Jasmeen S; Porter, Alexandra E

    2016-06-01

    On page 1310 J. S. Merzaban, A. E. Porter, and co-workers present fluorescently labeled RGD-targeted ZnO nanoparticles (NPs; green) for the targeted delivery of cytotoxic ZnO to integrin αvβ3 receptors expressed on triple negative breast cancer cells. Correlative light-electron microscopy shows that NPs dissolve into ionic Zn(2+) (blue) upon uptake and cause apoptosis (red) with intra-tumor heterogeneity, thereby providing a possible strategy for targeted breast cancer therapy. Cover design by Ivan Gromicho. PMID:27275627

  4. Methods to follow intracellular trafficking of cell-penetrating peptides.

    PubMed

    Pärnaste, Ly; Arukuusk, Piret; Zagato, Elisa; Braeckmans, Kevin; Langel, Ülo

    2016-01-01

    Cell-penetrating peptides (CPPs) are efficient vehicles to transport bioactive molecules into the cells. Despite numerous studies the exact mechanism by which CPPs facilitate delivery of cargo to its intracellular target is still debated. The current work presents methods that can be used for tracking CPP/pDNA complexes through endosomal transport and show the role of endosomal transport in the delivery of cargo. Separation of endosomal vesicles by differential centrifugation enables to pinpoint the localization of delivered cargo without labeling it and gives important quantitative information about pDNA trafficing in certain endosomal compartments. Single particle tracking (SPT) allows following individual CPP/cargo complex through endosomal path in live cells, using fluoresently labled cargo and green fluoresent protein expressing cells. These two different methods show similar results about tested NickFect/pDNA complexes intracellular trafficing. NF51 facilitates rapid internalization of complexes into the cells, prolongs their stay in early endosomes and promotes release to cytosol. NF1 is less capable to induce endosomal release and higher amount of complexes are routed to lysosomes for degradation. Our findings offer potential delivery vector for in vivo applications, NF51, where endosomal entrapment has been allayed. Furthermore, these methods are valuable tools to study other CPP-based delivery systems. PMID:26460120

  5. Calcium Imaging of AM Dyes Following Prolonged Incubation in Acute Neuronal Tissue

    PubMed Central

    Morley, John W.; Tapson, Jonathan; Breen, Paul P.; van Schaik, André

    2016-01-01

    Calcium-imaging is a sensitive method for monitoring calcium dynamics during neuronal activity. As intracellular calcium concentration is correlated to physiological and pathophysiological activity of neurons, calcium imaging with fluorescent indicators is one of the most commonly used techniques in neuroscience today. Current methodologies for loading calcium dyes into the tissue require prolonged incubation time (45–150 min), in addition to dissection and recovery time after the slicing procedure. This prolonged incubation curtails experimental time, as tissue is typically maintained for 6–8 hours after slicing. Using a recently introduced recovery chamber that extends the viability of acute brain slices to more than 24 hours, we tested the effectiveness of calcium AM staining following long incubation periods post cell loading and its impact on the functional properties of calcium signals in acute brain slices and wholemount retinae. We show that calcium dyes remain within cells and are fully functional >24 hours after loading. Moreover, the calcium dynamics recorded >24 hrs were similar to the calcium signals recorded in fresh tissue that was incubated for <4 hrs. These results indicate that long exposure of calcium AM dyes to the intracellular cytoplasm did not alter the intracellular calcium concentration, the functional range of the dye or viability of the neurons. This data extends our previous work showing that a custom recovery chamber can extend the viability of neuronal tissue, and reliable data for both electrophysiology and imaging can be obtained >24hrs after dissection. These methods will not only extend experimental time for those using acute neuronal tissue, but also may reduce the number of animals required to complete experimental goals. PMID:27183102

  6. Intracellular Parasite Invasion Strategies

    NASA Astrophysics Data System (ADS)

    Sibley, L. D.

    2004-04-01

    Intracellular parasites use various strategies to invade cells and to subvert cellular signaling pathways and, thus, to gain a foothold against host defenses. Efficient cell entry, ability to exploit intracellular niches, and persistence make these parasites treacherous pathogens. Most intracellular parasites gain entry via host-mediated processes, but apicomplexans use a system of adhesion-based motility called ``gliding'' to actively penetrate host cells. Actin polymerization-dependent motility facilitates parasite migration across cellular barriers, enables dissemination within tissues, and powers invasion of host cells. Efficient invasion has brought widespread success to this group, which includes Toxoplasma, Plasmodium, and Cryptosporidium.

  7. Two complementary approaches for intracellular delivery of exogenous enzymes

    PubMed Central

    Rust, Aleksander; Hassan, Hazirah H. A.; Sedelnikova, Svetlana; Niranjan, Dhevahi; Hautbergue, Guillaume; Abbas, Shaymaa A.; Partridge, Lynda; Rice, David; Binz, Thomas; Davletov, Bazbek

    2015-01-01

    Intracellular delivery of biologically active proteins remains a formidable challenge in biomedical research. Here we show that biomedically relevant enzymes can be delivered into cells using a new DNA transfection reagent, lipofectamine 3000, allowing assessment of their intracellular functions. We also show that the J774.2 macrophage cell line exhibits unusual intracellular uptake of structurally and functionally distinct enzymes providing a convenient, reagent-free approach for evaluation of intracellular activities of enzymes. PMID:26207613

  8. Two complementary approaches for intracellular delivery of exogenous enzymes.

    PubMed

    Rust, Aleksander; Hassan, Hazirah H A; Sedelnikova, Svetlana; Niranjan, Dhevahi; Hautbergue, Guillaume; Abbas, Shaymaa A; Partridge, Lynda; Rice, David; Binz, Thomas; Davletov, Bazbek

    2015-01-01

    Intracellular delivery of biologically active proteins remains a formidable challenge in biomedical research. Here we show that biomedically relevant enzymes can be delivered into cells using a new DNA transfection reagent, lipofectamine 3000, allowing assessment of their intracellular functions. We also show that the J774.2 macrophage cell line exhibits unusual intracellular uptake of structurally and functionally distinct enzymes providing a convenient, reagent-free approach for evaluation of intracellular activities of enzymes. PMID:26207613

  9. Histone Deacetylase Inhibitors Prolong Cardiac Repolarization through Transcriptional Mechanisms.

    PubMed

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

    2016-09-01

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

  10. Prolonged Starvation—A Dangerous Procedure

    PubMed Central

    Runcie, J.; Thomson, T. J.

    1970-01-01

    Experience with 18 obese patients who have undergone prolonged (60 days) therapeutic starvation shows that in general this is a safe procedure, but there are significant associated hazards, particularly a breakdown in electrolyte homoeostasis. The need for close biochemical control of such patients is stressed. PMID:5454322

  11. Intracellular Penetration and Activity of Gemifloxacin in Human Polymorphonuclear Leukocytes

    PubMed Central

    García, Isabel; Pascual, Alvaro; Ballesta, Sofía; Joyanes, Providencia; Perea, Evelio J.

    2000-01-01

    The intracellular penetration and activity of gemifloxacin in human polymorphonuclear leukocytes (PMN) were evaluated. Gemifloxacin reached intracellular concentrations eight times higher than extracellular concentrations. The uptake was rapid, reversible, and nonsaturable and was affected by environmental temperature, cell viability, and membrane stimuli. At therapeutic extracellular concentrations, gemifloxacin showed intracellular activity against Staphylococcus aureus. PMID:11036051

  12. Azelnidipine prevents cardiac dysfunction in streptozotocin-diabetic rats by reducing intracellular calcium accumulation, oxidative stress and apoptosis

    PubMed Central

    2011-01-01

    Background Numerous evidences suggest that diabetic heart is characterized by compromised ventricular contraction and prolonged relaxation attributable to multiple causative factors including calcium accumulation, oxidative stress and apoptosis. Therapeutic interventions to prevent calcium accumulation and oxidative stress could be therefore helpful in improving the cardiac function under diabetic condition. Methods This study was designed to examine the effect of long-acting calcium channel blocker (CCB), Azelnidipine (AZL) on contractile dysfunction, intracellular calcium (Ca2+) cycling proteins, stress-activated signaling molecules and apoptosis on cardiomyocytes in diabetes. Adult male Wistar rats were made diabetic by a single intraperitoneal (IP) injection of streptozotocin (STZ). Contractile functions were traced from live diabetic rats to isolated individual cardiomyocytes including peak shortening (PS), time-to-PS (TPS), time-to-relengthening (TR90), maximal velocity of shortening/relengthening (± dL/dt) and intracellular Ca2+ fluorescence. Results Diabetic heart showed significantly depressed PS, ± dL/dt, prolonged TPS, TR90 and intracellular Ca2+ clearing and showed an elevated resting intracellular Ca2+. AZL itself exhibited little effect on myocyte mechanics but it significantly alleviated STZ-induced myocyte contractile dysfunction. Diabetes increased the levels of superoxide, enhanced expression of the cardiac damage markers like troponin I, p67phox NADPH oxidase subunit, restored the levels of the mitochondrial superoxide dismutase (Mn-SOD), calcium regulatory proteins RyR2 and SERCA2a, and suppressed the levels of the anti-apoptotic Bcl-2 protein. All of these STZ-induced alterations were reconciled by AZL treatment. Conclusion Collectively, the data suggest beneficial effect of AZL in diabetic cardiomyopathy via altering intracellular Ca2+ handling proteins and preventing apoptosis by its antioxidant property. PMID:22054019

  13. Chloride Channels of Intracellular Membranes

    PubMed Central

    Edwards, John C.; Kahl, Christina R.

    2010-01-01

    Proteins implicated as intracellular chloride channels include the intracellular ClC proteins, the bestrophins, the cystic fibrosis transmembrane conductance regulator, the CLICs, and the recently described Golgi pH regulator. This paper examines current hypotheses regarding roles of intracellular chloride channels and reviews the evidence supporting a role in intracellular chloride transport for each of these proteins. PMID:20100480

  14. "The Show"

    ERIC Educational Resources Information Center

    Gehring, John

    2004-01-01

    For the past 16 years, the blue-collar city of Huntington, West Virginia, has rolled out the red carpet to welcome young wrestlers and their families as old friends. They have come to town chasing the same dream for a spot in what many of them call "The Show". For three days, under the lights of an arena packed with 5,000 fans, the state's best…

  15. Glutamate-induced intracellular calcium oscillations in astrocytes with confocal microscopy

    NASA Astrophysics Data System (ADS)

    Zhang, Yuan; Zhou, Wei; Liu, Xiuli; Zhu, Geng; Wu, Yuxiang; Luo, Qingming

    2006-02-01

    Changes in the intracellular Ca 2+ concentration ([Ca 2+]i) play a crucial role involved in the modulation of signal transduction, development, and plasticity in the CNS. Glial cells can respond to various stimuli with an increase in [Ca 2+]i. In this paper, we used confocal microscopy to study calcium transient induced by glutamate in cultured astrocytes. Firstly, 100 μM glutamate induced long-time intracellular calcium oscillations in astrocytes and only a single spike under calcium-free solution. When the concentration of glutamate decreased to 1 μM, only a single spike could be induced. It shows that intracellular calcium oscillations depend on agonist concentration and extracellular Ca 2+. Secondly, we investigated amplitude of responses under different stimulation. The amplitude of initial peak induced by 100 μM glutamate decreased in Ca 2+-free condition, whereas the duration of kinetics was prolonged. But both the amplitude and area of a single spike induced by 1 μM Glu decreased in Ca 2+-free condition. The results show that areaof peak is more accurate than amplitude to display transients of [Ca 2+]i. All results above suggest that astrocytes are not passive, they display diverse temporal and spatial increases in [Ca 2+]i in response to a variety of stimuli. These [Ca 2+]i increases provide a possible means for information coding.

  16. Prolonged hypothermia exposure diminishes neuroprotection for severe ischemic-hypoxic primary neurons.

    PubMed

    Gao, Xiao-Ya; Zhu, Shu-Zhen; Xiang, Wei; Huang, Kai-Bin; Hu, Ya-Fang; Gu, Yong; Pan, Su-Yue

    2016-04-01

    This study aimed to identify optimal mild hypothermic (MH) condition that would provide the best protection for neuronal cells undergoing severe ischemia and hypoxia. We also sought to determine if longer exposure to mild hypothermia would confer greater protection to severe ischemia and hypoxia in these cells. We designed a primary neuronal cell model for severe glucose and oxygen deprivation/reoxygenation (OGD/R) to simulate the hypoxic-ischemic condition of patients with severe stroke, trauma, or hypoxic-ischemic encephalopathy. We evaluated the viability of these neurons following 3 h of OGD/R and variable MH conditions including different temperatures and durations of OGD/R exposure. We further explored the effects of the optimal MH condition on several parts which are associated with mitochondrial apoptosis pathway: intracellular calcium, reactive oxygen species (ROS), and mitochondrial transmembrane potential (MTP). The results of this study showed that the apoptosis proportion (AP) and cell viability proportion (CVP) after OGD/R significantly varied depending on which MH condition cells were exposed to (p < 0.001). Further, our findings showed that prolonged MH reduced the neuroprotection to AP and CVP. We also determined that the optimal MH conditions (34 °C for 4.5 h) reduced intracellular calcium, ROS, and recovered MTP. These findings indicate that there is an optimal MH treatment strategy for severely hypoxia-ischemic neurons, prolonged duration might diminish the neuroprotection, and that MH treatment likely initiates neuroprotection by inhibiting the mitochondrial apoptosis pathway. PMID:26802735

  17. Physiology Of Prolonged Bed Rest

    NASA Technical Reports Server (NTRS)

    Greenleaf, John E.

    1991-01-01

    Report describes physiological effects of prolonged bed rest. Rest for periods of 24 hours or longer deconditions body to some extent; healing proceeds simultaneously with deconditioning. Report provides details on shifts in fluid electrolytes and loss of lean body mass, which comprises everything in body besides fat - that is, water, muscle, and bone. Based on published research.

  18. Measures that Prolong Work Life.

    ERIC Educational Resources Information Center

    Nusberg, Charlotte

    1986-01-01

    Discusses measures that have been adopted by France, Great Britain, Sweden, the Netherlands, the United States, and Japan to prolong the work life of older workers. Measures include job transfer and exemption, dismissal protection, retirement policies, and reintegration of unemployed older workers. (JOW)

  19. Prolonged abulia following putaminal hemorrhage.

    PubMed

    Nagaratnam, N; Fanella, S; Gopinath, S; Goodwin, A

    2001-01-01

    Abulia, akinetic mutism, and other conditions causing reduced activity and slowness are a continuum of severity of behavior. Unilateral lesions usually cause transient symptoms. This article describes a patient with prolonged abulia lasting 12 weeks after aspontaneous left putaminal hemorrhage. He developed seizures that could be a contributing factor. The pathophysiologic mechanisms are discussed. PMID:17903806

  20. Glycan modification of antigen alters its intracellular routing in dendritic cells, promoting priming of T cells

    PubMed Central

    Streng-Ouwehand, Ingeborg; Ho, Nataschja I; Litjens, Manja; Kalay, Hakan; Boks, Martine Annemarie; Cornelissen, Lenneke AM; Kaur Singh, Satwinder; Saeland, Eirikur; Garcia-Vallejo, Juan J; Ossendorp, Ferry A; Unger, Wendy WJ; van Kooyk, Yvette

    2016-01-01

    Antigen uptake by dendritic cells and intracellular routing of antigens to specific compartments is regulated by C-type lectin receptors that recognize glycan structures. We show that the modification of Ovalbumin (OVA) with the glycan-structure LewisX (LeX) re-directs OVA to the C-type lectin receptor MGL1. LeX-modification of OVA favored Th1 skewing of CD4+ T cells and enhanced cross-priming of CD8+ T cells. While cross-presentation of native OVA requires high antigen dose and TLR stimuli, LeX modification reduces the required amount 100-fold and obviates its dependence on TLR signaling. The OVA-LeX-induced enhancement of T cell cross-priming is MGL1-dependent as shown by reduced CD8+ effector T cell frequencies in MGL1-deficient mice. Moreover, MGL1-mediated cross-presentation of OVA-LeX neither required TAP-transporters nor Cathepsin-S and was still observed after prolonged intracellular storage of antigen in Rab11+LAMP1+ compartments. We conclude that controlled neo-glycosylation of antigens can crucially influence intracellular routing of antigens, the nature and strength of immune responses and should be considered for optimizing current vaccination strategies. DOI: http://dx.doi.org/10.7554/eLife.11765.001 PMID:26999763

  1. Propofol-associated QTc prolongation

    PubMed Central

    Scalese, Michael J.; Herring, Holly R.; Rathbun, R. Chris; Skrepnek, Grant H.; Ripley, Toni L.

    2016-01-01

    Objectives: Propofol is a preferred agent for sedation in patients in the intensive care unit (ICU) due, in part, to its established safety profile. Despite this, recent case reports have suggested a potential for prolongation of the corrected QT interval (QTc) in ICU patients receiving propofol, though limited empirical work has been conducted to evaluate this association. As such, the purpose of this study was to assess the relationship between propofol infusion and QTc prolongation in a historical cohort of ICU patients. Methods: A single-center, historical, observational, pre-post cohort analysis of medical records from admitted patients ⩾18 years old with cardiovascular disease was conducted, involving cases who received propofol infusion for ⩾3 hours with sequential electrocardiogram monitoring from 2006 to 2012. A multivariable, generalized linear model regression was employed to assess the primary outcome of on-propofol QTc interval (QTc2), controlling for various demographic and clinical factors. Results: A total of 96 patients met inclusion criteria, averaging 56.1 ± 14.1 years of age and 86.1 ± 25.0 kg, with 37.5% being female. A mean prolongation in QTc interval of 30.4 ± 55.5 ms (p < 0.001) was observed during the propofol infusion, with 43.8% of cases exhibiting an on-infusion QTc2 of ⩾ 500 ms. Regression analyses suggested that prolongation in on-propofol QTc was independently associated with baseline QTc interval and amiodarone use, while weight as inversely associated with QTc2 (p < 0.05). Conclusion: This historical cohort analysis of adult ICU patients receiving propofol suggests that on-infusion QTc prolongation was associated with increasing baseline QTc interval and with amiodarone use. Further research is needed to evaluate the clinical significance and cause-and-effect relationship between potential QTc changes and propofol use in the ICU. PMID:27298717

  2. Prolonged stimulus exposure reveals prolonged neurobehavioral response patterns.

    PubMed

    Johnson, Brett A; Woo, Cynthia C; Zeng, Yu; Xu, Zhe; Hingco, Edna E; Ong, Joan; Leon, Michael

    2010-05-15

    Although it has been shown repeatedly that minimum response times in sensory systems can be quite short, organisms more often continue to respond to sensory stimuli over considerably longer periods of time. The continuing response to sensory stimulation may be a more realistic assessment of natural sensory responses, so we determined for how long a stimulus would evoke a response in naïve, freely moving animals. Specifically, we determined for how long such rats responded to odorants during continuous passive exposures by monitoring their sniffing with whole-body plethysmography. We found that naïve rats continue to sniff odorants vigorously for up to 3 minutes, much longer than what has been reported for highly trained, highly motivated rats. Patterns of 2-deoxyglucose (2-DG) uptake in the glomerular layer of the rat olfactory bulb also were seen after only 1-5 minutes of odorant exposure, overlapping with the period of increased respiration to odorants. Moreover, these 2-DG uptake patterns closely resembled the patterns that emerge from prolonged odorant exposures, suggesting that activity mapping over prolonged periods can identify areas of activity that are present when rats are still attending and responding to odorant stimuli. Given these findings, it seems important to consider the possibility that prolonged exposure to other sensory stimuli will reveal more realistic neural response patterns. PMID:20232477

  3. Nanovehicular intracellular delivery systems.

    PubMed

    Prokop, Ales; Davidson, Jeffrey M

    2008-09-01

    This article provides an overview of principles and barriers relevant to intracellular drug and gene transport, accumulation and retention (collectively called as drug delivery) by means of nanovehicles (NV). The aim is to deliver a cargo to a particular intracellular site, if possible, to exert a local action. Some of the principles discussed in this article apply to noncolloidal drugs that are not permeable to the plasma membrane or to the blood-brain barrier. NV are defined as a wide range of nanosized particles leading to colloidal objects which are capable of entering cells and tissues and delivering a cargo intracelullarly. Different localization and targeting means are discussed. Limited discussion on pharmacokinetics and pharmacodynamics is also presented. NVs are contrasted to micro-delivery and current nanotechnologies which are already in commercial use. Newer developments in NV technologies are outlined and future applications are stressed. We also briefly review the existing modeling tools and approaches to quantitatively describe the behavior of targeted NV within the vascular and tumor compartments, an area of particular importance. While we list "elementary" phenomena related to different level of complexity of delivery to cancer, we also stress importance of multi-scale modeling and bottom-up systems biology approach. PMID:18200527

  4. Nanovehicular Intracellular Delivery Systems

    PubMed Central

    PROKOP, ALES; DAVIDSON, JEFFREY M.

    2013-01-01

    This article provides an overview of principles and barriers relevant to intracellular drug and gene transport, accumulation and retention (collectively called as drug delivery) by means of nanovehicles (NV). The aim is to deliver a cargo to a particular intracellular site, if possible, to exert a local action. Some of the principles discussed in this article apply to noncolloidal drugs that are not permeable to the plasma membrane or to the blood–brain barrier. NV are defined as a wide range of nanosized particles leading to colloidal objects which are capable of entering cells and tissues and delivering a cargo intracelullarly. Different localization and targeting means are discussed. Limited discussion on pharmacokinetics and pharmacodynamics is also presented. NVs are contrasted to micro-delivery and current nanotechnologies which are already in commercial use. Newer developments in NV technologies are outlined and future applications are stressed. We also briefly review the existing modeling tools and approaches to quantitatively describe the behavior of targeted NV within the vascular and tumor compartments, an area of particular importance. While we list “elementary” phenomena related to different level of complexity of delivery to cancer, we also stress importance of multi-scale modeling and bottom-up systems biology approach. PMID:18200527

  5. Metabolism of normothermic woodchucks during prolonged fasting.

    PubMed

    Reidy, Shannon P; Weber, Jean-Michel

    2004-12-01

    The energy metabolism of hibernators has not been characterized for normothermic fasting, and our goal was to quantify oxidative fuel selection of non-hibernating woodchucks Marmota monax during prolonged food deprivation. Indirect calorimetry and nitrogen excretion measurements were used to assess changes in metabolic rate (VO2), fuel selection and composition of nitrogen wastes, as well as seasonal differences. For reference, matching experiments were also performed on rabbits. The results show that woodchucks have a higher metabolic rate in summer (271 micromol O2 kg(-1) min(-1)) than in spring (200 micromol O2 kg(-1) min(-1)) and that fasting-induced metabolic depression is only possible in summer (-25% in 14 days). The metabolic rate of rabbits is high at all times (383 micromol O2 kg(-1) min(-1)), but they show a more rapid depression in response to fasting (-32% in 7 days). Woodchucks have a naturally low reliance on proteins in the fed state (accounting for 8% VO2) in spring; 17% VO2 in summer; vs 28% VO2 in rabbits) and are able to decrease it even further during fasting (spring, 5% VO2); summer, 6% VO2; vs 20% VO2 in rabbits). This study shows that, apart from their notorious capacity for hibernation, woodchucks are particularly well adapted for normothermic fasting. Their ability to cope with prolonged food deprivation is based on a series of integrated responses eliciting deep metabolic depression and a rapid change in fuel selection to spare limited protein reserves. Information presently available on prolonged fasting suggests that such an ability for metabolic depression, possibly down to minimal levels still compatible with normothermic life, may be common among mammals. In contrast, the extreme protein sparing demonstrated in woodchucks is a unique metabolic feature of fasting champions. PMID:15579548

  6. GABAAergic stimulation modulates intracellular protein arginine methylation.

    PubMed

    Denman, Robert B; Xie, Wen; Merz, George; Sung, Ying-Ju

    2014-06-20

    Changes in cytoplasmic pH are known to regulate diverse cellular processes and influence neuronal activities. In neurons, the intracellular alkalization is shown to occur after stimulating several channels and receptors. For example, it has previously demonstrated in P19 neurons that a sustained intracellular alkalinization can be mediated by the Na(+)/H(+) antiporter. In addition, the benzodiazepine binding subtypes of the γ-amino butyric acid type A (GABAA) receptor mediate a transient intracellular alkalinization when they are stimulated. Because the activities of many enzymes are sensitive to pH shift, here we investigate the effects of intracellular pH modulation resulted from stimulating GABAA receptor on the protein arginine methyltransferases (PRMT) activities. We show that the major benzodiazepine subtype (2α1, 2β2, 1γ2) is constitutively expressed in both undifferentiated P19 cells and retinoic acid (RA) differentiated P19 neurons. Furthermore stimulation with diazepam and, diazepam plus muscimol produce an intracellular alkalinization that can be detected ex vivo with the fluorescence dye. The alkalinization results in significant perturbation in protein arginine methylation activity as measured in methylation assays with specific protein substrates. Altered protein arginine methylation is also observed when cells are treated with the GABAA agonist muscimol but not an antagonist, bicuculline. These data suggest that pH-dependent and pH-independent methylation pathways can be activated by GABAAergic stimulation, which we verified using hippocampal slice preparations from a mouse model of fragile X syndrome. PMID:24793772

  7. Chemical development of intracellular protein heterodimerizers.

    PubMed

    Erhart, Dominik; Zimmermann, Mirjam; Jacques, Olivier; Wittwer, Matthias B; Ernst, Beat; Constable, Edwin; Zvelebil, Marketa; Beaufils, Florent; Wymann, Matthias P

    2013-04-18

    Cell activation initiated by receptor ligands or oncogenes triggers complex and convoluted intracellular signaling. Techniques initiating signals at defined starting points and cellular locations are attractive to elucidate the output of selected pathways. Here, we present the development and validation of a protein heterodimerization system based on small molecules cross-linking fusion proteins derived from HaloTags and SNAP-tags. Chemical dimerizers of HaloTag and SNAP-tag (HaXS) show excellent selectivity and have been optimized for intracellular reactivity. HaXS force protein-protein interactions and can translocate proteins to various cellular compartments. Due to the covalent nature of the HaloTag-HaXS-SNAP-tag complex, intracellular dimerization can be easily monitored. First applications include protein targeting to cytoskeleton, to the plasma membrane, to lysosomes, the initiation of the PI3K/mTOR pathway, and multiplexed protein complex formation in combination with the rapamycin dimerization system. PMID:23601644

  8. Biodegradable silicon nanoneedles delivering nucleic acids intracellularly induce localized in vivo neovascularization

    NASA Astrophysics Data System (ADS)

    Chiappini, C.; De Rosa, E.; Martinez, J. O.; Liu, X.; Steele, J.; Stevens, M. M.; Tasciotti, E.

    2015-05-01

    The controlled delivery of nucleic acids to selected tissues remains an inefficient process mired by low transfection efficacy, poor scalability because of varying efficiency with cell type and location, and questionable safety as a result of toxicity issues arising from the typical materials and procedures employed. High efficiency and minimal toxicity in vitro has been shown for intracellular delivery of nuclei acids by using nanoneedles, yet extending these characteristics to in vivo delivery has been difficult, as current interfacing strategies rely on complex equipment or active cell internalization through prolonged interfacing. Here, we show that a tunable array of biodegradable nanoneedles fabricated by metal-assisted chemical etching of silicon can access the cytosol to co-deliver DNA and siRNA with an efficiency greater than 90%, and that in vivo the nanoneedles transfect the VEGF-165 gene, inducing sustained neovascularization and a localized sixfold increase in blood perfusion in a target region of the muscle.

  9. Intracellular Sterol Dynamics

    PubMed Central

    Mesmin, Bruno; Maxfield, Frederick R.

    2009-01-01

    We review the cellular mechanisms implicated in cholesterol trafficking and distribution. Recent studies have provided new information about the distribution of sterols within cells, including analysis of its transbilayer distribution. The cholesterol interaction with other lipids and its engagement in various trafficking processes will determine its proper level in a specific membrane; making the cholesterol distribution uneven among the various intracellular organelles. The cholesterol content is important since cholesterol plays an essential role in membranes by controlling their physicochemical properties as well as key cellular events such as signal transduction and protein trafficking. Cholesterol movement between cellular organelles is highly dynamic, and can be achieved by vesicular and non-vesicular processes. Various studies have analyzed the proteins that play a significant role in these processes, giving us new information about the relative importance of these two trafficking pathways in cholesterol transport. Although still poorly characterized in many trafficking routes, several potential sterol transport proteins have been described in detail; as a result, molecular mechanisms for sterol transport among membranes start to be appreciated. PMID:19286471

  10. Human muscle function following prolonged eccentric exercise.

    PubMed

    Sargeant, A J; Dolan, P

    1987-01-01

    4 subjects performed repeated eccentric contractions with leg extensors during prolonged downhill walking (-25% gradient) at 6.44 km.h-1 until collapse due to muscle weakness (range of exercise duration 29 to 40 min). During the exercise oxygen uptake rose progressively from approximately 45% of the previously determined VO2max at 10 min to approximately 65% at the end of the exercise. Following the exercise there was an immediate, significant, and sustained reduction in maximal voluntary isometric contraction, and short term (anaerobic) power output measured concentrically on an isokinetic ergometer. These reductions in muscle function persisted for 96 hours post exercise, and were reflected by significant reductions in the tension generated at low frequency (20 Hz) relative to higher frequency (50 Hz) percutaneous stimulation of the quadriceps. All four subjects showed an increase in plasma levels of creatine kinase post eccentric exercise. Performing concentric contractions by walking uphill for one hour at a significantly greater metabolic cost failed to induce comparable reductions in muscle function. These results provide evidence for the consequences of prolonged eccentric work upon dynamic function which complements earlier reports of structural, enzymatic, and static function changes. PMID:3678226

  11. Moving frames and prolongation algebras

    NASA Technical Reports Server (NTRS)

    Estabrook, F. B.

    1982-01-01

    Differential ideals generated by sets of 2-forms which can be written with constant coefficients in a canonical basis of 1-forms are considered. By setting up a Cartan-Ehresmann connection, in a fiber bundle over a base space in which the 2-forms live, one finds an incomplete Lie algebra of vector fields in the fields in the fibers. Conversely, given this algebra (a prolongation algebra), one can derive the differential ideal. The two constructs are thus dual, and analysis of either derives properties of both. Such systems arise in the classical differential geometry of moving frames. Examples of this are discussed, together with examples arising more recently: the Korteweg-de Vries and Harrison-Ernst systems.

  12. INTRACELLULAR SIGNALING AND DEVELOPMENTAL NEUROTOXICITY.

    EPA Science Inventory

    A book chapter in ?Molecular Toxicology: Transcriptional Targets? reviewed the role of intracellular signaling in the developmental neurotoxicity of environmental chemicals. This chapter covered a number of aspects including the development of the nervous system, role of intrace...

  13. Overexpression of hsp27 Rescued Neuronal Cell Death and Reduction in Life- and Health-Span in Drosophila melanogaster Against Prolonged Exposure to Dichlorvos.

    PubMed

    Pandey, Ashutosh; Saini, Sanjay; Khatoon, Rehana; Sharma, Divya; Narayan, Gopeshwar; Kar Chowdhuri, Debapratim

    2016-07-01

    Long-term exposure to dichlorvos (O,O-dimethyl-2,2-dichlorovinyl phosphate (DDVP), an organophosphate pesticide) is reported to exert neurotoxicity, i.e., generation of reactive oxygen species (ROS), oxidative damage, and neuronal cell death along with life- and health-span reduction in nontarget organisms including humans. However, studies on genetic modulation towards neuroprotection against prolonged DDVP exposure are elusive. Hsp27 (a small heat shock protein) is involved in various cellular processes and thus has attained emphasis as a therapeutic target. We aimed to examine the protective effect of hsp27 overexpression against prolonged DDVP exposure using an in vivo model Drosophila melanogaster. Flies were exposed to 15.0 ng/ml DDVP for a prolonged period to examine neuronal cell death, locomotor performance, and lifespan. After prolonged exposure, cell death, ROS level, glutathione depletion, nicotinamide adenine dinucleotide phosphate level (NADPH), glucose-6-phosphate dehydrogenase (G6PD), and thioredoxin reductase (TrxR) activities were examined in fly brain tissues at different days of age (days 10, 20, and 30). Flies with ubiquitous overexpression of hsp27 showed better resistance (improved lifespan and locomotor performance) in comparison to that targeted to motor neurons and nervous system. These flies also exhibited lesser intracellular ROS level and glutathione depletion by restoring G6PD activity, NADPH level, and TrxR activity in their brains thereby resisted neuronal cell death. Conversely, hsp27 knockdown flies exhibited reversal of the above endpoints. The study evidenced the neuroprotective efficacy of hsp27 overexpression against prolonged DDVP exposure and favored Hsp27 as a therapeutic target towards achieving better organismal (including human) health against long-term chemical exposure. PMID:26033218

  14. Pharmacometabolomic Approach to Predict QT Prolongation in Guinea Pigs

    PubMed Central

    Lee, Hae Won; Lim, Mi-sun; Seong, Sook Jin; Seo, Jeong Ju; Kim, Eun-Jung; Kang, Wonku; Yoon, Young-Ran

    2013-01-01

    Drug-induced torsades de pointes (TdP), a life-threatening arrhythmia associated with prolongation of the QT interval, has been a significant reason for withdrawal of several medicines from the market. Prolongation of the QT interval is considered as the best biomarker for predicting the torsadogenic risk of a new chemical entity. Because of the difficulty assessing the risk for TdP during drug development, we evaluated the metabolic phenotype for predicting QT prolongation induced by sparfloxacin, and elucidated the metabolic pathway related to the QT prolongation. We performed electrocardiography analysis and liquid chromatography–mass spectroscopy-based metabolic profiling of plasma samples obtained from 15 guinea pigs after administration of sparfloxacin at doses of 33.3, 100, and 300 mg/kg. Principal component analysis and partial least squares modelling were conducted to select the metabolites that substantially contributed to the prediction of QT prolongation. QTc increased significantly with increasing dose (r = 0.93). From the PLS analysis, the key metabolites that showed the highest variable importance in the projection values (>1.5) were selected, identified, and used to determine the metabolic network. In particular, cytidine-5′-diphosphate (CDP), deoxycorticosterone, L-aspartic acid and stearic acid were found to be final metabolomic phenotypes for the prediction of QT prolongation. Metabolomic phenotypes for predicting drug-induced QT prolongation of sparfloxacin were developed and can be applied to cardiac toxicity screening of other drugs. In addition, this integrative pharmacometabolomic approach would serve as a good tool for predicting pharmacodynamic or toxicological effects caused by changes in dose. PMID:23593245

  15. Physiology of prolonged bed rest

    NASA Technical Reports Server (NTRS)

    Greenleaf, J. E.

    1988-01-01

    Bed rest has been a normal procedure used by physicians for centuries in the treatment of injury and disease. Exposure of patients to prolonged bed rest in the horizontal position induces adaptive deconditioning responses. While deconditioning responses are appropriate for patients or test subjects in the horizontal position, they usually result in adverse physiological responses (fainting, muscular weakness) when the patient assume the upright posture. These deconditioning responses result from reduction in hydrostatic pressure within the cardiovascular system, virtual elimination of longitudinal pressure on the long bones, some decrease in total body metabolism, changes in diet, and perhaps psychological impact from the different environment. Almost every system in the body is affected. An early stimulus is the cephalic shift of fluid from the legs which increases atrial pressure and induces compensatory responses for fluid and electrolyte redistribution. Without countermeasures, deterioration in strength and muscle function occurs within 1 wk while increased calcium loss may continue for months. Research should also focus on drug and carbohydrate metabolism.

  16. Imaging and controlling intracellular reactions: Lysosome transport as a function of diameter and the intracellular synthesis of conducting polymers

    NASA Astrophysics Data System (ADS)

    Payne, Christine

    2014-03-01

    Eukaryotic cells are the ultimate complex environment with intracellular chemical reactions regulated by the local cellular environment. For example, reactants are sequestered into specific organelles to control local concentration and pH, motor proteins transport reactants within the cell, and intracellular vesicles undergo fusion to bring reactants together. Current research in the Payne Lab in the School of Chemistry and Biochemistry at Georgia Tech is aimed at understanding and utilizing this complex environment to control intracellular chemical reactions. This will be illustrated using two examples, intracellular transport as a function of organelle diameter and the intracellular synthesis of conducting polymers. Using single particle tracking fluorescence microscopy, we measured the intracellular transport of lysosomes, membrane-bound organelles, as a function of diameter as they underwent transport in living cells. Both ATP-dependent active transport and diffusion were examined. As expected, diffusion scales with the diameter of the lysosome. However, active transport is unaffected suggesting that motor proteins are insensitive to cytosolic drag. In a second example, we utilize intracellular complexity, specifically the distinct micro-environments of different organelles, to carry out chemical reactions. We show that catalase, found in the peroxisomes of cells, can be used to catalyze the polymerization of the conducting polymer PEDOT:PSS. More importantly, we have found that a range of iron-containing biomolecules are suitable catalysts with different iron-containing biomolecules leading to different polymer properties. These experiments illustrate the advantage of intracellular complexity for the synthesis of novel materials.

  17. [Excessive energy drink consumption caused marked QT prolongation. Case report].

    PubMed

    Tomcsányi, János; Jávor, Kinga

    2015-10-25

    The authors report a case of a 22-year-old man with atypical chest pain after consumption of six energy drinks (1.5 liter containing 470 mg coffein) with vodka. On admission ECG showed marked QT/QTc prolongation (QT/QTc, 520/580 msec). Next day the QT/QTc returned to fully normal (QT/QTc, 360/430 msec). It was assumed that the patient had a silent long QT syndrome and that high dose of highly caffeinated energy drink triggered the (paradoxical) prolonged QT/QTc. The authors conclude that excessive energy drink intake with alcohol or during physical exercise should be avoided. PMID:26477618

  18. TgERK7 is involved in the intracellular proliferation of Toxoplasma gondii.

    PubMed

    Li, Zhong-Yuan; Wang, Ze-Dong; Huang, Si-Yang; Zhu, Xing-Quan; Liu, Quan

    2016-09-01

    Toxoplasma gondii uses a unique mechanism to fulfill its asexual life cycles by which the parasite can infect all the warm-blooded animals including humans. Mitogen-activated protein kinase (MAPK) or extracellular signal-regulated kinase (ERK) pathway widely existed in eukaryotic cells mediates the conversion of environmental stimuli to intracellular events such as proliferation and differentiation. Their counterparts have been identified in Apicomplexan parasites such as ERK7 in T. gondii. To confirm whether the unique mechanism of T. gondii is relevant to MAPK/ERK member, we created a mutant (ΔTgERK7) in GT1 tachyzoites using double homologous recombination method. Our results of virulence evaluation showed 100 % survival of all the ΔTgERK7-infected mice until 35 days post-challenge compared to no survival in wild-type GT1-infected group (10.6 ± 0.34 days). Furthermore, lower parasite loads were detected in the peritoneal fluid of ΔTgERK7-infected mice (P < 0.05). To ensure whether or not ERK7 gene knockout leads to the growth deficiency of T. gondii, the intracellular proliferation of ΔTgERK7 was also examined in vitro. Our data indicated that the proliferation of ΔTgERK7 parasites was significantly prolonged in comparison with wild-type GT1 tachyzoites (P < 0.05). Therefore, we concluded that TgERK7 is important for the intracellular proliferation of T. gondii, which further emphasized that MAPK/ERK derived from T. gondii participates in the regulation of the asexual life cycles to ensure the survival and reinfections of this parasite. PMID:27150970

  19. The CovS/CovR acid response regulator is required for intracellular survival of group B Streptococcus in macrophages.

    PubMed

    Cumley, Nicola J; Smith, Leanne M; Anthony, Mark; May, Robin C

    2012-05-01

    Group B Streptococcus (GBS) is a leading cause of neonatal meningitis and septicemia. The ability of this organism to survive inside phagocytic cells is poorly understood but thought to be an important step for the establishment of disease in the host. Here, we demonstrate that GBS shows prolonged survival within J774 macrophages and that the capacity to survive is not significantly changed across a diverse range of strains representing different serotypes, multilocus sequence types (MLST), and sites of clinical isolation. Using staining for the lysosome-associated membrane protein (LAMP) and by pharmacological inhibition of phagosome acidification, we demonstrate that streptococci reside in a phagosome and that acidification of the phagosome is required for GBS to survive intracellularly. Moreover, we show that the GBS two-component system CovS/CovR, which is the major acid response regulator in this organism, is required for survival inside the phagosome. PMID:22331428

  20. The CovS/CovR Acid Response Regulator Is Required for Intracellular Survival of Group B Streptococcus in Macrophages

    PubMed Central

    Cumley, Nicola J.; Smith, Leanne M.; Anthony, Mark

    2012-01-01

    Group B Streptococcus (GBS) is a leading cause of neonatal meningitis and septicemia. The ability of this organism to survive inside phagocytic cells is poorly understood but thought to be an important step for the establishment of disease in the host. Here, we demonstrate that GBS shows prolonged survival within J774 macrophages and that the capacity to survive is not significantly changed across a diverse range of strains representing different serotypes, multilocus sequence types (MLST), and sites of clinical isolation. Using staining for the lysosome-associated membrane protein (LAMP) and by pharmacological inhibition of phagosome acidification, we demonstrate that streptococci reside in a phagosome and that acidification of the phagosome is required for GBS to survive intracellularly. Moreover, we show that the GBS two-component system CovS/CovR, which is the major acid response regulator in this organism, is required for survival inside the phagosome. PMID:22331428

  1. Calcium Transients Closely Reflect Prolonged Action Potentials in iPSC Models of Inherited Cardiac Arrhythmia

    PubMed Central

    Spencer, C. Ian; Baba, Shiro; Nakamura, Kenta; Hua, Ethan A.; Sears, Marie A.F.; Fu, Chi-cheng; Zhang, Jianhua; Balijepalli, Sadguna; Tomoda, Kiichiro; Hayashi, Yohei; Lizarraga, Paweena; Wojciak, Julianne; Scheinman, Melvin M.; Aalto-Setälä, Katriina; Makielski, Jonathan C.; January, Craig T.; Healy, Kevin E.; Kamp, Timothy J.; Yamanaka, Shinya; Conklin, Bruce R.

    2014-01-01

    Summary Long-QT syndrome mutations can cause syncope and sudden death by prolonging the cardiac action potential (AP). Ion channels affected by mutations are various, and the influences of cellular calcium cycling on LQTS cardiac events are unknown. To better understand LQTS arrhythmias, we performed current-clamp and intracellular calcium ([Ca2+]i) measurements on cardiomyocytes differentiated from patient-derived induced pluripotent stem cells (iPS-CM). In myocytes carrying an LQT2 mutation (HERG-A422T), APs and [Ca2+]i transients were prolonged in parallel. APs were abbreviated by nifedipine exposure and further lengthened upon releasing intracellularly stored Ca2+. Validating this model, control iPS-CM treated with HERG-blocking drugs recapitulated the LQT2 phenotype. In LQT3 iPS-CM, expressing NaV1.5-N406K, APs and [Ca2+]i transients were markedly prolonged. AP prolongation was sensitive to tetrodotoxin and to inhibiting Na+-Ca2+ exchange. These results suggest that LQTS mutations act partly on cytosolic Ca2+ cycling, potentially providing a basis for functionally targeted interventions regardless of the specific mutation site. PMID:25254341

  2. Competing for Consciousness: Prolonged Mask Exposure Reduces Object Substitution Masking

    ERIC Educational Resources Information Center

    Goodhew, Stephanie C.; Visser, Troy A. W.; Lipp, Ottmar V.; Dux, Paul E.

    2011-01-01

    In object substitution masking (OSM) a sparse, temporally trailing 4-dot mask impairs target identification, even though it has different contours from, and does not spatially overlap with the target. Here, we demonstrate a previously unknown characteristic of OSM: Observers show reduced masking at prolonged (e.g., 640 ms) relative to intermediate…

  3. Recognition Memory Is Impaired in Children after Prolonged Febrile Seizures

    ERIC Educational Resources Information Center

    Martinos, Marina M.; Yoong, Michael; Patil, Shekhar; Chin, Richard F. M.; Neville, Brian G.; Scott, Rod C.; de Haan, Michelle

    2012-01-01

    Children with a history of a prolonged febrile seizure show signs of acute hippocampal injury on magnetic resonance imaging. In addition, animal studies have shown that adult rats who suffered febrile seizures during development reveal memory impairments. Together, these lines of evidence suggest that memory impairments related to hippocampal…

  4. Light adaptation of invertebrate photoreceptors: influence of intracellular pH buffering capacity.

    PubMed Central

    Bolsover, S R; Brown, J E

    1982-01-01

    1. The possible role of pH changes in mediating light adaptation in Limulus ventral photoreceptor cells was studied by intracellular injection of zwitterionic pH buffers. The intracellular concentration of buffer was estimated by inclusion of a radioactive marker in the injection solution. 2. The light-induced increase of intracellular Ca2+ concentration was monitored by intracellular aequorin. The light-induced increase of Ca2+ concentration was not markedly altered by injection of pH buffer to an intracellular concentration of about 200 mM. 3. The progressive decrease in responsiveness during intracellular ionophoretic injection of Ca2+ was not markedly altered by injection of pH buffer to an intracellular concentration of about 200 mM. 4. Photoreceptors of both Limulus and Balanus were impaled with two micropipettes and voltage clamped. Membrane current induced by a prolonged steady illumination declined from an early transient to a plateau. This delayed decline of current indicates a light-induced reduction of sensitivity (i.e. light adaptation). The wave forms were similar before and after injection of pH buffer to an intracellular concentration of about 200 mM. 5. We conclude that it is unlikely that a light-induced change of cytosolic pH mediates light adaptation in Limulus (and Balanus) photoreceptors. PMID:7175745

  5. A practical approach for intracellular protein delivery

    PubMed Central

    Biri, Stéphanie; Adib, Abdennaji; Erbacher, Patrick

    2007-01-01

    Protein delivery represents a powerful tool for experiments in live cells including studies of protein-protein interactions, protein interference with blocking antibodies, intracellular trafficking and protein or peptide biological functions. Most available reagents dedicated to the protein delivery allow efficient crossing of the plasma membrane. Nevertheless, the major disadvantage for these reagents is a weak release of the delivered protein into the cytoplasm. In this publication we demonstrate efficient protein delivery with a non-peptide based reagent, in human epithelial carcinoma HeLa cells and primary human skin fibroblasts. Using a fluorescent protein in combination with fluorescence microscopy and fluorescence-assisted cell sorting analysis, we show that the delivered protein is indeed released effectively in the cytoplasm, as expected for a dedicated carrier. Furthermore, we present a step-by-step method to optimize conditions for successful intracellular protein delivery. PMID:19002840

  6. Intracellular Pressure Dynamics in Blebbing Cells.

    PubMed

    Strychalski, Wanda; Guy, Robert D

    2016-03-01

    Blebs are pressure-driven protrusions that play an important role in cell migration, particularly in three-dimensional environments. A bleb is initiated when the cytoskeleton detaches from the cell membrane, resulting in the pressure-driven flow of cytosol toward the area of detachment and local expansion of the cell membrane. Recent experiments involving blebbing cells have led to conflicting hypotheses regarding the timescale of intracellular pressure propagation. The interpretation of one set of experiments supports a poroelastic model of the cytoplasm that leads to slow pressure equilibration when compared to the timescale of bleb expansion. A different study concludes that pressure equilibrates faster than the timescale of bleb expansion. To address this discrepancy, a dynamic computational model of the cell was developed that includes mechanics of and the interactions among the cytoplasm, the actin cortex, the cell membrane, and the cytoskeleton. The model results quantify the relationship among cytoplasmic rheology, pressure, and bleb expansion dynamics, and provide a more detailed picture of intracellular pressure dynamics. This study shows the elastic response of the cytoplasm relieves pressure and limits bleb size, and that both permeability and elasticity of the cytoplasm determine bleb expansion time. Our model with a poroelastic cytoplasm shows that pressure disturbances from bleb initiation propagate faster than the timescale of bleb expansion and that pressure equilibrates slower than the timescale of bleb expansion. The multiple timescales in intracellular pressure dynamics explain the apparent discrepancy in the interpretation of experimental results. PMID:26958893

  7. Intracellular auxin transport in pollen

    PubMed Central

    Dal Bosco, Cristina; Dovzhenko, Alexander; Palme, Klaus

    2012-01-01

    Cellular auxin homeostasis is controlled at many levels that include auxin biosynthesis, auxin metabolism, and auxin transport. In addition to intercellular auxin transport, auxin homeostasis is modulated by auxin flow through the endoplasmic reticulum (ER). PIN5, a member of the auxin efflux facilitators PIN protein family, was the first protein to be characterized as an intracellular auxin transporter. We demonstrated that PIN8, the closest member of the PIN family to PIN5, represents another ER-residing auxin transporter. PIN8 is specifically expressed in the male gametophyte and is located in the ER. By combining genetic, physiological, cellular and biochemical data we demonstrated a role for PIN8 in intracellular auxin homeostasis. Although our investigation shed light on intracellular auxin transport in pollen, the physiological function of PIN8 still remains to be elucidated. Here we discuss our data taking in consideration other recent findings. PMID:22990451

  8. Intracellular Acidosis Enhances the Excitability of Working Muscle

    NASA Astrophysics Data System (ADS)

    Pedersen, Thomas H.; Nielsen, Ole B.; Lamb, Graham D.; Stephenson, D. George

    2004-08-01

    Intracellular acidification of skeletal muscles is commonly thought to contribute to muscle fatigue. However, intracellular acidosis also acts to preserve muscle excitability when muscles become depolarized, which occurs with working muscles. Here, we show that this process may be mediated by decreased chloride permeability, which enables action potentials to still be propagated along the internal network of tubules in a muscle fiber (the T system) despite muscle depolarization. These results implicate chloride ion channels in muscle function and emphasize that intracellular acidosis of muscle has protective effects during muscle fatigue.

  9. Are steady magnetospheric convection events prolonged substorms?

    NASA Astrophysics Data System (ADS)

    Walach, M.-T.; Milan, S. E.

    2015-03-01

    Magnetospheric modes, including substorms, sawtooth events, and steady magnetospheric convection events, have in the past been described as different responses of the magnetosphere to coupling with the solar wind. Using previously determined event lists for sawtooth events, steady magnetospheric convection events, and substorms, we produce a statistical study of these event types to examine their similarities and behavior in terms of solar wind parameters, auroral brightness, open magnetospheric flux, and geomagnetic indices. A superposed epoch analysis shows that individual sawteeth show the same signatures as substorms but occur during more extreme cases of solar wind driving as well as geomagnetic activity. We also explore the limitations of current methods of identifying steady magnetospheric convection events and explain why some of those events are flagged inappropriately. We show that 58% of the steady magnetospheric convection events considered, as identified by criteria defined in previous studies are part of a prolonged version of substorms due to continued dayside driving during expansion phase. The remaining 42% are episodes of enhanced magnetospheric convection, occurring after extended periods of dayside driving.

  10. The Nod1, Nod2, and Rip2 Axis Contributes to Host Immune Defense against Intracellular Acinetobacter baumannii Infection

    PubMed Central

    Bist, Pradeep; Dikshit, Neha; Koh, Tse Hsien; Mortellaro, Alessandra; Tan, Thuan Tong

    2014-01-01

    Acinetobacter baumannii is a major extensively drug-resistant lethal human nosocomial bacterium. However, the host innate immune mechanisms controlling A. baumannii are not well understood. Although viewed as an extracellular pathogen, A. baumannii can also invade and survive intracellularly. However, whether host innate immune pathways sensing intracellular bacteria contribute to immunity against A. baumannii is not known. Here, we provide evidence for the first time that intracellular antibacterial innate immune receptors Nod1 and Nod2, and their adaptor Rip2, play critical roles in the sensing and clearance of A. baumannii by human airway epithelial cells in vitro. A. baumannii infection upregulated Rip2 expression. Silencing of Nod1, Nod2, and Rip2 expression profoundly increased intracellular invasion and prolonged the multiplication and survival of A. baumannii in lung epithelial cells. Notably, the Nod1/2-Rip2 axis did not contribute to the control of A. baumannii infection of human macrophages, indicating that they play cell type-specific roles. The Nod1/2-Rip2 axis was needed for A. baumannii infection-induced activation of NF-κB but not mitogen-activated protein kinases. Moreover, the Nod1/2-Rip2 axis was critical to induce optimal cytokine and chemokine responses to A. baumannii infection. Mechanistic studies showed that the Nod1/2 pathway contributed to the innate control of A. baumannii infection through the production of β-defensin 2 by airway epithelial cells. This study revealed new insights into the immune control of A. baumannii and may contribute to the development of effective immune therapeutics and vaccines against A. baumannii. PMID:24366254

  11. Liver cancer cells: targeting and prolonged-release drug carriers consisting of mesoporous silica nanoparticles and alginate microspheres

    PubMed Central

    Liao, Yu-Te; Liu, Chia-Hung; Yu, Jiashing; Wu, Kevin C-W

    2014-01-01

    A new microsphere consisting of inorganic mesoporous silica nanoparticles (MSNs) and organic alginate (denoted as MSN@Alg) was successfully synthesized by air-dynamic atomization and applied to the intracellular drug delivery systems (DDS) of liver cancer cells with sustained release and specific targeting properties. MSN@Alg microspheres have the advantages of MSN and alginate, where MSN provides a large surface area for high drug loading and alginate provides excellent biocompatibility and COOH functionality for specific targeting. Rhodamine 6G was used as a model drug, and the sustained release behavior of the rhodamine 6G-loaded MSN@Alg microspheres can be prolonged up to 20 days. For targeting therapy, the anticancer drug doxorubicin was loaded into MSN@Alg microspheres, and the (lysine)4-tyrosine-arginine-glycine-aspartic acid (K4YRGD) peptide was functionalized onto the surface of MSN@Alg for targeting liver cancer cells, hepatocellular carcinoma (HepG2). The results of the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay and confocal laser scanning microscopy indicate that the MSN@Alg microspheres were successfully uptaken by HepG2 without apparent cytotoxicity. In addition, the intracellular drug delivery efficiency was greatly enhanced (ie, 3.5-fold) for the arginine-glycine-aspartic acid (RGD)-labeled, doxorubicin-loaded MSN@Alg drug delivery system compared with the non-RGD case. The synthesized MSN@Alg microspheres show great potential as drug vehicles with high biocompatibility, sustained release, and targeting features for future intracellular DDS. PMID:24940057

  12. A bacteriophage endolysin that eliminates intracellular streptococci

    PubMed Central

    Shen, Yang; Barros, Marilia; Vennemann, Tarek; Gallagher, D Travis; Yin, Yizhou; Linden, Sara B; Heselpoth, Ryan D; Spencer, Dennis J; Donovan, David M; Moult, John; Fischetti, Vincent A; Heinrich, Frank; Lösche, Mathias; Nelson, Daniel C

    2016-01-01

    PlyC, a bacteriophage-encoded endolysin, lyses Streptococcus pyogenes (Spy) on contact. Here, we demonstrate that PlyC is a potent agent for controlling intracellular Spy that often underlies refractory infections. We show that the PlyC holoenzyme, mediated by its PlyCB subunit, crosses epithelial cell membranes and clears intracellular Spy in a dose-dependent manner. Quantitative studies using model membranes establish that PlyCB interacts strongly with phosphatidylserine (PS), whereas its interaction with other lipids is weak, suggesting specificity for PS as its cellular receptor. Neutron reflection further substantiates that PlyC penetrates bilayers above a PS threshold concentration. Crystallography and docking studies identify key residues that mediate PlyCB–PS interactions, which are validated by site-directed mutagenesis. This is the first report that a native endolysin can traverse epithelial membranes, thus substantiating the potential of PlyC as an antimicrobial for Spy in the extracellular and intracellular milieu and as a scaffold for engineering other functionalities. DOI: http://dx.doi.org/10.7554/eLife.13152.001 PMID:26978792

  13. A bacteriophage endolysin that eliminates intracellular streptococci.

    PubMed

    Shen, Yang; Barros, Marilia; Vennemann, Tarek; Gallagher, D Travis; Yin, Yizhou; Linden, Sara B; Heselpoth, Ryan D; Spencer, Dennis J; Donovan, David M; Moult, John; Fischetti, Vincent A; Heinrich, Frank; Lösche, Mathias; Nelson, Daniel C

    2016-01-01

    PlyC, a bacteriophage-encoded endolysin, lyses Streptococcus pyogenes (Spy) on contact. Here, we demonstrate that PlyC is a potent agent for controlling intracellular Spy that often underlies refractory infections. We show that the PlyC holoenzyme, mediated by its PlyCB subunit, crosses epithelial cell membranes and clears intracellular Spy in a dose-dependent manner. Quantitative studies using model membranes establish that PlyCB interacts strongly with phosphatidylserine (PS), whereas its interaction with other lipids is weak, suggesting specificity for PS as its cellular receptor. Neutron reflection further substantiates that PlyC penetrates bilayers above a PS threshold concentration. Crystallography and docking studies identify key residues that mediate PlyCB-PS interactions, which are validated by site-directed mutagenesis. This is the first report that a native endolysin can traverse epithelial membranes, thus substantiating the potential of PlyC as an antimicrobial for Spy in the extracellular and intracellular milieu and as a scaffold for engineering other functionalities. PMID:26978792

  14. Prolonged Speech and Modification of Stuttering: Perceptual, Acoustic, and Electroglottographic Data.

    ERIC Educational Resources Information Center

    Packman, Ann; And Others

    1994-01-01

    This study investigated changes in the speech patterns of young adult male subjects when stuttering was modified by deliberately prolonging speech. Three subjects showed clinically significant stuttering reductions when using prolonged speech to reduce their stuttering. Resulting speech was perceptually stutter free. Acoustic and…

  15. Stigma Prolongs Global HIV Epidemic Among Gays

    MedlinePlus

    ... gov/medlineplus/news/fullstory_159757.html Stigma Prolongs Global HIV Epidemic Among Gays High-risk men still ... some countries will repeal anti-gay laws. "The global epidemic of HIV in gay men is ongoing ...

  16. Prolonged partial epilepsy: a case report

    SciTech Connect

    Wilson, M.A.

    1980-11-01

    The case study of a patient with prolonged partial epilepsy is presented. There was a discrepancy between the extent of the abnormality seen on the radionuclide angiogram and that seen on the static brain scan.

  17. Intracellular Delivery System for Antibody–Peptide Drug Conjugates

    PubMed Central

    Berguig, Geoffrey Y; Convertine, Anthony J; Frayo, Shani; Kern, Hanna B; Procko, Erik; Roy, Debashish; Srinivasan, Selvi; Margineantu, Daciana H; Booth, Garrett; Palanca-Wessels, Maria Corinna; Baker, David; Hockenbery, David; Press, Oliver W; Stayton, Patrick S

    2015-01-01

    Antibodies armed with biologic drugs could greatly expand the therapeutic potential of antibody–drug conjugates for cancer therapy, broadening their application to disease targets currently limited by intracellular delivery barriers. Additional selectivity and new therapeutic approaches could be realized with intracellular protein drugs that more specifically target dysregulated pathways in hematologic cancers and other malignancies. A multifunctional polymeric delivery system for enhanced cytosolic delivery of protein drugs has been developed that incorporates endosomal-releasing activity, antibody targeting, and a biocompatible long-chain ethylene glycol component for optimized safety, pharmacokinetics, and tumor biodistribution. The pH-responsive polymeric micelle carrier, with an internalizing anti-CD22 monoclonal targeting antibody, effectively delivered a proapoptotic Bcl-2 interacting mediator (BIM) peptide drug that suppressed tumor growth for the duration of treatment and prolonged survival in a xenograft mouse model of human B-cell lymphoma. Antitumor drug activity was correlated with a mechanistic induction of the Bcl-2 pathway biomarker cleaved caspase-3 and a marked decrease in the Ki-67 proliferation biomarker. Broadening the intracellular target space by more effective delivery of protein/peptide drugs could expand the repertoire of antibody–drug conjugates to currently undruggable disease-specific targets and permit tailored drug strategies to stratified subpopulations and personalized medicines. PMID:25669432

  18. Primary Pulmonary Synovial Sarcoma Showing a Prolonged Survival with Multimodality Therapy.

    PubMed

    Ogino, Hirokazu; Hanibuchi, Masaki; Takizawa, Hiromitsu; Sakiyama, Shoji; Sumitomo, Hiroyuki; Iwamoto, Seiji; Ikushima, Hitoshi; Nakajima, Kohei; Nagahiro, Shinji; Yamago, Taito; Toyoda, Yuko; Bando, Yoshimi; Nishioka, Yasuhiko

    2016-01-01

    A 54-year-old man was referred to our hospital due to a mass shadow noted on a chest X-ray. Thoracoscopic lobectomy yielded a diagnosis of primary pulmonary synovial sarcoma according to the histology and SYT-SSX1 gene analyses. Five months after the thoracic surgery, he developed brain metastasis; therefore, we performed resection of the brain metastatic focus followed by radiotherapy. As a local recurrence in the thoracic cavity concurrently emerged, systemic chemotherapy was also administered. These observations indicated that a multidisciplinary approach may be useful against primary pulmonary synovial sarcoma, although there is presently no established therapeutic strategy due to its rarity and highly aggressive nature. PMID:26875964

  19. [A case of prolonged paroxysmal sympathetic hyperactivity].

    PubMed

    Yamamoto, Akiko; Ide, Shuhei; Iwasaki, Yuji; Kaga, Makiko; Arima, Masataka

    2016-03-01

    We report the case of a 4-year-old girl who presented with paroxysmal sympathetic hyperactivity (PSH), after developing severe hypoxic-ischemic-encephalopathy because of cardiopulmonary arrest. She showed dramatic paroxysmal sympathetic activity with dystonia. She was treated with wide variety of medications against PSH, which were found to be effective in previous studies. Among them, morphine, bromocriptine, propranolol, and clonidine were effective in reducing the frequency of her attacks while gabapentin, baclofen, dantrolene, and benzodiazepine were ineffective. Though the paroxysms decreased markedly after the treatment, they could not be completely controlled beyond 500 days. Following the treatment, levels of plasma catecholamines and their urinary metabolites decreased to normal during inter- paroxysms. However, once a paroxysm had recurred, these levels were again very high. This case study is considered significant for two rea- sons. One is that PSH among children have been rarely reported, and the other is that this case of prolonged PSH delineated the transition of plasma catecholamines during the treatment. The excitatory: inhibitory ratio (EIR) model proposed by Baguley was considered while dis- cussing drug sensitivity in this case. Accumulation of similar case studies will help establish more effective treatment strategies and elucidate the pathophysiology of PSH. PMID:27149743

  20. ROS and intracellular ion channels.

    PubMed

    Kiselyov, Kirill; Muallem, Shmuel

    2016-08-01

    Oxidative stress is a well-known driver of numerous pathological processes involving protein and lipid peroxidation and DNA damage. The resulting increase of pro-apoptotic pressure drives tissue damage in a host of conditions, including ischemic stroke and reperfusion injury, diabetes, death in acute pancreatitis and neurodegenerative diseases. Somewhat less frequently discussed, but arguably as important, is the signaling function of oxidative stress stemming from the ability of oxidative stress to modulate ion channel activity. The evidence for the modulation of the intracellular ion channels and transporters by oxidative stress is constantly emerging and such evidence suggests new regulatory and pathological circuits that can be explored towards new treatments for diseases in which oxidative stress is an issue. In this review we summarize the current knowledge on the effects of oxidative stress on the intracellular ion channels and transporters and their role in cell function. PMID:26995054

  1. Direct Measurement of Intracellular Pressure

    PubMed Central

    Petrie, Ryan J.; Koo, Hyun

    2014-01-01

    A method to directly measure the intracellular pressure of adherent, migrating cells is described in the Basic Protocol. This approach is based on the servo-null method where a microelectrode is introduced into the cell to directly measure the physical pressure of the cytoplasm. We also describe the initial calibration of the microelectrode as well as the application of the method to cells migrating inside three-dimensional (3D) extracellular matrix (ECM). PMID:24894836

  2. Stochastic models of intracellular transport

    NASA Astrophysics Data System (ADS)

    Bressloff, Paul C.; Newby, Jay M.

    2013-01-01

    The interior of a living cell is a crowded, heterogenuous, fluctuating environment. Hence, a major challenge in modeling intracellular transport is to analyze stochastic processes within complex environments. Broadly speaking, there are two basic mechanisms for intracellular transport: passive diffusion and motor-driven active transport. Diffusive transport can be formulated in terms of the motion of an overdamped Brownian particle. On the other hand, active transport requires chemical energy, usually in the form of adenosine triphosphate hydrolysis, and can be direction specific, allowing biomolecules to be transported long distances; this is particularly important in neurons due to their complex geometry. In this review a wide range of analytical methods and models of intracellular transport is presented. In the case of diffusive transport, narrow escape problems, diffusion to a small target, confined and single-file diffusion, homogenization theory, and fractional diffusion are considered. In the case of active transport, Brownian ratchets, random walk models, exclusion processes, random intermittent search processes, quasi-steady-state reduction methods, and mean-field approximations are considered. Applications include receptor trafficking, axonal transport, membrane diffusion, nuclear transport, protein-DNA interactions, virus trafficking, and the self-organization of subcellular structures.

  3. Use of in vitro methods to predict QT prolongation

    SciTech Connect

    Hammond, T.G. . E-mail: tim.hammond@astrazeneca.com; Pollard, C.E.

    2005-09-01

    The inhibition of the hERG-encoded potassium channel can lead to prolongation of the cardiac action potential-manifested as a prolongation of the QT interval on the ECG. Although QT interval prolongation is not dangerous per se, in a small percentage of cases, it is associated with a potentially fatal arrhythmia: Torsades de Pointes (TdP). This channel type is pharmacologically promiscuous, so many compounds have caused QT interval prolongation in man and this has led to drugs being withdrawn from the market following evidence of TdP. From a drug discovery perspective, focusing as early as possible on screening out hERG activity is important. Retrospective analysis of hERG potency versus clinical incidence of TdP suggests provisional safety margins that could be used as target values by medicinal chemists. Large safety margins will not always be possible; however, and in such circumstances, if the risk-benefit ratio still favours developing the compound, a pre-clinical assessment of the likelihood that any QT interval prolongation will or will not lead to TdP in man may be important. An isolated rabbit heart model of arrhythmia shows promise in this respect, based on a comparison of clinical data with that obtained from this assay. Specific regulatory guidance on this topic is still in the draft form but the pre-clinical document (ICH S7B) contains a largely useful perspective on how an integrated risk assessment could be formed using in vitro and in vivo assays. The role of this document is evolving however, since the draft clinical guideline (E14) suggests that irrespective of the pre-clinical data, a thorough clinical ECG study will be required at some point during development.

  4. Invasion of the Central Nervous System by Intracellular Bacteria

    PubMed Central

    Drevets, Douglas A.; Leenen, Pieter J. M.; Greenfield, Ronald A.

    2004-01-01

    Infection of the central nervous system (CNS) is a severe and frequently fatal event during the course of many diseases caused by microbes with predominantly intracellular life cycles. Examples of these include the facultative intracellular bacteria Listeria monocytogenes, Mycobacterium tuberculosis, and Brucella and Salmonella spp. and obligate intracellular microbes of the Rickettsiaceae family and Tropheryma whipplei. Unfortunately, the mechanisms used by intracellular bacterial pathogens to enter the CNS are less well known than those used by bacterial pathogens with an extracellular life cycle. The goal of this review is to elaborate on the means by which intracellular bacterial pathogens establish infection within the CNS. This review encompasses the clinical and pathological findings that pertain to the CNS infection in humans and includes experimental data from animal models that illuminate how these microbes enter the CNS. Recent experimental data showing that L. monocytogenes can invade the CNS by more than one mechanism make it a useful model for discussing the various routes for neuroinvasion used by intracellular bacterial pathogens. PMID:15084504

  5. Change in macrostructure and porosity of graphite on prolonged irradiation

    SciTech Connect

    Virgil'ev, Y.S.; Butyrin, G.M.; Kalyagina, I.P.; Nikishina, L.M.; Shurshakova, T.N.

    1986-02-01

    This work studies the variation in the microstructure of strongly irradiated reactor-grade graphite samples by mercury porosimetry, optical microscopy, and x-ray analysis. The chief characteristics of the samples are listed. Experimental study of the nature of porous and crystal structure of reactor graphite show that prolonged neutron irradiation at 360 and 1220 degrees K up to a luence of 10/sup 22/ neutrons/cm/sup 2/ causes marked irreversible changes in the graphite macrostructure.

  6. Mesangial cell hillocks. Nodular foci of exaggerated growth of cells and matrix in prolonged culture.

    PubMed Central

    Sterzel, R. B.; Lovett, D. H.; Foellmer, H. G.; Perfetto, M.; Biemesderfer, D.; Kashgarian, M.

    1986-01-01

    To examine the capability of glomerular mesangial cells (MCs) to produce extracellular matrix, the authors studied MCs in culture by light and electron microscopy as well as immunocytochemistry. MCs were obtained from isolated rat glomeruli and maintained up to 12 weeks in medium containing 20% fetal calf serum. MC outgrowth of primary culture and of up to three subcultures showed characteristic organization consisting of bands of elongated or stellate intertwined cells. After confluency at 10-16 days, MCs continued to grow in irregular multilayers. MCs produced extracellular matrix material within 2-4 days after plating, and large amounts of matrix accumulated with time. By 2-3 weeks, foci of exaggerated MC proliferation, matrix secretion, and necrotic cell debris formed nodular protrusions, which gradually produced large hillocks. Immunocytochemical studies of MC outgrowths were performed on culture plates or on sectioned material with the use of specific rabbit polyclonal antibodies to isolated matrix proteins and FITC-conjugated, affinity-purified second antibodies. Within 3 days of culture, MCs elaborated fibronectin and collagen Types I, III, IV, and V. With time, strands of matrix, notably in the central mass of hillocks, stained extensively for these constituents. Staining for laminin was less pronounced. Smooth muscle cell myosin was regularly found on distinct intracellular fibrils and in the extracellular material of hillocks. Electron microscopy revealed the hillocks to be composed of elongated cells on the surface and stellate cells intermingled with matrix and necrotic cell debris in the core. The results show that proliferating MCs can be maintained in homogeneous culture for a prolonged time period. MCs produce large amounts of the extracellular matrix proteins (Type IV and V collagen, fibronectin, laminin), which are found in normal glomeruli. Cultured MCs also produce interstitial collagen Types I and III. MC hillocks show the nodular accumulation

  7. Intracellular calcium levels can regulate Importin-dependent nuclear import

    SciTech Connect

    Kaur, Gurpreet; Ly-Huynh, Jennifer D.; Jans, David A.

    2014-07-18

    Highlights: • High intracellular calcium inhibits Impα/β1- or Impβ1-dependent nuclear protein import. • The effect of Ca{sup 2+} on nuclear import does not relate to changes in the nuclear pore. • High intracellular calcium can result in mislocalisation of Impβ1, Ran and RCC1. - Abstract: We previously showed that increased intracellular calcium can modulate Importin (Imp)β1-dependent nuclear import of SRY-related chromatin remodeling proteins. Here we extend this work to show for the first time that high intracellular calcium inhibits Impα/β1- or Impβ1-dependent nuclear protein import generally. The basis of this relates to the mislocalisation of the transport factors Impβ1 and Ran, which show significantly higher nuclear localization in contrast to various other factors, and RCC1, which shows altered subnuclear localisation. The results here establish for the first time that intracellular calcium modulates conventional nuclear import through direct effects on the nuclear transport machinery.

  8. Peptide modified gold nanoparticles for improved cellular uptake, nuclear transport, and intracellular retention

    NASA Astrophysics Data System (ADS)

    Yang, C.; Uertz, J.; Yohan, D.; Chithrani, B. D.

    2014-09-01

    Gold nanoparticles (GNPs) are being extensively used in cancer therapeutic applications due to their ability to act both as an anticancer drug carrier in chemotherapy and as a dose enhancer in radiotherapy. The therapeutic response can be further enhanced if nanoparticles (NPs) can be effectively targeted into the nucleus. Here, we present an uptake and removal of GNPs functionalized with three peptides. The first peptide (RGD peptide) enhanced the uptake, the second peptide (NLS peptide) facilitated the nuclear delivery, while the third one (pentapeptide) covered the rest of the surface and protected it from the binding of serum proteins onto the NP surface. The pentapeptide also stabilized the conjugated GNP complex. The peptide-capped GNPs showed a five-fold increase in NP uptake followed by effective nuclear localization. The fraction of NPs exocytosed was less for peptide-capped NPs as compared to citrate-capped ones. Enhanced uptake and prolonged intracellular retention of peptide-capped GNPs could allow NPs to perform their desired applications more efficiently in cells. These studies will provide guidelines for developing NPs for therapeutic applications, which will require ``controlling'' of the NP accumulation rate while maintaining low toxicity.Gold nanoparticles (GNPs) are being extensively used in cancer therapeutic applications due to their ability to act both as an anticancer drug carrier in chemotherapy and as a dose enhancer in radiotherapy. The therapeutic response can be further enhanced if nanoparticles (NPs) can be effectively targeted into the nucleus. Here, we present an uptake and removal of GNPs functionalized with three peptides. The first peptide (RGD peptide) enhanced the uptake, the second peptide (NLS peptide) facilitated the nuclear delivery, while the third one (pentapeptide) covered the rest of the surface and protected it from the binding of serum proteins onto the NP surface. The pentapeptide also stabilized the conjugated GNP

  9. Management of children with prolonged diarrhea

    PubMed Central

    Giannattasio, Antonietta; Guarino, Alfredo; Lo Vecchio, Andrea

    2016-01-01

    Prolonged diarrhea is usually defined as acute-onset diarrhea lasting 7 days or more, but less than 14 days. Its trend has been declining in recent years because of improvement in the management of acute diarrhea, which represents the ideal strategy to prevent prolonged diarrhea. The pathogenesis of prolonged diarrhea is multifactorial and essentially based on persistent mucosal damage due to specific infections or sequential infections with different pathogens, host-related factors including micronutrient and/or vitamin deficiency, undernutrition and immunodeficiency, high mucosal permeability due to previous infectious processes and nutrient deficiency with consequential malabsorption, and microbiota disruption. Infections seem to play a major role in causing prolonged diarrhea in both developing and developed areas. However, single etiologic pathogens have not been identified, and the pattern of agents varies according to settings, host risk factors, and previous use of antibiotics and other drugs. The management of prolonged diarrhea is complex. Because of the wide etiologic spectrum, diagnostic algorithms should take into consideration the age of the patient, clinical and epidemiological factors, and the nutritional status and should always include a search for enteric pathogens. Often, expensive laboratory evaluations are of little benefit in guiding therapy, and an empirical approach may be effective in the majority of cases. The presence or absence of weight loss is crucial for driving the initial management of prolonged diarrhea. If there is no weight loss, generally there is no need for further evaluation. If weight loss is present, empiric anti-infectious therapy or elimination diet may be considered once specific etiologies have been excluded. PMID:26962439

  10. Management of children with prolonged diarrhea.

    PubMed

    Giannattasio, Antonietta; Guarino, Alfredo; Lo Vecchio, Andrea

    2016-01-01

    Prolonged diarrhea is usually defined as acute-onset diarrhea lasting 7 days or more, but less than 14 days. Its trend has been declining in recent years because of improvement in the management of acute diarrhea, which represents the ideal strategy to prevent prolonged diarrhea. The pathogenesis of prolonged diarrhea is multifactorial and essentially based on persistent mucosal damage due to specific infections or sequential infections with different pathogens, host-related factors including micronutrient and/or vitamin deficiency, undernutrition and immunodeficiency, high mucosal permeability due to previous infectious processes and nutrient deficiency with consequential malabsorption, and microbiota disruption. Infections seem to play a major role in causing prolonged diarrhea in both developing and developed areas. However, single etiologic pathogens have not been identified, and the pattern of agents varies according to settings, host risk factors, and previous use of antibiotics and other drugs. The management of prolonged diarrhea is complex. Because of the wide etiologic spectrum, diagnostic algorithms should take into consideration the age of the patient, clinical and epidemiological factors, and the nutritional status and should always include a search for enteric pathogens. Often, expensive laboratory evaluations are of little benefit in guiding therapy, and an empirical approach may be effective in the majority of cases. The presence or absence of weight loss is crucial for driving the initial management of prolonged diarrhea. If there is no weight loss, generally there is no need for further evaluation. If weight loss is present, empiric anti-infectious therapy or elimination diet may be considered once specific etiologies have been excluded. PMID:26962439

  11. Intracellular replication of Staphylococcus aureus in mature phagolysosomes in macrophages precedes host cell death, and bacterial escape and dissemination.

    PubMed

    Flannagan, Ronald S; Heit, Bryan; Heinrichs, David E

    2016-04-01

    The success of Staphylococcus aureus as a pathogen is partly attributable to its ability to thwart host innate immune responses, which includes resisting the antimicrobial functions of phagocytes. Here, we have studied the interaction of methicillin-resistant S. aureus (MRSA) strain USA300 with murine RAW 264.7 and primary human macrophages using molecular imaging and single cell analysis to obtain an unprecedented understanding of the interaction between the macrophage and MRSA. Herein we demonstrate that macrophages fail to control intracellular infection by MRSA USA300 despite trafficking the bacteria into mature phagolysosomes. Using fluorescence-based proliferation assays we also show that intracellular staphylococci proliferate and that replication commences while the bacteria are residing in mature phagolysosomes hours after initial phagocytosis. Finally, live-cell fluorescence video microscopy allowed for unprecedented visual insight into the escape of MRSA from macrophages, demonstrating that the macrophages die through a pathway characterized by membrane blebbing and activation of caspase-3 followed by acquisition of the vital dye propidium iodide. Moreover, cell death precedes the emergence of MRSA from infected macrophages, and these events can be ablated by prolonged exposure of infected phagocytes to gentamicin. PMID:26408990

  12. Intracellular targeting with engineered proteins.

    PubMed

    Miersch, Shane; Sidhu, Sachdev S

    2016-01-01

    If the isolation, production, and clinical use of insulin marked the inception of the age of biologics as therapeutics, the convergence of molecular biology and combinatorial engineering techniques marked its coming of age. The first wave of recombinant protein-based drugs in the 1980s demonstrated emphatically that proteins could be engineered, formulated, and employed for clinical advantage. Yet despite the successes of protein-based drugs such as antibodies, enzymes, and cytokines, the druggable target space for biologics is currently restricted to targets outside the cell. Insofar as estimates place the number of proteins either secreted or with extracellular domains in the range of 8000 to 9000, this represents only one-third of the proteome and circumscribes the pathways that can be targeted for therapeutic intervention. Clearly, a major objective for this field to reach maturity is to access, interrogate, and modulate the majority of proteins found inside the cell. However, owing to the large size, complex architecture, and general cellular impermeability of existing protein-based drugs, this poses a daunting challenge. In recent years, though, advances on the two related fronts of protein engineering and drug delivery are beginning to bring this goal within reach. First, prompted by the restrictions that limit the applicability of antibodies, intense efforts have been applied to identifying and engineering smaller alternative protein scaffolds for the modulation of intracellular targets. In parallel, innovative solutions for delivering proteins to the intracellular space while maintaining their stability and functional activity have begun to yield successes. This review provides an overview of bioactive intrabodies and alternative protein scaffolds amenable to engineering for intracellular targeting and also outlines advances in protein engineering and formulation for delivery of functional proteins to the interior of the cell to achieve therapeutic action

  13. Intracellular targeting with engineered proteins

    PubMed Central

    Miersch, Shane; Sidhu, Sachdev S.

    2016-01-01

    If the isolation, production, and clinical use of insulin marked the inception of the age of biologics as therapeutics, the convergence of molecular biology and combinatorial engineering techniques marked its coming of age. The first wave of recombinant protein-based drugs in the 1980s demonstrated emphatically that proteins could be engineered, formulated, and employed for clinical advantage. Yet despite the successes of protein-based drugs such as antibodies, enzymes, and cytokines, the druggable target space for biologics is currently restricted to targets outside the cell. Insofar as estimates place the number of proteins either secreted or with extracellular domains in the range of 8000 to 9000, this represents only one-third of the proteome and circumscribes the pathways that can be targeted for therapeutic intervention. Clearly, a major objective for this field to reach maturity is to access, interrogate, and modulate the majority of proteins found inside the cell. However, owing to the large size, complex architecture, and general cellular impermeability of existing protein-based drugs, this poses a daunting challenge. In recent years, though, advances on the two related fronts of protein engineering and drug delivery are beginning to bring this goal within reach. First, prompted by the restrictions that limit the applicability of antibodies, intense efforts have been applied to identifying and engineering smaller alternative protein scaffolds for the modulation of intracellular targets. In parallel, innovative solutions for delivering proteins to the intracellular space while maintaining their stability and functional activity have begun to yield successes. This review provides an overview of bioactive intrabodies and alternative protein scaffolds amenable to engineering for intracellular targeting and also outlines advances in protein engineering and formulation for delivery of functional proteins to the interior of the cell to achieve therapeutic action

  14. Intracellular ion channels and cancer.

    PubMed

    Leanza, Luigi; Biasutto, Lucia; Managò, Antonella; Gulbins, Erich; Zoratti, Mario; Szabò, Ildikò

    2013-01-01

    Several types of channels play a role in the maintenance of ion homeostasis in subcellular organelles including endoplasmatic reticulum, nucleus, lysosome, endosome, and mitochondria. Here we give a brief overview of the contribution of various mitochondrial and other organellar channels to cancer cell proliferation or death. Much attention is focused on channels involved in intracellular calcium signaling and on ion fluxes in the ATP-producing organelle mitochondria. Mitochondrial K(+) channels (Ca(2+)-dependent BKCa and IKCa, ATP-dependent KATP, Kv1.3, two-pore TWIK-related Acid-Sensitive K(+) channel-3 (TASK-3)), Ca(2+) uniporter MCU, Mg(2+)-permeable Mrs2, anion channels (voltage-dependent chloride channel VDAC, intracellular chloride channel CLIC) and the Permeability Transition Pore (MPTP) contribute importantly to the regulation of function in this organelle. Since mitochondria play a central role in apoptosis, modulation of their ion channels by pharmacological means may lead to death of cancer cells. The nuclear potassium channel Kv10.1 and the nuclear chloride channel CLIC4 as well as the endoplasmatic reticulum (ER)-located inositol 1,4,5-trisphosphate (IP3) receptor, the ER-located Ca(2+) depletion sensor STIM1 (stromal interaction molecule 1), a component of the store-operated Ca(2+) channel and the ER-resident TRPM8 are also mentioned. Furthermore, pharmacological tools affecting organellar channels and modulating cancer cell survival are discussed. The channels described in this review are summarized on Figure 1. Overall, the view is emerging that intracellular ion channels may represent a promising target for cancer treatment. PMID:24027528

  15. Pharmacology of intracellular signalling pathways

    PubMed Central

    Nahorski, Stefan R

    2006-01-01

    This article provides a brief and somewhat personalized review of the dramatic developments that have occurred over the last 45 years in our understanding of intracellular signalling pathways associated with G-protein-coupled receptor activation. Signalling via cyclic AMP, the phosphoinositides and Ca2+ is emphasized and these systems have already been revealed as new pharmacological targets. The therapeutic benefits of most of such targets are, however, yet to be realized, but it is certain that the discipline of pharmacology needs to widen its boundaries to meet these challenges in the future. PMID:16402119

  16. A new PZT with prolonged exposure and Wuchang PZT catalogue.

    NASA Astrophysics Data System (ADS)

    Gao, Buxi; Li, Jingfeng; Hu, Yashe

    The most important improvement in the authors' PZT is that the stars with 11 mag. can be observed, because exposure time for dimmer stars is prolonged. The observational practice during last three years denotes that the method of prolonged exposure is very successful. The number of observed stars is increased about three times, and the precision is improved. As there are so many star images in the plate, a series of processes is suggested, which includes the process of predicting the positions of star images on plates, finding out the pairs of star images and their corresponding stars automatically, calculating the apparent positions of stars and giving the final observational results. The corrections of 289 stars (on 150 measured plates) are given. The results show that many errors of stellar positions in AGK 3R and AGK 3 are larger than 0″5.

  17. Prolonged and tunable residence time using reversible covalent kinase inhibitors

    PubMed Central

    Bradshaw, J. Michael; McFarland, Jesse M.; Paavilainen, Ville O.; Bisconte, Angelina; Tam, Danny; Phan, Vernon T.; Romanov, Sergei; Finkle, David; Shu, Jin; Patel, Vaishali; Ton, Tony; Li, Xiaoyan; Loughhead, David G.; Nunn, Philip A.; Karr, Dane E.; Gerritsen, Mary E.; Funk, Jens Oliver; Owens, Timothy D.; Verner, Erik; Brameld, Ken A.; Hill, Ronald J.; Goldstein, David M.; Taunton, Jack

    2015-01-01

    Drugs with prolonged, on-target residence time often show superior efficacy, yet general strategies for optimizing drug-target residence time are lacking. Here, we demonstrate progress toward this elusive goal by targeting a noncatalytic cysteine in Bruton's tyrosine kinase (BTK) with reversible covalent inhibitors. Utilizing an inverted orientation of the cysteine-reactive cyanoacrylamide electrophile, we identified potent and selective BTK inhibitors that demonstrate biochemical residence times spanning from minutes to 7 days. An inverted cyanoacrylamide with prolonged residence time in vivo remained bound to BTK more than 18 hours after clearance from the circulation. The inverted cyanoacrylamide strategy was further utilized to discover fibroblast growth factor receptor (FGFR) kinase inhibitors with residence times of several days, demonstrating generalizability of the approach. Targeting noncatalytic cysteines with inverted cyanoacrylamides may serve as a broadly applicable platform that facilitates “residence time by design”, the ability to modulate and improve the duration of target engagement in vivo. PMID:26006010

  18. Prolongation of Actions of Ca2+ Early in Phototransduction by 9-Demethylretinal

    PubMed Central

    Matthews, Hugh R.; Cornwall, M.C.; Crouch, R.K.

    2001-01-01

    During adaptation Ca2+ acts on a step early in phototransduction, which is normally available for only a brief period after excitation. To investigate the identity of this step, we studied the effect of the light-induced decline in intracellular Ca2+ concentration on the response to a bright flash in normal rods, and in rods bleached and regenerated with 11-cis 9-demethylretinal, which forms a photopigment with a prolonged photoactivated lifetime. Changes in cytoplasmic Ca2+ were opposed by rapid superfusion of the outer segment with a 0Na+/0Ca2+ solution designed to minimize Ca2+ fluxes across the surface membrane. After regeneration of a bleached rod with 9-demethlyretinal, the response in Ringer's to a 440-nm bright flash was prolonged in comparison with the unbleached control, and the response remained in saturation for 10–15s. If the dynamic fall in Ca2+i induced by the flash was delayed by stepping the outer segment to 0Na+/0Ca2+ solution just before the flash and returning it to Ringer's shortly before recovery, then the response saturation was prolonged further, increasing linearly by 0.41 ± 0.01 of the time spent in this solution. In contrast, even long exposures to 0Na+/0Ca2+ solution of rods containing native photopigment evoked only a modest response prolongation on the return to Ringer's. Furthermore, if the rod was preexposed to steady subsaturating light, thereby reducing the cytoplasmic calcium concentration, then the prolongation of the bright flash response evoked by 0Na+/0Ca2+ solution was reduced in a graded manner with increasing background intensity. These results indicate that altering the chromophore of rhodopsin prolongs the time course of the Ca2+-dependent step early in the transduction cascade so that it dominates response recovery, and suggest that it is associated with photopigment quenching by phosphorylation. PMID:11585850

  19. Modification of bursting in a Helix neuron by drugs influencing intracellular regulation of calcium level.

    PubMed

    Salánki, J; Budai, D; Véró, M

    1983-01-01

    The effect of ruthenium red, caffein and EGTA (ethyleneglycol tetraacetic acid) influencing intracellular Ca2+ level as well as that of pH-lowering was investigated on identified RPal neuron of Helix pomatia characterized by bimodal pacemaker (bursting) activity. Drugs were applied both extracellularly and intracellularly. Intracellular injection was performed from micropipettes by pressure. It was found that intracellular injection of ruthenium red, caffein, EGTA and pH-lowering caused immediate short hyperpolarization and suspension of bursting. The effect of caffein and lowering of pH was biphasic, hyperpolarization was followed by an increase of spiking. Following EGTA injection the amplitudes of interburst hyperpolarizing waves decreased, and prolongation of spikes occurred. Extracellular application of ruthenium red caused slight depolarization, while caffein produced mainly effects that were similar to those of the intracellular injection. Adding EGTA into the bath resulted in cessation of bursting, and later on also spike generation was blocked. All these effects could be eliminated by washing. It is concluded that Ca-influx during spiking cannot be considered as a single factor in maintaining bursting activity, nevertheless, intracellular binding and liberation of Ca depending on the cell metabolism should also be taken into consideration as a possible mechanism of burst regulation. PMID:6198869

  20. Intracellular azo decolorization is coupled with aerobic respiration by a Klebsiella oxytoca strain.

    PubMed

    Yu, Lei; Zhang, Xiao-Yu; Xie, Tian; Hu, Jin-Mei; Wang, Shi; Li, Wen-Wei

    2015-03-01

    Reduction of azo dye methyl red coupled with aerobic respiration by growing cultures of Klebsiella oxytoca GS-4-08 was investigated. In liquid media containing dye and 0.6 % glucose in a mineral salts base, 100 mg l(-1) of the dye are completely removed in 3 h under shaking conditions. The dye cannot be aerobically decolorized by strain GS-4-08 without extra carbon sources, indicating a co-metabolism process. Higher initial dye concentration prolonged the lag phase of the cell growth, but final cell concentrations of each batches reached a same level with range from 6.3 to 7.6 mg l(-1) after the dye adaption period. This strain showed stronger dye tolerance and decolorization ability than many reported strains. Furthermore, a new intracellular oxygen-insensitive azoreductase was isolated from this strain, and the specific activity of enzyme was 0.846 and 0.633 U mg(-1) protein in the presence of NADH and NADPH, respectively. N,N dimethyl-p-phenylenediamine and anthranilic acid were stoichiometrically released from MR dye, indicating the breakage of azo bonds accounts for the intracellular decolorization. Combining the characteristics of azoreductase, the stoichiometry of EMP, and TCA cycle, the electron transfer chain theory of aerobic respiration, and the possible mechanism of aerobic respiration coupled with azo reduction by K. oxytoca GS-4-08 are proposed. This study is expected to provide a sound theoretical basis for the development of the K. oxytoca strain in aerobic process for azo dye containing wastewaters. PMID:25343980

  1. A novel glyceryl monoolein-bearing cubosomes for gambogenic acid: Preparation, cytotoxicity and intracellular uptake.

    PubMed

    Luo, Qing; Lin, Tongyuan; Zhang, Cai Yuan; Zhu, Tingting; Wang, Lei; Ji, Zhaojie; Jia, Buyun; Ge, Tao; Peng, Daiyin; Chen, Weidong

    2015-09-30

    Lyotropic cubic liquid crystalline nanoparticles, also known as 'cubosomes', have been tested as effective carriers for a variety of drugs due to their ability to enhance delivery efficiency and reduced drug side effects. Cubosomes are colloidal carriers composed of biodegradable Glyceryl monooleate and F127. Being composed of well tolerable and physiological materials, these carriers are well tolerated, compatible and non-toxic. In this study, therefore, we developed a novel, water-soluble, glyceryl monooleate and F127 based multiblock copolymer for Gambogenic acid (GNA) by emulsion-evaporation and low temperature-solidification technique. Physicochemical properties, in vitro cytotoxicity, cellular uptake and in vivo pharmacokinetic of GNA-loaded cubosomes (GNA-Cubs) were investigated. The results revealed that GNA-Cubs were spherical or ellipsoidal monocellular by dynamic light scattering, meanwhile, 150-250nm in mean size with narrow polydispersity indexas determined by transmission electron microscopy. Small angle X-ray scattering indicated that GNA-Cubs retain the Pn3m cubic symmetry. Compared with GNA solution, GNA-Cubs exhibited markedly prolonged inhibitory activity in SMMC-7721 cells, as well as time-dependent increases in intra-cellular uptake. Furthermore, in vivo pharmacokinetic study showed that the Cmax values and the AUC of GNA-Cubs were higher than GNA solution approximately 1.2-fold and 9.1-fold, respectively. In conclusion, the results showed that the cubic liquid crystalline nanoparticles could be a potentially nanocarrier in the delivery of GNA for cancer therapy. PMID:26209071

  2. Neurological complications of prolonged hunger strike.

    PubMed

    Başoğlu, M; Yetimalar, Y; Gürgör, N; Büyükçatalbaş, S; Kurt, T; Seçil, Y; Yeniocak, A

    2006-10-01

    We investigated neurological findings in 41 prisoners (mean age: 28.6) who participated in a hunger strike between 2000 and 2002. All cases were evaluated using neuropsychological, neuroradiological, and electrophysiological methods. The total duration of fasting ranged from 130 to 324 days (mean 199 days). All cases had 200-600 mg/day thiamine orally for 60-294 days (mean 156) during the hunger strike, and had neurological findings consistent with Wernicke-Korsakoff syndrome. All 41 patients exhibited altered consciousness which lasted from 3 to 31 days. All patients also presented gaze-evoked horizontal nystagmus and truncal ataxia. Paralysis of lateral rectus muscles was found in 14. Amnesia was apparent in all cases. Abnormal nerve conduction study parameters were not found in the patient group, but the amplitude of compound muscle action potential of the median and fibular nerves and sensory nerve action potential amplitude of the sural nerve were lower than the control group, and distal motor latency of the posterior tibial nerve was significantly prolonged as compared with the control group. The latency of visual evoked potential was prolonged in 22 cases. Somatosensory evoked potential (P37) was prolonged but not statistically significant. Our most significant finding was that the effect of hunger was more prominent on the central nervous system than on the neuromuscular system, despite the fact that all patients were taking thiamine. In our opinion, partial recovery of neurological, and neurocognitive signs in prolonged hunger could be a result of permanent neurological injury. PMID:16987161

  3. S2k-Guideline "Prolonged Weaning".

    PubMed

    Schönhofer, B; Geiseler, J; Dellweg, D; Moerer, O; Barchfeld, T; Fuchs, H; Karg, O; Rosseau, S; Sitter, H; Weber-Carstens, S; Westhoff, M; Windisch, W

    2015-10-01

    All mechanically ventilated patients must be weaned from the ventilator at some stage. According to an International Consensus Conference the criteria for "prolonged weaning" are fulfilled if patients fail at least 3 weaning attempts (i. e. spontaneous breathing trial, SBT) or require more than 7 days of weaning after the first SBT. This occurs in about 15 - 20 % of patients.Because of the growing number of patients requiring prolonged weaning a German guideline on prolonged weaning has been developed. It is an initiative of the German Respiratory Society (Deutsche Gesellschaft für Pneumologie und Beatmungsmedizin e. V., DGP) in cooperation with other societies (see acknowledgement) engaged in the field chaired by the Association of Scientific and Medical Societies in Germany (Arbeitsgemeinschaft der Wissenschaftlichen Medizinischen Fachgesellschaften, AWMF).This guideline deals with the definition, epidemiology, weaning categories, underlying pathophysiology, therapeutic strategies, the weaning unit, transition to out-of-hospital ventilation and therapeutic recommendations for end of life care. This short version summarises recommendations on prolonged weaning from the German guideline. PMID:26444135

  4. Effects of Prolonged Deprivation on Learned Helplessness.

    ERIC Educational Resources Information Center

    Mal, Suraj; And Others

    1990-01-01

    Investigated influence of prolonged deprivation on responses to uncontrollable outcome among 104 Indian students in the tenth grade. Finds high-deprived and female students displayed greater helplessness than did their low-deprived and male counterparts. Females and high-deprives students attributed uncontrollable outcome more to internal, stable,…

  5. Intracellular α-Amylase of Streptococcus mutans

    PubMed Central

    Simpson, Christine L.; Russell, Roy R. B.

    1998-01-01

    Sequencing upstream of the Streptococcus mutans gene for a CcpA gene homolog, regM, revealed an open reading frame, named amy, with homology to genes encoding α-amylases. The deduced amino acid sequence showed a strong similarity (60% amino acid identity) to the intracellular α-amylase of Streptococcus bovis and, in common with this enzyme, lacked a signal sequence. Amylase activity was found only in S. mutans cell extracts, with no activity detected in culture supernatants. Inactivation of amy by insertion of an antibiotic resistance marker confirmed that S. mutans has a single α-amylase activity. The amylase activity was induced by maltose but not by starch, and no acid was produced from starch. S. mutans can, however, transport limit dextrins and maltooligosaccharides generated by salivary amylase, but inactivation of amy did not affect growth on these substrates or acid production. The amylase digested the glycogen-like intracellular polysaccharide (IPS) purified from S. mutans, but the amy mutant was able to digest and produce acid from IPS; thus, amylase does not appear to be essential for IPS breakdown. However, when grown on excess maltose, the amy mutant produced nearly threefold the amount of IPS produced by the parent strain. The role of Amy has not been established, but Amy appears to be important in the accumulation of IPS in S. mutans grown on maltose. PMID:9721315

  6. Intracellular alpha-amylase of Streptococcus mutans.

    PubMed

    Simpson, C L; Russell, R R

    1998-09-01

    Sequencing upstream of the Streptococcus mutans gene for a CcpA gene homolog, regM, revealed an open reading frame, named amy, with homology to genes encoding alpha-amylases. The deduced amino acid sequence showed a strong similarity (60% amino acid identity) to the intracellular alpha-amylase of Streptococcus bovis and, in common with this enzyme, lacked a signal sequence. Amylase activity was found only in S. mutans cell extracts, with no activity detected in culture supernatants. Inactivation of amy by insertion of an antibiotic resistance marker confirmed that S. mutans has a single alpha-amylase activity. The amylase activity was induced by maltose but not by starch, and no acid was produced from starch. S. mutans can, however, transport limit dextrins and maltooligosaccharides generated by salivary amylase, but inactivation of amy did not affect growth on these substrates or acid production. The amylase digested the glycogen-like intracellular polysaccharide (IPS) purified from S. mutans, but the amy mutant was able to digest and produce acid from IPS; thus, amylase does not appear to be essential for IPS breakdown. However, when grown on excess maltose, the amy mutant produced nearly threefold the amount of IPS produced by the parent strain. The role of Amy has not been established, but Amy appears to be important in the accumulation of IPS in S. mutans grown on maltose. PMID:9721315

  7. High-Throughput Intracellular Antimicrobial Susceptibility Testing of Legionella pneumophila

    PubMed Central

    Chiaraviglio, Lucius

    2015-01-01

    Legionella pneumophila is a Gram-negative opportunistic human pathogen that causes a severe pneumonia known as Legionnaires' disease. Notably, in the human host, the organism is believed to replicate solely within an intracellular compartment, predominantly within pulmonary macrophages. Consequently, successful therapy is predicated on antimicrobials penetrating into this intracellular growth niche. However, standard antimicrobial susceptibility testing methods test solely for extracellular growth inhibition. Here, we make use of a high-throughput assay to characterize intracellular growth inhibition activity of known antimicrobials. For select antimicrobials, high-resolution dose-response analysis was then performed to characterize and compare activity levels in both macrophage infection and axenic growth assays. Results support the superiority of several classes of nonpolar antimicrobials in abrogating intracellular growth. Importantly, our assay results show excellent correlations with prior clinical observations of antimicrobial efficacy. Furthermore, we also show the applicability of high-throughput automation to two- and three-dimensional synergy testing. High-resolution isocontour isobolograms provide in vitro support for specific combination antimicrobial therapy. Taken together, findings suggest that high-throughput screening technology may be successfully applied to identify and characterize antimicrobials that target bacterial pathogens that make use of an intracellular growth niche. PMID:26392509

  8. High-Throughput Intracellular Antimicrobial Susceptibility Testing of Legionella pneumophila.

    PubMed

    Chiaraviglio, Lucius; Kirby, James E

    2015-12-01

    Legionella pneumophila is a Gram-negative opportunistic human pathogen that causes a severe pneumonia known as Legionnaires' disease. Notably, in the human host, the organism is believed to replicate solely within an intracellular compartment, predominantly within pulmonary macrophages. Consequently, successful therapy is predicated on antimicrobials penetrating into this intracellular growth niche. However, standard antimicrobial susceptibility testing methods test solely for extracellular growth inhibition. Here, we make use of a high-throughput assay to characterize intracellular growth inhibition activity of known antimicrobials. For select antimicrobials, high-resolution dose-response analysis was then performed to characterize and compare activity levels in both macrophage infection and axenic growth assays. Results support the superiority of several classes of nonpolar antimicrobials in abrogating intracellular growth. Importantly, our assay results show excellent correlations with prior clinical observations of antimicrobial efficacy. Furthermore, we also show the applicability of high-throughput automation to two- and three-dimensional synergy testing. High-resolution isocontour isobolograms provide in vitro support for specific combination antimicrobial therapy. Taken together, findings suggest that high-throughput screening technology may be successfully applied to identify and characterize antimicrobials that target bacterial pathogens that make use of an intracellular growth niche. PMID:26392509

  9. Neuronal Recordings with Solid-Conductor Intracellular Nanoelectrodes (SCINEs)

    PubMed Central

    Angle, Matthew R.; Schaefer, Andreas T.

    2012-01-01

    Direct electrical recording of the neuronal transmembrane potential has been crucial to our understanding of the biophysical mechanisms subserving neuronal computation. Existing intracellular recording techniques, however, limit the accuracy and duration of such measurements by changing intracellular biochemistry and/or by damaging the plasma membrane. Here we demonstrate that nanoengineered electrodes can be used to record neuronal transmembrane potentials in brain tissue without causing these physiological perturbations. Using focused ion beam milling, we have fabricated Solid-Conductor Intracellular NanoElectrodes (SCINEs), from conventional tungsten microelectrodes. SCINEs have tips that are <300 nm in diameter for several micrometers, but can be easily handled and can be inserted into brain tissue. Performing simultaneous whole-cell patch recordings, we show that SCINEs can record action potentials (APs) as well as slower, subthreshold neuronal potentials without altering cellular properties. These results show a key role for nanotechnology in the development of new electrical recording techniques in neuroscience. PMID:22905231

  10. Motor-driven intracellular transport powers bacterial gliding motility.

    PubMed

    Sun, Mingzhai; Wartel, Morgane; Cascales, Eric; Shaevitz, Joshua W; Mignot, Tâm

    2011-05-01

    Protein-directed intracellular transport has not been observed in bacteria despite the existence of dynamic protein localization and a complex cytoskeleton. However, protein trafficking has clear potential uses for important cellular processes such as growth, development, chromosome segregation, and motility. Conflicting models have been proposed to explain Myxococcus xanthus motility on solid surfaces, some favoring secretion engines at the rear of cells and others evoking an unknown class of molecular motors distributed along the cell body. Through a combination of fluorescence imaging, force microscopy, and genetic manipulation, we show that membrane-bound cytoplasmic complexes consisting of motor and regulatory proteins are directionally transported down the axis of a cell at constant velocity. This intracellular motion is transmitted to the exterior of the cell and converted to traction forces on the substrate. Thus, this study demonstrates the existence of a conserved class of processive intracellular motors in bacteria and shows how these motors have been adapted to produce cell motility. PMID:21482768

  11. Carotid Baroreflex Function During Prolonged Exercise

    NASA Technical Reports Server (NTRS)

    Raven, P. B.

    1999-01-01

    Astronauts are often required to work (exercise) at moderate to high intensities for extended periods while performing extra-vehicular activities (EVA). Although the physiologic responses associated with prolonged exercise have been documented, the mechanisms involved in blood pressure regulation under these conditions have not yet been fully elucidated. An understanding of this issue is pertinent to the ability of humans to perform work in microgravity and complies with the emphasis of NASA's Space Physiology and Countermeasures Program. Prolonged exercise at a constant workload is know to result in a progressive decrease in mean arterial pressure (MAP) concomitant with a decrease in stroke volume and a compensatory increase in heart rate. The continuous decrease in MAP during the exercise, which is related to the thermoregulatory redistribution of circulating blood volume to the cutaneous circulation, raises the question as to whether there is a loss of baroreflex regulation of arterial blood pressure. We propose that with prolongation of the exercise to 60 minutes, progressive increases on central command reflect a progressive upward resetting of the carotid baroreflex (CBR) such that the operating point of the CBR is shifted to a pressure below the threshold of the reflex rendering it ineffectual in correcting the downward drift in MAP. In order to test this hypothesis, experiments have been designed to uncouple the global hemodynamic response to prolonged exercise from the central command mediated response via: (1) continuous maintenance of cardiac filling volume by intravenous infusion of a dextran solution; and (2) whole body surface cooling to counteract thermoregulatory cutaneous vasodialation. As the type of work (exercise) performed by astronauts is inherently arm and upper body dependent, we will also examine the physiologic responses to prolonged leg cycling and arm ergometry exercise in the supine positions with and without level lower body negative

  12. Slow recovery in desert perennial vegetation following prolonged human disturbance

    USGS Publications Warehouse

    Guo, Q.

    2004-01-01

    Questions: How long may it take for desert perennial vegetation to recover from prolonged human disturbance and how do different plant community variables (i.e. diversity, density and cover) change during the recovery process? Location: Sonoran Desert, Arizona, USA. Methods: Since protection from grazing from 1907 onwards, plant diversity, density and cover of perennial species were monitored intermittently on ten 10 m x 10 m permanent plots on Tumamoc Hill, Tucson, Arizona, USA. Results: The study shows an exceptionally slow recovery of perennial vegetation from prolonged heavy grazing and other human impacts. Since protection, overall species richness and habitat heterogeneity at the study site continued to increase until the 1960s when diversity, density and cover had been stabilized. During the same period, overall plant density and cover also increased. Species turnover increased gradually with time but no significant relation between any of the three community variables and precipitation or Palmer Drought Severity Index (PDSI) was detected. Conclusions: It took more than 50 yr for the perennial vegetation to recover from prolonged human disturbance. The increases in plant species richness, density, and cover of the perennial vegetation were mostly due to the increase of herbaceous species, especially palatable species. The lack of a clear relationship between environment (e.g. precipitation) and community variables suggests that site history and plant life history must be taken into account in examining the nature of vegetation recovery processes after disturbance.

  13. [Circulating immune complexes in acute and prolonged hepatitis A infection].

    PubMed

    Dautović-Krkić, Sajma; Gribajcević, Mehmed

    2002-01-01

    Level and dynamics activity of circulating immune complexes (CiC) and persistence CiC in the sera in the acute and prolonged HAV-infection was examined. In the same time we explored the relation of level and dynamics CiC compared with level, dynamics and persistence length ALT and IgM anti-HAV in sera, longitude excretion HAV Ag in stool and intensity patohistological damage in liver. Research have been undertaken in the prospected study on two groups with 90 patients in total: 60 patients with prolonged form of the hepatitis A, and 30 patients with HAV-infection with normal development. CiC was prescribe with fotometer in sediment of poliethilenglicol, and IgM anti HAV with ELISA technique. Ag-HAV in stool was prescribe with methodImmuno/electro/osmophoresis. Results of examination showed that high level values of CiC had present in all patients with HAV-infection, bat yet middle values of CiC had significantly higher in prolonged forms (p < 0.01). In a case of patients with PTHA CiC persistence almost three times longer than in HAV infection with normal development. The highest value of CiC have been found from one to two weeks after e peak ALT in HAV and in PTHA 4-6 weeks later. Persistence of elevated values CiC responded to the middle length persistence of Igm anti HAV-in the sera. PMID:12378858

  14. Resveratrol Improves Survival and Prolongs Life Following Hemorrhagic Shock

    PubMed Central

    Ayub, Ahmar; Poulose, Ninu; Raju, Raghavan

    2015-01-01

    Resveratrol has been shown to potentiate mitochondrial function and extend longevity; however, there is no evidence to support whether resveratrol can improve survival or prolong life following hemorrhagic shock. We sought to determine whether (a) resveratrol can improve survival following hemorrhage and resuscitation and (b) prolong life in the absence of resuscitation. Using a hemorrhagic injury (HI) model in the rat, we describe for the first time that the naturally occurring small molecule, resveratrol, may be an effective adjunct to resuscitation fluid. In a series of three sets of experiments we show that resveratrol administration during resuscitation improves survival following HI (p < 0.05), resveratrol and its synthetic mimic SRT1720 can significantly prolong life in the absence of resuscitation fluid (<30 min versus up to 4 h; p < 0.05), and resveratrol as well as SRT1720 restores left ventricular function following HI. We also found significant changes in the expression level of mitochondria-related transcription factors Ppar-α and Tfam, as well as Pgc-1α in the left ventricular tissues of rats subjected to HI and treated with resveratrol. The results indicate that resveratrol is a strong candidate adjunct to resuscitation following severe hemorrhage. PMID:25879628

  15. Prolonging dying is the same as prolonging living--one more response to Long.

    PubMed

    Kuhse, H; Singer, P

    1991-12-01

    In earlier publications, we had argued that Paul Ramsey is inconsistent because he simultaneously asserts that (i) 'all our days and years are of equal worth' and (ii) 'that it is permissible to refrain from prolonging the lives of some dying patients'. Thomas Long has suggested that we have not shown that Paul Ramsey is inconsistent. Ramsey and we, he holds, start from incommensurable metaphysical views: for Ramsey, the dying process has religious significance--God is calling his servant home. While it is normally a good thing to keep a patient alive, it would, for Ramsey, show deafness to God's call to keep a dying patient alive. It is true we do not share Paul Ramsey's religious views. It is, however, not necessary to rely on any particular metaphysical views to refute Ramsey's position. For Ramsey's view to be internally consistent, Ramsey would have to be able to distinguish between dying and non-dying patients. We examine some of Ramsey's examples and show that his practical judgements do not allow us to draw this distinction. This means that, contra Long, we hold fast to our charge that Ramsey's view is inconsistent. PMID:1787522

  16. Prolonged reorganization of thiol-capped Au nanoparticles layered structures

    NASA Astrophysics Data System (ADS)

    Kundu, Sarathi; Das, Kaushik; Konovalov, Oleg

    2013-09-01

    Prolonged reorganization behaviour of mono-, di-, tri- and multi-layer films of Au nanoparticles prepared by Langmuir-Blodgett method on hydrophobic Si(001) substrates have been studied by using X-ray scattering techniques. Out-of-plane study shows that although at the initial stage the reorganization occurs through the compaction of the films keeping the layered structure unchanged but finally all layered structures modify to monolayer structure. Due to this reorganization the Au density increases within the nanometer thick films. In-plane study shows that inside the reorganized films Au nanoparticles are distributed randomly and the particle size modifies as the metallic core of Au nanoparticles coalesces.

  17. Intracellular insulin processing is altered in monocytes from patients with type II diabetes mellitus

    SciTech Connect

    Trischitta, V.; Benzi, L.; Brunetti, A.; Cecchetti, P.; Marchetti, P.; Vigneri, R.; Navalesi, R.

    1987-05-01

    We studied total cell-associated A14-(/sup 125/I)insulin radioactivity (including surface-bound and internalized radioactivity), insulin internalization, and its intracellular degradation at 37 C in monocytes from nonobese type II untreated diabetic patients (n = 9) and normal subjects (n = 7). Total cell-associated radioactivity was decreased in diabetic patients (2.65 +/- 1.21% (+/- SD) vs. 4.47 +/- 1.04% of total radioactivity. Insulin internalization was also reduced in diabetic patients (34.0 +/- 6.8% vs. 59.0 +/- 11.3% of cell-associated radioactivity. Using high performance liquid chromatography six intracellular forms of radioactivity derived from A14-(/sup 125/I) insulin were identified; 10-20% of intracellular radioactivity had approximately 300,000 mol wt and was identified as radioactivity bound to the insulin receptor, and the remaining intracellular radioactivity included intact A14-(/sup 125/I)insulin, (/sup 125/I)iodide, or (/sup 125/I)tyrosine, and three intermediate compounds. A progressive reduction of intact insulin and a corresponding increase in iodine were found when the incubation time was prolonged. Intracellular insulin degradation was reduced in monocytes from diabetic patients; intracellular intact insulin was 65.6 +/- 18.1% vs. 37.4 +/- 18.0% of intracellular radioactivity after 2 min and 23.6 +/- 22.3% vs. 3.9 +/- 2.3% after 60 min in diabetic patients vs. normal subjects, respectively. In conclusion, 1) human monocytes internalize and degrade insulin in the intracellular compartment in a stepwise time-dependent manner; and 2) in monocytes from type II diabetic patients total cell-associated radioactivity, insulin internalization, and insulin degradation are significantly reduced. These defects may be related to the cellular insulin resistance present in these patients.

  18. Mechanisms of Obligatory Intracellular Infection with Anaplasma phagocytophilum

    PubMed Central

    Rikihisa, Yasuko

    2011-01-01

    Summary: Anaplasma phagocytophilum persists in nature by cycling between mammals and ticks. Human infection by the bite of an infected tick leads to a potentially fatal emerging disease called human granulocytic anaplasmosis. A. phagocytophilum is an obligatory intracellular bacterium that replicates inside mammalian granulocytes and the salivary gland and midgut cells of ticks. A. phagocytophilum evolved the remarkable ability to hijack the regulatory system of host cells. A. phagocytophilum alters vesicular traffic to create an intracellular membrane-bound compartment that allows replication in seclusion from lysosomes. The bacterium downregulates or actively inhibits a number of innate immune responses of mammalian host cells, and it upregulates cellular cholesterol uptake to acquire cholesterol for survival. It also upregulates several genes critical for the infection of ticks, and it prolongs tick survival at freezing temperatures. Several host factors that exacerbate infection have been identified, including interleukin-8 (IL-8) and cholesterol. Host factors that overcome infection include IL-12 and gamma interferon (IFN-γ). Two bacterial type IV secretion effectors and several bacterial proteins that associate with inclusion membranes have been identified. An understanding of the molecular mechanisms underlying A. phagocytophilum infection will foster the development of creative ideas to prevent or treat this emerging tick-borne disease. PMID:21734244

  19. Intracellular Ca-carbonate biomineralization is widespread in cyanobacteria.

    PubMed

    Benzerara, Karim; Skouri-Panet, Feriel; Li, Jinhua; Férard, Céline; Gugger, Muriel; Laurent, Thierry; Couradeau, Estelle; Ragon, Marie; Cosmidis, Julie; Menguy, Nicolas; Margaret-Oliver, Isabel; Tavera, Rosaluz; López-García, Purificación; Moreira, David

    2014-07-29

    Cyanobacteria have played a significant role in the formation of past and modern carbonate deposits at the surface of the Earth using a biomineralization process that has been almost systematically considered induced and extracellular. Recently, a deep-branching cyanobacterial species, Candidatus Gloeomargarita lithophora, was reported to form intracellular amorphous Ca-rich carbonates. However, the significance and diversity of the cyanobacteria in which intracellular biomineralization occurs remain unknown. Here, we searched for intracellular Ca-carbonate inclusions in 68 cyanobacterial strains distributed throughout the phylogenetic tree of cyanobacteria. We discovered that diverse unicellular cyanobacterial taxa form intracellular amorphous Ca-carbonates with at least two different distribution patterns, suggesting the existence of at least two distinct mechanisms of biomineralization: (i) one with Ca-carbonate inclusions scattered within the cell cytoplasm such as in Ca. G. lithophora, and (ii) another one observed in strains belonging to the Thermosynechococcus elongatus BP-1 lineage, in which Ca-carbonate inclusions lie at the cell poles. This pattern seems to be linked with the nucleation of the inclusions at the septum of the cells, showing an intricate and original connection between cell division and biomineralization. These findings indicate that intracellular Ca-carbonate biomineralization by cyanobacteria has been overlooked by past studies and open new perspectives on the mechanisms and the evolutionary history of intra- and extracellular Ca-carbonate biomineralization by cyanobacteria. PMID:25009182

  20. Intracellular Ca-carbonate biomineralization is widespread in cyanobacteria

    PubMed Central

    Benzerara, Karim; Skouri-Panet, Feriel; Li, Jinhua; Férard, Céline; Gugger, Muriel; Laurent, Thierry; Couradeau, Estelle; Ragon, Marie; Cosmidis, Julie; Menguy, Nicolas; Margaret-Oliver, Isabel; Tavera, Rosaluz; López-García, Purificación; Moreira, David

    2014-01-01

    Cyanobacteria have played a significant role in the formation of past and modern carbonate deposits at the surface of the Earth using a biomineralization process that has been almost systematically considered induced and extracellular. Recently, a deep-branching cyanobacterial species, Candidatus Gloeomargarita lithophora, was reported to form intracellular amorphous Ca-rich carbonates. However, the significance and diversity of the cyanobacteria in which intracellular biomineralization occurs remain unknown. Here, we searched for intracellular Ca-carbonate inclusions in 68 cyanobacterial strains distributed throughout the phylogenetic tree of cyanobacteria. We discovered that diverse unicellular cyanobacterial taxa form intracellular amorphous Ca-carbonates with at least two different distribution patterns, suggesting the existence of at least two distinct mechanisms of biomineralization: (i) one with Ca-carbonate inclusions scattered within the cell cytoplasm such as in Ca. G. lithophora, and (ii) another one observed in strains belonging to the Thermosynechococcus elongatus BP-1 lineage, in which Ca-carbonate inclusions lie at the cell poles. This pattern seems to be linked with the nucleation of the inclusions at the septum of the cells, showing an intricate and original connection between cell division and biomineralization. These findings indicate that intracellular Ca-carbonate biomineralization by cyanobacteria has been overlooked by past studies and open new perspectives on the mechanisms and the evolutionary history of intra- and extracellular Ca-carbonate biomineralization by cyanobacteria. PMID:25009182

  1. Intracellular Neural Recording with Pure Carbon Nanotube Probes

    PubMed Central

    Yoon, Inho; Hamaguchi, Kosuke; Borzenets, Ivan V.; Finkelstein, Gleb; Mooney, Richard; Donald, Bruce R.

    2013-01-01

    The computational complexity of the brain depends in part on a neuron’s capacity to integrate electrochemical information from vast numbers of synaptic inputs. The measurements of synaptic activity that are crucial for mechanistic understanding of brain function are also challenging, because they require intracellular recording methods to detect and resolve millivolt- scale synaptic potentials. Although glass electrodes are widely used for intracellular recordings, novel electrodes with superior mechanical and electrical properties are desirable, because they could extend intracellular recording methods to challenging environments, including long term recordings in freely behaving animals. Carbon nanotubes (CNTs) can theoretically deliver this advance, but the difficulty of assembling CNTs has limited their application to a coating layer or assembly on a planar substrate, resulting in electrodes that are more suitable for in vivo extracellular recording or extracellular recording from isolated cells. Here we show that a novel, yet remarkably simple, millimeter-long electrode with a sub-micron tip, fabricated from self-entangled pure CNTs can be used to obtain intracellular and extracellular recordings from vertebrate neurons in vitro and in vivo. This fabrication technology provides a new method for assembling intracellular electrodes from CNTs, affording a promising opportunity to harness nanotechnology for neuroscience applications. PMID:23840357

  2. Intracellular trafficking of VP22 in bovine herpesvirus-1 infected cells

    SciTech Connect

    Lobanov, Vladislav A.; Babiuk, Lorne A.; Drunen Littel-van den Hurk, Sylvia van

    2010-01-20

    The intracellular trafficking of different VP22-enhanced yellow fluorescent protein (EYFP) fusion proteins expressed by bovine herpesvirus-1 (BHV-1) recombinants was examined by live-cell imaging. Our results demonstrate that (i) the fusion of EYFP to the C terminus of VP22 does not alter the trafficking of the protein in infected cells, (ii) VP22 expressed during BHV-1 infection translocates to the nucleus through three different pathways, namely early mitosis-dependent nuclear translocation, late massive nuclear translocation that follows a prolonged cytoplasmic stage of the protein in non-mitotic cells, and accumulation of a small subset of VP22 in discrete dot-like nuclear domains during its early cytoplasmic stage, (iii) the addition of the SV40 large-T-antigen nuclear localization signal (NLS) to VP22-EYFP abrogates its early cytoplasmic stage, and (iv) the VP22 {sup 131}PRPR{sup 134} NLS is not required for the late massive nuclear translocation of the protein, but this motif is essential for the targeting of VP22 to discrete dot-like nuclear domains during the early cytoplasmic stage. These results show that the amount of VP22 in the nucleus is precisely regulated at different stages of BHV-1 infection and suggest that the early pathways of VP22 nuclear accumulation may be more relevant to the infection process as the late massive nuclear influx starts when most of the viral progeny has already emerged from the cell.

  3. VEGF-functionalized dextran has longer intracellular bioactivity than VEGF in endothelial cells.

    PubMed

    Maia, João; Vazão, Helena; Pedroso, Dora C S; Jesus, Catarina S H; Brito, Rui M M; Grãos, Mário; Gil, Maria H; Ferreira, Lino

    2012-09-10

    Herein, we report that VEGF-functionalized dextran (dexOx-VEGF) is comparatively superior to free VEGF in prolonging the phosphorylation of VEGF receptor 2 (VEGFR-2). Both dexOx-VEGF and free VEGF activate VEGFR-2, and the complexes are internalized into early endosomes (EEA1(+) vesicles) and then transported to lysosomes (Rab7(+) vesicles). However, after cell activation, dexOx-VEGF is preferentially colocalized in early endosomes where VEGF signaling is still active while free VEGF is preferentially transported to late endosomes or lysosomes. We further show that dexOx-VEGF after phosphorylation of VEGF receptor 2 induces an increase of intracellular Ca(2+) and activates VEGF downstream effectors such as Akt and extracellular signal-regulated kinase (ERK1/2) proteins. Under specific conditions, the activation level is different from the one observed for free VEGF, thus suggesting mechanistic differences, which is illustrated by cell migration and cord-like formation studies. DexOx-VEGF can be cross-linked with adipic acid dihydrazide to form a degradable gel, which in turn can be incorporated in a fibrin gel containing endothelial cells (ECs) to modulate their activity. We envision that these constructs might be beneficial to extend the pro-angiogenic activity of VEGF in ischemic tissues and to modulate the biological activity of vascular cells. PMID:22901277

  4. Severe bradycardia and prolonged hypotension in ciguatera.

    PubMed

    Chan, Thomas Yan Keung

    2013-06-01

    Ciguatera results when ciguatoxin-contaminated coral reef fish from tropical or subtropical waters are consumed. The clinical features that present in affected persons are mainly gastrointestinal, neurological, general, and much less commonly, cardiovascular. We report the case of a 50-year-old man who developed the characteristic combination of acute gastrointestinal and neurological symptoms after the consumption of an unidentified coral reef fish head. In addition to those symptoms, he developed dizziness, severe bradycardia (46 bpm) and prolonged hypotension, which required the administration of intravenous atropine and over three days of intravenous fluid replacement with dopamine infusion. Patients with ciguatera can develop severe bradycardia and prolonged hypotension. Physicians should recognise the possible cardiovascular complications of ciguatera and promptly initiate treatment with intravenous atropine, intravenous fluid replacement and inotropic therapy if such complications are observed. PMID:23665698

  5. Measurements of intracellular Ca2+ in dissociated type I cells of the rabbit carotid body.

    PubMed Central

    Biscoe, T J; Duchen, M R; Eisner, D A; O'Neill, S C; Valdeolmillos, M

    1989-01-01

    1. The carotid body chemoreceptors are stimulated in situ by cyanide (CN-), which mimics the effect of hypoxia. We have shown that CN- increases a calcium-dependent potassium conductance (gK(Ca)) in single type I cells dissociated from the carotid body of the rabbit. We have now used the Ca2(+)-sensitive fluorophore, Fura-2, to measure intracellular Ca2+ directly in single type I cells. 2. CN- reversibly increased [Ca2+]i from approximately 90 nM to a mean of approximately 200 nM. Some of this Ca2+ originated from an intracellular store, which was depleted by exposure to Ca2(+)-free solutions. Prolonged application of CN- caused a sustained increase in [Ca2+]i, suggesting that CN- impairs the removal or sequestration of Ca2+. 3. pHi measured with the dye BCECF (2,7-bis(2-carboxyethyl)-5(and-6)-carboxyfluorescein) did not change consistently in response to CN-, although pHi changed predictably in response to both ammonium chloride and to acidification of the superfusate with CO2. 4. Potassium-induced depolarization (35 mM-K+) caused a large, cadmium-sensitive rise in [Ca2+]i. The K(+)-induced Ca2+ load was used to study the regulation of [Ca2+]i. 5. The clearance of a Ca2+ load was slowed either by removal of [Na+]o or by application of CN-. This shows that both a Na+-Ca2+ exchange and an energy-dependent process or processes contribute to the regulation of [Ca2+]i. 6. Carbachol (CCh, 10-100 microM), which also hyperpolarizes type I cells, caused a small transient rise in [Ca2+]i, indicating release from an exhaustible intracellular pool. The response to CN- was unaffected by prior or continued exposure to CCh, suggesting that the two stimuli operate by distinct mechanisms. 7. The increased gK(Ca) seen in type I cells in response to CN- thus reflects a change in cellular Ca2+ homeostasis. The rise in [Ca2+]i presumably underlies the documented increase in transmitter release from the carotid body in response to CN-. If chemotransduction is a consequence of the

  6. Mobilization of intracellular calcium by intracellular flash photolysis of caged dihydrosphingosine in cultured neonatal rat sensory neurones.

    PubMed

    Ayar, A; Thatcher, N M; Zehavi, U; Trentham, D R; Scott, R H

    1998-01-01

    The ability of dihydrosphingosine to release Ca2+ from intracellular stores in neurones was investigated by combining the whole cell patch clamp technique with intracellular flash photolysis of caged, N-(2-nitrobenzyl)dihydrosphingosine. The caged dihydrosphingosine (100 microM) was applied to the intracellular environment via the CsCl-based patch pipette solution which also contained 0.3% dimethylformamide and 2 mM dithiothreitol. Cultured dorsal root ganglion neurones from neonatal rats were voltage clamped at -90 mV and inward whole cell Ca2+-activated currents were recorded in response to intracellular photorelease of dihydrosphingosine. Intracellular photorelease of dihydrosphingosine (about 5 microM) was achieved using a Xenon flash lamp. Inward Ca2+-activated currents were evoked in 50 out of 57 neurones, the mean delay to current activation following photolysis was 82+/-13 s. The responses were variable with neurones showing transient, oscillating or sustained inward currents. High voltage-activated Ca2+ currents evoked by 100 ms voltage step commands to 0 mV were not attenuated by photorelease of dihydrosphingosine. Controls showed that alone a flash from the Xenon lamp did not activate currents, and that the unphotolysed caged dihydrosphingosine, and intracellular photolysis of 2-(2-nitrobenzylamino) propanediol also did not evoke responses. The dihydrosphingosine current had a reversal potential of -11+/-3 mV (n = 11), and was carried by two distinct Cl- and cation currents which were reduced by 85% and about 20% following replacement of monovalent cations with N-methyl-D-glucamine or application of the Cl- channel blocker niflumic acid (10 microM) respectively. The responses to photoreleased dihydrosphingosine were inhibited by intracellular application of 20 mM EGTA, 10 microM ryanodine or extracellular application of 10 microM dantrolene, but persisted when Ca2+ free saline was applied to the extracellular environment. Intracellular application of

  7. Nanoconjugation prolongs endosomal signaling of the epidermal growth factor receptor and enhances apoptosis

    NASA Astrophysics Data System (ADS)

    Wu, L.; Xu, F.; Reinhard, B. M.

    2016-07-01

    It is becoming increasingly clear that intracellular signaling can be subject to strict spatial control. As the covalent attachment of a signaling ligand to a nanoparticle (NP) impacts ligand-receptor binding, uptake, and trafficking, nanoconjugation provides new opportunities for manipulating intracellular signaling in a controlled fashion. To establish the effect of nanoconjugation on epidermal growth factor (EGF) mediated signaling, we investigate here the intracellular fate of nanoconjugated EGF (NP-EGF) and its bound receptor (EGFR) by quantitative correlated darkfield/fluorescence microscopy and density-based endosomal fractionation. We demonstrate that nanoconjugation prolongs the dwell time of phosphorylated receptors in the early endosomes and that the retention of activated EGFR in the early endosomes is accompanied by an EGF mediated apoptosis at effective concentrations that do not induce apoptosis in the case of free EGF. Overall, these findings indicate nanoconjugation as a rational strategy for modifying signaling that acts by modulating the temporo-spatial distribution of the activated EGF-EGFR ligand-receptor complex.It is becoming increasingly clear that intracellular signaling can be subject to strict spatial control. As the covalent attachment of a signaling ligand to a nanoparticle (NP) impacts ligand-receptor binding, uptake, and trafficking, nanoconjugation provides new opportunities for manipulating intracellular signaling in a controlled fashion. To establish the effect of nanoconjugation on epidermal growth factor (EGF) mediated signaling, we investigate here the intracellular fate of nanoconjugated EGF (NP-EGF) and its bound receptor (EGFR) by quantitative correlated darkfield/fluorescence microscopy and density-based endosomal fractionation. We demonstrate that nanoconjugation prolongs the dwell time of phosphorylated receptors in the early endosomes and that the retention of activated EGFR in the early endosomes is accompanied by an EGF

  8. Brain glycogen decreases during prolonged exercise

    PubMed Central

    Matsui, Takashi; Soya, Shingo; Okamoto, Masahiro; Ichitani, Yukio; Kawanaka, Kentaro; Soya, Hideaki

    2011-01-01

    Abstract Brain glycogen could be a critical energy source for brain activity when the glucose supply from the blood is inadequate (hypoglycaemia). Although untested, it is hypothesized that during prolonged exhaustive exercise that induces hypoglycaemia and muscular glycogen depletion, the resultant hypoglycaemia may cause a decrease in brain glycogen. Here, we tested this hypothesis and also investigated the possible involvement of brain monoamines with the reduced levels of brain glycogen. For this purpose, we exercised male Wistar rats on a treadmill for different durations (30–120 min) at moderate intensity (20 m min−1) and measured their brain glycogen levels using high-power microwave irradiation (10 kW). At the end of 30 and 60 min of running, the brain glycogen levels remained unchanged from resting levels, but liver and muscle glycogen decreased. After 120 min of running, the glycogen levels decreased significantly by ∼37–60% in five discrete brain loci (the cerebellum 60%, cortex 48%, hippocampus 43%, brainstem 37% and hypothalamus 34%) compared to those of the sedentary control. The brain glycogen levels in all five regions after running were positively correlated with the respective blood and brain glucose levels. Further, in the cortex, the levels of methoxyhydroxyphenylglycol (MHPG) and 5-hydroxyindoleacetic acid (5-HIAA), potential involved in degradation of the brain glycogen, increased during prolonged exercise and negatively correlated with the glycogen levels. These results support the hypothesis that brain glycogen could decrease with prolonged exhaustive exercise. Increased monoamines together with hypoglycaemia should be associated with the development of decreased brain glycogen, suggesting a new clue towards the understanding of central fatigue during prolonged exercise. PMID:21521757

  9. Prolonged grief: setting the research agenda

    PubMed Central

    Rosner, Rita

    2015-01-01

    Background Prolonged grief disorder is proposed for the International Classification of Diseases (ICD-11), though it was rejected as a diagnosis for DSM-5. Objective This review outlines findings and defines important areas for future research viewed from a lifespan perspective. Results The development and psychometric evaluation of measures for the new diagnosis is paramount, specifically for children and adolescents. Treatments need to be adapted for specific subgroups and research findings have to be disseminated into various professional settings. PMID:25994020

  10. The Association between Job-Related Psychosocial Factors and Prolonged Fatigue among Industrial Employees in Taiwan

    PubMed Central

    Tang, Feng-Cheng; Li, Ren-Hau; Huang, Shu-Ling

    2016-01-01

    Background and Objectives Prolonged fatigue is common among employees, but the relationship between prolonged fatigue and job-related psychosocial factors is seldom studied. This study aimed (1) to assess the individual relations of physical condition, psychological condition, and job-related psychosocial factors to prolonged fatigue among employees, and (2) to clarify the associations between job-related psychosocial factors and prolonged fatigue using hierarchical regression when demographic characteristics, physical condition, and psychological condition were controlled. Methods A cross-sectional study was employed. A questionnaire was used to obtain information pertaining to demographic characteristics, physical condition (perceived physical health and exercise routine), psychological condition (perceived mental health and psychological distress), job-related psychosocial factors (job demand, job control, and workplace social support), and prolonged fatigue. Results A total of 3,109 employees were recruited. Using multiple regression with controlled demographic characteristics, psychological condition explained 52.0% of the variance in prolonged fatigue. Physical condition and job-related psychosocial factors had an adjusted R2 of 0.370 and 0.251, respectively. Hierarchical multiple regression revealed that, among job-related psychosocial factors, job demand and job control showed significant associations with fatigue. Conclusion Our findings highlight the role of job demand and job control, in addition to the role of perceived physical health, perceived mental health, and psychological distress, in workers’ prolonged fatigue. However, more research is required to verify the causation among all the variables. PMID:26930064

  11. Evidence of Health Risks Associated with Prolonged Standing at Work and Intervention Effectiveness

    PubMed Central

    Waters, Thomas R.; Dick, Robert B.

    2015-01-01

    Purpose Prolonged standing at work has been shown to be associated with a number of potentially serious health outcomes, such as lower back and leg pain, cardiovascular problems, fatigue, discomfort, and pregnancy related health outcomes. Recent studies have been conducted examining the relationship between these health outcomes and the amount of time spent standing while on the job. The purpose of this article was to provide a review of the health risks and interventions for workers and employers that are involved in occupations requiring prolonged standing. A brief review of recommendations by governmental and professional organizations for hours of prolonged standing is also included. Findings Based on our review of the literature, there seems to be ample evidence showing that prolonged standing at work leads to adverse health outcomes. Review of the literature also supports the conclusion that certain interventions are effective in reducing the hazards associated with prolonged standing. Suggested interventions include the use of floor mats, sit-stand workstations/chairs, shoes, shoe inserts and hosiery or stockings. Studies could be improved by using more precise definitions of prolonged standing (e.g., duration, movement restrictions, and type of work), better measurement of the health outcomes and more rigorous study protocols. Conclusion and Clinical Relevance Use of interventions and following suggested guidelines on hours of standing from governmental and professional organizations should reduce the health risks from prolonged standing. PMID:25041875

  12. Prolonging life: legal, ethical, and social dilemmas.

    PubMed

    Paulson, Steve; Comfort, Christopher P; Lee, Barbara Coombs; Shemie, Sam; Solomon, Mildred Z

    2014-11-01

    The ability of modern medicine to prolong life has raised a variety of difficult legal, ethical, and social issues on which reasonable minds can differ. Among these are the morality of euthanasia in cases of deep coma or irreversible injury, as well as the Dead Donor Rule with respect to organ harvesting and transplants. As science continues to refine and develop lifesaving technologies, questions remain as to how much medical effort and financial resources should be expended to prolong the lives of patients suspended between life and death. At what point should death be considered irreversible? What criteria should be used to determine when to withhold or withdraw life-prolonging treatments in cases of severe brain damage and terminal illness? To explore these complex dilemmas, Steve Paulson, executive producer and host of To the Best of Our Knowledge, moderated a discussion panel. Pediatrician Sam Shemie, hospice medical director Christopher P. Comfort, bioethicist Mildred Z. Solomon, and attorney Barbara Coombs Lee examined the underlying assumptions and considerations that ultimately shape individual and societal decisions surrounding these issues. The following is an edited transcript of the discussion that occurred November 12, 2013, 7:00-8:30 PM, at the New York Academy of Sciences in New York City. PMID:25079490

  13. Novel Waddlia Intracellular Bacterium in Artibeus intermedius Fruit Bats, Mexico

    PubMed Central

    Pierlé, Sebastián Aguilar; Morales, Cirani Obregón; Martínez, Leonardo Perea; Ceballos, Nidia Aréchiga; Rivero, Juan José Pérez; Díaz, Osvaldo López; Brayton, Kelly A.

    2015-01-01

    An intracellular bacterium was isolated from fruit bats (Artibeus intermedius) in Cocoyoc, Mexico. The bacterium caused severe lesions in the lungs and spleens of bats and intracytoplasmic vacuoles in cell cultures. Sequence analyses showed it is related to Waddlia spp. (order Chlamydiales). We propose to call this bacterium Waddlia cocoyoc. PMID:26583968

  14. Novel Waddlia Intracellular Bacterium in Artibeus intermedius Fruit Bats, Mexico.

    PubMed

    Pierlé, Sebastián Aguilar; Morales, Cirani Obregón; Martínez, Leonardo Perea; Ceballos, Nidia Aréchiga; Rivero, Juan José Pérez; Díaz, Osvaldo López; Brayton, Kelly A; Setién, Alvaro Aguilar

    2015-12-01

    An intracellular bacterium was isolated from fruit bats (Artibeus intermedius) in Cocoyoc, Mexico. The bacterium caused severe lesions in the lungs and spleens of bats and intracytoplasmic vacuoles in cell cultures. Sequence analyses showed it is related to Waddlia spp. (order Chlamydiales). We propose to call this bacterium Waddlia cocoyoc. PMID:26583968

  15. Proton-dependent zinc release from intracellular ligands.

    PubMed

    Kiedrowski, Lech

    2014-07-01

    In cultured cortical and hippocampal neurons when intracellular pH drops from 6.6 to 6.1, yet unclear intracellular stores release micromolar amounts of Zn(2+) into the cytosol. Mitochondria, acidic organelles, and/or intracellular ligands could release this Zn(2+) . Although exposure to the protonophore FCCP precludes reloading of the mitochondria and acidic organelles with Zn(2+) , FCCP failed to compromise the ability of the intracellular stores to repeatedly release Zn(2+) . Therefore, Zn(2+) -releasing stores were not mitochondria or acidic organelles but rather intracellular Zn(2+) ligands. To test which ligands might be involved, the rate of acid-induced Zn(2+) release from complexes with cysteine, glutathione, histidine, aspartate, glutamate, glycine, and carnosine was investigated; [Zn(2+) ] was monitored in vitro using the ratiometric Zn(2+) -sensitive fluorescent probe FuraZin-1. Carnosine failed to chelate Zn(2+) but did chelate Cu(2+) ; the remaining ligands chelated Zn(2+) and upon acidification were releasing it into the medium. However, when pH was decreasing from 6.6 to 6.1, only zinc-cysteine complexes rapidly accelerated the rate of Zn(2+) release. The zinc-cysteine complexes also released Zn(2+) when a histidine-modifying agent, diethylpyrocarbonate, was applied at pH 7.2. Since the cytosolic zinc-cysteine complexes can contain micromolar amounts of Zn(2+) , these complexes may represent the stores responsible for an acid-induced intracellular Zn(2+) release. This study aimed at identifying intracellular stores which release Zn(2+) when pHi drops from 6.6 to 6.1. It was found that these stores are not mitochondria or acidic organelles, but rather intracellular Zn(2+) ligands. When the pH was decreasing from 6.6 to 6.1, only zinc-cysteine complexes showed a rapid acceleration in the rate of Zn(2+) release. Therefore, the stores responsible for an acid-induced intracellular Zn(2+) release in neurons may be the cytosolic zinc-cysteine complexes

  16. A prolonged rhythmic midtemporal discharge in a child without seizures.

    PubMed

    Fawaz, Ahmad; Nasreddine, Wassim; Bustros, Stephanie; Kayed, Deeb Maxwell; Beydoun, Ahmad

    2015-04-01

    Rhythmic midtemporal discharge (RMTD) is one of the benign epileptiform variants, typically consisting of runs of 4-Hz to 7-Hz activity, lasting up to 10 seconds and maximal over the midtemporal area. We report a child who, during an admission for diagnostic closed-circuit television (CCTV) and electroencephalographic (EEG) monitoring, was found to have prolonged rhythmic monomorphic discharges, alternating over both midtemporal areas, with one of the discharges lasting up to 82 minutes. An analysis of the dominant frequency, during the longest discharge, showed that it was monomorphic throughout. On the basis of various features of these discharges, we concluded that they represented RMTD of unusual duration. PMID:24864322

  17. Adaptive prolonged postreproductive life span in killer whales.

    PubMed

    Foster, Emma A; Franks, Daniel W; Mazzi, Sonia; Darden, Safi K; Balcomb, Ken C; Ford, John K B; Croft, Darren P

    2012-09-14

    Prolonged life after reproduction is difficult to explain evolutionarily unless it arises as a physiological side effect of increased longevity or it benefits related individuals (i.e., increases inclusive fitness). There is little evidence that postreproductive life spans are adaptive in nonhuman animals. By using multigenerational records for two killer whale (Orcinus orca) populations in which females can live for decades after their final parturition, we show that postreproductive mothers increase the survival of offspring, particularly their older male offspring. This finding may explain why female killer whales have evolved the longest postreproductive life span of all nonhuman animals. PMID:22984064

  18. Fluorescence Ratio Imaging Of Dynamic Intracellular Signals

    NASA Astrophysics Data System (ADS)

    Harootunian, Alec T.; Kao, J. P.; Tsien, Roger Y.

    1989-12-01

    Traditional biochemical assays of cellular messengers require grinding up thousands or millions of cells for each data point. Such destructive measurements use up large amounts of tissue, have poor time resolution, and cannot assess heterogeneity between individual cells or dynamic spatial localizations. Recent technical advances now enable important ionic signals to be continuously imaged inside individual living cells with micron spatial resolution and subsecond time resolution. This methodology relies on the molecular engineering of indicator dyes whose fluorescence is strong and highly sensitive to ions such as Ca2+, H+, or Na+. Binding of these ions shifts the fluorescence excitation spectrum of the corresponding indicator. The ratio of excitation amplitudes at two wavelengths measures the free ion concentration while canceling out intensity variations due to nonuniform cell thickness or dye content. By rapidly alternating between the two ion-sensitive excitation wavelengths, a fluorescence microscope equipped with a low-light television camera and digital image processor can produce dynamic images of intracellular messenger levels. In many populations of cells traditionally assumed to be homogeneous, we find that neighboring individual cells can differ enormously in their cytosolic Ca2+ response to agonist stimulation, some ignoring the stimulus, others raising cytosolic Ca2+ transiently, others showing oscillations. Oscillations have been speculated to be important as a basis for frequency-coding of oscillations. Oscillations have been speculated to be important as a basis for frequency-coding of graded inputs; we are investigating the mechanism of their generation using light flashes to generate pulses of intracellular messengers. Spatial gradients of cytosolic Ca t+ within single cells have been observed in embryos during fertilization and development, neurons exposed to electrical or drug stimulation and in cytotoxic T lymphocytes during killing of target

  19. Secretome of obligate intracellular Rickettsia

    PubMed Central

    Gillespie, Joseph J.; Kaur, Simran J.; Rahman, M. Sayeedur; Rennoll-Bankert, Kristen; Sears, Khandra T.; Beier-Sexton, Magda; Azad, Abdu F.

    2014-01-01

    The genus Rickettsia (Alphaproteobacteria, Rickettsiales, Rickettsiaceae) is comprised of obligate intracellular parasites, with virulent species of interest both as causes of emerging infectious diseases and for their potential deployment as bioterrorism agents. Currently, there are no effective commercially available vaccines, with treatment limited primarily to tetracycline antibiotics, although others (e.g. josamycin, ciprofloxacin, chloramphenicol, and azithromycin) are also effective. Much of the recent research geared toward understanding mechanisms underlying rickettsial pathogenicity has centered on characterization of secreted proteins that directly engage eukaryotic cells. Herein, we review all aspects of the Rickettsia secretome, including six secretion systems, 19 characterized secretory proteins, and potential moonlighting proteins identified on surfaces of multiple Rickettsia species. Employing bioinformatics and phylogenomics, we present novel structural and functional insight on each secretion system. Unexpectedly, our investigation revealed that the majority of characterized secretory proteins have not been assigned to their cognate secretion pathways. Furthermore, for most secretion pathways, the requisite signal sequences mediating translocation are poorly understood. As a blueprint for all known routes of protein translocation into host cells, this resource will assist research aimed at uniting characterized secreted proteins with their apposite secretion pathways. Furthermore, our work will help in the identification of novel secreted proteins involved in rickettsial ‘life on the inside’. PMID:25168200

  20. Dynamics of intracellular information decoding

    NASA Astrophysics Data System (ADS)

    Kobayashi, Tetsuya J.; Kamimura, Atsushi

    2011-10-01

    A variety of cellular functions are robust even to substantial intrinsic and extrinsic noise in intracellular reactions and the environment that could be strong enough to impair or limit them. In particular, of substantial importance is cellular decision-making in which a cell chooses a fate or behavior on the basis of information conveyed in noisy external signals. For robust decoding, the crucial step is filtering out the noise inevitably added during information transmission. As a minimal and optimal implementation of such an information decoding process, the autocatalytic phosphorylation and autocatalytic dephosphorylation (aPadP) cycle was recently proposed. Here, we analyze the dynamical properties of the aPadP cycle in detail. We describe the dynamical roles of the stationary and short-term responses in determining the efficiency of information decoding and clarify the optimality of the threshold value of the stationary response and its information-theoretical meaning. Furthermore, we investigate the robustness of the aPadP cycle against the receptor inactivation time and intrinsic noise. Finally, we discuss the relationship among information decoding with information-dependent actions, bet-hedging and network modularity.

  1. Intracellular signalling during neutrophil recruitment.

    PubMed

    Mócsai, Attila; Walzog, Barbara; Lowell, Clifford A

    2015-08-01

    Recruitment of leucocytes such as neutrophils to the extravascular space is a critical step of the inflammation process and plays a major role in the development of various diseases including several cardiovascular diseases. Neutrophils themselves play a very active role in that process by sensing their environment and responding to the extracellular cues by adhesion and de-adhesion, cellular shape changes, chemotactic migration, and other effector functions of cell activation. Those responses are co-ordinated by a number of cell surface receptors and their complex intracellular signal transduction pathways. Here, we review neutrophil signal transduction processes critical for recruitment to the site of inflammation. The two key requirements for neutrophil recruitment are the establishment of appropriate chemoattractant gradients and the intrinsic ability of the cells to migrate along those gradients. We will first discuss signalling steps required for sensing extracellular chemoattractants such as chemokines and lipid mediators and the processes (e.g. PI3-kinase pathways) leading to the translation of extracellular chemoattractant gradients to polarized cellular responses. We will then discuss signal transduction by leucocyte adhesion receptors (e.g. tyrosine kinase pathways) which are critical for adhesion to, and migration through the vessel wall. Finally, additional neutrophil signalling pathways with an indirect effect on the neutrophil recruitment process, e.g. through modulation of the inflammatory environment, will be discussed. Mechanistic understanding of these pathways provide better understanding of the inflammation process and may point to novel therapeutic strategies for controlling excessive inflammation during infection or tissue damage. PMID:25998986

  2. The role of autophagy in the intracellular survival of Campylobacter concisus

    PubMed Central

    Burgos-Portugal, Jose A.; Mitchell, Hazel M.; Castaño-Rodríguez, Natalia; Kaakoush, Nadeem O.

    2014-01-01

    Campylobacter concisus is an emerging pathogen that has been associated with gastrointestinal diseases. Given the importance of autophagy for the elimination of intracellular bacteria and the subversion of this process by pathogenic bacteria, we investigated the role of autophagy in C. concisus intracellular survival. Gentamicin protection assays were employed to assess intracellular levels of C. concisus within Caco-2 cells, following autophagy induction and inhibition. To assess the interaction between C. concisus and autophagosomes, confocal microscopy, scanning electron microscopy, and transmission electron microscopy were employed. Expression levels of 84 genes involved in the autophagy process were measured using qPCR. Autophagy inhibition resulted in two- to four-fold increases in intracellular levels of C. concisus within Caco-2 cells, while autophagy induction resulted in a significant reduction in intracellular levels or bacterial clearance. C. concisus strains with low intracellular survival levels showed a dramatic increase in these levels upon autophagy inhibition. Confocal microscopy showed co-localization of the bacterium with autophagosomes, while transmission electron microscopy identified intracellular bacteria persisting within autophagic vesicles. Further, qPCR showed that following infection, 13 genes involved in the autophagy process were significantly regulated, and a further five showed borderline results, with an overall indication towards a dampening effect exerted by the bacterium on this process. Our data collectively indicates that while autophagy is important for the clearance of C. concisus, some strains may manipulate this process to benefit their intracellular survival. PMID:24918042

  3. Television Quiz Show Simulation

    ERIC Educational Resources Information Center

    Hill, Jonnie Lynn

    2007-01-01

    This article explores the simulation of four television quiz shows for students in China studying English as a foreign language (EFL). It discusses the adaptation and implementation of television quiz shows and how the students reacted to them.

  4. Inhibition of AMPK accentuates prolonged caloric restriction-induced change in cardiac contractile function through disruption of compensatory autophagy.

    PubMed

    Zheng, Qijun; Zhao, Kun; Han, Xuefeng; Huff, Anna F; Cui, Qin; Babcock, Sara A; Yu, Shiqiang; Zhang, Yingmei

    2015-02-01

    Prolonged caloric restriction often results in alteration in heart geometry and function although the underlying mechanism remains poorly defined. Autophagy, a conserved pathway for bulk degradation of intracellular proteins and organelles, preserves energy and nutrient in the face of caloric insufficiency. This study was designed to examine the role of AMPK in prolonged caloric restriction-induced change in cardiac homeostasis and the underlying mechanism(s) involved with a focus on autophagy. Wild-type (WT) and AMPK kinase dead (KD) mice were caloric restricted (by 40%) for 30 weeks. Echocardiographic, cardiomyocyte contractile and intracellular Ca²⁺ properties, autophagy and autophagy regulatory proteins were evaluated. Caloric restriction compromised echocardiographic indices (decreased ventricular mass, left ventricular diameters, and cardiac output), cardiomyocyte contractile and intracellular Ca²⁺ properties associated with upregulated autophagy (Beclin-1, Atg5 and LC3BII-to-LC3BI ratio), increased autophagy adaptor protein p62, elevated phosphorylation of AMPK and TSC1/2, depressed phosphorylation of mTOR and ULK1. Although AMPK inhibition did not affect cardiac mechanical function, autophagy and autophagy signaling proteins, it significantly accentuated caloric restriction-induced changes in myocardial contractile function and intracellular Ca²⁺ handling. Interestingly, AMPK inhibition reversed caloric restriction-induced changes in autophagy and autophagy signaling. AMPK inhibition led to dampened levels of Beclin-1, Atg 5 and LC3B ratio along with suppressed phosphorylation of AMPK and TSC1/2 as well as elevated phosphorylation of mTOR and ULK1. Taken together, these data suggest an indispensible role for AMPK in the maintenance of cardiac homeostasis under prolonged caloric restriction-induced pathological changes possibly through autophagy regulation. This article is part of a Special Issue entitled: Autophagy and protein quality control in

  5. Proton Fall or Bicarbonate Rise: GLYCOLYTIC RATE IN MOUSE ASTROCYTES IS PAVED BY INTRACELLULAR ALKALINIZATION.

    PubMed

    Theparambil, Shefeeq M; Weber, Tobias; Schmälzle, Jana; Ruminot, Ivàn; Deitmer, Joachim W

    2016-09-01

    Glycolysis is the primary step for major energy production in the cell. There is strong evidence suggesting that glucose consumption and rate of glycolysis are highly modulated by cytosolic pH/[H(+)], but those can also be stimulated by an increase in the intracellular [HCO3 (-)]. Because proton and bicarbonate shift concomitantly, it remained unclear whether enhanced glucose consumption and glycolytic rate were mediated by the changes in intracellular [H(+)] or [HCO3 (-)]. We have asked whether glucose metabolism is enhanced by either a fall in intracellular [H(+)] or a rise in intracellular [HCO3 (-)], or by both, in mammalian astrocytes. We have recorded intracellular glucose in mouse astrocytes using a FRET-based nanosensor, while imposing different intracellular [H(+)] and [CO2]/[HCO3 (-)]. Glucose consumption and glycolytic rate were augmented by a fall in intracellular [H(+)], irrespective of a concomitant rise or fall in intracellular [HCO3 (-)]. Transport of HCO3 (-) into and out of astrocytes by the electrogenic sodium bicarbonate cotransporter (NBCe1) played a crucial role in causing changes in intracellular pH and [HCO3 (-)], but was not obligatory for the pH-dependent changes in glucose metabolism. Our results clearly show that it is the cytosolic pH that modulates glucose metabolism in cortical astrocytes, and possibly also in other cell types. PMID:27422823

  6. [Prolonged or chronic fatigue of unknown origin].

    PubMed

    Favrat, Bernard; Guessous, Idris; Gonthier, Ariane; Cornuz, Jacques

    2015-04-22

    Although prolonged or chronic fatigue is a very common complaint in primary care medicine, a biomedical obvious cause is often not found. In such a case, for women between 18 and 50 years with a ferritin level of less than 50 µg/l in the absence of anaemia, an iron supplementation may be associated with an improvement in fatigue. Appropriate treatment is also important for depression, anxiety or insomnia. In other cases, the approach is essentially non-pharmacological in the form of lifestyle advice, empathy and cognitive behavioural therapy as well as progressive and adapted physical exercises. PMID:26072601

  7. Prolonged cataleptogenic effects of potentized homoeopathic drugs.

    PubMed

    Sukul, N C; Bala, S K; Bhattacharyya, B

    1986-01-01

    The four homoeopathic drugs, Gelsemium, Cannabis Indica, Graphites and Agaricus Muscarius, administered orally in 30th and 200th potencies on white rats, enhanced restraint-induced catalepsy in a similar manner to the two standard drugs pilocarpine and haloperidol (IP injection at 5 mg/kg). All the drugs tested differed from each other in the duration of cataleptogenic effect, which was more prolonged with Cannabis, Graphites and Agaricus than with Gelsemium and the two non-homoeopathic drugs used. The 200th potency of any homoeopathic drug tested acted longer than its 30th potency. PMID:3088660

  8. JC virus Reactivation During Prolonged Natalizumab Monotherapy for Multiple Sclerosis

    PubMed Central

    Chalkias, Spyridon; Dang, Xin; Bord, Evelyn; Stein, Marion C.; Kinkel, R. Philip; Sloane, Jacob A.; Donnelly, Maureen; Ionete, Carolina; Houtchens, Maria K.; Buckle, Guy J.; Batson, Stephanie; Koralnik, Igor J.

    2015-01-01

    Objective To determine the prevalence of JC virus (JCV) reactivation and JCV-specific cellular immune response during prolonged natalizumab treatment for multiple sclerosis (MS). Methods We enrolled 43 JCV-seropositive MS patients, including 32 on natalizumab monotherapy>18 months, 6 on interferon β-1a monotherapy>36 months and 5 untreated controls. We performed QPCR in cerebrospinal fluid (CSF), blood and urine for JCV DNA and we determined JCV-specific T cell responses using enzyme-linked immunosorbent spot (ELISpot) and intracellular cytokine staining (ICS) assays, ex vivo and after in vitro stimulation with JCV peptides. Results JCV DNA was detected in the CSF of 2/27 (7.4%) natalizumab-treated MS patients who had no symptoms or MRI lesions consistent with progressive multifocal leukoencephalopathy. JCV DNA was detected in blood of 12/43 (27.9%) and in urine of 11/43 (25.6%) subjects without difference between natalizumab-treated patients and controls. JC viral load was higher in CD34+ cells and in monocytes compared to other subpopulations. ICS was more sensitive than ELISpot, and JCV-specific T cell responses, mediated by both CD4+ and CD8+ T-lymphocytes, were detected more frequently after in vitro stimulation. JCV-specific CD4+ T-cells were detected ex vivo more frequently in MS patients with JCV DNA in CD34+ (p=0.05) and B cells (p=0.03). Interpretation Asymptomatic JCV reactivation may occur in CSF of natalizumab-treated MS patients. JCV DNA load is higher in circulating CD34+ cells and monocytes compared to other mononuclear cells, and JCV in blood might trigger a JCV-specific CD4+ T-cell response. JCV-specific cellular immune response is highly prevalent in all JCV-seropositive MS patients, regardless of treatment. PMID:24687904

  9. Nanoconjugation prolongs endosomal signaling of the epidermal growth factor receptor and enhances apoptosis.

    PubMed

    Wu, L; Xu, F; Reinhard, B M

    2016-07-14

    It is becoming increasingly clear that intracellular signaling can be subject to strict spatial control. As the covalent attachment of a signaling ligand to a nanoparticle (NP) impacts ligand-receptor binding, uptake, and trafficking, nanoconjugation provides new opportunities for manipulating intracellular signaling in a controlled fashion. To establish the effect of nanoconjugation on epidermal growth factor (EGF) mediated signaling, we investigate here the intracellular fate of nanoconjugated EGF (NP-EGF) and its bound receptor (EGFR) by quantitative correlated darkfield/fluorescence microscopy and density-based endosomal fractionation. We demonstrate that nanoconjugation prolongs the dwell time of phosphorylated receptors in the early endosomes and that the retention of activated EGFR in the early endosomes is accompanied by an EGF mediated apoptosis at effective concentrations that do not induce apoptosis in the case of free EGF. Overall, these findings indicate nanoconjugation as a rational strategy for modifying signaling that acts by modulating the temporo-spatial distribution of the activated EGF-EGFR ligand-receptor complex. PMID:27378391

  10. The Wordpath Show.

    ERIC Educational Resources Information Center

    Anderton, Alice

    The Intertribal Wordpath Society is a nonprofit educational corporation formed to promote the teaching, status, awareness, and use of Oklahoma Indian languages. The Society produces "Wordpath," a weekly 30-minute public access television show about Oklahoma Indian languages and the people who are teaching and preserving them. The show aims to…

  11. Review and Outcome of Prolonged Cardiopulmonary Resuscitation

    PubMed Central

    Youness, Houssein; Al Halabi, Tarek; Hussein, Hussein; Awab, Ahmed; Jones, Kellie; Keddissi, Jean

    2016-01-01

    The maximal duration of cardiopulmonary resuscitation (CPR) is unknown. We report a case of prolonged CPR. We have then reviewed all published cases with CPR duration equal to or more than 20 minutes. The objective was to determine the survival rate, the neurological outcome, and the characteristics of the survivors. Measurements and Main Results. The CPR data for 82 patients was reviewed. The median duration of CPR was 75 minutes. Patients mean age was 43 ± 21 years with no significant comorbidities. The main causes of the cardiac arrests were myocardial infarction (29%), hypothermia (21%), and pulmonary emboli (12%). 74% of the arrests were witnessed, with a mean latency to CPR of 2 ± 6 minutes and good quality chest compression provided in 96% of the cases. Adjunct therapy included extracorporeal membrane oxygenation (18%), thrombolysis (15.8%), and rewarming for hypothermia (19.5%). 83% were alive at 1 year, with full neurological recovery reported in 63 patients. Conclusion. Patients undergoing prolonged CPR can survive with good outcome. Young age, myocardial infarction, and potentially reversible causes of cardiac arrest such as hypothermia and pulmonary emboli predict a favorable result, especially when the arrest is witnessed and followed by prompt and good resuscitative efforts. PMID:26885387

  12. Prolonged Sleep under Stone Age Conditions

    PubMed Central

    Piosczyk, Hannah; Landmann, Nina; Holz, Johannes; Feige, Bernd; Riemann, Dieter; Nissen, Christoph; Voderholzer, Ulrich

    2014-01-01

    Study Objectives: We report on a unique experiment designed to investigate the impact of prehistoric living conditions on sleep-wake behavior. Methods: A group of five healthy adults were assessed during life in a Stone Age-like settlement over two months. Results: The most notable finding was that nocturnal time in bed and estimated sleep time, as measured by actigraphy, markedly increased during the experimental period compared to the periods prior to and following the experiment. These increases were primarily driven by a phase-advance shift of sleep onset. Subjective assessments of health and functioning did not reveal any relevant changes across the study. Conclusions: Our observations provide further evidence for the long-held belief that the absence of modern living conditions is associated with an earlier sleep phase and prolonged sleep duration. Commentary: A commentary on this article appears in this issue on page 723. Citation: Piosczyk H, Landmann N, Holz J, Feige B, Riemann D, Nissen C, Voderholzer U. Prolonged sleep under Stone Age conditions. J Clin Sleep Med 2014;10(7):719-722. PMID:25024647

  13. Autophagy and checkpoints for intracellular pathogen defense

    PubMed Central

    Paulus, Geraldine L.C.; Xavier, Ramnik J.

    2015-01-01

    Purpose of review Autophagy plays a crucial role in intracellular defense against various pathogens. Xenophagy is a form of selective autophagy that targets intracellular pathogens for degradation. In addition, several related yet distinct intracellular defense responses depend on autophagy-related (ATG) genes. This review gives an overview of these processes, pathogen strategies to subvert them, and their crosstalk with various cell death programs. Recent findings The recruitment of ATG proteins plays a key role in multiple intracellular defense programs, specifically xenophagy, LC3-associated phagocytosis (LAP), and the IFNγ-mediated elimination of pathogens such as Toxoplasma gondii and murine norovirus. Recent progress has revealed methods employed by pathogens to resist these intracellular defense mechanisms and/or persist in spite of them. The intracellular pathogen load can tip the balance between cell survival and cell death. Further, it was recently observed that LAP is indispensable for the efficient clearance of dying cells. Summary Autophagy-dependent and ATG gene-dependent pathways are essential in intracellular defense against a broad range of pathogens. PMID:25394238

  14. Prolonged grief disorder and depression in a German community sample.

    PubMed

    Schaal, Susanne; Richter, Anne; Elbert, Thomas

    2014-01-01

    The aims of this study were to examine rates and risk factors for prolonged grief and to investigate the association between prolonged grief and depression. The authors interviewed a heterogeneous bereaved sample of 61 Germans, 6 of whom had prolonged grief and depression, respectively. The 2 syndromes were strongly linked to one another. Risk factors for prolonged grief were being a woman and having high levels of religious beliefs and low levels of satisfaction with one's religious beliefs, emotional closeness to the deceased, and unanticipated loss. Symptoms of prolonged grief may endure years post-loss and often overlap with depression. PMID:24758218

  15. Stochastic resonance in an intracellular genetic perceptron.

    PubMed

    Bates, Russell; Blyuss, Oleg; Zaikin, Alexey

    2014-03-01

    Intracellular genetic networks are more intelligent than was first assumed due to their ability to learn. One of the manifestations of this intelligence is the ability to learn associations of two stimuli within gene-regulating circuitry: Hebbian-type learning within the cellular life. However, gene expression is an intrinsically noisy process; hence, we investigate the effect of intrinsic and extrinsic noise on this kind of intracellular intelligence. We report a stochastic resonance in an intracellular associative genetic perceptron, a noise-induced phenomenon, which manifests itself in noise-induced increase of response in efficiency after the learning event under the conditions of optimal stochasticity. PMID:24730883

  16. Visualization of Intracellular Tyrosinase Activity in vitro

    PubMed Central

    Setty, Subba Rao Gangi

    2016-01-01

    Melanocytes produce the melanin pigments in melanosomes and these organelles protect the skin against harmful ultraviolet rays. Tyrosinase is the key cuproenzyme which initiates the pigment synthesis using its substrate amino acid tyrosine or L-DOPA (L-3, 4-dihydroxyphenylalanine). Moreover, the activity of tyrosinase directly correlates to the cellular pigmentation. Defects in tyrosinase transport to melanosomes or mutations in the enzyme or reduced intracellular copper levels results in loss of tyrosinase activity in melanosomes, commonly observed in albinism. Here, we described a method to detect the intracellular activity of tyrosinase in mouse melanocytes. This protocol will visualize the active tyrosinase present in the intracellular vesicles or organelles including melanosomes. PMID:27231711

  17. QTc interval prolongation with vascular endothelial growth factor receptor tyrosine kinase inhibitors

    PubMed Central

    Ghatalia, P; Je, Y; Kaymakcalan, M D; Sonpavde, G; Choueiri, T K

    2015-01-01

    Background: Multi-targeted vascular endothelial growth factor receptor (VEGFR) tyrosine kinase inhibitors (TKIs) are known to cause cardiac toxicity, but the relative risk (RR) of QTc interval prolongation and serious arrhythmias associated with them are not reported. Methods: We conducted a trial-level meta-analysis of randomised phase II and III trials comparing arms with and without a US Food and Drug Administration-approved VEGFR TKI (sunitinib, sorafenib, pazopanib, axitinib, vandetanib, cabozantinib, ponatinib and regorafenib). A total of 6548 patients from 18 trials were selected. Statistical analyses were conducted to calculate the summary incidence, RR and 95% CIs. Results: The RR for all-grade and high-grade QTc prolongation for the TKI vs no TKI arms was 8.66 (95% CI 4.92–15.2, P<0.001) and 2.69 (95% CI 1.33–5.44, P=0.006), respectively, with most of the events being asymptomatic QTc prolongation. Respectively, 4.4% and 0.83% of patients exposed to VEGFR TKI had all-grade and high-grade QTc prolongation. On subgroup analysis, only sunitinib and vandetanib were associated with a statistically significant risk of QTc prolongation, with higher doses of vandetanib associated with a greater risk. The rate of serious arrhythmias including torsades de pointes did not seem to be higher with high-grade QTc prolongation. The risk of QTc prolongation was independent of the duration of therapy. Conclusions: In the largest study to date, we show that VEGFR TKI can be associated with QTc prolongation. Although most cases were of low clinical significance, it is unclear whether the same applies to patients treated off clinical trials. PMID:25349964

  18. A Holographic Road Show.

    ERIC Educational Resources Information Center

    Kirkpatrick, Larry D.; Rugheimer, Mac

    1979-01-01

    Describes the viewing sessions and the holograms of a holographic road show. The traveling exhibits, believed to stimulate interest in physics, include a wide variety of holograms and demonstrate several physical principles. (GA)

  19. Microsporidia Are Natural Intracellular Parasites of the Nematode Caenorhabditis elegans

    PubMed Central

    Troemel, Emily R; Félix, Marie-Anne; Whiteman, Noah K; Barrière, Antoine; Ausubel, Frederick M

    2008-01-01

    For decades the soil nematode Caenorhabditis elegans has been an important model system for biology, but little is known about its natural ecology. Recently, C. elegans has become the focus of studies of innate immunity and several pathogens have been shown to cause lethal intestinal infections in C. elegans. However none of these pathogens has been shown to invade nematode intestinal cells, and no pathogen has been isolated from wild-caught C. elegans. Here we describe an intracellular pathogen isolated from wild-caught C. elegans that we show is a new species of microsporidia. Microsporidia comprise a large class of eukaryotic intracellular parasites that are medically and agriculturally important, but poorly understood. We show that microsporidian infection of the C. elegans intestine proceeds through distinct stages and is transmitted horizontally. Disruption of a conserved cytoskeletal structure in the intestine called the terminal web correlates with the release of microsporidian spores from infected cells, and appears to be part of a novel mechanism by which intracellular pathogens exit from infected cells. Unlike in bacterial intestinal infections, the p38 MAPK and insulin/insulin-like growth factor (IGF) signaling pathways do not appear to play substantial roles in resistance to microsporidian infection in C. elegans. We found microsporidia in multiple wild-caught isolates of Caenorhabditis nematodes from diverse geographic locations. These results indicate that microsporidia are common parasites of C. elegans in the wild. In addition, the interaction between C. elegans and its natural microsporidian parasites provides a system in which to dissect intracellular intestinal infection in vivo and insight into the diversity of pathogenic mechanisms used by intracellular microbes. PMID:19071962

  20. Intracellular mGluR5 plays a critical role in neuropathic pain

    PubMed Central

    Vincent, Kathleen; Cornea, Virginia M.; Jong, Yuh-Jiin I.; Laferrière, André; Kumar, Naresh; Mickeviciute, Aiste; Fung, Jollee S. T.; Bandegi, Pouya; Ribeiro-da-Silva, Alfredo; O'Malley, Karen L.; Coderre, Terence J.

    2016-01-01

    Spinal mGluR5 is a key mediator of neuroplasticity underlying persistent pain. Although brain mGluR5 is localized on cell surface and intracellular membranes, neither the presence nor physiological role of spinal intracellular mGluR5 is established. Here we show that in spinal dorsal horn neurons >80% of mGluR5 is intracellular, of which ∼60% is located on nuclear membranes, where activation leads to sustained Ca2+ responses. Nerve injury inducing nociceptive hypersensitivity also increases the expression of nuclear mGluR5 and receptor-mediated phosphorylated-ERK1/2, Arc/Arg3.1 and c-fos. Spinal blockade of intracellular mGluR5 reduces neuropathic pain behaviours and signalling molecules, whereas blockade of cell-surface mGluR5 has little effect. Decreasing intracellular glutamate via blocking EAAT-3, mimics the effects of intracellular mGluR5 antagonism. These findings show a direct link between an intracellular GPCR and behavioural expression in vivo. Blockade of intracellular mGluR5 represents a new strategy for the development of effective therapies for persistent pain. PMID:26837579

  1. Imaging atrial arrhythmic intracellular calcium in intact heart

    PubMed Central

    Xie, Wenjun; Santulli, Gaetano; Guo, Xiaoxiao; Gao, Melanie; Chen, Bi-Xing; Marks, Andrew R.

    2014-01-01

    Abnormalities in intracellular Ca2+ signaling have been proposed to play an essential role in the pathophysiology of atrial arrhythmias. However, a direct observation of intracellular Ca2+ in atrial myocytes during atrial arrhythmias is lacking. Here, we have developed an ex vivo model of simultaneous Ca2+ imaging and electrocardiographic recording in cardiac atria. Using this system we were able to record atrial arrhythmic intracellular Ca2+ activities. Our results indicate that atrial arrhythmias can be tightly linked to intracellular Ca2+ waves and Ca2+ alternans. Moreover, we applied this strategy to analyze Ca2+ signals in the hearts of WT and knock-in mice harboring a ‘leaky’ type 2 ryanodine receptor (RyR2-R2474S). We showed that sarcoplasmic reticulum (SR) Ca2+ leak increases the susceptibility to Ca2+ alternans and Ca2+ waves increasing the incidence of atrial arrhythmias. Reduction of SR Ca2+ leak via RyR2 by acute treatment with S107 reduced both Ca2+ alternans and Ca2+ waves, and prevented atrial arrhythmias. PMID:24041536

  2. Optogenetic oligomerization of Rab GTPases regulates intracellular membrane trafficking.

    PubMed

    Nguyen, Mai Khanh; Kim, Cha Yeon; Kim, Jin Man; Park, Byung Ouk; Lee, Sangkyu; Park, Hyerim; Heo, Won Do

    2016-06-01

    Intracellular membrane trafficking, which is involved in diverse cellular processes, is dynamic and difficult to study in a spatiotemporal manner. Here we report an optogenetic strategy, termed light-activated reversible inhibition by assembled trap of intracellular membranes (IM-LARIAT), that uses various Rab GTPases combined with blue-light-induced hetero-interaction between cryptochrome 2 and CIB1. In this system, illumination induces a rapid and reversible intracellular membrane aggregation that disrupts the dynamics and functions of the targeted membrane. We applied IM-LARIAT to specifically perturb several Rab-mediated trafficking processes, including receptor transport, protein sorting and secretion, and signaling initiated from endosomes. We finally used this tool to reveal different functions of local Rab5-mediated and Rab11-mediated membrane trafficking in growth cones and soma of young hippocampal neurons. Our results show that IM-LARIAT is a versatile tool that can be used to dissect spatiotemporal functions of intracellular membranes in diverse systems. PMID:27065232

  3. Intracellular pH measurements using perfluorocarbon nanoemulsions.

    PubMed

    Patrick, Michael J; Janjic, Jelena M; Teng, Haibing; O'Hear, Meredith R; Brown, Cortlyn W; Stokum, Jesse A; Schmidt, Brigitte F; Ahrens, Eric T; Waggoner, Alan S

    2013-12-11

    We report the synthesis and formulation of unique perfluorocarbon (PFC) nanoemulsions enabling intracellular pH measurements in living cells via fluorescent microscopy and flow cytometry. These nanoemulsions are formulated to readily enter cells upon coincubation and contain two cyanine-based fluorescent reporters covalently bound to the PFC molecules, specifically Cy3-PFC and CypHer5-PFC conjugates. The spectral and pH-sensing properties of the nanoemulsions were characterized in vitro and showed the unaltered spectral behavior of dyes after formulation. In rat 9L glioma cells loaded with nanoemulsion, the local pH of nanoemulsions was longitudinally quantified using optical microscopy and flow cytometry and displayed a steady decrease in pH to a level of 5.5 over 3 h, indicating rapid uptake of nanoemulsion to acidic compartments. Overall, these reagents enable real-time optical detection of intracellular pH in living cells in response to pharmacological manipulations. Moreover, recent approaches for in vivo cell tracking using magnetic resonance imaging (MRI) employ intracellular PFC nanoemulsion probes to track cells using (19)F MRI. However, the intracellular fate of these imaging probes is poorly understood. The pH-sensing nanoemulsions allow the study of the fate of the PFC tracer inside the labeled cell, which is important for understanding the PFC cell loading dynamics, nanoemulsion stability and cell viability over time. PMID:24266634

  4. Intracellular pH measurements using perfluorocarbon nanoemulsions

    PubMed Central

    Patrick, Michael J.; Janjic, Jelena M.; Teng, Haibing; O’Hear, Meredith R.; Brown, Cortlyn W.; Stokum, Jesse A.; Schmidt, Brigitte F.; Ahrens, Eric T.; Waggoner, Alan S.

    2014-01-01

    We report the synthesis and formulation of unique perfluorocarbon (PFC) nanoemulsions enabling intracellular pH measurements in living cells via fluorescent microscopy and flow cytometry. These nanoemulsions are formulated to readily enter cells upon co-incubation and contain two cyanine-based fluorescent reporters covalently bound to the PFC molecules, specifically Cy3-PFC and CypHer5-PFC conjugates. The spectral and pH-sensing properties of the nanoemulsions where characterized in vitro and showed the unaltered spectral behavior of dyes after formulation. In rat 9L glioma cells loaded with nanoemulsion, the local pH of nanoemulsions was longitudinally quantified using optical microscopy and flow cytometry, and displayed a steady decrease in pH to a level of 5.5 over 3 hours, indicating rapid uptake of nanoemulsion to acidic compartments. Overall, these reagents enable real-time optical detection of intracellular pH in living cells in response to pharmacological manipulations. Moreover, recent approaches for in vivo cell tracking using magnetic resonance imaging (MRI) employ intracellular PFC nanoemulsion probes to track cells using 19F MRI. However, the intracellular fate of these imaging probes is poorly understood. The pH sensing nanoemulsions allow the study of the fate of the PFC tracer inside the labeled cell, which is important for understanding the PFC cell loading dynamics and nanoemulsion stability and cell viability over time. PMID:24266634

  5. Drug-induced QT interval prolongation: mechanisms and clinical management

    PubMed Central

    Nachimuthu, Senthil; Assar, Manish D.

    2012-01-01

    The prolonged QT interval is both widely seen and associated with the potentially deadly rhythm, Torsades de Pointes (TdP). While it can occur spontaneously in the congenital form, there is a wide array of drugs that have been implicated in the prolongation of the QT interval. Some of these drugs have either been restricted or withdrawn from the market due to the increased incidence of fatal polymorphic ventricular tachycardia. The list of drugs that cause QT prolongation continues to grow, and an updated list of specific drugs that prolong the QT interval can be found at www.qtdrugs.org. This review focuses on the mechanism of drug-induced QT prolongation, risk factors for TdP, culprit drugs, prevention and monitoring of prolonged drug-induced QT prolongation and treatment strategies. PMID:25083239

  6. HIF-1α-mediated upregulation of SERCA2b: The endogenous mechanism for alleviating the ischemia-induced intracellular Ca(2+) store dysfunction in CA1 and CA3 hippocampal neurons.

    PubMed

    Kopach, Olga; Maistrenko, Anastasiia; Lushnikova, Iryna; Belan, Pavel; Skibo, Galina; Voitenko, Nana

    2016-05-01

    Pyramidal neurons of the hippocampus possess differential susceptibility to the ischemia-induced damage with the highest vulnerability of CA1 and the lower sensitivity of CA3 neurons. This damage is triggered by Ca(2+)-dependent excitotoxicity and can result in a delayed cell death that might be potentially suspended through activation of endogenous neuroprotection with the hypoxia-inducible transcription factors (HIF). However, the molecular mechanisms of this neuroprotection remain poorly understood. Here we show that prolonged (30min) oxygen and glucose deprivation (OGD) in situ impairs intracellular Ca(2+) regulation in CA1 rather than in CA3 neurons with the differently altered expression of genes coding Ca(2+)-ATPases: the mRNA level of plasmalemmal Ca(2+)-ATPases (PMCA1 and PMCA2 subtypes) was downregulated in CA1 neurons, whereas the mRNA level of the endoplasmic reticulum Ca(2+)-ATPases (SERCA2b subtype) was increased in CA3 neurons at 4h of re-oxygenation after prolonged OGD. These demonstrate distinct susceptibility of CA1 and CA3 neurons to the ischemic impairments in intracellular Ca(2+) regulation and Ca(2+)-ATPase expression. Stabilization of HIF-1α by inhibiting HIF-1α hydroxylation prevented the ischemic decrease in both PMCA1 and PMCA2 mRNAs in CA1 neurons, upregulated the SERCA2b mRNA level and eliminated the OGD-induced Ca(2+) store dysfunction in these neurons. Cumulatively, these findings reveal the previously unknown HIF-1α-driven upregulation of Ca(2+)-ATPases as a mechanism opposing the ischemic impairments in intracellular Ca(2+) regulation in hippocampal neurons. The ability of HIF-1α to modulate expression of genes coding Ca(2+)-ATPases suggests SERCA2b as a novel target for HIF-1 and may provide potential implications for HIF-1α-stabilizing strategy in activating endogenous neuroprotection. PMID:26969192

  7. Intracellular activity of antibiotics against Staphylococcus aureus in a mouse peritonitis model.

    PubMed

    Sandberg, Anne; Hessler, Jonas H R; Skov, Robert L; Blom, Jens; Frimodt-Møller, Niels

    2009-05-01

    Antibiotic treatment of Staphylococcus aureus infections is often problematic due to the slow response to therapy and the high frequency of infection recurrence. The intracellular persistence of staphylococci has been recognized and could offer a good explanation for these treatment difficulties. Knowledge of the interplay between intracellular antibiotic activity and the overall outcome of infection is therefore important. Several intracellular in vitro models have been developed, but few experimental animal models have been published. The mouse peritonitis/sepsis model was used as the basic in vivo model exploring a quantitative ex vivo extra- and intracellular differentiation assay. The intracellular presence of S. aureus was documented by electron microscopy. Five antibiotics, dicloxacillin, cefuroxime, gentamicin, azithromycin, and rifampin (rifampicin), were tested in the new in vivo model; and the model was able to distinguish between their extra- and intracellular effects. The intracellular effects of the five antibiotics could be ranked as follows as the mean change in the log(10) number of CFU/ml (Delta log(10) CFU/ml) between treated and untreated mice after 4 h of treatment: dicloxacillin (3.70 Delta log(10) CFU/ml) > cefuroxime (3.56 Delta log(10) CFU/ml) > rifampin (1.86 Delta log(10) CFU/ml) > gentamicin (0.61 Delta log(10) CFU/ml) > azithromycin (0.21 Delta log(10) CFU/ml). We could also show that the important factors during testing of intracellular activity in vivo are the size, number, and frequency of doses; the time of exposure; and the timing between the start of infection and treatment. A poor correlation between the intracellular accumulation of the antibiotics and the actual intracellular effect was found. This stresses the importance of performing experimental studies, like those with the new in vivo model described here, to measure actual intracellular activity instead of making predictions based on cellular pharmacokinetic and MICs. PMID

  8. Polymer physics of intracellular phase transitions

    NASA Astrophysics Data System (ADS)

    Brangwynne, Clifford P.; Tompa, Peter; Pappu, Rohit V.

    2015-11-01

    Intracellular organelles are either membrane-bound vesicles or membrane-less compartments that are made up of proteins and RNA. These organelles play key biological roles, by compartmentalizing the cell to enable spatiotemporal control of biological reactions. Recent studies suggest that membrane-less intracellular compartments are multicomponent viscous liquid droplets that form via phase separation. Proteins that have an intrinsic tendency for being conformationally heterogeneous seem to be the main drivers of liquid-liquid phase separation in the cell. These findings highlight the relevance of classical concepts from the physics of polymeric phase transitions for understanding the assembly of intracellular membrane-less compartments. However, applying these concepts is challenging, given the heteropolymeric nature of protein sequences, the complex intracellular environment, and non-equilibrium features intrinsic to cells. This provides new opportunities for adapting established theories and for the emergence of new physics.

  9. Nanoparticles for intracellular-targeted drug delivery

    NASA Astrophysics Data System (ADS)

    Paulo, Cristiana S. O.; Pires das Neves, Ricardo; Ferreira, Lino S.

    2011-12-01

    Nanoparticles (NPs) are very promising for the intracellular delivery of anticancer and immunomodulatory drugs, stem cell differentiation biomolecules and cell activity modulators. Although initial studies in the area of intracellular drug delivery have been performed in the delivery of DNA, there is an increasing interest in the use of other molecules to modulate cell activity. Herein, we review the latest advances in the intracellular-targeted delivery of short interference RNA, proteins and small molecules using NPs. In most cases, the drugs act at different cellular organelles and therefore the drug-containing NPs should be directed to precise locations within the cell. This will lead to the desired magnitude and duration of the drug effects. The spatial control in the intracellular delivery might open new avenues to modulate cell activity while avoiding side-effects.

  10. Show What You Know

    ERIC Educational Resources Information Center

    Eccleston, Jeff

    2007-01-01

    Big things come in small packages. This saying came to the mind of the author after he created a simple math review activity for his fourth grade students. Though simple, it has proven to be extremely advantageous in reinforcing math concepts. He uses this activity, which he calls "Show What You Know," often. This activity provides the perfect…

  11. The Ozone Show.

    ERIC Educational Resources Information Center

    Mathieu, Aaron

    2000-01-01

    Uses a talk show activity for a final assessment tool for students to debate about the ozone hole. Students are assessed on five areas: (1) cooperative learning; (2) the written component; (3) content; (4) self-evaluation; and (5) peer evaluation. (SAH)

  12. Honored Teacher Shows Commitment.

    ERIC Educational Resources Information Center

    Ratte, Kathy

    1987-01-01

    Part of the acceptance speech of the 1985 National Council for the Social Studies Teacher of the Year, this article describes the censorship experience of this honored social studies teacher. The incident involved the showing of a videotape version of the feature film entitled "The Seduction of Joe Tynan." (JDH)

  13. Talk Show Science.

    ERIC Educational Resources Information Center

    Moore, Mitzi Ruth

    1992-01-01

    Proposes having students perform skits in which they play the roles of the science concepts they are trying to understand. Provides the dialog for a skit in which hot and cold gas molecules are interviewed on a talk show to study how these properties affect wind, rain, and other weather phenomena. (MDH)

  14. Stage a Water Show

    ERIC Educational Resources Information Center

    Frasier, Debra

    2008-01-01

    In the author's book titled "The Incredible Water Show," the characters from "Miss Alaineus: A Vocabulary Disaster" used an ocean of information to stage an inventive performance about the water cycle. In this article, the author relates how she turned the story into hands-on science teaching for real-life fifth-grade students. The author also…

  15. Showing What They Know

    ERIC Educational Resources Information Center

    Cech, Scott J.

    2008-01-01

    Having students show their skills in three dimensions, known as performance-based assessment, dates back at least to Socrates. Individual schools such as Barrington High School--located just outside of Providence--have been requiring students to actively demonstrate their knowledge for years. The Rhode Island's high school graduating class became…

  16. Influence of a Prolonged Tennis Match Play on Serve Biomechanics.

    PubMed

    Martin, Caroline; Bideau, Benoit; Delamarche, Paul; Kulpa, Richard

    2016-01-01

    The aim of this study was to quantify kinematic, kinetic and performance changes that occur in the serve throughout a prolonged tennis match play. Serves of eight male advanced tennis players were recorded with a motion capture system before, at mid-match, and after a 3-hour tennis match. Before and after each match, electromyographic data of 8 upper limb muscles obtained during isometric maximal voluntary contraction were compared to determine the presence of muscular fatigue. Vertical ground reaction forces, rating of perceived exertion, ball speed, and ball impact height were measured. Kinematic and upper limb kinetic variables were computed. The results show decrease in mean power frequency values for several upper limb muscles that is an indicator of local muscular fatigue. Decreases in serve ball speed, ball impact height, maximal angular velocities and an increase in rating of perceived exertion were also observed between the beginning and the end of the match. With fatigue, the majority of the upper limb joint kinetics decreases at the end of the match. No change in timing of maximal angular velocities was observed between the beginning and the end of the match. A prolonged tennis match play may induce fatigue in upper limb muscles, which decrease performance and cause changes in serve maximal angular velocities and joint kinetics. The consistency in timing of maximal angular velocities suggests that advanced tennis players are able to maintain the temporal pattern of their serve technique, in spite of the muscular fatigue development. PMID:27532421

  17. Cranberry anthocyanin extract prolongs lifespan of fruit flies.

    PubMed

    Wang, Lijun; Li, Yuk Man; Lei, Lin; Liu, Yuwei; Wang, Xiaobo; Ma, Ka Ying; Chen, Zhen-Yu

    2015-09-01

    Cranberry is an excellent source of dietary antioxidants. The present study investigated the effect of cranberry anthocyanin (CrA) extract on the lifespan of fruit flies with focus on its interaction with aging-related genes including superoxide dismutase (SOD), catalase (CAT), methuselah (MTH), insulin receptor (InR), target of rapamycin (TOR), hemipterus (Hep), and phosphoenolpyruvate carboxykinase (PEPCK). Results showed that diet containing 20mg/mL CrA could significantly prolong the mean lifespan of fruit flies by 10% compared with the control diet. This was accompanied by up-regulation of SOD1 and down-regulation of MTH, InR, TOR and PEPCK. The stress resistance test demonstrated that CrA could reduce the mortality rate induced by H2O2 but not by paraquat. It was therefore concluded that the lifespan-prolonging activity of CrA was most likely mediated by modulating the genes of SOD1, MTH, InR, TOR and PEPCK. PMID:26159161

  18. Dengue haemorrhagic fever with unusual prolonged thrombocytopaenia.

    PubMed

    Kamil, S M; Mohamad, N H; Narazah, M Y; Khan, F A

    2006-04-01

    We describe a case of dengue haemorrhagic fever with prolonged thrombocytopaenia. A 22-year-old Malay man with no prior illness presented with a history of fever and generalised macular rash of four days duration. Initial work-up suggested the diagnosis of dengue haemorrhagic fever based on thrombocytopaenia and positive dengue serology. Patient recovered from acute illness by day ten, and was discharged from the hospital with improving platelet count. He was then noted to have declining platelet count on follow-up and required another hospital admission on day 19 of his illness because of declining platelet count. The patient remained hospitalised till day 44 of his illness and managed with repeated platelet transfusion and supportive care till he recovered spontaneously. PMID:16572249

  19. Bilateral Scapulohumeral Ankylosis after Prolonged Mechanical Ventilation.

    PubMed

    van Lotten, Manon L; Schreinemakers, J Rieneke; van Noort, Arthur; Rademakers, Maarten V

    2016-09-01

    This case demonstrates a rarely reported bilateral scapulohumeral bony ankylosis. A young woman developed extensive heterotopic ossifications (HOs) in both shoulder joints after being mechanically ventilated for several months at the intensive care unit in a comatose status. She presented with a severe movement restriction of both shoulder joints. Surgical resection of the bony bridges was performed in 2 separate sessions with a significant improvement of shoulder function afterwards. No postoperative complications, pain, or recurrence of HOs were noted at 1-year follow-up. Mechanical ventilation, immobilization, neuromuscular blockage, and prolonged sedation are known risk factors for the development of HOs in the shoulder joints. Relatively early surgical resection of the HOs can be performed safely in contrary to earlier belief. Afterwards, nonsteroidal anti-inflammatory drugs and/or radiation therapy can be possible treatment modalities to prevent recurrence of HOs. PMID:27583120

  20. Bilateral Scapulohumeral Ankylosis after Prolonged Mechanical Ventilation

    PubMed Central

    Schreinemakers, J. Rieneke; van Noort, Arthur; Rademakers, Maarten V.

    2016-01-01

    This case demonstrates a rarely reported bilateral scapulohumeral bony ankylosis. A young woman developed extensive heterotopic ossifications (HOs) in both shoulder joints after being mechanically ventilated for several months at the intensive care unit in a comatose status. She presented with a severe movement restriction of both shoulder joints. Surgical resection of the bony bridges was performed in 2 separate sessions with a significant improvement of shoulder function afterwards. No postoperative complications, pain, or recurrence of HOs were noted at 1-year follow-up. Mechanical ventilation, immobilization, neuromuscular blockage, and prolonged sedation are known risk factors for the development of HOs in the shoulder joints. Relatively early surgical resection of the HOs can be performed safely in contrary to earlier belief. Afterwards, nonsteroidal anti-inflammatory drugs and/or radiation therapy can be possible treatment modalities to prevent recurrence of HOs. PMID:27583120

  1. Liver and kidney metabolism during prolonged starvation

    PubMed Central

    Owen, Oliver E.; Felig, Philip; Morgan, Alfred P.; Wahren, John; Cahill, George F.

    1969-01-01

    This study quantifies the concentrations of circulating insulin, growth hormone, glucose, free fatty acids, glycerol, β-hydroxybutyrate, acetoacetate, and alpha amino nitrogen in 11 obese subjects during prolonged starvation. The sites and estimated rates of gluconeogenesis and ketogenesis after 5-6 wk of fasting were investigated in five of the subjects. Blood glucose and insulin concentrations fell acutely during the 1st 3 days of fasting, and alpha amino nitrogen after 17 days. The concentration of free fatty acids, β-hydroxybutyrate, and acetoacetate did not reach a plateau until after 17 days. Estimated glucose production at 5-6 wk of starvation is reduced to approximately 86 g/24 hr. Of this amount the liver contributes about one-half and the kidney the remainder. Approximately all of the lactate, pyruvate, glycerol, and amino acid carbons which are removed by liver and kidney are converted into glucose, as evidenced by substrate balances across these organs. Images PMID:5773093

  2. Prolonging Microgravity on Parabolic Airplane Flights

    NASA Technical Reports Server (NTRS)

    Robinson, David W.

    2003-01-01

    Three techniques have been proposed to prolong the intervals of time available for microgravity experiments aboard airplanes flown along parabolic trajectories. Typically, a pilot strives to keep an airplane on such a trajectory during a nominal time interval as long as 25 seconds, and an experimental apparatus is released to float freely in the airplane cabin to take advantage of the microgravitational environment of the trajectory for as long as possible. It is usually not possible to maintain effective microgravity during the entire nominal time interval because random aerodynamic forces and fluctuations in pilot control inputs cause the airplane to deviate slightly from a perfect parabolic trajectory, such that the freely floating apparatus bumps into the ceiling, floor, or a wall of the airplane before the completion of the parabola.

  3. The evolution of prolonged life after reproduction.

    PubMed

    Croft, Darren P; Brent, Lauren J N; Franks, Daniel W; Cant, Michael A

    2015-07-01

    Why females of some species cease ovulation before the end of their natural lifespan is a longstanding evolutionary puzzle. For many species in captivity, post-reproductive life is simply an epiphenomenon of lengthened lifespan. Yet in natural populations of humans as well as some cetaceans and insects, reproductive senescence occurs much faster than somatic aging and females exhibit prolonged post-reproductive lifespans (PRLSs). Determining the mechanisms and functions that underpin PRLSs has proved a significant challenge. Here we bring together both classic and modern hypotheses proposed to explain PRLSs and discuss their application to both human and nonhuman animals. By taking an integrative and broad taxonomic approach we highlight the need to consider multiple interacting explanations for the evolution of PRLSs. PMID:25982154

  4. Effects of Prolonged Centrifugation on Orthostasis

    NASA Technical Reports Server (NTRS)

    Cohen, Malcolm M..; Hargens, A. R.; Yates, B. J.; Bowley, Susan M. (Technical Monitor)

    2000-01-01

    A feasibility study conducted on the Ames 20-G Human Centrifuge examined how well humans can maintain orthostatic tolerance during and after prolonged exposures to hypergravity. Three adult males lived for periods of 22 hours in the centrifuge while it was at rest (1.00 G), and while it rotated at 9.38 RPM to provide 1.25 G-total at the mean radius of 7.62 m. Two participants also experienced 22-hour habitation sessions at 11.46 RPM, which provided 1.50 G-total. Both before and after each habitation session, the participants were given gradual onset rate (GOR) acceleration profiles at 0.067 G/sec to determine their Gz tolerance. In addition, cardiovascular responses were compared while subjects were supine, siting, and standing at various times during the habitation (stand test), and cardiovascular responsiveness was determined using a lower body negative pressure tilt table (LBNPTT) at the beginning of the experiment and after each session. Post-Pre changes in G tolerance were -0.33 (mean) +/- 0.11 (std. error) Gz for habitation at 1.00 G, -0.02 +/- 0.12 Gz for habitation at 1.25 G, and +0.41 +/- 0.13 Gz for habitation at 1.50 G. Performance on the stand test generally improved with duration of habitation in hypergravity. Our results suggest that habitation in a confined chamber at 1.00 G reduces G tolerance and leads to lowered LBNPTT tolerance. Exposure to increased G in the centrifuge leads to enhanced performance on the stand test, and to increased GOR acceleration tolerance, but only when fluid balance is maintained; when motion sickness and negative fluid balance were observed, G tolerance was reduced. The data indicate that enhanced G tolerance can result from prolonged exposure to hypergravity, but that these changes are complex and depend on multiple underlying physiological processes.

  5. Intracellular Gene Expression Profile of Listeria monocytogenes †

    PubMed Central

    Chatterjee, Som Subhra; Hossain, Hamid; Otten, Sonja; Kuenne, Carsten; Kuchmina, Katja; Machata, Silke; Domann, Eugen; Chakraborty, Trinad; Hain, Torsten

    2006-01-01

    Listeria monocytogenes is a gram-positive, food-borne microorganism responsible for invasive infections with a high overall mortality. L. monocytogenes is among the very few microorganisms that can induce uptake into the host cell and subsequently enter the host cell cytosol by breaching the vacuolar membrane. We infected the murine macrophage cell line P388D1 with L. monocytogenes strain EGD-e and examined the gene expression profile of L. monocytogenes inside the vacuolar and cytosolic environments of the host cell by using whole-genome microarray and mutant analyses. We found that ∼17% of the total genome was mobilized to enable adaptation for intracellular growth. Intracellularly expressed genes showed responses typical of glucose limitation within bacteria, with a decrease in the amount of mRNA encoding enzymes in the central metabolism and a temporal induction of genes involved in alternative-carbon-source utilization pathways and their regulation. Adaptive intracellular gene expression involved genes that are associated with virulence, the general stress response, cell division, and changes in cell wall structure and included many genes with unknown functions. A total of 41 genes were species specific, being absent from the genome of the nonpathogenic Listeria innocua CLIP 11262 strain. We also detected 25 genes that were strain specific, i.e., absent from the genome of the previously sequenced L. monocytogenes F2365 serotype 4b strain, suggesting heterogeneity in the gene pool required for intracellular survival of L. monocytogenes in host cells. Overall, our study provides crucial insights into the strategy of intracellular survival and measures taken by L. monocytogenes to escape the host cell responses. PMID:16428782

  6. Intracellular recording of action potentials by nanopillar electroporation

    NASA Astrophysics Data System (ADS)

    Xie, Chong; Lin, Ziliang; Hanson, Lindsey; Cui, Yi; Cui, Bianxiao

    2012-03-01

    Action potentials have a central role in the nervous system and in many cellular processes, notably those involving ion channels. The accurate measurement of action potentials requires efficient coupling between the cell membrane and the measuring electrodes. Intracellular recording methods such as patch clamping involve measuring the voltage or current across the cell membrane by accessing the cell interior with an electrode, allowing both the amplitude and shape of the action potentials to be recorded faithfully with high signal-to-noise ratios. However, the invasive nature of intracellular methods usually limits the recording time to a few hours, and their complexity makes it difficult to simultaneously record more than a few cells. Extracellular recording methods, such as multielectrode arrays and multitransistor arrays, are non-invasive and allow long-term and multiplexed measurements. However, extracellular recording sacrifices the one-to-one correspondence between the cells and electrodes, and also suffers from significantly reduced signal strength and quality. Extracellular techniques are not, therefore, able to record action potentials with the accuracy needed to explore the properties of ion channels. As a result, the pharmacological screening of ion-channel drugs is usually performed by low-throughput intracellular recording methods. The use of nanowire transistors, nanotube-coupled transistors and micro gold-spine and related electrodes can significantly improve the signal strength of recorded action potentials. Here, we show that vertical nanopillar electrodes can record both the extracellular and intracellular action potentials of cultured cardiomyocytes over a long period of time with excellent signal strength and quality. Moreover, it is possible to repeatedly switch between extracellular and intracellular recording by nanoscale electroporation and resealing processes. Furthermore, vertical nanopillar electrodes can detect subtle changes in action

  7. Determination of Intracellular Vitrification Temperatures for Unicellular Micro Organisms under Conditions Relevant for Cryopreservation.

    PubMed

    Fonseca, Fernanda; Meneghel, Julie; Cenard, Stéphanie; Passot, Stéphanie; Morris, G John

    2016-01-01

    During cryopreservation ice nucleation and crystal growth may occur within cells or the intracellular compartment may vitrify. Whilst previous literature describes intracellular vitrification in a qualitative manner, here we measure the intracellular vitrification temperature of bacteria and yeasts under conditions relevant to cryopreservation, including the addition of high levels of permeating and nonpermeating additives and the application of rapid rates of cooling. The effects of growth conditions that are known to modify cellular freezing resistance on the intracellular vitrification temperature are also examined. For bacteria a plot of the activity on thawing against intracellular glass transition of the maximally freeze-concentrated matrix (Tg') shows that cells with the lowest value of intracellular Tg' survive the freezing process better than cells with a higher intracellular Tg'. This paper demonstrates the role of the physical state of the intracellular environment in determining the response of microbial cells to preservation and could be a powerful tool to be manipulated to allow the optimization of methods for the preservation of microorganisms. PMID:27055246

  8. Purification and Characterization of a Novel Intracellular Sucrase Enzyme of Leishmania donovani Promastigotes

    PubMed Central

    Singh, Arpita; Mandal, Debjani

    2016-01-01

    The promastigote stage of Leishmania resides in the sand fly gut, enriched with sugar molecules. Recently we reported that Leishmania donovani possesses a sucrose uptake system and a stable pool of intracellular sucrose metabolizing enzyme. In the present study, we purified the intracellular sucrase nearly to its homogeneity and compared it with the purified extracellular sucrase. The estimated size of intracellular sucrase is ~112 kDa by gel filtration chromatography, native PAGE, and substrate staining. However, in SDS-PAGE, the protein is resolved at ~56 kDa, indicating the possibility of a homodimer in its native state. The kinetics of purified intracellular sucrase shows its higher substrate affinity with a K m of 1.61 mM than the extracellular form having a K m of 4.4 mM. The highly specific activity of intracellular sucrase towards sucrose is optimal at pH 6.0 and at 30°C. In this report the purification and characterization of intracellular sucrase provide evidence that sucrase enzyme exists at least in two different forms in Leishmania donovani promastigotes. This intracellular sucrase may support further intracellular utilization of transported sucrose. PMID:27190649

  9. Assembly and composition of intracellular particles formed by Moloney murine leukemia virus.

    PubMed Central

    Hansen, M; Jelinek, L; Jones, R S; Stegeman-Olsen, J; Barklis, E

    1993-01-01

    Assembly of type C retroviruses such as Moloney murine leukemia virus (M-MuLV) ordinarily occurs at the plasma membranes of infected cells and absolutely requires the particle core precursor protein, Pr65gag. Previously we have shown that Pr65gag is membrane associated and that at least a portion of intracellular Pr65gag protein appears to be routed to the plasma membrane by a vesicular transport pathway. Here we show that intracellular particle formation can occur in M-MuLV-infected cells. M-MuLV immature particles were observed by electron microscopy budding into and within rough endoplasmic reticulum, Golgi, and vacuolar compartments. Biochemical fractionation studies indicated that intracellular Pr65gag was present in nonionic detergent-resistant complexes of greater than 150S. Additionally, viral RNA and polymerase functions appeared to be associated with intracellular particles, as were Gag-beta-galactosidase fusion proteins which have the capacity to be incorporated into virions. Immature intracellular particles in postnuclear lysates could be proteolytically processed in vitro to mature forms, while extracellular immature M-MuLV particles remained immature as long as 10 h during incubations. The occurrence of M-MuLV-derived intracellular particles demonstrates that Pr65gag can associate with intracellular membranes and indicates that if a plasma membrane Pr65gag receptor exists, it also can be found in other membrane compartments. These results support the hypothesis that intracellular particles may serve as a virus reservoir during in vivo infections. Images PMID:8350394

  10. Determination of Intracellular Vitrification Temperatures for Unicellular Micro Organisms under Conditions Relevant for Cryopreservation

    PubMed Central

    Fonseca, Fernanda; Meneghel, Julie; Cenard, Stéphanie; Passot, Stéphanie; Morris, G. John

    2016-01-01

    During cryopreservation ice nucleation and crystal growth may occur within cells or the intracellular compartment may vitrify. Whilst previous literature describes intracellular vitrification in a qualitative manner, here we measure the intracellular vitrification temperature of bacteria and yeasts under conditions relevant to cryopreservation, including the addition of high levels of permeating and nonpermeating additives and the application of rapid rates of cooling. The effects of growth conditions that are known to modify cellular freezing resistance on the intracellular vitrification temperature are also examined. For bacteria a plot of the activity on thawing against intracellular glass transition of the maximally freeze-concentrated matrix (Tg’) shows that cells with the lowest value of intracellular Tg’ survive the freezing process better than cells with a higher intracellular Tg’. This paper demonstrates the role of the physical state of the intracellular environment in determining the response of microbial cells to preservation and could be a powerful tool to be manipulated to allow the optimization of methods for the preservation of microorganisms. PMID:27055246

  11. Intracellular Mechanics of Migrating FibroblastsD⃞

    PubMed Central

    Kole, Thomas P.; Tseng, Yiider; Jiang, Ingjye; Katz, Joseph L.; Wirtz, Denis

    2005-01-01

    Cell migration is a highly coordinated process that occurs through the translation of biochemical signals into specific biomechanical events. The biochemical and structural properties of the proteins involved in cell motility, as well as their subcellular localization, have been studied extensively. However, how these proteins work in concert to generate the mechanical properties required to produce global motility is not well understood. Using intracellular microrheology and a fibroblast scratch-wound assay, we show that cytoskeleton reorganization produced by motility results in mechanical stiffening of both the leading lamella and the perinuclear region of motile cells. This effect is significantly more pronounced in the leading edge, suggesting that the mechanical properties of migrating fibroblasts are spatially coordinated. Disruption of the microtubule network by nocodazole treatment results in the arrest of cell migration and a loss of subcellular mechanical polarization; however, the overall mechanical properties of the cell remain mostly unchanged. Furthermore, we find that activation of Rac and Cdc42 in quiescent fibroblasts elicits mechanical behavior similar to that of migrating cells. We conclude that a polarized mechanics of the cytoskelton is essential for directed cell migration and is coordinated through microtubules. PMID:15483053

  12. Receptor Tyrosine Kinases with Intracellular Pseudokinase Domains

    PubMed Central

    Mendrola, Jeannine M.; Shi, Fumin; Park, Jin H.; Lemmon, Mark A.

    2013-01-01

    As with other groups of protein kinases, approximately 10% of the receptor tyrosine kinases (RTKs) in the human proteome contain intracellular pseudokinases that lack one or more conserved catalytically important residues. These include ErbB3, a member of the epidermal growth factor receptor (EGFR) family, and a series of unconventional Wnt receptors. We recently showed that, despite its reputation as a pseudokinase, the ErbB3 tyrosine kinase domain (TKD) does retain significant – albeit weak – kinase activity. This led us to suggest that a subgroup of RTKs may be able to signal even with very inefficient kinases. Recent work suggests that this is not the case, however. Other pseudokinase RTKs have not revealed significant kinase activity, and mutations that impair ErbB3’s weak kinase activity have not so far been found to exhibit signaling defects. These findings therefore point to models in which the TKDs of pseudokinase RTKs participate in receptor signaling by allosterically regulating associated kinases (such as ErbB3 regulation of ErbB2) and/or function as regulated ‘scaffolds’ for other intermolecular interactions central to signal propagation. Further structural and functional studies – particularly of the pseudokinase RTKs involved in Wnt signaling – are required to shed new light on these intriguing signaling mechanisms. PMID:23863174

  13. Taking in a Show.

    PubMed

    Boden, Timothy W

    2016-01-01

    Many medical practices have cut back on education and staff development expenses, especially those costs associated with conventions and conferences. But there are hard-to-value returns on your investment in these live events--beyond the obvious benefits of acquired knowledge and skills. Major vendors still exhibit their services and wares at many events, and the exhibit hall is a treasure-house of information and resources for the savvy physician or administrator. Make and stick to a purposeful plan to exploit the trade show. You can compare products, gain new insights and ideas, and even negotiate better deals with representatives anxious to realize returns on their exhibition investments. PMID:27249887

  14. Coexistence of amplitude and frequency modulations in intracellular calcium dynamics

    NASA Astrophysics Data System (ADS)

    de Pittà, Maurizio; Volman, Vladislav; Levine, Herbert; Pioggia, Giovanni; de Rossi, Danilo; Ben-Jacob, Eshel

    2008-03-01

    The complex dynamics of intracellular calcium regulates cellular responses to information encoded in extracellular signals. Here we study the encoding of these external signals in the context of the Li-Rinzel model. We show that by control of biophysical parameters the information can be encoded in amplitude modulation (AM), frequency modulation (FM), or mixed (AM and FM) modulation. We briefly discuss the possible implications of this role of information encoding for astrocytes.

  15. Short-Duration Spaceflight Does Not Prolong QTc Intervals in Male Astronauts

    NASA Technical Reports Server (NTRS)

    Mitchell, Brett M.; Meck, Janice V.

    2004-01-01

    Although ventricular dysrhythmias are not increased during, and QTc intervals are not prolonged after, short-duration (5 to 16 days) spaceflights, QTc intervals have not previously been reported during these shorter flights. Holter monitor recordings, obtained in 11 male astronauts who flew on shuttle missions ranging from 5 to 10 days, showed that QTc intervals did not change significantly 10 days before launch, on 2 separate days of spaceflight, and 2 days after landing. Taken together, these data and our previous report show that QTc interval prolongation occurs sometime between the 9th and 30th days of spaceflight.

  16. Not a "reality" show.

    PubMed

    Wrong, Terence; Baumgart, Erica

    2013-01-01

    The authors of the preceding articles raise legitimate questions about patient and staff rights and the unintended consequences of allowing ABC News to film inside teaching hospitals. We explain why we regard their fears as baseless and not supported by what we heard from individuals portrayed in the filming, our decade-long experience making medical documentaries, and the full un-aired context of the scenes shown in the broadcast. The authors don't and can't know what conversations we had, what documents we reviewed, and what protections we put in place in each televised scene. Finally, we hope to correct several misleading examples cited by the authors as well as their offhand mischaracterization of our program as a "reality" show. PMID:23631336

  17. Intracellular Zn(2+) Increase in Cardiomyocytes Induces both Electrical and Mechanical Dysfunction in Heart via Endogenous Generation of Reactive Nitrogen Species.

    PubMed

    Tuncay, Erkan; Turan, Belma

    2016-02-01

    Oxidants increase intracellular free Zn(2+) concentration ([Zn(2+)]i) in ventricular myocytes, which contributes to oxidant-induced alterations in excitation-contraction coupling (ECC). However, it is not clear whether increased [Zn(2+)]i in cardiomyocytes via increased reactive nitrogen species (RNS) has a role on heart function under pathological conditions, such as hyperglycemia. In this study, first we aimed to investigate the role of increased [Zn(2+)]i under in vitro condition in the development of both electrical and mechanical dysfunction of isolated papillary muscle strips from rat heart via exposed samples to a Zn(2+)-ionophore (Zn-pyrithione; 1 μM) for 20 min. Under simultaneous measurement of intracellular action potential and contractile activity in these preparations, Zn-pyrithione exposure caused marked prolongation in action potential repolarization phase and slowdown in both contraction and relaxation rates of twitch activity. Second, in order to demonstrate an association between increased [Zn(2+)]i and increased RNS, we monitored intracellular [Zn(2+)]i under an acute exposure of nitric oxide (NO) donor sodium nitroprusside, SNP, in freshly isolated quiescent cardiomyocytes loaded with FluoZin-3. Resting level of free Zn(2+) is significantly higher in cardiomyocytes under hyperglycemic condition compared to those of the controls, which seems to be associated with increased level of RNS production in hyperglycemic cardiomyocytes. Western blot analysis showed that Zn-pyrithione exposure induced a marked decrease in the activity of protein phosphatase 1 and 2A, member of macromolecular protein complex of cardiac ryanodine receptors, RyR2, besides significant increase in the phosphorylation level of extracellular signal-regulated kinase1/2 as a concentration-dependent manner. Overall, the present data demonstrated that there is a cross-relationship between increased RNS production and increased [Zn(2+)]i level in cardiomyocytes under pathological

  18. Glycolytic inhibition: effects on diastolic relaxation and intracellular calcium handling in hypertrophied rat ventricular myocytes.

    PubMed Central

    Kagaya, Y; Weinberg, E O; Ito, N; Mochizuki, T; Barry, W H; Lorell, B H

    1995-01-01

    We tested the hypothesis that glycolytic inhibition by 2-deoxyglucose causes greater impairment of diastolic relaxation and intracellular calcium handling in well-oxygenated hypertrophied adult rat myocytes compared with control myocytes. We simultaneously measured cell motion and intracellular free calcium concentration ([Ca2+]i) with indo-1 in isolated paced myocytes from aortic-banded rats and sham-operated rats. There was no difference in either the end-diastolic or peak-systolic [Ca2+]i between control and hypertrophied myocytes (97 +/- 18 vs. 105 +/- 15 nM, 467 +/- 92 vs. 556 +/- 67 nM, respectively). Myocytes were first superfused with oxygenated Hepes-buffered solution containing 1.2 mM CaCl2, 5.6 mM glucose, and 5 mM acetate, and paced at 3 Hz at 36 degrees C. Exposure to 20 mM 2-deoxyglucose as substitution of glucose for 15 min caused an upward shift of end-diastolic cell position in both control (n = 5) and hypertrophied myocytes (n = 10) (P < 0.001 vs. baseline), indicating an impaired extent of relaxation. Hypertrophied myocytes, however, showed a greater upward shift in end-diastolic cell position and slowing of relaxation compared with control myocytes (delta 144 +/- 28 vs. 55 +/- 15% of baseline diastolic position, P < 0.02). Exposure to 2-deoxyglucose increased end-diastolic [Ca2+]i in both groups (P < 0.001 vs. baseline), but there was no difference between hypertrophied and control myocytes (218 +/- 38 vs. 183 +/- 29 nM, respectively). The effects of 2-deoxyglucose were corroborated in isolated oxygenated perfused hearts in which glycolytic inhibition which caused severe elevation of isovolumic diastolic pressure and prolongation of relaxation in the hypertrophied hearts compared with controls. In summary, the inhibition of the glycolytic pathway impairs diastolic relaxation to a greater extent in hypertrophied myocytes than in control myocytes even in well-oxygenated conditions. The severe impairment of diastolic relaxation induced by 2

  19. Grace's story: prolonged incestuous abuse from childhood into adulthood.

    PubMed

    Salter, Michael

    2013-02-01

    Some sexually abused women in mental health settings are reporting prolonged incest and yet little is known about the circumstances that enable fathers to sexually abuse their daughters over a period of decades. This article draws from the life history of Grace, a woman who survived prolonged incest, in order to document and analyze the interplay of familial, social, and political factors that entrap girls and women within prolonged incestuous abuse. PMID:23420835

  20. A geometric interpretation of prolongation by means of connections

    NASA Astrophysics Data System (ADS)

    Bracken, Paul

    2010-11-01

    A geometric interpretation of prolongation can be formulated by using the theory of connections. A fiber bundle can be established which is composed of a base manifold and variables which span a prolongation space. A particular connection is introduced in terms of these coordinates. This provides a very different way of viewing the technique and for introducing prolongation algebras as well as generating integrable equations in a novel way.

  1. Public medical shows.

    PubMed

    Walusinski, Olivier

    2014-01-01

    In the second half of the 19th century, Jean-Martin Charcot (1825-1893) became famous for the quality of his teaching and his innovative neurological discoveries, bringing many French and foreign students to Paris. A hunger for recognition, together with progressive and anticlerical ideals, led Charcot to invite writers, journalists, and politicians to his lessons, during which he presented the results of his work on hysteria. These events became public performances, for which physicians and patients were transformed into actors. Major newspapers ran accounts of these consultations, more like theatrical shows in some respects. The resultant enthusiasm prompted other physicians in Paris and throughout France to try and imitate them. We will compare the form and substance of Charcot's lessons with those given by Jules-Bernard Luys (1828-1897), Victor Dumontpallier (1826-1899), Ambroise-Auguste Liébault (1823-1904), Hippolyte Bernheim (1840-1919), Joseph Grasset (1849-1918), and Albert Pitres (1848-1928). We will also note their impact on contemporary cinema and theatre. PMID:25273491

  2. Inhibition of spontaneous recovery of fear by mGluR5 after prolonged extinction training.

    PubMed

    Mao, Sheng-Chun; Chang, Chih-Hua; Wu, Chia-Chen; Orejarena, M Juliana; Orejanera, Maria Juliana; Manzoni, Olivier J; Gean, Po-Wu

    2013-01-01

    Fear behavior is vital for survival and involves learning contingent associations of non-threatening cues with aversive stimuli. In contrast, excessive levels of fear can be maladaptive and lead to anxiety disorders. Generally, extensive sessions of extinction training correlates with reduced spontaneous recovery. The molecular mechanisms underlying the long-term inhibition of fear recovery following repeated extinction training are not fully understood. Here we show that in rats, prolonged extinction training causes greater reduction in both fear-potentiated startle and spontaneous recovery. This effect was specifically blocked by metabotropic glutamate receptor 5 (mGluR5), but not by mGluR1 antagonists and by a protein synthesis inhibitor. Similar inhibition of memory recovery following prolonged extinction training was also observed in mice. In agreement with the instrumental role of mGluR5 in the prolonged inhibition of fear recovery, we found that FMR1-/- mice which exhibit enhanced mGluR5-mediated signaling exhibit lower spontaneous recovery of fear after extinction training than wild-type littermates. At the molecular level, we discovered that prolonged extinction training reversed the fear conditioning-induced increase in surface expression of GluR1, AMPA/NMDA ratio, postsynaptic density-95 (PSD-95) and synapse-associated protein-97 (SAP97). Accordingly, delivery of Tat-GluR2(3Y), a synthetic peptide that blocks AMPA receptor endocytosis, inhibited prolonged extinction training-induced inhibition of fear recovery. Together, our results demonstrate that prolonged extinction training results in the mGluR5-dependent long-term inhibition of fear recovery. This effect may involve the degradation of original memory and may explain the beneficial effects of prolonged exposure therapy for the treatment of phobias. PMID:23555716

  3. The Great Cometary Show

    NASA Astrophysics Data System (ADS)

    2007-01-01

    its high spatial and spectral resolution, it was possible to zoom into the very heart of this very massive star. In this innermost region, the observations are dominated by the extremely dense stellar wind that totally obscures the underlying central star. The AMBER observations show that this dense stellar wind is not spherically symmetric, but exhibits a clearly elongated structure. Overall, the AMBER observations confirm that the extremely high mass loss of Eta Carinae's massive central star is non-spherical and much stronger along the poles than in the equatorial plane. This is in agreement with theoretical models that predict such an enhanced polar mass-loss in the case of rapidly rotating stars. ESO PR Photo 06c/07 ESO PR Photo 06c/07 RS Ophiuchi in Outburst Several papers from this special feature focus on the later stages in a star's life. One looks at the binary system Gamma 2 Velorum, which contains the closest example of a star known as a Wolf-Rayet. A single AMBER observation allowed the astronomers to separate the spectra of the two components, offering new insights in the modeling of Wolf-Rayet stars, but made it also possible to measure the separation between the two stars. This led to a new determination of the distance of the system, showing that previous estimates were incorrect. The observations also revealed information on the region where the winds from the two stars collide. The famous binary system RS Ophiuchi, an example of a recurrent nova, was observed just 5 days after it was discovered to be in outburst on 12 February 2006, an event that has been expected for 21 years. AMBER was able to detect the extension of the expanding nova emission. These observations show a complex geometry and kinematics, far from the simple interpretation of a spherical fireball in extension. AMBER has detected a high velocity jet probably perpendicular to the orbital plane of the binary system, and allowed a precise and careful study of the wind and the shockwave

  4. Slow recovery in desert perennial vegetation following prolonged human disturbance

    USGS Publications Warehouse

    Guo, Q.

    2004-01-01

    The study shows an exceptionally long-term recovery of perennial vegetation from prolonged heavy grazing and other human impacts. Since protection in 1906, overall species richness and habitat heterogeneity at the study site continued to increase until the 1960s when diversity, density and cover stabilized. During the same period, overall plant density and cover also increased. Species turnover increased gradually with time but no significant relation between any of the three community variables and precipitation or Palmer Drought Severity Index (PDSI) was detected. The increases in plant species richness, density, and cover of the perennial vegetation were mostly due to the increase of herbaceous species, especially palatable species. The lack of clear relationship between environment (e.g., precipitation) and community variables suggests that site history and plant life history must be taken into account in examining the nature of vegetation recovery process after disturbances.

  5. Targeting intracellular compartments by magnetic polymeric nanoparticles.

    PubMed

    Kocbek, Petra; Kralj, Slavko; Kreft, Mateja Erdani; Kristl, Julijana

    2013-09-27

    Superparamagnetic iron oxide nanoparticles (SPIONs) show a great promise for a wide specter of bioapplications, due to their characteristic magnetic properties exhibited only in the presence of magnetic field. Their advantages in the fields of magnetic drug targeting and imaging are well established and their safety is assumed, since iron oxide nanoparticles have already been approved for in vivo application, however, according to many literature reports the bare metal oxide nanoparticles may cause toxic effects on treated cells. Therefore, it is reasonable to prevent the direct interactions between metal oxide core and surrounding environment. In the current research ricinoleic acid coated maghemite nanoparticles were successfully synthesized, characterized and incorporated in the polymeric matrix, resulting in nanosized magnetic polymeric particles. The carrier system was shown to exhibit superparamagnetic properties and was therefore responsive towards external magnetic field. Bioevaluation using T47-D breast cancer cells confirmed internalization of magnetic polymeric nanoparticles (MNPs) and their intracellular localization in various subcellular compartments, depending on presence/absence of external magnetic field. However, the number of internalized MNPs observed by fluorescent and transmission electron microscopy was relatively low, making such way of targeting effective only for delivery of highly potent drugs. The scanning electron microscopy of treated cells revealed that MNPs influenced the cell adhesion, when external magnetic field was applied, and that treatment resulted in damaged apical plasma membrane right after exposure to the magnetic carrier. On the other hand, MNPs showed only reversibly reduced cellular metabolic activity in concentrations up to 200 μg/ml and, in the tested concentration the cell cycle distribution was within the normal range, indicating safety of the established magnetic carrier system for the treated cells. PMID:23603023

  6. Cell-Permeable MR Contrast Agents with Increased Intracellular Retention

    PubMed Central

    Endres, Paul J.; MacRenaris, Keith W.; Vogt, Stefan; Meade, Thomas J.

    2009-01-01

    Magnetic resonance imaging (MRI) is a technique used in both clinical and experimental settings to produce high resolution images of opaque organisms without ionizing radiation. Currently, MR imaging is augmented by contrast agents and the vast majority these small molecule Gd(III) chelates are confined to the extracellular regions. As a result, contrast agents are confined to vascular regions reducing their ability to provide information about cell physiology or molecular pathology. We have shown that polypeptides of arginine have the capacity to transport Gd(III) contrast agents across cell membranes. However, this transport is not unidirectional and once inside the cell the arginine-modified contrast agents efflux rapidly, decreasing the intracellular Gd(III) concentration and corresponding MR image intensity. By exploiting the inherent disulfide reducing environment of cells, thiol compounds, Gd(III)-DOTA-SS-Arg8 and Gd(III)-DTPA-SS-Arg8, are cleaved from their cell penetrating peptide transduction domains upon cell internalization. This reaction prolongs the cell-associated lifetime of the chelated Gd(III) by cleaving it from the cell transduction domain. PMID:18803414

  7. Gluteal compartment syndrome after prolonged immobilisation.

    PubMed

    Liu, H L; Wong, David S Y

    2009-04-01

    Muscles in the gluteal region are confined by distinct fascial attachments which can potentially result in compartment syndrome. A 74-year-old chronic drinker was admitted to the medical ward after being found drunk on the street. He noticed acute painful swelling of the right side of his buttock the following morning and recalled a slip and fall prior to his blackout. The whole right half of the buttock was tense with erythematous overlying skin. Examination revealed sciatic nerve palsy and myoglobinuria. Emergency fasciotomy and debridement were performed. Intra-operative pressure measurement confirmed a grossly elevated intra-compartmental pressure. Gluteal compartment syndrome is an extremely rare condition and has only been scantily documented previously in case reports. Early diagnosis is crucial but delay recognition is common from lack of knowledge of the condition and readily results in permanent sciatic nerve injury and acute renal shutdown from myoglobinuria. Awareness of the condition, early diagnosis and prompt exploration provide the only chance of avoiding these devastating consequences. Acute swelling diffusely affecting the whole or one side of the buttock, a history of trauma and prolonged local pressure impingement associated with pain out of proportion to the clinical signs should raise a suspicion of this rare condition. PMID:19423461

  8. Breathing during prolonged exercise in humans.

    PubMed Central

    Kearon, M C; Summers, E; Jones, N L; Campbell, E J; Killian, K J

    1991-01-01

    1. Six normal subjects cycled to endurance or for 60 min at four work rates (WR 1-4): mean of 34% working capacity (93 watts for 60 min); 43% (120 watts for 56 min); 63% (177 watts for 37 min); and 84% (233 watts for 12 min), to determine how breathing pattern and dyspnoea change during prolonged activity. Four to six minutes were allowed to establish steady state and subsequent changes were considered to be endurance related. 2. Dyspnoea (Borg scale, 0-10) increased with the duration of activity at all work rates. 3. Ventilation (VE) did not change at WR1; increased from 44 to 47 l min-1 at WR2; from 60 to 88 l min-1 at WR3; and from 111 to 132 l min-1 at WR4. Dyspnoea was significantly and independently related to ventilation and duration of activity: dyspnoea = 0.004 VE1.36 time 0.25 (r = 0.81; partial F 202 and 26 respectively). 4. Inspiratory resistance did not increase at any work rate. Dynamic elastance remained constant during WR1, WR2 and WR3 but increased from 7.4 to 9.1 cmH2O l-1 during WR4. 5. Peak inspiratory pressure did not increase, and the increase in VE was accomplished by an increased breathing frequency without change in duty cycle. 6. Duration of activity is an important contributor to dyspnoea independent of changes in respiratory muscle contractile activity. PMID:1798038

  9. Prolonged and recurrent fevers in children.

    PubMed

    Marshall, Gary S

    2014-01-01

    Some children referred for prolonged fever are actually not having elevated temperatures; the approach here requires dissection of the history and correction of health misperceptions. Others have well-documented fevers associated with clinical, laboratory, or epidemiologic findings that should point to a specific diagnosis. "Fever-of-Unknown-Origin" (FUO) is the clinical scenario of daily fever for ≥ 14 days that defies explanation after a careful history, physical examination, and basic laboratory tests. The diagnostic approach requires a meticulous fever diary, serial clinical and laboratory evaluations, vigilance for the appearance of new signs and symptoms, and targeted investigations; the pace of the work-up is determined by the severity of the illness. Approximately half of children with FUO will have a self-limited illness and will never have a specific diagnosis made; the other half will ultimately be found to have, in order, infectious, inflammatory, or neoplastic conditions. Irregular, intermittent, recurrent fevers in the well-appearing child are likely to be sequential viral illnesses. Monogenic autoinflammatory diseases should be considered in those who do not fit the picture of recurrent infections and who do not have hallmarks of immune deficiency. Stereotypical febrile illnesses that recur with clockwork periodicity should raise the possibilities of cyclic neutropenia, if the cycle is approximately 21 days, or periodic fever, aphthous stomatitis, pharyngitis, and adenitis (PFAPA) syndrome, the most common periodic fever in childhood. PMID:24120354

  10. Intracellular pH Modulates Autophagy and Mitophagy.

    PubMed

    Berezhnov, Alexey V; Soutar, Marc P M; Fedotova, Evgeniya I; Frolova, Maria S; Plun-Favreau, Helene; Zinchenko, Valery P; Abramov, Andrey Y

    2016-04-15

    The specific autophagic elimination of mitochondria (mitophagy) plays the role of quality control for this organelle. Deregulation of mitophagy leads to an increased number of damaged mitochondria and triggers cell death. The deterioration of mitophagy has been hypothesized to underlie the pathogenesis of several neurodegenerative diseases, most notably Parkinson disease. Although some of the biochemical and molecular mechanisms of mitochondrial quality control are described in detail, physiological or pathological triggers of mitophagy are still not fully characterized. Here we show that the induction of mitophagy by the mitochondrial uncoupler FCCP is independent of the effect of mitochondrial membrane potential but dependent on acidification of the cytosol by FCCP. The ionophore nigericin also reduces cytosolic pH and induces PINK1/PARKIN-dependent and -independent mitophagy. The increase of intracellular pH with monensin suppresses the effects of FCCP and nigericin on mitochondrial degradation. Thus, a change in intracellular pH is a regulator of mitochondrial quality control. PMID:26893374

  11. Multicomponent Supramolecular Polymers as a Modular Platform for Intracellular Delivery.

    PubMed

    Bakker, Maarten H; Lee, Cameron C; Meijer, E W; Dankers, Patricia Y W; Albertazzi, Lorenzo

    2016-02-23

    Supramolecular polymers are an emerging family of nanosized structures with potential use in materials chemistry and medicine. Surprisingly, application of supramolecular polymers in the field of drug delivery has received only limited attention. Here, we explore the potential of PEGylated 1,3,5-benzenetricarboxamide (BTA) supramolecular polymers for intracellular delivery. Exploiting the unique modular approach of supramolecular chemistry, we can coassemble neutral and cationic BTAs and control the overall properties of the polymer by simple monomer mixing. Moreover, this platform offers a versatile approach toward functionalization. The core can be efficiently loaded with a hydrophobic guest molecule, while the exterior can be electrostatically complexed with siRNA. It is demonstrated that both compounds can be delivered in living cells, and that they can be combined to enable a dual delivery strategy. These results show the advantages of employing a modular system and pave the way for application of supramolecular polymers in intracellular delivery. PMID:26811943

  12. Estimating the biophysical properties of neurons with intracellular calcium dynamics

    NASA Astrophysics Data System (ADS)

    Ye, Jingxin; Rozdeba, Paul J.; Morone, Uriel I.; Daou, Arij; Abarbanel, Henry D. I.

    2014-06-01

    We investigate the dynamics of a conductance-based neuron model coupled to a model of intracellular calcium uptake and release by the endoplasmic reticulum. The intracellular calcium dynamics occur on a time scale that is orders of magnitude slower than voltage spiking behavior. Coupling these mechanisms sets the stage for the appearance of chaotic dynamics, which we observe within certain ranges of model parameter values. We then explore the question of whether one can, using observed voltage data alone, estimate the states and parameters of the voltage plus calcium (V+Ca) dynamics model. We find the answer is negative. Indeed, we show that voltage plus another observed quantity must be known to allow the estimation to be accurate. We show that observing both the voltage time course V (t) and the intracellular Ca time course will permit accurate estimation, and from the estimated model state, accurate prediction after observations are completed. This sets the stage for how one will be able to use a more detailed model of V+Ca dynamics in neuron activity in the analysis of experimental data on individual neurons as well as functional networks in which the nodes (neurons) have these biophysical properties.

  13. Direct measurement of intracellular free Ca2+ in rat peritoneal macrophages: correlation with oxygen-radical production.

    PubMed Central

    Hallett, M B; Campbell, A K

    1983-01-01

    A novel method has been developed, based on osmotic lysis of intracellular pinocytotic vesicles, to introduce the Ca2+-activated photoprotein obelin into the cytoplasm of rat peritoneal macrophages. The change in osmolarity of the incubating medium necessary to induce lysis of the pinocytotic vesicles did not significantly affect the viability or responsiveness of the cells. The method enabled on average 3 fl of external medium to be introduced into each cell. Macrophages loaded with photoprotein had a resting intracellular Ca2+ concentration of 0.24 +/- 0.02 microM, calculated from the obelin consumption rate. The calcium ionophore, A23187, induced a prolonged rise in intracellular Ca2+ and also stimulated oxygen-radical production, monitored by luminol-dependent chemiluminescence. The chemotactic peptide, N-formyl-methionyl-leucyl-phenylalanine, 1 microM, produced a transient increase in cytoplasmic Ca2+ which reached a plateau of 1.2 +/- 0.64 microM (n = 7) and declined with a half-time of approximately 40 sec. Unopsonized particles, latex beads (diameter = 1 micron), did not produce any detectable rise in intracellular Ca2+. Incorporation of a calcium chelator EGTA-ethylene-glycol-bis-(aminoethylether) tetra-acetate--into the cytoplasm abolished the transient intracellular Ca2+ rise induced by chemotactic peptide. Oxygen-radical production was also abolished. However, oxygen radical production induced by unopsonized particles was unaffected by intracellular EGTA. It was concluded that oxygen-radical production detected by chemiluminescence can be triggered by a rise in intracellular Ca2+. Chemotactic peptide induces oxygen-radical production by this mechanism. However, unopsonized particles induce oxygen-radical production by a mechanism independent of a rise in intracellular Ca2+. Images Figure 1 PMID:6414943

  14. Intracellular proton access in a Cl(-)/H(+) antiporter.

    PubMed

    Lim, Hyun-Ho; Shane, Tania; Miller, Christopher

    2012-01-01

    Chloride-transporting membrane proteins of the CLC family appear in two distinct mechanistic flavors: H(+)-gated Cl(-) channels and Cl(-)/H(+) antiporters. Transmembrane H(+) movement is an essential feature of both types of CLC. X-ray crystal structures of CLC antiporters show the Cl(-) ion pathway through these proteins, but the H(+) pathway is known only inferentially by two conserved glutamate residues that act as way-stations for H(+) in its path through the protein. The extracellular-facing H(+) transfer glutamate becomes directly exposed to aqueous solution during the transport cycle, but the intracellular glutamate E203, Glu(in), is buried within the protein. Two regions, denoted "polar" and "interfacial," at the intracellular surface of the bacterial antiporter CLC-ec1 are examined here as possible pathways by which intracellular aqueous protons gain access to Glu(in). Mutations at multiple residues of the polar region have little effect on antiport rates. In contrast, mutation of E202, a conserved glutamate at the protein-water boundary of the interfacial region, leads to severe slowing of the Cl(-)/H(+) antiport rate. An X-ray crystal structure of E202Y, the most strongly inhibited of these substitutions, shows an aqueous portal leading to Glu(in) physically blocked by cross-subunit interactions; moreover, this mutation has only minimal effect on a monomeric CLC variant, which necessarily lacks such interactions. The several lines of experiments presented argue that E202 acts as a water-organizer that creates a proton conduit connecting intracellular solvent with Glu(in). PMID:23239938

  15. Drug- and non-drug-associated QT interval prolongation

    PubMed Central

    van Noord, Charlotte; Eijgelsheim, Mark; Stricker, Bruno H Ch

    2010-01-01

    Sudden cardiac death is among the most common causes of cardiovascular death in developed countries. The majority of sudden cardiac deaths are caused by acute ventricular arrhythmia following repolarization disturbances. An important risk factor for repolarization disturbances is use of QT prolonging drugs, probably partly explained by gene–drug interactions. In this review, we will summarize QT interval physiology, known risk factors for QT prolongation, including drugs and the contribution of pharmacogenetics. The long QT syndrome can be congenital or acquired. The congenital long QT syndrome is caused by mutations in ion channel subunits or regulatory protein coding genes and is a rare monogenic disorder with a mendelian pattern of inheritance. Apart from that, several common genetic variants that are associated with QT interval duration have been identified. Acquired QT prolongation is more prevalent than the congenital form. Several risk factors have been identified with use of QT prolonging drugs as the most frequent cause. Most drugs that prolong the QT interval act by blocking hERG-encoded potassium channels, although some drugs mainly modify sodium channels. Both pharmacodynamic as well as pharmacokinetic mechanisms may be responsible for QT prolongation. Pharmacokinetic interactions often involve drugs that are metabolized by cytochrome P450 enzymes. Pharmacodynamic gene–drug interactions are due to genetic variants that potentiate the QT prolonging effect of drugs. QT prolongation, often due to use of QT prolonging drugs, is a major public health issue. Recently, common genetic variants associated with QT prolongation have been identified. Few pharmacogenetic studies have been performed to establish the genetic background of acquired QT prolongation but additional studies in this newly developing field are warranted. PMID:20642543

  16. Effect of prolonged exposure to organic solvents on the active site environment of subtilisin Carlsberg

    PubMed Central

    Bansal, Vibha; Delgado, Yamixa; Fasoli, Ezio; Ferrer, Amaris; Griebenow, Kai; Secundo, Francesco; Barletta, Gabriel L

    2010-01-01

    The potential of enzyme catalysis as a tool for organic synthesis is nowadays indisputable, as is the fact that organic solvents affect an enzyme’s activity, selectivity and stability. Moreover, it was recently realized that an enzyme’s initial activity is substantially decreased after prolonged exposure to organic media, an effect that further hampers their potential as catalysts for organic synthesis. Regrettably, the mechanistic reasons for these effects are still debatable. In the present study we have made an attempt to explain the reasons behind the partial loss of enzyme activity on prolonged exposure to organic solvents. Fluorescence spectroscopic studies of the serine protease subtilisin Carlsberg chemically modified with polyethylene glycol (PEG-SC) and inhibited with a Dancyl fluorophore, and dissolved in two organic solvents (acetonitrile and 1,4-dioxane) indicate that when the enzyme is initially introduced into these solvents, the active site environment is similar to that in water; however prolonged exposure to the organic medium causes this environment to resemble that of the solvent in which the enzyme is dissolved. Furthermore, kinetic studies show a reduction on both Vmax and KM as a result of prolonged exposure to the solvents. One interpretation of these results is that during this prolonged exposure to organic solvents the active-site fluorescent label inhibitor adopts a different binding conformation. Extrapolating this to an enzymatic reaction we argue that substrates bind in a less catalytically favorable conformation after the enzyme has been exposed to organic media for several hours. PMID:20414456

  17. Global warming prolongs the thermal stratification of dimictic lake Mondsee.

    NASA Astrophysics Data System (ADS)

    Blatterer, Hubert; Luger, Martin

    2013-04-01

    The pre-alpine Lake Mondsee is situated at the northern margin of the European Alps (47° 49´N, 13° 24´E) in the Salzkammergut lake district of Upper Austria at a sea level of 481 m. The lake has a surface area of 14,21 km² and a maximum water depth of 68 m (volume is 500 Mio m³ and theoretical water retention time is 1,8 years). Sediment samples confirm oligotrophic conditions as historical reference status of the lake. From 1970 to 1985 the lake suffered from severe eutrophication leading to cyanobacterial blooms (Planctothrix rubescens). Reduction of nutrient load in the course of improved sewage treatment resulted in re-oligotrophication from 1985 to about 2000. Currently, lake Mondsee is assessed mesotrophic and the biological quality elements "phytoplankton" and "macrophytes" classify the lake in the "moderate ecological status". According to the Water Framework Directive, a key initiative throughout the EU, the aim is to improve water quality and reach the "good ecological status". Temperature data of the Lake have been measured since the 30ies of the last century in varying intervals. In the present study (1991 - 2009) water temperature measured at the deepest point of the lake shows an increase in average surface temperature (0 - 5 m) of about 2 °C over the last two decades. The increase is less pronounced in deeper water layers and almost not visible below 15 m depth. Due to global change and rising temperatures stratification is starting earlier in the season and is prolonged from formerly end of November to the middle or even end of December. Thus, between 1999 and 2011 in several years the stratification period was extended for 5 weeks. During stratification oxygen depletion occurs in the depth of lakes and prolonged stratification results in increased areas of oxygen depletion. The oxygen concentration controls the phosphorus release of lake sediments. Therefore prolonged stratification results in increased internal phosphorus load of the lake

  18. Macrophage defense mechanisms against intracellular bacteria

    PubMed Central

    Weiss, Günter; Schaible, Ulrich E

    2015-01-01

    Macrophages and neutrophils play a decisive role in host responses to intracellular bacteria including the agent of tuberculosis (TB), Mycobacterium tuberculosis as they represent the forefront of innate immune defense against bacterial invaders. At the same time, these phagocytes are also primary targets of intracellular bacteria to be abused as host cells. Their efficacy to contain and eliminate intracellular M. tuberculosis decides whether a patient initially becomes infected or not. However, when the infection becomes chronic or even latent (as in the case of TB) despite development of specific immune activation, phagocytes have also important effector functions. Macrophages have evolved a myriad of defense strategies to combat infection with intracellular bacteria such as M. tuberculosis. These include induction of toxic anti-microbial effectors such as nitric oxide and reactive oxygen intermediates, the stimulation of microbe intoxication mechanisms via acidification or metal accumulation in the phagolysosome, the restriction of the microbe's access to essential nutrients such as iron, fatty acids, or amino acids, the production of anti-microbial peptides and cytokines, along with induction of autophagy and efferocytosis to eliminate the pathogen. On the other hand, M. tuberculosis, as a prime example of a well-adapted facultative intracellular bacterium, has learned during evolution to counter-balance the host's immune defense strategies to secure survival or multiplication within this otherwise hostile environment. This review provides an overview of innate immune defense of macrophages directed against intracellular bacteria with a focus on M. tuberculosis. Gaining more insights and knowledge into this complex network of host-pathogen interaction will identify novel target sites of intervention to successfully clear infection at a time of rapidly emerging multi-resistance of M. tuberculosis against conventional antibiotics. PMID:25703560

  19. Endosomal escape: a bottleneck in intracellular delivery.

    PubMed

    Shete, Harshad K; Prabhu, Rashmi H; Patravale, Vandana B

    2014-01-01

    With advances in therapeutic science, apart from drugs, newer bioactive moieties like oligonucleotides, proteins, peptides, enzymes and antibodies are constantly being introduced for the betterment of therapeutic efficacy. These moieties have intracellular components of the cells like cytoplasm and nucleus as one of their pharmacological sites for exhibiting therapeutic activity. Despite their promising efficacy, their intracellular bioavailability has been critically hampered leading to failure in the treatment of numerous diseases and disorders. The endosomal uptake pathway is known to be a rate-limiting barrier for such systems. Bioactive molecules get trapped in the endosomal vesicles and degraded in the lysosomal compartment, necessitating the need for effective strategies that facilitate the endosomal escape and enhance the cytosolic bioavailability of bioactives. Microbes like viruses and bacteria have developed their innate mechanistic tactics to translocate their genome and toxins by efficiently penetrating the host cell membrane. Understanding this mechanism and exploring it further for intracellular delivery has opened new avenues to surmount the endosomal barrier. These strategies include membrane fusion, pore formation and proton sponge effects. On the other hand, progress in designing a novel smart polymeric carrier system that triggers endosomal escape by undergoing modulations in the intracellular milieu has further led to an improvement in intracellular delivery. These comprise pH, enzyme and temperature-induced modulators, synthetic cationic lipids and photo-induced physical disruption. Each of the aforementioned strategies has its own unique mechanism to escape the endosome. This review recapitulates the numerous strategies designed to surmount the bottleneck of endosomal escape and thereby achieve successful intracellular uptake of bioactives. PMID:24730275

  20. Intracellular potassium stabilizes human ether-à-go-go-related gene channels for export from endoplasmic reticulum.

    PubMed

    Wang, Lu; Dennis, Adrienne T; Trieu, Phan; Charron, Francois; Ethier, Natalie; Hebert, Terence E; Wan, Xiaoping; Ficker, Eckhard

    2009-04-01

    Several therapeutic compounds have been identified that prolong the QT interval on the electrocardiogram and cause torsade de pointes arrhythmias not by direct block of the cardiac potassium channel human ether-à-go-go-related gene (hERG) but via disruption of hERG trafficking to the cell surface membrane. One example of a clinically important compound class that potently inhibits hERG trafficking are cardiac glycosides. We have shown previously that inhibition of hERG trafficking by cardiac glycosides is initiated via direct block of Na(+)/K(+) pumps and not via off-target interactions with hERG or any other protein. However, it was not known how pump inhibition at the cell surface is coupled to hERG processing in the endoplasmic reticulum. Here, we show that depletion of intracellular K(+)-either indirectly after long-term exposure to cardiac glycosides or directly after exposure to gramicidin in low sodium media-is sufficient to disrupt hERG trafficking. In K(+)-depleted cells, hERG trafficking can be restored by permeating K(+) or Rb(+) ions, incubation at low temperature, exposure to the pharmacological chaperone astemizole, or specific mutations in the selectivity filter of hERG. Our data suggest a novel mechanism for drug-induced trafficking inhibition in which cardiac glycosides produce a [K(+)](i)-mediated conformational defect directly in the hERG channel protein. PMID:19139152

  1. Intracellular Potassium Stabilizes Human Ether-à-go-go-Related Gene Channels for Export from Endoplasmic ReticulumS⃞

    PubMed Central

    Wang, Lu; Dennis, Adrienne T.; Trieu, Phan; Charron, Francois; Ethier, Natalie; Hebert, Terence E.; Wan, Xiaoping; Ficker, Eckhard

    2009-01-01

    Several therapeutic compounds have been identified that prolong the QT interval on the electrocardiogram and cause torsade de pointes arrhythmias not by direct block of the cardiac potassium channel human ether-à-go-go-related gene (hERG) but via disruption of hERG trafficking to the cell surface membrane. One example of a clinically important compound class that potently inhibits hERG trafficking are cardiac glycosides. We have shown previously that inhibition of hERG trafficking by cardiac glycosides is initiated via direct block of Na+/K+ pumps and not via off-target interactions with hERG or any other protein. However, it was not known how pump inhibition at the cell surface is coupled to hERG processing in the endoplasmic reticulum. Here, we show that depletion of intracellular K+—either indirectly after long-term exposure to cardiac glycosides or directly after exposure to gramicidin in low sodium media—is sufficient to disrupt hERG trafficking. In K+-depleted cells, hERG trafficking can be restored by permeating K+ or Rb+ ions, incubation at low temperature, exposure to the pharmacological chaperone astemizole, or specific mutations in the selectivity filter of hERG. Our data suggest a novel mechanism for drug-induced trafficking inhibition in which cardiac glycosides produce a [K+]i-mediated conformational defect directly in the hERG channel protein. PMID:19139152

  2. Multiplexed imaging of intracellular protein networks.

    PubMed

    Grecco, Hernán E; Imtiaz, Sarah; Zamir, Eli

    2016-08-01

    Cellular functions emerge from the collective action of a large number of different proteins. Understanding how these protein networks operate requires monitoring their components in intact cells. Due to intercellular and intracellular molecular variability, it is important to monitor simultaneously multiple components at high spatiotemporal resolution. However, inherent trade-offs narrow the boundaries of achievable multiplexed imaging. Pushing these boundaries is essential for a better understanding of cellular processes. Here the motivations, challenges and approaches for multiplexed imaging of intracellular protein networks are discussed. © 2016 International Society for Advancement of Cytometry. PMID:27183498

  3. Peroxisome is a reservoir of intracellular calcium.

    PubMed

    Raychaudhury, Bikramjit; Gupta, Shreedhara; Banerjee, Shouvik; Datta, Salil C

    2006-07-01

    We have examined fura 2-loaded purified peroxisomes under confocal microscope to prove that this mammalian organelle is a store of intracellular calcium pool. Presence of calcium channel and vanadate sensitive Ca(2+)-ATPase in the purified peroxisomal membrane has been demonstrated. We have further observed that machineries to maintain calcium pool in this mammalian organelle are impaired during infection caused by Leishmania donovani. Results reveal that peroxisomes have a merit to play a significant role in the metabolism of intracellular calcium. PMID:16713100

  4. Expert position paper on prolonged dual antiplatelet therapy in secondary prevention following myocardial infarction.

    PubMed

    Weiss, Thomas W; Aichinger, Josef; Huber, Kurt; Speidl, Walter S; Watzinger, Norbert; Zweiker, Robert; Alber, Hannes F

    2016-06-01

    The protective effect of dual antiplatelet therapy (DAPT) following acute coronary syndrome is undisputed, but its duration is subject of debate. Several studies show that prolonged therapy provides a clinical benefit in patients following acute coronary syndrome. The aim of this position paper authored by Austrian experts is to outline the current evidence and provide an overview of recent studies. It is also intended to serve as a practical guide to identify those patients who may benefit from prolonged DAPT. PMID:27278134

  5. Prolonged QTc interval in association with medium-chain acyl-coenzyme A dehydrogenase deficiency.

    PubMed

    Wiles, Jason R; Leslie, Nancy; Knilans, Timothy K; Akinbi, Henry

    2014-06-01

    Medium-chain acyl-coenzyme A dehydrogenase (MCAD) deficiency is the most common disorder of mitochondrial fatty acid oxidation. We report a term male infant who presented at 3 days of age with hypoglycemia, compensated metabolic acidosis, hypocalcemia, and prolonged QTc interval. Pregnancy was complicated by maternal premature atrial contractions and premature ventricular contractions. Prolongation of the QTc interval resolved after correction of metabolic derangements. The newborn screen was suggestive for MCAD deficiency, a diagnosis that was confirmed on genetic analysis that showed homozygosity for the disease-associated missense A985G mutation in the ACADM gene. This is the first report of acquired prolonged QTc in a neonate with MCAD deficiency, and it suggests that MCAD deficiency should be considered in the differential diagnoses of acute neonatal illnesses associated with electrocardiographic abnormality. We review the clinical presentation and diagnosis of MCAD deficiency in neonates. PMID:24799540

  6. Development of a Decision Support System for Analysis and Solutions of Prolonged Standing in the Workplace

    PubMed Central

    Halim, Isa; Arep, Hambali; Kamat, Seri Rahayu; Abdullah, Rohana; Omar, Abdul Rahman; Ismail, Ahmad Rasdan

    2014-01-01

    Background Prolonged standing has been hypothesized as a vital contributor to discomfort and muscle fatigue in the workplace. The objective of this study was to develop a decision support system that could provide systematic analysis and solutions to minimize the discomfort and muscle fatigue associated with prolonged standing. Methods The integration of object-oriented programming and a Model Oriented Simultaneous Engineering System were used to design the architecture of the decision support system. Results Validation of the decision support system was carried out in two manufacturing companies. The validation process showed that the decision support system produced reliable results. Conclusion The decision support system is a reliable advisory tool for providing analysis and solutions to problems related to the discomfort and muscle fatigue associated with prolonged standing. Further testing of the decision support system is suggested before it is used commercially. PMID:25180141

  7. Arrhythmogenic consequences of intracellular calcium waves.

    PubMed

    Xie, Lai-Hua; Weiss, James N

    2009-09-01

    Intracellular Ca(2+) (Ca(i)(2+)) waves are known to cause delayed afterdepolarizations (DADs), which have been associated with arrhythmias in cardiac disease states such as heart failure, catecholaminergic polymorphic ventricular tachycardia, and digitalis toxicity. Here we show that, in addition to DADs, Ca(i)(2+) waves also have other consequences relevant to arrhythmogenesis, including subcellular spatially discordant alternans (SDA, in which the amplitude of the local Ca(i)(2+) transient alternates out of phase in different regions of the same cell), sudden repolarization changes promoting the dispersion of refractoriness, and early afterdepolarizations (EADs). Ca(i)(2+) was imaged using a charge-coupled device-based system in fluo-4 AM-loaded isolated rabbit ventricular myocytes paced at constant or incrementally increasing rates, using either field stimulation, current clamp, or action potential (AP) clamp. Ca(i)(2+) waves were induced by Bay K 8644 (50 nM) + isoproterenol (100 nM), or low temperature. When pacing was initiated during a spontaneous Ca(i)(2+) wave, SDA occurred abruptly and persisted during pacing. Similarly, during rapid pacing, SDA typically arose suddenly from spatially concordant alternans, due to an abrupt phase reversal of the subcellular Ca(i)(2+) transient in a region of the myocyte. Ca(i)(2+) waves could be visualized interspersed with AP-triggered Ca(i)(2+) transients, producing a rich variety of subcellular Ca(i)(2+) transient patterns. With free-running APs, complex Ca(i)(2+) release patterns were associated with DADs, EADs, and sudden changes in AP duration. These findings link Ca(i)(2+) waves directly to a variety of arrhythmogenic phenomena relevant to the intact heart. PMID:19561309

  8. Neuromuscular adaptations during submaximal prolonged cycling.

    PubMed

    Castronovo, A M; De Marchis, C; Bibbo, D; Conforto, S; Schmid, M; D'Alessio, T

    2012-01-01

    This study aims at evaluating the neuromuscular adaptations occurring during submaximal prolonged cycling tasks. In particular, we want to assess changes in surface electromyographic (sEMG) signal recorded during a pedaling task, performed by six subjects on a cycle-simulator at a constant power output, until voluntary exhaustion. Task failure was defined as the instant the subject was no longer able to maintain the required task. Electromyographic activity was recorded from eight muscles of the dominant leg and burst characteristics of sEMG signals were analyzed in order to assess the changes in muscle activity level produced by the occurrence of neuromuscular fatigue. In particular, three features were extracted from the sEMG signal for each burst: amplitude, location of the maxima and mean profile of the burst envelope. We have reported an increase in the amplitude parameter for all subjects only for Vastii while bi-articular muscles presented a high variability among subjects. Also the location of the maximal values of the mean envelope of the bursts was found to change when considering bi-articular or mono-articular muscles. The envelope profile was found not to be subject to alterations when comparing the end of the task with the beginning. We speculated that neuromuscular fatigue induces changes essentially in the mono-articular muscles which produce power. This phenomenon is highly correlated with the adopted pedaling strategy which, being not constrained, induces subjects to express the maximal power in the downstroke phase, related to knee extension and involving mainly mono-articular muscles. PMID:23366709

  9. Changes in Intracellular Free Calcium Concentration during Illumination of Invertebrate Photoreceptors

    PubMed Central

    Brown, J. E.; Blinks, J. R.

    1974-01-01

    Aequorin, which luminesces in the presence of calcium, was injected into photoreceptor cells of Limulus ventral eye. A bright light stimulus elicited a large increase in aequorin luminescence, the aequorin response, indicating a rise of intracellular calcium ion concentration, Cai. The aequorin response reached a maximum after the peak of the electrical response of the photoreceptor, decayed during a prolonged stimulus, and returned to an undetectable level in the dark. Reduction of Cao reduced the amplitude of the aequorin response by a factor no greater than 3. Raising Cao increased the amplitude of the aequorin response. The aequorin response became smaller when membrane voltage was clamped to successively more positive values. These results indicate that the stimulus-induced rise of Cai may be due in part to a light-induced influx of Ca and in part to release of Ca from an intracellular store. Our findings are consistent with the hypothesis that a rise in Cai is a step in the sequence of events underlying light-adaptation in Limulus ventral photoreceptors. Aequorin was also injected into photoreceptors of Balanus. The aequorin responses were similar to those recorded from Limulus cells in all but two ways: (a) A large sustained aequorin luminescence was measured during a prolonged stimulus, and (b) removal of extracellular calcium reduced the aequorin response to an undetectable level. PMID:4155426

  10. Intracellular Zn2+ accumulation enhances suppression of synaptic activity following spreading depolarization.

    PubMed

    Carter, Russell E; Seidel, Jessica L; Lindquist, Britta E; Sheline, Christian T; Shuttleworth, C William

    2013-06-01

    Spreading depolarization (SD) is a feed-forward wave that propagates slowly throughout brain tissue and recovery from SD involves substantial metabolic demand. Presynaptic Zn(2+) release and intracellular accumulation occurs with SD, and elevated intracellular Zn(2+) ([Zn(2+) ]i ) can impair cellular metabolism through multiple pathways. We tested here whether increased [Zn(2+) ]i could exacerbate the metabolic challenge of SD, induced by KCl, and delay recovery in acute murine hippocampal slices. [Zn(2+) ]i loading prior to SD, by transient ZnCl2 application with the Zn(2+) ionophore pyrithione (Zn/Pyr), delayed recovery of field excitatory post-synaptic potentials (fEPSPs) in a concentration-dependent manner, prolonged DC shifts, and significantly increased extracellular adenosine accumulation. These effects could be due to metabolic inhibition, occurring downstream of pyruvate utilization. Prolonged [Zn(2+) ]i accumulation prior to SD was required for effects on fEPSP recovery and consistent with this, endogenous synaptic Zn(2+) release during SD propagation did not delay recovery from SD. The effects of exogenous [Zn(2+) ]i loading were also lost in slices preconditioned with repetitive SDs, implying a rapid adaptation. Together, these results suggest that [Zn(2+) ]i loading prior to SD can provide significant additional challenge to brain tissue, and could contribute to deleterious effects of [Zn(2+) ]i accumulation in a range of brain injury models. PMID:23495967

  11. Prolonged Activity of the Pestiviral RNase Erns as an Interferon Antagonist after Uptake by Clathrin-Mediated Endocytosis

    PubMed Central

    Zürcher, Christoph; Sauter, Kay-Sara; Mathys, Veronika; Wyss, Fabienne

    2014-01-01

    ABSTRACT The RNase activity of the envelope glycoprotein Erns of the pestivirus bovine viral diarrhea virus (BVDV) is required to block type I interferon (IFN) synthesis induced by single-stranded RNA (ssRNA) and double-stranded RNA (dsRNA) in bovine cells. Due to the presence of an unusual membrane anchor at its C terminus, a significant portion of Erns is also secreted. In addition, a binding site for cell surface glycosaminoglycans is located within the C-terminal region of Erns. Here, we show that the activity of soluble Erns as an IFN antagonist is not restricted to bovine cells. Extracellularly applied Erns protein bound to cell surface glycosaminoglycans and was internalized into the cells within 1 h of incubation by an energy-dependent mechanism that could be blocked by inhibitors of clathrin-dependent endocytosis. Erns mutants that lacked the C-terminal membrane anchor retained RNase activity but lost most of their intracellular activity as an IFN antagonist. Surprisingly, once taken up into the cells, Erns remained active and blocked dsRNA-induced IFN synthesis for several days. Thus, we propose that Erns acts as an enzymatically active decoy receptor that degrades extracellularly added viral RNA mainly in endolysosomal compartments that might otherwise activate intracellular pattern recognition receptors (PRRs) in order to maintain a state of innate immunotolerance. IMPORTANCE The pestiviral RNase Erns was previously shown to inhibit viral ssRNA- and dsRNA-induced interferon (IFN) synthesis. However, the localization of Erns at or inside the cells, its species specificity, and its mechanism of interaction with cell membranes in order to block the host's innate immune response are still largely unknown. Here, we provide strong evidence that the pestiviral RNase Erns is taken up within minutes by clathrin-mediated endocytosis and that this uptake is mostly dependent on the glycosaminoglycan binding site located within the C-terminal end of the protein

  12. Molecular and photosynthetic responses to prolonged darkness and subsequent acclimation to re-illumination in the diatom Phaeodactylum tricornutum.

    PubMed

    Nymark, Marianne; Valle, Kristin C; Hancke, Kasper; Winge, Per; Andresen, Kjersti; Johnsen, Geir; Bones, Atle M; Brembu, Tore

    2013-01-01

    Photosynthetic diatoms that live suspended throughout the water column will constantly be swept up and down by vertical mixing. When returned to the photic zone after experiencing longer periods in darkness, mechanisms exist that enable the diatoms both to survive sudden light exposure and immediately utilize the available energy in photosynthesis and growth. We have investigated both the response to prolonged darkness and the re-acclimation to moderate intensity white irradiance (E = 100 µmol m(-2) s(-1)) in the diatom Phaeodactylum tricornutum, using an integrated approach involving global transcriptional profiling, pigment analyses, imaging and photo-physiological measurements. The responses were studied during continuous white light, after 48 h of dark treatment and after 0.5 h, 6 h, and 24 h of re-exposure to the initial irradiance. The analyses resulted in several intriguing findings. Dark treatment of the cells led to 1) significantly decreased nuclear transcriptional activity, 2) distinct intracellular changes, 3) fixed ratios of the light-harvesting pigments despite a decrease in the total cell pigment pool, and 4) only a minor drop in photosynthetic efficiency (Φ(PSII_max)). Re-introduction of the cells to the initial light conditions revealed 5) distinct expression profiles for nuclear genes involved in photosynthesis and those involved in photoprotection, 6) rapid rise in photosynthetic parameters (α and rETR(max)) within 0.5 h of re-exposure to light despite a very modest de novo synthesis of photosynthetic compounds, and 7) increasingly efficient resonance energy transfer from fucoxanthin chlorophyll a/c-binding protein complexes to photosystem II reaction centers during the first 0.5 h, supporting the observations stated in 6). In summary, the results show that despite extensive transcriptional, metabolic and intracellular changes, the ability of cells to perform photosynthesis was kept intact during the length of the experiment. We conclude

  13. Molecular and Photosynthetic Responses to Prolonged Darkness and Subsequent Acclimation to Re-Illumination in the Diatom Phaeodactylum tricornutum

    PubMed Central

    Nymark, Marianne; Valle, Kristin C.; Hancke, Kasper; Winge, Per; Andresen, Kjersti; Johnsen, Geir; Bones, Atle M.; Brembu, Tore

    2013-01-01

    Photosynthetic diatoms that live suspended throughout the water column will constantly be swept up and down by vertical mixing. When returned to the photic zone after experiencing longer periods in darkness, mechanisms exist that enable the diatoms both to survive sudden light exposure and immediately utilize the available energy in photosynthesis and growth. We have investigated both the response to prolonged darkness and the re-acclimation to moderate intensity white irradiance (E = 100 µmol m−2 s−1) in the diatom Phaeodactylum tricornutum, using an integrated approach involving global transcriptional profiling, pigment analyses, imaging and photo-physiological measurements. The responses were studied during continuous white light, after 48 h of dark treatment and after 0.5 h, 6 h, and 24 h of re-exposure to the initial irradiance. The analyses resulted in several intriguing findings. Dark treatment of the cells led to 1) significantly decreased nuclear transcriptional activity, 2) distinct intracellular changes, 3) fixed ratios of the light-harvesting pigments despite a decrease in the total cell pigment pool, and 4) only a minor drop in photosynthetic efficiency (ΦPSII_max). Re-introduction of the cells to the initial light conditions revealed 5) distinct expression profiles for nuclear genes involved in photosynthesis and those involved in photoprotection, 6) rapid rise in photosynthetic parameters (α and rETRmax) within 0.5 h of re-exposure to light despite a very modest de novo synthesis of photosynthetic compounds, and 7) increasingly efficient resonance energy transfer from fucoxanthin chlorophyll a/c-binding protein complexes to photosystem II reaction centers during the first 0.5 h, supporting the observations stated in 6). In summary, the results show that despite extensive transcriptional, metabolic and intracellular changes, the ability of cells to perform photosynthesis was kept intact during the length of the experiment. We conclude that

  14. QT prolongation and torsades de pointes with psychotropic agents

    PubMed Central

    Desai, Nagaraj; Venkatesh, Chilkunda Raviprakash; Kumar, Shambu Sunil

    2015-01-01

    The unexpected and catastrophic cardiovascular effects of psychotropic drugs are well described albeit uncommon. The list of drugs which have been associated with prolonging QT interval and hence potentially causing Torsades de pointes is exhaustive. The insight into the plausible mechanisms are largely unclear. However, the practical implications of anticipating and recognizing QT prolongation cannot be overemphasized. PMID:26600587

  15. Epidemiology of prolonged testicular infections with bovine viral diarrhea virus.

    PubMed

    Givens, M Daniel; Riddell, Kay P; Edmondson, Misty A; Walz, Paul H; Gard, Julie A; Zhang, Yijing; Galik, Patricia K; Brodersen, Bruce W; Carson, Robert L; Stringfellow, David A

    2009-10-20

    Previously, bovine viral diarrhea virus (BVDV) had been found in prolonged testicular infections following acute infection of immunocompetent bulls. The primary purpose of this research was to evaluate the production and maintenance of prolonged testicular infections after exposure to BVDV of seronegative bulls in varying circumstances. The secondary objective was to initiate assessment of the potential for transmission of BVDV via semen of bulls exhibiting a prolonged testicular infection. In total, 10 research trials were conducted. The first trial examined the duration of detectable virus in semen after intranasal inoculation of peri-pubertal bulls. The second to fifth trials examined the potential for prolonged testicular infections resulting from natural exposure of seronegative bulls to persistently infected heifers. In the last five trials, the potential for viral transmission from bulls exhibiting prolonged testicular infections to a small number of exposed animals (n=28) was evaluated. Results of this research demonstrated that prolonged testicular infections could result in detection of viral RNA in semen for 2.75 years with infectious virus grown from testicular tissue 12.5 months after viral exposure. A type 1b strain of BVDV caused prolonged testicular infection after natural exposure of seronegative bulls to a persistently infected heifer. However, transmission of BVDV to susceptible animals was not detected in the final five trials of this research. In conclusion, BVDV can persist in testicular tissue after acute infection for several years, but the potential for viral transmission from these prolonged testicular infections appears to be low. PMID:19473788

  16. Prolonged Field Care Working Group Fluid Therapy Recommendations.

    PubMed

    Baker, Benjamin L; Powell, Doug; Riesberg, Jamie; Keenan, Sean

    2016-01-01

    The Prolonged Field Care Working Group concurs that fresh whole blood (FWB) is the fluid of choice for patients in hemorrhagic shock, and the capability to transfuse FWB should be a basic skill set for Special Operations Forces (SOF) Medics. Prolonged field care (PFC) must also address resuscitative and maintenance fluid requirements in nonhemorrhagic conditions. PMID:27045508

  17. Impact of Prolonged Exacerbation Recovery in Chronic Obstructive Pulmonary Disease

    PubMed Central

    Law, Martin; Kowlessar, Beverly; Singh, Richa; Brill, Simon E.; Allinson, James P.; Wedzicha, Jadwiga A.

    2015-01-01

    Rationale: Exacerbations are important and heterogeneous events in the natural history of chronic obstructive pulmonary disease (COPD). Objectives: To examine the consequences of prolonged exacerbation recovery in patients with COPD. Methods: A cohort of 384 patients with COPD (FEV1 % predicted 45.8 [SD, 16.6] and a median exacerbation rate of 2.13 per year [interquartile range, 1.0–3.2]) were followed for 1,039 days (interquartile range, 660–1,814) between October 1995 and January 2013. Patients recorded daily worsening of respiratory symptoms and peak expiratory flow (PEF), and when stable underwent spirometry every 3 months, and completed the St. George’s Respiratory Questionnaire annually. Exacerbations were diagnosed as 2 consecutive days with one major symptom plus another respiratory symptom. Exacerbation duration was defined as the time from onset to the day preceding 2 consecutive symptom-free days and recovery in PEF as return to preexacerbation levels. Measurements and Main Results: A total of 351 patients had one or more exacerbations. Patients with a longer symptom duration (mean, 14.5 d) had a worse St. George’s Respiratory Questionnaire total score (0.2 units per 1 day; P = 0.040). A longer symptomatic duration was associated with a shorter interval between exacerbation recovery and onset of the next exacerbation (hazard ratio, 1.004; P = 0.013). For 257 (7.3%) exacerbations, PEF did not recover within 99 days. These exacerbations were associated with symptoms of a viral infection (cold and sore throat). Patients with these nonrecovered exacerbations showed a 10.8 ml/yr (P < 0.001) faster decline in FEV1. Conclusions: Prolonged exacerbation symptomatic duration is associated with poorer health status and a greater risk of a new event. Exacerbations where lung function does not recover are associated with symptoms of viral infections and accelerated decline in FEV1. PMID:26151174

  18. Hypothyroidism prolongs corpus luteum function in the pregnant rat.

    PubMed

    Hapon, María Belén; Motta, Alicia B; Ezquer, Marcelo; Bonafede, Melisa; Jahn, Graciela A

    2007-01-01

    It has been shown that hypothyroidism in the rat produces a prolongation of pregnancy associated with a delay in the fall of circulating progesterone (P4) at term. The aim of the present work is to determine whether the delayed P4 decline in hypothyroid mother rats is due to a retarded induction of P4 degradation to 20alphaOH P4 or to a stimulation of its synthesis, and to investigate the possible mechanisms that may underlie the altered luteal function. We determined by RIA the circulating profile of the hormones (TSH, PRL, LH, P4, PGF2alpha, and PGE2) involved in luteal regulation at the end of pregnancy and, by semiquantitative RT-PCR, the expression of factors involved in P4 synthesis (CytP450scc, StAR, 3betaHSD, PRLR) and metabolism (20alphaHSD, PGF2alphaR, iNOS and COX2). Our results show that the delay in P4 decline and parturition is the resultant of retarded luteal regression, caused by a combination of decreases in luteolytic factors, mainly luteal PGF2alpha, iNOS mRNA expression and also circulating LH, and increased synthesis or action of luteotrophic factors, such as luteal and circulating PGE2 and circulating PRL. All these changes may be direct causes of the decreased 20alphaHSD mRNA and protein (measured by western blot analysis) expression, which in the presence of unchanged expression of the factors involved in P4 synthesis results in elevated luteal and circulating P4 that prolonged pregnancy and also may favor longer survival of the corpus luteum. PMID:17244746

  19. Single-Prolonged Stress Induces Endoplasmic Reticulum - Dependent Apoptosis in the Hippocampus in a Rat Model of Post-Traumatic Stress Disorder

    PubMed Central

    Han, Fang; Yan, Shengnan; Shi, YuXiu

    2013-01-01

    Background Our previous research indicated that apoptosis induced atrophy in the hippocampus of post-traumatic stress disorder (PTSD) rats. Endoplasmic reticulum (ER) stress-induced apoptosis has been implicated in the development of several disorder diseases. The aim of this study was to investigate whether endoplasmic reticulum-related pathway is involved in single-prolonged stress (SPS) induces apoptosis in the hippocampus of PTSD rats by examining the expression levels of three important indicators in the ER-related apoptotic pathway: Glucose-regulated protein (GRP) 78, caspase-12 and Ca2+/CaM/CaMkinaseIIα (CaMkIIα). Methods Wistar rats were sacrificed at 1, 4 and 7 days after SPS. SPS is a reliable animal model of PTSD. The apoptotic cells in the hippocampus were assessed by TUNEL method and transmission electron microscopy (TEM). Free intracellular Ca2+ concentration was measured. GRP78 expression was examined by immunohistochemistry, western blotting and RT-PCR. mRNA of caspase-12 and CaM/CaMkIIα were determined by RT-PCR. Results Our results showed that apoptotic cells were increased in the SPS rats. TEM analysis revealed characteristic morphological changes of apoptosis in these cells. We observed that GRP78 was significantly up-regulated during early PTSD, and then recovered at 7 days after SPS. By RT-PCR, we observed that the change in caspase-12 expression level was similar to that in GRP78. Moreover, the free intracellular Ca2+ concentration was significantly higher at 1 day after SPS and decreased in 7 days. CaM expression increased significantly, while CaMKIIα expression decreased significantly in the hippocampus at 1 day after SPS. Conclusion SPS induced change in the expression levels of GRP78, caspase-12 and Ca2+/CaM/CaMkIIα in the hippocampus of PTSD rats indicated that the endoplasmic reticulum pathway may be involved in PTSD-induced apoptosis. PMID:23894451

  20. The Camperdown Program: Outcomes of a New Prolonged-Speech Treatment Model.

    ERIC Educational Resources Information Center

    O'Brian, Sue; Onslow, Mark; Cream, Angela; Packman, Ann

    2003-01-01

    This paper examines a prolonged speech treatment model for stuttering, the Camperdown Program. Sixteen participants showed minimal or no stuttering in everyday speaking situations for up to 12 months after entering the program's maintenance phase, with speech rates in the normal range. Results were achieved in a mean of 20 hours of clinic…

  1. Prolonged Exposure Treatment of Chronic PTSD in Juvenile Sex Offenders: Promising Results from Two Case Studies

    ERIC Educational Resources Information Center

    Hunter, John A.

    2010-01-01

    Prolonged exposure (PE) was used to treat chronic PTSD secondary to severe developmental trauma in two adolescent male sex offenders referred for residential sex offender treatment. Both youth were treatment resistant prior to initiation of PE and showed evidence of long-standing irritability and depression/anxiety. Clinical observation and…

  2. Shensong Yangxin capsules prevent ischemic arrhythmias by prolonging action potentials and alleviating Ca2+ overload.

    PubMed

    Zhao, Yixiu; Gao, Feng; Zhang, Yong; Wang, Hongtao; Zhu, Jiuxin; Chang, Liping; Du, Zhimin; Zhang, Yan

    2016-06-01

    Shensong Yangxin capsules (SSYX) are an effective traditional Chinese medicine that has been used to treat coronary heart disease clinically. The present study aimed to establish whether SSYX prevent ischemic arrhythmias in rats, and to explore the underlying mechanisms. Male rats were pretreated with distilled water, SSYX and amiodarone for one week. Acute myocardial ischemia (AMI) was performed to induce ischemic arrhythmias. The incidence and severity of ischemic arrhythmias were evaluated. The action potential, transient outward K+ current (Ito) and inward rectifier K+ current (IK1) of rat cardiomyocytes were measured using the patch‑clamp technique. The intracellular Ca2+ concentration of the cardiomyocytes was measured using a laser scanning confocal microscope. The results revealed that SSYX lowered the incidence of arrhythmia markedly during AMI. Furthermore, SSYX delayed the appearance, and reduced the severity, of ischemic arrhythmias compared with the control. In addition, SSYX markedly decreased the ratio of the myocardial infarction region to the whole heart. In an in vitro study, SSYX prolonged the action potential duration of rat cardiomyocytes, and inhibited Ito and IK1 markedly. Additionally, SSYX inhibited Ca2+ elevation induced by KCl in cardiomyocytes. These results suggested that SSYX prevents ischemic arrhythmia, and the underlying mechanism responsible for this process may include prolonging the action potential and alleviating Ca2+ overload. PMID:27122298

  3. Secretory vesicle rebound hyperacidification and increased quantal size due to prolonged methamphetamine exposure

    PubMed Central

    Markov, Dmitriy; Mosharov, Eugene V.; Setlik, Wanda; Gershon, Michael D.; Sulzer, David

    2009-01-01

    Acute exposure to amphetamines collapses secretory vesicle pH gradients, which increases cytosolic catecholamine levels while decreases the quantal size of catecholamine release during fusion events. Amphetamine and methamphetamine, however, are retained in tissues over long durations. We used optical and electron microscopic probes to measure the effects of long-term methamphetamine exposure on secretory vesicle pH, and amperometry and intracellular patch electrochemistry to observe the effects on neurosecretion and cytosolic catecholamines in cultured rat chromaffin cells. In contrast to acute methamphetamine effects, exposure to the drug for 6–48 h at 10 μM and higher concentrations produced a concentration-dependent rebound hyperacidification of secretory vesicles. At 5–10 μM levels, methamphetamine increased the quantal size and reinstated exocytotic catecholamine release, although very high (>100 μM) levels of the drug, while continuing to produce rebound hyperacidification, did not increase quantal size. Secretory vesicle rebound hyperacidification was temperature dependent with optimal response at ~ 37°C, was not blocked by the transcription inhibitor, puromycin, and appears to be a general compensatory response to prolonged exposure with membranophilic weak bases, including amphetamines, methylphenidate, cocaine, and ammonia. Thus, under some conditions of prolonged exposure, amphetamines and other weak bases can enhance, rather than deplete, the vesicular release of catecholamines via a compensatory response resulting in vesicle acidification. PMID:19014382

  4. Nanoparticle tumor localization, disruption of autophagosomal trafficking, and prolonged drug delivery improve survival in peritoneal mesothelioma.

    PubMed

    Liu, Rong; Colby, Aaron H; Gilmore, Denis; Schulz, Morgan; Zeng, Jialiu; Padera, Robert F; Shirihai, Orian; Grinstaff, Mark W; Colson, Yolonda L

    2016-09-01

    The treatment outcomes for malignant peritoneal mesothelioma are poor and associated with high co-morbidities due to suboptimal drug delivery. Thus, there is an unmet need for new approaches that concentrate drug at the tumor for a prolonged period of time yielding enhanced antitumor efficacy and improved metrics of treatment success. A paclitaxel-loaded pH-responsive expansile nanoparticle (PTX-eNP) system is described that addresses two unique challenges to improve the outcomes for peritoneal mesothelioma. First, following intraperitoneal administration, eNPs rapidly and specifically localize to tumors. The rate of eNP uptake by tumors is an order of magnitude faster than the rate of uptake in non-malignant cells; and, subsequent accumulation in autophagosomes and disruption of autophagosomal trafficking leads to prolonged intracellular retention of eNPs. The net effect of these combined mechanisms manifests as rapid localization to intraperitoneal tumors within 4 h of injection and persistent intratumoral retention for >14 days. Second, the high tumor-specificity of PTX-eNPs leads to delivery of greater than 100 times higher concentrations of drug in tumors compared to PTX alone and this is maintained for at least seven days following administration. As a result, overall survival of animals with established mesothelioma more than doubled when animals were treated with multiple doses of PTX-eNPs compared to equivalent dosing with PTX or non-responsive PTX-loaded nanoparticles. PMID:27343465

  5. Directed antigen delivery as a vaccine strategy for an intracellular bacterial pathogen

    NASA Astrophysics Data System (ADS)

    Bouwer, H. G. Archie; Alberti-Segui, Christine; Montfort, Megan J.; Berkowitz, Nathan D.; Higgins, Darren E.

    2006-03-01

    We have developed a vaccine strategy for generating an attenuated strain of an intracellular bacterial pathogen that, after uptake by professional antigen-presenting cells, does not replicate intracellularly and is readily killed. However, after degradation of the vaccine strain within the phagolysosome, target antigens are released into the cytosol for endogenous processing and presentation for stimulation of CD8+ effector T cells. Applying this strategy to the model intracellular pathogen Listeria monocytogenes, we show that an intracellular replication-deficient vaccine strain is cleared rapidly in normal and immunocompromised animals, yet antigen-specific CD8+ effector T cells are stimulated after immunization. Furthermore, animals immunized with the intracellular replication-deficient vaccine strain are resistant to lethal challenge with a virulent WT strain of L. monocytogenes. These studies suggest a general strategy for developing safe and effective, attenuated intracellular replication-deficient vaccine strains for stimulation of protective immune responses against intracellular bacterial pathogens. CD8+ T cell | replication-deficient | Listeria monocytogenes

  6. Evaluation of Intracellular Signaling Downstream Chimeric Antigen Receptors

    PubMed Central

    Karlsson, Hannah; Svensson, Emma; Gigg, Camilla; Jarvius, Malin; Olsson-Strömberg, Ulla; Savoldo, Barbara; Dotti, Gianpietro; Loskog, Angelica

    2015-01-01

    CD19-targeting CAR T cells have shown potency in clinical trials targeting B cell leukemia. Although mainly second generation (2G) CARs carrying CD28 or 4-1BB have been investigated in patients, preclinical studies suggest that third generation (3G) CARs with both CD28 and 4-1BB have enhanced capacity. However, little is known about the intracellular signaling pathways downstream of CARs. In the present work, we have analyzed the signaling capacity post antigen stimulation in both 2G and 3G CARs. 3G CAR T cells expanded better than 2G CAR T cells upon repeated stimulation with IL-2 and autologous B cells. An antigen-driven accumulation of CAR+ cells was evident post antigen stimulation. The cytotoxicity of both 2G and 3G CAR T cells was maintained by repeated stimulation. The phosphorylation status of intracellular signaling proteins post antigen stimulation showed that 3G CAR T cells had a higher activation status than 2G. Several proteins involved in signaling downstream the TCR were activated, as were proteins involved in the cell cycle, cell adhesion and exocytosis. In conclusion, 3G CAR T cells had a higher degree of intracellular signaling activity than 2G CARs which may explain the increased proliferative capacity seen in 3G CAR T cells. The study also indicates that there may be other signaling pathways to consider when designing or evaluating new generations of CARs. PMID:26700307

  7. Evaluation of Intracellular Signaling Downstream Chimeric Antigen Receptors.

    PubMed

    Karlsson, Hannah; Svensson, Emma; Gigg, Camilla; Jarvius, Malin; Olsson-Strömberg, Ulla; Savoldo, Barbara; Dotti, Gianpietro; Loskog, Angelica

    2015-01-01

    CD19-targeting CAR T cells have shown potency in clinical trials targeting B cell leukemia. Although mainly second generation (2G) CARs carrying CD28 or 4-1BB have been investigated in patients, preclinical studies suggest that third generation (3G) CARs with both CD28 and 4-1BB have enhanced capacity. However, little is known about the intracellular signaling pathways downstream of CARs. In the present work, we have analyzed the signaling capacity post antigen stimulation in both 2G and 3G CARs. 3G CAR T cells expanded better than 2G CAR T cells upon repeated stimulation with IL-2 and autologous B cells. An antigen-driven accumulation of CAR+ cells was evident post antigen stimulation. The cytotoxicity of both 2G and 3G CAR T cells was maintained by repeated stimulation. The phosphorylation status of intracellular signaling proteins post antigen stimulation showed that 3G CAR T cells had a higher activation status than 2G. Several proteins involved in signaling downstream the TCR were activated, as were proteins involved in the cell cycle, cell adhesion and exocytosis. In conclusion, 3G CAR T cells had a higher degree of intracellular signaling activity than 2G CARs which may explain the increased proliferative capacity seen in 3G CAR T cells. The study also indicates that there may be other signaling pathways to consider when designing or evaluating new generations of CARs. PMID:26700307

  8. Effect of ticlopidine ex vivo on platelet intracellular calcium mobilization

    SciTech Connect

    Derian, C.K.; Friedman, P.A.

    1988-04-01

    The antiplatelet compound ticlopidine exerts its potent inhibitory activity through an as yet undetermined mechanism(s). The goal of this study was to determine the effect, if any, of ticlopidine ex vivo on platelet calcium mobilization. Ticlopidine inhibited ADP-induced platelet aggregation by 50-80%. In the presence of 1 mM EGTA, ticlopidine inhibited ADP- and thrombin-stimulated increases in (Ca2+)i in fura-2 loaded platelets. We evaluated further the effect of ticlopidine on calcium mobilization by examining both agonist-stimulated formation of inositol trisphosphate in intact platelets and the ability of inositol trisphosphate to release /sup 45/Ca from intracellular sites in permeabilized cells. We show here that while ticlopidine significantly affected agonist-induced intracellular calcium mobilization in intact platelets, the drug was without effect on agonist-stimulated formation of inositol trisphosphate in intact platelets and on inositol trisphosphate-induced /sup 45/Ca release in saponin-permeabilized platelets. Our study demonstrates that ticlopidine exerts at least part of its effect via inhibition of intracellular calcium mobilization but that its site of action remains to be determined.

  9. Gamma Band Activity in the RAS-intracellular mechanisms

    PubMed Central

    Garcia-Rill, E.; Kezunovic, N.; D’Onofrio, S.; Luster, B.; Hyde, J.; Bisagno, V.; Urbano, F.J.

    2014-01-01

    Gamma band activity participates in sensory perception, problem solving, and memory. This review considers recent evidence showing that cells in the reticular activating system (RAS) exhibit gamma band activity, and describes the intrinsic membrane properties behind such manifestation. Specifically, we discuss how cells in the mesopontine pedunculopontine nucleus (PPN), intralaminar parafascicular nucleus (Pf), and pontine Subcoeruleus nucleus dorsalis (SubCD) all fire in the gamma band range when maximally activated, but no higher. The mechanisms involve high threshold, voltage-dependent P/Q-type calcium channels or sodium-dependent subthreshold oscillations. Rather than participating in the temporal binding of sensory events as in the cortex, gamma band activity in the RAS may participate in the processes of preconscious awareness, and provide the essential stream of information for the formulation of many of our actions. We address three necessary next steps resulting from these discoveries, an intracellular mechanism responsible for maintaining gamma band activity based on persistent G-protein activation, separate intracellular pathways that differentiate between gamma band activity during waking vs during REM sleep, and an intracellular mechanism responsible for the dysregulation in gamma band activity in schizophrenia. These findings open several promising research avenues that have not been thoroughly explored. What are the effects of sleep or REM sleep deprivation on these RAS mechanisms? Are these mechanisms involved in memory processing during waking and/or during REM sleep? Does gamma band processing differ during waking vs REM sleep after sleep or REM sleep deprivation? PMID:24309750

  10. Intracellular replication is essential for the virulence of Salmonella typhimurium.

    PubMed

    Leung, K Y; Finlay, B B

    1991-12-15

    Salmonella typhimurium is a facultative intracellular parasite, capable of penetrating, surviving, and multiplying within diverse eukaryotic cell types, including epithelial and phagocytic cells. We have been studying intracellular replication of S. typhimurium and found that it is essential in the pathogenesis of this bacterium. A total of 45,000 independent mini-Mu MudJ transposon mutants in S. typhimurium SL1344 were screened in Madin-Darby canine kidney (MDCK) epithelial cells with a beta-lactam, cefotaxime, to enrich for mutants defective for intracellular replication. Ten different auxotrophic (purine, pyrimidine, purine/methionine, and valine/isoleucine) and three prototrophic replication-defective mutants (Rep-) were identified. All Rep- mutants showed no differences in aerobic and anaerobic growth patterns, motility, serum sensitivity, mouse macrophage survival, iron uptake, and phosphate requirements. All Rep- mutants were unable to multiply inside MDCK, HeLa, and Caco-2 epithelial cells. When required nutrients for various auxotrophs were supplemented, auxotrophs then replicated inside MDCK cells. Although the parental strain multiplies in large vacuoles inside MDCK cells that distort the host cells, MDCK cells infected with the Rep- mutants appeared relatively normal and few bacteria were seen inside vacuoles. The purine auxotrophs and the three prototrophic Rep- mutants were highly attenuated in mice, and oral and intraperitoneal LD50 levels were 3 to 4 orders of magnitude higher than the wild type level. The three prototrophs were invasive and persisted in the murine organs such as livers and spleens for at least 3 weeks. Therefore, these prototrophic genes are needed for intracellular replication and are essential to the virulence of S. typhimurium. PMID:1763061

  11. Evaluation of two novel methods for assessing intracellular oxygen

    NASA Astrophysics Data System (ADS)

    Williams, Catrin F.; Kombrabail, M.; Vijayalakshmi, K.; White, Nick; Krishnamoorthy, G.; Lloyd, David

    2012-08-01

    The ability to resolve the spatio-temporal complexity of intracellular O2 distribution is the ‘Holy Grail’ of cellular physiology. In an effort to obtain a non-invasive approach of mapping intracellular O2 tensions, two methods of phosphorescent lifetime imaging microscopy were examined in the current study. These were picosecond time-resolved epiphosphorescence microscopy (single 0.5 µm focused spot) and two-photon confocal laser scanning microscopy with pinhole shifting. Both methods utilized nanoparticle-embedded Ru complex (45 nm diameter) as the phosphorescent probe, excited using pulsed outputs of Ti-sapphire Tsunami lasers (710-1050 nm). The former method used a 1 ps pulse width excitation beam with vertical polarization via a dichroic mirror (610 nm, XF43) and a 20× objective (NA 0.55, Nikon). Transmitted luminescence (1-2 × 104 counts s-1) was collected and time-correlated single photon counted decay times measured. Alternatively, an unmodified Zeiss LSM510 Confocal NLO microscope with 40× objective (NA 1.3) used successively shifted pinhole positions to collect image data from the lagging trail of the raster scan. Images obtained from two-photon excitation of a yeast (Schizosaccharomyces pombe) and a flagellate fish parasite (Spironucleus vortens), electroporated with Ru complex, indicated the intracellular location and magnitude of O2 gradients, thus confirming the feasibility of optical mapping under different external O2 concentrations. Both methods gave similar lifetimes for Ru complex phosphorescence under aerobic and anaerobic gas phases. Estimation of O2 tensions within individual fibroblasts (human dermal fibroblast (HDF)) and mammary adenocarcinoma (MCF-7) cells was possible using epiphosphorescence microscopy. MCF-7 cells showed lower intracellular O2 concentrations than HDF cells, possibly due to higher metabolic rates in the former. Future work should involve construction of higher resolution 3D maps of Ru coordinate complex lifetime

  12. Membranes, mechanics, and intracellular transport

    NASA Astrophysics Data System (ADS)

    Parthasarathy, Raghuveer

    2012-10-01

    Cellular membranes are remarkable materials -- self-assembled, flexible, two-dimensional fluids. Understanding how proteins manipulate membrane curvature is crucial to understanding the transport of cargo in cells, yet the mechanical activities of trafficking proteins remain poorly understood. Using an optical-trap based assay involving dynamic deformation of biomimetic membranes, we have examined the behavior of Sar1, a key component of the COPII family of transport proteins. We find that Sar1 from yeast (S. cerevisiae) lowers membrane rigidity by up to 100% as a function of its concentration, thereby lowering the energetic cost of membrane deformation. Human Sar1 proteins can also lower the mechanical rigidity of the membranes to which they bind. However, unlike the yeast proteins, the rigidity is not a monotonically decreasing function of concentration but rather shows increased rigidity and decreased mobility at high concentrations that implies interactions between proteins. In addition to describing this study of membrane mechanics, I'll also discuss some topics relevant to a range of biophysical investigations, such as the insights provided by imaging methods and open questions in the dynamics of multicellular systems.

  13. Prediction of Prolonged Hemodynamic Instability During Carotid Angioplasty and Stenting

    PubMed Central

    Rhim, Jong Kook; Park, Jeong Jin; Choi, Hyuk Jai; Cho, Young Dae; Sheen, Seung Hun; Jang, Kyung-Sool

    2016-01-01

    Purpose The aim of this study was to assess the risk factors of prolonged hemodynamic instability (HDI) after carotid angioplasty and stenting (CAS). Herein, a simplified predictive scoring system for prolonged HDI is proposed. Materials and Methods Sixty-six patients who had CAS from 2011 to 2016 at a single institution were evaluated. Prolonged HDI was defined as systolic blood pressure >160 mm Hg or <90 mm Hg or heart rate <50 beats/min, lasting over 30 minutes despite medical treatments. For the study, clinical data and radiologic data, including plaque morphology and stenosis were analyzed. Results Prolonged HDI was observed in 21 patients (31.8%). Multivariable analysis revealed that calcification (OR, 6.726; p=0.006), eccentric stenosis (OR, 3.645; p=0.047) and extensive plaque distribution (OR, 7.169; p=0.006) were related to prolonged HDI. According to these results, a simplified scoring scale was proposed based on the summation of points: 2 points for calcified plaque, 2 points for extensive plaque distribution, and 1 point for eccentric stenosis. The percentages of prolonged HDI according to the total score were as follows: score 0, 8.7%; score 1, 20.0%; score 2, 38.5%; score 3, 72.7%; score 4, 66.7%; score 5, 100%. From the analysis, the total score in patients with prolonged HDI was significantly higher than those without prolonged HDI (p<0.001). Conclusion Prolonged HDI can be associated with calcification of plaque, eccentric stenosis and extensive plaque distribution, and a simplified scoring system enables prediction of prolonged HDI according to our cohort. PMID:27621949

  14. Intracellular sodium homeostasis in rat hippocampal astrocytes.

    PubMed Central

    Rose, C R; Ransom, B R

    1996-01-01

    1. We determined the intracellular Na+ concentration ([Na+]i) and mechanisms of its regulation in cultured rat hippocampal astrocytes using fluorescence ratio imaging of the Na+ indicator SBFI-AM (acetoxymethylester of sodium-binding benzofuran isophthalate, 10 microM). Dye signal calibration within the astrocytes showed that the ratiometric dye signal changed monotonically with changes in [Na+]i from 0 to 140 nM. The K+ sensitivity of the dye was negligible; intracellular pH changes, however, slightly affected the 'Na+' signal. 2. Baseline [Na+]i was 14.6 +/- 4.9 mM (mean +/- S.D.) in CO2/HCO3(-)-containing saline with 3 mM K+. Removal of extracellular Na+ decreased [Na+]i in two phases: a rapid phase of [Na+]i reduction (0.58 +/- 0.32 mM min-1) followed by a slower phase (0.15 +/- 0.09 mM min-1). 3. Changing from CO2/HCO3(-)-free to CO2/HCO3(-)-buffered saline resulted in a transient increase in [Na+]i of approximately 5 mM, suggesting activation of inward Na(+)-HCO3- cotransport by CO2/HCO3-. During furosemide (frusemide, 1 mM) or bumetanide (50 microM) application, a slow decrease in [Na+]i of approximately 2 mM was observed, indicating a steady inward transport of Na+ via Na(+)-K(+)-2Cl- cotransport under control conditions. Tetrodotoxin (100 microM) did not influence [Na+]i in the majority of cells (85%), suggesting that influx of Na+ through voltage-gated Na+ channels contributed to baseline [Na+]i in only a small subpopulation of hippocampal astrocytes. 4. Blocking Na+, K(+)-ATPase activity with cardiac glycosides (ouabain or strophanthidin, 1 mM) or removal of extracellular K+ led to an increase in [Na+]i of about 2 and 4 mM min-1, respectively. This indicated that Na+, K(+)-ATPase activity was critical in maintaining low [Na+]i in the face of a steep electrochemical gradient, which would favour a much higher [Na+]i. 5. Elevation of extracellular K+ concentration ([K+]o) by as little as 1 mM (from 3 to 4 mM) resulted in a rapid and reversible decrease in

  15. Nanoscale-Tipped High-Aspect-Ratio Vertical Microneedle Electrodes for Intracellular Recordings.

    PubMed

    Kubota, Yoshihiro; Oi, Hideo; Sawahata, Hirohito; Goryu, Akihiro; Ando, Yoriko; Numano, Rika; Ishida, Makoto; Kawano, Takeshi

    2016-06-01

    Intracellular recording nanoscale electrode devices provide the advantages of a high spatial resolution and high sensitivity. However, the length of nanowire/nanotube-based nanoelectrodes is currently limited to <10 μm long due to fabrication issues for high-aspect-ratio nanoelectrodes. The concept reported here can address the technological limitations by fabricating >100 μm long nanoscale-tipped electrodes, which show intracellular recording capability. PMID:27062044

  16. Histoplasma capsulatum surmounts obstacles to intracellular pathogenesis.

    PubMed

    Garfoot, Andrew L; Rappleye, Chad A

    2016-02-01

    The fungal pathogen Histoplasma capsulatum causes respiratory and disseminated disease, even in immunocompetent hosts. In contrast to opportunistic pathogens, which are readily controlled by phagocytic cells, H. capsulatum yeasts are able to infect macrophages, survive antimicrobial defenses, and proliferate as an intracellular pathogen. In this review, we discuss some of the molecular mechanisms that enable H. capsulatum yeasts to overcome obstacles to intracellular pathogenesis. H. capsulatum yeasts gain refuge from extracellular obstacles such as antimicrobial lung surfactant proteins by engaging the β-integrin family of phagocytic receptors to promote entry into macrophages. In addition, H. capsulatum yeasts conceal immunostimulatory β-glucans to avoid triggering signaling receptors such as the β-glucan receptor Dectin-1. H. capsulatum yeasts counteract phagocyte-produced reactive oxygen species by expression of oxidative stress defense enzymes including an extracellular superoxide dismutase and an extracellular catalase. Within the phagosome, H. capsulatum yeasts block phagosome acidification, acquire essential metals such as iron and zinc, and utilize de novo biosynthesis pathways to overcome nutritional limitations. These mechanisms explain how H. capsulatum yeasts avoid and negate macrophage defense strategies and establish a hospitable intracellular niche, making H. capsulatum a successful intracellular pathogen of macrophages. PMID:26235362

  17. KRIT1 Regulates the Homeostasis of Intracellular Reactive Oxygen Species

    PubMed Central

    Goitre, Luca; Balzac, Fiorella; Degani, Simona; Degan, Paolo; Marchi, Saverio; Pinton, Paolo; Retta, Saverio Francesco

    2010-01-01

    KRIT1 is a gene responsible for Cerebral Cavernous Malformations (CCM), a major cerebrovascular disease characterized by abnormally enlarged and leaky capillaries that predispose to seizures, focal neurological deficits, and fatal intracerebral hemorrhage. Comprehensive analysis of the KRIT1 gene in CCM patients has suggested that KRIT1 functions need to be severely impaired for pathogenesis. However, the molecular and cellular functions of KRIT1 as well as CCM pathogenesis mechanisms are still research challenges. We found that KRIT1 plays an important role in molecular mechanisms involved in the maintenance of the intracellular Reactive Oxygen Species (ROS) homeostasis to prevent oxidative cellular damage. In particular, we demonstrate that KRIT1 loss/down-regulation is associated with a significant increase in intracellular ROS levels. Conversely, ROS levels in KRIT1−/− cells are significantly and dose-dependently reduced after restoration of KRIT1 expression. Moreover, we show that the modulation of intracellular ROS levels by KRIT1 loss/restoration is strictly correlated with the modulation of the expression of the antioxidant protein SOD2 as well as of the transcriptional factor FoxO1, a master regulator of cell responses to oxidative stress and a modulator of SOD2 levels. Furthermore, we show that the KRIT1-dependent maintenance of low ROS levels facilitates the downregulation of cyclin D1 expression required for cell transition from proliferative growth to quiescence. Finally, we demonstrate that the enhanced ROS levels in KRIT1−/− cells are associated with an increased cell susceptibility to oxidative DNA damage and a marked induction of the DNA damage sensor and repair gene Gadd45α, as well as with a decline of mitochondrial energy metabolism. Taken together, our results point to a new model where KRIT1 limits the accumulation of intracellular oxidants and prevents oxidative stress-mediated cellular dysfunction and DNA damage by enhancing the

  18. Identifying the translational gap in the evaluation of drug-induced QTc interval prolongation

    PubMed Central

    Chain, Anne SY; Dubois, Vincent FS; Danhof, Meindert; Sturkenboom, Miriam CJM; Della Pasqua, Oscar

    2013-01-01

    Aims Given the similarities in QTc response between dogs and humans, dogs are used in pre-clinical cardiovascular safety studies. The objective of our investigation was to characterize the PKPD relationships and identify translational gaps across species following the administration of three compounds known to cause QTc interval prolongation, namely cisapride, d, l-sotalol and moxifloxacin. Methods Pharmacokinetic and pharmacodynamic data from experiments in conscious dogs and clinical trials were included in this analysis. First, pharmacokinetic modelling and deconvolution methods were applied to derive drug concentrations at the time of each QT measurement. A Bayesian PKPD model was then used to describe QT prolongation, allowing discrimination of drug-specific effects from other physiological factors known to alter QT interval duration. A threshold of ≥10 ms was used to explore the probability of prolongation after drug administration. Results A linear relationship was found to best describe the pro-arrhythmic effects of cisapride, d,l-sotalol and moxifloxacin both in dogs and in humans. The drug-specific parameter (slope) in dogs was statistically significantly different from humans. Despite such differences, our results show that the probability of QTc prolongation ≥10 ms in dogs nears 100% for all three compounds at the therapeutic exposure range in humans. Conclusions Our findings indicate that the slope of PKPD relationship in conscious dogs may be used as the basis for the prediction of drug-induced QTc prolongation in humans. Furthermore, the risk of QTc prolongation can be expressed in terms of the probability associated with an increase ≥10 ms, allowing direct inferences about the clinical relevance of the pro-arrhythmic potential of a molecule. PMID:23351036

  19. QTc interval prolongation and torsade de pointes associated with second-generation antipsychotics and antidepressants: a comprehensive review.

    PubMed

    Hasnain, Mehrul; Vieweg, W Victor R

    2014-10-01

    We comprehensively reviewed published literature to determine whether it supported the link between corrected QT (QTc) interval prolongation and torsade de pointes (TdP) for the 11 second-generation antipsychotics and seven second-generation antidepressants commonly implicated in these complications. Using PubMed and EMBASE, we identified four thorough QT studies (one each for iloperidone, ziprasidone, citalopram, and escitalopram), 40 studies specifically designed to assess QTc interval prolongation or TdP, 58 publications based on data from efficacy and safety trials, 18 toxicology studies, and 102 case reports. Thorough QT studies, QTc prolongation-specific studies, and studies based on efficacy and safety trials did not link drug-associated QTc interval prolongation with TdP. They only showed that the drugs reviewed caused varying degrees of QTc interval prolongation, and even that information was not clear and consistent enough to stratify individual drugs for this risk. The few toxicology studies provided valuable information but their findings are pertinent only to situations of drug overdose. Case reports were most informative about the drug-QTc interval prolongation-TdP link. At least one additional well established risk factor for QTc prolongation was present in 92.2 % of case reports. Of the 28 cases of TdP, six (21.4 %) experienced it with QTc interval <500 ms; 75 % of TdP cases occurred at therapeutic doses. There is little evidence that drug-associated QTc interval prolongation by itself is sufficient to predict TdP. Future research needs to improve its precision and broaden its scope to better understand the factors that facilitate or attenuate progression of drug-associated QTc interval prolongation to TdP. PMID:25168784

  20. Intracellular NAD+ depletion enhances bortezomib-induced anti-myeloma activity

    PubMed Central

    Cagnetta, Antonia; Calimeri, Teresa; Acharya, Chirag; Fulciniti, Mariateresa; Tai, Yu-Tzu; Hideshima, Teru; Chauhan, Dharminder; Zhong, Mike Y.; Patrone, Franco; Nencioni, Alessio; Gobbi, Marco; Richardson, Paul; Munshi, Nikhil

    2013-01-01

    We recently demonstrated that Nicotinamide phosphoribosyltransferase (Nampt) inhibition depletes intracellular NAD+ content leading, to autophagic multiple myeloma (MM) cell death. Bortezomib has remarkably improved MM patient outcome, but dose-limiting toxicities and development of resistance limit its long-term utility. Here we observed higher Nampt messenger RNA levels in bortezomib-resistant patient MM cells, which correlated with decreased overall survival. We demonstrated that combining the NAD+ depleting agent FK866 with bortezomib induces synergistic anti-MM cell death and overcomes bortezomib resistance. This effect is associated with (1) activation of caspase-8, caspase-9, caspase-3, poly (ADP-ribose) polymerase, and downregulation of Mcl-1; (2) enhanced intracellular NAD+ depletion; (3) inhibition of chymotrypsin-like, caspase-like, and trypsin-like proteasome activities; (4) inhibition of nuclear factor κB signaling; and (5) inhibition of angiogenesis. Furthermore, Nampt knockdown significantly enhances the anti-MM effect of bortezomib, which can be rescued by ectopically overexpressing Nampt. In a murine xenograft MM model, low-dose combination FK866 and Bortezomib is well tolerated, significantly inhibits tumor growth, and prolongs host survival. Taken together, these findings indicate that intracellular NAD+ level represents a major determinant in the ability of bortezomib to induce apoptosis in MM cells and provide proof of concept for the combination with FK866 as a new strategy to enhance sensitivity or overcome resistance to bortezomib. PMID:23823317

  1. Intracellular Retention of ABL Kinase Inhibitors Determines Commitment to Apoptosis in CML Cells

    PubMed Central

    Dziadosz, Marek; Schnöder, Tina; Heidel, Florian; Schemionek, Mirle; Melo, Junia V.; Kindler, Thomas; Müller-Tidow, Carsten; Koschmieder, Steffen; Fischer, Thomas

    2012-01-01

    Clinical development of imatinib in CML established continuous target inhibition as a paradigm for successful tyrosine kinase inhibitor (TKI) therapy. However, recent reports suggested that transient potent target inhibition of BCR-ABL by high-dose TKI (HD-TKI) pulse-exposure is sufficient to irreversibly commit cells to apoptosis. Here, we report a novel mechanism of prolonged intracellular TKI activity upon HD-TKI pulse-exposure (imatinib, dasatinib) in BCR-ABL-positive cells. Comprehensive mechanistic exploration revealed dramatic intracellular accumulation of TKIs which closely correlated with induction of apoptosis. Cells were rescued from apoptosis upon HD-TKI pulse either by repetitive drug wash-out or by overexpression of ABC-family drug transporters. Inhibition of ABCB1 restored sensitivity to HD-TKI pulse-exposure. Thus, our data provide evidence that intracellular drug retention crucially determines biological activity of imatinib and dasatinib. These studies may refine our current thinking on critical requirements of TKI dose and duration of target inhibition for biological activity of TKIs. PMID:22815843

  2. Prostaglandin E2 inhibits apoptosis in human neutrophilic polymorphonuclear leukocytes: role of intracellular cyclic AMP levels.

    PubMed

    Ottonello, L; Gonella, R; Dapino, P; Sacchetti, C; Dallegri, F

    1998-08-01

    Human neutrophilic polymorphonuclear leukocytes (neutrophils) are terminally differentiated cells that die by undergoing apoptosis. At present, the intracellular pathways governing this process are only partially known. In particular, although the adenylate cyclase-dependent generation of cyclic AMP (cAMP) has been implicated in the triggering of apoptosis in lymphoid cells, the role of the intracellular cAMP pathway in neutrophil apoptosis remains controversial. In the present study, we found that two cAMP-elevating agents, prostaglandin E2 (PGE2) and the phosphodiesterase type IV inhibitor RO 20-1724, inhibit neutrophil apoptosis without inducing cell necrosis. When administered in combination, PGE2 and RO 20-1724 displayed additive effects. Moreover, neutrophil apoptosis was inhibited by a membrane-permeable analog of cAMP, dibutyryl-cAMP, in a dose-dependent manner. Finally, treatment of neutrophils with the protein kinase A inhibitor H-89 prevented PGE2- and RO 20-1724-induced inhibition of cell apoptosis. In conclusion, taking into account that PGE2 and other cAMP-elevating agents are well known downregulators of neutrophil functions, our results suggest that conditions favoring a state of functional rest, such as intracellular cAMP elevation, prolong the life span of neutrophils by delaying apoptosis. PMID:9694511

  3. Intracellular NAD⁺ depletion enhances bortezomib-induced anti-myeloma activity.

    PubMed

    Cagnetta, Antonia; Cea, Michele; Calimeri, Teresa; Acharya, Chirag; Fulciniti, Mariateresa; Tai, Yu-Tzu; Hideshima, Teru; Chauhan, Dharminder; Zhong, Mike Y; Patrone, Franco; Nencioni, Alessio; Gobbi, Marco; Richardson, Paul; Munshi, Nikhil; Anderson, Kenneth C

    2013-08-15

    We recently demonstrated that Nicotinamide phosphoribosyltransferase (Nampt) inhibition depletes intracellular NAD⁺ content leading, to autophagic multiple myeloma (MM) cell death. Bortezomib has remarkably improved MM patient outcome, but dose-limiting toxicities and development of resistance limit its long-term utility. Here we observed higher Nampt messenger RNA levels in bortezomib-resistant patient MM cells, which correlated with decreased overall survival. We demonstrated that combining the NAD⁺ depleting agent FK866 with bortezomib induces synergistic anti-MM cell death and overcomes bortezomib resistance. This effect is associated with (1) activation of caspase-8, caspase-9, caspase-3, poly (ADP-ribose) polymerase, and downregulation of Mcl-1; (2) enhanced intracellular NAD⁺ depletion; (3) inhibition of chymotrypsin-like, caspase-like, and trypsin-like proteasome activities; (4) inhibition of nuclear factor κB signaling; and (5) inhibition of angiogenesis. Furthermore, Nampt knockdown significantly enhances the anti-MM effect of bortezomib, which can be rescued by ectopically overexpressing Nampt. In a murine xenograft MM model, low-dose combination FK866 and Bortezomib is well tolerated, significantly inhibits tumor growth, and prolongs host survival. Taken together, these findings indicate that intracellular NAD⁺ level represents a major determinant in the ability of bortezomib to induce apoptosis in MM cells and provide proof of concept for the combination with FK866 as a new strategy to enhance sensitivity or overcome resistance to bortezomib. PMID:23823317

  4. Intra-ChIP: studying gene regulation in an intracellular pathogen.

    PubMed

    Hanson, Brett R; Tan, Ming

    2016-08-01

    Intracellular bacteria that reside within a host cell use a variety of strategies to exploit this unique niche. While these organisms are technically challenging to study in the context of an infected host cell, recent advances have led to an improved understanding of how the intracellular environment impacts bacterial gene expression. We recently demonstrated that chromatin immunoprecipitation (ChIP) can be used to quantify transcription factor binding in the obligate intracellular pathogen Chlamydia trachomatis within infected cells. Furthermore, we showed it was possible to experimentally modulate transcription factor binding while simultaneously measuring changes in transcription. Here we discuss these findings as well as other recent work that has used ChIP to study intracellular pathogens within infected cells. We also discuss technical considerations associated with this approach and its possible future applications. PMID:26886234

  5. Focal inhibitory seizure with prolonged deficit in adult Sturge-Weber syndrome.

    PubMed

    Aupy, Jerome; Bonnet, Charlotte; Arnould, Jean-Simon; Fernandez, Philippe; Marchal, Cecile; Zanotti-Fregonara, Paolo

    2015-09-01

    Sturge-Weber syndrome is a sporadic congenital neurocutaneous disorder often related to varying degrees of motor impairment. The phenomenon of prolonged ictal paresis is a rare seizure sign that can be due to lesions affecting the centro-parietal lobe. Focal inhibitory motor seizures can be difficult to differentiate from other clinical entities such as stroke, migraine or postictal paresis. We describe the case of a 40-year-old patient suffering from Sturge-Weber syndrome, admitted due to prolonged right-sided hemiparesis following a usual seizure. Repeated EEGs during the prolonged deficit showed only intermittent left fronto-parietal sharp waves. (99m)Tc HMPAO-brain SPECT performed seven days after the last seizure showed a vast area of parieto-occipital hyperperfusion in the left hemisphere. Aggressive antiepileptic therapy dramatically improved the clinical symptoms and scintigraphic images, which corroborated the diagnosis of ictal paresis. This case highlights the role of SPECT in the evaluation of Sturge-Weber syndrome, not only to investigate progressive neurological deterioration, but also exacerbation of seizures or prolonged neurological deficits. In fact, it may be possible to document ongoing epileptic activity using SPECT, despite a non-contributory EEG, which may be of help in adapting a therapeutic strategy. PMID:26235101

  6. Translation control during prolonged mTORC1 inhibition mediated by 4E-BP3.

    PubMed

    Tsukumo, Yoshinori; Alain, Tommy; Fonseca, Bruno D; Nadon, Robert; Sonenberg, Nahum

    2016-01-01

    Targeting mTORC1 is a highly promising strategy in cancer therapy. Suppression of mTORC1 activity leads to rapid dephosphorylation of eIF4E-binding proteins (4E-BP1-3) and subsequent inhibition of mRNA translation. However, how the different 4E-BPs affect translation during prolonged use of mTOR inhibitors is not known. Here we show that the expression of 4E-BP3, but not that of 4E-BP1 or 4E-BP2, is transcriptionally induced during prolonged mTORC1 inhibition in vitro and in vivo. Mechanistically, our data reveal that 4E-BP3 expression is controlled by the transcription factor TFE3 through a cis-regulatory element in the EIF4EBP3 gene promoter. CRISPR/Cas9-mediated EIF4EBP3 gene disruption in human cancer cells mitigated the inhibition of translation and proliferation caused by prolonged treatment with mTOR inhibitors. Our findings show that 4E-BP3 is an important effector of mTORC1 and a robust predictive biomarker of therapeutic response to prolonged treatment with mTOR-targeting drugs in cancer. PMID:27319316

  7. Translation control during prolonged mTORC1 inhibition mediated by 4E-BP3

    PubMed Central

    Tsukumo, Yoshinori; Alain, Tommy; Fonseca, Bruno D.; Nadon, Robert; Sonenberg, Nahum

    2016-01-01

    Targeting mTORC1 is a highly promising strategy in cancer therapy. Suppression of mTORC1 activity leads to rapid dephosphorylation of eIF4E-binding proteins (4E-BP1–3) and subsequent inhibition of mRNA translation. However, how the different 4E-BPs affect translation during prolonged use of mTOR inhibitors is not known. Here we show that the expression of 4E-BP3, but not that of 4E-BP1 or 4E-BP2, is transcriptionally induced during prolonged mTORC1 inhibition in vitro and in vivo. Mechanistically, our data reveal that 4E-BP3 expression is controlled by the transcription factor TFE3 through a cis-regulatory element in the EIF4EBP3 gene promoter. CRISPR/Cas9-mediated EIF4EBP3 gene disruption in human cancer cells mitigated the inhibition of translation and proliferation caused by prolonged treatment with mTOR inhibitors. Our findings show that 4E-BP3 is an important effector of mTORC1 and a robust predictive biomarker of therapeutic response to prolonged treatment with mTOR-targeting drugs in cancer. PMID:27319316

  8. Modulation of iron metabolism by iron chelation regulates intracellular calcium and increases sensitivity to doxorubicin

    PubMed Central

    Yalcintepe, Leman; Halis, Emre

    2016-01-01

    Increased intracellular iron levels can both promote cell proliferation and death, as such; iron has a “two-sided effect” in the delicate balance of human health. Though the role of iron in the development of cancer remains unclear, investigations of iron chelators as anti-tumor agents have revealed promising results. Here, we investigated the influence of iron and desferrioxamine (DFO), the iron chelating agent on intracellular calcium in a human leukemia cell line, K562. Iron uptake is associated with increased reactive oxygen species (ROS) generation. Therefore, we showed that iron also caused dose-dependent ROS generation in K562 cells. The measurement of intracellular calcium was determined using Furo-2 with a fluorescence spectrophotometer. The iron delivery process to the cytoplasmic iron pool was examined by monitoring the fluorescence of cells loaded with calcein-acetoxymethyl. Our data showed that iron increased intracellular calcium, and this response was 8 times higher when cells were incubated with DFO. K562 cells with DFO caused a 3.5 times increase of intracellular calcium in the presence of doxorubicin (DOX). In conclusion, DFO induces intracellular calcium and increases their sensitivity to DOX, a chemotherapeutic agent. PMID:26773173

  9. Probing the metabolic water contribution to intracellular water using oxygen isotope ratios of PO4

    NASA Astrophysics Data System (ADS)

    Li, Hui; Yu, Chan; Wang, Fei; Chang, Sae Jung; Yao, Jun; Blake, Ruth E.

    2016-05-01

    Knowledge of the relative contributions of different water sources to intracellular fluids and body water is important for many fields of study, ranging from animal physiology to paleoclimate. The intracellular fluid environment of cells is challenging to study due to the difficulties of accessing and sampling the contents of intact cells. Previous studies of multicelled organisms, mostly mammals, have estimated body water composition—including metabolic water produced as a byproduct of metabolism—based on indirect measurements of fluids averaged over the whole organism (e.g., blood) combined with modeling calculations. In microbial cells and aquatic organisms, metabolic water is not generally considered to be a significant component of intracellular water, due to the assumed unimpeded diffusion of water across cell membranes. Here we show that the 18O/16O ratio of PO4 in intracellular biomolecules (e.g., DNA) directly reflects the O isotopic composition of intracellular water and thus may serve as a probe allowing direct sampling of the intracellular environment. We present two independent lines of evidence showing a significant contribution of metabolic water to the intracellular water of three environmentally diverse strains of bacteria. Our results indicate that ˜30–40% of O in PO4 comprising DNA/biomass in early stationary phase cells is derived from metabolic water, which bolsters previous results and also further suggests a constant metabolic water value for cells grown under similar conditions. These results suggest that previous studies assuming identical isotopic compositions for intracellular/extracellular water may need to be reconsidered.

  10. Probing the metabolic water contribution to intracellular water using oxygen isotope ratios of PO4.

    PubMed

    Li, Hui; Yu, Chan; Wang, Fei; Chang, Sae Jung; Yao, Jun; Blake, Ruth E

    2016-05-24

    Knowledge of the relative contributions of different water sources to intracellular fluids and body water is important for many fields of study, ranging from animal physiology to paleoclimate. The intracellular fluid environment of cells is challenging to study due to the difficulties of accessing and sampling the contents of intact cells. Previous studies of multicelled organisms, mostly mammals, have estimated body water composition-including metabolic water produced as a byproduct of metabolism-based on indirect measurements of fluids averaged over the whole organism (e.g., blood) combined with modeling calculations. In microbial cells and aquatic organisms, metabolic water is not generally considered to be a significant component of intracellular water, due to the assumed unimpeded diffusion of water across cell membranes. Here we show that the (18)O/(16)O ratio of PO4 in intracellular biomolecules (e.g., DNA) directly reflects the O isotopic composition of intracellular water and thus may serve as a probe allowing direct sampling of the intracellular environment. We present two independent lines of evidence showing a significant contribution of metabolic water to the intracellular water of three environmentally diverse strains of bacteria. Our results indicate that ∼30-40% of O in PO4 comprising DNA/biomass in early stationary phase cells is derived from metabolic water, which bolsters previous results and also further suggests a constant metabolic water value for cells grown under similar conditions. These results suggest that previous studies assuming identical isotopic compositions for intracellular/extracellular water may need to be reconsidered. PMID:27170190

  11. Intracellular energetic units in red muscle cells.

    PubMed Central

    Saks, V A; Kaambre, T; Sikk, P; Eimre, M; Orlova, E; Paju, K; Piirsoo, A; Appaix, F; Kay, L; Regitz-Zagrosek, V; Fleck, E; Seppet, E

    2001-01-01

    The kinetics of regulation of mitochondrial respiration by endogenous and exogenous ADP in muscle cells in situ was studied in skinned cardiac and skeletal muscle fibres. Endogenous ADP production was initiated by addition of MgATP; under these conditions the respiration rate and ADP concentration in the medium were dependent on the calcium concentration, and 70-80% of maximal rate of respiration was achieved at ADP concentration below 20 microM in the medium. In contrast, when exogenous ADP was added, maximal respiration rate was observed only at millimolar concentrations. An exogenous ADP-consuming system consisting of pyruvate kinase (PK; 20-40 units/ml) and phosphoenolpyruvate (PEP; 5 mM), totally suppressed respiration activated by exogenous ADP, but the respiration maintained by endogenous ADP was not suppressed by more than 20-40%. Creatine (20 mM) further activated respiration in the presence of ATP and PK+PEP. Short treatment with trypsin (50-500 nM for 5 min) decreased the apparent K(m) for exogenous ADP from 300-350 microM to 50-60 microM, increased inhibition of respiration by PK+PEP system up to 70-80%, with no changes in MgATPase activity and maximal respiration rates. Electron-microscopic observations showed detachment of mitochondria and disordering of the regular structure of the sarcomere after trypsin treatment. Two-dimensional electrophoresis revealed a group of at least seven low-molecular-mass proteins in cardiac skinned fibres which were very sensitive to trypsin and not present in glycolytic fibres, which have low apparent K(m) for exogenous ADP. It is concluded that, in oxidative muscle cells, mitochondria are incorporated into functional complexes ('intracellular energetic units') with adjacent ADP-producing systems in myofibrils and in sarcoplasmic reticulum, probably due to specific interaction with cytoskeletal elements responsible for mitochondrial distribution in the cell. It is suggested that these complexes represent the basic

  12. Persistently Active Microbial Molecules Prolong Innate Immune Tolerance In Vivo

    PubMed Central

    Lu, Mingfang; Varley, Alan W.; Munford, Robert S.

    2013-01-01

    Measures that bolster the resolution phase of infectious diseases may offer new opportunities for improving outcome. Here we show that inactivation of microbial lipopolysaccharides (LPS) can be required for animals to recover from the innate immune tolerance that follows exposure to Gram-negative bacteria. When wildtype mice are exposed to small parenteral doses of LPS or Gram-negative bacteria, their macrophages become reprogrammed (tolerant) for a few days before they resume normal function. Mice that are unable to inactivate LPS, in contrast, remain tolerant for several months; during this time they respond sluggishly to Gram-negative bacterial challenge, with high mortality. We show here that prolonged macrophage reprogramming is maintained in vivo by the persistence of stimulatory LPS molecules within the cells' in vivo environment, where naïve cells can acquire LPS via cell-cell contact or from the extracellular fluid. The findings provide strong evidence that inactivation of a stimulatory microbial molecule can be required for animals to regain immune homeostasis following parenteral exposure to bacteria. Measures that disable microbial molecules might enhance resolution of tissue inflammation and help restore innate defenses in individuals recovering from many different infectious diseases. PMID:23675296

  13. The meteoric rise of regulated intracellular proteolysis.

    PubMed

    Mayer, R J

    2000-11-01

    It is often the case in biology that research into breaking things down lags behind research into synthesizing them, and this is certainly true for intracellular proteolysis. Now that we recognize that intracellular proteolysis, triggered by attaching multiple copies of a small protein called ubiquitin to target proteins, is fundamental to life, it is hard to believe that 20 years ago this field was little more than a backwater of biochemistry studied by a handful of laboratories. Among the few were Avram Hershko, Aaron Ciechanover and Alexander Varshavsky, who were recently awarded the Albert Lasker award for basic medical research for discovering the importance of protein degradation in cellular physiology. This Timeline traces how they and their collaborators triggered the rapid movement of ubiquitin-mediated proteolysis to centre stage. PMID:11253367

  14. The intracellular dynamic of protein palmitoylation

    PubMed Central

    Salaun, Christine; Greaves, Jennifer

    2010-01-01

    S-palmitoylation describes the reversible attachment of fatty acids (predominantly palmitate) onto cysteine residues via a labile thioester bond. This posttranslational modification impacts protein functionality by regulating membrane interactions, intracellular sorting, stability, and membrane micropatterning. Several recent findings have provided a tantalizing insight into the regulation and spatiotemporal dynamics of protein palmitoylation. In mammalian cells, the Golgi has emerged as a possible super-reaction center for the palmitoylation of peripheral membrane proteins, whereas palmitoylation reactions on post-Golgi compartments contribute to the regulation of specific substrates. In addition to palmitoylating and depalmitoylating enzymes, intracellular palmitoylation dynamics may also be controlled through interplay with distinct posttranslational modifications, such as phosphorylation and nitrosylation. PMID:21187327

  15. Dynamics of gradient formation by intracellular shuttling

    NASA Astrophysics Data System (ADS)

    Berezhkovskii, Alexander M.; Shvartsman, Stanislav Y.

    2015-08-01

    A number of important cellular functions rely on the formation of intracellular protein concentration gradients. Experimental studies discovered a number of mechanisms for the formation of such gradients. One of the mechanisms relies on the intracellular shuttling of a protein that interconverts between the two states with different diffusivities, under the action of two enzymes, one of which is localized to the plasma membrane, whereas the second is uniformly distributed in the cytoplasm. Recent work reported an analytical solution for the steady state gradient in this mechanism, obtained in the framework of a one-dimensional reaction-diffusion model. Here, we study the dynamics in this model and derive analytical expressions for the Laplace transforms of the time-dependent concentration profiles in terms of elementary transcendental functions. Inverting these transforms numerically, one can obtain time-dependent concentration profiles of the two forms of the protein.

  16. Dynamics of gradient formation by intracellular shuttling

    SciTech Connect

    Berezhkovskii, Alexander M.; Shvartsman, Stanislav Y.

    2015-08-21

    A number of important cellular functions rely on the formation of intracellular protein concentration gradients. Experimental studies discovered a number of mechanisms for the formation of such gradients. One of the mechanisms relies on the intracellular shuttling of a protein that interconverts between the two states with different diffusivities, under the action of two enzymes, one of which is localized to the plasma membrane, whereas the second is uniformly distributed in the cytoplasm. Recent work reported an analytical solution for the steady state gradient in this mechanism, obtained in the framework of a one-dimensional reaction-diffusion model. Here, we study the dynamics in this model and derive analytical expressions for the Laplace transforms of the time-dependent concentration profiles in terms of elementary transcendental functions. Inverting these transforms numerically, one can obtain time-dependent concentration profiles of the two forms of the protein.

  17. Toward Intracellular Targeted Delivery of Cancer Therapeutics

    PubMed Central

    Pandya, Hetal; Debinski, Waldemar

    2013-01-01

    A number of anti-cancer drugs have their targets localized to particular intracellular compartments. These drugs reach the targets mainly through diffusion, dependent on biophysical and biochemical forces that allow cell penetration. This means that both cancer cells and normal cells will be subjected to such diffusion; hence many of these drugs, like chemotherapeutics, are potentially toxic and the concentration achieved at the site of their action is often suboptimal. The same relates to radiation that indiscriminately affects normal and diseased cells. However, nature-designed systems enable compounds present in the extracellular environment to end up inside the cell and even travel to more specific intracellular compartments. For example, viruses and bacterial toxins can more or less specifically recognize eukaryotic cells, enter these cells, and direct some protein portions to designated intracellular areas. These phenomena have led to creative thinking, such as employing viruses or bacterial toxins for cargo delivery to cells and, more specifically, to cancer cells. Proteins can be genetically engineered in order to not only mimic what viruses and bacterial toxins can do, but also to add new functions, extending or changing the intracellular routes. It is possible to make conjugates or, more preferably, single-chain proteins that recognize cancer cells and deliver cargo inside the cells, even to the desired subcellular compartment. These findings offer new opportunities to deliver drugs/labels only to cancer cells and only to their site of action within the cells. The development of such dual-specificity vectors for targeting cancer cells is an attractive and potentially safer and more efficacious way of delivering drugs. We provide examples of this approach for delivering brain cancer therapeutics, using a specific biomarker on glioblastoma tumor cells. PMID:22671766

  18. Chemo May Prolong Lives of Some Brain Cancer Patients

    MedlinePlus

    ... 158167.html Chemo May Prolong Lives of Some Brain Cancer Patients: Study Those with slow-growing gliomas ... the lives of people with certain slow-growing brain tumors, a new study finds. The findings come ...

  19. Error Propagation Analysis for Quantitative Intracellular Metabolomics

    PubMed Central

    Tillack, Jana; Paczia, Nicole; Nöh, Katharina; Wiechert, Wolfgang; Noack, Stephan

    2012-01-01

    Model-based analyses have become an integral part of modern metabolic engineering and systems biology in order to gain knowledge about complex and not directly observable cellular processes. For quantitative analyses, not only experimental data, but also measurement errors, play a crucial role. The total measurement error of any analytical protocol is the result of an accumulation of single errors introduced by several processing steps. Here, we present a framework for the quantification of intracellular metabolites, including error propagation during metabolome sample processing. Focusing on one specific protocol, we comprehensively investigate all currently known and accessible factors that ultimately impact the accuracy of intracellular metabolite concentration data. All intermediate steps are modeled, and their uncertainty with respect to the final concentration data is rigorously quantified. Finally, on the basis of a comprehensive metabolome dataset of Corynebacterium glutamicum, an integrated error propagation analysis for all parts of the model is conducted, and the most critical steps for intracellular metabolite quantification are detected. PMID:24957773

  20. Dual responsive nanogels for intracellular doxorubicin delivery.

    PubMed

    Asadi, Hamed; Khoee, Sepideh

    2016-09-10

    Nanosized polymeric delivery systems that encapsulate drug molecules and release them in response to a specific intracellular stimulus are of promising interest for cancer therapy. Here, we demonstrated a simple and fast synthetic protocol of redox-responsive nanogels with high drug encapsulation efficiency and stability. The prepared nanogels displayed narrow size distributions and versatility of surface modification. The polymer precursor of these nanogels is based on a random copolymer that contains oligoethyleneglycol (OEG) and pyridyldisulfide (PDS) units as side-chain functionalities. The nanogels were prepared through a lock-in strategy in aqueous media via self cross-linking of PDS groups. By changing polymer concentration, we could control the size of nanogels in range of 80-115nm. The formed nanogels presented high doxorubicin (DOX) encapsulation efficiency (70% (w/w)) and displayed pH and redox-controlled drug release triggered by conditions mimicking the reducible intracellular environment. The nanogels displayed an excellent cytocompatibility and were effectively endocytosed by A2780CP ovarian cancer cells, which make them promising nanomaterials for the efficient intracellular delivery of anticancer drugs. PMID:27444549

  1. Invasion and Intracellular Survival by Protozoan Parasites

    PubMed Central

    Sibley, L. David

    2013-01-01

    Summary Intracellular parasitism has arisen only a few times during the long ancestry of protozoan parasites including in diverse groups such as microsporidians, kinetoplastids, and apicomplexans. Strategies used to gain entry differ widely from injection (e.g. microsporidians), active penetration of the host cell (e.g. Toxoplasma), recruitment of lysosomes to a plasma membrane wound (e.g. Trypanosoma cruzi), to host cell-mediated phagocytosis (e.g. Leishmania). The resulting range of intracellular niches is equally diverse ranging from cytosolic (e.g. T. cruzi) to residing within a nonfusigenic vacuole (e.g. Toxoplasma, Encephalitizoon) or a modified phagolysosome (e.g. Leishmania). These lifestyle choices influence access to nutrients, interaction with host cell signaling pathways, and detection by pathogen recognition systems. As such, intracellular life requires a repertoire of adaptations to assure entry-exit from the cell, as well as to thwart innate immune mechanisms and prevent clearance. Elucidating these pathways at the cellular and molecular level may identify key steps that can be targeted to reduce parasite survival or augment immunological responses and thereby prevent disease. PMID:21349087

  2. Antithrombotic activities of ferulic acid via intracellular cyclic nucleotide signaling.

    PubMed

    Hong, Qian; Ma, Zeng-Chun; Huang, Hao; Wang, Yu-Guang; Tan, Hong-Ling; Xiao, Cheng-Rong; Liang, Qian-De; Zhang, Han-Ting; Gao, Yue

    2016-04-15

    Ferulic acid (FA) produces protective effects against cardiovascular dysfunctions. However, the mechanisms of FA is still not known. Here we examined the antithrombotic effects of FA and its potential mechanisms. Anticoagulation assays and platelet aggregation was evaluated in vitro and in vivo. Thromboxane B2 (TXB2), cyclic adenosine monophosphate(cAMP), and cyclic guanosine monophosphate (cGMP) was determined using enzyme immunoassay kits. Nitric oxide (NO) production was measured using the Griess reaction. Protein expression was detected by Western blotting analysis. Oral administration of FA prevented death caused by pulmonary thrombosis and prolonged the tail bleeding and clotting time in mice,while, it did not alter the coagulation parameters, including the activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT). In addition, FA (50-200µM) dose-dependently inhibited platelet aggregation induced by various platelet agonists, including adenosine diphosphate (ADP), thrombin, collagen, arachidonic acid (AA), and U46619. Further, FA attenuated intracellular Ca(2)(+) mobilization and TXB2 production induced by the platelet agonists. FA increased the levels of cAMP and cGMP and phosphorylated vasodilator-stimulated phosphoprotein (VASP) while decreased phospho-MAPK (mitogen-activated protein kinase) and phosphodiesterase (PDE) in washed rat platelets, VASP is a substrate of cyclic nucleotide and PDE is an enzyme family responsible for hydrolysis of cAMP/cGMP. These results suggest that antithrombotic activities of FA may be regulated by inhibition of platelet aggregation, rather than through inhibiting the release of thromboplastin or formation of thrombin. The mechanism of this action may involve activation of cAMP and cGMP signaling. PMID:26948317

  3. Prolongation of RBC survival in the hypophysectomized rat.

    NASA Technical Reports Server (NTRS)

    Landaw, S. A.; Bristol, S. K.

    1971-01-01

    Red blood cell (RBC) survival was prolonged in hypophysectomized rats. While the rate of random hemolysis was decreased in some hypophysectomized hosts, in all directly injected and cross-transfused hypophysectomized rat hosts, there was a significant prolongation of the phase of senescent death. In contrast, RBCs from hypophysectomized donors survived normally in normal hosts. These experiments are further evidence of a relationship between RBC aging and metabolic rate, and suggest an intimate involvement with the calorigenic hormones.

  4. N-acetyl cysteine prolonged the developmental ability of mouse two-cell embryos against oxidative stress at refrigerated temperatures.

    PubMed

    Horikoshi, Yuka; Takeo, Toru; Nakagata, Naomi

    2016-06-01

    Cold storage of two-cell embryos at refrigerated temperatures is a useful means to ship genetically engineered mice. We previously reported that M2 medium maintained the developmental ability of two-cell embryos for 48 h at 4 °C, and offspring were obtained from embryos transported by a courier service under refrigerated temperatures. The limitation of 48 h practically restricts the shipping destination of the embryos. To enhance the applicability of the cold-storage technique, prolonging the time to maintain developmental ability of the embryos is required. Oxidative stress may be a cause of the declining developmental ability of cold-stored embryos. However, the effect of oxidative stress on developmental ability of embryos has not been investigated. We examined intracellular glutathione (GSH) levels of cold-stored two-cell embryos to evaluate the effect of oxidative and investigated the efficacy of adding N-acetyl cysteine (NAC) to the preservation medium on the developmental ability of cold-stored embryos and transported two-cell embryos at refrigerated temperatures. Intracellular GSH levels of two-cell embryos decreased by cold storage for longer than 72 h, whereas NAC recovered this reduction and improved the developmental ability of embryos cold-stored for 96 h. In the transport experiment, the developmental rate of transported two-cell embryos to offspring was increased by adding NAC to the preservation medium. We found that NAC prolonged the storage period of two-cell embryos and maintained the developmental ability by alleviating the reduction of intracellular GSH. These findings will improve the technique of cold-storage of two-cell embryos to facilitate efficient transport of genetically engineered mice worldwide. PMID:27164059

  5. Antimicrobial agents-associated with QT interval prolongation.

    PubMed

    Bril, Fernando; Gonzalez, Claudio Daniel; Di Girolamo, Guillermo

    2010-01-01

    QT interval prolongation is one of the most important causes of withdrawal of drugs from the market, due to its association with Torsades de Pointes (TdP), a potentially fatal arrhythmia. Although many antimicrobial drugs are capable of inducing this type of arrhythmia, the importance of this effect is usually underestimated. Macrolides, quinolones, azoles, pentamidine, protease inhibitors, antimalarial drugs and cotrimoxazole are the anti-infective agents more frequently associated with this adverse effect. Despite the fact that the risk of QT prolongation and TdP under single antimicrobial therapy is low, these drugs are so extensively used that sporadic cases of this arrhythmia are reported. Moreover, antimicrobial drugs are susceptible to pharmacokinetic and pharmacodynamic interactions with other drugs, which may increase the risk of this arrhythmia. Therefore, physicians must be familiar with not only the antimicrobial drugs capable of producing QT interval prolongation, but also their potential interactions. In addition, patient's specific risk factors of prolonging QT interval or producing TdP must be taken into account. This article reviews the role of anti-infective drugs in QT prolongation, focusing on QT prolongation mechanisms, potential drug interactions, and patients' predisposing factors to this arrhythmia. PMID:20210724

  6. Prolonged dry episodes over Northeast China during the period 1961-2012

    NASA Astrophysics Data System (ADS)

    Kong, Qinqin; Ge, Quansheng; Zheng, Jingyun; Xi, Jianchao

    2015-11-01

    Spatial and temporal changes of prolonged dry episodes, defined by the 90th percentile of consecutive no-rain days (<1 mm/day) in both the wet (May to September) and dry seasons (October to April), were investigated using daily precipitation data of 72 meteorological stations over Northeast China from 1961 to 2012. Increasing trends are detected for the prolonged dry episode duration at 94.4 % of the stations in the wet season, and 26.5 % of these increasing trends are statistically significant at the 5 % level. In the dry season, 77.8 % of the stations show decreasing trends, and 32.1 % of these decreasing trends are significant at the 5 % level. The drought trend in the wet season detected by the prolonged dry episode duration is more prominent than that detected by the total number of no-rain days or the Standard Precipitation Index. The ratio between the prolonged dry episode duration and total number of no-rain days exhibits a significant (at the 5 % level) increase of 8.3 %/52 years in the wet season and a significant decrease (at the 5 % level) of nearly 11 %/52 years. An overall dry (before 1980), wet (during the 1980s), and dry (after 1990) tendency in the wet season is detected at the regional average level. Inter-annual variations of the prolonged dry episode duration at individual stations show four patterns. The most predominant pattern is similar to the regional average condition but with larger amplitude and mainly occurring over the northern part of Northeast China. Our study provides a more in-depth case to monitor droughts caused by extremely long dry spells and supports studies over other regions.

  7. Hippocampal abnormalities after prolonged febrile convulsion: a longitudinal MRI study.

    PubMed

    Scott, Rod C; King, Martin D; Gadian, David G; Neville, Brian G R; Connelly, Alan

    2003-11-01

    Mesial temporal sclerosis (MTS) is the most common lesion in patients who require epilepsy surgery, and approximately 50% of patients with MTS have a history of prolonged febrile convulsion (PFC) in childhood. The latter led to the hypothesis that convulsive status epilepticus, including PFC, can cause MTS. Our recently published data on children investigated within 5 days of a PFC showed that children investigated by MRI within 48 h of a PFC had large hippocampal volumes and prolongation of T2 relaxation time. Patients investigated >48 h from a PFC had large hippocampal volumes and normal T2 relaxation time. These data are strongly suggestive of hippocampal oedema that is resolving within 5 days of a PFC, but do not exclude the possibility of a pre-existing hippocampal lesion. Fourteen children from the original study had follow-up investigations carried out 4-8 months after the acute investigations. Of the 14 patients, four have had further seizures. Two had short febrile convulsions, one had PFC and one had non-febrile seizures. There was a significant reduction in hippocampal volume and T2 relaxation time between the first and second investigations, and there is now no difference in hippocampal volume or T2 relaxation time in patients compared with a control population. Moreover, there is a significant increase in hippocampal volume asymmetry in patients at follow-up when compared with initial data. Five out of 14 patients had asymmetry outside the 95th percentile for control subjects and, of these, three had one hippocampal volume outside the lower 95% prediction limit for control subjects. A reduction in hippocampal volume or T2 relaxation time, into or below the normal range between the first and second scans, indicates that the earlier findings are temporary and are strongly suggestive of hippocampal oedema as the abnormality in the initial investigations. The change in hippocampal symmetry in the patient group is consistent with injury and neuronal loss

  8. Altered intracellular pH regulation in cells with high levels of P-glycoprotein expression.

    PubMed

    Young, Gregory; Reuss, Luis; Altenberg, Guillermo A

    2011-01-01

    P-glycoprotein is an ATP-binding-cassette transporter that pumps many structurally unrelated drugs out of cells through an ATP-dependent mechanism. As a result, multidrug-resistant cells that overexpress P-glycoprotein have reduced intracellular steady-state levels of a variety of chemotherapeutic agents. In addition, increased cytosolic pH has been a frequent finding in multidrug-resistant cells that express P-glycoprotein, and it has been proposed that this consequence of P-glycoprotein expression may contribute to the lower intracellular levels of chemotherapeutic agents. In these studies, we measured intracellular pH and the rate of acid extrusion in response to an acid load in two cells with very different levels of P-glycoprotein expression: V79 parental cells and LZ-8 multidrug resistant cells. Compared to the wild-type V79 cells, LZ-8 cells have a lower intracellular pH and a slower recovery of intracellular pH after an acid load. The data also show that LZ-8 cells have reduced ability to extrude acid, probably due to a decrease in Na(+)/H(+) exchanger activity. The alterations in intracellular pH and acid extrusion in LZ-8 cells are reversed by 24-h exposure to the multidrug-resistance modulator verapamil. The lower intracellular pH in LZ-8 indicates that intracellular alkalinization is not necessary for multidrug resistance. The reversal by verapamil of the decreased acid-extrusion suggests that P-glycoprotein can affect other membrane transport mechanism. PMID:22003434

  9. In Vivo Epithelial Wound Repair Requires Mobilization of Endogenous Intracellular and Extracellular Calcium*

    PubMed Central

    Aihara, Eitaro; Hentz, Courtney L.; Korman, Abraham M.; Perry, Nicholas P. J.; Prasad, Vikram; Shull, Gary E.; Montrose, Marshall H.

    2013-01-01

    We report that a localized intracellular and extracellular Ca2+ mobilization occurs at the site of microscopic epithelial damage in vivo and is required to mediate tissue repair. Intravital confocal/two-photon microscopy continuously imaged the surgically exposed stomach mucosa of anesthetized mice while photodamage of gastric epithelial surface cells created a microscopic lesion that healed within 15 min. Transgenic mice with an intracellular Ca2+-sensitive protein (yellow cameleon 3.0) report that intracellular Ca2+ selectively increases in restituting gastric epithelial cells adjacent to the damaged cells. Pretreatment with U-73122, indomethacin, 2-aminoethoxydiphenylborane, or verapamil inhibits repair of the damage and also inhibits the intracellular Ca2+ increase. Confocal imaging of Fura-Red dye in luminal superfusate shows a localized extracellular Ca2+ increase at the gastric surface adjacent to the damage that temporally follows intracellular Ca2+ mobilization. Indomethacin and verapamil also inhibit the luminal Ca2+ increase. Intracellular Ca2+ chelation (1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid/acetoxymethyl ester, BAPTA/AM) fully inhibits intracellular and luminal Ca2+ increases, whereas luminal calcium chelation (N-(2-hydroxyetheyl)-ethylendiamin-N,N,N′-triacetic acid trisodium, HEDTA) blocks the increase of luminal Ca2+ and unevenly inhibits late-phase intracellular Ca2+ mobilization. Both modes of Ca2+ chelation slow gastric repair. In plasma membrane Ca-ATPase 1+/− mice, but not plasma membrane Ca-ATPase 4−/− mice, there is slowed epithelial repair and a diminished gastric surface Ca2+ increase. We conclude that endogenous Ca2+, mobilized by signaling pathways and transmembrane Ca2+ transport, causes increased Ca2+ levels at the epithelial damage site that are essential to gastric epithelial cell restitution in vivo. PMID:24121509

  10. An efficient system for intracellular delivery of beta-lactam antibiotics to overcome bacterial resistance

    PubMed Central

    Abed, Nadia; Saïd-Hassane, Fatouma; Zouhiri, Fatima; Mougin, Julie; Nicolas, Valérie; Desmaële, Didier; Gref, Ruxandra; Couvreur, Patrick

    2015-01-01

    The “Golden era” of antibiotics is definitely an old story and this is especially true for intracellular bacterial infections. The poor intracellular bioavailability of antibiotics reduces the efficency of many treatments and thereby promotes resistances. Therefore, the development of nanodevices coupled with antibiotics that are capable of targeting and releasing the drug into the infected-cells appears to be a promising solution to circumvent these complications. Here, we took advantage of two natural terpenes (farnesyl and geranyl) to design nanodevices for an efficient intracellular delivery of penicillin G. The covalent linkage between the terpene moieties and the antibiotic leads to formation of prodrugs that self-assemble to form nanoparticles with a high drug payload between 55–63%. Futhermore, the addition of an environmentally-sensitive bond between the antibiotic and the terpene led to an efficient antibacterial activity against the intracellular pathogen Staphylococcus aureus with reduced intracellular replication of about 99.9% compared to untreated infected cells. Using HPLC analysis, we demonstrated and quantified the intracellular release of PenG when this sensitive-bond (SB) was present on the prodrug, showing the success of this technology to deliver antibiotics directly into cells. PMID:26311631

  11. Importance of Branched-Chain Amino Acid Utilization in Francisella Intracellular Adaptation

    PubMed Central

    Gesbert, Gael; Ramond, Elodie; Tros, Fabiola; Dairou, Julien; Frapy, Eric; Barel, Monique

    2014-01-01

    Intracellular bacterial pathogens have adapted their metabolism to optimally utilize the nutrients available in infected host cells. We recently reported the identification of an asparagine transporter required specifically for cytosolic multiplication of Francisella. In the present work, we characterized a new member of the major super family (MSF) of transporters, involved in isoleucine uptake. We show that this transporter (here designated IleP) plays a critical role in intracellular metabolic adaptation of Francisella. Inactivation of IleP severely impaired intracellular F. tularensis subsp. novicida multiplication in all cell types tested and reduced bacterial virulence in the mouse model. To further establish the importance of the ileP gene in F. tularensis pathogenesis, we constructed a chromosomal deletion mutant of ileP (ΔFTL_1803) in the F. tularensis subsp. holarctica live vaccine strain (LVS). Inactivation of IleP in the F. tularensis LVS provoked comparable intracellular growth defects, confirming the critical role of this transporter in isoleucine uptake. The data presented establish, for the first time, the importance of isoleucine utilization for efficient phagosomal escape and cytosolic multiplication of Francisella and suggest that virulent F. tularensis subspecies have lost their branched-chain amino acid biosynthetic pathways and rely exclusively on dedicated uptake systems. This loss of function is likely to reflect an evolution toward a predominantly intracellular life style of the pathogen. Amino acid transporters should be thus considered major players in the adaptation of intracellular pathogens. PMID:25332124

  12. Intracellular autocrine VEGF signaling promotes EBDC cell proliferation, which can be inhibited by Apatinib.

    PubMed

    Peng, Sui; Zhang, Yanyan; Peng, Hong; Ke, Zunfu; Xu, Lixia; Su, Tianhong; Tsung, Allan; Tohme, Samer; Huang, Hai; Zhang, Qiuyang; Lencioni, Riccardo; Zeng, Zhirong; Peng, Baogang; Chen, Minhu; Kuang, Ming

    2016-04-10

    Tumor cells produce vascular endothelial growth factor (VEGF) which can interact with membrane or cytoplasmic VEGF receptors (VEGFRs) to promote cell growth. We aimed to investigate the role of extracellular/intracellular autocrine VEGF signaling and Apatinib, a highly selective VEGFR2 inhibitor, in extrahepatic bile duct cancer (EBDC). We found conditioned medium or recombinant human VEGF treatment promoted EBDC cell proliferation through a phospholipase C-γ1-dependent pathway. This pro-proliferative effect was diminished by VEGF, VEGFR1 or VEGFR2 neutralizing antibodies, but more significantly suppressed by intracellular VEGFR inhibitor. The rhVEGF induced intracellular VEGF signaling by promoting nuclear accumulation of pVEGFR1/2 and enhancing VEGF promoter activity, mRNA and protein expression. Internal VEGFR2 inhibitor Apatinib significantly inhibited intracellular VEGF signaling, suppressed cell proliferation in vitro and delayed xenograft tumor growth in vivo, while anti-VEGF antibody Bevacizumab showed no effect. Clinically, overexpression of pVEGFR1 and pVEGFR2 was significantly correlated with poorer overall survival (P = .007 and P = .020, respectively). In conclusion, the intracellular autocrine VEGF loop plays a predominant role in VEGF-induced cell proliferation. Apatinib is an effective intracellular VEGF pathway blocker that presents a great therapeutic potential in EBDC. PMID:26805764

  13. Roles of mitochondria and temperature in the control of intracellular calcium in adult rat sensory neurons.

    PubMed

    Kang, S H; Carl, A; McHugh, J M; Goff, H R; Kenyon, J L

    2008-04-01

    We recorded Ca2+ current and intracellular Ca2+ ([Ca2+](i)) in isolated adult rat dorsal root ganglion (DRG) neurons at 20 and 30 degrees C. In neurons bathed in tetraethylammonium and dialyzed with cesium, warming reduced resting [Ca2+](i) from 87 to 49 nM and the time constant of the decay of [Ca2+](i) transients (tau(r)) from 1.3 to 0.99s (Q(10)=1.4). The Buffer Index, the ratio between Ca2+ influx and Delta[Ca2+](i) (f I(ca)d(t)/Delta[Ca2+]i) , increased two- to threefold with warming. Neither inhibition of the plasma membrane Ca2+ -ATPase by intracellular sodium orthovanadate nor inhibition of Ca2+ uptake by the endoplasmic reticulum by thapsigargin plus ryanodine were necessary for the effects of warming on these parameters. In contrast, inhibition of the mitochondrial Ca2+ uniporter by intracellular ruthenium red largely reversed the effects of warming. Carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP, 500 nM) increased resting [Ca2+](i) at 30 degrees C. Ten millimolar intracellular sodium prolonged the recovery of [Ca2+](i) transients to 10-40s. This effect was reversed by an inhibitor of mitochondrial Na(+)/Ca2+ -exchange (CGP 37157, 10 microM). Thus, mitochondrial Ca2+ uptake is necessary for the temperature-dependent increase in Ca2+ buffering and mitochondrial Ca2+ fluxes contribute to the control of [Ca2+](i) between 50 and 150 nM at 30 degrees C. PMID:17716728

  14. Prolonged Second Stage of Labor and Levator Ani Muscle Injuries

    PubMed Central

    Marsoosi, Vajihe; Jamal, Ashraf; Eslamian, Laleh; Oveisi, Sonia; Abotorabi, Shokohossadat

    2015-01-01

    Objective: To determine the effect of pregnancy and vaginal delivery on the pelvic floor and levatorani morphology and function. Methods: Design. Cross-sectional study. Setting. Tertiary care teaching hospital. Population. 75 primigravid women were recruited for assessment at 6 weeks postpartum compared with 25 nulliparous women. Hiatal morphology and levator ani muscle avulsion were assessed by 4-dimensional translabial ultrasound examination. The volume achievement obtained by ultrasound was performed in supine position with empty bladder at rest, on maximum Valsalva maneuver, and on maximum pelvic floor muscle contraction. Main Outcome Measures. Hiatal diameter and area were measured at the plane of minimal hiatal dimension as defined in the midsagittal plane and Levator avulsion was assessed. Results: There were significant differences in hiatal area morphology at rest, on Valsalva maneuver and during contraction of muscles among the study groups, but there was no difference in pelvic diameter at rest, on Valsalva maneuver, and during contraction. There were 21 cases of puborectalis avulsion (42%) with no significant difference between non-progressive labor (8 cases) and Normal Vaginal Delivery (NVD) (13 cases) groups. Conclusions: The results of the present study showed that non-progressive labor is the main risk factor for pelvic muscle injuries, indicating the necessity of a better management and timely cesareans in women with prolonged second stage of labor. PMID:25560352

  15. Cardiovascular time courses during prolonged immersed static apnoea.

    PubMed

    Perini, Renza; Gheza, Alberto; Moia, Christian; Sponsiello, Nicola; Ferretti, Guido

    2010-09-01

    To define the dynamics of cardiovascular adjustments to apnoea during immersion, beat-to-beat heart rate (HR) and systolic (SBP) and diastolic (DBP) blood pressures were recorded in six divers during and after prolonged apnoeas while resting fully immersed in 27 degrees C water. Apnoeas lasted 215 +/- 35 s. Compared to control values, HR decreased by 20 beats min(-1) and SBP and DBP increased by 23 and 17 mmHg, respectively, in the initial 20 +/- 3 s (phase I). Both HR and BP remained stable during the following 92 +/- 15 s (phase II). Subsequently, during the final 103 +/- 29 s, SBP and DBP increased linearly to values about 60% higher than control, whereas HR remained unchanged (phase III). Cardiac output (Q') decreased by 35% in phase I and did not further change in phases II and III. Compared to control, total peripheral resistances were twice and three times higher than control, respectively, at the end of phases I and III. After resumption of breathing, HR and BP returned to control values in 5 and 30 s, respectively. The time courses of cardiovascular adjustments to immersed breath-holding indicated that cardiac response took place only at the beginning of apnoea. In contrast, vascular responses showed two distinct adjustments. This pattern suggests that the chronotropic control via the baroreflex is modified during apnoea. These cardiovascular changes during immersed static apnoea are in agreement with those already reported for static dry apnoeas. PMID:20458594

  16. Injuries can prolong lifespan in Drosophila melanogaster males.

    PubMed

    Henten, Anne Marie Vestergaard; Loeschcke, Volker; Pedersen, Jørgen Granfeldt; Leisner, Jørgen J; Sarup, Pernille

    2016-04-01

    Previous studies have shown that a range of different stresses can increase mean lifespan. Here we investigated the effect of injuries and bacterial inoculation on mean lifespan in lines selected for increased longevity and their controls. The three lines from each selection regime were subjected to one of five treatments ranging from control, over perforating the cuticle with a sterile needle, to inoculating with peptidoglycan or one of two strains of live bacteria. The flies were subjected to the infection stress at two ages and the experiment was conducted on both males and females of replicate lines of each selection regime. The individual lines and sexes differed in response to the treatment. However, overall the sterile injury of young males resulted in prolonged mean lifespan from both selection regimes, whereas inoculating had no additional effect to stabbing with a sterile needle. In middle-aged males only treatment with peptidoglycan had a significant hormetic effect and this was only in longevity-selected flies. In females only one of the tested contrasts was significant and here the effect of the treatment was to reduce average lifespan. As could be expected, the results showed a significant interaction between the effects of sex and infection on survival. PMID:26564163

  17. The intracellular distribution of inositol polyphosphates in HL60 promyeloid cells.

    PubMed Central

    Stuart, J A; Anderson, K L; French, P J; Kirk, C J; Michell, R H

    1994-01-01

    1. HL60 promyeloid cells contain high intracellular concentrations of inositol polyphosphates, notably inositol 1,3,4,5,6-pentakisphosphate (InsP5) and inositol hexakisphosphate (InsP6). To determine their intracellular location(s), we studied the release of inositol (poly)phosphates, of ATP, and of cytosolic and granule-enclosed enzymes from cells permeabilized by four different methods. 2. When cells were treated with digitonin, all of the inositol phosphates were released in parallel with the cytosolic constituents. Most of the InsP5 and InsP6 was released before significant permeabilization of azurophil granules. 3. Similar results were obtained from cells preloaded with ethylene glycol and permeabilized by osmotic lysis. 4. Electroporation at approximately 500 V/cm caused rapid release of free inositol. Higher field strengths provoked release of most of the ATP, InsP5 and InsP6, but only slight release of the intracellular enzymes. Multiple discharges released approximately 80-90% of total InsP5 and InsP6. In the absence of bivalent-cation chelators, InsP5 and InsP6 were released less readily than ATP. 5. Treatment of cells with Staphylococcus aureus alpha-toxin caused quantitative release of inositol and ATP, without release of intracellular enzymes. However, inositol phosphates were released much less readily than inositol or ATP. Even after prolonged incubation with a high concentration of alpha-toxin, only approximately 50-70% of InsP2, InsP3 and InsP4 and < or = 20% of InsP5 and InsP6 were released, indicating that the high charge or large hydrated radius of InsP5 and InsP6 might limit their release through small toxin-induced pores. 6. These results indicate that most intracellular inositol metabolites are either in, or in rapid exchange with, the cytosolic compartment of HL60 cells. However, they leave open the possibility that a small proportion of cellular InsP5 and InsP6 (< or = 10-20%) might be in some intracellular bound form. Images Figure 2 PMID

  18. Legionella pneumophila requires polyamines for optimal intracellular growth.

    PubMed

    Nasrallah, Gheyath K; Riveroll, Angela L; Chong, Audrey; Murray, Lois E; Lewis, P Jeffrey; Garduño, Rafael A

    2011-09-01

    The Gram-negative intracellular pathogen Legionella pneumophila replicates in a membrane-bound compartment known as the Legionella-containing vacuole (LCV), into which it abundantly releases its chaperonin, HtpB. To determine whether HtpB remains within the LCV or reaches the host cell cytoplasm, we infected U937 human macrophages and CHO cells with L. pneumophila expressing a translocation reporter consisting of the Bordetella pertussisa denylate cyclase fused to HtpB. These infections led to increased cyclic AMP levels, suggesting that HtpB reaches the host cell cytoplasm. To identify potential functions of cytoplasmic HtpB, we expressed it in the yeast Saccharomyces cerevisiae, where HtpB induced pseudohyphal growth. A yeast-two-hybrid screen showed that HtpB interacted with S-adenosylmethionine decarboxylase (SAMDC), an essential yeast enzyme (encoded by SPE2) that is required for polyamine biosynthesis. Increasing the copy number of SPE2 induced pseudohyphal growth in S. cerevisiae; thus, we speculated that (i) HtpB induces pseudohyphal growth by activating polyamine synthesis and (ii) L. pneumophila may require exogenous polyamines for growth. A pharmacological inhibitor of SAMDC significantly reduced L. pneumophila replication in L929 mouse cells and U937 macrophages, whereas exogenously added polyamines moderately favored intracellular growth, confirming that polyamines and host SAMDC activity promote L. pneumophila proliferation. Bioinformatic analysis revealed that most known enzymes required for polyamine biosynthesis in bacteria (including SAMDC) are absent in L. pneumophila, further suggesting a need for exogenous polyamines. We hypothesize that HtpB may function to ensure a supply of polyamines in host cells, which are required for the optimal intracellular growth of L. pneumophila. PMID:21742865

  19. Factors Influencing Prolonged ICU Stay After Open Heart Surgery

    PubMed Central

    Azarfarin, Rasoul; Ashouri, Nasibeh; Totonchi, Ziae; Bakhshandeh, Hooman; Yaghoubi, Alireza

    2014-01-01

    Background: There are different risk factors that affect the intensive care unit (ICU) stay after cardiac surgery. Objectives: The aim of this study was to evaluate possible risk factors influencing prolonged ICU stay in a large referral hospital. Patients and Methods: We conducted a case-control study to determinate causes of prolonged ICU stay in 280 adult patients undergoing cardiac surgery in a tertiary care center for cardiovascular patients, Tehran, Iran. These patients were divided into two groups according to ICU stay ≤ 96 and > 96 hours. We evaluated perioperative risk factors of ICU stay > 96 hours. Results: Among the 280 patients studied, 184 (65.7%) had stayed ≤ 96 hours and 96 (34.3%) had stayed > 96 hours in ICU. Frequency of prolonged ICU stay was 34.2% in patients undergoing coronary artery bypass graft (CABG), 30.8% in patients with valve surgery, and 44.8% in patients with CABG plus valve surgery. Patients with > 96 hours of ICU stay received more blood transfusion and intravenous inotropes. They also had longer anesthesia, cardiopulmonary bypass, and postoperative intubation time. There were higher incidence of postoperative tamponade, re-exploration, re-intubation, hemodialysis, and hypotension in this group (P < 0.05 for all comparisons). Conclusions: In this study, about one-third of patients had prolonged ICU stay. Factors influencing prolonged ICU stay were medical and some non-medical factors. In the present study, up to 30% of the patients had a prolonged ICU stay of > 96 hours. Additional data from well-designed investigations are needed for further assessment of the factors influencing prolonged ICU stay after cardiac surgery. PMID:25785249

  20. Reactivity of organism in prolonged space flights

    NASA Technical Reports Server (NTRS)

    Vasilyev, P. V.

    1980-01-01

    An analysis of published data are presented as well as the results of experiments which show that the state of weightlessness and hypodynamia result in a reduced orthostatic and vestibular resistance, increased sensitivity to infections, decreased endurance of accelerations and physical exercises, and altered reactivity of the organism to drugs. Various consequences of weightlessness on the human body, especially weightlessness combined with other factors linked to long space flights are also considered.

  1. From prolonging life to prolonging working life: Tackling unemployment among liver-transplant recipients.

    PubMed

    Åberg, Fredrik

    2016-04-14

    Return to active and productive life is a key goal of modern liver transplantation (LT). Despite marked improvements in quality of life and functional status, a substantial proportion of LT recipients are unable to resume gainful employment. Unemployment forms a threat to physical and psychosocial health, and impairs LT cost-utility through lost productivity. In studies published after year 2000, the average post-LT employment rate is 37%, ranging from 22% to 55% by study. Significant heterogeneity exists among studies. Nonetheless, these employment rates are lower than in the general population and kidney-transplant population. Most consistent employment predictors include pre-LT employment status, male gender, functional/health status, and subjective work ability. Work ability is impaired by physical fatigue and depression, but affected also by working conditions and society. Promotion of post-LT employment is hampered by a lack of interventional studies. Prevention of pre-LT disability by effective treatment of (minimal) hepatic encephalopathy, maintaining mobility, and planning work adjustments early in the course of chronic liver disease, as well as timely post-LT physical rehabilitation, continuous encouragement, self-efficacy improvements, and depression management are key elements of successful employment-promoting strategies. Prolonging LT recipients' working life would further strengthen the success of transplantation, and this is likely best achieved through multidisciplinary efforts ideally starting even before LT candidacy. PMID:27076755

  2. From prolonging life to prolonging working life: Tackling unemployment among liver-transplant recipients

    PubMed Central

    Åberg, Fredrik

    2016-01-01

    Return to active and productive life is a key goal of modern liver transplantation (LT). Despite marked improvements in quality of life and functional status, a substantial proportion of LT recipients are unable to resume gainful employment. Unemployment forms a threat to physical and psychosocial health, and impairs LT cost-utility through lost productivity. In studies published after year 2000, the average post-LT employment rate is 37%, ranging from 22% to 55% by study. Significant heterogeneity exists among studies. Nonetheless, these employment rates are lower than in the general population and kidney-transplant population. Most consistent employment predictors include pre-LT employment status, male gender, functional/health status, and subjective work ability. Work ability is impaired by physical fatigue and depression, but affected also by working conditions and society. Promotion of post-LT employment is hampered by a lack of interventional studies. Prevention of pre-LT disability by effective treatment of (minimal) hepatic encephalopathy, maintaining mobility, and planning work adjustments early in the course of chronic liver disease, as well as timely post-LT physical rehabilitation, continuous encouragement, self-efficacy improvements, and depression management are key elements of successful employment-promoting strategies. Prolonging LT recipients’ working life would further strengthen the success of transplantation, and this is likely best achieved through multidisciplinary efforts ideally starting even before LT candidacy. PMID:27076755

  3. Impact of Prolonged Fraction Delivery Times Simulating IMRT on Cultured Nasopharyngeal Carcinoma Cell Killing

    SciTech Connect

    Zheng Xiaokang; Chen Longhua; Wang Wenjun; Ye Feng; Liu Jiabing; Li Qisheng; Sun Henwen

    2010-12-01

    Purpose: To determine the impact of prolonged fraction delivery times (FDTs) simulating intensity-modulated radiotherapy (IMRT) on cultured nasopharyngeal carcinoma (NPC) cell killing. Methods and Material: Cultured NPC cell lines CNE1 and CNE2 were used in this study. The biological effectiveness of fractionated irradiation protocols simulating conventional external beam radiotherapy and IMRT (FDT of 15, 36, and 50 minutes) was estimated with standard colony assay, and the differences in cell surviving fractions after irradiation with different protocols were tested by use of the paired t test. The impact degree of prolonged FDTs (from 8 to 50 minutes) on cell killing was also assessed by the dose-modifying factors, which were estimated by comparing the effectiveness of intermittently delivered 2 Gy with that of continuously delivered 1.5 to 2 Gy. Results: The cell surviving fractions of both CNE1 and CNE2 after fractionated irradiation simulating IMRT were higher than those simulating conventional external beam radiotherapy (p < 0.05). The dose-modifying factors for a fraction dose of 2 Gy increased from 1.05 to 1.18 for CNE1 and from 1.05 to 1.11 for CNE2 with the FDT being prolonged from 15 to 50 minutes. Conclusions: This study showed that the prolonged FDTs simulating IMRT significantly decreased the cell killing in both CNE1 and CNE2 cell lines, and these negative effects increased with the FDT being prolonged from 15 to 50 minutes. These effects, if confirmed by in vivo and clinical studies, need to be considered in designing IMRT treatments for NPC.

  4. A study on ice crystal formation behavior at intracellular freezing of plant cells using a high-speed camera.

    PubMed

    Ninagawa, Takako; Eguchi, Akemi; Kawamura, Yukio; Konishi, Tadashi; Narumi, Akira

    2016-08-01

    Intracellular ice crystal formation (IIF) causes several problems to cryopreservation, and it is the key to developing improved cryopreservation techniques that can ensure the long-term preservation of living tissues. Therefore, the ability to capture clear intracellular freezing images is important for understanding both the occurrence and the IIF behavior. The authors developed a new cryomicroscopic system that was equipped with a high-speed camera for this study and successfully used this to capture clearer images of the IIF process in the epidermal tissues of strawberry geranium (Saxifraga stolonifera Curtis) leaves. This system was then used to examine patterns in the location and formation of intracellular ice crystals and to evaluate the degree of cell deformation because of ice crystals inside the cell and the growing rate and grain size of intracellular ice crystals at various cooling rates. The results showed that an increase in cooling rate influenced the formation pattern of intracellular ice crystals but had less of an effect on their location. Moreover, it reduced the degree of supercooling at the onset of intracellular freezing and the degree of cell deformation; the characteristic grain size of intracellular ice crystals was also reduced, but the growing rate of intracellular ice crystals was increased. Thus, the high-speed camera images could expose these changes in IIF behaviors with an increase in the cooling rate, and these are believed to have been caused by an increase in the degree of supercooling. PMID:27343136

  5. Decoding of intracellular calcium spike trains

    NASA Astrophysics Data System (ADS)

    Prank, K.; Läer, L.; von zur Mühlen, A.; Brabant, G.; Schöfl, C.

    1998-04-01

    Cells respond to external signals, such as hormonal stimuli, by generating repetitive spikes in the intracellular free-calcium concentration ([Ca2+]i). These [Ca2+]i spikes, which can be modulated in their frequency and amplitude, regulate diverse cellular processes. Experimentally, [Ca2+]i can be assessed continuously in contrast to cellular responses represented by the activation of proteins. We propose a mathematical model that allows for the on-line decoding of [Ca2+]i spike trains into cellular responses represented by the activation of proteins.

  6. Chemokine receptor internalization and intracellular trafficking.

    PubMed

    Neel, Nicole F; Schutyser, Evemie; Sai, Jiqing; Fan, Guo-Huang; Richmond, Ann

    2005-12-01

    The internalization and intracellular trafficking of chemokine receptors have important implications for the cellular responses elicited by chemokine receptors. The major pathway by which chemokine receptors internalize is the clathrin-mediated pathway, but some receptors may utilize lipid rafts/caveolae-dependent internalization routes. This review discusses the current knowledge and controversies regarding these two different routes of endocytosis. The functional consequences of internalization and the regulation of chemokine receptor recycling will also be addressed. Modifications of chemokine receptors, such as palmitoylation, ubiquitination, glycosylation, and sulfation, may also impact trafficking, chemotaxis and signaling. Finally, this review will cover the internalization and trafficking of viral and decoy chemokine receptors. PMID:15998596

  7. Imaging Intracellular Fluorescent Proteins at Nanometer Resolution

    NASA Astrophysics Data System (ADS)

    Betzig, Eric; Patterson, George H.; Sougrat, Rachid; Lindwasser, O. Wolf; Olenych, Scott; Bonifacino, Juan S.; Davidson, Michael W.; Lippincott-Schwartz, Jennifer; Hess, Harald F.

    2006-09-01

    We introduce a method for optically imaging intracellular proteins at nanometer spatial resolution. Numerous sparse subsets of photoactivatable fluorescent protein molecules were activated, localized (to ~2 to 25 nanometers), and then bleached. The aggregate position information from all subsets was then assembled into a superresolution image. We used this method-termed photoactivated localization microscopy-to image specific target proteins in thin sections of lysosomes and mitochondria; in fixed whole cells, we imaged vinculin at focal adhesions, actin within a lamellipodium, and the distribution of the retroviral protein Gag at the plasma membrane.

  8. Kinesin superfamily motor proteins and intracellular transport.

    PubMed

    Hirokawa, Nobutaka; Noda, Yasuko; Tanaka, Yosuke; Niwa, Shinsuke

    2009-10-01

    Intracellular transport is fundamental for cellular function, survival and morphogenesis. Kinesin superfamily proteins (also known as KIFs) are important molecular motors that directionally transport various cargos, including membranous organelles, protein complexes and mRNAs. The mechanisms by which different kinesins recognize and bind to specific cargos, as well as how kinesins unload cargo and determine the direction of transport, have now been identified. Furthermore, recent molecular genetic experiments have uncovered important and unexpected roles for kinesins in the regulation of such physiological processes as higher brain function, tumour suppression and developmental patterning. These findings open exciting new areas of kinesin research. PMID:19773780

  9. Macrophage cell death upon intracellular bacterial infection

    PubMed Central

    Lai, Xin-He; Xu, Yunsheng; Chen, Xiao-Ming; Ren, Yi

    2015-01-01

    Macrophage-pathogen interaction is a complex process and the outcome of this tag-of-war for both sides is to live or die. Without attempting to be comprehensive, this review will discuss the complexity and significance of the interaction outcomes between macrophages and some facultative intracellular bacterial pathogens as exemplified by Francisella, Salmonella, Shigella and Yersinia. Upon bacterial infection, macrophages can die by a variety of ways, such as apoptosis, autophagic cell death, necrosis, necroptosis, oncosis, pyronecrosis, pyroptosis etc, which is the focus of this review. PMID:26690967

  10. Psidium guajava and Piper betle Leaf Extracts Prolong Vase Life of Cut Carnation (Dianthus caryophyllus) Flowers

    PubMed Central

    Rahman, M. M.; Ahmad, S. H.; Lgu, K. S.

    2012-01-01

    The effect of leaf extracts of Psidium guajava and Piper betle on prolonging vase life of cut carnation flowers was studied. “Carola” and “Pallas Orange” carnation flowers, at bud stage, were pulsed 24 hours with a floral preservative. Then, flowers were placed in a vase solution containing sprite and a “germicide” (leaf extracts of P. guajava and P. betle, 8-HQC, or a copper coin). Flowers treated with 8-HQC, copper coin, and leaf extracts had longer vase life, larger flower diameter, and higher rate of water uptake compared to control (tap water). The leaf extracts of P. guajava and P. betle showed highest antibacterial and antifungal activities compared to the other treatments. Both showed similar effects on flower quality as the synthetic germicide, 8-HQC. Therefore, these extracts are likely natural germicides to prolong vase life of cut flowers. PMID:22619568

  11. Effects of prolonged exposure to space flight factors for 175 days on lettuce seeds

    NASA Astrophysics Data System (ADS)

    Nevzgodina, L. V.; Maximova, E. N.; Akatov, Yu. A.

    We have studied the effects of prolonged (up to 175 days) exposure of Lactuca sativa seeds to space flight factors, including primary cosmic radiation heavy ions. The data obtained evidence a significant fourfold increase ofs pontaneous mutagenesis in seeds both with regard to the total number of aberrant cells as well as the formation of single cells with multiple aberrations. Comparison of the present experiment with earlier works shows that the frequency of such aberrations increases with the duration of the flight.

  12. Effects of prolonged exposure to space flight factors for 175 days on lettuce seeds

    SciTech Connect

    Nevzgodina, L.V.; Maximova, E.N.; Akatov, Yu.A.

    1981-01-01

    The effects of prolonged (up to 175 days) exposure of Lactuca sativa seeds to space flight factors, including primary cosmic radiation heavy ions have been studied. The data obtained evidence a significant fourfold increase of spontaneous mutagenesis in seeds both with regard to the total number of aberrant cells as well as the formation of single cells with multiple aberrations. Comparison of the present experiment with earlier works shows that the frequency of such aberrations increases with the duration of the flight.

  13. Treatment of Experimental Candida Sepsis with a Janus Kinase Inhibitor Controls Inflammation and Prolongs Survival.

    PubMed

    Tsirigotis, P; Papanikolaou, N; Elefanti, A; Konstantinou, P; Gkirkas, K; Rontogianni, D; Siafakas, N; Karakitsos, P; Roilides, E; Dimitriadis, G; Zerva, L; Meletiadis, J

    2015-12-01

    Janus kinases (JAK) are intracellular tyrosine kinases that transduce cytokine-mediated signals to the nucleus, promoting gene expression. Cytokines play a major role in microbial sepsis, which is often associated with uncontrolled inflammation leading to death. JAK inhibitors have been used for the treatment of several autoimmune diseases by modulating immune response, but they have never been tested against microbial sepsis. Ruxolitinib is a small-molecule inhibitor of JAK1/2 proteins, which are involved in the downstream signaling pathway of the vast majority of proinflammatory and anti-inflammatory cytokines. We therefore studied the effect of ruxolitinib in a mouse model of sepsis due to Candida albicans. When ruxolitinib therapy (50 mg/kg [of body weight]/day) was started 1 day before infection, the median survival time was reduced by 3 days, the fungal loads in all organs were higher, the inflammation was significantly less, and serum tumor necrosis factor alpha (TNF-α) and interleukin 10 (IL-10) levels and IL-10/TNF-α ratios were higher than in controls. When ruxolitinib therapy (50 to 1.5 mg/kg/day) was started 1 day after infection, an inverted-U relationship was found, with 6.25 mg/kg/day prolonging median survival time by 6 days, resulting in similar fungal loads, less inflammation, and similar cytokine levels but higher IL-10/TNF-α ratios than the controls. The optimal dose of ruxolitinib controlled infection and prolonged survival with less inflammation than in control animals. Administration of JAK inhibitors may be a promising therapeutic adjunct that needs further investigation. PMID:26369979

  14. Prolonged calcium influx after termination of light-induced calcium release in invertebrate photoreceptors

    PubMed Central

    Nasi, Enrico

    2009-01-01

    In microvillar photoreceptors, light stimulates the phospholipase C cascade and triggers an elevation of cytosolic Ca2+ that is essential for the regulation of both visual excitation and sensory adaptation. In some organisms, influx through light-activated ion channels contributes to the Ca2+ increase. In contrast, in other species, such as Lima, Ca2+ is initially only released from an intracellular pool, as the light-sensitive conductance is negligibly permeable to calcium ions. As a consequence, coping with sustained stimulation poses a challenge, requiring an alternative pathway for further calcium mobilization. We observed that after bright or prolonged illumination, the receptor potential of Lima photoreceptors is followed by the gradual development of an after-depolarization that decays in 1–4 minutes. Under voltage clamp, a graded, slow inward current (Islow) can be reproducibly elicited by flashes that saturate the photocurrent, and can reach a peak amplitude in excess of 200 pA. Islow obtains after replacing extracellular Na+ with Li+, guanidinium, or N-methyl-d-glucamine, indicating that it does not reflect the activation of an electrogenic Na/Ca exchange mechanism. An increase in membrane conductance accompanies the slow current. Islow is impervious to anion replacements and can be measured with extracellular Ca2+ as the sole permeant species; Ba can substitute for Ca2+ but Mg2+ cannot. A persistent Ca2+ elevation parallels Islow, when no further internal release takes place. Thus, this slow current could contribute to sustained Ca2+ mobilization and the concomitant regulation of the phototransduction machinery. Although reminiscent of the classical store depletion–operated calcium influx described in other cells, Islow appears to diverge in some significant aspects, such as its large size and insensitivity to SKF96365 and lanthanum; therefore, it may reflect an alternative mechanism for prolonged increase of cytosolic calcium in photoreceptors. PMID

  15. Intracellular membrane traffic at high resolution

    PubMed Central

    van Weering, Jan R.T.; Brown, Edward; Sharp, Thomas H.; Mantell, Judith; Cullen, Peter J.

    2014-01-01

    I. Abstract Membrane traffic between organelles is essential for a multitude of processes that maintain cell homeostasis. Many steps in these tightly regulated trafficking pathways take place in microdomains on the membranes of organelles, which require analysis at nanometer resolution. Electron Microscopy (EM) can visualize these processes in detail and is mainly responsible for our current view of morphology on the subcellular level. This review discusses how EM can be applied to solve many questions of intracellular membrane traffic, with a focus on the endosomal system. We describe the expansion of the technique from purely morphological analysis to cryo-immuno-EM, Correlative Light Electron Microscopy (CLEM) and 3D electron tomography. In this review we go into some technical details of these various techniques. Furthermore, we provide a full protocol for immunolabeling on Lowicryl sections of high-pressure frozen cells as well as a detailed description of a simple CLEM method that can be applied to answer many membrane trafficking questions. We believe that these EM-based techniques are important tools to expand our understanding of the molecular details of endosomal sorting and intracellular membrane traffic in general. PMID:20869541

  16. Strategies for Intracellular Survival of Burkholderia pseudomallei.

    PubMed

    Allwood, Elizabeth M; Devenish, Rodney J; Prescott, Mark; Adler, Ben; Boyce, John D

    2011-01-01

    Burkholderia pseudomallei is the causative agent of melioidosis, a disease with high mortality that is prevalent in tropical regions of the world. A key component of the pathogenesis of melioidosis is the ability of B. pseudomallei to enter, survive, and replicate within mammalian host cells. For non-phagocytic cells, bacterial adhesins have been identified both on the bacterial surface and associated with Type 4 pili. Cell invasion involves components of one or more of the three Type 3 Secretion System clusters, which also mediate, at least in part, the escape of bacteria from the endosome into the cytoplasm, where bacteria move by actin-based motility. The mechanism of actin-based motility is not clearly understood, but appears to differ from characterized mechanisms in other bacterial species. A small proportion of intracellular bacteria is targeted by host cell autophagy, involving direct recruitment of LC3 to endosomes rather than through uptake by canonical autophagosomes. However, the majority of bacterial cells are able to circumvent autophagy and other intracellular defense mechanisms such as the induction of inducible nitric oxide synthase, and then replicate in the cytoplasm and spread to adjacent cells via membrane fusion, resulting in the formation of multi-nucleated giant cells. A potential role for host cell ubiquitin in the autophagic response to bacterial infection has recently been proposed. PMID:22007185

  17. Small Peptide Recognition Sequence for Intracellular Sorting

    PubMed Central

    Pandey, Kailash N.

    2010-01-01

    Increasing evidence indicate that complex arrays of short signals and recognition peptide sequence ensure accurate trafficking and distribution of transmembrane receptors and/or proteins and their ligands into intracellular compartments. Internalization and subsequent trafficking of cell-surface receptors into the cell interior is mediated by specific short-sequence peptide signals within the cytoplasmic domains of these receptor proteins. The short signals usually consist of small linear amino acid sequences, which are recognized by adaptor coat proteins along the endocytic and sorting pathways. In recent years, much has been learned about the function and mechanisms of endocytic pathways responsible for the trafficking and molecular sorting of membrane receptors and their ligands into intracellular compartments, however, the significance and scope of the short sequence motifs in these cellular events is not well understood. Here a particular emphasis has been given to the functions of short-sequence signal motifs responsible for the itinerary and destination of membrane receptors and proteins moving into subcellular compartments. PMID:20817434

  18. Intracellular Calcium Dysregulation: Implications for Alzheimer's Disease

    PubMed Central

    Magi, Simona; Castaldo, Pasqualina; Macrì, Maria Loredana; Maiolino, Marta; Matteucci, Alessandra; Bastioli, Guendalina; Gratteri, Santo; Lariccia, Vincenzo

    2016-01-01

    Alzheimer's Disease (AD) is a neurodegenerative disorder characterized by progressive neuronal loss. AD is associated with aberrant processing of the amyloid precursor protein, which leads to the deposition of amyloid-β plaques within the brain. Together with plaques deposition, the hyperphosphorylation of the microtubules associated protein tau and the formation of intraneuronal neurofibrillary tangles are a typical neuropathological feature in AD brains. Cellular dysfunctions involving specific subcellular compartments, such as mitochondria and endoplasmic reticulum (ER), are emerging as crucial players in the pathogenesis of AD, as well as increased oxidative stress and dysregulation of calcium homeostasis. Specifically, dysregulation of intracellular calcium homeostasis has been suggested as a common proximal cause of neural dysfunction in AD. Aberrant calcium signaling has been considered a phenomenon mainly related to the dysfunction of intracellular calcium stores, which can occur in both neuronal and nonneuronal cells. This review reports the most recent findings on cellular mechanisms involved in the pathogenesis of AD, with main focus on the control of calcium homeostasis at both cytosolic and mitochondrial level. PMID:27340665

  19. Mucolipins: Intracellular TRPML1-3 Channels

    PubMed Central

    Cheng, Xiping; Shen, Dongbiao; Samie, Mohammad; Xu, Haoxing

    2010-01-01

    The mucolipin family of Transient Receptor Potential (TRPML) proteins is predicted to encode ion channels expressed in intracellular endosomes and lysosomes. Loss-of-function mutations of human TRPML1 cause type IV mucolipidosis (ML4), a childhood neurodegenerative disease. Meanwhile, gain-of-function mutations in the mouse TRPML3 result in the varitint-waddler (Va) phenotype with hearing and pigmentation defects. The broad spectrum phenotypes of ML4 and Va appear to result from certain aspects of endosomal/lysosomal dysfunction. Lysosomes, traditionally believed to be the terminal “recycling center” for biological “garbage”, are now known to play indispensable roles in intracellular signal transduction and membrane trafficking. Studies employing animal models and cell lines in which TRPML genes have been genetically disrupted or depleted have uncovered roles of TRPMLs in multiple cellular functions including membrane trafficking, signal transduction, and organellar ion homeostasis. Physiological assays of mammalian cell lines in which TRPMLs are heterologously over-expressed have revealed the channel properties of TRPMLs in mediating cation (Ca2+/Fe2+) efflux from endosomes and lysosomes in response to unidentified cellular cues. This review aims to summarize these recent advances in the TRPML field and to correlate the channel properties of endolysosomal TRPMLs with their biological functions. We will also discuss the potential cellular mechanisms by which TRPML deficiency leads to neurodegeneration. PMID:20074572

  20. Intracellular accumulation of ethanol in yeast

    SciTech Connect

    Loueiro, V.; Ferreira, H.G.

    1983-09-01

    Ethanol produced in the course of a batch fermentation by Saccharomyces cerevisiae or added from the outside, affects adversely the specific rate of growth of the yeast population, its viability, its specific rate of fermentation, and the specific rates of the uptake of sugar and amino acids. The underlying mechanisms are many and include irreversible denaturation and hyperbolic noncompetitive inhibition of glycolytic enzymes, the exponential noncompetitive inhibition of glucose, maltose, and ammonium transport, the depression of the optimum and the maximum temperature for growth, the increase of the minimum temperature for growth, and the enhancement of thermal death and petite mutation. Nagodawithana and Steinkraus reported that added ethanol was less toxic for S. cerevisiae than ethanol produced by the yeast. The death rates were lower in the presence of added ethanol than those measured at similar external ethanol concentrations endogenously produced. They proposed that, due to an unbalance between the rates of production and the net outflux of ethanol, there would be an intracellular accumulation of ethanol which in turn would explain the apparently greater inhibitory potency of endogenously produced ethanol present in the medium. This hypothesis was supported by the findings of several authors who reported that the intracellular concentration of ethanol, in the course of batch fermentation, is much higher than its concentration in the extracellular medium. The present work is an attempt to clarify this matter. (Refs. 32).

  1. Cytoskeletal Network Morphology Regulates Intracellular Transport Dynamics.

    PubMed

    Ando, David; Korabel, Nickolay; Huang, Kerwyn Casey; Gopinathan, Ajay

    2015-10-20

    Intracellular transport is essential for maintaining proper cellular function in most eukaryotic cells, with perturbations in active transport resulting in several types of disease. Efficient delivery of critical cargos to specific locations is accomplished through a combination of passive diffusion and active transport by molecular motors that ballistically move along a network of cytoskeletal filaments. Although motor-based transport is known to be necessary to overcome cytoplasmic crowding and the limited range of diffusion within reasonable timescales, the topological features of the cytoskeletal network that regulate transport efficiency and robustness have not been established. Using a continuum diffusion model, we observed that the time required for cellular transport was minimized when the network was localized near the nucleus. In simulations that explicitly incorporated network spatial architectures, total filament mass was the primary driver of network transit times. However, filament traps that redirect cargo back to the nucleus caused large variations in network transport. Filament polarity was more important than filament orientation in reducing average transit times, and transport properties were optimized in networks with intermediate motor on and off rates. Our results provide important insights into the functional constraints on intracellular transport under which cells have evolved cytoskeletal structures, and have potential applications for enhancing reactions in biomimetic systems through rational transport network design. PMID:26488648

  2. Stochastic models of intracellular calcium signals

    NASA Astrophysics Data System (ADS)

    Rüdiger, Sten

    2014-01-01

    Cellular signaling operates in a noisy environment shaped by low molecular concentrations and cellular heterogeneity. For calcium release through intracellular channels-one of the most important cellular signaling mechanisms-feedback by liberated calcium endows fluctuations with critical functions in signal generation and formation. In this review it is first described, under which general conditions the environment makes stochasticity relevant, and which conditions allow approximating or deterministic equations. This analysis provides a framework, in which one can deduce an efficient hybrid description combining stochastic and deterministic evolution laws. Within the hybrid approach, Markov chains model gating of channels, while the concentrations of calcium and calcium binding molecules (buffers) are described by reaction-diffusion equations. The article further focuses on the spatial representation of subcellular calcium domains related to intracellular calcium channels. It presents analysis for single channels and clusters of channels and reviews the effects of buffers on the calcium release. For clustered channels, we discuss the application and validity of coarse-graining as well as approaches based on continuous gating variables (Fokker-Planck and chemical Langevin equations). Comparison with recent experiments substantiates the stochastic and spatial approach, identifies minimal requirements for a realistic modeling, and facilitates an understanding of collective channel behavior. At the end of the review, implications of stochastic and local modeling for the generation and properties of cell-wide release and the integration of calcium dynamics into cellular signaling models are discussed.

  3. Intracellular imaging of Qdots-labeled DNA in cyanobacteria.

    PubMed

    Loukanov, Alexandre; Zhelyazkov, Veselin; Hihara, Yukako; Nakabayashi, Seiichiro

    2016-05-01

    In this contribution, they have attempted to develop a labeling technique for in vivo imaging of functionally active plasmid DNA in cyanobacterial cells through its decoration with semiconductor quantum dots (Qdots) as fluorescent nanoprobes. For that purpose biotinylated plasmid slr2060 DNA was conjugated with Qdots-streptavidine. The intact DNA was visualized in a single green color by light microscopy. These Qdots-DNA conjugates were capable of expressing the acyltransferase enzyme. Qdots-DNA conjugates and confocal microscope imaging technique were adopted to visualize the gene transport across the membrane of the live cyanobacteria cell in real time. Long-term kinetic study enabled to reveal the steps of extracellular and intracellular microenvironment for plasmid transportation into the live cell. To confirm these processes a confocal microscope and indicator plate assay test were applied in tandem. In this contribution, Qdots-labeled plasmid DNA was utilized for the first time for long-term intracellular imaging studies in cyanobacteria species PCC6803. The results showed that the Qdots-labeled plasmid DNA detection could be used as a powerful labeling technique for visualization of exogenous DNA entry and tracking into living cells by confocal microscopy. Microsc. Res. Tech. 79:447-452, 2016. © 2016 Wiley Periodicals, Inc. PMID:26957226

  4. Multistability and dynamic transitions of intracellular Min protein patterns.

    PubMed

    Wu, Fabai; Halatek, Jacob; Reiter, Matthias; Kingma, Enzo; Frey, Erwin; Dekker, Cees

    2016-01-01

    Cells owe their internal organization to self-organized protein patterns, which originate and adapt to growth and external stimuli via a process that is as complex as it is little understood. Here, we study the emergence, stability, and state transitions of multistable Min protein oscillation patterns in live Escherichia coli bacteria during growth up to defined large dimensions. De novo formation of patterns from homogenous starting conditions is observed and studied both experimentally and in simulations. A new theoretical approach is developed for probing pattern stability under perturbations. Quantitative experiments and simulations show that, once established, Min oscillations tolerate a large degree of intracellular heterogeneity, allowing distinctly different patterns to persist in different cells with the same geometry. Min patterns maintain their axes for hours in experiments, despite imperfections, expansion, and changes in cell shape during continuous cell growth. Transitions between multistable Min patterns are found to be rare events induced by strong intracellular perturbations. The instances of multistability studied here are the combined outcome of boundary growth and strongly nonlinear kinetics, which are characteristic of the reaction-diffusion patterns that pervade biology at many scales. PMID:27279643

  5. Intracellular iron concentration of neurons with and without perineuronal nets

    NASA Astrophysics Data System (ADS)

    Fiedler, Anja; Reinert, Tilo; Morawski, Markus; Brückner, Gert; Arendt, Thomas; Butz, Tilman

    2007-07-01

    Neurodegenerative diseases like Parkinson's disease, Alzheimer's disease and Huntington's disease are characterized by abnormally high concentrations of iron in the affected brain areas. Iron is believed to contribute to oxidative stress by catalysing radical generation and subsequently causing neuronal death. Interestingly, subpopulations of neurons are less vulnerable against degeneration. One of these subpopulations possesses a specialized extracellular matrix arranged as a perineuronal net (PN), a structure with poorly understood functions. In order to differentiate between neurons with and without PN according to their iron concentrations we have performed a μPIXE study at the Leipzig LIPSION laboratory. PN-ensheathed neurons in selected brain areas were detected by lectin-histochemical staining with Wisteria floribunda agglutinin (WFA). The staining was intensified by DAB- nickel by an established method enabling the visualisation of the PNs by nuclear microscopy. The cellular concentration of iron in the rat brain was about 1 mmol/l (ca. 30 μg/g dw). First results of subcellular analysis showed that the intracellular iron concentration of PN-ensheathed neurons tends to be slightly increased in comparison to neurons without PNs. The difference in intracellular iron concentrations could be an effect of the PNs.

  6. Structural rearrangement of the intracellular domains during AMPA receptor activation.

    PubMed

    Zachariassen, Linda G; Katchan, Ljudmila; Jensen, Anna G; Pickering, Darryl S; Plested, Andrew J R; Kristensen, Anders S

    2016-07-01

    α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) are ligand-gated ion channels that mediate the majority of fast excitatory neurotransmission in the central nervous system. Despite recent advances in structural studies of AMPARs, information about the specific conformational changes that underlie receptor function is lacking. Here, we used single and dual insertion of GFP variants at various positions in AMPAR subunits to enable measurements of conformational changes using fluorescence resonance energy transfer (FRET) in live cells. We produced dual CFP/YFP-tagged GluA2 subunit constructs that had normal activity and displayed intrareceptor FRET. We used fluorescence lifetime imaging microscopy (FLIM) in live HEK293 cells to determine distinct steady-state FRET efficiencies in the presence of different ligands, suggesting a dynamic picture of the resting state. Patch-clamp fluorometry of the double- and single-insert constructs showed that both the intracellular C-terminal domain (CTD) and the loop region between the M1 and M2 helices move during activation and the CTD is detached from the membrane. Our time-resolved measurements revealed unexpectedly complex fluorescence changes within these intracellular domains, providing clues as to how posttranslational modifications and receptor function interact. PMID:27313205

  7. Intracellular Trafficking of Clostridium perfringens Iota-Toxin b

    PubMed Central

    Umezaki, Mariko; Tashiro, Ryo; Oda, Masataka; Kobayashi, Keiko; Shibutani, Masahiro; Takagishi, Teruhisa; Ishidoh, Kazumi; Fukuda, Mitsunori; Sakurai, Jun

    2012-01-01

    Clostridium perfringens iota-toxin is composed of an enzymatic component (Ia) and a binding component (Ib). Ib binds to a cell surface receptor, undergoes oligomerization in lipid rafts, and binds Ia. The resulting complex is then endocytosed. Here, we show the intracellular trafficking of iota-toxin. After the binding of the Ib monomer with cells at 4°C, oligomers of Ib formed at 37°C and later disappeared. Immunofluorescence staining of Ib revealed that the internalized Ib was transported to early endosomes. Some Ib was returned to the plasma membrane through recycling endosomes, whereas the rest was transported to late endosomes and lysosomes for degradation. Degraded Ib was delivered to the plasma membrane by an increase in the intracellular Ca2+ concentration caused by Ib. Bafilomycin A1, an endosomal acidification inhibitor, caused the accumulation of Ib in endosomes, and both nocodazole and colchicine, microtubule-disrupting agents, restricted Ib's movement in the cytosol. These results indicated that an internalized Ia and Ib complex was delivered to early endosomes and that subsequent delivery of Ia to the cytoplasm occurs mainly in early endosomes. Ib was either sent back to the plasma membranes through recycling endosomes or transported to late endosomes and lysosomes for degradation. Degraded Ib was transported to plasma membranes. PMID:22825447

  8. Origins of intracellular calcium mobilization evoked by infrared laser stimulation

    NASA Astrophysics Data System (ADS)

    Olsovsky, Cory A.; Tolstykh, Gleb P.; Ibey, Bennett L.; Beier, Hope T.

    2015-03-01

    Cellular delivery of pulsed IR laser energy has been shown to stimulate action potentials in neurons. The mechanism for this stimulation is not completely understood. Certain hypotheses suggest the rise in temperature from IR exposure could activate temperature- or pressure-sensitive channels, or create pores in the cellular outer membrane. Studies using intensity-based Ca2+-responsive dyes show changes in Ca2+ levels after various IR stimulation parameters; however, determination of the origin of this signal proved difficult. An influx of larger, typically plasma-membrane-impermeant ions has been demonstrated, which suggests that Ca2+ may originate from the external solution. However, activation of intracellular signaling pathways, possibly indicating a more complex role of increasing Ca2+ concentration, has also been shown. By usingCa2+ sensitive dye Fura-2 and a high-speed ratiometric imaging system that rapidly alternates the excitation wavelengths, we have quantified the Ca2+ mobilization in terms of influx from the external solution and efflux from intracellular organelles. CHO-K1 cells, which lack voltage-gated Ca2+ channels, and NG-108 neuroblastoma cells, which do not produce action potentials in an early undifferentiated state, are used to determine the origin of the Ca2+ signals and investigate the role these mechanisms may play in IR neural stimulation.

  9. An Intracellular Transcriptomic Atlas of the Giant Coenocyte Caulerpa taxifolia

    PubMed Central

    Ranjan, Aashish; Townsley, Brad T.; Ichihashi, Yasunori; Sinha, Neelima R.; Chitwood, Daniel H.

    2015-01-01

    Convergent morphologies have arisen in plants multiple times. In non-vascular and vascular land plants, convergent morphology in the form of roots, stems, and leaves arose. The morphology of some green algae includes an anchoring holdfast, stipe, and leaf-like fronds. Such morphology occurs in the absence of multicellularity in the siphonous algae, which are single cells. Morphogenesis is separate from cellular division in the land plants, which although are multicellular, have been argued to exhibit properties similar to single celled organisms. Within the single, macroscopic cell of a siphonous alga, how are transcripts partitioned, and what can this tell us about the development of similar convergent structures in land plants? Here, we present a de novo assembled, intracellular transcriptomic atlas for the giant coenocyte Caulerpa taxifolia. Transcripts show a global, basal-apical pattern of distribution from the holdfast to the frond apex in which transcript identities roughly follow the flow of genetic information in the cell, transcription-to-translation. The analysis of the intersection of transcriptomic atlases of a land plant and Caulerpa suggests the recurrent recruitment of transcript accumulation patterns to organs over large evolutionary distances. Our results not only provide an intracellular atlas of transcript localization, but also demonstrate the contribution of transcript partitioning to morphology, independent from multicellularity, in plants. PMID:25569326

  10. Estradiol induces functional inactivation of p53 by intracellular redistribution.

    PubMed

    Molinari, A M; Bontempo, P; Schiavone, E M; Tortora, V; Verdicchio, M A; Napolitano, M; Nola, E; Moncharmont, B; Medici, N; Nigro, V; Armetta, I; Abbondanza, C; Puca, G A

    2000-05-15

    Estrogen treatment of MCF-7 cells grown in serum-free medium induced a modification of the intracellular distribution of p53 protein. Western blot analysis and immunofluorescence staining showed that p53 was localized in the nucleus of untreated cell and that after 48 h of hormone treatment, it was mostly localized in the cytoplasm. This effect was blocked by the antiestrogen ICI182,780. Intracellular redistribution of p53 was correlated to a reduced expression of the WAF1/CIP1 gene product and to the presence of degradation fragments of p53 in the cytosol. Estradiol treatment prevented the growth inhibition induced by oligonucleotide transfection, simulating DNA damage. This observation indicated that the wild-type p53 gene product present in the MCF-7 cell could be inactivated by estradiol through nuclear exclusion to permit the cyclin-dependent phosphorylation events leading to the G1-S transition. In addition, the estradiol-induced inactivation of p53 could be involved in the tumorigenesis of estrogen-dependent neoplasm. PMID:10825127

  11. A Transcriptional Reporter of Intracellular Ca2+ in Drosophila

    PubMed Central

    Riabinina, Olena; Li, Jiefu; Potter, Christopher J

    2015-01-01

    Intracellular Ca2+ is a widely used neuronal activity indicator. Here we describe a transcriptional reporter of intracellular Ca2+ (TRIC) in Drosophila, which uses a binary expression system to report Ca2+-dependent interactions between calmodulin and its target peptide. We show that in vitro assays predict in vivo properties of TRIC, and that TRIC signals in sensory systems depend on neuronal activity. TRIC can quantitatively monitor neuronal responses that change slowly, such as those of neuropeptide F-expressing neurons to sexual deprivation and neuroendocrine pars intercerebralis (PI) cells to food and arousal. Furthermore, TRIC-induced expression of a neuronal silencer in nutrient activated cells enhanced stress resistance, providing proof-of-principle that TRIC can be used for circuit manipulation. Thus, TRIC facilitates the monitoring and manipulation of neuronal activity, especially those reflecting slow changes in physiological states that are poorly captured by existing methods. TRIC’s modular design should enable optimization and adaptation to other organisms. PMID:25961791

  12. Prolonged Instability Prior to a Regime Shift

    PubMed Central

    Spanbauer, Trisha L.; Allen, Craig R.; Angeler, David G.; Eason, Tarsha; Fritz, Sherilyn C.; Garmestani, Ahjond S.; Nash, Kirsty L.; Stone, Jeffery R.

    2014-01-01

    Regime shifts are generally defined as the point of ‘abrupt’ change in the state of a system. However, a seemingly abrupt transition can be the product of a system reorganization that has been ongoing much longer than is evident in statistical analysis of a single component of the system. Using both univariate and multivariate statistical methods, we tested a long-term high-resolution paleoecological dataset with a known change in species assemblage for a regime shift. Analysis of this dataset with Fisher Information and multivariate time series modeling showed that there was a∼2000 year period of instability prior to the regime shift. This period of instability and the subsequent regime shift coincide with regional climate change, indicating that the system is undergoing extrinsic forcing. Paleoecological records offer a unique opportunity to test tools for the detection of thresholds and stable-states, and thus to examine the long-term stability of ecosystems over periods of multiple millennia. PMID:25280010

  13. Ultrastructural localization of intracellular calcium stores by a new cytochemical method.

    PubMed

    Poenie, M; Epel, D

    1987-09-01

    We describe a new cytochemical method for ultrastructural localization of intracellular calcium stores. This method uses fluoride ions for in situ precipitation of intracellular calcium during fixation. Comparisons made using oxalate, antimonate, or fluoride showed that fluoride was clearly superior for intracellular calcium localization in eggs of the sea urchin Strongylocentrotus purpuratus. Whereas oxalate generally gave no intracellular precipitate and antimonate gave copious but random precipitate, three prominent calcium stores were detected using fluoride: the tubular endoplasmic reticulum, the cortical granules, and large, clear, acidic vesicles of unknown function. The mitochondria of these eggs generally showed no detectable calcium deposits. X-ray spectra confirmed the presence of calcium in the fluoride precipitates, although in some cases magnesium was also detected. Rat skeletal muscle and sea urchin sperm were used to test the reliability of the fluoride method for calcium localization. In rat skeletal muscle, most fluoride precipitate was confined to the sarcoplasmic reticulum. Using sea urchin sperm, which transport calcium into the mitochondria after exposure to egg jelly to induce the acrosome reaction, the expected result was also obtained. Before the acrosome reaction, sperm mitochondria contain no detectable calcium-containing precipitate. Within 4 min after induction of the acrosome reaction, the expected result was also obtained. Before the acrosome reaction, sperm mitochondria displayed many foci of calcium-containing precipitate. The use of fluoride for intracellular calcium localization therefore appears to be a substantial improvement over previous cytochemical methods. PMID:3611737

  14. Extrinsic periodic information interpolates between monostable and bistable states in intracellular calcium dynamics

    NASA Astrophysics Data System (ADS)

    Lin, Ling; Duan, Wei-Long

    2015-06-01

    Extrinsic periodic information including physiological cyclical and circadian replacement would affect inevitably a real cell, in this paper we investigate the effect of extrinsic periodic information on intracellular calcium dynamics by means of second-order algorithm for stochastic simulation colored noises. By simulating time evolutions and stationary probability distribution of intracellular Ca2+ concentrations, the results show: (i) intracellular calcium oscillation between cytosol and calcium store shows synchronous and anti-synchronous oscillation as intensity and frequency of extrinsic periodic information vary; (ii) extrinsic periodic information interpolates stability from bistable state → monostable state → bistable state → monostable state as frequency of extrinsic periodic information increases; (iii) extrinsic periodic information interpolates stability from monostable state → bistable state as intensity of extrinsic periodic information increases.

  15. Silicon chips detect intracellular pressure changes in living cells

    NASA Astrophysics Data System (ADS)

    Gómez-Martínez, Rodrigo; Hernández-Pinto, Alberto M.; Duch, Marta; Vázquez, Patricia; Zinoviev, Kirill; de La Rosa, Enrique J.; Esteve, Jaume; Suárez, Teresa; Plaza, José A.

    2013-07-01

    The ability to measure pressure changes inside different components of a living cell is important, because it offers an alternative way to study fundamental processes that involve cell deformation. Most current techniques such as pipette aspiration, optical interferometry or external pressure probes use either indirect measurement methods or approaches that can damage the cell membrane. Here we show that a silicon chip small enough to be internalized into a living cell can be used to detect pressure changes inside the cell. The chip, which consists of two membranes separated by a vacuum gap to form a Fabry-Pérot resonator, detects pressure changes that can be quantified from the intensity of the reflected light. Using this chip, we show that extracellular hydrostatic pressure is transmitted into HeLa cells and that these cells can endure hypo-osmotic stress without significantly increasing their intracellular hydrostatic pressure.

  16. Drug-induced QT interval prolongation and torsades de pointes

    PubMed Central

    Tisdale, James E.

    2016-01-01

    Torsades de pointes (TdP) is a life-threatening arrhythmia associated with prolongation of the corrected QT (QTc) interval on the electrocardiogram. More than 100 drugs available in Canada, including widely used antibiotics, antidepressants, cardiovascular drugs and many others, may cause QTc interval prolongation and TdP. Risk factors for TdP include QTc interval >500 ms, increase in QTc interval ≥60 ms from the pretreatment value, advanced age, female sex, acute myocardial infarction, heart failure with reduced ejection fraction, hypokalemia, hypomagnesemia, hypocalcemia, bradycardia, treatment with diuretics and elevated plasma concentrations of QTc interval–prolonging drugs due to drug interactions, inadequate dose adjustment of renally eliminated drugs in patients with kidney disease and rapid intravenous administration. Pharmacokinetic drug interactions associated with the highest risk of TdP include antifungal agents, macrolide antibiotics (except azithromycin) and drugs to treat human immunodeficiency virus interacting with amiodarone, disopyramide, dofetilide or pimozide. Other important pharmacokinetic interactions include antidepressants (bupropion, duloxetine, fluoxetine, paroxetine) interacting with flecainide, quinidine or thioridazine. Pharmacists play an important role in minimizing the risk of drug-induced QTc interval prolongation and TdP through knowledge of drugs that are associated with a known or possible risk of TdP, individualized assessment of risk of drug-induced QTc interval prolongation, awareness of drug interactions most likely to result in TdP and attention to dose reduction of renally eliminated QTc interval-prolonging drugs in patients with kidney disease. Treatment of hemodynamically stable TdP consists of discontinuation of the offending drug(s), correction of electrolyte abnormalities and administration of intravenous magnesium sulfate 1 to 2 g. PMID:27212965

  17. Prolonged QT interval and reduced heart rate variability in patients with uncomplicated essential hypertension.

    PubMed

    Maule, Simona; Rabbia, Franco; Perni, Valentina; Tosello, Francesco; Bisbocci, Daniela; Mulatero, Paolo; Veglio, Franco

    2008-11-01

    A prolonged QT interval is a risk factor for ischemic heart disease in hypertensive subjects. Heart rate variability (HRV) is both an index of autonomic function and an important prognostic factor in several diseases. The aim of the present study was to evaluate the relation between a prolonged QT interval and autonomic nervous system function in patients with untreated uncomplicated essential hypertension. Two hundred and fifteen untreated patients with essential hypertension underwent a Holter ECG equipped with software dedicated to HRV and QT analyses. Nine percent of the patients showed a corrected QT (QTc) >or=440 ms. The HRV indexes in the time domain (SDNN, SDNN index, RMSSD, and pNN50) were significantly reduced in the patients with a prolonged QTc compared to those with a normal QTc (SDNN 24 h: 126.4+/-29.9 vs. 143.9+/-35.4 ms, p=0.02; SDNN index [nighttime]: 85.9+/-32.4 vs. 115.5+/-36.7 ms, p=0.0006; RMSSD 24 h: 22.2+/-7.7 vs. 31.2+/-13.0 ms, p=0.0007; pNN50 24 h: 4.4+/-4.9 vs. 9.7+/-8.4%, p=0.0006). The linear correlation analysis between QTc length and HRV parameters showed a significant negative correlation with all the time-domain indexes. Such a correlation was maintained for RMSSD 24 h, pNN50 24 h and SDNN index (nighttime) after correction for gender and age. The present study shows that, even prior to the development of cardiac hypertensive disease, a prolongation of the QTc and a reduced HRV, both markers of cardiovascular risk, coexist in a proportion of patients with untreated essential hypertension. Further studies are warranted to evaluate whether the combination of such markers can identify hypertensive patients at risk for life-threatening arrhythmias and sudden death. (Hypertens Res 2008; 31: 2003-2010). PMID:19098371

  18. Spontaneous hepatic hemorrhage secondary to prolonged use of oral contraceptives.

    PubMed

    Jaffar, Reema; Pechet, Liberto; Whalen, Giles Francis; Banner, Barbara F

    2010-05-15

    Oral contraceptive pills (OCP) are the most commonly used form of contraception throughout the United States of America. The prolonged usage of oral contraceptives leads to a variety of complications, ranging from subclinical modifications of liver function tests to the development of benign and malignant tumors of the liver. Spontaneous hepatic hemorrhage secondary to oral contraceptive use was only reported once in the early 1980s. We report a case of spontaneous hepatic hemorrhage secondary to prolonged ingestion of combined OCPs followed by multiple pulmonary emboli without underlying thrombophilia. PMID:19577854

  19. Interventions in the prolongation of reproductive life in women.

    PubMed

    Barlow, David H

    2011-03-01

    Women may seek to prolong their reproductive span for a variety of reasons. For many this implies reproduction at a late age, possibly driven by lifestyle decisions, but for others affected by a natural or a cancer treatment-induced premature ovarian failure it may simply mean seeking to achieve the normal reproductive span. The range of interventions now available to address the issue of prolonging reproductive life has never been greater, although several of the approaches discussed remain in the realm of future application through being dependent on ongoing scientific developments. PMID:21401623

  20. Changes in the human blood coagulating system during prolonged hypokinesia

    NASA Technical Reports Server (NTRS)

    Filatova, L. M.; Anashkin, O. D.

    1978-01-01

    Changes in the coagulating system of the blood were studied in six subjects during prolonged hypokinesia. Thrombogenic properties of the blood rose in all cases on the 8th day. These changes are explained by stress reaction due to unusual conditions for a healthy person. Changes in the blood coagulating system in the group subjected to physical exercise and without it ran a practically parallel course. Apparently physical exercise is insufficient to prevent such changes that appear in the coagulating system of the blood during prolonged hypokinesia.

  1. Risk assessment of drug-induced QT prolongation

    PubMed Central

    Isbister, Geoffrey K

    2015-01-01

    SUMMARY Drugs can cause prolongation of the QT interval, alone or in combination, potentially leading to fatal arrhythmias such as torsades de pointes. When prescribing drugs that prolong the QT interval, the balance of benefit versus harm should always be considered. Readouts from automated ECG machines are unreliable. The QT interval should be measured manually. Changes in heart rate influence the absolute QT interval. Heart rate correction formulae are inaccurate, particularly for fast and slow heart rates. The QT nomogram, a plot of QT interval versus heart rate, can be used as a risk assessment tool to detect an abnormal QT interval. PMID:26648606

  2. Behavioral evidence of prolonged interhemispheric transfer time among psychopathic offenders

    PubMed Central

    Hiatt, Kristina D.; Newman, Joseph P.

    2009-01-01

    Several lines of evidence suggest the possibility of abnormal interhemispheric communication in psychopathy, but there have been few direct empirical studies. To address this gap in the literature, we examined one important aspect of interhemispheric communication, the efficiency with which information is transferred across the corpus callosum. Using Poffenberger's (1912) paradigm for estimating interhemispheric transfer time (IHTT) from simple motor responses to lateralized stimuli, we found a substantially prolonged IHTT among psychopathic criminals relative to nonpsychopathic criminals. This prolonged IHTT was somewhat more pronounced when participants were using their right hand to respond. This study provides initial behavioral evidence of slowed interhemispheric transfer in psychopathy. PMID:17484594

  3. Interleukin-1 deficiency prolongs ovarian lifespan in mice

    PubMed Central

    Uri-Belapolsky, Shiri; Shaish, Aviv; Eliyahu, Efrat; Grossman, Hadas; Levi, Mattan; Chuderland, Dana; Ninio-Many, Lihi; Hasky, Noa; Shashar, David; Almog, Tal; Kandel-Kfir, Michal; Harats, Dror; Shalgi, Ruth; Kamari, Yehuda

    2014-01-01

    Oocyte endowment dwindles away during prepubertal and adult life until menopause occurs, and apoptosis has been identified as a central mechanism responsible for oocyte elimination. A few recent reports suggest that uncontrolled inflammation may adversely affect ovarian reserve. We tested the possible role of the proinflammatory cytokine IL-1 in the age-related exhaustion of ovarian reserve using IL-1α and IL-1β–KO mice. IL-1α–KO mice showed a substantially higher pregnancy rate and litter size compared with WT mice at advanced age. The number of secondary and antral follicles was significantly higher in 2.5-mo-old IL-1α–KO ovaries compared with WT ovaries. Serum anti-Müllerian hormone, a putative marker of ovarian reserve, was markedly higher in IL-1α–KO mice from 2.5 mo onward, along with a greater ovarian response to gonadotropins. IL-1β–KO mice displayed a comparable but more subtle prolongation of ovarian lifespan compared with IL-1α–KO mice. The protein and mRNA of both IL-1α and IL-1β mice were localized within the developing follicles (oocytes and granulosa cells), and their ovarian mRNA levels increased with age. Molecular analysis revealed decreased apoptotic signaling [higher B-cell lymphoma 2 (BCL-2) and lower BCL-2–associated X protein levels], along with a marked attenuation in the expression of genes coding for the proinflammatory cytokines IL-1β, IL-6, and TNF-α in ovaries of IL-1α–KO mice compared with WT mice. Taken together, IL-1 emerges as an important participant in the age-related exhaustion of ovarian reserve in mice, possibly by enhancing the expression of inflammatory genes and promoting apoptotic pathways. PMID:25114230

  4. Central and regional hemodynamics in prolonged space flights

    NASA Astrophysics Data System (ADS)

    Gazenko, O. G.; Shulzhenko, E. B.; Turchaninova, V. F.; Egorov, A. D.

    This paper presents the results of measuring central and regional (head, forearm, calf) hemodynamics at rest and during provocative tests by the method of tetrapolar rheography in the course of Salyut-6-Soyuz and Salyut-7-Soyuz missions. The measurements were carried out during short-term (19 man-flights of 7 days in duration) and long-term (21 man-flights of 65-237 days in duration) manned missions. At rest, stroke volume (SV) and cardiac output (CO) as well as heart rate (HR) decreased insignificantly (in short-term flights) or remained essentially unchanged (in long-term flights). In prolonged flights CO increased significantly in response to exercise tests due to an increase in HR and the lack of changes in SV. After exercise tests SV and CO decreased as compared to the preflight level. During lower body negative pressure (LBNP) tests HR and CO were slightly higher than preflight. Changes in regional hemodynamics included a distinct decrease of pulse blood filling (PBF) of the calf, a reduction of the tone of large vessels of the calf and small vessels of the forearm. Head examination (in the region of the internal carotid artery) showed a decrease of PBF of the left hemisphere (during flight months 2-8) and a distinct decline of the tone of small vessels, mainly, in the right hemisphere. During LBNP tests the tone of pre- and postcapillary vessels of the brain returned to normal while PBF of the right and left hemisphere vessels declined. It has been shown that regional circulation variations depend on the area examined and are induced by a rearrangement of total hemodynamics of the human body in microgravity. This paper reviews the data concerning changes in central and regional circulation of men in space flights of different duration.

  5. Depollution potential of three macrophytes: exudated, wall-bound and intracellular peroxidase activities plus intracellular phenol concentrations.

    PubMed

    Larue, Camille; Korboulewsky, Nathalie; Wang, Runying; Mévy, Jean-Philippe

    2010-10-01

    The aim of this study was to investigate the potential role of three macrophyte species (Iris pseudacorus, Typha latifolia and Phragmites australis) for detoxication of xenobiotics, and to study their variations with seasons or concentrations of sewage sludge from the food industry. For this purpose, some aspects of the green liver concept were explored through peroxidase measurements in three compartments in roots: intracellular, cell wall and extracellular. In addition, phenol concentrations were also measured in order to assess heavy metal detoxication potential. Enzyme activities and phenol concentrations were overall lower in winter according to the phenological stages and some sludge effects occurred. Results show that P. australis roots exuded and contained more peroxidase in all seasons: 17 U/g (1373 U/g protein), 0.8 U/g (613 U/g protein) and 4.8 U/g (1329 U/g protein) in intracellular compartments, cell wall and exudates, respectively. In contrast, the highest phenol concentration was found in I. pseudacorus roots: 3.58 mg eq. [corrected] gallic acid/g. Hence, in constructed wetlands, P. australis is suitable for organic waste water treatment, while I. pseudacorus should be used in the case of waters highly charged with heavy metals. PMID:20570142

  6. QTc interval prolongation in HIV-infected patients: a case–control study by 24-hour Holter ECG recording

    PubMed Central

    2012-01-01

    Background Aim of the study was to assess QTc interval by a 24-hour ECG recording in a group of HIV-infected individuals with a basal prolonged QTc. The risk factors associated with QTc prolongation and the indices of cardiovascular autonomic control were also evaluated. Methods A case–control study was performed using as cases 32 HIV-infected patients with prolonged (>440 msec) QTc interval as assessed by Holter ECG, and as controls 64 HIV-infected subjects with normal QTc interval. Autonomic function was evaluated by heart rate variability analysis during 24-hour recording. Results Duration of HIV disease was significantly longer among cases than among controls (p=0.04). Waist/hip ratio was also higher among cases than among controls (p=0.05). Frequency domain analysis showed the absence of physiologic decrease of low frequency (LF) in the night period in both cases and controls. The LF night in cases showed a statistically significant reduction when compared with controls (p=0.007). Conclusions In our study group, QTc interval prolongation was associated with a longer duration of HIV infection and with a greater waist/hip ratio. HIV patients with QTc interval prolongation and with a longer duration of HIV infection were more likely to have an impairment of parasympathetic and sympathetic cardiac component. PMID:23259665

  7. The control of intracellular glycerol in Saccharomyces cerevisiae influences osmotic stress response and resistance to increased temperature.

    PubMed

    Siderius, M; Van Wuytswinkel, O; Reijenga, K A; Kelders, M; Mager, W H

    2000-06-01

    Glycerol has been demonstrated to serve as the major osmolyte of Saccharomyces cerevisiae. Consistently, mutant strains gpd1gpd2 and gpp1gpp2, which are devoid of the main glycerol biosynthesis pathway, have been shown to be osmosensitive. In addition, the primary hyperosmotic stress response is affected in these strains. Hog1p phosphorylation turned out to be prolonged and osmostress-induced gene expression is delayed compared with the kinetics observed in wild-type cells. A hog1 deletion strain was previously found to contain lower internal glycerol and therefore displays an osmosensitive phenotype. Here, we show that the osmosensitivity of hog1 is suppressed by growth at 37 degrees C. We reasoned that this temperature-remedial osmoresistance might be caused by a higher intracellular glycerol level at the elevated temperature. This hypothesis was confirmed by measurement of the glycerol concentration, which was shown to be similar for wild type and hog1 cells only at elevated growth temperatures. In agreement with this finding, hog1 cells containing an fps1 allele, encoding a constitutively open glycerol channel, have lost their temperature-remedial osmoresistance. Furthermore, gpd1gpd2 and gpp1gpp2 strains were found to be temperature sensitive. The growth defect of these strains could be suppressed by adding external glycerol. In conclusion, the ability to control glycerol levels influences proper osmostress-induced signalling and the cellular potential to grow at elevated temperatures. These data point to an important, as yet unidentified, role of glycerol in cellular functioning. PMID:10931288

  8. Alk7 Depleted Mice Exhibit Prolonged Cardiac Repolarization and Are Predisposed to Ventricular Arrhythmia

    PubMed Central

    Ying, Shaozhen; Cao, Hong; Hu, He; Wang, Xin; Tang, Yanhong; Huang, Congxin

    2016-01-01

    We aimed to investigate the role of activin receptor-like kinase (ALK7) in regulating cardiac electrophysiology. Here, we showed that Alk7-/- mice exhibited prolonged QT intervals in telemetry ECG recordings. Furthermore, Langendorff-perfused Alk7-/- hearts had significantly longer action potential duration (APD) and greater incidence of ventricular arrhythmia (AV) induced by burst pacing. Using whole-cell patch clamp, we found that the densities of repolarizing K+ currents Ito and IK1 were profoundly reduced in Alk7-/- ventricular cardiomyocytes. Mechanistically, the expression of Kv4.2 (a major subunit of Ito carrying channel) and KCHIP2 (a key accessory subunit of Ito carrying channel), was markedly decreased in Alk7-/- hearts. These findings suggest that endogenous expression of ALK7 is necessary to maintain repolarizing K+ currents in ventricular cardiomyocytes, and finally prevent action potential prolongation and ventricular arrhythmia. PMID:26882027

  9. Atomistic study of grain boundary sink strength under prolonged electron irradiation.

    SciTech Connect

    Zhang, Y.; Huang, H.; Millett, P. C.; Tonks, M.; Wolf, D.; Phillpot, S. R.

    2012-01-01

    Grain boundaries (GBs) can act as either sinks or sources of the point defects that are produced in large numbers under irradiation damage. In polycrystalline materials, as the grain size decreases, more of the point defects resulting from irradiation damage annihilate at GBs. It is unknown, however, whether the GB sink efficiency will saturate after prolonged defect annihilation, particularly when the grain size is of nanoscale dimensions. Using a combination of molecular dynamics (MD) simulation and rate theory, the authors show that high-energy GBs in body-centered-cubic (BCC) Mo do not saturate as sinks of point defects. The MD simulations serve to provide direct measurement of defect evolution, and the rate theory serves both to test whether grain boundary sink strength is constant during prolonged defect annihilation, and to extend the MD results to realistic defect production rates.

  10. Effect of Prolonged Moderate Exercise on the Changes of Nonneuronal Cells in Early Myocardial Infarction.

    PubMed

    Rinaldi, Barbara; Guida, Francesca; Furiano, Anna; Donniacuo, Maria; Luongo, Livio; Gritti, Giulia; Urbanek, Konrad; Messina, Giovanni; Maione, Sabatino; Rossi, Francesco; de Novellis, Vito

    2015-01-01

    Myocardial infarction (MI) is one of the leading causes of death in developed countries and it is characterized by several associated symptomatologies and poor quality of life. Recent data showed a possible interaction between infarction and brain inflammation and activity. Previous studies have demonstrated the beneficial effect of exercise training on deterioration in cardiac function after MI. In this study we analyzed in sedentary and trained rats the microglia and astrocytes 48 hours after MI in PVN, thalamus, prefrontal cortex, and hippocampus through immunofluorescence approach. We found significant changes in specific microglia phenotypes in the brain areas analyzed together with astrocytes activation. Prolonged exercise normalized these morphological changes of microglia and astrocytes in the prefrontal cortex, hippocampus, and thalamus but not in the PVN. Our data suggest that there is an early brain reaction to myocardial infarction induction, involving nonneuronal cells, that is attenuated by the prolonged exercise. PMID:26266053

  11. Effect of Prolonged Moderate Exercise on the Changes of Nonneuronal Cells in Early Myocardial Infarction

    PubMed Central

    Rinaldi, Barbara; Guida, Francesca; Furiano, Anna; Donniacuo, Maria; Luongo, Livio; Gritti, Giulia; Urbanek, Konrad; Messina, Giovanni; Maione, Sabatino; Rossi, Francesco; de Novellis, Vito

    2015-01-01

    Myocardial infarction (MI) is one of the leading causes of death in developed countries and it is characterized by several associated symptomatologies and poor quality of life. Recent data showed a possible interaction between infarction and brain inflammation and activity. Previous studies have demonstrated the beneficial effect of exercise training on deterioration in cardiac function after MI. In this study we analyzed in sedentary and trained rats the microglia and astrocytes 48 hours after MI in PVN, thalamus, prefrontal cortex, and hippocampus through immunofluorescence approach. We found significant changes in specific microglia phenotypes in the brain areas analyzed together with astrocytes activation. Prolonged exercise normalized these morphological changes of microglia and astrocytes in the prefrontal cortex, hippocampus, and thalamus but not in the PVN. Our data suggest that there is an early brain reaction to myocardial infarction induction, involving nonneuronal cells, that is attenuated by the prolonged exercise. PMID:26266053

  12. VIEW SHOWING WEST ELEVATION, EAST SIDE OF MEYER AVENUE. SHOWS ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    VIEW SHOWING WEST ELEVATION, EAST SIDE OF MEYER AVENUE. SHOWS 499-501, MUNOZ HOUSE (AZ-73-37) ON FAR RIGHT - Antonio Bustamente House, 485-489 South Meyer Avenue & 186 West Kennedy Street, Tucson, Pima County, AZ

  13. 15. Detail showing lower chord pinconnected to vertical member, showing ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    15. Detail showing lower chord pin-connected to vertical member, showing floor beam riveted to extension of vertical member below pin-connection, and showing brackets supporting cantilevered sidewalk. View to southwest. - Selby Avenue Bridge, Spanning Short Line Railways track at Selby Avenue between Hamline & Snelling Avenues, Saint Paul, Ramsey County, MN

  14. Glycosaminoglycans: Sorting determinants in intracellular protein traffic.

    PubMed

    Mihov, Deyan; Spiess, Martin

    2015-11-01

    Intracellular transport of proteins to their appropriate destinations is crucial for the maintenance of cellular integrity and function. Sorting information is contained either directly in the amino acid sequence or in a protein's post-translational modifications. Glycosaminoglycans (GAGs) are characteristic modifications of proteoglycans. GAGs are long unbranched polysaccharide chains with unique structural and functional properties also contributing to protein sorting in various ways. By deletion or insertion of GAG attachment sites it has been shown that GAGs affect polarized sorting in epithelial cells, targeting to and storage in secretory granules, and endocytosis. Most recently, the role of GAGs as signals for rapid trans-Golgi-to-cell surface transport, dominant over the cytosolic sorting motifs in the core protein, was demonstrated. Here, we provide an overview on existing data on the roles of GAGs on protein and proteoglycan trafficking. PMID:26327396

  15. Intracellular pH in Sperm Physiology

    PubMed Central

    Nishigaki, Takuya; José, Omar; González-Cota, Ana Laura; Romero, Francisco; Treviño, Claudia L.; Darszon, Alberto

    2014-01-01

    Intracellular pH (pHi) regulation is essential for cell function. Notably, several unique sperm ion transporters and enzymes whose elimination causes infertility are either pHi dependent or somehow related to pHi regulation. Amongst them are: CatSper, a Ca2+ channel; Slo3, a K+ channel; the sperm-specific Na+/H+ exchanger and the soluble adenylyl cyclase. It is thus clear that pHi regulation is of the utmost importance for sperm physiology. This review briefly summarizes the key components involved in pHi regulation, their characteristics and participation in fundamental sperm functions such as motility, maturation and the acrosome reaction. PMID:24887564

  16. Impaired intracellular trafficking defines early Parkinson's disease

    PubMed Central

    Hunn, Benjamin H.M.; Cragg, Stephanie J.; Bolam, J. Paul; Spillantini, Maria-Grazia; Wade-Martins, Richard

    2015-01-01

    Parkinson's disease (PD) is an insidious and incurable neurodegenerative disease, and represents a significant cost to individuals, carers, and ageing societies. It is defined at post-mortem by the loss of dopamine neurons in the substantia nigra together with the presence of Lewy bodies and Lewy neurites. We examine here the role of α-synuclein and other cellular transport proteins implicated in PD and how their aberrant activity may be compounded by the unique anatomy of the dopaminergic neuron. This review uses multiple lines of evidence from genetic studies, human tissue, induced pluripotent stem cells, and refined animal models to argue that prodromal PD can be defined as a disease of impaired intracellular trafficking. Dysfunction of the dopaminergic synapse heralds trafficking impairment. PMID:25639775

  17. Intracellular signalling by C-peptide.

    PubMed

    Hills, Claire E; Brunskill, Nigel J

    2008-01-01

    C-peptide, a cleavage product of the proinsulin molecule, has long been regarded as biologically inert, serving merely as a surrogate marker for insulin release. Recent findings demonstrate both a physiological and protective role of C-peptide when administered to individuals with type I diabetes. Data indicate that C-peptide appears to bind in nanomolar concentrations to a cell surface receptor which is most likely to be G-protein coupled. Binding of C-peptide initiates multiple cellular effects, evoking a rise in intracellular calcium, increased PI-3-kinase activity, stimulation of the Na(+)/K(+) ATPase, increased eNOS transcription, and activation of the MAPK signalling pathway. These cell signalling effects have been studied in multiple cell types from multiple tissues. Overall these observations raise the possibility that C-peptide may serve as a potential therapeutic agent for the treatment or prevention of long-term complications associated with diabetes. PMID:18382618

  18. Intracellular dynamics with the phase microscope Airyscan

    NASA Astrophysics Data System (ADS)

    Tychinsky, Vladimir P.; Perevedentseva, Elena V.; Vyshenskaia, Tatiana V.; Kufal, Georgy E.

    1997-12-01

    Investigation of intracellular dynamics of Allium cepa inner epidermal cells are described. The applicability of the method for quantitative estimation of spatio-temporal phase fluctuations and the effect due to external factors is discussed. The analysis of time-sampled series allows one to detect the regions of various motility in cytoplasm. The intense Fourier-spectra harmonics in 0.2 - 8 Hz interval were observed inside a cell wall and cytoplasm. Regularly spaced 2- to 4-s long batches of 100-ms pulses at cell-wall sites are recorded. The phase-fluctuation intensity decreased and the frequencies of certain harmonics were shifted with lowering temperature. The advantages and specific features of the method are discussed.

  19. Optogenetic control of intracellular signaling pathways

    PubMed Central

    Zhang, Kai; Cui, Bianxiao

    2014-01-01

    Cells employ a plethora of signaling pathways to make their life-and-death decisions. Extensive genetic, biochemical, and physiological studies have led to the accumulation of knowledge about signaling components and their interactions within signaling networks. These conventional approaches, though useful, lack the ability to control the spatial and temporal aspects of signaling processes. The recently emerged optogenetic tools open up exciting opportunities by enabling signaling regulation with superior temporal and spatial resolution, easy delivery, rapid reversibility, fewer off-target side effects, and the ability to dissect complex signaling networks. Here we review recent achievements in using light to control intracellular signaling pathways, and discuss future prospects for the field, including integration of new genetic approaches into optogenetics. PMID:25529484

  20. Intracellular domoic acid production in Pseudo-nitzschia multistriata isolated from the Gulf of Naples (Tyrrhenian Sea, Italy).

    PubMed

    Amato, Alberto; Lüdeking, Alexander; Kooistra, Wiebe H C F

    2010-01-01

    Twenty-six Pseudo-nitzschia multistriata cultures were tested for intracellular domoic acid production and fourteen were found to be toxic. Four suboptimal growth conditions were compared with conditions observed to be optimal to explore possible triggers for intracellular domoic acid production. Silica- and phosphate-limitation and low light treatment induced elevated toxin concentrations whereas high temperature appeared to suppress it. Inheritance of the toxin-production ability was investigated by measuring intracellular toxin content in a total of thirty-nine F(1) strains from two different crosses. Results showed radical differences in domoic acid levels among the F(1) offspring from the same parents. PMID:19615395

  1. Crude oil exposures reveal roles for intracellular calcium cycling in haddock craniofacial and cardiac development

    NASA Astrophysics Data System (ADS)

    Sørhus, Elin; Incardona, John P.; Karlsen, Ørjan; Linbo, Tiffany; Sørensen, Lisbet; Nordtug, Trond; van der Meeren, Terje; Thorsen, Anders; Thorbjørnsen, Maja; Jentoft, Sissel; Edvardsen, Rolf B.; Meier, Sonnich

    2016-08-01

    Recent studies have shown that crude oil exposure affects cardiac development in fish by disrupting excitation-contraction (EC) coupling. We previously found that eggs of Atlantic haddock (Melanogrammus aeglefinus) bind dispersed oil droplets, potentially leading to more profound toxic effects from uptake of polycyclic aromatic hydrocarbons (PAHs). Using lower concentrations of dispersed crude oil (0.7–7 μg/L ∑PAH), here we exposed a broader range of developmental stages over both short and prolonged durations. We quantified effects on cardiac function and morphogenesis, characterized novel craniofacial defects, and examined the expression of genes encoding potential targets underlying cardiac and craniofacial defects. Because of oil droplet binding, a 24-hr exposure was sufficient to create severe cardiac and craniofacial abnormalities. The specific nature of the craniofacial abnormalities suggests that crude oil may target common craniofacial and cardiac precursor cells either directly or indirectly by affecting ion channels and intracellular calcium in particular. Furthermore, down-regulation of genes encoding specific components of the EC coupling machinery suggests that crude oil disrupts excitation-transcription coupling or normal feedback regulation of ion channels blocked by PAHs. These data support a unifying hypothesis whereby depletion of intracellular calcium pools by crude oil-derived PAHs disrupts several pathways critical for organogenesis in fish.

  2. Chemical Analysis of Drug Biocrystals: A Role for Counterion Transport Pathways in Intracellular Drug Disposition.

    PubMed

    Keswani, Rahul K; Baik, Jason; Yeomans, Larisa; Hitzman, Chuck; Johnson, Allison M; Pawate, Ashtamurthy S; Kenis, Paul J A; Rodriguez-Hornedo, Nair; Stringer, Kathleen A; Rosania, Gus R

    2015-07-01

    In mammals, highly lipophilic small molecule chemical agents can accumulate as inclusions within resident tissue macrophages. In this context, we characterized the biodistribution, chemical composition, and structure of crystal-like drug inclusions (CLDIs) formed by clofazimine (CFZ), a weakly basic lipophilic drug. With prolonged oral dosing, CFZ exhibited a significant partitioning with respect to serum and fat due to massive bioaccumulation and crystallization in the liver and spleen. The NMR, Raman, and powder X-ray diffraction (p-XRD) spectra of CLDIs isolated from the spleens of CFZ-treated mice matched the spectra of pure, CFZ hydrochloride crystals (CFZ-HCl). Elemental analysis revealed a 237-fold increase in chlorine content in CLDIs compared to untreated tissue samples and a 5-fold increase in chlorine content compared to CFZ-HCl, suggesting that the formation of CLDIs occurs through a chloride mediated crystallization mechanism. Single crystal analysis revealed that CFZ-HCl crystals had a densely packed orthorhombic lattice configuration. In vitro, CFZ-HCl formed at a pH of 4-5 only if chloride ions were present at sufficiently high concentrations (>50:1 Cl(-)/CFZ), indicating that intracellular chloride transport mechanisms play a key role in the formation of CLDIs. While microscopy and pharmacokinetic analyses clearly revealed crystallization and intracellular accumulation of the drug in vivo, the chemical and structural characterization of CLDIs implicates a concentrative, chloride transport mechanism, paralleling and thermodynamically stabilizing the massive bioaccumulation of a weakly basic drug. PMID:25926092

  3. Crude oil exposures reveal roles for intracellular calcium cycling in haddock craniofacial and cardiac development.

    PubMed

    Sørhus, Elin; Incardona, John P; Karlsen, Ørjan; Linbo, Tiffany; Sørensen, Lisbet; Nordtug, Trond; van der Meeren, Terje; Thorsen, Anders; Thorbjørnsen, Maja; Jentoft, Sissel; Edvardsen, Rolf B; Meier, Sonnich

    2016-01-01

    Recent studies have shown that crude oil exposure affects cardiac development in fish by disrupting excitation-contraction (EC) coupling. We previously found that eggs of Atlantic haddock (Melanogrammus aeglefinus) bind dispersed oil droplets, potentially leading to more profound toxic effects from uptake of polycyclic aromatic hydrocarbons (PAHs). Using lower concentrations of dispersed crude oil (0.7-7 μg/L ∑PAH), here we exposed a broader range of developmental stages over both short and prolonged durations. We quantified effects on cardiac function and morphogenesis, characterized novel craniofacial defects, and examined the expression of genes encoding potential targets underlying cardiac and craniofacial defects. Because of oil droplet binding, a 24-hr exposure was sufficient to create severe cardiac and craniofacial abnormalities. The specific nature of the craniofacial abnormalities suggests that crude oil may target common craniofacial and cardiac precursor cells either directly or indirectly by affecting ion channels and intracellular calcium in particular. Furthermore, down-regulation of genes encoding specific components of the EC coupling machinery suggests that crude oil disrupts excitation-transcription coupling or normal feedback regulation of ion channels blocked by PAHs. These data support a unifying hypothesis whereby depletion of intracellular calcium pools by crude oil-derived PAHs disrupts several pathways critical for organogenesis in fish. PMID:27506155

  4. Chemical Analysis of Drug Biocrystals: A Role for Counterion Transport Pathways in Intracellular Drug Disposition

    PubMed Central

    Keswani, Rahul K.; Baik, Jason; Yeomans, Larisa; Hitzman, Chuck; Johnson, Allison; Pawate, Ashtamurthy; Kenis, Paul J. A.; Rodriguez-Hornedo, Nair; Stringer, Kathleen A.; Rosania, Gus R.

    2015-01-01

    In mammals, highly lipophilic small molecule chemical agents can accumulate as inclusions within resident tissue macrophages. In this context, we characterized the biodistribution, chemical composition and structure of crystal-like drug inclusions (CLDIs) formed by clofazimine (CFZ), a weakly basic lipophilic drug. With prolonged oral dosing, CFZ exhibited a significant partitioning with respect to serum and fat due to massive bioaccumulation and crystallization in the liver and spleen. The NMR, Raman and Powder X-ray diffraction (p-XRD) spectra of CLDIs isolated from the spleens of CFZ-treated mice matched the spectra of pure, CFZ hydrochloride crystals (CFZ-HCl). Elemental analysis revealed a 237-fold increase in chlorine content in CLDIs compared to untreated tissue samples, and a five-fold increase in chlorine content compared to CFZ-HCl, suggesting that the formation of CLDIs occurs through a chloride mediated crystallization mechanism. Single crystal analysis revealed that CFZ-HCl crystals had a densely-packed orthorhombic lattice configuration. In vitro, CFZ-HCl formed at a pH of 4–5 only if chloride ions were present at sufficiently high concentrations (>50:1 Cl−:CFZ), indicating that intracellular chloride transport mechanisms play a key role in the formation of CLDIs. While microscopy and pharmacokinetic analyses clearly revealed crystallization and intracellular accumulation of the drug in vivo, the chemical and structural characterization of CLDIs implicates a concentrative, chloride transport mechanism, paralleling and thermodynamically stabilizing, the massive bioaccumulation of a weakly basic drug. PMID:25926092

  5. Crude oil exposures reveal roles for intracellular calcium cycling in haddock craniofacial and cardiac development

    PubMed Central

    Sørhus, Elin; Incardona, John P.; Karlsen, Ørjan; Linbo, Tiffany; Sørensen, Lisbet; Nordtug, Trond; van der Meeren, Terje; Thorsen, Anders; Thorbjørnsen, Maja; Jentoft, Sissel; Edvardsen, Rolf B.; Meier, Sonnich

    2016-01-01

    Recent studies have shown that crude oil exposure affects cardiac development in fish by disrupting excitation-contraction (EC) coupling. We previously found that eggs of Atlantic haddock (Melanogrammus aeglefinus) bind dispersed oil droplets, potentially leading to more profound toxic effects from uptake of polycyclic aromatic hydrocarbons (PAHs). Using lower concentrations of dispersed crude oil (0.7–7 μg/L ∑PAH), here we exposed a broader range of developmental stages over both short and prolonged durations. We quantified effects on cardiac function and morphogenesis, characterized novel craniofacial defects, and examined the expression of genes encoding potential targets underlying cardiac and craniofacial defects. Because of oil droplet binding, a 24-hr exposure was sufficient to create severe cardiac and craniofacial abnormalities. The specific nature of the craniofacial abnormalities suggests that crude oil may target common craniofacial and cardiac precursor cells either directly or indirectly by affecting ion channels and intracellular calcium in particular. Furthermore, down-regulation of genes encoding specific components of the EC coupling machinery suggests that crude oil disrupts excitation-transcription coupling or normal feedback regulation of ion channels blocked by PAHs. These data support a unifying hypothesis whereby depletion of intracellular calcium pools by crude oil-derived PAHs disrupts several pathways critical for organogenesis in fish. PMID:27506155

  6. Diet-consumer nitrogen isotope fractionation for prolonged fasting arthropods.

    PubMed

    Mizota, Chitoshi; Yamanaka, Toshiro

    2011-12-01

    Nitrogen acquisition for cellular metabolism during diapause is a primary concern for herbivorous arthropods. Analyses of naturally occurring stable isotopes of nitrogen help elucidate the mechanism. Relevant articles have cited (58 times up to mid-June 2011) anomalously elevated δ(15)N (per mil deviation of (15)N/(14)N, relative to atmospheric nitrogen=0 ‰) values (diet-consumer nitrogen isotope fractionation; up to 12 ‰) for a prolonged fasting raspberry beetle (Byturus tomentosus Degeer (Coleoptera: Byturidae)), which feeds on red raspberries (Rubus idaeus: δ(15)N= ~ +2 ‰). Biologists have hypothesised that extensive recycling of amino acid nitrogen is responsible for the prolonged fasting. Since this hypothesis was proposed in 1995, scientists have integrated biochemical and molecular knowledge to support the mechanism of prolonged diapausing of animals. To test the validity of the recycling hypothesis, we analysed tissue nitrogen isotope ratios for four Japanese arthropods: the shield bug Parastrachia japonensis Scott (Hemiptera: Cydnidae), the burrower bug Canthophorus niveimarginatus Scott (Hemiptera: Cydnidae), leaf beetle Gastrophysa atrocyanea Motschulsky (Coleoptera: Chrysomelidae) and the Japanese oak silkworm Antheraea yamamai (Lepidoptera: Saturniidae), all of which fast for more than 6 months as part of their life-history strategy. Resulting diet-consumer nitrogen isotope discrimination during fasting ranged from 0 to 7‰, as in many commonly known terrestrial arthropods. We conclude that prolonged fasting of arthropods does not always result in anomalous diet-consumer nitrogen isotope fractionation, since the recycling process is closed or nearly closed with respect to nitrogen isotopes. PMID:22166153

  7. Delirium in Prolonged Hospitalized Patients in the Intensive Care Unit

    PubMed Central

    Vahedian Azimi, Amir; Ebadi, Abbas; Ahmadi, Fazlollah; Saadat, Soheil

    2015-01-01

    Background: Prolonged hospitalization in the intensive care unit (ICU) can impose long-term psychological effects on patients. One of the most significant psychological effects from prolonged hospitalization is delirium. Objectives: The aim of this study was to assess the effect of prolonged hospitalization of patients and subsequent delirium in the intensive care unit. Patients and Methods: This conventional content analysis study was conducted in the General Intensive Care Unit of the Shariati Hospital of Tehran University of Medical Sciences, from the beginning of 2013 to 2014. All prolonged hospitalized patients and their families were eligible participants. From the 34 eligible patients and 63 family members, the final numbers of actual patients and family members were 9 and 16, respectively. Several semi-structured interviews were conducted face-to-face with patients and their families in a private room and data were gathered. Results: Two main themes from two different perspectives emerged, 'patients' perspectives' (experiences during ICU hospitalization) and 'family members' perspectives' (supportive-communicational experiences). The main results of this study focused on delirium, Patients' findings were described as pleasant and unpleasant, factual and delusional experiences. Conclusions: Family members are valuable components in the therapeutic process of delirium. Effective use of family members in the delirium caring process can be considered to be one of the key non-medical nursing components in the therapeutic process. PMID:26290854

  8. Preservation of human performance capacity under prolonged space flight conditions

    NASA Technical Reports Server (NTRS)

    Yeremin, A. V.; Bogdashevskiy, R. M.; Baburin, Y. F.

    1975-01-01

    Prophylactic measures directed toward preservation of health and maintenance of the performance ability of a man during prolonged space flight stress center on the selection of optimum work and rest cycles, physical exercises, the use of pharmacological agents, conditioning of the cardiovascular apparatus, etc. A specially selected set of hormone and pharmacological preparations is recommended to stimulate hemopoiesis.

  9. Community Use of Intranasal Midazolam for Managing Prolonged Seizures

    ERIC Educational Resources Information Center

    Kyrkou, Margaret; Harbord, Michael; Kyrkou, Nicole; Kay, Debra; Coulthard, Kingsley

    2006-01-01

    Background: Until a few years ago, rectal diazepam (RD) was the only option available to parents and carers managing prolonged seizures. However, its use in the community was limited due to the requirement for privacy, and because education staff in South Australia are not permitted to carry out invasive procedures. Method: Following a literature…

  10. Efficacy of an Emotion-Focused Treatment for Prolonged Fatigue

    ERIC Educational Resources Information Center

    Schutte, Nicola S.; Malouff, John M.; Brown, Rhonda F.

    2008-01-01

    Previous research findings have suggested a relationship between less adaptive emotional functioning and fatigue. The present study used a research design involving multiple baselines across participants to evaluate the efficacy of a new emotion-focused treatment for prolonged fatigue delivered in a cognitive behavioral therapy framework. The 13…

  11. Economic viability of beef cattle grazing systems under prolonged drought

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Prolonged drought in the Southern Great Plains of the USA in recent years has raised concerns about vulnerability of beef cattle grazing systems under adverse climate change. To help address the economic viability of beef grazing operations in the Southern Great Plains, this paper provides an econom...

  12. The effects of prolonged exposure to weightlessness on postural equilibrium

    NASA Technical Reports Server (NTRS)

    Homick, J. L.; Reschke, M. F.; Miller, E. F., II

    1977-01-01

    A postflight postural equilibrium rail tests on spacecrews was used to prove a pronounced decrement in ability to maintain an upright posture after prolonged exposure to weightlessness. Support for the hypothesis that central neural reorganization occurs in response to environmental change is obtained when postflight decrease in stability on the rails and the time course for recovery are compared with preflight performance.

  13. Single Prolonged Stress Disrupts Retention of Extinguished Fear in Rats

    ERIC Educational Resources Information Center

    Knox, Dayan; George, Sophie A.; Fitzpatrick, Christopher J.; Rabinak, Christine A.; Maren, Stephen; Liberzon, Israel

    2012-01-01

    Clinical research has linked post-traumatic stress disorder (PTSD) with deficits in fear extinction. However, it is not clear whether these deficits result from stress-related changes in the acquisition or retention of extinction or in the regulation of extinction memories by context, for example. In this study, we used the single prolonged stress…

  14. Fatal bacteremic melioidosis in patients with prolonged neutropenia.

    PubMed

    Sridhar, Siddharth; Teng, Jade L L; Lau, Susanna K P; Woo, Patrick C Y

    2016-03-01

    Melioidosis, an infection with an expanding geographic range, is extremely rare in neutropenic patients. We report bacteremic melioidosis (ST-70 and ST-660) in 2 patients with prolonged neutropenia, who succumbed despite appropriate antibiotics. Clinicians should be aware of this emerging infection in neutropenic patients residing in or returning from endemic areas. PMID:26712267

  15. QTc prolongation with antipsychotics: is routine ECG monitoring recommended?

    PubMed

    Shah, Asim A; Aftab, Awais; Coverdale, John

    2014-05-01

    Whether or not QTc interval should be routinely monitored in patients receiving antipsychotics is a controversial issue, given logistic and fiscal dilemmas. There is a link between antipsychotic medications and prolongation of QTc interval, which is associated with an increased risk of torsade de pointes (TdP). Our goal is to provide clinically practical guidelines for monitoring QTc intervals in patients being treated with antipsychotics. We provide an overview of the pathophysiology of the QT interval, its relationship to TdP, and a discussion of the QT prolonging effects of antipsychotics. A literature search for articles relevant to the QTc prolonging effects of antipsychotics and TdP was conducted utilizing the databases PubMed and Embase with various combinations of search words. The overall risk of TdP and sudden death associated with antipsychotics has been observed to be low. Medications, genetics, gender, cardiovascular status, pathological conditions, and electrolyte disturbances have been found to be related to prolongation of the QTc interval. We conclude that, while electrocardiogram (ECG) monitoring is useful when administering antipsychotic medications in the presence of co-existing risk factors, it is not mandatory to perform ECG monitoring as a prerequisite in the absence of cardiac risk factors. An ECG should be performed if the initial evaluation suggests increased cardiac risk or if the antipsychotic to be prescribed has been established to have an increased risk of TdP and sudden death. PMID:24847993

  16. Elevated Rates of Prolonged Grief Disorder in African Americans

    ERIC Educational Resources Information Center

    Goldsmith, B.; Morrison, R. S.; Vanderwerker, L. C.; Prigerson, H. G.

    2008-01-01

    The prevalence of Prolonged Grief Disorder (PGD) in non-Whites is currently unknown. This study was performed to explore the prevalence of PGD in African Americans (AAs). Multivariable analysis of two studies of recently bereaved individuals found AAs to have significantly higher rates of PGD than Whites (21% [14 of 66] vs. 12% [55 of 471],…

  17. Intrinsic motivation and amotivation in first episode and prolonged psychosis.

    PubMed

    Luther, Lauren; Lysaker, Paul H; Firmin, Ruth L; Breier, Alan; Vohs, Jenifer L

    2015-12-01

    The deleterious functional implications of motivation deficits in psychosis have generated interest in examining dimensions of the construct. However, there remains a paucity of data regarding whether dimensions of motivation differ over the course of psychosis. Therefore, this study examined two motivation dimensions, trait-like intrinsic motivation, and the negative symptom of amotivation, and tested the impact of illness phase on the 1) levels of these dimensions and 2) relationship between these dimensions. Participants with first episode psychosis (FEP; n=40) and prolonged psychosis (n=66) completed clinician-rated measures of intrinsic motivation and amotivation. Analyses revealed that when controlling for group differences in gender and education, the FEP group had significantly more intrinsic motivation and lower amotivation than the prolonged psychosis group. Moreover, intrinsic motivation was negatively correlated with amotivation in both FEP and prolonged psychosis, but the magnitude of the relationship did not statistically differ between groups. These findings suggest that motivation deficits are more severe later in the course of psychosis and that low intrinsic motivation may be partially independent of amotivation in both first episode and prolonged psychosis. Clinically, these results highlight the importance of targeting motivation in early intervention services. PMID:26386901

  18. Preferences for Prolonging Life: A Prospect Theory Approach

    ERIC Educational Resources Information Center

    Winter, Laraine; Lawton, M. Powell; Ruckdeschel, Katy

    2003-01-01

    Kahneman and Tversky's (1979) Prospect theory was tested as a model of preferences for prolonging life under various hypothetical health statuses. A sample of 384 elderly people living in congregate housing (263 healthy, 131 frail) indicated how long (if at all) they would want to live under each of nine hypothetical health conditions (e.g.,…

  19. Can we predict the intracellular metabolic state of a cell based on extracellular metabolite data?

    PubMed

    Granucci, Ninna; Pinu, Farhana R; Han, Ting-Li; Villas-Boas, Silas G

    2015-12-01

    The analysis of extracellular metabolites presents many technical advantages over the analysis of intracellular compounds, which made this approach very popular in recent years as a high-throughput tool to assess the metabolic state of microbial cells. However, very little effort has been made to determine the actual relationship between intracellular and extracellular metabolite levels. The secretion of intracellular metabolites has been traditionally interpreted as a consequence of an intracellular metabolic overflow, which is based on the premise that for a metabolite to be secreted, it must be over-produced inside the cell. Therefore, we expect to find a secreted metabolite at increased levels inside the cells. Here we present a time-series metabolomics study of Saccharomyces cerevisiae growing on a glucose-limited chemostat with parallel measurements of intra- and extracellular metabolites. Although most of the extracellular metabolites were also detected in the intracellular samples and showed a typical metabolic overflow behaviour, we demonstrate that the secretion of many metabolites could not be explained by the metabolic overflow theory. PMID:26400772

  20. Gαi3-Dependent Inhibition of JNK Activity on Intracellular Membranes

    PubMed Central

    Bastin, Guillaume; Yang, Jin Ye; Heximer, Scott P.

    2015-01-01

    Heterotrimeric G-protein signaling has been shown to modulate a wide variety of intracellular signaling pathways, including the mitogen-activated protein kinase (MAPK) family. The activity of one MAPK family class, c-Jun N-terminal kinases (JNKs), has been traditionally linked to the activation of G-protein coupled receptors (GPCRs) at the plasma membrane. Using a unique set of G-protein signaling tools developed in our laboratory, we show that subcellular domain-specific JNK activity is inhibited by the activation of Gαi3, the Gαi isoform found predominantly within intracellular membranes, such as the endoplasmic reticulum (ER)–Golgi interface, and their associated vesicle pools. Regulators of intracellular Gαi3, including activator of G-protein signaling 3 (AGS3) and the regulator of G-protein signaling protein 4 (RGS4), have a marked impact on the regulation of JNK activity. Together, these data support the existence of unique intracellular signaling complexes that control JNK activity deep within the cell. This work highlights some of the cellular pathways that are regulated by these intracellular complexes and identifies potential strategies for their regulation in mammalian cells. PMID:26389115

  1. Intracellular Phosphate Dynamics in Muscle Measured by Magnetic Resonance Spectroscopy during Hemodialysis.

    PubMed

    Lemoine, Sandrine; Fournier, Thomas; Kocevar, Gabriel; Belloi, Amélie; Normand, Gabrielle; Ibarrola, Danielle; Sappey-Marinier, Dominique; Juillard, Laurent

    2016-07-01

    Of the 600-700 mg inorganic phosphate (Pi) removed during a 4-hour hemodialysis session, a maximum of 10% may be extracted from the extracellular space. The origin of the other 90% of removed phosphate is unknown. This study tested the hypothesis that the main source of phosphate removed during hemodialysis is the intracellular compartment. Six binephrectomized pigs each underwent one 3-hour hemodialysis session, during which the extracorporeal circulation blood flow was maintained between 100 and 150 ml/min. To determine in vivo phosphate metabolism, we performed phosphorous ((31)P) magnetic resonance spectroscopy using a 1.5-Tesla system and a surface coil placed over the gluteal muscle region. (31)P magnetic resonance spectra (repetition time =10 s; echo time =0.35 ms) were acquired every 160 seconds before, during, and after dialysis. During the dialysis sessions, plasma phosphate concentrations decreased rapidly (-30.4 %; P=0.003) and then, plateaued before increasing approximately 30 minutes before the end of the sessions; 16 mmol phosphate was removed in each session. When extracellular phosphate levels plateaued, intracellular Pi content increased significantly (11%; P<0.001). Moreover, βATP decreased significantly (P<0.001); however, calcium levels remained balanced. Results of this study show that intracellular Pi is the source of Pi removed during dialysis. The intracellular Pi increase may reflect cellular stress induced by hemodialysis and/or strong intracellular phosphate regulation. PMID:26561642

  2. Intracellular trafficking of superparamagnetic iron oxide nanoparticles conjugated with TAT peptide: 3-dimensional electron tomography analysis

    SciTech Connect

    Nair, Baiju G.; Fukuda, Takahiro; Mizuki, Toru; Hanajiri, Tatsuro; Maekawa, Toru

    2012-05-18

    Highlights: Black-Right-Pointing-Pointer We study the intracellular localisation of TAT-SPIONs using 3-D electron tomography. Black-Right-Pointing-Pointer 3-D images of TAT-SPIONs in a cell are clearly shown. Black-Right-Pointing-Pointer Release of TAT-SPIONs from endocytic vesicles into the cytoplasm is clearly shown. -- Abstract: Internalisation of nanoparticles conjugated with cell penetrating peptides is a promising approach to various drug delivery applications. Cell penetrating peptides such as transactivating transcriptional activator (TAT) peptides derived from HIV-1 proteins are effective intracellular delivery vectors for a wide range of nanoparticles and pharmaceutical agents thanks to their amicable ability to enter cells and minimum cytotoxicity. Although different mechanisms of intracellular uptake and localisation have been proposed for TAT conjugated nanoparticles, it is necessary to visualise the particles on a 3-D plane in order to investigate the actual intracellular uptake and localisation. Here, we study the intracellular localisation and trafficking of TAT peptide conjugated superparamagnetic ion oxide nanoparticles (TAT-SPIONs) using 3-D electron tomography. 3-D tomograms clearly show the location of TAT-SPIONs in a cell and their slow release from the endocytic vesicles into the cytoplasm. The present methodology may well be utilised for further investigations of the behaviours of nanoparticles in cells and eventually for the development of nano drug delivery systems.

  3. Plasticity of Total and Intracellular Phosphorus Quotas in Microcystis aeruginosa Cultures and Lake Erie Algal Assemblages.

    PubMed

    Saxton, Matthew A; Arnold, Robert J; Bourbonniere, Richard A; McKay, Robert Michael L; Wilhelm, Steven W

    2012-01-01

    Blooms of the potentially toxic cyanobacterium Microcystis are common events globally, and as a result significant resources continue to be dedicated to monitoring and controlling these events. Recent studies have shown that a significant proportion of total cell-associated phosphorus (P) in marine phytoplankton can be surface adsorbed; as a result studies completed to date do not accurately report the P demands of these organisms. In this study we measure the total cell-associated and intracellular P as well as growth rates of two toxic strains of Microcystis aeruginosa Kütz grown under a range of P concentrations. The results show that the intracellular P pool in Microcystis represents a percentage of total cell-associated P (50-90%) similar to what has been reported for actively growing algae in marine systems. Intracellular P concentrations (39-147 fg cell(-1)) generally increased with increasing P concentrations in the growth medium, but growth rate and the ratio of total cell-associated to intracellular P remained generally stable. Intracellular P quotas and growth rates in cells grown under the different P treatments illustrate the ability of this organism to successfully respond to changes in ambient P loads, and thus have implications for ecosystem scale productivity models employing P concentrations to predict algal bloom events. PMID:22279445

  4. Membrane contact with oviductal epithelium modulates the intracellular calcium concentration of equine spermatozoa in vitro.

    PubMed

    Dobrinski, I; Smith, T T; Suarez, S S; Ball, B A

    1997-04-01

    Interaction of equine spermatozoa with oviductal epithelial cells (OEC) prolongs sperm viability and maintains low intracellular calcium concentration ([Ca2+]i) in spermatozoa. Experiments were designed to investigate 1) whether release of spermatozoa from OEC in vitro is associated with elevated [Ca2+]i and 2) whether soluble products from OEC or direct membrane contact between spermatozoa and OEC mediates the effects of OEC on sperm [Ca2+]i. In the first experiment, changes in [Ca2+]i in spermatozoa loaded with indo-1 acetoxymethylester were determined in motile spermatozoa released from OEC monolayers after 4 h of culture compared to [Ca2+]i in spermatozoa still attached to OEC. In addition, [Ca2+]i was determined in spermatozoa incubated with OEC-conditioned medium for 6 h compared to that in spermatozoa incubated in control medium. [Ca2+]i was higher in motile spermatozoa released from OEC than in spermatozoa still attached to OEC after 4 h of incubation. Incubation in OEC-conditioned medium resulted in lower sperm [Ca2+]i only at 4 h of incubation, but not at 0.5, 2, or 6 h of incubation. In the second experiment, a suspension of apical plasma membrane vesicles (AMV) isolated from isthmic oviductal epithelium was used to study the specific effect of sperm contact with OEC membranes on sperm viability, capacitation, and [Ca2+]i. Direct membrane contact between spermatozoa and AMV prolonged sperm viability, delayed capacitation, and maintained low [Ca2+]i in spermatozoa. These results indicated that membrane contact between equine spermatozoa and OEC is required to maintain low [Ca2+]i, delay capacitation, and prolong viability of spermatozoa in vitro. Modulation of capacitation rate for spermatozoa stored in the isthmic sperm reservoir might ensure the availability of a competent sperm population at the time of fertilization. PMID:9096866

  5. Dexamethasone enhances insulin-like growth factor-I effects on skeletal muscle cell proliferation. Role of specific intracellular signaling pathways.

    PubMed Central

    Giorgino, F; Smith, R J

    1995-01-01

    IGF-I stimulation of cell proliferation and c-Fos expression in skeletal muscle cells is markedly enhanced by dexamethasone. The effect of dexamethasone is not mediated by changes in IGF-binding proteins, as evidenced by similar effects of dexamethasone on the actions of insulin, PDGF-BB, and the IGF-I analogue long R3IGF-I. Dexamethasone also does not alter autocrine IGF-II secretion by muscle cells. To investigate the mechanism of the augmentation of IGF-I action, the effects of dexamethasone on intracellular IGF-I signaling pathways were determined. In dexamethasone-treated cells, the levels of IGF-I receptor tyrosine phosphorylation and receptor-associated phosphatidylinositol 3-kinase activity were increased. Dexamethasone-treated cells also showed increased and prolonged tyrosine phosphorylation of the Shc proteins. In contrast, dexamethasone decreased both tyrosine phosphorylation and expression of insulin receptor substrate 1 (IRS-1) and IRS-1-associated phosphatidylinositol 3-kinase activity. Thus, distinct signaling pathways activated by the IGF-I receptor in skeletal muscle cells are differentially regulated by dexamethasone. Potentiation of IGF-I action correlates with increased IGF-I receptor-associated phosphatidylinositol 3-kinase activity and tyrosine phosphorylation of Shc, but appears to be independent of activation of the IRS-1/phosphatidylinositol 3-kinase signaling pathway. Images PMID:7544807

  6. The effect of photoinitiators on intracellular AKT signaling pathway in tissue engineering application

    PubMed Central

    Xu, Leyuan; Sheybani, Natasha; Yeudall, W. Andrew; Yang, Hu

    2015-01-01

    Free-radical photopolymerization initiated by photoinitiators is an important method to make tissue engineering scaffolds. To advance understanding of photoinitiator cytocompatibility, we examined three photoinitiators including 2,2-dimethoxy-2-phenylacetophenone (DMPA), Irgacure 2959 (I-2959), and eosin Y photoinitiating system (EY) in terms of their effects on viability of HN4 cells and expression levels of intracellular AKT and its phosphorylated form p-AKT. Our results show that the photoinitiators and their UV-exposed counterparts affect intracellular AKT signaling, which can be used in conjunction with cell viability for cytocompatibility assessment of photoinitiators. PMID:25709809

  7. In Vitro and Intracellular Activities of Peptide Deformylase Inhibitor GSK1322322 against Legionella pneumophila Isolates

    PubMed Central

    Dubois, Jacques; Dubois, Maïtée; Martel, Jean-François; Aubart, Kelly

    2014-01-01

    GSK1322322, a novel peptide deformylase inhibitor currently in development as an oral and intravenous agent for the treatment of hospitalized community-acquired bacterial pneumonia, showed poor in vitro activity against a panel of 50 Legionella pneumophila strains, with MICs ranging from 1 to 16 μg/ml and an MIC90 of 16 μg/ml, but very potent intracellular activity, with the minimum extracellular concentrations capable of inhibiting intracellular proliferation (MIECs) ranging from 0.12 to 2 μg/ml and 98% of the strains being inhibited by concentrations of ≤1 μg/ml. PMID:25348534

  8. Prolonged auditory brainstem responses in infants with autism.

    PubMed

    Miron, Oren; Ari-Even Roth, Daphne; Gabis, Lidia V; Henkin, Yael; Shefer, Shahar; Dinstein, Ilan; Geva, Ronny

    2016-06-01

    Numerous studies have attempted to identify early physiological abnormalities in infants and toddlers who later develop autism spectrum disorder (ASD). One potential measure of early neurophysiology is the auditory brainstem response (ABR), which has been reported to exhibit prolonged latencies in children with ASD. We examined whether prolonged ABR latencies appear in infancy, before the onset of ASD symptoms, and irrespective of hearing thresholds. To determine how early in development these differences appear, we retrospectively examined clinical ABR recordings of infants who were later diagnosed with ASD. Of the 118 children in the participant pool, 48 were excluded due to elevated ABR thresholds, genetic aberrations, or old testing age, leaving a sample of 70 children: 30 of which were tested at 0-3 months, and 40 were tested at toddlerhood (1.5-3.5 years). In the infant group, the ABR wave-V was significantly prolonged in those who later developed ASD as compared with case-matched controls (n = 30). Classification of infants who later developed ASD and case-matched controls using this measure enabled accurate identification of ASD infants with 80% specificity and 70% sensitivity. In the group of toddlers with ASD, absolute and interpeak latencies were prolonged compared to clinical norms. Findings indicate that ABR latencies are significantly prolonged in infants who are later diagnosed with ASD irrespective of their hearing thresholds; suggesting that abnormal responses might be detected soon after birth. Further research is needed to determine if ABR might be a valid marker for ASD risk. Autism Res 2016, 9: 689-695. © 2015 The Authors Autism Research published by Wiley Periodicals, Inc. on behalf of International Society for Autism Research. PMID:26477791

  9. Imaging intracellular viscosity by a new molecular rotor suitable for phasor analysis of fluorescence lifetime.

    PubMed

    Battisti, Antonella; Panettieri, Silvio; Abbandonato, Gerardo; Jacchetti, Emanuela; Cardarelli, Francesco; Signore, Giovanni; Beltram, Fabio; Bizzarri, Ranieri

    2013-07-01

    The arsenal of fluorescent probes tailored to functional imaging of cells is rapidly growing and benefits from recent developments in imaging strategies. Here, we present a new molecular rotor, which displays strong absorption in the green region of the spectrum, very little solvatochromism, and strong emission sensitivity to local viscosity. The emission increase is paralleled by an increase in emission lifetime. Owing to its concentration-independent nature, fluorescence lifetime is particularly suitable to image environmental properties, such as viscosity, at the intracellular level. Accordingly, we demonstrate that intracellular viscosity measurements can be efficiently carried out by lifetime imaging with our probe and phasor analysis, an efficient method for measuring lifetime-related properties (e.g., bionalyte concentration or local physicochemical features) in living cells. Notably, we show that it is possible to monitor the partition of our probe into different intracellular regions/organelles and to follow mitochondrial de-energization upon oxidative stress. PMID:23780224

  10. Economic Game Theory to Model the Attenuation of Virulence of an Obligate Intracellular Bacterium

    PubMed Central

    Tago, Damian; Meyer, Damien F.

    2016-01-01

    Diseases induced by obligate intracellular pathogens have a large burden on global human and animal health. Understanding the factors involved in the virulence and fitness of these pathogens contributes to the development of control strategies against these diseases. Based on biological observations, a theoretical model using game theory is proposed to explain how obligate intracellular bacteria interact with their host. The equilibrium in such a game shows that the virulence and fitness of the bacterium is host-triggered and by changing the host's defense system to which the bacterium is confronted, an evolutionary process leads to an attenuated strain. Although, the attenuation procedure has already been conducted in practice in order to develop an attenuated vaccine (e.g., with Ehrlichia ruminantium), there was a lack of understanding of the theoretical basis behind this process. Our work provides a model to better comprehend the existence of different phenotypes and some underlying evolutionary mechanisms for the virulence of obligate intracellular bacteria. PMID:27610355

  11. Raman Chemical Imaging of Chromate Reduction Sites in a Single Bacterium Using Intracellularly Grown Gold Nanoislands

    PubMed Central

    Ravindranath, Sandeep P.; Henne, Kristene L.; Thompson, Dorothea K.; Irudayaraj, Joseph

    2011-01-01

    Imagingon act live molecular events within micro-organisms at single cell resolution would deliver valuable mechanistic information much needed in understanding key biological processes. We present a surface-enhanced Raman (SERS) chemical imaging strategy as a first step towards exploring the intracellular bioreduction pockets of toxic chromate in Shewanella. In order to achieve this, we take advantage of an innate reductive mechanism in bacteria of reducing gold ions into intracellular gold nanoislands which provide the necessary enhancement for SERS imaging. We show that SERS has the sensitivity and selectivity not only to identify, but also to differentiate between the two stable valence forms of chromate in cells. The imaging platform was used to understand intracellular metal reductiivities in a ubiquitous metal-reducing organism Shewanella oneidensis MR-1, by mapping Chromate reduction. PMID:21634405

  12. Economic Game Theory to Model the Attenuation of Virulence of an Obligate Intracellular Bacterium.

    PubMed

    Tago, Damian; Meyer, Damien F

    2016-01-01

    Diseases induced by obligate intracellular pathogens have a large burden on global human and animal health. Understanding the factors involved in the virulence and fitness of these pathogens contributes to the development of control strategies against these diseases. Based on biological observations, a theoretical model using game theory is proposed to explain how obligate intracellular bacteria interact with their host. The equilibrium in such a game shows that the virulence and fitness of the bacterium is host-triggered and by changing the host's defense system to which the bacterium is confronted, an evolutionary process leads to an attenuated strain. Although, the attenuation procedure has already been conducted in practice in order to develop an attenuated vaccine (e.g., with Ehrlichia ruminantium), there was a lack of understanding of the theoretical basis behind this process. Our work provides a model to better comprehend the existence of different phenotypes and some underlying evolutionary mechanisms for the virulence of obligate intracellular bacteria. PMID:27610355

  13. Engineering of bacterial exotoxins for highly efficient and receptor-specific intracellular delivery of diverse cargos.

    PubMed

    Ryou, Jeong-Hyun; Sohn, Yoo-Kyoung; Hwang, Da-Eun; Park, Woo-Yong; Kim, Nury; Heo, Won-Do; Kim, Mi-Young; Kim, Hak-Sung

    2016-08-01

    The intracellular delivery of proteins with high efficiency in a receptor-specific manner is of great significance in molecular medicine and biotechnology, but remains a challenge. Herein, we present the development of a highly efficient and receptor-specific delivery platform for protein cargos by combining the receptor binding domain of Escherichia coli Shiga-like toxin and the translocation domain of Pseudomonas aeruginosa exotoxin A. We demonstrated the utility and efficiency of the delivery platform by showing a cytosolic delivery of diverse proteins both in vitro and in vivo in a receptor-specific manner. In particular, the delivery system was shown to be effective for targeting an intracellular protein and consequently suppressing the tumor growth in xenograft mice. The present platform can be widely used for intracellular delivery of diverse functional macromolecules with high efficiency in a receptor-specific manner. Biotechnol. Bioeng. 2016;113: 1639-1646. © 2016 Wiley Periodicals, Inc. PMID:26773973

  14. Self-Assembly of Multi-nanozymes to Mimic an Intracellular Antioxidant Defense System.

    PubMed

    Huang, Yanyan; Liu, Zhen; Liu, Chaoqun; Ju, Enguo; Zhang, Yan; Ren, Jinsong; Qu, Xiaogang

    2016-06-01

    In this work, for the first time, we constructed a novel multi-nanozymes cooperative platform to mimic intracellular antioxidant enzyme-based defense system. V2 O5 nanowire served as a glutathione peroxidase (GPx) mimic while MnO2 nanoparticle was used to mimic superoxide dismutase (SOD) and catalase (CAT). Dopamine was used as a linker to achieve the assembling of the nanomaterials. The obtained V2 O5 @pDA@MnO2 nanocomposite could serve as one multi-nanozyme model to mimic intracellular antioxidant enzyme-based defense procedure in which, for example SOD, CAT, and GPx co-participate. In addition, through assembling with dopamine, the hybrid nanocomposites provided synergistic antioxidative effect. Importantly, both in vitro and in vivo experiments demonstrated that our biocompatible system exhibited excellent intracellular reactive oxygen species (ROS) removal ability to protect cell components against oxidative stress, showing its potential application in inflammation therapy. PMID:27098681

  15. Serotonin increases intracellular Ca2+ transients in voltage-clamped sensory neurons of Aplysia californica.

    PubMed Central

    Boyle, M B; Klein, M; Smith, S J; Kandel, E R

    1984-01-01

    Noxious stimulation of the tail of Aplysia californica produces behavioral sensitization; it enhances several related defensive reflexes. This reflex enhancement involves heterosynaptic facilitation of transmitter release from sensory neurons of the reflex. The facilitation is stimulated by serotonin (5-HT) and involves suppression of a 5-HT-sensitive K+ current (the S current). Suppression of the S current broadens the action potential of the sensory neurons and is thought to enhance transmitter release by prolonging entry of Ca2+ in the presynaptic terminals. We now report a component of enhanced Ca2+ accumulation that is independent of changes in spike shape. We have measured intracellular free Ca2+ transients during long depolarizing steps in voltage-clamped sensory neuron cell bodies injected with the Ca2+-sensitive dye arsenazo III. The free Ca2+ transients elicited by a range of depolarizing voltage-clamp steps increase in amplitude by 75% following application of 5-HT. Since it is observed under voltage-clamp conditions, this increase in the free Ca2+ transients is not merely secondary to the changes in K+ current but must reflect an additional mechanism, an intrinsic change in the handling of Ca2+ by the cell. We have not yet determined whether this change in Ca2+ handling reflects an increase in Ca2+ influx, a reduction in intracellular Ca2+ uptake, or a release of Ca2+ from intracellular stores. Regardless of the underlying mechanism, however, it seems possible that the enhancement of Ca2+ accumulation and the reduction in K+ current act synergistically in producing short-term presynaptic facilitation. Alternatively, this additional modulation of Ca2+ by 5-HT might contribute to processes such as classical conditioning or long-term sensitization that may depend on Ca2+. PMID:6594707

  16. Plasmodesmata dynamics are coordinated by intracellular signaling pathways

    PubMed Central

    Brunkard, Jacob O.; Runkel, Anne M.; Zambryski, Patricia C.

    2013-01-01

    Membrane-lined channels called plasmodesmata (PD) connect the cytoplasts of adjacent plant cells across the cell wall, permitting intercellular movement of small molecules, proteins, and RNA. Recent genetic screens for mutants with altered PD transport identified genes suggesting that chloroplasts play crucial roles in coordinating PD transport. Complementing this discovery, studies manipulating expression of PD-localized proteins imply that changes in PD transport strongly impact chloroplast biology. Ongoing efforts to find genes that control root and stomatal development reveal the critical role of PD in enforcing tissue patterning, and newly discovered PD-localized proteins show that PD influence development, intracellular signaling, and defense against pathogens. Together, these studies demonstrate that PD function and formation are tightly integrated with plant physiology. PMID:23978390

  17. Poking cells for efficient vector-free intracellular delivery

    NASA Astrophysics Data System (ADS)

    Wang, Ying; Yang, Yang; Yan, Li; Kwok, So Ying; Li, Wei; Wang, Zhigang; Zhu, Xiaoyue; Zhu, Guangyu; Zhang, Wenjun; Chen, Xianfeng; Shi, Peng

    2014-07-01

    Techniques for introducing foreign molecules and materials into living cells are of great value in cell biology research. A major barrier for intracellular delivery is to cross the cell membrane. Here we demonstrate a novel platform utilizing diamond nanoneedle arrays to facilitate efficient vector-free cytosolic delivery. Using our technique, cellular membrane is deformed by an array of nanoneedles with a force on the order of a few nanonewtons. We show that this technique is applicable to deliver a broad range of molecules and materials into different types of cells, including primary neurons in adherent culture. Especially, for delivering plasmid DNAs into neurons, our technique produces at least eightfold improvement (~45% versus ~1-5%) in transfection efficiency with a dramatically shorter experimental protocol, when compared with the commonly used lipofection approach. It is anticipated that our technique will greatly benefit basic research in cell biology and also a wide variety of clinical applications.

  18. Intracellular sensing of complement C3 activates cell autonomous immunity

    PubMed Central

    Tam, Jerry C.H.; Bidgood, Susanna R.; McEwan, William A.; James, Leo C.

    2014-01-01

    Pathogens traverse multiple barriers during infection including cell membranes. Here we show that during this transition pathogens carry covalently attached complement C3 into the cell, triggering immediate signalling and effector responses. Sensing of C3 in the cytosol activates MAVS-dependent signalling cascades and induces proinflammatory cytokine secretion. C3 also flags viruses for rapid proteasomal degradation, thereby preventing their replication. This system can detect both viral and bacterial pathogens but is antagonized by enteroviruses, such as rhinovirus and poliovirus, which cleave C3 using their 3C protease. The antiviral Rupintrivir inhibits 3C protease and prevents C3 cleavage, rendering enteroviruses susceptible to intracellular complement sensing. Thus, complement C3 allows cells to detect and disable pathogens that have invaded the cytosol. PMID:25190799

  19. Intracellular localization of the calcium- and calmodulin antagonist fendiline.

    PubMed

    Weyhenmeyer, R; Gross, M; Maurer-Schultze, B

    1989-02-01

    Microautoradiograpic studies of mouse heart sections were performed to evaluate intracellular localization of the antianginal drug fendiline (Sensit). 15 microCi 3H-fendiline/g b. w. (0.5 mg/kg b.w.) were injected intravenously. 10 min p.a. the heart was removed and either frozen in acetone/dry ice or, after perfusion in situ with dextran 40 and formaldehyde, fixed in formaldehyde. Frozen, paraffin-embedded, and semithin epoxy resin sections were prepared and coated with Ilford K2 emulsion. After 7 to 30 days exposure and development in amidol silver grains were counted above cells, nuclei and extracellular space. The results show that fendiline is able to enter myocardial cells. PMID:2730688

  20. Intracellular Transport and Kinesin Superfamily Proteins: Structure, Function and Dynamics

    NASA Astrophysics Data System (ADS)

    Hirokawa, N.; Takemura, R.

    Using various molecular cell biological and molecular genetic approaches, we identified kinesin superfamily proteins (KIFs) and characterized their significant functions in intracellular transport, which is fundamental for cellular morphogenesis, functioning, and survival. We showed that KIFs not only transport various membranous organelles, proteins complexes and mRNAs fundamental for cellular functions but also play significant roles in higher brain functions such as memory and learning, determination of important developmental processes such as left-right asymmetry formation and brain wiring. We also elucidated that KIFs recognize and bind to their specific cargoes using scaffolding or adaptor protein complexes. Concerning the mechanism of motility, we discovered the simplest unique monomeric motor KIF1A and determined by molecular biophysics, cryoelectron microscopy and X-ray crystallography that KIF1A can move on a microtubule processively as a monomer by biased Brownian motion and by hydolyzing ATP.

  1. Strategies of Intracellular Pathogens for Obtaining Iron from the Environment

    PubMed Central

    Leon-Sicairos, Nidia; Reyes-Cortes, Ruth; Guadrón-Llanos, Alma M.; Madueña-Molina, Jesús; Leon-Sicairos, Claudia; Canizalez-Román, Adrian

    2015-01-01

    Most microorganisms are destroyed by the host tissues through processes that usually involve phagocytosis and lysosomal disruption. However, some organisms, called intracellular pathogens, are capable of avoiding destruction by growing inside macrophages or other cells. During infection with intracellular pathogenic microorganisms, the element iron is required by both the host cell and the pathogen that inhabits the host cell. This minireview focuses on how intracellular pathogens use multiple strategies to obtain nutritional iron from the intracellular environment in order to use this element for replication. Additionally, the implications of these mechanisms for iron acquisition in the pathogen-host relationship are discussed. PMID:26120582

  2. Stimulation of catecholamine secretion from cultured chromaffin cells by an ionophore-mediated rise in intracellular sodium.

    PubMed

    Suchard, S J; Lattanzio, F A; Rubin, R W; Pressman, B C

    1982-09-01

    The significance of intracellular Na+ concentration in catecholamine secretion of cultured bovine adrenal chromaffin cells was investigated using the monovalent carboxylic ionophore monensin. This ionophore, which is known to mediate a one-for-one exchange of intracellular K+ for extracellular Na+, induces a slow, prolonged release of catecholamines which, at 6 h, amounts of 75-90% of the total catecholamines; carbachol induces a rapid pulse of catecholamine secretion of 25-35%. Although secretory granule numbers appear to be qualitatively reduced after carbachol, multiple carbachol, or Ba2+ stimulation, overall granule distribution remains similar to that in untreated cells. Monensin-stimulated catecholamine release requires extracellular Na+ but not Ca2+ whereas carbachol-stimulated catecholamine release requires extracellular Ca2+ and is partially dependent on extracellular Na+. Despite its high selectivity for monovalent ions, monensin is considerably more effective in promoting catecholamine secretion than the divalent ionophores, A23187 and ionomycin, which mediate a more direct entry of extracellular Ca2+ into the cell. We propose that the monensin-stimulated increase in intracellular Na+ levels causes an increase in the availability of intracellular Ca2+ which, in turn, stimulates exocytosis. This hypothesis is supported by the comparable stimulation of catecholamine release by ouabain which inhibits the outwardly directed Na+ pump and thus permits intracellular Na+ to accumulate. The relative magnitudes of the secretion elicited by monensin, carbachol, and the calcium ionophores, are most consistent with the hypothesis that, under normal physiological conditions, Na+ acts by decreasing the propensity of Ca2+-sequestering sites to bind the Ca2+ that enters the cell as a result of acetylcholine stimulation. PMID:7130269

  3. Mycobacterium simiae and Mycobacterium avium-M. intracellulare mixed infection in acquired immune deficiency syndrome.

    PubMed Central

    Lévy-Frébault, V; Pangon, B; Buré, A; Katlama, C; Marche, C; David, H L

    1987-01-01

    Acquired immune deficiency syndrome was diagnosed in a 43-year-old man, born and living in Congo. The patient presented a disseminated infection caused by mycobacteria which were recovered from blood, jejunal fluid, and duodenal and rectal biopsies. Identification, according to conventional tests and mycolate profile determination, showed that Mycobacterium avium-M. intracellulare and M. simiae were both involved. Images PMID:3793869

  4. Control of Francisella tularensis Intracellular Growth by Pulmonary Epithelial Cells

    PubMed Central

    Maggio, Savannah; Takeda, Kazuyo; Stark, Felicity; Meierovics, Anda I.; Yabe, Idalia; Cowley, Siobhan C.

    2015-01-01

    The virulence of F. tularensis is often associated with its ability to grow in macrophages, although recent studies show that Francisella proliferates in multiple host cell types, including pulmonary epithelial cells. Thus far little is known about the requirements for killing of F. tularensis in the non-macrophage host cell types that support replication of this organism. Here we sought to address this question through the use of a murine lung epithelial cell line (TC-1 cells). Our data show that combinations of the cytokines IFN-γ, TNF, and IL-17A activated murine pulmonary epithelial cells to inhibit the intracellular growth of the F. tularensis Live Vaccine Strain (LVS) and the highly virulent F. tularensis Schu S4 strain. Although paired combinations of IFN-γ, TNF, and IL-17A all significantly controlled LVS growth, simultaneous treatment with all three cytokines had the greatest effect on LVS growth inhibition. In contrast, Schu S4 was more resistant to cytokine-induced growth effects, exhibiting significant growth inhibition only in response to all three cytokines. Since one of the main antimicrobial mechanisms of activated macrophages is the release of reactive nitrogen intermediates (RNI) via the activity of iNOS, we investigated the role of RNI and iNOS in Francisella growth control by pulmonary epithelial cells. NOS2 gene expression was significantly up-regulated in infected, cytokine-treated pulmonary epithelial cells in a manner that correlated with LVS and Schu S4 growth control. Treatment of LVS-infected cells with an iNOS inhibitor significantly reversed LVS killing in cytokine-treated cultures. Further, we found that mouse pulmonary epithelial cells produced iNOS during in vivo respiratory LVS infection. Overall, these data demonstrate that lung epithelial cells produce iNOS both in vitro and in vivo, and can inhibit Francisella intracellular growth via reactive nitrogen intermediates. PMID:26379269

  5. Intracellular zinc distribution in mitochondria, ER and the Golgi apparatus

    PubMed Central

    Lu, Qiping; Haragopal, Hariprakash; Slepchenko, Kira G; Stork, Christian; Li, Yang V

    2016-01-01

    Zinc (Zn2+) is required for numerous cellular functions. As such, the homeostasis and distribution of intracellular zinc can influence cellular metabolism and signaling. However, the exact distribution of free zinc within live cells remains elusive. Previously we showed the release of zinc from thapsigargin/IP3-sensitive endoplasmic reticulum (ER) storage in cortical neurons. In the present study, we investigated if other cellular organelles also contain free chelatable zinc and function as organelle storage for zinc. To identify free zinc within the organelles, live cells were co-stained with Zinpyr-1, a zinc fluorescent dye, and organelle-specific fluorescent dyes (MitoFluor Red 589: mitochondria; ER Tracker Red: endoplasmic reticulum; BODIPY TR ceramide: Golgi apparatus; Syto Red 64: nucleus). We examined organelles that represent potential storing sites for intracellular zinc. We showed that zinc fluorescence staining was co-localized with MitoFluor Red 589, ER Tracker Red, and BODIPY TR ceramide respectively, suggesting the presence of free zinc in mitochondria, endoplasmic reticulum, and the Golgi apparatus. On the other hand, cytosol and nucleus had nearly no detectable zinc fluorescence. It is known that nucleus contains high amount of zinc binding proteins that have high zinc binding affinity. The absence of zinc fluorescence suggests that there is little free zinc in these two regions. It also indicates that the zinc fluorescence detected in mitochondria, ER and Golgi apparatus represents free chelatable zinc. Taken together, our results support that these organelles are potential zinc storing organelles during cellular zinc homeostasis. PMID:27186321

  6. 28. MAP SHOWING LOCATION OF ARVFS FACILITY AS BUILT. SHOWS ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    28. MAP SHOWING LOCATION OF ARVFS FACILITY AS BUILT. SHOWS LINCOLN BOULEVARD, BIG LOST RIVER, AND NAVAL REACTORS FACILITY. F.C. TORKELSON DRAWING NUMBER 842-ARVFS-101-2. DATED OCTOBER 12, 1965. INEL INDEX CODE NUMBER: 075 0101 851 151969. - Idaho National Engineering Laboratory, Advanced Reentry Vehicle Fusing System, Scoville, Butte County, ID

  7. 8. Detail showing concrete abutment, showing substructure of bridge, specifically ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    8. Detail showing concrete abutment, showing substructure of bridge, specifically west side of arch and substructure. - Presumpscot Falls Bridge, Spanning Presumptscot River at Allen Avenue extension, 0.75 mile west of U.S. Interstate 95, Falmouth, Cumberland County, ME

  8. Transient increase of interleukin-1β after prolonged febrile seizures promotes adult epileptogenesis through long-lasting upregulating endocannabinoid signaling

    PubMed Central

    Feng, Bo; Tang, Yangshun; Chen, Bin; Xu, Cenglin; Wang, Yi; Dai, Yunjian; Wu, Dengchang; Zhu, Junmin; Wang, Shuang; Zhou, Yudong; Shi, Liyun; Hu, Weiwei; Zhang, Xia; Chen, Zhong

    2016-01-01

    It remains unclear how infantile febrile seizures (FS) enhance adult seizure susceptibility. Here we showed that the transient increase of interleukin-1β (IL-1β) after prolonged FS promoted adult seizure susceptibility, which was blocked by interleukin-1 receptor antagonist (IL-1Ra) within a critical time window. Postnatal administered IL-1β alone mimicked the effect of FS on adult seizure susceptibility. IL-1R1 knockout mice were not susceptible to adult seizure after prolonged FS or IL-1β treatment. Prolonged FS or early-life IL-1β treatment increased the expression of cannabinoid type 1 receptor (CB1R) for over 50 days, which was blocked by IL-1Ra or was absent in IL-1R1 knockout mice. CB1R antagonist, knockdown and endocannabinoid synthesis inhibitor abolished FS or IL-1β-enhanced seizure susceptibility. Thus, this work identifies a pathogenic role of postnatal IL-1β/IL-1R1 pathway and subsequent prolonged prominent increase of endocannabinoid signaling in adult seizure susceptibility following prolonged FS, and highlights IL-1R1 as a potential therapeutic target for preventing the development of epilepsy after infantile FS. PMID:26902320

  9. Management of psychosis and agitation in medical-surgical patients who have or are at risk for prolonged QT interval.

    PubMed

    Ries, Rose; Sayadipour, Amirali

    2014-09-01

    We review the literature on management of psychosis and agitation in medical-surgical patients who have or are at risk for prolonged QT interval, a risk factor for torsade de pointes (TdP), and we describe our protocols for treating these patients. We searched PubMed and PsycInfo for relevant studies and found few papers describing options for treating psychosis and agitation in these patients. Prolonged QTc interval has been more often associated with low-potency phenothiazines such as thioridazine; however, it may occur with high potency typical antipsychotics such as fluphenazine and haloperidol as well as with atypical antipsychotics such as quetiapine, risperidone, olanzapine, iloperidone, and particularly ziprasidone. Antipsychotics for which no association with prolonged QTc interval has been shown include lurasidone, clozapine, and aripiprazole. For patients who have risk factors for prolonged QTc interval but whose electrocardiograms do not show this, reasonable first choices include oral or intramuscular olanzapine or aripiprazole, followed by risperidone and quetiapine or oral or intramuscular haloperidol. For those who have prolonged QTc but that measures less than 500 ms, we limit the use of antipsychotics to aripiprazole, olanzapine, risperidone, or quetiapine. Finally, for patients who have a QTc of 500 ms or greater, we rely on aripiprazole, valproate, trazodone, and benzodiazepines. PMID:25226194

  10. Cognitive Changes During Prolonged Exposure versus Prolonged Exposure Plus Cognitive Restructuring in Female Assault Survivors with Posttraumatic Stress Disorder

    ERIC Educational Resources Information Center

    Foa, Edna B.; Rauch, Sheila A. M.

    2004-01-01

    The authors report on changes in cognitions related to posttraumatic stress disorder (PTSD) among 54 female survivors of sexual and nonsexual assault with chronic PTSD who completed either prolonged exposure alone or in combination with cognitive restructuring. Treatment included 9-12 weekly sessions, and assessment was conducted at pretreatment,…

  11. Effects of prolonged head-down bed rest on working memory

    PubMed Central

    Liu, Qing; Zhou, Renlai; Zhao, Xin; Oei, Tian Po S

    2015-01-01

    Background The weightlessness caused by prolonged bed rest results in changes in cerebral circulation and thus, brain functions, which is of interest. Methods We investigated the effects of 45-day, −6° head-down bed rest, which stimulated microgravity, on working memory in 16 healthy male participants. The 2-back task was used to test the working memory variations on the 2nd day before bed rest (R−2); on the 11th (R11), 20th (R20), 32nd (R32), and 40th (R40) days of bed rest; and on the eighth day after bed rest (R+8). The cognitive response and the physiological reactivity (such as galvanic skin response, heart rate, and heart rate variability) under the 2-back task were recorded simultaneously. Results The results showed that compared with R−2, on the R+8, the participants’ galvanic skin response increased significantly, and the high frequency of heart rate variability (HF), low frequency of heart rate variability (LF), and reaction time in the 2-back task decreased significantly. There were positive correlations between the participants’ reaction time of working memory and the LF/HF under head-down bed rest (at R11, R20, and R32). Conclusion The results suggested that the prolonged head-down bed rest may have a detrimental effect on individual physiology and working memory. Physiology indices, such as galvanic skin response and heart rate variability, were sensitive to the prolonged bed rest. PMID:25848281

  12. Prolonged maternal separation disturbs the serotonergic system during early brain development.

    PubMed

    Ohta, Ken-Ichi; Miki, Takanori; Warita, Katsuhiko; Suzuki, Shingo; Kusaka, Takashi; Yakura, Tomiko; Liu, Jun-Qian; Tamai, Motoki; Takeuchi, Yoshiki

    2014-04-01

    Early life stress interrupts brain development through the disturbance of various neurotransmitter and neurotrophic factor activities, but the details remain unclear. In the current study, we focused on the serotonergic system, which plays a critical role in brain development, and examined the time-dependent influence of prolonged maternal separation on male Sprague-Dawley rats. The rats were separated from their dams for 3h twice-daily during postnatal days (PDs) 2-20. The influence of prolonged maternal separation was analyzed on PDs 7, 14, 21, and 28 using HPLC to assess concentrations of serotonin and 5-hydroxyindoleacetic acid and using real-time RT-PCR to measure mRNA expression of the serotonin 1A and 2A receptors in various brain regions. HPLC revealed imbalance between serotonin and 5-hydroxyindoleacetic acid in midbrain raphe nuclei, the amygdala, the hippocampus, and the medial prefrontal cortex (mPFC) on PDs 7 and 14. Furthermore, real-time RT-PCR showed attenuation of mRNA expression of the serotonin 1A receptor in the hippocampus and the mPFC and of the serotonin 2A receptor only in the mPFC on PDs 7 and 14. The observed alterations returned to control levels after maternal separation ended. These findings suggest that the early life stress of prolonged maternal separation disturbs the serotonergic system during a crucial period of brain development, which might in part be responsible for emotional abnormalities later in life. PMID:24184298

  13. Relative axial myopia induced by prolonged light exposure in C57BL/6 mice.

    PubMed

    Zhou, Xiangtian; An, Jianhong; Wu, Xiaomin; Lu, Runxia; Huang, Qinzhu; Xie, Ruozhong; Jiang, Liqin; Qu, Jia

    2010-01-01

    Ambient lighting is essential for ocular development in many species, however, disruption in diurnal lighting cycle can affect the development in refraction and axial growth of the eye. This study investigated the effects of prolonged daily lighting on refraction and various optical components of the eye by raising C57BL/6 mice under three different light/dark cycles (18/6, 12/12 and 6/18). Egr-1 mRNA expression, apoptosis and histology of the retina and size of the scleral fibrils were evaluated in these three lighting cycles. Results showed that there was a trend of myopic development, increasing vitreous chamber depth and thinning of the retina in eyes from 6/18 to 18/6 groups. Retinal Egr-1 mRNA expression and diameter of scleral fibrils were reduced with the prolongation of daily lighting from 6/18 to 18/6. However, retinal apoptosis was not detected in all the groups. These results suggest that prolonged lighting can induce axial myopia in inbred mice. This model, which uses mice with similar genetic backgrounds, provides an alternative to the currently available models and therefore is useful for evaluation of refractive errors caused by changes in environmental illumination. PMID:19912561

  14. Resilience and vulnerability: prolonged grief in the bereaved spouses of marital partners who died of AIDS.

    PubMed

    Yu, Nancy Xiaonan; Chan, Cecilia L W; Zhang, Jianxin; Stewart, Sunita M

    2016-04-01

    Spousal bereavement is closely linked to prolonged grief, that is, significant adjustment symptoms that last for more than six months after the loss. This article focused on potential risk and protective factors that may influence bereavement outcomes. Participants in this study were surviving spouses of individuals who died of acquired immune deficiency syndrome (AIDS). These participants were themselves living with human immunodeficiency syndrome. In this cross-sectional study, 120 bereaved participants completed measures of grief, quality of dying and death of the deceased, negative conceptions of death resulting from AIDS, death attitudes, and personal resilience. The results showed that one-third (35.0%) of the bereaved participants reported grief levels above the prolonged grief cut-off scores, and can be categorized as the "prolonged grief" group. Although quality of dying and death was not associated with the intensity of grief, negative conceptions of death from AIDS, fear of death and resilience independently predicted grief symptoms in the regression models. Our findings provide insight into the grief process for the surviving spouse of AIDS victims in rural China. Since resilience is malleable, developing resilience interventions to enhance adjustment to bereavement may be a promising direction in grief counselling and therapies. PMID:26573556

  15. Prolonged Influenza Virus Shedding and Emergence of Antiviral Resistance in Immunocompromised Patients and Ferrets

    PubMed Central

    van der Vries, Erhard; Stittelaar, Koert J.; van Amerongen, Geert; Veldhuis Kroeze, Edwin J. B.; de Waal, Leon; Fraaij, Pieter L. A.; Meesters, Roland J.; Luider, Theo M.; van der Nagel, Bart; Koch, Birgit; Vulto, Arnold G.; Schutten, Martin; Osterhaus, Albert D. M. E.

    2013-01-01

    Immunocompromised individuals tend to suffer from influenza longer with more serious complications than otherwise healthy patients. Little is known about the impact of prolonged infection and the efficacy of antiviral therapy in these patients. Among all 189 influenza A virus infected immunocompromised patients admitted to ErasmusMC, 71 were hospitalized, since the start of the 2009 H1N1 pandemic. We identified 11 (15%) cases with prolonged 2009 pandemic virus replication (longer than 14 days), despite antiviral therapy. In 5 out of these 11 (45%) cases oseltamivir resistant H275Y viruses emerged. Given the inherent difficulties in studying antiviral efficacy in immunocompromised patients, we have infected immunocompromised ferrets with either wild-type, or oseltamivir-resistant (H275Y) 2009 pandemic virus. All ferrets showed prolonged virus shedding. In wild-type virus infected animals treated with oseltamivir, H275Y resistant variants emerged within a week after infection. Unexpectedly, oseltamivir therapy still proved to be partially protective in animals infected with resistant virus. Immunocompromised ferrets offer an attractive alternative to study efficacy of novel antiviral therapies. PMID:23717200

  16. Prolonged post-hyperventilation apnea in two young adults with hyperventilation syndrome

    PubMed Central

    2013-01-01

    Background The prognosis of hyperventilation syndrome (HVS) is generally good. However, it is important to proceed with care when treating HVS because cases of death following hyperventilation have been reported. This paper was done to demonstrate the clinical risk of post-hyperventilation apnea (PHA) in patients with HVS. Case presentation We treated two patients with HVS who suffered from PHA. The first, a 21-year-old woman, had a maximum duration of PHA of about 3.5 minutes and an oxygen saturation (SpO2) level of 60%. The second patient, a 22-year-old woman, had a maximum duration of PHA of about 3 minutes and an SpO2 level of 66%. Both patients had loss of consciousness and cyanosis. Because there is no widely accepted regimen for treating patients with prolonged PHA related to HVS, we administered artificial ventilation to both patients using a bag mask and both recovered without any after effects. Conclusion These cases show that some patients with HVS develop prolonged PHA or severe hypoxia, which has been shown to lead to death in some cases. Proper treatment must be given to patients with HVS who develop PHA to protect against this possibility. If prolonged PHA or severe hypoxemia arises, respiratory assistance using a bag mask must be done immediately. PMID:23594702

  17. Prolonged Particulate Hexavalent Chromium Exposure Suppresses Homologous Recombination Repair in Human Lung Cells.

    PubMed

    Browning, Cynthia L; Qin, Qin; Kelly, Deborah F; Prakash, Rohit; Vanoli, Fabio; Jasin, Maria; Wise, John Pierce

    2016-09-01

    Genomic instability is one of the primary models of carcinogenesis and a feature of almost all cancers. Homologous recombination (HR) repair protects against genomic instability by maintaining high genomic fidelity during the repair of DNA double strand breaks. The defining step of HR repair is the formation of the Rad51 nucleofilament, which facilitates the search for a homologous sequence and invasion of the template DNA strand. Particulate hexavalent chromium (Cr(VI)), a human lung carcinogen, induces DNA double strand breaks and chromosome instability. Since the loss of HR repair increases Cr(VI)-induced chromosome instability, we investigated the effect of extended Cr(VI) exposure on HR repair. We show acute (24 h) Cr(VI) exposure induces a normal HR repair response. In contrast, prolonged (120 h) exposure to particulate Cr(VI) inhibited HR repair and Rad51 nucleofilament formation. Prolonged Cr(VI) exposure had a profound effect on Rad51, evidenced by reduced protein levels and Rad51 mislocalization to the cytoplasm. The response of proteins involved in Rad51 nuclear import and nucleofilament formation displayed varying responses to prolonged Cr(VI) exposure. BRCA2 formed nuclear foci after prolonged Cr(VI) exposure, while Rad51C foci formation was suppressed. These results suggest that particulate Cr(VI), a major chemical carcinogen, inhibits HR repair by targeting Rad51, causing DNA double strand breaks to be repaired by a low fidelity, Rad51-independent repair pathway. These results further enhance our understanding of the underlying mechanism of Cr(VI)-induced chromosome instability and thus, carcinogenesis. PMID:27449664

  18. Liposomes ameliorate Crizotinib- and Nilotinib-induced inhibition of the cardiac IKr channel and QTc prolongation.

    PubMed

    Shopp, George M; Helson, Lawrence; Bouchard, Annie; Salvail, Dany; Majeed, Muhammad

    2014-09-01

    Crizotinib (Xalkori®) and nilotinib (Tasigna®) are tyrosine kinase inhibitors approved for the treatment of non-small cell lung cancer and chronic myeloid leukemia, respectively. Both have been shown to result in electrocardiogram rate-corrected Q-wave T-wave interval (QTc) prolongation in humans and animals. Liposomes have been shown to ameliorate drug-induced effects on the cardiac-delayed rectifier K(+) current (IKr, KV11.1), coded by the human ether-a-go-go-related gene (hERG). This study was undertaken to determine if liposomes would also decrease the effect of crizotinib and nilotinib on the IKr channel. Crizotinib and nilotinib were tested in an in vitro IKr assay using human embryonic kidney (HEK) 293 cells stably transfected with the hERG. Dose-responses were determined and the 50% inhibitory concentrations (IC50s) were calculated. When the HEK 293 cells were treated with crizotinib or nilotinib that were mixed with liposomes, there was a significant decrease in the IKr channel inhibitory effects of these two drugs. When isolated, rabbit hearts were exposed to crizotinib or nilotinib, there were significant increases in QTc prolongation. Mixing either of the drugs with liposomes ameliorated the effects of the drugs. Rabbits dosed intravenously (IV) with crizotinib or nilotinib showed QTc prolongation. When liposomes were injected prior to crizotinib or nilotinib, the liposomes decreased the effects on the QTc interval. The use of liposomal encapsulated QT-prolongation agents, or giving liposomes in combination with drugs, may decrease their cardiac liability. PMID:25202051

  19. Adenovirus-mediated gene transfer to ciliated airway epithelia requires prolonged incubation time.

    PubMed Central

    Zabner, J; Zeiher, B G; Friedman, E; Welsh, M J

    1996-01-01

    The efficiency of adenovirus-mediated gene transfer to airway epithelia will be an important factor in determining whether recombinant adenoviruses can be developed as vectors for transferring cystic fibrosis transmembrane conductance regulator (CFTR) cDNA to patients with cystic fibrosis. Current understanding of the biology of CF lung disease suggests that vectors should express transgene in mature, ciliated airway epithelia. We evaluated the efficiency of adenovirus-mediated gene transfer to primary cultures of normal and CF human airway epithelia. Our studies showed that the airway cells developed from an undifferentiated epithelium with markers characteristic of basal cells and a surface covered by short microvilli 3 days after seeding to a mature epithelium whose apical surface was covered with cilia by 10 to 14 days. The ability of adenovirus vectors to express a reporter gene and to correct defective cyclic AMP-stimulated Cl- transport in CF epithelia was correlated inversely with the state of differentiation. However, the inefficiency of adenovirus-mediated gene transfer could be partially corrected when the contact time between vector and epithelium was prolonged. After prolonged contact, we observed complete correction of the CF Cl- transport defect in differentiated CF airway epithelia in culture and of the Cl- transport defect in the nasal epithelia of mice homozygous for the deltaF508 mutation. The fact that gene transfer to airway epithelia required prolonged incubation with vector contrasts with the rapid infection observed in cell models such as 293 and HeLa cells, which are commonly used to study adenovirus infection. Gene transfer observed after prolonged incubation may result from mechanisms different from those that mediate infection of 293 cells. These observations suggest that interventions that either increase the contact time or alter the epithelium or the vector may be required to facilitate gene transfer to ciliated respiratory epithelia

  20. Changes over time in the quality of life, prolonged grief and family strain of family caregivers of patients in vegetative state: A pilot study.

    PubMed

    Bastianelli, Alessia; Gius, Erminio; Cipolletta, Sabrina

    2016-05-01

    This study explored changes over time and in the internal standards of the quality of life, prolonged grief and family strain of informal caregivers of patients in vegetative state. Data obtained from 52 caregivers showed high levels of prolonged grief and family strain, and low quality of life. A decrease of caregivers' quality of life and an increase of family strain were found by adopting a response shift procedure. Only prolonged grief did not change during time. Clinical intervention with the caregivers of vegetative state patients should be differentiated on the basis of the duration of the caring experience. PMID:24984718

  1. Effects of calcium and magnesium on the frequency of miniature end-plate potentials during prolonged tetanization

    PubMed Central

    Hurlbut, W. P.; Longenecker, H. B.; Mauro, Alexander

    1971-01-01

    1. End-plate potentials (e.p.p.s) and miniature end-plate potentials (min.e.p.p.s) were recorded intracellularly from the cutaneous pectoris nerve-muscle preparation of the frog during prolonged stimulation at low frequencies (5/sec—50/sec). 2. When Ca was present in the bathing solution, the quantum content of the e.p.p. and the frequency of occurrence of the min.e.p.p.s gradually increased during the period of stimulation. During the first few minutes of stimulation, the min.e.p.p. frequency increased linearly with time, and the rate of increase was dependent on the Ca concentration of the bathing solution. However, Mg had no effect on this Ca-dependent increase in min.e.p.p. frequency. 3. A large maintained increase in min.e.p.p. frequency also occurred during prolonged stimulation in solutions that contained no added Ca and 1-2 mM-EGTA. Under these conditions the increase in min.e.p.p. frequency was dependent on the Mg concentration of the bathing solution and was exponential in time. 4. It is suggested that the rise in min.e.p.p. frequency is caused by an accumulation of Ca or Mg ions in the nerve terminal, and it is suggested that these ions enter the terminal at relatively non-specific sites distinct from the Ca-specific sites that trigger the `phasic' release of transmitter. PMID:5316661

  2. Planning a Successful Tech Show

    ERIC Educational Resources Information Center

    Nikirk, Martin

    2011-01-01

    Tech shows are a great way to introduce prospective students, parents, and local business and industry to a technology and engineering or career and technical education program. In addition to showcasing instructional programs, a tech show allows students to demonstrate their professionalism and skills, practice public presentations, and interact…

  3. Hey Teacher, Your Personality's Showing!

    ERIC Educational Resources Information Center

    Paulsen, James R.

    1977-01-01

    A study of 30 fourth, fifth, and sixth grade teachers and 300 of their students showed that a teacher's age, sex, and years of experience did not relate to students' mathematics achievement, but that more effective teachers showed greater "freedom from defensive behavior" than did less effective teachers. (DT)

  4. What Do Blood Tests Show?

    MedlinePlus

    ... shows the ranges for blood glucose levels after 8 to 12 hours of fasting (not eating). It shows the normal range and the abnormal ranges that are a sign of prediabetes or diabetes. Plasma Glucose Results (mg/dL)* Diagnosis 70 to 99 ...

  5. Coseismic thermal pressurization can prolong recurrence intervals of earthquake cycle

    NASA Astrophysics Data System (ADS)

    Mitsui, Y.; Hirahara, K.

    2008-12-01

    Earthquake is a short-lived event, while it needs a very long preparation period. The transition is rapid but seamless. We should correlate physics governing during the short-term earthquake period with that governing during the long-term preparation period. Brace and Byerlee [1966] proposed that stick-slip behavior is a mechanism for earthquakes from this standpoint. Following the proposition, lots of researchers have executed numerical simulations of a spring- slider system in order to interpret the earthquake cyclicity (e.g., Gu et al. [1991]). For such researches, it is necessary to use a constitutive law of friction on an interface between a slider and ground. By way of example, a rate- and state- dependent friction law (Dieterich [1979]) has been widely used, because it can represent frictional healing during the interseismic period. Despite the previous extensive studies, there is a dearth of information on roles of pore fluid. The pore fluid existence within a fault zone dramatically changes the frictional property via reduction of normal stress (Brace and Martin [1968]). Further, the pore fluid pressure may evolve and affect every aspect of earthquakes. Here, we try to add a new perspective to the earthquake cyclicity. It is an effect of short-term temporal change of the pore pressure, due to the coseismic thermal pressurization (hereinafter called TP). TP is a short-lived physical mechanism that frictional heating at a fluid-saturated fault pressurizes the pore fluid within the fault zone (Sibson [1973]). It can greatly affect the fault constitutive relation (Andrews [2002]) and the dynamic propagation of the earthquake rupture (Bizzarri and Cocco [2006]). In this presentation, we show that the short-lived TP is again a significant mechanism for the earthquake cyclicity, using the spring-slider system with the rate- and state- dependent law in a 1-D elastic body. If the shear zone thickness is smaller than several decimeters, TP can greatly prolong the

  6. Polynitroxyl-albumin (PNA) plus tempol attenuate lung capillary leak elicited by prolonged intestinal ischemia and reperfusion(1).

    PubMed

    Zhang, S; Li, H; Ma, L; Trimble, C E; Kuppusamy, P; Hsia, C J; Carden, D L

    2000-07-01

    Stable nitroxyl radicals (nitroxides) are potential antioxidant drugs, and we have previously reported that linking nitroxide to biological macromolecules can improve therapeutic activity in at least two ways. First, polynitroxylated compounds such as polynitroxyl human serum albumin (PNA) are a novel class of high molecular weight, extracellular antioxidants. Second, compounds such as PNA can prolong the half-life of free (unbound, low molecular weight) nitroxides such as 4-hydroxy-2,2,6, 6-tetramethylpiperidine-N-oxyl (Tempol) in vivo. Unlike PNA, Tempol can readily access the intracellular compartment. Thus PNA can act alone in the extracellular compartment, or in concert with Tempol, to provide additional antioxidant protection within cells. In this study, we compared the abilities of PNA, Tempol, and the combination of PNA + Tempol to prevent lung microvascular injury secondary to prolonged gut ischemia (I, 120 min) and reperfusion (R, 20 min) in the rat. Pulmonary capillary filtration coefficient (K(f,c)) and lung neutrophil retention (tissue myeloperoxidase activity, MPO) were measured in normal, isolated rat lungs perfused with blood harvested from I/R rats. Blood donor rats were treated with drug during ischemia. Gut I/R resulted in a marked increase in pulmonary capillary coefficient and lung MPO. PNA + Tempol, but not PNA alone or Tempol alone, at the doses used, prevented the development of lung leak. None of the treatments had an effect on lung neutrophil retention. Anti-inflammatory therapeutic activity appeared to correlate with blood Tempol level: in the presence of PNA, blood Tempol levels were maintained in the 50-100 microM range vs. essentially undetectable levels shortly after Tempol was administered alone. In this model of lung injury secondary to prolonged gut I/R, lung capillary leak was prevented when the membrane-permeable compound Tempol was maintained in its active, free radical state by PNA. PMID:10962204

  7. ZnO Nanostructure-Based Intracellular Sensor.

    PubMed

    Asif, Muhammad H; Danielsson, Bengt; Willander, Magnus

    2015-01-01

    Recently ZnO has attracted much interest because of its usefulness for intracellular measurements of biochemical species by using its semiconducting, electrochemical, catalytic properties and for being biosafe and biocompatible. ZnO thus has a wide range of applications in optoelectronics, intracellular nanosensors, transducers, energy conversion and medical sciences. This review relates specifically to intracellular electrochemical (glucose and free metal ion) biosensors based on functionalized zinc oxide nanowires/nanorods. For intracellular measurements, the ZnO nanowires/nanorods were grown on the tip of a borosilicate glass capillary (0.7 µm in diameter) and functionalized with membranes or enzymes to produce intracellular selective metal ion or glucose sensors. Successful intracellular measurements were carried out using ZnO nanowires/nanorods grown on small tips for glucose and free metal ions using two types of cells, human fat cells and frog oocytes. The sensors in this study were used to detect real-time changes of metal ions and glucose across human fat cells and frog cells using changes in the electrochemical potential at the interface of the intracellular micro-environment. Such devices are helpful in explaining various intracellular processes involving ions and glucose. PMID:26007730

  8. ZnO Nanostructure-Based Intracellular Sensor

    PubMed Central

    Asif, Muhammad H.; Danielsson, Bengt; Willander, Magnus

    2015-01-01

    Recently ZnO has attracted much interest because of its usefulness for intracellular measurements of biochemical species by using its semiconducting, electrochemical, catalytic properties and for being biosafe and biocompatible. ZnO thus has a wide range of applications in optoelectronics, intracellular nanosensors, transducers, energy conversion and medical sciences. This review relates specifically to intracellular electrochemical (glucose and free metal ion) biosensors based on functionalized zinc oxide nanowires/nanorods. For intracellular measurements, the ZnO nanowires/nanorods were grown on the tip of a borosilicate glass capillary (0.7 µm in diameter) and functionalized with membranes or enzymes to produce intracellular selective metal ion or glucose sensors. Successful intracellular measurements were carried out using ZnO nanowires/nanorods grown on small tips for glucose and free metal ions using two types of cells, human fat cells and frog oocytes. The sensors in this study were used to detect real-time changes of metal ions and glucose across human fat cells and frog cells using changes in the electrochemical potential at the interface of the intracellular micro-environment. Such devices are helpful in explaining various intracellular processes involving ions and glucose. PMID:26007730

  9. Assessment of Methods for the Intracellular Blockade of GABAA Receptors.

    PubMed

    Atherton, Laura A; Burnell, Erica S; Mellor, Jack R

    2016-01-01

    Selective blockade of inhibitory synaptic transmission onto specific neurons is a useful tool for dissecting the excitatory and inhibitory synaptic components of ongoing network activity. To achieve this, intracellular recording with a patch solution capable of blocking GABAA receptors has advantages over other manipulations, such as pharmacological application of GABAergic antagonists or optogenetic inhibition of populations of interneurones, in that the majority of inhibitory transmission is unaffected and hence the remaining network activity preserved. Here, we assess three previously described methods to block inhibition: intracellular application of the molecules picrotoxin, 4,4'-dinitro-stilbene-2,2'-disulphonic acid (DNDS) and 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS). DNDS and picrotoxin were both found to be ineffective at blocking evoked, monosynaptic inhibitory postsynaptic currents (IPSCs) onto mouse CA1 pyramidal cells. An intracellular solution containing DIDS and caesium fluoride, but lacking nucleotides ATP and GTP, was effective at decreasing the amplitude of IPSCs. However, this effect was found to be independent of DIDS, and the absence of intracellular nucleotides, and was instead due to the presence of fluoride ions in this intracellular solution, which also blocked spontaneously occurring IPSCs during hippocampal sharp waves. Critically, intracellular fluoride ions also caused a decrease in both spontaneous and evoked excitatory synaptic currents and precluded the inclusion of nucleotides in the intracellular solution. Therefore, of the methods tested, only fluoride ions were effective for intracellular blockade of IPSCs but this approach has additional cellular effects reducing its selectivity and utility. PMID:27501143

  10. Functional Analysis and Intracellular Localization of Rice Cryptochromes

    PubMed Central

    Matsumoto, Nanako; Hirano, Tomoharu; Iwasaki, Toshisuke; Yamamoto, Naoki

    2003-01-01

    Blue-light-receptor cryptochrome (CRY), which mediates cotyledon expansion, increased accumulation of anthocyanin, and inhibition of hypocotyl elongation, was first identified in Arabidopsis. Two Arabidopsis cryptochromes (AtCRY1 and AtCRY2) have been reported to be localized to the nucleus. However, there is no information on the cryptochromes in monocotyledons. In this study, we isolated two cryptochrome cDNAs, OsCRY1 and OsCRY2, from rice (Oryza sativa) plants. The deduced amino acid sequences of OsCRY1 and OsCRY2 have a photolyase-like domain in their N termini and are homologous to AtCRY1. To investigate the function of OsCRY1, we overexpressed a green fluorescence protein-OsCRY1 fusion gene in Arabidopsis and assessed the phenotypes of the resulting transgenic plants. When the seedlings were germinated in the dark, no discernible effect was observed. However, light-germinated seedlings showed pronounced inhibition of hypocotyl elongation and increased accumulation of anthocyanin. These phenotypes were induced in a blue-light-dependent manner, indicating that OsCRY1 functions as a blue-light-receptor cryptochrome. We also examined the intracellular localization of green fluorescence protein-OsCRY1 in the transgenic plants. It was localized to both the nucleus and the cytoplasm. We identified two nuclear localization domains in the primary structure of OsCRY1. We discuss the relationship between the function and intracellular localization of rice cryptochromes by using additional data obtained with OsCRY2. PMID:14657402

  11. Axotomy Depletes Intracellular Calcium Stores in Primary Sensory Neurons

    PubMed Central

    Rigaud, Marcel; Gemes, Geza; Weyker, Paul D.; Cruikshank, James M.; Kawano, Takashi; Wu, Hsiang-En; Hogan, Quinn H.

    2010-01-01

    Background The cellular mechanisms of neuropathic pain are inadequately understood. Previous investigations have revealed disrupted Ca2+ signaling in primary sensory neurons after injury. We therefore examined the effect of injury on intracellular Ca2+ stores of the endoplasmic reticulum, which critically regulate the Ca2+ signal and neuronal function. Methods Intracellular Ca2+ levels were measured with Fura-2 or mag-Fura-2 microfluorometry in axotomized fifth lumbar (L5) dorsal root ganglion neurons and adjacent L4 neurons isolated from hyperalgesic rats following L5 spinal nerve ligation, compared to neurons from control animals. Results Endoplasmic reticulum Ca2+ stores released by the ryanodine-receptor agonist caffeine decreased by 46% in axotomized small neurons. This effect persisted in Ca2+-free bath solution that removes the contribution of store-operated membrane Ca2+ channels, and after blockade of both the mitochondrial, sarco-endoplasmic Ca2+-ATPase, and the plasma membrane Ca2+ ATPase pathways. Ca2+ released by the sarco-endoplasmic Ca2+-ATPase blocker thapsigargin and by the Ca2+-ionophore ionomycin was also diminished by 25% and 41%, respectively. In contrast to control neurons, Ca2+ stores in axotomized neurons were not expanded by neuronal activation by K+ depolarization, and the proportionate rate of refilling by sarco-endoplasmic Ca2+-ATPase was normal. Luminal Ca2+ concentration was also reduced by 38% in axotomized neurons in permeabilized neurons. The adjacent neurons of the L4 dorsal root ganglia showed modest and inconsistent changes after L5 spinal nerve ligation. Conclusions Painful nerve injury leads to diminished releasable endoplasmic reticulum Ca2+ stores and a reduced luminal Ca2+ concentration. Depletion of Ca2+ stores may contribute to the pathogenesis of neuropathic pain. PMID:19602958

  12. Endothelial tubes assemble from intracellular vacuoles in vivo.

    PubMed

    Kamei, Makoto; Saunders, W Brian; Bayless, Kayla J; Dye, Louis; Davis, George E; Weinstein, Brant M

    2006-07-27

    The formation of epithelial tubes is crucial for the proper development of many different tissues and organs, and occurs by means of a variety of different mechanisms. Morphogenesis of seamless, properly patterned endothelial tubes is essential for the development of a functional vertebrate circulatory system, but the mechanism of vascular lumenization in vivo remains unclear. Evidence dating back more than 100 years has hinted at an important function for endothelial vacuoles in lumen formation. More than 25 years ago, in some of the first endothelial cell culture experiments in vitro, Folkman and Haudenschild described "longitudinal vacuoles" that "appeared to be extruded and connected from one cell to the next", observations confirmed and extended by later studies in vitro showing that intracellular vacuoles arise from integrin-dependent and cdc42/Rac1-dependent pinocytic events downstream of integrin-extracellular-matrix signalling interactions. Despite compelling data supporting a model for the assembly of endothelial tubes in vitro through the formation and fusion of vacuoles, conclusive evidence in vivo has been lacking, primarily because of difficulties associated with imaging the dynamics of subcellular endothelial vacuoles deep within living animals. Here we use high-resolution time-lapse two-photon imaging of transgenic zebrafish to examine how endothelial tubes assemble in vivo, comparing our results with time-lapse imaging of human endothelial-cell tube formation in three-dimensional collagen matrices in vitro. Our results provide strong support for a model in which the formation and intracellular and intercellular fusion of endothelial vacuoles drives vascular lumen formation. PMID:16799567

  13. Biomechanics and Intracellular Dynamics of Vascular Endothelial Cells

    NASA Astrophysics Data System (ADS)

    Ou-Yang, H. Daniel

    2004-03-01

    Understanding the internal mechanical properties of living cells is essential to gain insight to basic cellular functions ranging from cellular signal transduction, intracellular traffics to cell motility. Vascular endothelial cells form a single cell layer that lines all blood vessels and serves to regulate exchanges between the blood stream and the surrounding tissues. Endothelial cells are one of the most studied cell types because of their roles in cardiovascular diseases and the linkage between their growth control and strategies of cancer treatments. This talk reports the application of a novel methodology by which scientists can explore cellular functions and study cytoskeleton dynamics of living cells at the subcellular level with minimal invasion. The methodology is based on the realization that optical tweezers can be used to measure the mechanical properties of the cytoskeleton in the vicinity of organelles and cellular structures. Optical tweezers is a technique based on the physics that dielectric materials, such as silica beads, latex particles or protein aggregates are attracted to and thus trapped at the focal point of a tightly focused laser beam in an aqueous medium. It has been shown that viscoelasticity can be determined from the movements of the trapped object in an oscillating optical tweezers. Applying the oscillating tweezers to intracellular cellular structures, we were able to determine the frequency dependent mechanical properties of the interior of cultured bovine endothelial cells. In contrast to the viscoelastic behavior expected of a network of cytoskelatal proteins, we found unusually large fluctuations in both elastic and loss moduli of the cell interior. More surprisingly, both mechanical moduli showed rhythmic behavior with a periodicity in the range of 20 - 30 seconds in healthy living cells. The rhythm could be altered by drug treatments, and the amplitude of the fluctuations diminished when cells were depleted of nutrients

  14. Intracellular protein interaction mapping with FRET hybrids

    PubMed Central

    You, Xia; Nguyen, Annalee W.; Jabaiah, Abeer; Sheff, Mark A.; Thorn, Kurt S.; Daugherty, Patrick S.

    2006-01-01

    A quantitative methodology was developed to identify protein interactions in a broad range of cell types by using FRET between fluorescent proteins. Genetic fusions of a target receptor to a FRET acceptor and a large library of candidate peptide ligands to a FRET donor enabled high-throughput optical screening for optimal interaction partners in the cytoplasm of Escherichia coli. Flow cytometric screening identified a panel of peptide ligands capable of recognizing the target receptors in the intracellular environment. For both SH3 and PDZ domain-type target receptors, physiologically meaningful consensus sequences were apparent among the isolated ligands. The relative dissociation constants of interacting partners could be measured directly by using a dilution series of cell lysates containing FRET hybrids, providing a previously undescribed high-throughput approach to rank the affinity of many interaction partners. FRET hybrid interaction screening provides a powerful tool to discover protein ligands in the cellular context with potential applications to a wide variety of eukaryotic cell types. PMID:17130455

  15. On the Computing Potential of Intracellular Vesicles

    PubMed Central

    Mayne, Richard; Adamatzky, Andrew

    2015-01-01

    Collision-based computing (CBC) is a form of unconventional computing in which travelling localisations represent data and conditional routing of signals determines the output state; collisions between localisations represent logical operations. We investigated patterns of Ca2+-containing vesicle distribution within a live organism, slime mould Physarum polycephalum, with confocal microscopy and observed them colliding regularly. Vesicles travel down cytoskeletal ‘circuitry’ and their collisions may result in reflection, fusion or annihilation. We demonstrate through experimental observations that naturally-occurring vesicle dynamics may be characterised as a computationally-universal set of Boolean logical operations and present a ‘vesicle modification’ of the archetypal CBC ‘billiard ball model’ of computation. We proceed to discuss the viability of intracellular vesicles as an unconventional computing substrate in which we delineate practical considerations for reliable vesicle ‘programming’ in both in vivo and in vitro vesicle computing architectures and present optimised designs for both single logical gates and combinatorial logic circuits based on cytoskeletal network conformations. The results presented here demonstrate the first characterisation of intracelluar phenomena as collision-based computing and hence the viability of biological substrates for computing. PMID:26431435

  16. Intracellular pH of symbiotic dinoflagellates

    NASA Astrophysics Data System (ADS)

    Gibbin, E. M.; Davy, S. K.

    2013-09-01

    Intracellular pH (pHi) is likely to play a key role in maintaining the functional success of cnidarian-dinoflagellate symbiosis, yet until now the pHi of the symbiotic dinoflagellates (genus Symbiodinium) has never been quantified. Flow cytometry was used in conjunction with the ratiometric fluorescent dye BCECF to monitor changes in pHi over a daily light/dark cycle. The pHi of Symbiodinium type B1 freshly isolated from the model sea anemone Aiptasia pulchella was 7.25 ± 0.01 (mean ± SE) in the light and 7.10 ± 0.02 in the dark. A comparable effect of irradiance was seen across a variety of cultured Symbiodinium genotypes (types A1, B1, E1, E2, F1, and F5) which varied between pHi 7.21-7.39 in the light and 7.06-7.14 in the dark. Of note, there was a significant genotypic difference in pHi, irrespective of irradiance.

  17. An intracellular anion channel critical for pigmentation

    PubMed Central

    Bellono, Nicholas W; Escobar, Iliana E; Lefkovith, Ariel J; Marks, Michael S; Oancea, Elena

    2014-01-01

    Intracellular ion channels are essential regulators of organellar and cellular function, yet the molecular identity and physiological role of many of these channels remains elusive. In particular, no ion channel has been characterized in melanosomes, organelles that produce and store the major mammalian pigment melanin. Defects in melanosome function cause albinism, characterized by vision and pigmentation deficits, impaired retinal development, and increased susceptibility to skin and eye cancers. The most common form of albinism is caused by mutations in oculocutaneous albinism II (OCA2), a melanosome-specific transmembrane protein with unknown function. Here we used direct patch-clamp of skin and eye melanosomes to identify a novel chloride-selective anion conductance mediated by OCA2 and required for melanin production. Expression of OCA2 increases organelle pH, suggesting that the chloride channel might regulate melanin synthesis by modulating melanosome pH. Thus, a melanosomal anion channel that requires OCA2 is essential for skin and eye pigmentation. DOI: http://dx.doi.org/10.7554/eLife.04543.001 PMID:25513726

  18. Backbone Dynamics Of Intracellular Lipid Binding Proteins

    NASA Astrophysics Data System (ADS)

    Gutiérrez-González, Luis H.

    2005-04-01

    The family of intracellular lipid binding proteins (iLBPs) comprises a group of homologous 14-15 kDa proteins that specifically bind and facilitate the transport of fatty acids, bile acids, retinoids or eicosanoids. Members of this family include several types of fatty acid binding proteins (FABPs), ileal lipid binding protein, cellular retinoic acid binding proteins and cellular retinoid binding proteins. As a contribution to understanding the structure-function relationship in this protein family, the solution structure and backbone dynamics of human epidermal-type FABP (E-FABP) determined by NMR spectroscopy are reported. Moreover, hydrogen/deuterium exchange experiments indicated a direct correlation between the stability of the hydrogen-bonding network in the β-sheet structure and the conformational exchange in the millisecond-to-microsecond time range. The features of E-FABP backbone dynamics discussed in the present study are compared with those obtained for other phylogenetically related proteins. A strong interdependence with the overall protein stability and possibly also with the ligand-binding affinity for members of the lipid-binding protein family is shown.

  19. Intracellular trafficking of heat shock factor 2.

    PubMed

    Le Goff, Pascale; Le Dréan, Yves; Le Péron, Christine; Le Jossic-Corcos, Catherine; Ainouche, Abdelkadder; Michel, Denis

    2004-04-01

    HSF2 is an enigmatic member of the heat shock factor family, identified in the homeotherm classes of birds and mammals. We report the characterization of HSF2 from an evolutionary ancient vertebrate, the fish rainbow trout (rtHSF2). rtHSF2 appears closely related to its mammalian counterparts at structural and functional levels. The conservation of the distinctive features of HSF2 from fish to human suggests that it should ensure important biological functions, not redundant with those of HSF1. Proteasome inhibition, reported as a potent stimulator of HSF2, leads to the stabilization and to a striking nuclear trafficking of rtHSF2-GFP fusion protein. Upon treatment with the proteasome inhibitor MG132, rtHSF2-GFP accumulates into PML nuclear bodies (NBs) independently of its sumoylation and, if expressed at moderate level, moves to nucleoli. The translocation of rtHSF2-GFP from NBs to nucleoli is greatly favored by overexpression of the heat shock protein Hsp70. The mammalian counterpart mouse HSF2 (mHSF2) also exhibited changes in intracellular distribution upon MG132 treatment. mHSF2 partitioned between a juxtanuclear area that we characterized as an aggresome and the nucleoli. These relocalizations are likely to reflect common structural changes of mouse and trout HSF2 upon activation. PMID:15023536

  20. Characterizations of intracellular arsenic in a bacterium

    NASA Astrophysics Data System (ADS)

    Wolfe-Simon, F.; Yannone, S. M.; Tainer, J. A.

    2011-12-01

    Life requires a key set of chemical elements to sustain growth. Yet, a growing body of literature suggests that microbes can alter their nutritional requirements based on the availability of these chemical elements. Under limiting conditions for one element microbes have been shown to utilize a variety of other elements to serve similar functions often (but not always) in similar molecular structures. Well-characterized elemental exchanges include manganese for iron, tungsten for molybdenum and sulfur for phosphorus or oxygen. These exchanges can be found in a wide variety of biomolecules ranging from protein to lipids and DNA. Recent evidence suggested that arsenic, as arsenate or As(V), was taken up and incorporated into the cellular material of the bacterium GFAJ-1. The evidence was interpreted to support As(V) acting in an analogous role to phosphate. We will therefore discuss our ongoing efforts to characterize intracellular arsenate and how it may partition among the cellular fractions of the microbial isolate GFAJ-1 when exposed to As(V) in the presence of various levels of phosphate. Under high As(V) conditions, cells express a dramatically different proteome than when grown given only phosphate. Ongoing studies on the diversity and potential role of proteins and metabolites produced in the presence of As(V) will be reported. These investigations promise to inform the role and additional metabolic potential for As in biology. Arsenic assimilation into biomolecules contributes to the expanding set of chemical elements utilized by microbes in unusual environmental niches.

  1. Histone Acetylation Regulates Intracellular pH

    PubMed Central

    McBrian, Matthew A.; Behbahan, Iman Saramipoor; Ferrari, Roberto; Su, Trent; Huang, Ta-Wei; Li, Kunwu; Hong, Candice S.; Christofk, Heather R.; Vogelauer, Maria; Seligson, David B.; Kurdistani, Siavash K.

    2014-01-01

    SUMMARY Differences in global levels of histone acetylation occur in normal and cancer cells, although the reason why cells regulate these levels has been unclear. Here we demonstrate a role for histone acetylation in regulating intracellular pH (pHi). As pHi decreases, histones are globally deacetylated by histone deacetylases (HDACs), and the released acetate anions are coexported with protons out of the cell by monocarboxylate transporters (MCTs), preventing further reductions in pHi. Conversely, global histone acetylation increases as pHi rises, such as when resting cells are induced to proliferate. Inhibition of HDACs or MCTs decreases acetate export and lowers pHi, particularly compromising pHi maintenance in acidic environments. Global deacetylation at low pH is reflected at a genomic level by decreased abundance and extensive redistribution of acetylation throughout the genome. Thus, acetylation of chromatin functions as a rheostat to regulate pHi with important implications for mechanism of action and therapeutic use of HDAC inhibitors. PMID:23201122

  2. An intracellular anion channel critical for pigmentation.

    PubMed

    Bellono, Nicholas W; Escobar, Iliana E; Lefkovith, Ariel J; Marks, Michael S; Oancea, Elena

    2014-01-01

    Intracellular ion channels are essential regulators of organellar and cellular function, yet the molecular identity and physiological role of many of these channels remains elusive. In particular, no ion channel has been characterized in melanosomes, organelles that produce and store the major mammalian pigment melanin. Defects in melanosome function cause albinism, characterized by vision and pigmentation deficits, impaired retinal development, and increased susceptibility to skin and eye cancers. The most common form of albinism is caused by mutations in oculocutaneous albinism II (OCA2), a melanosome-specific transmembrane protein with unknown function. Here we used direct patch-clamp of skin and eye melanosomes to identify a novel chloride-selective anion conductance mediated by OCA2 and required for melanin production. Expression of OCA2 increases organelle pH, suggesting that the chloride channel might regulate melanin synthesis by modulating melanosome pH. Thus, a melanosomal anion channel that requires OCA2 is essential for skin and eye pigmentation. PMID:25513726

  3. A viral peptide for intracellular delivery

    NASA Astrophysics Data System (ADS)

    Falanga, Annarita; Tarallo, Rossella; Cantisani, Marco; Della Pepa, Maria Elena; Galdiero, Massimiliano; Galdiero, Stefania

    2012-10-01

    Biological membranes represent a critical hindrance for administering active molecules which are often unable to reach their designated intracellular target sites. In order to overcome this barrier-like behavior not easily circumvented by many pharmacologically-active molecules, synthetic transporters have been exploited to promote cellular uptake. Linking or complexing therapeutic molecules to peptides that can translocate through the cellular membranes could enhance their internal delivery, and consequently, a higher amount of active compound would reach the site of action. Use of cell penetrating peptides (CPPs) is one of the most promising strategy to efficiently translocate macromolecules through the plasma membrane, and have attracted a lot of attention. New translocating peptides are continuously described and in the present review, we will focus on viral derived peptides, and in particular a peptide (gH625) derived from the herpes simplex virus type 1 (HSV-1) glycoprotein H (gH) that has proved to be a useful delivery vehicle due to its intrinsic properties of inducing membrane perturbation.

  4. Intracellular sphingosine releases calcium from lysosomes

    PubMed Central

    Höglinger, Doris; Haberkant, Per; Aguilera-Romero, Auxiliadora; Riezman, Howard; Porter, Forbes D; Platt, Frances M; Galione, Antony; Schultz, Carsten

    2015-01-01

    To elucidate new functions of sphingosine (Sph), we demonstrate that the spontaneous elevation of intracellular Sph levels via caged Sph leads to a significant and transient calcium release from acidic stores that is independent of sphingosine 1-phosphate, extracellular and ER calcium levels. This photo-induced Sph-driven calcium release requires the two-pore channel 1 (TPC1) residing on endosomes and lysosomes. Further, uncaging of Sph leads to the translocation of the autophagy-relevant transcription factor EB (TFEB) to the nucleus specifically after lysosomal calcium release. We confirm that Sph accumulates in late endosomes and lysosomes of cells derived from Niemann-Pick disease type C (NPC) patients and demonstrate a greatly reduced calcium release upon Sph uncaging. We conclude that sphingosine is a positive regulator of calcium release from acidic stores and that understanding the interplay between Sph homeostasis, calcium signaling and autophagy will be crucial in developing new therapies for lipid storage disorders such as NPC. DOI: http://dx.doi.org/10.7554/eLife.10616.001 PMID:26613410

  5. Intracellular sphingosine releases calcium from lysosomes.

    PubMed

    Höglinger, Doris; Haberkant, Per; Aguilera-Romero, Auxiliadora; Riezman, Howard; Porter, Forbes D; Platt, Frances M; Galione, Antony; Schultz, Carsten

    2015-01-01

    To elucidate new functions of sphingosine (Sph), we demonstrate that the spontaneous elevation of intracellular Sph levels via caged Sph leads to a significant and transient calcium release from acidic stores that is independent of sphingosine 1-phosphate, extracellular and ER calcium levels. This photo-induced Sph-driven calcium release requires the two-pore channel 1 (TPC1) residing on endosomes and lysosomes. Further, uncaging of Sph leads to the translocation of the autophagy-relevant transcription factor EB (TFEB) to the nucleus specifically after lysosomal calcium release. We confirm that Sph accumulates in late endosomes and lysosomes of cells derived from Niemann-Pick disease type C (NPC) patients and demonstrate a greatly reduced calcium release upon Sph uncaging. We conclude that sphingosine is a positive regulator of calcium release from acidic stores and that understanding the interplay between Sph homeostasis, calcium signaling and autophagy will be crucial in developing new therapies for lipid storage disorders such as NPC. PMID:26613410

  6. Apoptotic cell death induced by intracellular proteolysis.

    PubMed

    Williams, M S; Henkart, P A

    1994-11-01

    To mimic the injection of granzymes into target cells by cytotoxic lymphocytes or the activation of endogenous proteases in programmed cell death, the proteases chymotrypsin, proteinase K, or trypsin were loaded into the cytoplasm of several different cell types using the osmotic lysis of pinosomes technique. Internalization of these proteases caused cell lysis within several hours, accompanied by extensive nuclear damage in most but not all combinations of target cells and proteases. This nuclear damage, quantitated by DNA release from nuclei, was associated with apoptotic features including DNA fragmentation into nucleosomal ladders, chromatin condensation, nuclear fragmentation, and membrane blebbing. Agents reported to block programmed cell death, including aurintricarboxylic acid, inhibitors of energy metabolism, and protein or RNA synthesis, failed to block this protease-induced death, although some inhibited nuclear damage. In separate experiments, introduction of staphylococcal nuclease into cells led to near complete (at least 75% of total) nucleosomal DNA fragmentation within 6 to 8 h. Condensation of chromatin did not accompany this fragmentation to the same extent, and there was approximately a 10-h lag between half-maximal DNA fragmentation and 50% loss of membrane integrity. The results suggest that activation of intracellular proteases during cell death by any molecular pathway could give rise to apoptotic morphology and DNA fragmentation. PMID:7930626

  7. On the Computing Potential of Intracellular Vesicles.

    PubMed

    Mayne, Richard; Adamatzky, Andrew

    2015-01-01

    Collision-based computing (CBC) is a form of unconventional computing in which travelling localisations represent data and conditional routing of signals determines the output state; collisions between localisations represent logical operations. We investigated patterns of Ca2+-containing vesicle distribution within a live organism, slime mould Physarum polycephalum, with confocal microscopy and observed them colliding regularly. Vesicles travel down cytoskeletal 'circuitry' and their collisions may result in reflection, fusion or annihilation. We demonstrate through experimental observations that naturally-occurring vesicle dynamics may be characterised as a computationally-universal set of Boolean logical operations and present a 'vesicle modification' of the archetypal CBC 'billiard ball model' of computation. We proceed to discuss the viability of intracellular vesicles as an unconventional computing substrate in which we delineate practical considerations for reliable vesicle 'programming' in both in vivo and in vitro vesicle computing architectures and present optimised designs for both single logical gates and combinatorial logic circuits based on cytoskeletal network conformations. The results presented here demonstrate the first characterisation of intracelluar phenomena as collision-based computing and hence the viability of biological substrates for computing. PMID:26431435

  8. Prolonged Ventricular Asystole: A Rare Adverse Effect of Hydrocodone Use

    PubMed Central

    Sudhakaran, Sivakumar; Surani, Saherish S.; Surani, Salim R.

    2014-01-01

    Patient: Female, 56 Final Diagnosis: Ventricular asystole Symptoms: Dizziness, headache, near-syncope, weakness Medication: — Clinical Procedure: — Specialty: Cardiology Objective: Unusual clinical course Background: Prolonged ventricular asystole is a rare vagal reaction caused by hydrocodone use. Sinus bradycardia is a characteristic presentation of the vasovagal response; examples of other presentations include arrest or atrioventricular block. Physicians need to be aware of ventricular asystole due to vagally-mediated atrioventricular block caused by hydrocodone or other opiates. Case Report: We present a case of prolonged ventricular asystole in a young patient due to a vasovagal reaction caused by the hydrocodone found in the hydrocodone/acetaminophen combination. Conclusions: Ventricular asystole can be a rare complication of hydrocodone found in hydrocodone/acetaminophen. Physicians need to be aware of this adverse effect, rather then resorting to expensive diagnostic interventions. PMID:25330933

  9. Prolonged Remission in Neuromyelitis Optica Following Cessation of Rituximab Treatment.

    PubMed

    Weinfurtner, Kelley; Graves, Jennifer; Ness, Jayne; Krupp, Lauren; Milazzo, Maria; Waubant, Emmanuelle

    2015-09-01

    Neuromyelitis optica is an autoimmune disease characterized by acute episodes of transverse myelitis and optic neuritis. Several small, open-label studies suggest rituximab, a monoclonal antibody against CD20, prevents relapses in neuromyelitis optica; however, there is little consensus on timing or duration of treatment. Here we report four patients with severe relapsing neuromyelitis optica who were stabilized on rituximab and, after discontinuing treatment, continued to experience prolonged remission of their disease. Remission ranged from 4.5 to 10.5 years total, including 3 to 9 years off all therapies. The patients had sustained clinical responses despite normal B-lymphocyte levels and, in at least 2 patients, continued seropositivity for aquaporin-4 antibodies. These cases suggest that rituximab may induce prolonged remission in certain neuromyelitis optica patients, and they highlight the need for further elucidation of rituximab's mechanism in neuromyelitis optica. PMID:25387545

  10. Deep, prolonged torpor by pregnant, free-ranging bats

    NASA Astrophysics Data System (ADS)

    Willis, Craig K. R.; Brigham, R. Mark; Geiser, Fritz

    2006-02-01

    Many mammals save energy during food shortage or harsh weather using controlled reductions in body temperature and metabolism called torpor. However, torpor slows offspring growth, and reproductive individuals are thought to avoid using it because of reduced fitness resulting from delayed offspring development. We tested this hypothesis by investigating torpor during reproduction in hoary bats ( Lasiurus cinereus, Vespertilionidae) in southern Canada. We recorded deep, prolonged torpor bouts, which meet the definition for hibernation, by pregnant females. Prolonged torpor occurred during spring storms. When conditions improved females aroused and gave birth within several days. Our observations imply a fitness advantage of torpor in addition to energy conservation because reduced foetal growth rate could delay parturition until conditions are more favourable for lactation and neonatal survival.

  11. How to prolong the patency of vascular access.

    PubMed

    Glazer, Sidney; Saint, Lindsey; Shenoy, Surendra

    2015-01-01

    Prolonging the patency and limiting the complications of a functioning hemodialysis (HD) access require a multidisciplinary approach. It begins with careful access planning that is executed and continually reinforced by physicians and facility staff encouraging active patient participation. Vascular access (VA) dysfunctions identified by regular monitoring and surveillance need further evaluation. Color duplex ultrasound is evolving as the primary tool to evaluate functional implication of the structural problems in the VA. While ease of scheduling makes endovascular management attractive, definitive surgical management provides better longevity and should be used when indicated. Timing of intervention and selection of technique depend on optimal use of available expertise and the nature of the problem. Avoiding a bridging HD catheter should be a priority while prolonging access patency and improving patient safety. PMID:25676299

  12. Intracellular cleavable poly(2-dimethylaminoethyl methacrylate) functionalized mesoporous silica nanoparticles for efficient siRNA delivery in vitro and in vivo

    NASA Astrophysics Data System (ADS)

    Lin, Daoshu; Cheng, Qiang; Jiang, Qian; Huang, Yuanyu; Yang, Zheng; Han, Shangcong; Zhao, Yuning; Guo, Shutao; Liang, Zicai; Dong, Anjie

    2013-05-01

    A low cytotoxicity and high efficiency delivery system with the advantages of low cost and facile fabrication is needed for the application of small interfering RNA (siRNA) delivery both in vitro and in vivo. For these prerequisites, cationic polymer-mesoporous silica nanoparticles (ssCP-MSNs) were prepared by surface functionalized mesoporous silica nanoparticles with disulfide bond cross-linked poly(2-dimethylaminoethyl methacrylate) (PDMAEMA). In vitro and in vivo evaluations were performed. The synthesized ssCP-MSNs are 100-150 nm in diameter with a pore size of 10 nm and a positively charged surface with a high zeta potential of 27 mV. Consequently, the ssCP-MSNs showed an excellent binding capacity for siRNA, and an enhancement in the cell uptake and cytosolic availability of siRNA. Furthermore, the intracellular reducing cleavage of the disulfide bonds cross-linking the PDMAEMA segments led to intracellular cleavage of PDMAEMA from ssCP-MSNs, which facilitated the intracellular triggered release of siRNA. Therefore, promoted RNA interference was observed in HeLa-Luc cells, which was equal to that of Lipofectamine 2000. Significantly, compared to Lipofectamine 2000, the ssCP-MSNs were more biocompatible, with low cytotoxicity (even non-cytotoxicity) and promotion of cell proliferation to HeLa-Luc cells. The in vivo systemic distribution studies certified that ssCP-MSNs/siRNA could prolong the duration of siRNA in vivo, and that they accumulated in the adrenal gland, liver, lung, spleen, kidney, heart and thymus after intravenous injection. Encouragingly, with the ability to deliver siRNA to a tumor, ssCP-MSNs/siRNA showed a tumor suppression effect in the HeLa-Luc xenograft murine model after intravenous injection. Therefore, the ssCP-MSNs cationic polymer-mesoporous silica nanoparticles with low cytotoxicity are promising for siRNA delivery.A low cytotoxicity and high efficiency delivery system with the advantages of low cost and facile fabrication is needed

  13. Reduced insulin secretion function is associated with pancreatic islet redistribution of cell adhesion molecules (CAMS) in diabetic mice after prolonged high-fat diet.

    PubMed

    Falcão, Viviane Tannuri F L; Maschio, Daniela A; de Fontes, Camila Calvo; Oliveira, Ricardo B; Santos-Silva, Junia C; Almeida, Anna Carolina Soares; Vanzela, Emerielle C; Cartaxo, Maria Tereza; Carvalho, Carolina P F; Collares-Buzato, Carla Beatriz

    2016-07-01

    Intercellular junctions play a role in regulating islet cytoarchitecture, insulin biosynthesis and secretion. In this study, we investigated the animal metabolic state as well as islet histology and cellular distribution/expression of CAMs and F-actin in the endocrine pancreas of C57BL/6/JUnib mice fed a high-fat diet (HFd) for a prolonged time period (8 months). Mice fed a HFd became obese and type 2 diabetic, displaying significant peripheral insulin resistance, hyperglycemia and moderate hyperinsulinemia. Isolated islets of HFd-fed mice displayed a significant impairment of glucose-induced insulin secretion associated with a diminished frequency of intracellular calcium oscillations compared with control islets. No marked change in islet morphology and cytoarchitecture was observed; however, HFd-fed mice showed higher beta cell relative area in comparison with controls. As shown by immunohistochemistry, ZO-1, E-, N-cadherins, α- and β-catenins were expressed at the intercellular contact site of endocrine cells, while VE-cadherin, as well as ZO-1, was found at islet vascular compartment. Redistribution of N-, E-cadherins and α-catenin (from the contact region to the cytoplasm in endocrine cells) associated with increased submembranous F-actin cell level as well as increased VE-cadherin islet immunolabeling was observed in diabetic mice. Increased gene expression of VE-cadherin and ZO-1, but no change for the other proteins, was observed in islets of diabetic mice. Only in the case of VE-cadherin, a significant increase in islet content of this CAM was detected by immunoblotting in diabetic mice. In conclusion, CAMs are expressed by endocrine and endothelial cells of pancreatic islets. The distribution/expression of N-, E- and VE-cadherins as well as α-catenin and F-actin is significantly altered in islet cells of obese and diabetic mice. PMID:27020567

  14. Regulation of intracellular heme trafficking revealed by subcellular reporters.

    PubMed

    Yuan, Xiaojing; Rietzschel, Nicole; Kwon, Hanna; Walter Nuno, Ana Beatriz; Hanna, David A; Phillips, John D; Raven, Emma L; Reddi, Amit R; Hamza, Iqbal

    2016-08-30

    Heme is an essential prosthetic group in proteins that reside in virtually every subcellular compartment performing diverse biological functions. Irrespective of whether heme is synthesized in the mitochondria or imported from the environment, this hydrophobic and potentially toxic metalloporphyrin has to be trafficked across membrane barriers, a concept heretofore poorly understood. Here we show, using subcellular-targeted, genetically encoded hemoprotein peroxidase reporters, that both extracellular and endogenous heme contribute to cellular labile heme and that extracellular heme can be transported and used in toto by hemoproteins in all six subcellular compartments examined. The reporters are robust, show large signal-to-background ratio, and provide sufficient range to detect changes in intracellular labile heme. Restoration of reporter activity by heme is organelle-specific, with the Golgi and endoplasmic reticulum being important sites for both exogenous and endogenous heme trafficking. Expression of peroxidase reporters in Caenorhabditis elegans shows that environmental heme influences labile heme in a tissue-dependent manner; reporter activity in the intestine shows a linear increase compared with muscle or hypodermis, with the lowest heme threshold in neurons. Our results demonstrate that the trafficking pathways for exogenous and endogenous heme are distinct, with intrinsic preference for specific subcellular compartments. We anticipate our results will serve as a heuristic paradigm for more sophisticated studies on heme trafficking in cellular and whole-animal models. PMID:27528661

  15. Hormone supply of the organism in prolonged emotional stress

    NASA Technical Reports Server (NTRS)

    Amiragova, M. G.; Stulnikov, B. V.; Svirskaya, R. I.

    1980-01-01

    The effect of prolonged emotional stress of varying genesis on the hormonal function of the pancreas, thyroid gland, and adrenal cortex was studied. The amount of the hormonal secretion was found to depend on the type of adaptation activity and its duration. High secretion of the hormones observed outside the adaptation activity was examined as an index of the phase transition of defense reactions to the phase of overstress.

  16. Prolonged Soil Frost Affects Hydraulics and Phenology of Apple Trees.

    PubMed

    Beikircher, Barbara; Mittmann, Claudia; Mayr, Stefan

    2016-01-01

    Restoration of an adequate water supply in spring is a prerequisite for survival of angiosperm trees in temperate regions. Trees must re-establish access to soil water and recover xylem functionality. We thus hypothesized that prolonged soil frost impairs recovery and affects hydraulics and phenology of Malus domestica var. 'Golden Delicious.' To test this hypothesis, over two consecutive winters the soil around some trees was insulated to prolong soil frosting, From mid-winter to early summer, the level of native embolism, the water and starch contents of wood, bark and buds were quantified at regular intervals and findings correlated with various phenological parameters, xylogenesis and fine root growth. The findings confirm that prolonged soil frost affects tree hydraulics and phenology but the severity of the effect depends on the climatic conditions. In both study years, percentage loss of hydraulic conductivity (PLC) decreased from about 70% at the end of winter to about 10% in May. Thereby, xylem refilling strongly coincided with a decrease of starch in wood and bark. Also treated trees were able to restore their hydraulic system by May but, in the warm spring of 2012, xylem refilling, the increases in water content and starch depolymerization were delayed. In contrast, in the cold spring of 2013 only small differences between control and treated trees were observed. Prolongation of soil frost also led to a delay in phenology, xylogenesis, and fine root growth. We conclude that reduced water uptake from frozen or cold soils impairs refilling and thus negatively impacts tree hydraulics and growth of apple trees in spring. Under unfavorable circumstances, this may cause severe winter damage or even dieback. PMID:27379146

  17. [Karyological analysis of hybridoma cells after prolonged cultivation].

    PubMed

    Zhil'tsova, M A; Trofimova, M N; Novikov, V V

    1989-06-01

    The karyological properties, the level of monoclonal antibody production and the proliferative properties of hybridoma strains after their prolonged passage in vivo and in vitro have been studied. Hybridoma EKO-G-2 having the supertetraploid set of chromosomes has proved to be a more stable antibody producer and to possess better proliferative properties. The suggestion has been made that the stability of antibody production is linked with the surplus number of chromosomal copies. PMID:2800799

  18. The unpredictability of prolonged activation of stress response pathways

    PubMed Central

    Lamech, Lilian T.

    2015-01-01

    In response to stress, cellular compartments activate signaling pathways that mediate transcriptional programs to promote survival and reestablish homeostasis. Manipulation of the magnitude and duration of the activation of stress responses has been proposed as a strategy to prevent or repair the damage associated with aging or degenerative diseases. However, as these pathways likely evolved to respond specifically to transient perturbations, the unpredictability of prolonged activation should be considered. PMID:26101215

  19. Deep vein thrombosis following prolonged kneeling: a case report.

    PubMed

    van Beeck, J Looringh; Versfeld, K; Ehrlich, R

    2014-06-01

    This report describes a fibreglass mould maker in the yacht building industry who developed a deep vein thrombosis (DVT) after 6 weeks of working in a kneeling position. We propose that his prolonged kneeling combined with constrictive knee pad straps caused vascular compression, precipitating his DVT. A hypercoagulability diathesis was suspected but not confirmed. Operator and employer education, modified work practices and strapless knee pads are suggested as possible preventive measures. PMID:24727563

  20. Prolonged Soil Frost Affects Hydraulics and Phenology of Apple Trees

    PubMed Central

    Beikircher, Barbara; Mittmann, Claudia; Mayr, Stefan

    2016-01-01

    Restoration of an adequate water supply in spring is a prerequisite for survival of angiosperm trees in temperate regions. Trees must re-establish access to soil water and recover xylem functionality. We thus hypothesized that prolonged soil frost impairs recovery and affects hydraulics and phenology of Malus domestica var. ‘Golden Delicious.’ To test this hypothesis, over two consecutive winters the soil around some trees was insulated to prolong soil frosting, From mid-winter to early summer, the level of native embolism, the water and starch contents of wood, bark and buds were quantified at regular intervals and findings correlated with various phenological parameters, xylogenesis and fine root growth. The findings confirm that prolonged soil frost affects tree hydraulics and phenology but the severity of the effect depends on the climatic conditions. In both study years, percentage loss of hydraulic conductivity (PLC) decreased from about 70% at the end of winter to about 10% in May. Thereby, xylem refilling strongly coincided with a decrease of starch in wood and bark. Also treated trees were able to restore their hydraulic system by May but, in the warm spring of 2012, xylem refilling, the increases in water content and starch depolymerization were delayed. In contrast, in the cold spring of 2013 only small differences between control and treated trees were observed. Prolongation of soil frost also led to a delay in phenology, xylogenesis, and fine root growth. We conclude that reduced water uptake from frozen or cold soils impairs refilling and thus negatively impacts tree hydraulics and growth of apple trees in spring. Under unfavorable circumstances, this may cause severe winter damage or even dieback. PMID:27379146

  1. Intracellular ethanol accumulation in Saccharomyces cerevisiae during fermentation.

    PubMed

    D'Amore, T; Panchal, C J; Stewart, G G

    1988-01-01

    An intracellular accumulation of ethanol in Saccharomyces cerevisiae was observed during the early stages of fermentation (3 h). However, after 12 h of fermentation, the intracellular and extracellular ethanol concentrations were similar. Increasing the osmotic pressure of the medium caused an increase in the ratio of intracellular to extracellular ethanol concentrations at 3 h of fermentation. As in the previous case, the intracellular and extracellular ethanol concentrations were similar after 12 h of fermentation. Increasing the osmotic pressure also caused a decrease in yeast cell growth and fermentation activities. However, nutrient supplementation of the medium increased the extent of growth and fermentation, resulting in complete glucose utilization, even though intracellular ethanol concentrations were unaltered. These results suggest that nutrient limitation is a major factor responsible for the decreased growth and fermentation activities observed in yeast cells at higher osmotic pressures. PMID:3278685

  2. Control of Intracellular Calcium Signaling as a Neuroprotective Strategy

    PubMed Central

    Duncan, R. Scott; Goad, Daryl L.; Grillo, Michael A.; Kaja, Simon; Payne, Andrew J.; Koulen, Peter

    2010-01-01

    Both acute and chronic degenerative diseases of the nervous system reduce the viability and function of neurons through changes in intracellular calcium signaling. In particular, pathological increases in the intracellular calcium concentration promote such pathogenesis. Disease involvement of numerous regulators of intracellular calcium signaling located on the plasma membrane and intracellular organelles has been documented. Diverse groups of chemical compounds targeting ion channels, G-protein coupled receptors, pumps and enzymes have been identified as potential neuroprotectants. The present review summarizes the discovery, mechanisms and biological activity of neuroprotective molecules targeting proteins that control intracellular calcium signaling to preserve or restore structure and function of the nervous system. Disease relevance, clinical applications and new technologies for the identification of such molecules are being discussed. PMID:20335972

  3. Host metabolism regulates intracellular growth of Trypanosoma cruzi.

    PubMed

    Caradonna, Kacey L; Engel, Juan C; Jacobi, David; Lee, Chih-Hao; Burleigh, Barbara A

    2013-01-16

    Metabolic coupling of intracellular pathogens with host cells is essential for successful colonization of the host. Establishment of intracellular infection by the protozoan Trypanosoma cruzi leads to the development of human Chagas' disease, yet the functional contributions of the host cell toward the infection process remain poorly characterized. Here, a genome-scale functional screen identified interconnected metabolic networks centered around host energy production, nucleotide metabolism, pteridine biosynthesis, and fatty acid oxidation as key processes that fuel intracellular T. cruzi growth. Additionally, the host kinase Akt, which plays essential roles in various cellular processes, was critical for parasite replication. Targeted perturbations in these host metabolic pathways or Akt-dependent signaling pathways modulated the parasite's replicative capacity, highlighting the adaptability of this intracellular pathogen to changing conditions in the host. These findings identify key cellular process regulating intracellular T. cruzi growth and illuminate the potential to leverage host pathways to limit T. cruzi infection. PMID:23332160

  4. Host metabolism regulates intracellular growth of Trypanosoma cruzi

    PubMed Central

    Caradonna, Kacey L.; Engel, Juan C.; Jacobi, David; Lee, Chih-Hao; Burleigh, Barbara A.

    2012-01-01

    SUMMARY Metabolic coupling of intracellular pathogens with host cells is essential for successful colonization of the host. Establishment of intracellular infection by the protozoan Trypanosoma cruzi leads to the development of human Chagas disease, yet the functional contributions of the host cell toward the infection process remain poorly characterized. Here, a genome-scale functional screen identified interconnected metabolic networks centered around host energy production, nucleotide metabolism, pteridine biosynthesis, and fatty acid oxidation as key processes that fuel intracellular T. cruzi growth. Additionally, the host kinase Akt, which plays essential roles in various cellular processes, was critical for parasite replication. Targeted perturbations in these host metabolic pathways or Akt-dependent signaling pathways modulated the parasite’s replicative capacity, highlighting the adaptability of this intracellular pathogen to changing conditions in the host. These findings identify key cellular process regulating intracellular T. cruzi growth and illuminate the potential to leverage host pathways to limit T. cruzi infection. PMID:23332160

  5. Satellite Movie Shows Erika Dissipate

    NASA Video Gallery

    This animation of visible and infrared imagery from NOAA's GOES-West satellite from Aug. 27 to 29 shows Tropical Storm Erika move through the Eastern Caribbean Sea and dissipate near eastern Cuba. ...

  6. Is prolonged grief distinct from bereavement-related posttraumatic stress?

    PubMed

    Golden, Ann-Marie J; Dalgleish, Tim

    2010-07-30

    Prolonged grief disorder (PGD) (previously called complicated grief (CG)) is proposed as a distinct post loss syndrome, with its own core symptoms. A key issue concerning the diagnostic validity of PGD is whether it can reliably be distinguished from related psychiatric outcomes following bereavement. This study therefore sought to determine whether the core symptoms of PGD could be distinguished from those of bereavement-related anxiety, depression and posttraumatic stress disorder (PTSD). Data were derived from a community sample of 223 bereaved adults in Croatia. PGD symptomatology was measured using the Revised Inventory of Complicated Grief. Depression and anxiety symptoms were measured using the Beck Depression and Anxiety Inventories, respectively. The intrusion and avoidance symptoms of PTSD were assessed using the Revised Impact of Event Scale. The distinctiveness of the five symptom clusters was examined using principal component analysis (PCA). Symptoms of prolonged grief, depression, anxiety, PTSD-intrusion, and PTSD-avoidance clustered together into five distinct factors. These results support the phenomenological distinctiveness of prolonged grief symptoms, from those of bereavement-related anxiety, depression and, for the first time, PTSD. PMID:20493535

  7. Targeted Delivery of Antibiotics to Intracellular Chlamydial Infections using PLGA Nanoparticles

    PubMed Central

    Toti, Udaya S.; Guru, Bharath R.; Hali, Mirabela; McPharlin, Christopher; Wykes, Susan M.; Panyam, Jayanth; Whittum-Hudson, Judith A.

    2011-01-01

    Chlamydia trachomatis and C pneumoniae are intracellular bacterial pathogens that have been shown to cause, or are strongly associated with, diverse chronic diseases. Persistent infections by both organisms are refractory to antibiotic therapy. The lack of therapeutic efficacy results from the attenuated metabolic rate of persistently infecting chlamydiae in combination with the modest intracellular drug concentrations achievable by normal delivery of antibiotics to the inclusions within which chlamydiae reside in the host cell cytoplasm. In this research, we evaluated whether nanoparticles formulated using the biodegradable poly(d-L-lactide-co-glycolide) (PLGA) polymer can enhance the delivery of antibiotics to the chlamydial inclusion complexes. We initially studied the trafficking of PLGA nanoparticles in Chlamydia-infected cells. We then evaluated nanoparticles for the delivery of antibiotics to the inclusions. Intracellular trafficking studies show that PLGA nanoparticles efficiently concentrate in inclusions in both acutely and persistently infected cells. Further, encapsulation of rifampin and azithromycin antibiotics in PLGA nanoparticles enhanced the effectiveness of the antibiotics in reducing microbial burden. Combination of rifampin and azithromycin was more effective than the individual drugs. Overall, our studies show that PLGA nanoparticles can be effective carriers for targeted delivery of antibiotics to intracellular chlamydial infections. PMID:21652065

  8. Intracellular in vitro probe acylcarnitine assay for identifying deficiencies of carnitine transporter and carnitine palmitoyltransferase-1.

    PubMed

    Purevsuren, Jamiyan; Kobayashi, Hironori; Hasegawa, Yuki; Yamada, Kenji; Takahashi, Tomoo; Takayanagi, Masaki; Fukao, Toshiyuki; Fukuda, Seiji; Yamaguchi, Seiji

    2013-02-01

    Mitochondrial fatty acid oxidation (FAO) disorders are caused by defects in one of the FAO enzymes that regulates cellular uptake of fatty acids and free carnitine. An in vitro probe acylcarnitine (IVP) assay using cultured cells and tandem mass spectrometry is a tool to diagnose enzyme defects linked to most FAO disorders. Extracellular acylcarnitine (AC) profiling detects carnitine palmitoyltransferase-2, carnitine acylcarnitine translocase, and other FAO deficiencies. However, the diagnosis of primary carnitine deficiency (PCD) or carnitine palmitoyltransferase-1 (CPT1) deficiency using the conventional IVP assay has been hampered by the presence of a large amount of free carnitine (C0), a key molecule deregulated by these deficiencies. In the present study, we developed a novel IVP assay for the diagnosis of PCD and CPT1 deficiency by analyzing intracellular ACs. When exogenous C0 was reduced, intracellular C0 and total AC in these deficiencies showed specific profiles clearly distinguishable from other FAO disorders and control cells. Also, the ratio of intracellular to extracellular C0 levels showed a significant difference in cells with these deficiencies compared with control. Hence, intracellular AC profiling using the IVP assay under reduced C0 conditions is a useful method for diagnosing PCD or CPT1 deficiency. PMID:23143007

  9. Free-Standing Kinked Silicon Nanowires for Probing Inter- and Intracellular Force Dynamics.

    PubMed

    Zimmerman, John F; Murray, Graeme F; Wang, Yucai; Jumper, John M; Austin, Jotham R; Tian, Bozhi

    2015-08-12

    Silicon nanowires (SiNWs) have emerged as a new class of materials with important applications in biology and medicine with current efforts having focused primarily on using substrate bound SiNW devices. However, developing devices capable of free-standing inter- and intracellular operation is an important next step in designing new synthetic cellular materials and tools for biophysical characterization. To demonstrate this, here we show that label free SiNWs can be internalized in multiple cell lines, forming robust cytoskeletal interfaces, and when kinked can serve as free-standing inter- and intracellular force probes capable of continuous extended (>1 h) force monitoring. Our results show that intercellular interactions exhibit ratcheting like behavior with force peaks of ∼69.6 pN/SiNW, while intracellular force peaks of ∼116.9 pN/SiNW were recorded during smooth muscle contraction. To accomplish this, we have introduced a simple single-capture dark-field/phase contrast optical imaging modality, scatter enhanced phase contrast (SEPC), which enables the simultaneous visualization of both cellular components and inorganic nanostructures. This approach demonstrates that rationally designed devices capable of substrate-independent operation are achievable, providing a simple and scalable method for continuous inter- and intracellular force dynamics studies. PMID:26192816

  10. Polarized exocyst-mediated vesicle fusion directs intracellular lumenogenesis within the C. elegans excretory cell

    PubMed Central

    Armenti, Stephen T.; Chan, Emily; Nance, Jeremy

    2015-01-01

    Lumenogenesis of small seamless tubes occurs through intracellular membrane growth and directed vesicle fusion events. Within the C. elegans excretory cell, which forms seamless intracellular tubes (canals) that mediate osmoregulation, lumens grow in length and diameter when vesicles fuse with the expanding lumenal surface. Here, we show that lumenal vesicle fusion depends on the small GTPase RAL-1, which localizes to vesicles and acts through the exocyst vesicle-tethering complex. Loss of either the exocyst or RAL-1 prevents excretory canal lumen extension. Within the excretory canal and other polarized cells, the exocyst co-localizes with the PAR polarity proteins PAR-3, PAR-6 and PKC-3. Using early embryonic cells to determine the functional relationships between the exocyst and PAR proteins, we show that RAL-1 recruits the exocyst to the membrane, while PAR proteins concentrate membrane-localized exocyst proteins to a polarized domain. These findings reveal that RAL-1 and the exocyst direct the polarized vesicle fusion events required for intracellular lumenogenesis of the excretory cell, suggesting mechanistic similarities in the formation of topologically distinct multicellular and intracellular lumens. PMID:25102190

  11. Polarized exocyst-mediated vesicle fusion directs intracellular lumenogenesis within the C. elegans excretory cell.

    PubMed

    Armenti, Stephen T; Chan, Emily; Nance, Jeremy

    2014-10-01

    Lumenogenesis of small seamless tubes occurs through intracellular membrane growth and directed vesicle fusion events. Within the Caenorhabditis elegans excretory cell, which forms seamless intracellular tubes (canals) that mediate osmoregulation, lumens grow in length and diameter when vesicles fuse with the expanding lumenal surface. Here, we show that lumenal vesicle fusion depends on the small GTPase RAL-1, which localizes to vesicles and acts through the exocyst vesicle-tethering complex. Loss of either the exocyst or RAL-1 prevents excretory canal lumen extension. Within the excretory canal and other polarized cells, the exocyst co-localizes with the PAR polarity proteins PAR-3, PAR-6 and PKC-3. Using early embryonic cells to determine the functional relationships between the exocyst and PAR proteins, we show that RAL-1 recruits the exocyst to the membrane, while PAR proteins concentrate membrane-localized exocyst proteins to a polarized domain. These findings reveal that RAL-1 and the exocyst direct the polarized vesicle fusion events required for intracellular lumenogenesis of the excretory cell, suggesting mechanistic similarities in the formation of topologically distinct multicellular and intracellular lumens. PMID:25102190

  12. Depletion of Rab32 decreases intracellular lipid accumulation and induces lipolysis through enhancing ATGL expression in hepatocytes.

    PubMed

    Li, Qing; Wang, Jun; Wan, Ying; Chen, Dongfeng

    2016-03-18

    Nonalcoholic fatty liver disease (NAFLD) is a disease caused by the accumulation of lipids in hepatocytes. To date, however, the pathogenesis of NAFLD is still unclear. Recent studies have shown that Rab GTPases, a major protein family in vesicle trafficking, are associated with intracellular lipid accumulation. Here, we show that Rab32, the only Rab GTPase located in mitochondria, participates in hepatic steatosis. Ablation of Rab32 can decrease intracellular lipid accumulation in hepatocytes (HepG2, L02). Further studying the possible mechanism, we found that knockdown of Rab32 can enhance lipolysis instead of lipogenesis via inducing the expression of adipose triglyceride lipase (ATGL), a key enzyme on the surface of lipid droplets which has been proved to be significant in controlling intracellular lipid accumulation. Co-immunoprecipitation shows that Rab32 and ATGL are not directly associated. These findings suggest that knockdown of Rab32 indirectly affects lipolysis through increasing the expression of ATGL. Taken together, our study reveals that Rab32 can participate in regulating intracellular lipid accumulation and that knockdown of Rab32 can decrease intracellular lipid accumulation in hepatocytes. We also demonstrated that ablation of Rab32 can induce intracellular lipolysis by enhancing the expression of ATGL. PMID:26882978

  13. National Orange Show Photovoltaic Demonstration

    SciTech Connect

    Dan Jimenez Sheri Raborn, CPA; Tom Baker

    2008-03-31

    National Orange Show Photovoltaic Demonstration created a 400KW Photovoltaic self-generation plant at the National Orange Show Events Center (NOS). The NOS owns a 120-acre state fairground where it operates an events center and produces an annual citrus fair known as the Orange Show. The NOS governing board wanted to employ cost-saving programs for annual energy expenses. It is hoped the Photovoltaic program will result in overall savings for the NOS, help reduce the State's energy demands as relating to electrical power consumption, improve quality of life within the affected grid area as well as increase the energy efficiency of buildings at our venue. In addition, the potential to reduce operational expenses would have a tremendous effect on the ability of the NOS to service its community.

  14. Harmonization of the intracellular cytokine staining assay.

    PubMed

    Welters, Marij J P; Gouttefangeas, Cécile; Ramwadhdoebe, Tamara H; Letsch, Anne; Ottensmeier, Christian H; Britten, Cedrik M; van der Burg, Sjoerd H

    2012-07-01

    Active immunotherapy for cancer is an accepted treatment modality aiming to reinforce the T-cell response to cancer. T-cell reactivity is measured by various assays and used to guide the clinical development of immunotherapeutics. However, data obtained across different institutions may vary substantially making comparative conclusions difficult. The Cancer Immunotherapy Immunoguiding Program organizes proficiency panels to identify key parameters influencing the outcome of commonly used T-cell assays followed by harmonization. Our successes with IFNγ-ELISPOT and peptide HLA multimer analysis have led to the current study on intracellular cytokine staining (ICS). We report the results of three successive panels evaluating this assay. At the beginning, 3 out of 9 participants (33 %) were able to detect >6 out of 8 known virus-specific T-cell responses in peripheral blood of healthy individuals. This increased to 50 % of the laboratories in the second phase. The reported percentages of cytokine-producing T cells by the different laboratories were highly variable with coefficients of variation well over 60 %. Variability could partially be explained by protocol-related differences in background cytokine production leading to sub-optimal signal-to-noise ratios. The large number of protocol variables prohibited identification of prime guidelines to harmonize the assays. In addition, the gating strategy used to identify reactive T cells had a major impact on assay outcome. Subsequent harmonization of the gating strategy considerably reduced the variability within the group of participants. In conclusion, we propose that first basic guidelines should be applied for gating in ICS experiments before harmonizing assay protocol variables. PMID:22714399

  15. Use of photoproteins as intracellular calcium indicators.

    PubMed Central

    Blinks, J R

    1990-01-01

    The calcium-regulated photoproteins, of which aequorin is the best known, continue to be one of the most useful groups of intracellular Ca2+ indicators. They are self-contained bioluminescent systems that emit blue light in the presence of Ca2+ ions, can readily be purified intact, and are nontoxic when introduced into foreign cells. They have been used successfully as Ca2+ indicators in almost every kind of cell, but are most widely used in muscle cells because of their relative freedom from motion artifacts. Photoproteins have also been used in conjunction with microscopic image intensification to localize Ca2+ in cells. Their large molecular size makes them difficult to introduce into cells, but once there, they have the advantage of staying in the cytoplasm. Aequorin can be microinjected satisfactorily into single cells of almost any size, but a number of alternative methods for introducing photoproteins into cells have been developed in recent years. Disadvantages of the photoproteins for some applications include the nonlinear relation between [Ca2+] and light intensity, the modest speed with which they respond to sudden changes in [Ca2+], and the fact the Mg2+ antagonizes the effect of Ca2+. Native photoproteins consist of a mixture of isospecies, and there are differences in Ca2+ sensitivity and in kinetic properties--both among photoproteins and among the isospecies of a given photoprotein. The genes for several of the isospecies of aequorin have been cloned and expressed in E. coli. It seems reasonable to hope that genetic engineering techniques may soon make it possible to consider using, as Ca2+ indicators, rare isospecies or rare photoproteins that have optimal properties for particular applications. PMID:2190821

  16. Purification and characterization of an intracellular peroxidase from Streptomyces cyaneus

    SciTech Connect

    Mliki, A.; Zimmermann, W. )

    1992-03-01

    Peroxidases play an important role in the oxidation of a large number of aromatic compounds, including recalcitrant substances. An intracellular peroxidase (EC 1.11.1.7) from Streptomyces cyaneus was purified to homogeneity. The enzyme had a molecular weight of 185,000 and was composed of two subunits of equal size. It had an isoelectric point of 6.1. The enzyme had a peroxidase activity toward o-dianisidine with a K{sub m} of 17.8 {mu}M and a pH optimum of 5.0. It also showed catalase activity with a K{sub m} of 2.07 mM H{sub 2}O{sub 2} and a pH optimum of 8.0. The purified enzyme did not catalyze C{alpha}-C{beta} bond cleavage of 1,3-dihydroxy-2-(2-methoxyphenoxy)-1-(4-ethoxy-3-methoxyphenyl) propane, a nonphenolic dimeric lignin model compound. The spectrum of te peroxidase showed a soret band at 405 nm, which disappeared after reduction with sodium dithionite, indicating that the enzyme is a hemoprotein. Testing the effects of various inhibitors on the enzyme activity showed that it is a bifunctional enzyme having catalase and peroxidase activities.

  17. Creating Slide Show Book Reports.

    ERIC Educational Resources Information Center

    Taylor, Harriet G.; Stuhlmann, Janice M.

    1995-01-01

    Describes the use of "Kid Pix 2" software by fourth grade students to develop slide-show book reports. Highlights include collaboration with education majors from Louisiana State University, changes in attitudes of the education major students and elementary students, and problems with navigation and disk space. (LRW)

  18. Producing Talent and Variety Shows.

    ERIC Educational Resources Information Center

    Szabo, Chuck

    1995-01-01

    Identifies key aspects of producing talent shows and outlines helpful hints for avoiding pitfalls and ensuring a smooth production. Presents suggestions concerning publicity, scheduling, and support personnel. Describes types of acts along with special needs and problems specific to each act. Includes a list of resources. (MJP)

  19. Restricting expression prolongs expression of foreign genes introduced into animals by retroviruses.

    PubMed

    Pinto, V B; Prasad, S; Yewdell, J; Bennink, J; Hughes, S H

    2000-11-01

    If foreign genes are ubiquitously expressed in mice using a viral vector, expression is abrogated by CD8(+) cells in 2 to 4 weeks. However, if the expression of the genes is confined to skeletal muscle cells, the CD8(+) T-cell response is much weaker and expression is maintained for more than 6 weeks. These data show that restricting the expression of foreign genes to skeletal muscle cells and presumably to other cells that are inefficient at antigen presentation can prolong the expression of a foreign gene product. PMID:11024149

  20. Prolonged respiratory illness after single overnight continuous positive airway pressure humidification: endotoxin as the suspect.

    PubMed

    Raymond, Lawrence W; Barkley, John E; Langley, Ricky; Sautter, Robert

    2009-12-01

    A patient developed a prolonged respiratory illness after a single overnight use of tap water to humidify air supplied by a constant positive airway pressure (CPAP) device, which she had previously used for six years without difficulty. During those years, she used only distilled water for this purpose, as instructed by her sleep specialist. Analysis of the well water supplying her home showed no microorganisms, metals or other analytes likely to have caused her illness, but endotoxin was found at concentrations well above that recommended by the U.S. Pharmacopeia, as a maximum in water which may be inhaled as an aerosol. PMID:20016436

  1. Visual reaction times during prolonged angular acceleration parallel the subjective perception of rotation

    NASA Technical Reports Server (NTRS)

    Mattson, D. L.

    1975-01-01

    The effect of prolonged angular acceleration on choice reaction time to an accelerating visual stimulus was investigated, with 10 commercial airline pilots serving as subjects. The pattern of reaction times during and following acceleration was compared with the pattern of velocity estimates reported during identical trials. Both reaction times and velocity estimates increased at the onset of acceleration, declined prior to the termination of acceleration, and showed an aftereffect. These results are inconsistent with the torsion-pendulum theory of semicircular canal function and suggest that the vestibular adaptation is of central origin.

  2. Analysis of Intracellular Glucose at Single Cells Using Electrochemiluminescence Imaging.

    PubMed

    Xu, Jingjing; Huang, Peiyuan; Qin, Yu; Jiang, Dechen; Chen, Hong-Yuan

    2016-05-01

    Here, luminol electrochemiluminescence was first applied to analyze intracellular molecules, such as glucose, at single cells. The individual cells were retained in cell-sized microwells on a gold coated indium tin oxide (ITO) slide, which were treated with luminol, triton X-100, and glucose oxidase simultaneously. The broken cellular membrane in the presence of triton X-100 released intracellular glucose into the microwell and reacted with glucose oxidase to generate hydrogen peroxide, which induced luminol luminescence under positive potential. To achieve fast analysis, the luminescences from 64 individual cells on one ITO slide were imaged in 60 s using a charge-coupled device (CCD). More luminescence was observed at all the microwells after the introduction of triton X-100 and glucose oxidase suggested that intracellular glucose was detected at single cells. The starvation of cells to decrease intracellular glucose produced less luminescence, which confirmed that our luminescence intensity was correlated with the concentration of intracellular glucose. Large deviations in glucose concentration at observed single cells revealed high cellular heterogeneity in intracellular glucose for the first time. This developed electrochemiluminescence assay will be potentially applied for fast analysis of more intracellular molecules in single cells to elucidate cellular heterogeneity. PMID:27094779

  3. Magic Carpet Shows Its Colors

    NASA Technical Reports Server (NTRS)

    2004-01-01

    The upper left image in this display is from the panoramic camera on the Mars Exploration Rover Spirit, showing the 'Magic Carpet' region near the rover at Gusev Crater, Mars, on Sol 7, the seventh martian day of its journey (Jan. 10, 2004). The lower image, also from the panoramic camera, is a monochrome (single filter) image of a rock in the 'Magic Carpet' area. Note that colored portions of the rock correlate with extracted spectra shown in the plot to the side. Four different types of materials are shown: the rock itself, the soil in front of the rock, some brighter soil on top of the rock, and some dust that has collected in small recesses on the rock face ('spots'). Each color on the spectra matches a line on the graph, showing how the panoramic camera's different colored filters are used to broadly assess the varying mineral compositions of martian rocks and soils.

  4. Antimicrobial susceptibility and molecular characterization of Mycobacterium intracellulare in China.

    PubMed

    Zhao, Xiuqin; Wang, Yufeng; Pang, Yu

    2014-10-01

    Mycobacterium avium complex (MAC) is the most common non-tuberculosis mycobacterial pathogen isolated from respiratory samples, mainly including two species, Mycobacterium avium (M. avium) and Mycobacterium intracellulare (M. intracellulare). Although these two species belong to the same group, M. avium and M. intracellulare reveal significantly differences in pathogenicity and biology. Nevertheless, little is known regarding the drug resistant details profile of M. avium or M. intracellulare instead of MAC. Here, we examined the antimicrobial susceptibility profiles of 52 clinical M. intracellulare isolates against fourteen antimicrobial agents, which are widely selected for the treatment of nontuberculous mycobacteria (NTM) infection. The drug susceptibility test revealed that clarithromycin (47/52, 90.4%), rifampicin (41/52, 78.8%) and capreomycin (40/52, 76.9%) revealed highly antimicrobial activities against M. intracellulare isolates in vitro. Furthermore, all clarithromycin resistant isolates harbored mutations in the 23S rRNA gene, and the percentage of amikacin resistant ones with mutation in the rrs gene is 62.5% (10/16). The Hunter-Gaston Discriminatory Index (HGDI) value for the 16-loci Variable Number of Tandem Repeat (VNTR) typing of M. intracellulare isolates was 0.994, and M. intracellulare resistance to moxifloxacin was significantly more commonly found in clustered strains than in nonclustered strains (χ(2)=5.551, P=0.040). In conclusion, our data demonstrated that clarithromycin and capreomycin revealed highly antimicrobial activities against M. intracellulare isolates, and clarithromycin and amikacin resistance could be detected more readily and rapidly using molecular scanning of corresponding drug target than conventional drug susceptibility testing. We also found that infection by clustered strains was significantly associated with resistance to moxifloxacin. PMID:25131955

  5. A charge-adaptive nanosystem for prolonged and enhanced in vivo antibiotic delivery.

    PubMed

    Chu, Liping; Gao, Honglin; Cheng, Tangjian; Zhang, Yumin; Liu, Jinjian; Huang, Fan; Yang, Cuihong; Shi, Linqi; Liu, Jianfeng

    2016-05-01

    Herein we report on a charge-adaptive nanosystem for prolonged and enhanced in vivo antibiotic delivery. The nanocarrier achieves acid-dependent charge conversion, thus prolonging the circulation time and enhancing antibiotic accumulation in subcutaneous inflammation models. PMID:27077219

  6. Intracellular trafficking of silicon particles and logic-embedded vectors

    NASA Astrophysics Data System (ADS)

    Ferrati, Silvia; Mack, Aaron; Chiappini, Ciro; Liu, Xuewu; Bean, Andrew J.; Ferrari, Mauro; Serda, Rita E.

    2010-08-01

    Mesoporous silicon particles show great promise for use in drug delivery and imaging applications as carriers for second-stage nanoparticles and higher order particles or therapeutics. Modulation of particle geometry, surface chemistry, and porosity allows silicon particles to be optimized for specific applications such as vascular targeting and avoidance of biological barriers commonly found between the site of drug injection and the final destination. In this study, the intracellular trafficking of unloaded carrier silicon particles and carrier particles loaded with secondary iron oxide nanoparticles was investigated. Following cellular uptake, membrane-encapsulated silicon particles migrated to the perinuclear region of the cell by a microtubule-driven mechanism. Surface charge, shape (spherical and hemispherical) and size (1.6 and 3.2 μm) of the particle did not alter the rate of migration. Maturation of the phagosome was associated with an increase in acidity and acquisition of markers of late endosomes and lysosomes. Cellular uptake of iron oxide nanoparticle-loaded silicon particles resulted in sorting of the particles and trafficking to unique destinations. The silicon carriers remained localized in phagosomes, while the second stage iron oxide nanoparticles were sorted into multi-vesicular bodies that dissociated from the phagosome into novel membrane-bound compartments. Release of iron from the cells may represent exocytosis of iron oxide nanoparticle-loaded vesicles. These results reinforce the concept of multi-functional nanocarriers, in which different particles are able to perform specific tasks, in order to deliver single- or multi-component payloads to specific sub-cellular compartments.Mesoporous silicon particles show great promise for use in drug delivery and imaging applications as carriers for second-stage nanoparticles and higher order particles or therapeutics. Modulation of particle geometry, surface chemistry, and porosity allows silicon

  7. Availability of human induced pluripotent stem cell-derived cardiomyocytes in assessment of drug potential for QT prolongation

    SciTech Connect

    Nozaki, Yumiko; Honda, Yayoi; Tsujimoto, Shinji; Watanabe, Hitoshi; Kunimatsu, Takeshi; Funabashi, Hitoshi

    2014-07-01

    Field potential duration (FPD) in human-induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs), which can express QT interval in an electrocardiogram, is reported to be a useful tool to predict K{sup +} channel and Ca{sup 2+} channel blocker effects on QT interval. However, there is no report showing that this technique can be used to predict multichannel blocker potential for QT prolongation. The aim of this study is to show that FPD from MEA (Multielectrode array) of hiPS-CMs can detect QT prolongation induced by multichannel blockers. hiPS-CMs were seeded onto MEA and FPD was measured for 2 min every 10 min for 30 min after drug exposure for the vehicle and each drug concentration. I{sub Kr} and I{sub Ks} blockers concentration-dependently prolonged corrected FPD (FPDc), whereas Ca{sup 2+} channel blockers concentration-dependently shortened FPDc. Also, the multichannel blockers Amiodarone, Paroxetine, Terfenadine and Citalopram prolonged FPDc in a concentration dependent manner. Finally, the I{sub Kr} blockers, Terfenadine and Citalopram, which are reported to cause Torsade de Pointes (TdP) in clinical practice, produced early afterdepolarization (EAD). hiPS-CMs using MEA system and FPDc can predict the effects of drug candidates on QT interval. This study also shows that this assay can help detect EAD for drugs with TdP potential. - Highlights: • We focused on hiPS-CMs to replace in vitro assays in preclinical screening studies. • hiPS-CMs FPD is useful as an indicator to predict drug potential for QT prolongation. • MEA assay can help detect EAD for drugs with TdP potentials. • MEA assay in hiPS-CMs is useful for accurately predicting drug TdP risk in humans.

  8. Type 2 Diabetes Induces Prolonged P-wave Duration without Left Atrial Enlargement.

    PubMed

    Li, Bin; Pan, Yilong; Li, Xiaodong

    2016-04-01

    Prolonged P-wave duration has been observed in diabetes. However, the underlying mechanisms remain unclear. The aim of this study was to elucidate the possible mechanisms. A rat model of type 2 diabetes mellitus (T2DM) was used. P-wave durations were obtained using surface electrocardiography and sizes of the left atrium were determined using echocardiography. Cardiac inward rectifier K(+) currents (Ik1), Na(+) currents (INa), and action potentials were recorded from isolated left atrial myocytes using patch clamp techniques. Left atrial tissue specimens were analyzed for total connexin-40 (Cx40) and connexin-43 (Cx43) expression levels on western-blots. Specimens were also analyzed for Cx40 and Cx43 distribution and interstitial fibrosis by immunofluorescent and Masson trichrome staining, respectively. The mean P-wave duration was longer in T2DM rats than in controls; however, the mean left atrial sizes of each group of rats were similar. The densities of Ik1 and INa were unchanged in T2DM rats compared to controls. The action potential duration was longer in T2DM rats, but there was no significant difference in resting membrane potential or action potential amplitude compared to controls. The expression level of Cx40 protein was significantly lower, but Cx43 was unaltered in T2DM rats. However, immunofluorescent labeling of Cx43 showed a significantly enhanced lateralization. Staining showed interstitial fibrosis was greater in T2DM atrial tissue. Prolonged P-wave duration is not dependent on the left atrial size in rats with T2DM. Dysregulation of Cx40 and Cx43 protein expression, as well as fibrosis, might partly account for the prolongation of P-wave duration in T2DM. PMID:27051235

  9. Early Rehabilitation Therapy Is Beneficial for Patients With Prolonged Mechanical Ventilation After Coronary Artery Bypass Surgery.

    PubMed

    Dong, Zehua; Yu, Bangxu; Zhang, Quanfang; Pei, Haitao; Xing, Jinyan; Fang, Wei; Sun, Yunbo; Song, Zhen

    2016-01-01

    We investigated the effects of early rehabilitation therapy on prolonged mechanically ventilated patients after coronary artery bypass surgery (CABG).A total of 106 patients who underwent CABG between June 2012 and May 2015 were enrolled and randomly assigned into an early rehabilitation group (53 cases) and a control group (53 cases). The rehabilitation therapy consisted of 6 steps including head up, transferring from supination to sitting, sitting on the edge of bed, sitting in a chair, transferring from sitting to standing, and walking along a bed. The patients received rehabilitation therapy in the intensive care unit (ICU) after CABG in the early rehabilitation group. The control group patients received rehabilitation therapy after leaving the ICU.The results showed that the early rehabilitation therapy could significantly decrease the duration of mechanical ventilation (early rehabilitation group: 8.1 ± 3.3 days; control group: 13.9 ± 4.1 days, P < 0.01), hospital stay (early rehabilitation group: 22.0 ± 3.8 days; control group: 29.1 ± 4.6 days, P < 0.01), and ICU stay (early rehabilitation group: 11.7 ± 3.2 days; control group: 18.3 ± 4.2 days, P < 0.01) for patients requiring more than 72 hours prolonged mechanical ventilation. The results of Kaplan-Meier analysis showed that the proportions of patients remaining on mechanical ventilation in the early rehabilitation group were larger than that in the control group after 7 days of rehabilitation therapy (logrank test: P < 0.01). The results provide evidence for supporting the application of early rehabilitation therapy in patients requiring prolonged mechanical ventilation after CABG. PMID:26973269

  10. Facilitated physiological adaptation to prolonged circadian disruption through dietary supplementation with essence of chicken.

    PubMed

    Wu, Tao; Yao, Cencen; Tsang, Fai; Huang, Liangfeng; Zhang, Wanjing; Jiang, Jianguo; Mao, Youxiang; Shao, Yujian; Kong, Boda; Singh, Paramjeet; Fu, Zhengwei

    2015-01-01

    Synchrony between circadian and metabolic processes is critical to the maintenance of energy homeostasis. Studies on essence of chicken (EC), a chicken meat extract rich in proteins, amino acids and peptides, showed its effectiveness in alleviating fatigue and promoting metabolism. A recent study revealed that it facilitated the re-entrainment of clock genes (Bmal1, Cry1, Dec1, Per1 and Per2) in the pineal gland and liver in a rat model of circadian disruption. Here, we investigated the role of EC-facilitated circadian synchrony in the maintenance of the energy homeostasis using a mouse model of prolonged circadian disruption. Prolonged circadian disruption (12 weeks) resulted in hepatic maladaptation, manifested by a mild but significant (p < 0.05) hepatomegaly, accompanied by disturbed hepatic lipid metabolism and liver injury (indicated by increased circulating hepatic enzymes). Evidently, there was marked elevations of hepatic inflammatory mediators (interleukin-1beta and interleukin-6), suggesting an underlying inflammation leading to the hepatic injury and functional impairment. Importantly, the disruption paradigm caused the decoupling between key metabolic regulators (e.g. mTOR and AMPK) and hepatic clock genes (Per1, Cry1, Dec1, Bmal1). Further, we showed that the loss of circadian synchrony between the master and hepatic clock genes (Per1, Cry1, Dec1, Bmal1) could be the underlying cause of the maladaptation. When supplemented with EC, the functional impairment and inflammation were abolished. The protective effects could be linked to its effectiveness in maintaining the synchrony between the master and hepatic clocks, and the resultant improved coupling of the circadian oscillators (Per1, Cry1, Dec1, Bmal1) and metabolic regulators (mTOR, AMPK). Overall, EC supplementation promoted the physiological adaptation to the prolonged circadian disruption through facilitation of endogenous circadian synchrony and the coupling of circadian oscillators and

  11. Type 2 Diabetes Induces Prolonged P-wave Duration without Left Atrial Enlargement

    PubMed Central

    2016-01-01

    Prolonged P-wave duration has been observed in diabetes. However, the underlying mechanisms remain unclear. The aim of this study was to elucidate the possible mechanisms. A rat model of type 2 diabetes mellitus (T2DM) was used. P-wave durations were obtained using surface electrocardiography and sizes of the left atrium were determined using echocardiography. Cardiac inward rectifier K+ currents (Ik1), Na+ currents (INa), and action potentials were recorded from isolated left atrial myocytes using patch clamp techniques. Left atrial tissue specimens were analyzed for total connexin-40 (Cx40) and connexin-43 (Cx43) expression levels on western-blots. Specimens were also analyzed for Cx40 and Cx43 distribution and interstitial fibrosis by immunofluorescent and Masson trichrome staining, respectively. The mean P-wave duration was longer in T2DM rats than in controls; however, the mean left atrial sizes of each group of rats were similar. The densities of Ik1 and INa were unchanged in T2DM rats compared to controls. The action potential duration was longer in T2DM rats, but there was no significant difference in resting membrane potential or action potential amplitude compared to controls. The expression level of Cx40 protein was significantly lower, but Cx43 was unaltered in T2DM rats. However, immunofluorescent labeling of Cx43 showed a significantly enhanced lateralization. Staining showed interstitial fibrosis was greater in T2DM atrial tissue. Prolonged P-wave duration is not dependent on the left atrial size in rats with T2DM. Dysregulation of Cx40 and Cx43 protein expression, as well as fibrosis, might partly account for the prolongation of P-wave duration in T2DM. PMID:27051235

  12. The Intracellular Domain of the Coxsackievirus and Adenovirus Receptor Differentially Influences Adenovirus Entry

    PubMed Central

    Loustalot, Fabien

    2015-01-01

    ABSTRACT The coxsackievirus and adenovirus receptor (CAR) is a cell adhesion molecule used as a docking molecule by some adenoviruses (AdVs) and group B coxsackieviruses. We previously proposed that the preferential transduction of neurons by canine adenovirus type 2 (CAV-2) is due to CAR-mediated internalization. Our proposed pathway of CAV-2 entry is in contrast to that of human AdV type 5 (HAdV-C5) in nonneuronal cells, where internalization is mediated by auxiliary receptors such as integrins. We therefore asked if in fibroblast-like cells the intracellular domain (ICD) of CAR plays a role in the internalization of the CAV-2 fiber knob (FKCAV), CAV-2, or HAdV-C5 when the capsid cannot engage integrins. Here, we show that in fibroblast-like cells, the CAR ICD is needed for FKCAV entry and efficient CAV-2 transduction but dispensable for HAdV-C5 and an HAdV-C5 capsid lacking the RGD sequence (an integrin-interacting motif) in the penton. Moreover, the deletion of the CAR ICD further impacts CAV-2 intracellular trafficking, highlighting the crucial role of CAR in CAV-2 intracellular dynamics. These data demonstrate that the CAR ICD contains sequences important for the recruitment of the endocytic machinery that differentially influences AdV cell entry. IMPORTANCE Understanding how viruses interact with the host cell surface and reach the intracellular space is of crucial importance for applied and fundamental virology. Here, we compare the role of a cell adhesion molecule (CAR) in the internalization of adenoviruses that naturally infect humans and Canidae. We show that the intracellular domain of CAR differentially regulates AdV entry and trafficking. Our study highlights the mechanistic differences that a receptor can have for two viruses from the same family. PMID:26136571

  13. ENVITEC shows off air technologies

    SciTech Connect

    McIlvaine, R.W.

    1995-08-01

    The ENVITEC International Trade Fair for Environmental Protection and Waste Management Technologies, held in June in Duesseldorf, Germany, is the largest air pollution exhibition in the world and may be the largest environmental technology show overall. Visitors saw thousands of environmental solutions from 1,318 companies representing 29 countries and occupying roughly 43,000 square meters of exhibit space. Many innovations were displayed under the category, ``thermal treatment of air pollutants.`` New technologies include the following: regenerative thermal oxidizers; wet systems for removing pollutants; biological scrubbers;electrostatic precipitators; selective adsorption systems; activated-coke adsorbers; optimization of scrubber systems; and air pollution monitors.

  14. Effects of extremely low frequency electromagnetic fields on intracellular calcium transients in cardiomyocytes.

    PubMed

    Wei, Jinhong; Sun, Junqing; Xu, Hao; Shi, Liang; Sun, Lijun; Zhang, Jianbao

    2015-03-01

    Calcium transients play an essential role in cardiomyocytes and electromagnetic fields (EMF) and affect intracellular calcium levels in many types of cells. Effects of EMF on intracellular calcium transients in cardiomyocytes are not well studied. The aim of this study was to assess whether extremely low frequency electromagnetic fields (ELF-EMF) could affect intracellular calcium transients in cardiomyocytes. Cardiomyocytes isolated from neonatal Sprague-Dawley rats were exposed to rectangular-wave pulsed ELF-EMF at four different frequencies (15 Hz, 50 Hz, 75 Hz and 100 Hz) and at a flux density of 2 mT. Intracellular calcium concentration ([Ca(2+)]i) was measured using Fura-2/AM and spectrofluorometry. Perfusion of cardiomyocytes with a high concentration of caffeine (10 mM) was carried out to verify the function of the cardiac Na(+)/Ca(2+) exchanger (NCX) and the activity of sarco(endo)-plasmic reticulum Ca(2+)-ATPase (SERCA2a). The results showed that ELF-EMF enhanced the activities of NCX and SERCA2a, increased [Ca(2+)]i baseline level and frequency of calcium transients in cardiomyocytes and decreased the amplitude of calcium transients and calcium level in sarcoplasmic reticulum. These results indicated that ELF-EMF can regulate calcium-associated activities in cardiomyocytes. PMID:24499289

  15. Nanoparticle PEBBLE Sensors for Quantitative Nanomolar Imaging of Intracellular Free Calcium Ions

    PubMed Central

    Si, Di; Epstein, Tamir; Lee, Yong-Eun Koo; Kopelman, Raoul

    2011-01-01

    Ca2+ is a universal second messenger and plays a major role in intracellular signaling, metabolism and a wide range of cellular processes. To date, one of the most successful approaches for intracellular Ca2+ measurement involves introduction of optically sensitive Ca2+ indicators into living cells, combined with digital imaging microscopy. However, the use of free Ca2+ indicators for intracellular sensing and imaging has several limitations, such as nonratiometric measurement for the most sensitive indicators, cytotoxicity of the indicators, interference from non-specific binding caused by cellular biomacromolecules, challenging calibration and unwanted sequestration of the indicator molecules. These problems are minimized when the Ca2+ indicators are encapsulated inside porous and inert polyacrylamide nanoparticles. We present PEBBLE nanosensors encapsulated with rhodamine based Ca2+ fluorescence indicators. The here presented rhod-2 containing PEBBLEs show a stable sensing range at near-neutral pH (pH 6–9). Due to the protection of the PEBBLE matrix, the interference of protein non-specific binding to the indicator is minimal. The rhod-2 PEBBLEs give a nanomolar dynamic sensing range for both in-solution (Kd = 478 nM) and intracellular (Kd = 293 nM) measurements. These nanosensors are a useful quantitative tool for the measurement and imaging of the cytosolic nanomolar free Ca2+ levels. PMID:22122409

  16. Intracellular reduction/activation of a disulfide switch in thiosemicarbazone iron chelators.

    PubMed

    Akam, Eman A; Chang, Tsuhen M; Astashkin, Andrei V; Tomat, Elisa

    2014-10-01

    Iron scavengers (chelators) offer therapeutic opportunities in anticancer drug design by targeting the increased demand for iron in cancer cells as compared to normal cells. Prochelation approaches are expected to avoid systemic iron depletion as chelators are liberated under specific intracellular conditions. In the strategy described herein, a disulfide linkage is employed as a redox-directed switch within the binding unit of an antiproliferative thiosemicarbazone prochelator, which is activated for iron coordination following reduction to the thiolate chelator. In glutathione redox buffer, this reduction event occurs at physiological concentrations and half-cell potentials. Consistent with concurrent reduction and activation, higher intracellular thiol concentrations increase cell susceptibility to prochelator toxicity in cultured cancer cells. The reduction of the disulfide switch and intracellular iron chelation are confirmed in cell-based assays using calcein as a fluorescent probe for paramagnetic ions. The resulting low-spin Fe(III) complex is identified in intact Jurkat cells by EPR spectroscopy measurements, which also document a decreased concentration of active ribonucleotide reductase following exposure to the prochelator. Cell viability and fluorescence-based assays show that the iron complex presents low cytotoxicity and does not participate in intracellular redox chemistry, indicating that this antiproliferative chelation strategy does not rely on the generation of reactive oxygen species. PMID:25100578

  17. On-demand intracellular amplification of chemoradiation with cancer-specific plasmonic nanobubbles

    PubMed Central

    Lukianova-Hleb, Ekaterina Y; Wu, Xiangwei; Torchilin, Vladimir P; Lapotko, Dmitri O

    2014-01-01

    Chemoradiation-resistant cancers limit treatment efficacy and safety. We show here the cancer cell–specific, on-demand intracellular amplification of chemotherapy and chemoradiation therapy via gold nanoparticle– and laser pulse–induced mechanical intracellular impact. Cancer aggressiveness promotes the clustering of drug nanocarriers and gold nanoparticles in cancer cells. This cluster, upon exposure to a laser pulse, generates a plasmonic nanobubble, the mechanical explosion that destroys the host cancer cell or ejects the drug into its cytoplasm by disrupting the liposome and endosome. The same cluster locally amplifies external X-rays. Intracellular synergy of the mechanical impact of plasmonic nanobubble, ejected drug and amplified X-rays improves the efficacy of standard chemoradiation in resistant and aggressive head and neck cancer by 100-fold in vitro and 17-fold in vivo, reduces the effective entry doses of drugs and X-rays to 2–6% of their clinical doses and efficiently spares normal cells. The developed quadrapeutics technology combines four clinically validated components and transforms a standard macrotherapy into an intracellular on-demand theranostic microtreatment with radically amplified therapeutic efficacy and specificity. PMID:24880615

  18. On-demand intracellular amplification of chemoradiation with cancer-specific plasmonic nanobubbles.

    PubMed

    Lukianova-Hleb, Ekaterina Y; Ren, Xiaoyang; Sawant, Rupa R; Wu, Xiangwei; Torchilin, Vladimir P; Lapotko, Dmitri O

    2014-07-01

    Chemoradiation-resistant cancers limit treatment efficacy and safety. We show here the cancer cell-specific, on-demand intracellular amplification of chemotherapy and chemoradiation therapy via gold nanoparticle- and laser pulse-induced mechanical intracellular impact. Cancer aggressiveness promotes the clustering of drug nanocarriers and gold nanoparticles in cancer cells. This cluster, upon exposure to a laser pulse, generates a plasmonic nanobubble, the mechanical explosion that destroys the host cancer cell or ejects the drug into its cytoplasm by disrupting the liposome and endosome. The same cluster locally amplifies external X-rays. Intracellular synergy of the mechanical impact of plasmonic nanobubble, ejected drug and amplified X-rays improves the efficacy of standard chemoradiation in resistant and aggressive head and neck cancer by 100-fold in vitro and 17-fold in vivo, reduces the effective entry doses of drugs and X-rays to 2-6% of their clinical doses and efficiently spares normal cells. The developed quadrapeutics technology combines four clinically validated components and transforms a standard macrotherapy into an intracellular on-demand theranostic microtreatment with radically amplified therapeutic efficacy and specificity. PMID:24880615

  19. Exogenous control over intracellular acidification: Enhancement via proton caged compounds coupled to gold nanoparticles.

    PubMed

    Carbone, Marilena; Sabbatella, Gianfranco; Antonaroli, Simonetta; Remita, Hynd; Orlando, Viviana; Biagioni, Stefano; Nucara, Alessandro

    2015-11-01

    The pH regulation has a fundamental role in several intracellular processes and its variation via exogenous compounds is a potential tool for intervening in the intracellular processes. Proton caged compounds (PPCs) release protons upon UV irradiation and may efficiently provoke intracellular on-command acidification. Here, we explore the intracellular pH variation, when purposely synthesized PCCs are coupled to gold nanoparticles (AuNPs) and dosed to HEK-293 cells. We detected the acidification process caused by the UV irradiation by monitoring the intensity of the asymmetric stretching mode of the CO(2) molecule at 2343 cm(-1). The comparison between free and AuNPs functionalized proton caged compound demonstrates a highly enhanced CO(2) yield, hence pH variation, in the latter case. Finally, PCC functionalized AuNPs were marked with a purposely synthesized fluorescent marker and dosed to HEK-293 cells. The corresponding fluorescence optical images show green grains throughout the whole cytoplasm. PMID:26235337

  20. Thymosin {beta}4 promotes the migration of endothelial cells without intracellular Ca{sup 2+} elevation

    SciTech Connect

    Selmi, Anna; Malinowski, Mariusz; Brutkowski, Wojciech; Bednarek, Radoslaw; Cierniewski, Czeslaw S.

    2012-08-15

    Numerous studies have demonstrated the effects of T{beta}4 on cell migration, proliferation, apoptosis and inflammation after exogenous treatment, but the mechanism by which T{beta}4 functions is still unclear. Previously, we demonstrated that incubation of endothelial cells with T{beta}4 induced synthesis and secretion of various proteins, including plasminogen activator inhibitor type 1 and matrix metaloproteinases. We also showed that T{beta}4 interacts with Ku80, which may operate as a novel receptor for T{beta}4 and mediates its intracellular activity. In this paper, we provide evidence that T{beta}4 induces cellular processes without changes in the intracellular Ca{sup 2+} concentration. External treatment of HUVECs with T{beta}4 and its mutants deprived of the N-terminal tetrapeptide AcSDKP (T{beta}4{sub AcSDKPT/4A}) or the actin-binding sequence KLKKTET (T{beta}4{sub KLKKTET/7A}) resulted in enhanced cell migration and formation of tubular structures in Matrigel. Surprisingly, the increased cell motility caused by T{beta}4 was not associated with the intracellular Ca{sup 2+} elevation monitored with Fluo-4 NW or Fura-2 AM. Therefore, it is unlikely that externally added T{beta}4 induces HUVEC migration via the surface membrane receptors known to generate Ca{sup 2+} influx. Our data confirm the concept that externally added T{beta}4 must be internalized to induce intracellular mechanisms supporting endothelial cell migration.