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  1. Inverse Stochastic Resonance in Cerebellar Purkinje Cells.

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    Anatoly Buchin

    2016-08-01

    Full Text Available Purkinje neurons play an important role in cerebellar computation since their axons are the only projection from the cerebellar cortex to deeper cerebellar structures. They have complex internal dynamics, which allow them to fire spontaneously, display bistability, and also to be involved in network phenomena such as high frequency oscillations and travelling waves. Purkinje cells exhibit type II excitability, which can be revealed by a discontinuity in their f-I curves. We show that this excitability mechanism allows Purkinje cells to be efficiently inhibited by noise of a particular variance, a phenomenon known as inverse stochastic resonance (ISR. While ISR has been described in theoretical models of single neurons, here we provide the first experimental evidence for this effect. We find that an adaptive exponential integrate-and-fire model fitted to the basic Purkinje cell characteristics using a modified dynamic IV method displays ISR and bistability between the resting state and a repetitive activity limit cycle. ISR allows the Purkinje cell to operate in different functional regimes: the all-or-none toggle or the linear filter mode, depending on the variance of the synaptic input. We propose that synaptic noise allows Purkinje cells to quickly switch between these functional regimes. Using mutual information analysis, we demonstrate that ISR can lead to a locally optimal information transfer between the input and output spike train of the Purkinje cell. These results provide the first experimental evidence for ISR and suggest a functional role for ISR in cerebellar information processing.

  2. Fear conditioning-related changes in cerebellar Purkinje cell activities in goldfish

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    Yoshida Masayuki

    2012-10-01

    Full Text Available Abstract Background Fear conditioning-induced changes in cerebellar Purkinje cell responses to a conditioned stimulus have been reported in rabbits. It has been suggested that synaptic long-term potentiation and the resulting increases in firing rates of Purkinje cells are related to the acquisition of conditioned fear in mammals. However, Purkinje cell activities during acquisition of conditioned fear have not been analysed, and changes in Purkinje cell activities throughout the development of conditioned fear have not yet been investigated. In the present study, we tracked Purkinje cell activities throughout a fear conditioning procedure and aimed to elucidate further how cerebellar circuits function during the acquisition and expression of conditioned fear. Methods Activities of single Purkinje cells in the corpus cerebelli were tracked throughout a classical fear conditioning procedure in goldfish. A delayed conditioning paradigm was used with cardiac deceleration as the conditioned response. Conditioning-related changes of Purkinje cell responses to a conditioned stimulus and unconditioned stimulus were examined. Results The majority of Purkinje cells sampled responded to the conditioned stimulus by either increasing or decreasing their firing rates before training. Although there were various types of conditioning-related changes in Purkinje cells, more than half of the cells showed suppressed activities in response to the conditioned stimulus after acquisition of conditioned fear. Purkinje cells that showed unconditioned stimulus-coupled complex-spike firings also exhibited conditioning-related suppression of simple-spike responses to the conditioned stimulus. A small number of Purkinje cells showed increased excitatory responses in the acquisition sessions. We found that the magnitudes of changes in the firing frequencies of some Purkinje cells in response to the conditioned stimulus correlated with the magnitudes of the conditioned

  3. A signal processing analysis of Purkinje cells in vitro

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    Ze'ev R Abrams

    2010-05-01

    Full Text Available Cerebellar Purkinje cells in vitro fire recurrent sequences of Sodium and Calcium spikes. Here, we analyze the Purkinje cell using harmonic analysis, and our experiments reveal that its output signal is comprised of three distinct frequency bands, which are combined using Amplitude and Frequency Modulation (AM/FM. We find that the three characteristic frequencies - Sodium, Calcium and Switching – occur in various combinations in all waveforms observed using whole-cell current clamp recordings. We found that the Calcium frequency can display a frequency doubling of its frequency mode, and the Switching frequency can act as a possible generator of pauses that are typically seen in Purkinje output recordings. Using a reversibly photo-switchable kainate receptor agonist, we demonstrate the external modulation of the Calcium and Switching frequencies. These experiments and Fourier analysis suggest that the Purkinje cell can be understood as a harmonic signal oscillator, enabling a higher level of interpretation of Purkinje signaling based on modern signal processing techniques.

  4. Modulation, plasticity and pathophysiology of the parallel fiber-Purkinje cell synapse

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    Eriola Hoxha

    2016-11-01

    Full Text Available The parallel fiber-Purkinje cell synapse represents the point of maximal signal divergence in the cerebellar cortex with an estimated number of about 60 billion synaptic contacts in the rat and 100,000 billions in humans. At the same time, the Purkinje cell dendritic tree is a site of remarkable convergence of more than 100,000 parallel fiber synapses. Parallel fibers activity generates fast postsynaptic currents via AMPA receptors, and slower signals, mediated by mGlu1 receptors, resulting in Purkinje cell depolarization accompanied by sharp calcium elevation within dendritic regions. Long-term depression and long-term potentiation have been widely described for the parallel fiber-Purkinje cell synapse and have been proposed as mechanisms for motor learning. The mechanisms of induction for LTP and LTD involve different signaling mechanisms within the presynaptic terminal and/or at the postsynaptic site, promoting enduring modification in the neurotransmitter release and change in responsiveness to the neurotransmitter. The parallel fiber-Purkinje cell synapse is finely modulated by several neurotransmitters, including serotonin, noradrenaline, and acetylcholine. The ability of these neuromodulators to gate LTP and LTD at the parallel fiber-Purkinje cell synapse could, at least in part, explain their effect on cerebellar-dependent learning and memory paradigms. Overall, these findings have important implications for understanding the cerebellar involvement in a series of pathological conditions, ranging from ataxia to autism. For example, parallel fiber-Purkinje cell synapse dysfunctions have been identified in several murine models of spinocerebellar ataxia (SCA types 1, 3, 5 and 27. In some cases, the defect is specific for the AMPA receptor signaling (SCA27, while in others the mGlu1 pathway is affected (SCA1, 3, 5. Interestingly, the parallel fiber-Purkinje cell synapse has been shown to be hyper-functional in a mutant mouse model of autism

  5. Regularity, variability and bi-stability in the activity of cerebellar purkinje cells.

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    Rokni, Dan; Tal, Zohar; Byk, Hananel; Yarom, Yosef

    2009-01-01

    Recent studies have demonstrated that the membrane potential of Purkinje cells is bi-stable and that this phenomenon underlies bi-modal simple spike firing. Membrane potential alternates between a depolarized state, that is associated with spontaneous simple spike firing (up state), and a quiescent hyperpolarized state (down state). A controversy has emerged regarding the relevance of bi-stability to the awake animal, yet recordings made from behaving cat Purkinje cells have demonstrated that at least 50% of the cells exhibit bi-modal firing. The robustness of the phenomenon in vitro or in anaesthetized systems on the one hand, and the controversy regarding its expression in behaving animals on the other hand suggest that state transitions are under neuronal control. Indeed, we have recently demonstrated that synaptic inputs can induce transitions between the states and suggested that the role of granule cell input is to control the states of Purkinje cells rather than increase or decrease firing rate gradually. We have also shown that the state of a Purkinje cell does not only affect its firing but also the waveform of climbing fiber-driven complex spikes and the associated calcium influx. These findings call for a reconsideration of the role of Purkinje cells in cerebellar function. In this manuscript we review the recent findings on Purkinje cell bi-stability and add some analyses of its effect on the regularity and variability of Purkinje cell activity.

  6. Regularity, variabilty and bi-stability in the activity of cerebellar Purkinje cells

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    Dan Rokni

    2009-11-01

    Full Text Available Recent studies have demonstrated that the membrane potential of Purkinje cells is bi-stable and that this phenomenon underlies bi-modal simple spike firing. Membrane potential alternates between a depolarized state, that is associated with spontaneous simple spike firing (up state, and a quiescent hyperpolarized state (down state. A controversy has emerged regarding the relevance of bi-stability to the awake animal, yet recordings made from behaving cat Purkinje cells have demonstrated that at least 50% of the cells exhibit bi-modal firing. The robustness of the phenomenon in-vitro or in anaesthetized systems on the one hand, and the controversy regarding its expression in behaving animals on the other hand suggest that state transitions are under neuronal control. Indeed, we have recently demonstrated that synaptic inputs can induce transitions between the states and suggested that the role of granule cell input is to control the states of Purkinje cells rather than increase or decrease firing rate gradually. We have also shown that the state of a Purkinje cell does not only affect its firing but also the waveform of climbing fiber-driven complex spikes and the associated calcium influx. These findings call for a reconsideration of the role of Purkinje cells in cerebellar function. In this manuscript we review the recent findings on Purkinje cell bi-stability and add some analyses of its effect on the regularity and variability of Purkinje cell activity.

  7. Calcium Imaging Reveals Coordinated Simple Spike Pauses in Populations of Cerebellar Purkinje Cells

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    Jorge E. Ramirez

    2016-12-01

    Full Text Available The brain’s control of movement is thought to involve coordinated activity between cerebellar Purkinje cells. The results reported here demonstrate that somatic Ca2+ imaging is a faithful reporter of Na+-dependent “simple spike” pauses and enables us to optically record changes in firing rates in populations of Purkinje cells in brain slices and in vivo. This simultaneous calcium imaging of populations of Purkinje cells reveals a striking spatial organization of pauses in Purkinje cell activity between neighboring cells. The source of this organization is shown to be the presynaptic gamma-Aminobutyric acid producing (GABAergic network, and blocking ionotropic gamma-Aminobutyric acid receptor (GABAARs abolishes the synchrony. These data suggest that presynaptic interneurons synchronize (inactivity between neighboring Purkinje cells, and thereby maximize their effect on downstream targets in the deep cerebellar nuclei.

  8. The volume of Purkinje cells decreases in the cerebellum of acrylamide-intoxicated rats, but no cells are lost

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    Larsen, Jytte Overgaard; Tandrup, T; Braendgaard, H

    1994-01-01

    The effects of acrylamide intoxication on the numbers of granule and Purkinje cells and the volume of Purkinje cell perikarya have been evaluated with stereological methods. The analysis was carried out in the cerebella of rats that had received a dose of 33.3 mg/kg acrylamide, twice a week, for 7.......5 weeks. The total numbers of cerebellar granule and Purkinje cells were estimated using the optical fractionator and the mean volume of the Purkinje cell perikarya was estimated with the vertical rotator technique. The volumes of the molecular layer, the granular cell layer and the white matter were...... estimated using the Cavalieri principle. The mean weight of the cerebellum of the intoxicated rats was 7% lower than that of the control rats (2P = 0.001). The numbers of the Purkinje cells and granule cells were the same in both groups, but the mean volume of the perikarya of the Purkinje cells...

  9. Transient developmental Purkinje cell axonal torpedoes in healthy and ataxic mouse cerebellum

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    Lovisa Ljungberg

    2016-11-01

    Full Text Available Information is carried out of the cerebellar cortical microcircuit via action potentials propagated along Purkinje cell axons. In several human neurodegenerative diseases, focal axonal swellings on Purkinje cells – known as torpedoes – have been associated with Purkinje cell loss. Interestingly, torpedoes are also reported to appear transiently during development in rat cerebellum. The function of Purkinje cell axonal torpedoes in health as well as in disease is poorly understood. We investigated the properties of developmental torpedoes in the postnatal mouse cerebellum of wildtype and transgenic mice. We found that Purkinje cell axonal torpedoes transiently appeared on axons of Purkinje neurons, with the largest number of torpedoes observed at postnatal day 11 (P11. This was after peak developmental apoptosis had occurred, when Purkinje cell counts in a lobule were static, suggesting that most developmental torpedoes appear on axons of neurons that persist into adulthood. We found that developmental torpedoes were not associated with a presynaptic GABAergic marker, indicating that they are not synapses. They were seldom found at axonal collateral branch points, and lacked microglia enrichment, suggesting that they are unlikely to be involved in axonal refinement. Interestingly, we found several differences between developmental torpedoes and disease-related torpedoes: developmental torpedoes occured largely on myelinated axons, and were not associated with changes in basket cell innervation on their parent soma. Disease-related torpedoes are typically reported to contain neurofilament; while the majority of developmental torpedoes did as well, a fraction of smaller developmental torpedoes did not. These differences indicate that developmental torpedoes may not be functionally identical to disease-related torpedoes. To study this further, we used a mouse model of spinocerebellar ataxia type 6 (SCA6, and found elevated disease

  10. A note on the definition and the development of cerebellar purkinje cell zones

    NARCIS (Netherlands)

    J. Voogd (Jan)

    2012-01-01

    textabstractThe definition of Purkinje cell zones by their white matter comprtments, their physiological properties, and their molecular identity and the birthdate of their Purkinje cells will be reviewed.

  11. A Note on the Definition and the Development of Cerebellar Purkinje Cell Zones

    OpenAIRE

    Voogd, J.

    2012-01-01

    textabstractThe definition of Purkinje cell zones by their white matter comprtments, their physiological properties, and their molecular identity and the birthdate of their Purkinje cells will be reviewed.

  12. Activity-Dependent Plasticity of Spike Pauses in Cerebellar Purkinje Cells

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    Giorgio Grasselli

    2016-03-01

    Full Text Available The plasticity of intrinsic excitability has been described in several types of neurons, but the significance of non-synaptic mechanisms in brain plasticity and learning remains elusive. Cerebellar Purkinje cells are inhibitory neurons that spontaneously fire action potentials at high frequencies and regulate activity in their target cells in the cerebellar nuclei by generating a characteristic spike burst-pause sequence upon synaptic activation. Using patch-clamp recordings from mouse Purkinje cells, we find that depolarization-triggered intrinsic plasticity enhances spike firing and shortens the duration of spike pauses. Pause plasticity is absent from mice lacking SK2-type potassium channels (SK2−/− mice and in occlusion experiments using the SK channel blocker apamin, while apamin wash-in mimics pause reduction. Our findings demonstrate that spike pauses can be regulated through an activity-dependent, exclusively non-synaptic, SK2 channel-dependent mechanism and suggest that pause plasticity—by altering the Purkinje cell output—may be crucial to cerebellar information storage and learning.

  13. Physiological and pharmacological properties of Purkinje cells in rat cerebellum degranulated by postnatal x irradiation

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    Woodward, D.J.; Hoffer, B.J.; Altman, J.

    1974-01-01

    Elimination of most granule, basket, and stellate interneurons in the rat cerebellum was achieved by repeated doses of low level x irradiation applied during the first two weeks of postnatal life. Purkinje neurons in these rats, studied when adults, exhibited sustained spiking activity in Halothane anesthetized preparations. Mean firing rates were 35 to 40/sec, no different from normal. Spontaneous bursts presumed to be generated by climbing fiber synaptic activity differed from normal by often consisting of full sized spikes rather than characteristic inactivation responses. Intracellularly observed correlates of bursts consisted of epsp's of several discretely different amplitudes appearing independently in time. Stimulation of white matter revealed evidence for, a) graded synaptic excitation of Purkinje cells indicating more than one converging excitatory synapse, and b) inhibitory actions on Purkinje cells either through a few remaining inhibitory interneurons or through Purkinje cell recurrent collaterals. Iontophoretic drug application studies showed normal chemosensitivity of the Purkinje cell membrane, i.e., excitation by flutamate and inhibition by gamma-amino butyric acid, serotonin, norepinephrine, and 3'5' cyclic AMP. These studies indicate considerable autonomy of Purkinje cell ontogenesis in the absence of normal interneuronal input. A unique synaptic relation only rarely found in normal cerebellum is the innervation of single Purkinje cells by more than one climbing fiber. (U.S.)

  14. The Knockout of Secretin in Cerebellar Purkinje Cells Impairs Mouse Motor Coordination and Motor Learning

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    Zhang, Li; Chung, Sookja Kim; Chow, Billy Kwok Chong

    2014-01-01

    Secretin (SCT) was first considered to be a gut hormone regulating gastrointestinal functions when discovered. Recently, however, central actions of SCT have drawn intense research interest and are supported by the broad distribution of SCT in specific neuronal populations and by in vivo physiological studies regarding its role in water homeostasis and food intake. The direct action of SCT on a central neuron was first discovered in cerebellar Purkinje cells in which SCT from cerebellar Purkinje cells was found to potentiate GABAergic inhibitory transmission from presynaptic basket cells. Because Purkinje neurons have a major role in motor coordination and learning functions, we hypothesize a behavioral modulatory function for SCT. In this study, we successfully generated a mouse model in which the SCT gene was deleted specifically in Purkinje cells. This mouse line was tested together with SCT knockout and SCT receptor knockout mice in a full battery of behavioral tasks. We found that the knockout of SCT in Purkinje neurons did not affect general motor ability or the anxiety level in open field tests. However, knockout mice did exhibit impairments in neuromuscular strength, motor coordination, and motor learning abilities, as shown by wire hanging, vertical climbing, and rotarod tests. In addition, SCT knockout in Purkinje cells possibly led to the delayed development of motor neurons, as supported by the later occurrence of key neural reflexes. In summary, our data suggest a role in motor coordination and motor learning for SCT expressed in cerebellar Purkinje cells. PMID:24356714

  15. A new approach for determining phase response curves reveals that Purkinje cells can act as perfect integrators.

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    Elena Phoka

    2010-04-01

    Full Text Available Cerebellar Purkinje cells display complex intrinsic dynamics. They fire spontaneously, exhibit bistability, and via mutual network interactions are involved in the generation of high frequency oscillations and travelling waves of activity. To probe the dynamical properties of Purkinje cells we measured their phase response curves (PRCs. PRCs quantify the change in spike phase caused by a stimulus as a function of its temporal position within the interspike interval, and are widely used to predict neuronal responses to more complex stimulus patterns. Significant variability in the interspike interval during spontaneous firing can lead to PRCs with a low signal-to-noise ratio, requiring averaging over thousands of trials. We show using electrophysiological experiments and simulations that the PRC calculated in the traditional way by sampling the interspike interval with brief current pulses is biased. We introduce a corrected approach for calculating PRCs which eliminates this bias. Using our new approach, we show that Purkinje cell PRCs change qualitatively depending on the firing frequency of the cell. At high firing rates, Purkinje cells exhibit single-peaked, or monophasic PRCs. Surprisingly, at low firing rates, Purkinje cell PRCs are largely independent of phase, resembling PRCs of ideal non-leaky integrate-and-fire neurons. These results indicate that Purkinje cells can act as perfect integrators at low firing rates, and that the integration mode of Purkinje cells depends on their firing rate.

  16. A novel approach to non-biased systematic random sampling: a stereologic estimate of Purkinje cells in the human cerebellum.

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    Agashiwala, Rajiv M; Louis, Elan D; Hof, Patrick R; Perl, Daniel P

    2008-10-21

    Non-biased systematic sampling using the principles of stereology provides accurate quantitative estimates of objects within neuroanatomic structures. However, the basic principles of stereology are not optimally suited for counting objects that selectively exist within a limited but complex and convoluted portion of the sample, such as occurs when counting cerebellar Purkinje cells. In an effort to quantify Purkinje cells in association with certain neurodegenerative disorders, we developed a new method for stereologic sampling of the cerebellar cortex, involving calculating the volume of the cerebellar tissues, identifying and isolating the Purkinje cell layer and using this information to extrapolate non-biased systematic sampling data to estimate the total number of Purkinje cells in the tissues. Using this approach, we counted Purkinje cells in the right cerebella of four human male control specimens, aged 41, 67, 70 and 84 years, and estimated the total Purkinje cell number for the four entire cerebella to be 27.03, 19.74, 20.44 and 22.03 million cells, respectively. The precision of the method is seen when comparing the density of the cells within the tissue: 266,274, 173,166, 167,603 and 183,575 cells/cm3, respectively. Prior literature documents Purkinje cell counts ranging from 14.8 to 30.5 million cells. These data demonstrate the accuracy of our approach. Our novel approach, which offers an improvement over previous methodologies, is of value for quantitative work of this nature. This approach could be applied to morphometric studies of other similarly complex tissues as well.

  17. Dose response relationship of disturbed migration of Purkinje cells in the cerebellum due to X-irradiation

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    Darmanto, W.; Inouye, Minoru; Hayasaka, Shizu; Takagishi, Yoshiko; Aolad, H.; Murata, Yoshiharu

    1998-01-01

    Pregnant rats were exposed to 2.0, 2.25 or 2.5 Gy X-irradiation on gestation day 21. Pups were sacrificed 12 hr after exposure, and on postnatal day 5 (P5), P7 and P9. Their cerebella were observed immunohistochemically using anti-inositol 1,4,5 triphosphate (IP3) receptor antibody to identify Purkinje cells. These cells were disturbed to migrate and remained in the internal granular layer and white matter of the cerebellum. They had short dendrites, and some showed an abnormal direction of dendrites in rats exposed to 2.25 or 2.5 Gy. Alignment of Purkinje cells was also disturbed when examined either on P5, P7 or P9 especially by doses of 2.25 and 2.5 Gy. There was a relationship between X-ray doses and the number of cells piling up in the Purkinje cell layer of the cerebellum. The dose-response relationship with the number of ectopic Purkinje cells was noted in the anterior lobes of the cerebellum. (author)

  18. Quantitative neuroanatomy of all Purkinje cells with light sheet microscopy and high-throughput image analysis

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    Ludovico eSilvestri

    2015-05-01

    Full Text Available Characterizing the cytoarchitecture of mammalian central nervous system on a brain-wide scale is becoming a compelling need in neuroscience. For example, realistic modeling of brain activity requires the definition of quantitative features of large neuronal populations in the whole brain. Quantitative anatomical maps will also be crucial to classify the cytoarchtitectonic abnormalities associated with neuronal pathologies in a high reproducible and reliable manner. In this paper, we apply recent advances in optical microscopy and image analysis to characterize the spatial distribution of Purkinje cells across the whole cerebellum. Light sheet microscopy was used to image with micron-scale resolution a fixed and cleared cerebellum of an L7-GFP transgenic mouse, in which all Purkinje cells are fluorescently labeled. A fast and scalable algorithm for fully automated cell identification was applied on the image to extract the position of all the fluorescent Purkinje cells. This vectorized representation of the cell population allows a thorough characterization of the complex three-dimensional distribution of the neurons, highlighting the presence of gaps inside the lamellar organization of Purkinje cells, whose density is believed to play a significant role in autism spectrum disorders. Furthermore, clustering analysis of the localized somata permits dividing the whole cerebellum in groups of Purkinje cells with high spatial correlation, suggesting new possibilities of anatomical partition. The quantitative approach presented here can be extended to study the distribution of different types of cell in many brain regions and across the whole encephalon, providing a robust base for building realistic computational models of the brain, and for unbiased morphological tissue screening in presence of pathologies and/or drug treatments.

  19. Increased protein kinase C gamma activity induces Purkinje cell pathology in a mouse model of spinocerebellar ataxia 14.

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    Ji, Jingmin; Hassler, Melanie L; Shimobayashi, Etsuko; Paka, Nagendher; Streit, Raphael; Kapfhammer, Josef P

    2014-10-01

    Spinocerebellar ataxias (SCAs) are hereditary diseases leading to Purkinje cell degeneration and cerebellar dysfunction. Most forms of SCA are caused by expansion of CAG repeats similar to other polyglutamine disorders such as Huntington's disease. In contrast, in the autosomal dominant SCA-14 the disease is caused by mutations in the protein kinase C gamma (PKCγ) gene which is a well characterized signaling molecule in cerebellar Purkinje cells. The study of SCA-14, therefore, offers the unique opportunity to reveal the molecular and pathological mechanism eventually leading to Purkinje cell dysfunction and degeneration. We have created a mouse model of SCA-14 in which PKCγ protein with a mutation found in SCA-14 is specifically expressed in cerebellar Purkinje cells. We find that in mice expressing the mutated PKCγ protein the morphology of Purkinje cells in cerebellar slice cultures is drastically altered and mimics closely the morphology seen after pharmacological PKC activation. Similar morphological abnormalities were seen in localized areas of the cerebellum of juvenile transgenic mice in vivo. In adult transgenic mice there is evidence for some localized loss of Purkinje cells but there is no overall cerebellar atrophy. Transgenic mice show a mild cerebellar ataxia revealed by testing on the rotarod and on the walking beam. Our findings provide evidence for both an increased PKCγ activity in Purkinje cells in vivo and for pathological changes typical for cerebellar disease thus linking the increased and dysregulated activity of PKCγ tightly to the development of cerebellar disease in SCA-14 and possibly also in other forms of SCA. Copyright © 2014 Elsevier Inc. All rights reserved.

  20. Impaired succinic dehydrogenase activity of rat Purkinje cell mitochondria during aging.

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    Fattoretti, P; Bertoni-Freddari, C; Caselli, U; Paoloni, R; Meier-Ruge, W

    1998-03-16

    The perikaryal Purkinje cell mitochondria positive to the copper ferrocyanide histochemical reaction for succinic dehydrogenase (SDH) have been investigated by means of semiautomatic morphometric methods in rats of 3, 12 and 24 months of age. The number of organelles/microm3 of Purkinje cell cytoplasm (Numeric density: Nv), the average mitochondrial volume (V) and the mitochondrial volume fraction (Volume density: Vv) were the ultrastructural parameters taken into account. Nv was significantly higher at 12 than at 3 and 24 months of age. V was significantly decreased at 12 and 24 months of age, but no difference was envisaged between adult and old rats. Vv was significantly decreased in old animals vs. the other age groups. In young and old rats, the percentage of organelles larger than 0.32 microm3 was 13.5 and 11%, respectively, while these enlarged mitochondria accounted for less than 1% in the adult group. Since SDH activity is of critical importance when energy demand is high, the marked decrease of Vv supports an impaired capacity of the old Purkinje cells to match actual energy supply at sustained transmission of the nervous impulse. However, the high percentage of enlarged organelles found in old rats may witness a morphofunctional compensatory response.

  1. Red sorrel (Hibiscus Sabdariffa) prevents the ethanol-induced deficits of Purkinje cells in the cerebellum.

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    Suryanti, S; Partadiredja, G; Atthobari, J

    2015-01-01

    The present study is aimed at investigating the possible protective effects of H. sabdariffa on ethanol-elicited deficits of motor coordination and estimated total number of the Purkinje cells of the cerebellums of adolescent male Wistar rats. Forty male Wistar rats aged 21 days were divided into five groups. Na/wtr group was given water orally and injected with normal saline intra peritoneally (ip). Eth/wtr group was given water orally and ethanol (ip). Another three experimental groups (Eth/Hsab) were given different dosages of H. sabdariffa and ethanol (ip). All groups were treated intermittently for the total period of treatment of two weeks. The motor coordination of rats was tested prior and subsequent to the treatments. The rats were euthanized, and their cerebellums were examined. The total number of Purkinje cells was estimated using physical fractionator method. Upon revolving drum test, the number of falls of rats increased following ethanol treatment. There was no significant difference between the total number of falls prior and subsequent to treatment in all Eth/Hsab groups. The estimated total number of Purkinje cells in Eth/Hsab groups was higher than in Eth/wtr group. H. sabdariffa may prevent the ethanol-induced deficits of motor coordination and estimated total number of Purkinje cells of the cerebellums in adolescent rats (Tab. 3, Fig. 1, Ref. 42).

  2. Selective Transgenic Expression of Mutant Ubiquitin in Purkinje Cell Stripes in the Cerebellum.

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    Verheijen, Bert M; Gentier, Romina J G; Hermes, Denise J H P; van Leeuwen, Fred W; Hopkins, David A

    2017-06-01

    The ubiquitin-proteasome system (UPS) is one of the major mechanisms for protein breakdown in cells, targeting proteins for degradation by enzymatically conjugating them to ubiquitin molecules. Intracellular accumulation of ubiquitin-B +1 (UBB +1 ), a frameshift mutant of ubiquitin-B, is indicative of a dysfunctional UPS and has been implicated in several disorders, including neurodegenerative disease. UBB +1 -expressing transgenic mice display widespread labeling for UBB +1 in brain and exhibit behavioral deficits. Here, we show that UBB +1 is specifically expressed in a subset of parasagittal stripes of Purkinje cells in the cerebellar cortex of a UBB +1 -expressing mouse model. This expression pattern is reminiscent of that of the constitutively expressed Purkinje cell antigen HSP25, a small heat shock protein with neuroprotective properties.

  3. Sensorimotor-correlated discharge recorded from ensembles of cerebellar Purkinje cells varies across the estrous cycle of the rat.

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    Smith, S S

    1995-09-01

    1. In the present study, locomotor-correlated activity of cerebellar Purkinje cells, recorded using arrays of microwires chronically implanted in adult female rats, was examined across estrous-cycle-associated fluctuations in endogenous sex steroids. Ongoing studies from this laboratory have shown that systemic and local administration of the sex steroid 17 beta-estradiol (E2) augments excitatory responses of cerebellar Purkinje cells to iontophoretically applied glutamate, recorded in vivo from anesthetized female rats. In addition, this steroid potentiated discharge correlated with limb movement. For the present study, extracellular single-unit activity was recorded from as many as 5-11 Purkinje cells simultaneously during treadmill locomotion paradigms. Motor modulation of activity was recorded across three to five consecutive estrous cycles from behaviorally identified cohorts of neurons to test the hypothesis that fluctuations in endogenous sex steroids alter motor modulation of Purkinje cell discharge. 2. Locomotor-associated discharge correlated with treadmill locomotion was increased by a mean of 47% on proestrus, when E2 levels are elevated, relative to diestrus 1. These changes in discharge rate during treadmill locomotion were of significantly greater magnitude than corresponding cyclic alterations in discharge during stationary periods. 3. Correlations with the circadian cycle were also significant, because peak levels of locomotor-associated discharge on the night of behavioral estrus, following elevations in circulating E2, were on average 67% greater than corresponding discharge recorded during the light (proestrus). 4. Alterations in the step cycle were also observed across the estrous cycle: significant decreases in the duration of the flexion phase (by 265 ms, P estrus compared with diestrus. 5. When recorded on estrus, Purkinje cell discharge correlated with the stance or flexion phase of the step cycle was greater in magnitude and preceded the

  4. Dendritic Kv3.3 potassium channels in cerebellar purkinje cells regulate generation and spatial dynamics of dendritic Ca2+ spikes.

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    Zagha, Edward; Manita, Satoshi; Ross, William N; Rudy, Bernardo

    2010-06-01

    Purkinje cell dendrites are excitable structures with intrinsic and synaptic conductances contributing to the generation and propagation of electrical activity. Voltage-gated potassium channel subunit Kv3.3 is expressed in the distal dendrites of Purkinje cells. However, the functional relevance of this dendritic distribution is not understood. Moreover, mutations in Kv3.3 cause movement disorders in mice and cerebellar atrophy and ataxia in humans, emphasizing the importance of understanding the role of these channels. In this study, we explore functional implications of this dendritic channel expression and compare Purkinje cell dendritic excitability in wild-type and Kv3.3 knockout mice. We demonstrate enhanced excitability of Purkinje cell dendrites in Kv3.3 knockout mice, despite normal resting membrane properties. Combined data from local application pharmacology, voltage clamp analysis of ionic currents, and assessment of dendritic Ca(2+) spike threshold in Purkinje cells suggest a role for Kv3.3 channels in opposing Ca(2+) spike initiation. To study the physiological relevance of altered dendritic excitability, we measured [Ca(2+)](i) changes throughout the dendritic tree in response to climbing fiber activation. Ca(2+) signals were specifically enhanced in distal dendrites of Kv3.3 knockout Purkinje cells, suggesting a role for dendritic Kv3.3 channels in regulating propagation of electrical activity and Ca(2+) influx in distal dendrites. These findings characterize unique roles of Kv3.3 channels in dendrites, with implications for synaptic integration, plasticity, and human disease.

  5. The dynamic relationship between cerebellar Purkinje cell simple spikes and the spikelet number of complex spikes.

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    Burroughs, Amelia; Wise, Andrew K; Xiao, Jianqiang; Houghton, Conor; Tang, Tianyu; Suh, Colleen Y; Lang, Eric J; Apps, Richard; Cerminara, Nadia L

    2017-01-01

    Purkinje cells are the sole output of the cerebellar cortex and fire two distinct types of action potential: simple spikes and complex spikes. Previous studies have mainly considered complex spikes as unitary events, even though the waveform is composed of varying numbers of spikelets. The extent to which differences in spikelet number affect simple spike activity (and vice versa) remains unclear. We found that complex spikes with greater numbers of spikelets are preceded by higher simple spike firing rates but, following the complex spike, simple spikes are reduced in a manner that is graded with spikelet number. This dynamic interaction has important implications for cerebellar information processing, and suggests that complex spike spikelet number may maintain Purkinje cells within their operational range. Purkinje cells are central to cerebellar function because they form the sole output of the cerebellar cortex. They exhibit two distinct types of action potential: simple spikes and complex spikes. It is widely accepted that interaction between these two types of impulse is central to cerebellar cortical information processing. Previous investigations of the interactions between simple spikes and complex spikes have mainly considered complex spikes as unitary events. However, complex spikes are composed of an initial large spike followed by a number of secondary components, termed spikelets. The number of spikelets within individual complex spikes is highly variable and the extent to which differences in complex spike spikelet number affects simple spike activity (and vice versa) remains poorly understood. In anaesthetized adult rats, we have found that Purkinje cells recorded from the posterior lobe vermis and hemisphere have high simple spike firing frequencies that precede complex spikes with greater numbers of spikelets. This finding was also evident in a small sample of Purkinje cells recorded from the posterior lobe hemisphere in awake cats. In addition

  6. The Sodium-Potassium Pump Controls the Intrinsic Firing of the Cerebellar Purkinje Neuron

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    Forrest, Michael D.; Wall, Mark J.; Press, Daniel A.; Feng, Jianfeng

    2012-01-01

    In vitro, cerebellar Purkinje cells can intrinsically fire action potentials in a repeating trimodal or bimodal pattern. The trimodal pattern consists of tonic spiking, bursting, and quiescence. The bimodal pattern consists of tonic spiking and quiescence. It is unclear how these firing patterns are generated and what determines which firing pattern is selected. We have constructed a realistic biophysical Purkinje cell model that can replicate these patterns. In this model, Na+/K+ pump activity sets the Purkinje cell's operating mode. From rat cerebellar slices we present Purkinje whole cell recordings in the presence of ouabain, which irreversibly blocks the Na+/K+ pump. The model can replicate these recordings. We propose that Na+/K+ pump activity controls the intrinsic firing mode of cerbellar Purkinje cells. PMID:23284664

  7. The effect of the timing of prenatal exposure to x-irradiation on Purkinje cell numbers in rat cerebellum

    International Nuclear Information System (INIS)

    Miki, T.; Satriotomo, I.; Matsumoto, Y.; Kuma, H.; Takeuchi, Y.; Gu

    2003-01-01

    Full text: Prenatal exposure of the developing brain to X-irradiation is known to cause various deleterious consequences. We have examined the effects of prenatal X-irradiation on the development of the cerebellum. Wistar rats were exposed to 1.5 Gy X-irradiation either on the 14, 15 or 16th day of gestation (E14, E15, E16). Sham-irradiated animals were used as controls. At seven postnatal weeks of age, male rats were deeply anesthetized and killed by intracardiac perfusion with 2.5 % glutaraldehyde in 0.1 M phosphate buffer. The unbiased stereological procedure known as the fractionator method was used to estimate the total number of Purkinje cells in the cerebellum. Body and cerebellar weights from E14 and E15, but not E16 irradiated rats showed significant deficits compared to control animals. Rats irradiated on E16 and control rats had about 285,100 - 304,800 Purkinje cells in the cerebellum. There was no significant difference between these values. However, E14 and E15 irradiated animals had about 117,500 and 196,300 Purkinje cells, respectively. These estimates were significantly different from those observed in both control and E16 irradiated rats. Given that the phase of division of Purkinje cell progenitors is mainly between E14-E15 and the phase of differentiation and migration is between E16-E20, it is concluded that the vulnerable period of the Purkinje cells to X-irradiation closely overlaps the phase of division of progenitors

  8. Inositol Hexakisphosphate Kinase-3 Regulates the Morphology and Synapse Formation of Cerebellar Purkinje Cells via Spectrin/Adducin

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    Fu, Chenglai; Xu, Jing; Li, Ruo-Jing; Crawford, Joshua A.; Khan, A. Basit; Ma, Ting Martin; Cha, Jiyoung Y.; Snowman, Adele M.; Pletnikov, Mikhail V.

    2015-01-01

    The inositol hexakisphosphate kinases (IP6Ks) are the principal enzymes that generate inositol pyrophosphates. There are three IP6Ks (IP6K1, 2, and 3). Functions of IP6K1 and IP6K2 have been substantially delineated, but little is known of IP6K3's role in normal physiology, especially in the brain. To elucidate functions of IP6K3, we generated mice with targeted deletion of IP6K3. We demonstrate that IP6K3 is highly concentrated in the brain in cerebellar Purkinje cells. IP6K3 physiologically binds to the cytoskeletal proteins adducin and spectrin, whose mutual interactions are perturbed in IP6K3-null mutants. Consequently, IP6K3 knock-out cerebella manifest abnormalities in Purkinje cell structure and synapse number, and the mutant mice display deficits in motor learning and coordination. Thus, IP6K3 is a major determinant of cytoskeletal disposition and function of cerebellar Purkinje cells. SIGNIFICANCE STATEMENT We identified and cloned a family of three inositol hexakisphosphate kinases (IP6Ks) that generate the inositol pyrophosphates, most notably 5-diphosphoinositol pentakisphosphate (IP7). Of these, IP6K3 has been least characterized. In the present study we generated IP6K3 knock-out mice and show that IP6K3 is highly expressed in cerebellar Purkinje cells. IP6K3-deleted mice display defects of motor learning and coordination. IP6K3-null mice manifest aberrations of Purkinje cells with a diminished number of synapses. IP6K3 interacts with the cytoskeletal proteins spectrin and adducin whose altered disposition in IP6K3 knock-out mice may mediate phenotypic features of the mutant mice. These findings afford molecular/cytoskeletal mechanisms by which the inositol polyphosphate system impacts brain function. PMID:26245967

  9. Chronic treadmill exercise in rats delicately alters the Purkinje cell structure to improve motor performance and toxin resistance in the cerebellum.

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    Huang, Tung-Yi; Lin, Lung-Sheng; Cho, Keng-Chi; Chen, Shean-Jen; Kuo, Yu-Min; Yu, Lung; Wu, Fong-Sen; Chuang, Jih-Ing; Chen, Hsiun-Ing; Jen, Chauying J

    2012-09-01

    Although exercise usually improves motor performance, the underlying cellular changes in the cerebellum remain to be elucidated. This study aimed to investigate whether and how chronic treadmill exercise in young rats induced Purkinje cell changes to improve motor performance and rendered the cerebellum less vulnerable to toxin insults. After 1-wk familiarization of treadmill running, 6-wk-old male Wistar rats were divided into exercise and sedentary groups. The exercise group was then subjected to 8 wk of exercise training at moderate intensity. The rotarod test was carried out to evaluate motor performance. Purkinje cells in cerebellar slices were visualized by lucifer yellow labeling in single neurons and by calbindin immunostaining in groups of neurons. Compared with sedentary control rats, exercised rats not only performed better in the rotarod task, but also showed finer Purkinje cell structure (higher dendritic volume and spine density with the same dendritic field). The exercise-improved cerebellar functions were further evaluated by monitoring the long-lasting effects of intraventricular application of OX7-saporin. In the sedentary group, OX7-saporin treatment retarded the rotarod performance and induced ∼60% Purkinje cell loss in 3 wk. As a comparison, the exercise group showed much milder injuries in the cerebellum by the same toxin treatment. In conclusion, exercise training in young rats increased the dendritic density of Purkinje cells, which might play an important role in improving the motor performance. Furthermore, as Purkinje cells in the exercise group were relatively toxin resistant, the exercised rats showed good motor performance, even under toxin-treated conditions.

  10. Changes in Purkinje cell simple spike encoding of reach kinematics during adaption to a mechanical perturbation.

    Science.gov (United States)

    Hewitt, Angela L; Popa, Laurentiu S; Ebner, Timothy J

    2015-01-21

    The cerebellum is essential in motor learning. At the cellular level, changes occur in both the simple spike and complex spike firing of Purkinje cells. Because simple spike discharge reflects the main output of the cerebellar cortex, changes in simple spike firing likely reflect the contribution of the cerebellum to the adapted behavior. Therefore, we investigated in Rhesus monkeys how the representation of arm kinematics in Purkinje cell simple spike discharge changed during adaptation to mechanical perturbations of reach movements. Monkeys rapidly adapted to a novel assistive or resistive perturbation along the direction of the reach. Adaptation consisted of matching the amplitude and timing of the perturbation to minimize its effect on the reach. In a majority of Purkinje cells, simple spike firing recorded before and during adaptation demonstrated significant changes in position, velocity, and acceleration sensitivity. The timing of the simple spike representations change within individual cells, including shifts in predictive versus feedback signals. At the population level, feedback-based encoding of position increases early in learning and velocity decreases. Both timing changes reverse later in learning. The complex spike discharge was only weakly modulated by the perturbations, demonstrating that the changes in simple spike firing can be independent of climbing fiber input. In summary, we observed extensive alterations in individual Purkinje cell encoding of reach kinematics, although the movements were nearly identical in the baseline and adapted states. Therefore, adaption to mechanical perturbation of a reaching movement is accompanied by widespread modifications in the simple spike encoding. Copyright © 2015 the authors 0270-6474/15/351106-19$15.00/0.

  11. Prophylactic role of melatonin against radiation induced damage in mouse cerebellum with special reference to Purkinje cells

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    Sisodia, Rashmi; Kumari, Seema; Verma, Rajesh Kumar; Bhatia, A L [Neurobiology Laboratory, Department of Zoology, University of Rajasthan, Jaipur 302004 (India)

    2006-06-15

    Melatonin, a hormone with a proven antioxidative efficacy, crosses all morphophysiological barriers, including the blood-brain barrier, and distributes throughout the cell. The present study is an attempt to investigate the prophylactic influence of a chronic low level of melatonin against an acute radiation induced oxidative stress in the cerebellum of Swiss albino mice, with special reference to Purkinje cells. After 15 days of treatment the mice were sacrificed at various intervals from 1 to 30 days. Biochemical parameters included lipid peroxidation (LPO) and glutathione (GSH) levels as the endpoints. The quantitative study included alterations in number and volume of Purkinje cells. Swiss albino mice were orally administered a very low dose of melatonin (0.25 mg/mouse/day) for 15 consecutive days before single exposure to 4 Gy gamma radiation. Melatonin checked the augmented levels of LPO, by approximately 55%, by day 30 day post-exposure. Radiation induced depleted levels of GSH could be raised by 68.9% by day 30 post-exposure. Radiation exposure resulted in a reduction of the volume of Purkinje cells and their total number. The administration of melatonin significantly protected against the radiation induced decreases in Purkinje cell volume and number. Results indicate the antioxidative properties of melatonin resulting in its prophylactic property against radiation induced biochemical and cellular alterations in the cerebellum. The findings support the idea that melatonin may be used as an anti-irradiation drug due to its potent free radical scavenging and antioxidative efficacy.

  12. Abnormal nuclear envelope in the cerebellar Purkinje cells and impaired motor learning in DYT11 myoclonus-dystonia mouse models.

    Science.gov (United States)

    Yokoi, Fumiaki; Dang, Mai T; Yang, Guang; Li, Jindong; Doroodchi, Atbin; Zhou, Tong; Li, Yuqing

    2012-02-01

    Myoclonus-dystonia (M-D) is a movement disorder characterized by myoclonic jerks with dystonia. DYT11 M-D is caused by mutations in SGCE which codes for ɛ-sarcoglycan. SGCE is maternally imprinted and paternally expressed. Abnormal nuclear envelope has been reported in mouse models of DYT1 generalized torsion dystonia. However, it is not known whether similar alterations occur in DYT11 M-D. We developed a mouse model of DYT11 M-D using paternally inherited Sgce heterozygous knockout (Sgce KO) mice and reported that they had myoclonus and motor coordination and learning deficits in the beam-walking test. However, the specific brain regions that contribute to these phenotypes have not been identified. Since ɛ-sarcoglycan is highly expressed in the cerebellar Purkinje cells, here we examined the nuclear envelope in these cells using a transmission electron microscope and found that they are abnormal in Sgce KO mice. Our results put DYT11 M-D in a growing family of nuclear envelopathies. To analyze the effect of loss of ɛ-sarcoglycan function in the cerebellar Purkinje cells, we produced paternally inherited cerebellar Purkinje cell-specific Sgce conditional knockout (Sgce pKO) mice. Sgce pKO mice showed motor learning deficits, while they did not show abnormal nuclear envelope in the cerebellar Purkinje cells, robust motor deficits, or myoclonus. The results suggest that ɛ-sarcoglycan in the cerebellar Purkinje cells contributes to the motor learning, while loss of ɛ-sarcoglycan in other brain regions may contribute to nuclear envelope abnormality, myoclonus and motor coordination deficits. Copyright © 2011 Elsevier B.V. All rights reserved.

  13. Spiral-wave dynamics in a mathematical model of human ventricular tissue with myocytes and Purkinje fibers.

    Science.gov (United States)

    Nayak, Alok Ranjan; Panfilov, A V; Pandit, Rahul

    2017-02-01

    We present systematic numerical studies of the possible effects of the coupling of human endocardial and Purkinje cells at cellular and two-dimensional tissue levels. We find that the autorhythmic-activity frequency of the Purkinje cell in a composite decreases with an increase in the coupling strength; this can even eliminate the autorhythmicity. We observe a delay between the beginning of the action potentials of endocardial and Purkinje cells in a composite; such a delay increases as we decrease the diffusive coupling, and eventually a failure of transmission occurs. An increase in the diffusive coupling decreases the slope of the action-potential-duration-restitution curve of an endocardial cell in a composite. By using a minimal model for the Purkinje network, in which we have a two-dimensional, bilayer tissue, with a layer of Purkinje cells on top of a layer of endocardial cells, we can stabilize spiral-wave turbulence; however, for a sparse distribution of Purkinje-ventricular junctions, at which these two layers are coupled, we can also obtain additional focal activity and many complex transient regimes. We also present additional effects resulting from the coupling of Purkinje and endocardial layers and discuss the relation of our results to the studies performed in anatomically accurate models of the Purkinje network.

  14. Crista Supraventricularis Purkinje Network and Its Relation to Intraseptal Purkinje Network.

    Science.gov (United States)

    De Almeida, Marcos C; Araujo, Mayssa; Duque, Mathias; Vilhena, Virginia

    2017-10-01

    Using transparent specimens with a dual color injection, microscopy, and computer tomography, this report shows that the right and left ventricular subendocardial Purkinje networks are connected by an extensive septal network in the bovine heart. The septal network is present along the entire septum except at a free zone below ventricular valves. Being the only communication of the basal right septum with the right free wall, the supraventricular crest is an enigmatic but not, by any means, hidden muscular structure. It is one of the last structures to be activated in human heart. It is shown here that the supraventricular crest Purkinje network connects the anterosuperior right ventricular basal free wall Purkinje network to anterior right ventricular basal septal Purkinje network. It is suggested that the stimulus initiated at middle left ventricular endocardium will activate the supraventricular crest. The intraseptal connection found between the basal left ventricular subendocardial septal Purkinje network and the right ventricular basal septal Purkinje network is, probably, the pathway for the stimulus. An anatomic basis is provided to explain why the inflow tract contracts earlier than the outflow tract in the right ventricle systole. Anat Rec, 2017. © 2017 Wiley Periodicals, Inc. Anat Rec, 300:1793-1801, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  15. Organization of spinocerebellar projection map in three types of agranular cerebellum: Purkinje cells vs. granule cells as organizer element

    International Nuclear Information System (INIS)

    Arsenio Nunes, M.L.; Sotelo, C.; Wehrle, R.

    1988-01-01

    The organization of the spinocerebellar projection was analysed by the anterograde axonal WGA-HRP (horseradish peroxidase-wheat germ agglutinin conjugate) tracing method in three different types of agranular cerebellar cortex either induced experimentally by X-irradiation or occurring spontaneously in weaver (wv/wv) and staggerer (sg/sg) mutant mice. The results of this study show that in the X-irradiated rat and weaver mouse, in both of which the granule cells are directly affected and die early in development, the spinal axons reproduce, with few differences, the normal spinocerebellar pattern. Conversely, in staggerer mouse, in which the Purkinje cells are intrinsically affected and granule neurons do not seem to be primarily perturbed by the staggerer gene action, the spinocerebellar organization is severely modified. These findings appear somewhat paradoxical because if granule cells, the synaptic targets of mossy spinocerebellar fibers, were necessary for the organization of spinocerebellar projection, the staggerer cerebellum would exhibit a much more normal projectional map than the weaver and the X-irradiated cerebella. It is, therefore, obvious that granule cells, and even specific synaptogenesis, are not essential for the establishment of the normal spinocerebellar topography. On the other hand, the fact that the Purkinje cells are primarily affected in the unique agranular cortex in which the spinocerebellar organization is severely modified suggests that these neurons could be the main element in the organization of the spinocerebellar projection map. This hypothesis is discussed in correlation with already-reported findings on the zonation of the cerebellar cortex by biochemically different clusters of Purkinje cells

  16. Structural study of Purkinje cell axonal torpedoes in essential tremor.

    Science.gov (United States)

    Louis, Elan D; Yi, Hong; Erickson-Davis, Cordelia; Vonsattel, Jean-Paul G; Faust, Phyllis L

    2009-02-06

    Essential tremor (ET) is one of the most common neurological diseases. A basic understanding of its neuropathology is now emerging. Aside from Purkinje cell loss, a prominent finding is an abundance of torpedoes (rounded swellings of Purkinje cell axons). Such swellings often result from the mis-accumulation of cell constituents. Identifying the basic nature of these accumulations is an important step in understanding the underlying disease process. Torpedoes, only recently identified in ET, have not yet been characterized ultrastructurally. Light and electron microscopy were used to characterize the structural constituents of torpedoes in ET. Formalin-fixed cerebellar cortical tissue from four prospectively collected ET brains was sectioned and immunostained with a monoclonal phosphorylated neurofilament antibody (SMI-31, Covance, Emeryville, CA). Using additional sections from three ET brains, torpedoes were assessed using electron microscopy. Immunoreactivity for phosphorylated neurofilament protein revealed clear labeling of torpedoes in each case. Torpedoes were strongly immunoreactive; in many instances, two or more torpedoes were noted in close proximity to one another. On electron microscopy, torpedoes were packed with randomly arranged 10-12nm neurofilaments. Mitochondria and smooth endoplasmic reticulum were abundant as well, particularly at the periphery of the torpedo. We demonstrated that the torpedoes in ET represent the mis-accumulation of disorganized neurofilaments and other organelles. It is not known where in the pathogenic cascade these accumulations occur (i.e., whether these accumulations are the primary event or a secondary/downstream event) and this deserves further study.

  17. Responses of Purkinje cells in the oculomotor vermis of monkeys during smooth pursuit eye movements and saccades: comparison with floccular complex.

    Science.gov (United States)

    Raghavan, Ramanujan T; Lisberger, Stephen G

    2017-08-01

    We recorded the responses of Purkinje cells in the oculomotor vermis during smooth pursuit and saccadic eye movements. Our goal was to characterize the responses in the vermis using approaches that would allow direct comparisons with responses of Purkinje cells in another cerebellar area for pursuit, the floccular complex. Simple-spike firing of vermis Purkinje cells is direction selective during both pursuit and saccades, but the preferred directions are sufficiently independent so that downstream circuits could decode signals to drive pursuit and saccades separately. Complex spikes also were direction selective during pursuit, and almost all Purkinje cells showed a peak in the probability of complex spikes during the initiation of pursuit in at least one direction. Unlike the floccular complex, the preferred directions for simple spikes and complex spikes were not opposite. The kinematics of smooth eye movement described the simple-spike responses of vermis Purkinje cells well. Sensitivities were similar to those in the floccular complex for eye position and considerably lower for eye velocity and acceleration. The kinematic relations were quite different for saccades vs. pursuit, supporting the idea that the contributions from the vermis to each kind of movement could contribute independently in downstream areas. Finally, neither the complex-spike nor the simple-spike responses of vermis Purkinje cells were appropriate to support direction learning in pursuit. Complex spikes were not triggered reliably by an instructive change in target direction; simple-spike responses showed very small amounts of learning. We conclude that the vermis plays a different role in pursuit eye movements compared with the floccular complex. NEW & NOTEWORTHY The midline oculomotor cerebellum plays a different role in smooth pursuit eye movements compared with the lateral, floccular complex and appears to be much less involved in direction learning in pursuit. The output from the

  18. Bergmann glia and the recognition molecule CHL1 organize GABAergic axons and direct innervation of Purkinje cell dendrites.

    Directory of Open Access Journals (Sweden)

    Fabrice Ango

    2008-04-01

    Full Text Available The geometric and subcellular organization of axon arbors distributes and regulates electrical signaling in neurons and networks, but the underlying mechanisms have remained elusive. In rodent cerebellar cortex, stellate interneurons elaborate characteristic axon arbors that selectively innervate Purkinje cell dendrites and likely regulate dendritic integration. We used GFP BAC transgenic reporter mice to examine the cellular processes and molecular mechanisms underlying the development of stellate cell axons and their innervation pattern. We show that stellate axons are organized and guided towards Purkinje cell dendrites by an intermediate scaffold of Bergmann glial (BG fibers. The L1 family immunoglobulin protein Close Homologue of L1 (CHL1 is localized to apical BG fibers and stellate cells during the development of stellate axon arbors. In the absence of CHL1, stellate axons deviate from BG fibers and show aberrant branching and orientation. Furthermore, synapse formation between aberrant stellate axons and Purkinje dendrites is reduced and cannot be maintained, leading to progressive atrophy of axon terminals. These results establish BG fibers as a guiding scaffold and CHL1 a molecular signal in the organization of stellate axon arbors and in directing their dendritic innervation.

  19. Signals and Circuits in the Purkinje Neuron

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    Ze'ev R Abrams

    2011-09-01

    Full Text Available Purkinje neurons in the cerebellum have over 100,000 inputs organized in an orthogonal geometry, and a single output channel. As the sole output of the cerebellar cortex layer, their complex firing pattern has been associated with motor control and learning. As such they have been extensively modeled and measured using tools ranging from electrophysiology and neuroanatomy, to dynamic systems and artificial intelligence methods. However, there is an alternative approach to analyze and describe the neuronal output of these cells using concepts from Electrical Engineering, particularly signal processing and digital/analog circuits. By viewing the Purkinje neuron as an unknown circuit to be reverse-engineered, we can use the tools that provide the foundations of today’s integrated circuits and communication systems to analyze the Purkinje system at the circuit level. We use Fourier transforms to analyze and isolate the inherent frequency modes in the Purkinje neuron and define 3 unique frequency ranges associated with the cells’ output. Comparing the Purkinje neuron to a signal generator that can be externally modulated adds an entire level of complexity to the functional role of these neurons both in terms of data analysis and information processing, relying on Fourier analysis methods in place of statistical ones. We also re-describe some of the recent literature in the field, using the nomenclature of signal processing. Furthermore, by comparing the experimental data of the past decade with basic electronic circuitry, we can resolve the outstanding controversy in the field, by recognizing that the Purkinje neuron can act as a multivibrator circuit.

  20. Geranylgeranyltransferase I is essential for dendritic development of cerebellar Purkinje cells

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    Wu Kong-Yan

    2010-06-01

    Full Text Available Abstract Background During cerebellar development, Purkinje cells (PCs form the most elaborate dendritic trees among neurons in the brain, but the mechanism regulating PC arborization remains largely unknown. Geranylgeranyltransferase I (GGT is a prenyltransferase that is responsible for lipid modification of several signaling proteins, such as Rho family small GTPase Rac1, which has been shown to be involved in neuronal morphogenesis. Here we show that GGT plays an important role in dendritic development of PCs. Results We found that GGT was abundantly expressed in the developing rat cerebellum, in particular molecular layer (ML, the region enriched with PC dendrites. Inhibition or down-regulation of GGT using small interference RNA (siRNA inhibited dendritic development of PCs. In contrast, up-regulation of GGT promoted dendritic arborization of PCs. Furthermore, neuronal depolarization induced by high K+ or treatment with brain-derived neurotrophic factor (BDNF promoted membrane association of Rac1 and dendritic development of PCs in cultured cerebellar slices. The effect of BDNF or high K+ was inhibited by inhibition or down-regulation of GGT. Conclusion Our results indicate that GGT plays an important role in Purkinje cell development, and suggest a novel role of GGT in neuronal morphogenesis in vivo.

  1. Reminiscing about Jan Evangelista Purkinje: a pioneer of modern experimental physiology.

    Science.gov (United States)

    Cavero, Icilio; Guillon, Jean-Michel; Holzgrefe, Henry H

    2017-12-01

    This article reminisces about the life and key scientific achievements of Jan Evangelista Purkinje (1787-1869), a versatile 19th century Czech pioneer of modern experimental physiology. In 1804, after completing senior high school, Purkinje joined the Piarist monk order, but, after a 3-yr novitiate, he gave up the religious calling "to deal more freely with science." In 1818, he earned a Medical Doctor degree from Prague University by defending a dissertation on intraocular phenomena observed in oneself. In 1823, Purkinje became a Physiology and Pathology professor at the Prussian Medical University in Breslau, where he innovated the traditional teaching methods of physiology. Purkinje's contributions to physiology were manifold: accurate descriptions of various visual phenomena (e.g., Purkinje-Sanson images, Purkinje phenomenon), discovery of the terminal network of the cardiac conduction system (Purkinje fibers), identification of cerebellar neuronal bodies (Purkinje cells), formulation of the vertigo law (Purkinje's law), discovery of criteria to classify human fingerprints, etc. In 1850, Purkinje accepted and held until his death the Physiology chair at Prague Medical Faculty. During this period, he succeeded in introducing the Czech idiom (in addition to long-established German and Latin) as a Medical Faculty teaching language. Additionally, as a zealous Czech patriot, he actively contributed to the naissance and consolidation of a national Czech identity conscience. Purkinje was a trend-setting scientist who, throughout his career, worked to pave the way for the renovation of physiology from a speculative discipline, ancilla of anatomy, into a factual, autonomous science committed to the discovery of mechanisms governing in-life functions. Copyright © 2017 the American Physiological Society.

  2. Sensory processing and corollary discharge effects in posterior caudal lobe Purkinje cells in a weakly electric mormyrid fish.

    Science.gov (United States)

    Alviña, Karina; Sawtell, Nathaniel B

    2014-07-15

    Although it has been suggested that the cerebellum functions to predict the sensory consequences of motor commands, how such predictions are implemented in cerebellar circuitry remains largely unknown. A detailed and relatively complete account of predictive mechanisms has emerged from studies of cerebellum-like sensory structures in fish, suggesting that comparisons of the cerebellum and cerebellum-like structures may be useful. Here we characterize electrophysiological response properties of Purkinje cells in a region of the cerebellum proper of weakly electric mormyrid fish, the posterior caudal lobe (LCp), which receives the same mossy fiber inputs and projects to the same target structures as the electrosensory lobe (ELL), a well-studied cerebellum-like structure. We describe patterns of simple spike and climbing fiber activation in LCp Purkinje cells in response to motor corollary discharge, electrosensory, and proprioceptive inputs and provide evidence for two functionally distinct Purkinje cell subtypes within LCp. Protocols that induce rapid associative plasticity in ELL fail to induce plasticity in LCp, suggesting differences in the adaptive functions of the two structures. Similarities and differences between LCp and ELL are discussed in light of these results. Copyright © 2014 the American Physiological Society.

  3. Tissue Plasminogen Activator Induction in Purkinje Neurons After Cerebellar Motor Learning

    Science.gov (United States)

    Seeds, Nicholas W.; Williams, Brian L.; Bickford, Paula C.

    1995-12-01

    The cerebellar cortex is implicated in the learning of complex motor skills. This learning may require synaptic remodeling of Purkinje cell inputs. An extracellular serine protease, tissue plasminogen activator (tPA), is involved in remodeling various nonneural tissues and is associated with developing and regenerating neurons. In situ hybridization showed that expression of tPA messenger RNA was increased in the Purkinje neurons of rats within an hour of their being trained for a complex motor task. Antibody to tPA also showed the induction of tPA protein associated with cerebellar Purkinje cells. Thus, the induction of tPA during motor learning may play a role in activity-dependent synaptic plasticity.

  4. TACTILE STIMULATION EVOKES LONG-LASTING POTENTIATION OF PURKINJE CELL DISCHARGE IN VIVO

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    Ramakrishnan eKanchipuram

    2016-02-01

    Full Text Available In the cerebellar network, a precise relationship between plasticity and neuronal discharge has been predicted. However, the potential generation of persistent changes in PC spike discharge as a consequence of plasticity following natural stimulation patterns has not been clearly determined. Here we show that facial tactile stimuli organized in theta-patterns can induce stereotyped NMDA and GABA-A receptor-dependent changes in Purkinje cell (PCs and molecular layer interneuron (MLIs firing: invariably, all PCs showed a long-lasting increase (spike-related potentiation or SR-P and MLIs a long-lasting decrease (spike-related suppression or SR-S in baseline activity and spike response probability. These observations suggests that natural sensory stimulation engages multiple long-term plastic changes that are distributed along the mossy fiber – parallel fiber pathway and operate synergistically to potentiate spike generation in PCs. In contrast, theta-pattern electrical stimulation of PFs indistinctly induced SR-P and SR-S both in PCs and MLIs, suggesting that natural sensory stimulation preordinates plasticity upstream of the PF-PC synapse. All these effects occurred in the absence of complex spike changes, supporting the theoretical prediction that Purkinje cell activity is potentiated when the mossy fiber - parallel fiber system is activated in the absence of conjunctive climbing fiber activity.

  5. Improved motor performance in Dyt1 ΔGAG heterozygous knock-in mice by cerebellar Purkinje-cell specific Dyt1 conditional knocking-out.

    Science.gov (United States)

    Yokoi, Fumiaki; Dang, Mai Tu; Li, Yuqing

    2012-05-01

    Early-onset generalized torsion dystonia (dystonia 1) is an inherited movement disorder caused by mutations in DYT1 (TOR1A), which codes for torsinA. Most patients have a 3-base pair deletion (ΔGAG) in one allele of DYT1, corresponding to a loss of a glutamic acid residue (ΔE) in the C-terminal region of the protein. Functional alterations in basal ganglia circuits and the cerebellum have been reported in dystonia. Pharmacological manipulations or mutations in genes that result in functional alterations of the cerebellum have been reported to have dystonic symptoms and have been used as phenotypic rodent models. Additionally, structural lesions in the abnormal cerebellar circuits, such as cerebellectomy, have therapeutic effects in these models. A previous study has shown that the Dyt1 ΔGAG heterozygous knock-in (KI) mice exhibit motor deficits in the beam-walking test. Both Dyt1 ΔGAG heterozygous knock-in (KI) and Dyt1 Purkinje cell-specific knockout (Dyt1 pKO) mice exhibit dendritic alterations of cerebellar Purkinje cells. Here, Dyt1 pKO mice exhibited significantly less slip numbers in the beam-walking test, suggesting better motor performance than control littermates, and normal gait. Furthermore, Dyt1 ΔGAG KI/Dyt1 pKO double mutant mice exhibited significantly lower numbers of slips than Dyt1 ΔGAG heterozygous KI mice, suggesting Purkinje-cell specific knockout of Dyt1 wild-type (WT) allele in Dyt1 ΔGAG heterozygous KI mice rescued the motor deficits. The results suggest that molecular lesions of torsinA in Purkinje cells by gene therapy or intervening in the signaling pathway downstream of the cerebellar Purkinje cells may rescue motor symptoms in dystonia 1. Copyright © 2012 Elsevier B.V. All rights reserved.

  6. Dendritic excitability modulates dendritic information processing in a purkinje cell model.

    Science.gov (United States)

    Coop, Allan D; Cornelis, Hugo; Santamaria, Fidel

    2010-01-01

    Using an electrophysiological compartmental model of a Purkinje cell we quantified the contribution of individual active dendritic currents to processing of synaptic activity from granule cells. We used mutual information as a measure to quantify the information from the total excitatory input current (I(Glu)) encoded in each dendritic current. In this context, each active current was considered an information channel. Our analyses showed that most of the information was encoded by the calcium (I(CaP)) and calcium activated potassium (I(Kc)) currents. Mutual information between I(Glu) and I(CaP) and I(Kc) was sensitive to different levels of excitatory and inhibitory synaptic activity that, at the same time, resulted in the same firing rate at the soma. Since dendritic excitability could be a mechanism to regulate information processing in neurons we quantified the changes in mutual information between I(Glu) and all Purkinje cell currents as a function of the density of dendritic Ca (g(CaP)) and Kca (g(Kc)) conductances. We extended our analysis to determine the window of temporal integration of I(Glu) by I(CaP) and I(Kc) as a function of channel density and synaptic activity. The window of information integration has a stronger dependence on increasing values of g(Kc) than on g(CaP), but at high levels of synaptic stimulation information integration is reduced to a few milliseconds. Overall, our results show that different dendritic conductances differentially encode synaptic activity and that dendritic excitability and the level of synaptic activity regulate the flow of information in dendrites.

  7. Heat Shock Protein Beta-1 Modifies Anterior to Posterior Purkinje Cell Vulnerability in a Mouse Model of Niemann-Pick Type C Disease.

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    Chan Chung

    2016-05-01

    Full Text Available Selective neuronal vulnerability is characteristic of most degenerative disorders of the CNS, yet mechanisms underlying this phenomenon remain poorly characterized. Many forms of cerebellar degeneration exhibit an anterior-to-posterior gradient of Purkinje cell loss including Niemann-Pick type C1 (NPC disease, a lysosomal storage disorder characterized by progressive neurological deficits that often begin in childhood. Here, we sought to identify candidate genes underlying vulnerability of Purkinje cells in anterior cerebellar lobules using data freely available in the Allen Brain Atlas. This approach led to the identification of 16 candidate neuroprotective or susceptibility genes. We demonstrate that one candidate gene, heat shock protein beta-1 (HSPB1, promoted neuronal survival in cellular models of NPC disease through a mechanism that involved inhibition of apoptosis. Additionally, we show that over-expression of wild type HSPB1 or a phosphomimetic mutant in NPC mice slowed the progression of motor impairment and diminished cerebellar Purkinje cell loss. We confirmed the modulatory effect of Hspb1 on Purkinje cell degeneration in vivo, as knockdown by Hspb1 shRNA significantly enhanced neuron loss. These results suggest that strategies to promote HSPB1 activity may slow the rate of cerebellar degeneration in NPC disease and highlight the use of bioinformatics tools to uncover pathways leading to neuronal protection in neurodegenerative disorders.

  8. Apoptosis of Purkinje and granular cells of the cerebellum following chronic ethanol intake.

    Science.gov (United States)

    Oliveira, Suelen A; Chuffa, Luiz Gustavo A; Fioruci-Fontanelli, Beatriz Aparecida; Lizarte Neto, Fermino Sanches; Novais, Paulo Cezar; Tirapelli, Luiz Fernando; Oishi, Jorge Camargo; Takase, Luiz Fernando; Stefanini, Maira Aparecida; Martinez, Marcelo; Martinez, Francisco Eduardo

    2014-12-01

    Ethanol alters motricity, learning, cognition, and cellular metabolism in the cerebellum. We evaluated the effect of ethanol on apoptosis in Golgi, Purkinje, and granule cells of the cerebellum in adult rats. There were two groups of 20 rats: a control group that did not consume ethanol and an experimental group of UChA rats that consumed ethanol at 10% (cerebellum of adult UChA rats.

  9. Dendritic and axonic fields of Purkinje cells in developing and X-irradiated rat cerebellum. A comparative study using intracellular staining with horseradish peroxidase

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    Crepel, F; Delhaye-Bouchaud, N; Dupont, J L [Paris-5 Univ., 75 (France); Sotelo, C [Hopital Foch, 92 - Suresnes (France). Centre Medico-Chirurgical

    1980-01-01

    Intracellular staining of cerebellar Purkinje cells with horseradish peroxidase was achieved in normal developing rats (8-13 days old), in normal adult rats and in adult rats in which the cerebellum had been degranulated by X-ray treatment. The mono- and multiple innervation of Purkinje cells by climbing fibres was electrophysiologically determined and correlated with their dendritic pattern and axonal field. In immature rats, considerable variations in dendritic arborization were observed between cells at the same age, according to their position in the vermis. In adult X-irradiated animals, a large variety of dendritic shapes was found, confirming previous anatomical data, but no obvious correlation was found between the morphology of the dendrites of Purkinje cells and their synaptic investment by climbing fibres. As regards the axonal field, the adult branching pattern of recurrent axon collaterals was almost established by postnatal day 8, except for some cells which exhibited richer recurrent collaterals. On the other hand, in X-irradiated animals, profuse plexuses were the rule and they originated either from one collateral stem, or from several collaterals, also independently of the number of afferent climbing fibres. The existence of these enlarged recurrent collateral plexuses can be explained by the persistence of an immature stage, and certainly also by the collateral sprouting following the largely impaired innervation of the terminal field during development. These results emphasize the role of the cellular interactions that occur during Purkinje cell growth in the formation of both its axonal and dendritic fields.

  10. An expandable embryonic stem cell-derived Purkinje neuron progenitor population that exhibits in vivo maturation in the adult mouse cerebellum

    NARCIS (Netherlands)

    G.A. Higuera (Gustavo A.); Iaffaldano, G. (Grazia); Bedar, M. (Meiwand); G. Shpak (Guy); R. Broersen (Robin); S.T. Munshi (Shashini T.); Dupont, C. (Catherine); J.H. Gribnau (Joost); F.M.S. Vrij (Femke); S.A. Kushner (Steven); C.I. de Zeeuw (Chris)

    2017-01-01

    textabstractThe directed differentiation of patient-derived induced pluripotent stem cells into cell-type specific neurons has inspired the development of therapeutic discovery for neurodegenerative diseases. Many forms of ataxia result from degeneration of cerebellar Purkinje cells, but thus far it

  11. A spiking network model of cerebellar Purkinje cells and molecular layer interneurons exhibiting irregular firing

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    William eLennon

    2014-12-01

    Full Text Available While the anatomy of the cerebellar microcircuit is well studied, how it implements cerebellar function is not understood. A number of models have been proposed to describe this mechanism but few emphasize the role of the vast network Purkinje cells (PKJs form with the molecular layer interneurons (MLIs – the stellate and basket cells. We propose a model of the MLI-PKJ network composed of simple spiking neurons incorporating the major anatomical and physiological features. In computer simulations, the model reproduces the irregular firing patterns observed in PKJs and MLIs in vitro and a shift toward faster, more regular firing patterns when inhibitory synaptic currents are blocked. In the model, the time between PKJ spikes is shown to be proportional to the amount of feedforward inhibition from an MLI on average. The two key elements of the model are: (1 spontaneously active PKJs and MLIs due to an endogenous depolarizing current, and (2 adherence to known anatomical connectivity along a parasagittal strip of cerebellar cortex. We propose this model to extend previous spiking network models of the cerebellum and for further computational investigation into the role of irregular firing and MLIs in cerebellar learning and function.

  12. An expandable embryonic stem cell-derived Purkinje neuron progenitor population that exhibits in vivo maturation in the adult mouse cerebellum

    NARCIS (Netherlands)

    Higuera, Gustavo A; Iaffaldano, Grazia; Bedar, Meiwand; Shpak, Guy; Broersen, Robin; Munshi, Shashini T; Dupont, Catherine; Gribnau, Joost; de Vrij, Femke M S; Kushner, Steven A; De Zeeuw, Chris I

    2017-01-01

    The directed differentiation of patient-derived induced pluripotent stem cells into cell-type specific neurons has inspired the development of therapeutic discovery for neurodegenerative diseases. Many forms of ataxia result from degeneration of cerebellar Purkinje cells, but thus far it has not

  13. Dendritic and axonic fields of Purkinje cells in developing and X-irradiated rat cerebellum. A comparative study using intracellular staining with horseradish peroxidase

    International Nuclear Information System (INIS)

    Crepel, F.; Delhaye-Bouchaud, N.; Dupont, J.L.; Sotelo, C.

    1980-01-01

    Intracellular staining of cerebellar Purkinje cells with horseradish peroxidase was achieved in normal developing rats (8-13 days old), in normal adult rats and in adult rats in which the cerebellum had been degranulated by X-ray treatment. The mono- and multiple innervation of Purkinje cells by climbing fibres was electrophysiologically determined and correlated with their dendritic pattern and axonal field. In immature rats, considerable variations in dendritic arborization were observed between cells at the same age, according to their position in the vermis. In adult X-irradiated animals, a large variety of dendritic shapes was found, confirming previous anatomical data, but no obvious correlation was found between the morphology of the dendrites of Purkinje cells and their synaptic investment by climbing fibres. As regards the axonal field, the adult branching pattern of recurrent axon collaterals was almost established by postnatal day 8, except for some cells which exhibited richer recurrent collaterals. On the other hand, in X-irradiated animals, profuse plexuses were the rule and they originated either from one collateral stem, or from several collaterals, also independently of the number of afferent climbing fibres. The existence of these enlarged recurrent collateral plexuses can be explained by the persistence of an immature stage, and certainly also by the collateral sprouting following the largely impaired innervation of the terminal field during development. These results emphasize the role of the cellular interactions that occur during Purkinje cell growth in the formation of both its axonal and dendritic fields. (author)

  14. A Simple Mathematical Model Inspired by the Purkinje Cells: From Delayed Travelling Waves to Fractional Diffusion.

    Science.gov (United States)

    Dipierro, Serena; Valdinoci, Enrico

    2018-07-01

    Recently, several experiments have demonstrated the existence of fractional diffusion in the neuronal transmission occurring in the Purkinje cells, whose malfunctioning is known to be related to the lack of voluntary coordination and the appearance of tremors. Also, a classical mathematical feature is that (fractional) parabolic equations possess smoothing effects, in contrast with the case of hyperbolic equations, which typically exhibit shocks and discontinuities. In this paper, we show how a simple toy-model of a highly ramified structure, somehow inspired by that of the Purkinje cells, may produce a fractional diffusion via the superposition of travelling waves that solve a hyperbolic equation. This could suggest that the high ramification of the Purkinje cells might have provided an evolutionary advantage of "smoothing" the transmission of signals and avoiding shock propagations (at the price of slowing a bit such transmission). Although an experimental confirmation of the possibility of such evolutionary advantage goes well beyond the goals of this paper, we think that it is intriguing, as a mathematical counterpart, to consider the time fractional diffusion as arising from the superposition of delayed travelling waves in highly ramified transmission media. The case of a travelling concave parabola with sufficiently small curvature is explicitly computed. The new link that we propose between time fractional diffusion and hyperbolic equation also provides a novelty with respect to the usual paradigm relating time fractional diffusion with parabolic equations in the limit. This paper is written in such a way as to be of interest to both biologists and mathematician alike. In order to accomplish this aim, both complete explanations of the objects considered and detailed lists of references are provided.

  15. Posterior cerebellar Purkinje cells in an SCA5/SPARCA1 mouse model are especially vulnerable to the synergistic effect of loss of β-III spectrin and GLAST.

    Science.gov (United States)

    Perkins, Emma M; Suminaite, Daumante; Clarkson, Yvonne L; Lee, Sin Kwan; Lyndon, Alastair R; Rothstein, Jeffrey D; Wyllie, David J A; Tanaka, Kohichi; Jackson, Mandy

    2016-10-15

    Clinical phenotypes of spinocerebellar ataxia type-5 (SCA5) and spectrin-associated autosomal recessive cerebellar ataxia type-1 (SPARCA1) are mirrored in mice lacking β-III spectrin (β-III-/-). One function of β-III spectrin is the stabilization of the Purkinje cell-specific glutamate transporter EAAT4 at the plasma membrane. In β-III-/- mice EAAT4 levels are reduced from an early age. In contrast levels of the predominant cerebellar glutamate transporter GLAST, expressed in Bergmann glia, only fall progressively from 3 months onwards. Here we elucidated the roles of these two glutamate transporters in cerebellar pathogenesis mediated through loss of β-III spectrin function by studying EAAT4 and GLAST knockout mice as well as crosses of both with β-III-/- mice. Our data demonstrate that EAAT4 loss, but not abnormal AMPA receptor composition, in young β-III-/- mice underlies early Purkinje cell hyper-excitability and that subsequent loss of GLAST, superimposed on the earlier deficiency of EAAT4, is responsible for Purkinje cell loss and progression of motor deficits. Yet the loss of GLAST appears to be independent of EAAT4 loss, highlighting that other aspects of Purkinje cell dysfunction underpin the pathogenic loss of GLAST. Finally, our results demonstrate that Purkinje cells in the posterior cerebellum of β-III-/- mice are most susceptible to the combined loss of EAAT4 and GLAST, with degeneration of proximal dendrites, the site of climbing fibre innervation, most pronounced. This highlights the necessity for efficient glutamate clearance from these regions and identifies dysregulation of glutamatergic neurotransmission particularly within the posterior cerebellum as a key mechanism in SCA5 and SPARCA1 pathogenesis.

  16. Axonal propagation of simple and complex spikes in cerebellar Purkinje neurons.

    Science.gov (United States)

    Khaliq, Zayd M; Raman, Indira M

    2005-01-12

    In cerebellar Purkinje neurons, the reliability of propagation of high-frequency simple spikes and spikelets of complex spikes is likely to regulate inhibition of Purkinje target neurons. To test the extent to which a one-to-one correspondence exists between somatic and axonal spikes, we made dual somatic and axonal recordings from Purkinje neurons in mouse cerebellar slices. Somatic action potentials were recorded with a whole-cell pipette, and the corresponding axonal signals were recorded extracellularly with a loose-patch pipette. Propagation of spontaneous and evoked simple spikes was highly reliable. At somatic firing rates of approximately 200 spikes/sec, 375 Hz during somatic hyperpolarizations that silenced spontaneous firing to approximately 150 Hz during spontaneous activity. The probability of propagation of individual spikelets could be described quantitatively as a saturating function of spikelet amplitude, rate of rise, or preceding interspike interval. The results suggest that ion channels of Purkinje axons are adapted to produce extremely short refractory periods and that brief bursts of forward-propagating action potentials generated by complex spikes may contribute transiently to inhibition of postsynaptic neurons.

  17. Transferences of Purkinje systems

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    W. F. Harris

    2011-12-01

    Full Text Available The transferences of heterocentric astigmatic Purkinje systems are special: submatrices B and C, that is, the disjugacy and the divergence of the system, are symmetric and submatrix D (the divarication is the transpose of submatrix A (the dilation.  It is the primary purpose of this paper to provide a proof.  The paper also derives other relationships among the fundamental properties and compact expressions for the transference and optical axis locator of a Purkinje system. (S Afr Optom 2011 70(2 57-60

  18. The Phospholipase D2 Knock Out Mouse Has Ectopic Purkinje Cells and Suffers from Early Adult-Onset Anosmia.

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    Matthieu M Vermeren

    Full Text Available Phospholipase D2 (PLD2 is an enzyme that produces phosphatidic acid (PA, a lipid messenger molecule involved in a number of cellular events including, through its membrane curvature properties, endocytosis. The PLD2 knock out (PLD2KO mouse has been previously reported to be protected from insult in a model of Alzheimer's disease. We have further analysed a PLD2KO mouse using mass spectrophotometry of its lipids and found significant differences in PA species throughout its brain. We have examined the expression pattern of PLD2 which allowed us to define which region of the brain to analyse for defect, notably PLD2 was not detected in glial-rich regions. The expression pattern lead us to specifically examine the mitral cells of olfactory bulbs, the Cornus Amonis (CA regions of the hippocampus and the Purkinje cells of the cerebellum. We find that the change to longer PA species correlates with subtle architectural defect in the cerebellum, exemplified by ectopic Purkinje cells and an adult-onset deficit of olfaction. These observations draw parallels to defects in the reelin heterozygote as well as the effect of high fat diet on olfaction.

  19. Downregulation of the Glial GLT1 Glutamate Transporter and Purkinje Cell Dysfunction in a Mouse Model of Myotonic Dystrophy

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    Géraldine Sicot

    2017-06-01

    Full Text Available Brain function is compromised in myotonic dystrophy type 1 (DM1, but the underlying mechanisms are not fully understood. To gain insight into the cellular and molecular pathways primarily affected, we studied a mouse model of DM1 and brains of adult patients. We found pronounced RNA toxicity in the Bergmann glia of the cerebellum, in association with abnormal Purkinje cell firing and fine motor incoordination in DM1 mice. A global proteomics approach revealed downregulation of the GLT1 glutamate transporter in DM1 mice and human patients, which we found to be the result of MBNL1 inactivation. GLT1 downregulation in DM1 astrocytes increases glutamate neurotoxicity and is detrimental to neurons. Finally, we demonstrated that the upregulation of GLT1 corrected Purkinje cell firing and motor incoordination in DM1 mice. Our findings show that glial defects are critical in DM1 brain pathophysiology and open promising therapeutic perspectives through the modulation of glutamate levels.

  20. Purkinje Cell Compartmentation in the Cerebellum of the Lysosomal Acid Phosphatase 2 Mutant Mouse (Nax - Naked-Ataxia Mutant Mouse)

    Science.gov (United States)

    Bailey, Karen; Rahimi Balaei, Maryam; Mannan, Ashraf; Del Bigio, Marc R.; Marzban, Hassan

    2014-01-01

    The Acp2 gene encodes the beta subunit of lysosomal acid phosphatase, which is an isoenzyme that hydrolyzes orthophosphoric monoesters. In mice, a spontaneous mutation in Acp2 results in severe cerebellar defects. These include a reduced size, abnormal lobulation, and an apparent anterior cerebellar disorder with an absent or hypoplastic vermis. Based on differential gene expression in the cerebellum, the mouse cerebellar cortex can normally be compartmentalized anteroposteriorly into four transverse zones and mediolaterally into parasagittal stripes. In this study, immunohistochemistry was performed using various Purkinje cell compartmentation markers to examine their expression patterns in the Acp2 mutant. Despite the abnormal lobulation and anterior cerebellar defects, zebrin II and PLCβ4 showed similar expression patterns in the nax mutant and wild type cerebellum. However, fewer stripes were found in the anterior zone of the nax mutant, which could be due to a lack of Purkinje cells or altered expression of the stripe markers. HSP25 expression was uniform in the central zone of the nax mutant cerebellum at around postnatal day (P) 18–19, suggesting that HSP25 immunonegative Purkinje cells are absent or delayed in stripe pattern expression compared to the wild type. HSP25 expression became heterogeneous around P22–23, with twice the number of parasagittal stripes in the nax mutant compared to the wild type. Aside from reduced size and cortical disorganization, both the posterior zone and nodular zone in the nax mutant appeared less abnormal than the rest of the cerebellum. From these results, it is evident that the anterior zone of the nax mutant cerebellum is the most severely affected, and this extends beyond the primary fissure into the rostral central zone/vermis. This suggests that ACP2 has critical roles in the development of the anterior cerebellum and it may regulate anterior and central zone compartmentation. PMID:24722417

  1. Releasing dentate nucleus cells from Purkinje cell inhibition generates output from the cerebrocerebellum.

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    Takahiro Ishikawa

    Full Text Available The cerebellum generates its vast amount of output to the cerebral cortex through the dentate nucleus (DN that is essential for precise limb movements in primates. Nuclear cells in DN generate burst activity prior to limb movement, and inactivation of DN results in cerebellar ataxia. The question is how DN cells become active under intensive inhibitory drive from Purkinje cells (PCs. There are two excitatory inputs to DN, mossy fiber and climbing fiber collaterals, but neither of them appears to have sufficient strength for generation of burst activity in DN. Therefore, we can assume two possible mechanisms: post-inhibitory rebound excitation and disinhibition. If rebound excitation works, phasic excitation of PCs and a concomitant inhibition of DN cells should precede the excitation of DN cells. On the other hand, if disinhibition plays a primary role, phasic suppression of PCs and activation of DN cells should be observed at the same timing. To examine these two hypotheses, we compared the activity patterns of PCs in the cerebrocerebellum and DN cells during step-tracking wrist movements in three Japanese monkeys. As a result, we found that the majority of wrist-movement-related PCs were suppressed prior to movement onset and the majority of wrist-movement-related DN cells showed concurrent burst activity without prior suppression. In a minority of PCs and DN cells, movement-related increases and decreases in activity, respectively, developed later. These activity patterns suggest that the initial burst activity in DN cells is generated by reduced inhibition from PCs, i.e., by disinhibition. Our results indicate that suppression of PCs, which has been considered secondary to facilitation, plays the primary role in generating outputs from DN. Our findings provide a new perspective on the mechanisms used by PCs to influence limb motor control and on the plastic changes that underlie motor learning in the cerebrocerebellum.

  2. Oligodendrocyte ablation affects the coordinated interaction between granule and Purkinje neurons during cerebellum development

    International Nuclear Information System (INIS)

    Collin, Ludovic; Doretto, Sandrine; Malerba, Monica; Ruat, Martial; Borrelli, Emiliana

    2007-01-01

    Oligodendrocytes (OLs) are the glial cells of the central nervous system (CNS) classically known to be devoted to the formation of myelin sheaths around most axons of the vertebrate brain. We have addressed the role of these cells during cerebellar development, by ablating OLs in vivo. Previous analyses had indicated that OL ablation during the first six postnatal days results into a striking cerebellar phenotype, whose major features are a strong reduction of granule neurons and aberrant Purkinje cells development. These two cell types are highly interconnected during cerebellar development through the production of molecules that help their proliferation, differentiation and maintenance. In this article, we present data showing that OL ablation has major effects on the physiology of Purkinje (PC) and granule cells (GC). In particular, OL ablation results into a reduction of sonic hedgehog (Shh), Brain Derived Neurotrophic Factor (BDNF), and Reelin (Rln) expression. These results indicate that absence of OLs profoundly alters the normal cerebellar developmental program

  3. Early increase and late decrease of purkinje cell dendritic spine density in prion-infected organotypic mouse cerebellar cultures.

    Science.gov (United States)

    Campeau, Jody L; Wu, Gengshu; Bell, John R; Rasmussen, Jay; Sim, Valerie L

    2013-01-01

    Prion diseases are infectious neurodegenerative diseases associated with the accumulation of protease-resistant prion protein, neuronal loss, spongiform change and astrogliosis. In the mouse model, the loss of dendritic spines is one of the earliest pathological changes observed in vivo, occurring 4-5 weeks after the first detection of protease-resistant prion protein in the brain. While there are cell culture models of prion infection, most do not recapitulate the neuropathology seen in vivo. Only the recently developed prion organotypic slice culture assay has been reported to undergo neuronal loss and the development of some aspects of prion pathology, namely small vacuolar degeneration and tubulovesicular bodies. Given the rapid replication of prions in this system, with protease-resistant prion protein detectable by 21 days, we investigated whether the dendritic spine loss and altered dendritic morphology seen in prion disease might also develop within the lifetime of this culture system. Indeed, six weeks after first detection of protease-resistant prion protein in tga20 mouse cerebellar slice cultures infected with RML prion strain, we found a statistically significant loss of Purkinje cell dendritic spines and altered dendritic morphology in infected cultures, analogous to that seen in vivo. In addition, we found a transient but statistically significant increase in Purkinje cell dendritic spine density during infection, at the time when protease-resistant prion protein was first detectable in culture. Our findings support the use of this slice culture system as one which recapitulates prion disease pathology and one which may facilitate study of the earliest stages of prion disease pathogenesis.

  4. Heterogeneity of Purkinje cell simple spike-complex spike interactions: zebrin- and non-zebrin-related variations.

    Science.gov (United States)

    Tang, Tianyu; Xiao, Jianqiang; Suh, Colleen Y; Burroughs, Amelia; Cerminara, Nadia L; Jia, Linjia; Marshall, Sarah P; Wise, Andrew K; Apps, Richard; Sugihara, Izumi; Lang, Eric J

    2017-08-01

    Cerebellar Purkinje cells (PCs) generate two types of action potentials, simple and complex spikes. Although they are generated by distinct mechanisms, interactions between the two spike types exist. Zebrin staining produces alternating positive and negative stripes of PCs across most of the cerebellar cortex. Thus, here we compared simple spike-complex spike interactions both within and across zebrin populations. Simple spike activity undergoes a complex modulation preceding and following a complex spike. The amplitudes of the pre- and post-complex spike modulation phases were correlated across PCs. On average, the modulation was larger for PCs in zebrin positive regions. Correlations between aspects of the complex spike waveform and simple spike activity were found, some of which varied between zebrin positive and negative PCs. The implications of the results are discussed with regard to hypotheses that complex spikes are triggered by rises in simple spike activity for either motor learning or homeostatic functions. Purkinje cells (PCs) generate two types of action potentials, called simple and complex spikes (SSs and CSs). We first investigated the CS-associated modulation of SS activity and its relationship to the zebrin status of the PC. The modulation pattern consisted of a pre-CS rise in SS activity, and then, following the CS, a pause, a rebound, and finally a late inhibition of SS activity for both zebrin positive (Z+) and negative (Z-) cells, though the amplitudes of the phases were larger in Z+ cells. Moreover, the amplitudes of the pre-CS rise with the late inhibitory phase of the modulation were correlated across PCs. In contrast, correlations between modulation phases across CSs of individual PCs were generally weak. Next, the relationship between CS spikelets and SS activity was investigated. The number of spikelets/CS correlated with the average SS firing rate only for Z+ cells. In contrast, correlations across CSs between spikelet numbers and the

  5. [Hering, Vintschgau and the problem of Purkinje's succession].

    Science.gov (United States)

    Sablik, K

    1989-01-01

    The problem of Jan Evangelista Purkinje's succession will be presented according to the results of archival research. The Ministery of Cult and Education in Vienna, and especially Karl Rokitansky, who was the adviser for medical education, in 1867 created a new professorship and Institute for Physiology, beside Purkinje and his Institute. Maximilian Vintschgau was to assist the world-famous 80 years old Purkinje but was not permitted to teach the whole field of physiology and to examine students. The fact that the professors of the Prague Medical Faculty in 1868 started to remove the restrictions for Vintschgau with the argument of academic freedom and in 1869 tried to keep the second institute for the future, is not yet mentioned in the literature. Discussions about the problems of the Czech language and its use in physiological lectures were scarcely mentioned by the Ministery: if one day there should be a Czech-speaking lecturer, the problem would be solved. Unfortunately Purkinje had no genuine pupil in Prague, and after his death, Vintschgau was provisional director of the Institute for half a year. In this situation Rokitansky decided that there should only be one institute for physiology in Prague. The Medical Faculty wanted to have Hermann Helmholtz to succeed Purkinje, but Helmholtz refused to come. Ewald Hering, who was nominated in the second place by the Faculty, accepted the call. Vintschgau had only rank four, third was Conrad Eckhard from Giessen. The Ministery in Vienna, however, made a special decision: The Medical Faculty of Innsbruck was founded in 1869, and there was not professor for physiology at the beginning of 1870. The candidates of the Insbruck Medical Faculty were neglected in favour of Vintschgau, who was considered to be a trustworthy Austrian patriot. Hering and Vintschgau became professors on March 6, 1870, and Hering started his work in Prague in a new institute in the "Wenzelsbad".

  6. Systematic Regional Variations in Purkinje Cell Spiking Patterns

    Science.gov (United States)

    Xiao, Jianqiang; Cerminara, Nadia L.; Kotsurovskyy, Yuriy; Aoki, Hanako; Burroughs, Amelia; Wise, Andrew K.; Luo, Yuanjun; Marshall, Sarah P.; Sugihara, Izumi; Apps, Richard; Lang, Eric J.

    2014-01-01

    In contrast to the uniform anatomy of the cerebellar cortex, molecular and physiological studies indicate that significant differences exist between cortical regions, suggesting that the spiking activity of Purkinje cells (PCs) in different regions could also show distinct characteristics. To investigate this possibility we obtained extracellular recordings from PCs in different zebrin bands in crus IIa and vermis lobules VIII and IX in anesthetized rats in order to compare PC firing characteristics between zebrin positive (Z+) and negative (Z−) bands. In addition, we analyzed recordings from PCs in the A2 and C1 zones of several lobules in the posterior lobe, which largely contain Z+ and Z− PCs, respectively. In both datasets significant differences in simple spike (SS) activity were observed between cortical regions. Specifically, Z− and C1 PCs had higher SS firing rates than Z+ and A2 PCs, respectively. The irregularity of SS firing (as assessed by measures of interspike interval distribution) was greater in Z+ bands in both absolute and relative terms. The results regarding systematic variations in complex spike (CS) activity were less consistent, suggesting that while real differences can exist, they may be sensitive to other factors than the cortical location of the PC. However, differences in the interactions between SSs and CSs, including the post-CS pause in SSs and post-pause modulation of SSs, were also consistently observed between bands. Similar, though less strong trends were observed in the zonal recordings. These systematic variations in spontaneous firing characteristics of PCs between zebrin bands in vivo, raises the possibility that fundamental differences in information encoding exist between cerebellar cortical regions. PMID:25144311

  7. Systematic regional variations in Purkinje cell spiking patterns.

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    Jianqiang Xiao

    Full Text Available In contrast to the uniform anatomy of the cerebellar cortex, molecular and physiological studies indicate that significant differences exist between cortical regions, suggesting that the spiking activity of Purkinje cells (PCs in different regions could also show distinct characteristics. To investigate this possibility we obtained extracellular recordings from PCs in different zebrin bands in crus IIa and vermis lobules VIII and IX in anesthetized rats in order to compare PC firing characteristics between zebrin positive (Z+ and negative (Z- bands. In addition, we analyzed recordings from PCs in the A2 and C1 zones of several lobules in the posterior lobe, which largely contain Z+ and Z- PCs, respectively. In both datasets significant differences in simple spike (SS activity were observed between cortical regions. Specifically, Z- and C1 PCs had higher SS firing rates than Z+ and A2 PCs, respectively. The irregularity of SS firing (as assessed by measures of interspike interval distribution was greater in Z+ bands in both absolute and relative terms. The results regarding systematic variations in complex spike (CS activity were less consistent, suggesting that while real differences can exist, they may be sensitive to other factors than the cortical location of the PC. However, differences in the interactions between SSs and CSs, including the post-CS pause in SSs and post-pause modulation of SSs, were also consistently observed between bands. Similar, though less strong trends were observed in the zonal recordings. These systematic variations in spontaneous firing characteristics of PCs between zebrin bands in vivo, raises the possibility that fundamental differences in information encoding exist between cerebellar cortical regions.

  8. Cerebellar Nuclear Neurons Use Time and Rate Coding to Transmit Purkinje Neuron Pauses.

    Science.gov (United States)

    Sudhakar, Shyam Kumar; Torben-Nielsen, Benjamin; De Schutter, Erik

    2015-12-01

    Neurons of the cerebellar nuclei convey the final output of the cerebellum to their targets in various parts of the brain. Within the cerebellum their direct upstream connections originate from inhibitory Purkinje neurons. Purkinje neurons have a complex firing pattern of regular spikes interrupted by intermittent pauses of variable length. How can the cerebellar nucleus process this complex input pattern? In this modeling study, we investigate different forms of Purkinje neuron simple spike pause synchrony and its influence on candidate coding strategies in the cerebellar nuclei. That is, we investigate how different alignments of synchronous pauses in synthetic Purkinje neuron spike trains affect either time-locking or rate-changes in the downstream nuclei. We find that Purkinje neuron synchrony is mainly represented by changes in the firing rate of cerebellar nuclei neurons. Pause beginning synchronization produced a unique effect on nuclei neuron firing, while the effect of pause ending and pause overlapping synchronization could not be distinguished from each other. Pause beginning synchronization produced better time-locking of nuclear neurons for short length pauses. We also characterize the effect of pause length and spike jitter on the nuclear neuron firing. Additionally, we find that the rate of rebound responses in nuclear neurons after a synchronous pause is controlled by the firing rate of Purkinje neurons preceding it.

  9. Cerebellar Nuclear Neurons Use Time and Rate Coding to Transmit Purkinje Neuron Pauses

    Science.gov (United States)

    Sudhakar, Shyam Kumar; Torben-Nielsen, Benjamin; De Schutter, Erik

    2015-01-01

    Neurons of the cerebellar nuclei convey the final output of the cerebellum to their targets in various parts of the brain. Within the cerebellum their direct upstream connections originate from inhibitory Purkinje neurons. Purkinje neurons have a complex firing pattern of regular spikes interrupted by intermittent pauses of variable length. How can the cerebellar nucleus process this complex input pattern? In this modeling study, we investigate different forms of Purkinje neuron simple spike pause synchrony and its influence on candidate coding strategies in the cerebellar nuclei. That is, we investigate how different alignments of synchronous pauses in synthetic Purkinje neuron spike trains affect either time-locking or rate-changes in the downstream nuclei. We find that Purkinje neuron synchrony is mainly represented by changes in the firing rate of cerebellar nuclei neurons. Pause beginning synchronization produced a unique effect on nuclei neuron firing, while the effect of pause ending and pause overlapping synchronization could not be distinguished from each other. Pause beginning synchronization produced better time-locking of nuclear neurons for short length pauses. We also characterize the effect of pause length and spike jitter on the nuclear neuron firing. Additionally, we find that the rate of rebound responses in nuclear neurons after a synchronous pause is controlled by the firing rate of Purkinje neurons preceding it. PMID:26630202

  10. Effect of gabazine on sensory stimulation train evoked response in mouse cerebellar Purkinje cells.

    Science.gov (United States)

    Bing, Yan-Hua; Jin, Wen-Zhe; Sun, Lei; Chu, Chun-Ping; Qiu, De-Lai

    2015-02-01

    Cerebellar Purkinje cells (PCs) respond to sensory stimulation via climbing fiber and mossy fiber-granule cell pathways, and generate motor-related outputs according to internal rules of integration and computation. However, the dynamic properties of sensory information processed by PC in mouse cerebellar cortex are currently unclear. In the present study, we examined the effects of the gamma-aminobutyric acid receptor A (GABA(A)) antagonist, gabazine, on the stimulation train on the simple spike firing of PCs by electrophysiological recordings method. Our data showed that the output of cerebellar PCs could be significantly affected by all pulses of the low-frequency (0.25 -2 Hz) sensory stimulation train, but only by the 1st and 2nd pulses of the high-frequency (≥ 4 Hz) sensory stimulation train. In the presence of gabazine (20 μM), each pulse of 1 Hz facial stimulation evoked simple spike firing in the PCs, but only the 1st and 2nd pulses of 4 Hz stimulation induced an increase in simple spike firing of the PCs. These results indicated that GABAA receptor-mediated inhibition did not significantly affect the frequency properties of sensory stimulation evoked responses in the mouse cerebellar PCs.

  11. Maturation of Cerebellar Purkinje Cell Population Activity during Postnatal Refinement of Climbing Fiber Network

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    Jean-Marc Good

    2017-11-01

    Full Text Available Neural circuits undergo massive refinements during postnatal development. In the developing cerebellum, the climbing fiber (CF to Purkinje cell (PC network is drastically reshaped by eliminating early-formed redundant CF to PC synapses. To investigate the impact of CF network refinement on PC population activity during postnatal development, we monitored spontaneous CF responses in neighboring PCs and the activity of populations of nearby CF terminals using in vivo two-photon calcium imaging. Population activity is highly synchronized in newborn mice, and the degree of synchrony gradually declines during the first postnatal week in PCs and, to a lesser extent, in CF terminals. Knockout mice lacking P/Q-type voltage-gated calcium channel or glutamate receptor δ2, in which CF network refinement is severely impaired, exhibit an abnormally high level of synchrony in PC population activity. These results suggest that CF network refinement is a structural basis for developmental desynchronization and maturation of PC population activity.

  12. Administration of memantine during ethanol withdrawal in neonatal rats: effects on long-term ethanol-induced motor incoordination and cerebellar Purkinje cell loss.

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    Idrus, Nirelia M; McGough, Nancy N H; Riley, Edward P; Thomas, Jennifer D

    2011-02-01

    Alcohol consumption during pregnancy can damage the developing fetus, illustrated by central nervous system dysfunction and deficits in motor and cognitive abilities. Binge drinking has been associated with an increased risk of fetal alcohol spectrum disorders, likely due to increased episodes of ethanol withdrawal. We hypothesized that overactivity of the N-methyl-D-aspartate (NMDA) receptor during ethanol withdrawal leads to excitotoxic cell death in the developing brain. Consistent with this, administration of NMDA receptor antagonists (e.g., MK-801) during withdrawal can attenuate ethanol's teratogenic effects. The aim of this study was to determine whether administration of memantine, an NMDA receptor antagonist, during ethanol withdrawal could effectively attenuate ethanol-related deficits, without the adverse side effects associated with other NMDA receptor antagonists. Sprague-Dawley pups were exposed to 6.0 g/kg ethanol or isocaloric maltose solution via intubation on postnatal day 6, a period of brain development equivalent to a portion of the 3rd trimester. Twenty-four and 36 hours after ethanol, subjects were injected with 0, 10, or 15 mg/kg memantine, totaling doses of 0, 20, or 30 mg/kg. Motor coordination was tested on a parallel bar task and the total number of cerebellar Purkinje cells was estimated using unbiased stereology. Alcohol exposure induced significant parallel bar motor incoordination and reduced Purkinje cell number. Memantine administration significantly attenuated both ethanol-associated motor deficits and cerebellar cell loss in a dose-dependent manner. Memantine was neuroprotective when administered during ethanol withdrawal. These data provide further support that ethanol withdrawal contributes to fetal alcohol spectrum disorders. Copyright © 2010 by the Research Society on Alcoholism.

  13. Effects of gadolinium-based contrast agents on thyroid hormone receptor action and thyroid hormone-induced cerebellar Purkinje cell morphogenesis

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    Noriyuki Koibuchi

    2016-08-01

    Full Text Available Gadolinium (Gd-based contrast agents (GBCAs are used in diagnostic imaging to enhance the quality of magnetic resonance imaging or angiography. After intravenous injection, GBCAs can accumulate in the brain. Thyroid hormones (THs are critical to the development and functional maintenance of the central nervous system. TH actions in brain are mainly exerted through nuclear TH receptors (TRs. We examined the effects of GBCAs on TR-mediated transcription in CV-1 cells using transient transfection-based reporter assay and thyroid hormone-mediated cerebellar Purkinje cell morphogenesis in primary culture. We also measured the cellular accumulation and viability of Gd after representative GBCA treatments in cultured CV-1 cells. Both linear (Gd-diethylene triamine pentaacetic acid-bis methyl acid, Gd-DTPA-BMA and macrocyclic (Gd-tetraazacyclododecane tetraacetic acid, Gd-DOTA GBCAs were accumulated without inducing cell death in CV-1 cells. In contrast, Gd chloride (GdCl3 treatment induced approximately 100 times higher Gd accumulation and significantly reduced the number of cells. Low doses of Gd-DTPA-BMA (10−8–10−6 M augmented TR-mediated transcription, but the transcription was suppressed at higher dose (10−5 – 10−4 M, with decreased β-galactosidase activity indicating cellular toxicity. TR-mediated transcription was not altered by Gd-DOTA or GdCl3, but the latter induced a significant reduction in β-galactosidase activity at high doses, indicating cellular toxicity. In cerebellar cultures, the dendrite arborization of Purkinje cells induced by 10-9 M T4 was augmented by low-dose Gd-DTPA-BMA (10−7 M but was suppressed by higher dose (10−5 M. Such augmentation by low-dose Gd-DTPA-BMA was not observed with 10-9 M T3, probably because of the greater dendrite arborization by T3; however, the arborization by T3 was suppressed by a higher dose of Gd-DTPA-BMA (10-5 M as seen in T4 treatment. The effect of Gd-DOTA on dendrite arborization

  14. Caspase-mediated apoptosis induction in zebrafish cerebellar Purkinje neurons.

    Science.gov (United States)

    Weber, Thomas; Namikawa, Kazuhiko; Winter, Barbara; Müller-Brown, Karina; Kühn, Ralf; Wurst, Wolfgang; Köster, Reinhard W

    2016-11-15

    The zebrafish is a well-established model organism in which to study in vivo mechanisms of cell communication, differentiation and function. Existing cell ablation methods are either invasive or they rely on the cellular expression of prokaryotic enzymes and the use of antibiotic drugs as cell death-inducing compounds. We have recently established a novel inducible genetic cell ablation system based on tamoxifen-inducible Caspase 8 activity, thereby exploiting mechanisms of cell death intrinsic to most cell types. Here, we prove its suitability in vivo by monitoring the ablation of cerebellar Purkinje cells (PCs) in transgenic zebrafish that co-express the inducible caspase and a fluorescent reporter. Incubation of larvae in tamoxifen for 8 h activated endogenous Caspase 3 and cell death, whereas incubation for 16 h led to the near-complete loss of PCs by apoptosis. We observed synchronous cell death autonomous to the PC population and phagocytosing microglia in the cerebellum, reminiscent of developmental apoptosis in the forebrain. Thus, induction of apoptosis through targeted activation of caspase by tamoxifen (ATTAC TM ) further expands the repertoire of genetic tools for conditional interrogation of cellular functions. © 2016. Published by The Company of Biologists Ltd.

  15. Intraocular lens alignment from purkinje and Scheimpflug imaging.

    Science.gov (United States)

    Rosales, Patricia; De Castro, Alberto; Jiménez-Alfaro, Ignacio; Marcos, Susana

    2010-11-01

    The improved designs of intraocular lenses (IOLs) implanted during cataract surgery demand understanding of the possible effects of lens misalignment on optical performance. In this review, we describe the implementation, set-up and validation of two methods to measure in vivo tilt and decentration of IOLs, one based on Purkinje imaging and the other on Scheimpflug imaging. The Purkinje system images the reflections of an oblique collimated light source on the anterior cornea and anterior and posterior IOL surfaces and relies on the well supported assumption of the linearity of the Purkinje images with respect to IOL tilt and decentration. Scheimpflug imaging requires geometrical distortion correction and image processing techniques to retrieve the pupillary axis, IOL axis and pupil centre from the three-dimensional anterior segment image of the eye. Validation of the techniques using a physical eye model indicates that IOL tilt is estimated within an accuracy of 0.261 degree and decentration within 0.161 mm. Measurements on patients implanted with aspheric IOLs indicate that IOL tilt and decentration tend to be mirror symmetric between left and right eyes. The average tilt was 1.54 degrees and the average decentration was 0.21 mm. Simulated aberration patterns using custom models of the patients eyes, built using anatomical data of the anterior cornea and foveal position, the IOL geometry and the measured IOL tilt and decentration predict the experimental wave aberrations measured using laser ray tracing aberrometry on the same eyes. This reveals a relatively minor contribution of IOL tilt and decentration on the higher-order aberrations of the normal pseudophakic eye.

  16. Motor learning induces plastic changes in Purkinje cell dendritic spines in the rat cerebellum.

    Science.gov (United States)

    González-Tapia, D; González-Ramírez, M M; Vázquez-Hernández, N; González-Burgos, I

    2017-12-14

    The paramedian lobule of the cerebellum is involved in learning to correctly perform motor skills through practice. Dendritic spines are dynamic structures that regulate excitatory synaptic stimulation. We studied plastic changes occurring in the dendritic spines of Purkinje cells from the paramedian lobule of rats during motor learning. Adult male rats were trained over a 6-day period using an acrobatic motor learning paradigm; the density and type of dendritic spines were determined every day during the study period using a modified version of the Golgi method. The learning curve reflected a considerable decrease in the number of errors made by rats as the training period progressed. We observed more dendritic spines on days 2 and 6, particularly more thin spines on days 1, 3, and 6, fewer mushroom spines on day 3, fewer stubby spines on day 1, and more thick spines on days 4 and 6. The initial stage of motor learning may be associated with fast processing of the underlying synaptic information combined with an apparent "silencing" of memory consolidation processes, based on the regulation of the neuronal excitability. Copyright © 2017 Sociedad Española de Neurología. Publicado por Elsevier España, S.L.U. All rights reserved.

  17. A coupled 3D-1D numerical monodomain solver for cardiac electrical activation in the myocardium with detailed Purkinje network

    Science.gov (United States)

    Vergara, Christian; Lange, Matthias; Palamara, Simone; Lassila, Toni; Frangi, Alejandro F.; Quarteroni, Alfio

    2016-03-01

    We present a model for the electrophysiology in the heart to handle the electrical propagation through the Purkinje system and in the myocardium, with two-way coupling at the Purkinje-muscle junctions. In both the subproblems the monodomain model is considered, whereas at the junctions a resistor element is included that induces an orthodromic propagation delay from the Purkinje network towards the heart muscle. We prove a sufficient condition for convergence of a fixed-point iterative algorithm to the numerical solution of the coupled problem. Numerical comparison of activation patterns is made with two different combinations of models for the coupled Purkinje network/myocardium system, the eikonal/eikonal and the monodomain/monodomain models. Test cases are investigated for both physiological and pathological activation of a model left ventricle. Finally, we prove the reliability of the monodomain/monodomain coupling on a realistic scenario. Our results underlie the importance of using physiologically realistic Purkinje-trees with propagation solved using the monodomain model for simulating cardiac activation.

  18. Selective loss of Purkinje cells in a patient with anti-gliadin-antibody-positive autoimmune cerebellar ataxia

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    Hasegawa Akira

    2011-02-01

    Full Text Available Abstract The patient was an 84-year-old woman who had the onset of truncal ataxia at age 77 and a history of Basedow's disease. Her ataxic gait gradually deteriorated. She could not walk without support at age 81 and she was admitted to our hospital at age 83. Gaze-evoked nystagmus and dysarthria were observed. Mild ataxia was observed in all limbs. Her deep tendon reflex and sense of position were normal. IgA anti-gliadin antibody, IgG anti-gliadin antibody, anti-SS-A/Ro antibody, anti-SS-B/La antibody and anti-TPO antibody were positive. A conventional brain MRI did not show obvious cerebellar atrophy. However, MRI voxel based morphometry (VBM and SPECT-eZIS revealed cortical cerebellar atrophy and reduced cerebellar blood flow. IVIg treatment was performed and was moderately effective. After her death at age 85, the patient was autopsied. Neuropathological findings were as follows: selective loss of Purkinje cells; no apparent degenerative change in the efferent pathways, such as the dentate nuclei or vestibular nuclei; no prominent inflammatory reaction. From these findings, we diagnosed this case as autoimmune cerebellar atrophy associated with gluten ataxia. All 3 autopsies previously reported on gluten ataxia have noted infiltration of inflammatory cells in the cerebellum. In this case, we postulated that the infiltration of inflammatory cells was not found because the patient's condition was based on humoral immunity. The clinical conditions of gluten ataxia have not yet been properly elucidated, but are expected to be revealed as the number of autopsied cases increases.

  19. Purkinje Cell Signaling Deficits in Animal Models of Ataxia

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    Eriola Hoxha

    2018-04-01

    Full Text Available Purkinje cell (PC dysfunction or degeneration is the most frequent finding in animal models with ataxic symptoms. Mutations affecting intrinsic membrane properties can lead to ataxia by altering the firing rate of PCs or their firing pattern. However, the relationship between specific firing alterations and motor symptoms is not yet clear, and in some cases PC dysfunction precedes the onset of ataxic signs. Moreover, a great variety of ionic and synaptic mechanisms can affect PC signaling, resulting in different features of motor dysfunction. Mutations affecting Na+ channels (NaV1.1, NaV1.6, NaVβ4, Fgf14 or Rer1 reduce the firing rate of PCs, mainly via an impairment of the Na+ resurgent current. Mutations that reduce Kv3 currents limit the firing rate frequency range. Mutations of Kv1 channels act mainly on inhibitory interneurons, generating excessive GABAergic signaling onto PCs, resulting in episodic ataxia. Kv4.3 mutations are responsible for a complex syndrome with several neurologic dysfunctions including ataxia. Mutations of either Cav or BK channels have similar consequences, consisting in a disruption of the firing pattern of PCs, with loss of precision, leading to ataxia. Another category of pathogenic mechanisms of ataxia regards alterations of synaptic signals arriving at the PC. At the parallel fiber (PF-PC synapse, mutations of glutamate delta-2 (GluD2 or its ligand Crbl1 are responsible for the loss of synaptic contacts, abolishment of long-term depression (LTD and motor deficits. At the same synapse, a correct function of metabotropic glutamate receptor 1 (mGlu1 receptors is necessary to avoid ataxia. Failure of climbing fiber (CF maturation and establishment of PC mono-innervation occurs in a great number of mutant mice, including mGlu1 and its transduction pathway, GluD2, semaphorins and their receptors. All these models have in common the alteration of PC output signals, due to a variety of mechanisms affecting incoming

  20. Climbing fiber-Purkinje cell synaptic pathology in tremor and cerebellar degenerative diseases

    Science.gov (United States)

    Lin, Chi-Ying; Wang, Jie; Sims, Peter A.; Pan, Ming-Kai; Liou, Jyun-you; Lee, Danielle; Tate, William J.; Kelly, Geoffrey C.; Louis, Elan D.; Faust, Phyllis L.

    2017-01-01

    Changes in climbing fiber-Purkinje cell (CF-PC) synaptic connections have been found in the essential tremor (ET) cerebellum, and these changes are correlated with tremor severity. Whether these postmortem changes are specific to ET remains to be investigated. We assessed CF-PC synaptic pathology in the postmortem cerebellum across a range of degenerative movement disorders [10 Parkinson’s disease (PD) cases, 10 multiple system atrophy (MSA) cases, 10 spinocerebellar ataxia type 1 (SCA1) cases, and 20 ET cases] and 25 controls. We observed differences in terms of CF pathological features across these disorders. Specifically, PD cases and ET cases both had more CFs extending into the parallel fiber (PF) territory, but ET cases had more complex branching and increased length of CFs in the PF territory along with decreased CF synaptic density compared to PD cases. MSA cases and SCA1 cases had the most severely reduced CF synaptic density and a marked paucity of CFs extending into the PF territory. Furthermore, CFs in a subset of MSA cases formed collateral branches parallel to the PC layer, a feature not seen in other diagnostic groups. Using unsupervised cluster analysis, the cases and controls could all be categorized into four clusters based on the CF pathology and features of PC pathology, including counts of PCs and their axonal torpedoes. ET cases and PD cases co-segregated into two clusters, whereas SCA1 cases and MSA cases formed another cluster, separate from the control cluster. Interestingly, the presence of resting tremor seemed to be the clinical feature that separated the cases into the two ET-PD clusters. In conclusion, our study demonstrates that these degenerative movement disorders seem to differ with respect to the pattern of CF synaptic pathology they exhibit. It remains to be determined how these differences contribute to the clinical presentations of these diseases. PMID:27704282

  1. Calcium microdomains near R-type calcium channels control the induction of presynaptic LTP at parallel fiber to Purkinje cell synapses

    Science.gov (United States)

    Myoga, Michael H.; Regehr, Wade G.

    2011-01-01

    R-type calcium channels in postsynaptic spines signal through functional calcium microdomains to regulate a calcium-calmodulin sensitive potassium channel that in turn regulates postsynaptic hippocampal LTP. Here we ask whether R-type calcium channels in presynaptic terminals also signal through calcium microdomains to control presynaptic LTP. We focus on presynaptic LTP at parallel fiber to Purkinje cell synapses in the cerebellum (PF-LTP), which is mediated by calcium/calmodulin-stimulated adenylyl cyclases. Although most presynaptic calcium influx is through N-type and P/Q-type calcium channels, blocking these channels does not disrupt PF-LTP, but blocking R-type calcium channels does. Moreover, global calcium signaling cannot account for the calcium dependence of PF-LTP because R-type channels contribute modestly to overall calcium entry. These findings indicate that within presynaptic terminals, R-type calcium channels produce calcium microdomains that evoke presynaptic LTP at moderate frequencies that do not greatly increase global calcium levels,. PMID:21471358

  2. Rates of incorporation of radioactive molecules during the cell cycle

    International Nuclear Information System (INIS)

    Gray, J.W.; Pallavicini, M.G.; George, Y.S.; Groppi, V.; Look, M.; Dean, P.N.

    1981-01-01

    We report measurements of the incorporation of radioactive molecules during short labeling periods, as a function of cell-cycle stage, using a cell-sorter-based technique that does not require cell synchronization. We have determined: (1) tritiated thymidine ( 3 H-TdR) incorporation throughout S-phase in Lewis lung tumor cells in vitro both before and after treatment with cytosine arabinoside; (2) 3 H-TdR incorporation throughout S-phase in KHT tumor cells in vitro and in vivo; (3) 3 H-TdR incorporation throughout S-phase in Chinese hamster ovary cells and compared it with DNA synthesis throughout S-phase; (4) a mathematical expression describing 3 H-TdR incorporation throughout S-phase in Chinese hamster M3-1 cells; and (5) the simultaneous incorporation of 3 H-TdR and 35 S-methionine as they are related to cell size and DNA content in S49 mouse lymphoma cells. In asynchronously growing cells in vitro and in vivo, 3 H-TdR incorporation was generally low in early and late S-phase and highest in mid-S-phase. However, in Lewis lung tumor cells treated with cytosine arabinoside 3 H-TdR incorporation was highest in early and late S-phase and lowest in mid-S-phase. Incorporation of 35 S-methionine increased continuously with cell size and DNA content. Incorporation of 3 H-TdR in CHO cells was proportional to DNA synthesis

  3. [Computer-assisted measurement of ocular misalignment in infants and young children using the digital Purkinje reflection pattern procedure].

    Science.gov (United States)

    Barry, J C; Effert, R; Kaupp, A; Kleine, M; Reim, M

    1994-02-01

    A digital image recording and processing system is presented that allows a quick diagnosis of microstrabismus in non-cooperative children. It is thus particularly suited for screening purposes. The Purkinje Reflection Pattern Evaluation (RPE) method is used: three small flashes are used to produce the desired Purkinje images. Two horizontal rows of the three 1st Purkinje images (anterior corneal reflections) and of the three 4th Purkinje images (posterior crystalline lens reflections) stemming from the three light sources form the characteristic Purkinje image reflection pattern. Each eye's position is calculated from the shift between the upper and lower rows of reflections by means of two simple formulae. From the angles obtained in binocular fixation and monocular fixation the manifest angle of strabismus corresponding to the angle measured in the simultaneous prism-and-cover test is computed. The measurement is performed at a fixation distance of 50 cm under natural viewing conditions. To obtain a picture one only has to get the child's attention for a short moment. The primary position is triggered with the fixation light, which is operated by a switch. The digital image recording is done with a hand-held device comprising two miniaturized video cameras, three photo flashes and a fixation light that is operated manually. An IBM-compatible PC equipped with a hard disk and two frame grabbers was adapted for the storage and processing of the pictures. The pictures are evaluated interactively in a few minutes on the workstation's monitor immediately after the measurement. To this end specially designed menu-driven software was implemented. Examples of the measuring procedure and clinical results in infants with microtropic highlight the potential of the system as a screening apparatus and for the exact measurement of small and large squint angles. Usually even 1-year-old children can cooperate well enough to get good-quality pictures in binocular fixation. The new

  4. Differential association of GABAB receptors with their effector ion channels in Purkinje cells.

    Science.gov (United States)

    Luján, Rafael; Aguado, Carolina; Ciruela, Francisco; Cózar, Javier; Kleindienst, David; de la Ossa, Luis; Bettler, Bernhard; Wickman, Kevin; Watanabe, Masahiko; Shigemoto, Ryuichi; Fukazawa, Yugo

    2018-04-01

    Metabotropic GABA B receptors mediate slow inhibitory effects presynaptically and postsynaptically through the modulation of different effector signalling pathways. Here, we analysed the distribution of GABA B receptors using highly sensitive SDS-digested freeze-fracture replica labelling in mouse cerebellar Purkinje cells. Immunoreactivity for GABA B1 was observed on presynaptic and, more abundantly, on postsynaptic compartments, showing both scattered and clustered distribution patterns. Quantitative analysis of immunoparticles revealed a somato-dendritic gradient, with the density of immunoparticles increasing 26-fold from somata to dendritic spines. To understand the spatial relationship of GABA B receptors with two key effector ion channels, the G protein-gated inwardly rectifying K + (GIRK/Kir3) channel and the voltage-dependent Ca 2+ channel, biochemical and immunohistochemical approaches were performed. Co-immunoprecipitation analysis demonstrated that GABA B receptors co-assembled with GIRK and Ca V 2.1 channels in the cerebellum. Using double-labelling immunoelectron microscopic techniques, co-clustering between GABA B1 and GIRK2 was detected in dendritic spines, whereas they were mainly segregated in the dendritic shafts. In contrast, co-clustering of GABA B1 and Ca V 2.1 was detected in dendritic shafts but not spines. Presynaptically, although no significant co-clustering of GABA B1 and GIRK2 or Ca V 2.1 channels was detected, inter-cluster distance for GABA B1 and GIRK2 was significantly smaller in the active zone than in the dendritic shafts, and that for GABA B1 and Ca V 2.1 was significantly smaller in the active zone than in the dendritic shafts and spines. Thus, GABA B receptors are associated with GIRK and Ca V 2.1 channels in different subcellular compartments. These data provide a better framework for understanding the different roles played by GABA B receptors and their effector ion channels in the cerebellar network.

  5. Plasticity of Cerebellar Purkinje Cells in Behavioral Training of Body Balance Control

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    Ray X. Lee

    2015-08-01

    Full Text Available Neural responses to sensory inputs caused by self-generated movements (reafference and external passive stimulation (exafference differ in various brain regions. The ability to differentiate such sensory information can lead to movement execution with better accuracy. However, how sensory responses are adjusted in regard to this distinguishability during motor learning is still poorly understood. The cerebellum has been hypothesized to analyze the functional significance of sensory information during motor learning, and is thought to be a key region of reafference computation in the vestibular system. In this study, we investigated Purkinje cell (PC spike trains as cerebellar cortical output when rats learned to balance on a suspended dowel. Rats progressively reduced the amplitude of body swing and made fewer foot slips during a 5-min balancing task. Both PC simple (SSs; 17 of 26 and complex spikes (CSs; 7 of 12 were found to code initially on the angle of the heads with respect to a fixed reference. Using periods with comparable degrees of movement, we found that such SS coding of information in most PCs (10 of 17 decreased rapidly during balance learning. In response to unexpected perturbations and under anesthesia, SS coding capability of these PCs recovered. By plotting SS and CS firing frequencies over 15-s time windows in double-logarithmic plots, a negative correlation between SS and CS was found in awake, but not anesthetized, rats. PCs with prominent SS coding attenuation during motor learning showed weaker SS-CS correlation. Hence, we demonstrate that neural plasticity for filtering out sensory reafference from active motion occurs in the cerebellar cortex in rats during balance learning. SS-CS interaction may contribute to this rapid plasticity as a form of receptive field plasticity in the cerebellar cortex between two receptive maps of sensory inputs from the external world and of efference copies from the will center for

  6. Antioxidant supplementation upregulates calbindin expression in cerebellar Purkinje cells of rat pups subjected to post natal exposure to sodium arsenite.

    Science.gov (United States)

    Dhar, Pushpa; Kaushal, Parul; Kumar, Pavan

    2018-07-01

    Optimal cytoplasmic calcium (Ca 2+ ) levels have been associated with adequate cell functioning and neuronal survival. Altered intracellular Ca 2+ levels following impaired Ca 2+ homeostasis could induce neuronal degeneration or even cell death. There are reports of arsenite induced oxidative stress and the associated disturbances in intracellular calcium homeostasis. The present study focused on determining the strategies that would modulate tissue redox status and calcium binding protein (CaBP) (Calbindin D28k-CB) expression affected adversely by sodium arsenite (NaAsO 2 ) exposure (postnatal) of rat pups. NaAsO 2 alone or along with antioxidants (AOXs) (alpha lipoic acid or curcumin) was administered by intraperitoneal (i.p.) route from postnatal day (PND) 1-21 (covering rapid brain growth period - RBGP) to experimental groups and animals receiving sterile water by the same route served as the controls. At the end of the experimental period, the animals were subjected to euthanasia and the cerebellar tissue obtained therefrom was processed for immunohistochemical localization and western blot analysis of CB protein. CB was diffusely expressed in cell body as well as dendritic processes of Purkinje cells (PCs) along the PC Layer (PCL) in all cerebellar folia of the control and the experimental animals. The multilayered pattern of CB +ve cells along with their downregulated expression and low packing density was significantly evident in the arsenic (iAs) alone exposed group as against the controls and AOX supplemented groups. The observations are suggestive of AOX induced restoration of CaBP expression in rat cerebellum following early postnatal exposure to NaAsO 2 . Copyright © 2018 Elsevier B.V. All rights reserved.

  7. Synaptic responses evoked by tactile stimuli in Purkinje cells in mouse cerebellar cortex Crus II in vivo.

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    Chun-Ping Chu

    Full Text Available Sensory stimuli evoke responses in cerebellar Purkinje cells (PCs via the mossy fiber-granule cell pathway. However, the properties of synaptic responses evoked by tactile stimulation in cerebellar PCs are unknown. The present study investigated the synaptic responses of PCs in response to an air-puff stimulation on the ipsilateral whisker pad in urethane-anesthetized mice.Thirty-three PCs were recorded from 48 urethane-anesthetized adult (6-8-week-old HA/ICR mice by somatic or dendritic patch-clamp recording and pharmacological methods. Tactile stimulation to the ipsilateral whisker pad was delivered by an air-puff through a 12-gauge stainless steel tube connected with a pressurized injection system. Under current-clamp conditions (I = 0, the air-puff stimulation evoked strong inhibitory postsynaptic potentials (IPSPs in the somata of PCs. Application of SR95531, a specific GABA(A receptor antagonist, blocked IPSPs and revealed stimulation-evoked simple spike firing. Under voltage-clamp conditions, tactile stimulation evoked a sequence of transient inward currents followed by strong outward currents in the somata and dendrites in PCs. Application of SR95531 blocked outward currents and revealed excitatory postsynaptic currents (EPSCs in somata and a temporal summation of parallel fiber EPSCs in PC dendrites. We also demonstrated that PCs respond to both the onset and offset of the air-puff stimulation.These findings indicated that tactile stimulation induced asynchronous parallel fiber excitatory inputs onto the dendrites of PCs, and failed to evoke strong EPSCs and spike firing in PCs, but induced the rapid activation of strong GABA(A receptor-mediated inhibitory postsynaptic currents in the somata and dendrites of PCs in the cerebellar cortex Crus II in urethane-anesthetized mice.

  8. Incorporation of mammalian actin into microfilaments in plant cell nucleus

    Directory of Open Access Journals (Sweden)

    Paves Heiti

    2004-04-01

    Full Text Available Abstract Background Actin is an ancient molecule that shows more than 90% amino acid homology between mammalian and plant actins. The regions of the actin molecule that are involved in F-actin assembly are largely conserved, and it is likely that mammalian actin is able to incorporate into microfilaments in plant cells but there is no experimental evidence until now. Results Visualization of microfilaments in onion bulb scale epidermis cells by different techniques revealed that rhodamine-phalloidin stained F-actin besides cytoplasm also in the nuclei whereas GFP-mouse talin hybrid protein did not enter the nuclei. Microinjection of fluorescently labeled actin was applied to study the presence of nuclear microfilaments in plant cells. Ratio imaging of injected fluorescent rabbit skeletal muscle actin and phalloidin staining of the microinjected cells showed that mammalian actin was able to incorporate into plant F-actin. The incorporation occurred preferentially in the nucleus and in the perinuclear region of plant cells whereas part of plant microfilaments, mostly in the periphery of cytoplasm, did not incorporate mammalian actin. Conclusions Microinjected mammalian actin is able to enter plant cell's nucleus, whereas incorporation of mammalian actin into plant F-actin occurs preferentially in the nucleus and perinuclear area.

  9. Absence of Rapid Propagation through the Purkinje Network as a Potential Cause of Line Block in the Human Heart with Left Bundle Branch Block.

    Science.gov (United States)

    Okada, Jun-Ichi; Washio, Takumi; Nakagawa, Machiko; Watanabe, Masahiro; Kadooka, Yoshimasa; Kariya, Taro; Yamashita, Hiroshi; Yamada, Yoko; Momomura, Shin-Ichi; Nagai, Ryozo; Hisada, Toshiaki; Sugiura, Seiryo

    2018-01-01

    Background: Cardiac resynchronization therapy is an effective device therapy for heart failure patients with conduction block. However, a problem with this invasive technique is the nearly 30% of non-responders. A number of studies have reported a functional line of block of cardiac excitation propagation in responders. However, this can only be detected using non-contact endocardial mapping. Further, although the line of block is considered a sign of responders to therapy, the mechanism remains unclear. Methods: Herein, we created two patient-specific heart models with conduction block and simulated the propagation of excitation based on a cellmodel of electrophysiology. In one model with a relatively narrow QRS width (176 ms), we modeled the Purkinje network using a thin endocardial layer with rapid conduction. To reproduce a wider QRS complex (200 ms) in the second model, we eliminated the Purkinje network, and we simulated the endocardial mapping by solving the inverse problem according to the actual mapping system. Results: We successfully observed the line of block using non-contact mapping in the model without the rapid propagation of excitation through the Purkinje network, although the excitation in the wall propagated smoothly. This model of slow conduction also reproduced the characteristic properties of the line of block, including dense isochronal lines and fractionated local electrocardiograms. Further, simulation of ventricular pacing from the lateral wall shifted the location of the line of block. By contrast, in the model with the Purkinje network, propagation of excitation in the endocardial map faithfully followed the actual propagation in the wall, without showing the line of block. Finally, switching the mode of propagation between the two models completely reversed these findings. Conclusions: Our simulation data suggest that the absence of rapid propagation of excitation through the Purkinje network is the major cause of the functional line

  10. Calbindin-D28k is a more reliable marker of human Purkinje cells than standard Nissl stains: a stereological experiment.

    Science.gov (United States)

    Whitney, Elizabeth R; Kemper, Thomas L; Rosene, Douglas L; Bauman, Margaret L; Blatt, Gene J

    2008-02-15

    In a study of human Purkinje cell (PC) number, a striking mismatch between the number of PCs observed with the Nissl stain and the number of PCs immunopositive for calbindin-D28k (CB) was identified in 2 of the 10 brains examined. In the remaining eight brains this mismatch was not observed. Further, in these eight brains, analysis of CB immunostained sections counterstained with the Nissl stain revealed that more than 99% Nissl stained PCs were also immunopositive for CB. In contrast, in the two discordant brains, only 10-20% of CB immunopositive PCs were also identified with the Nissl stain. Although this finding was unexpected, a historical survey of the literature revealed that Spielmeyer [Spielmeyer W. Histopathologie des nervensystems. Julius Springer: Berlin; 1922. p. 56-79] described human cases with PCs that lacked the expected Nissl staining intensity, an important historical finding and critical issue when studying postmortem human brains. The reason for this failure in Nissl staining is not entirely clear, but it may result from premortem circumstances since it is not accounted for by postmortem delay or processing variables. Regardless of the exact cause, these observations suggest that Nissl staining may not be a reliable marker for PCs and that CB is an excellent alternative marker.

  11. Chemical ablation of the Purkinje system causes early termination and activation rate slowing of long-duration ventricular fibrillation in dogs.

    Science.gov (United States)

    Dosdall, Derek J; Tabereaux, Paul B; Kim, Jong J; Walcott, Gregory P; Rogers, Jack M; Killingsworth, Cheryl R; Huang, Jian; Robertson, Peter G; Smith, William M; Ideker, Raymond E

    2008-08-01

    Endocardial mapping has suggested that Purkinje fibers may play a role in the maintenance of long-duration ventricular fibrillation (LDVF). To determine the influence of Purkinje fibers on LDVF, we chemically ablated the Purkinje system with Lugol solution and recorded endocardial and transmural activation during LDVF. Dog hearts were isolated and perfused, and the ventricular endocardium was exposed and treated with Lugol solution (n = 6) or normal Tyrode solution as a control (n = 6). The left anterior papillary muscle endocardium was mapped with a 504-electrode (21 x 24) plaque with electrodes spaced 1 mm apart. Transmural activation was recorded with a six-electrode plunge needle on each side of the plaque. Ventricular fibrillation (VF) was induced, and perfusion was halted. LDVF spontaneously terminated sooner in Lugol-ablated hearts than in control hearts (4.9 +/- 1.5 vs. 9.2 +/- 3.2 min, P = 0.01). After termination of VF, both the control and Lugol hearts were typically excitable, but only short episodes of VF could be reinduced. Endocardial activation rates were similar during the first 2 min of LDVF for Lugol-ablated and control hearts but were significantly slower in Lugol hearts by 3 min. In control hearts, the endocardium activated more rapidly than the epicardium after 4 min of LDVF with wave fronts propagating most often from the endocardium to epicardium. No difference in transmural activation rate or wave front direction was observed in Lugol hearts. Ablation of the subendocardium hastens VF spontaneous termination and alters VF activation sequences, suggesting that Purkinje fibers are important in the maintenance of LDVF.

  12. His-Purkinje system-related incessant ventricular tachycardia arising from the left coronary cusp

    Directory of Open Access Journals (Sweden)

    Eiji Sato, MD

    2014-08-01

    Full Text Available We describe the case of a 23-year-old woman who had His-Purkinje system-related incessant ventricular tachycardia with a narrow QRS configuration. The ventricular tachycardia was ablated successfully in the left coronary cusp where the earliest endocardial activation had been recorded. We hypothesize that a remnant of the subaortic conducting tissue was the source of the ventricular arrhythmias.

  13. Inflammation-induced reversible switch of the neuron-specific enolase promoter from Purkinje neurons to Bergmann glia.

    Science.gov (United States)

    Sawada, Yusuke; Konno, Ayumu; Nagaoka, Jun; Hirai, Hirokazu

    2016-06-13

    Neuron-specific enolase (NSE) is a glycolytic isoenzyme found in mature neurons and cells of neuronal origin. Injecting adeno-associated virus serotype 9 (AAV9) vectors carrying the NSE promoter into the cerebellar cortex is likely to cause the specific transduction of neuronal cells, such as Purkinje cells (PCs) and interneurons, but not Bergmann glia (BG). However, we found BG-predominant transduction without PC transduction along a traumatic needle tract for viral injection. The enhancement of neuroinflammation by the co-application of lipopolysaccharide (LPS) with AAV9 significantly expanded the BG-predominant area concurrently with the potentiated microglial activation. The BG-predominant transduction was gradually replaced by the PC-predominant transduction as the neuroinflammation dissipated. Experiments using glioma cell cultures revealed significant activation of the NSE promoter due to glucose deprivation, suggesting that intracellularly stored glycogen is metabolized through the glycolytic pathway for energy. Activation of the glycolytic enzyme promoter in BG concurrently with inactivation in PC may have pathophysiological significance for the production of lactate in activated BG and the utilization of lactate, which is provided by the BG-PC lactate shuttle, as a primary energy resource in injured PCs.

  14. Role of the Purkinje-Muscle Junction on the Ventricular Repolarization Heterogeneity in the Healthy and Ischemic Ovine Ventricular Myocardium

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    Marine E. Martinez

    2018-06-01

    Full Text Available Alteration of action potential duration (APD heterogeneity contributes to arrhythmogenesis. Purkinje-muscle junctions (PMJs present differential electrophysiological properties including longer APD. The goal of this study was to determine if Purkinje-related or myocardial focal activation modulates ventricular repolarization differentially in healthy and ischemic myocardium. Simultaneous epicardial (EPI and endocardial (ENDO optical mapping was performed on sheep left ventricular (LV wedges with intact free-running Purkinje network (N = 7. Preparations were paced on either ENDO or EPI surfaces, or the free-running Purkinje fibers (PFs, mimicking normal activation. EPI and ENDO APDs were assessed for each pacing configuration, before and after (7 min of the onset of no-flow ischemia. Experiments were supported by simulations. In control conditions, maximal APD was found at endocardial PMJ sites. We observed a significant transmural APD gradient for PF pacing with PMJ APD = 347 ± 41 ms and EPI APD = 273 ± 36 ms (p < 0.001. A similar transmural gradient was observed when pacing ENDO (49 ± 31 ms; p = 0.005. However, the gradient was reduced when pacing EPI (37 ± 20 ms; p = 0.005. Global dispersion of repolarization was the most pronounced for EPI pacing. In ischemia, both ENDO and EPI APD were reduced (p = 0.005 and the transmural APD gradient (109 ± 55 ms was increased when pacing ENDO compared to control condition or when pacing EPI (p < 0.05. APD maxima remained localized at functional PMJs during ischemia. Local repolarization dispersion was significantly higher at the PMJ than at other sites. The results were consistent with simulations. We found that the activation sequence modulates repolarization heterogeneity in the ischemic sheep LV. PMJs remain active following ischemia and exert significant influence on local repolarization patterns.

  15. The Secreted Protein C1QL1 and Its Receptor BAI3 Control the Synaptic Connectivity of Excitatory Inputs Converging on Cerebellar Purkinje Cells

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    Séverine M. Sigoillot

    2015-02-01

    Full Text Available Precise patterns of connectivity are established by different types of afferents on a given target neuron, leading to well-defined and non-overlapping synaptic territories. What regulates the specific characteristics of each type of synapse, in terms of number, morphology, and subcellular localization, remains to be understood. Here, we show that the signaling pathway formed by the secreted complement C1Q-related protein C1QL1 and its receptor, the adhesion-GPCR brain angiogenesis inhibitor 3 (BAI3, controls the stereotyped pattern of connectivity established by excitatory afferents on cerebellar Purkinje cells. The BAI3 receptor modulates synaptogenesis of both parallel fiber and climbing fiber afferents. The restricted and timely expression of its ligand C1QL1 in inferior olivary neurons ensures the establishment of the proper synaptic territory for climbing fibers. Given the broad expression of C1QL and BAI proteins in the developing mouse brain, our study reveals a general mechanism contributing to the formation of a functional brain.

  16. Developmental disorders of the brain can be caused by PCBs; low doses of hydroxy-PCBs disrupt thyroid hormone-dependent dendrite formation from Purkinje neurons in culture

    Energy Technology Data Exchange (ETDEWEB)

    Kuroda, Y; Kimura-Kuroda, J [Tokyo Metropol. Inst. for Neuroscience, Tokyo (Japan); Nagata, I [CREST/ JST, Tokyo (Japan)

    2004-09-15

    Exposure to some environmental chemicals during the perinatal period causes developmental disorders of the brain. Cognitive impairment and hyperactivity in infants were reported in Taiwan, known as Yu-cheng incidents caused by the accidental contamination of polychlorinated biphenyls (PCBs). Together with recent experimental data, Kuroda proposes a hypothesis that spatio-temporal disruptions of developing neuronal circuits by PCB exposure can cause the comobidity of learning disorders (LD), attention deficit hyperactivity disorder (ADHD) and autsm with the co-exposure to other environmental chemicals. PCBs and hydroxylated PCBs (OH-PCBs) have similar chemical structures to thyroid hormones (TH), thyroxine (T4) and triiodothyronine (T3). TH deficiency in the perinatal period causes cretinism children with severe cognitive and mental retardation. In primate model, Rice demonstrates that postnatal exposure to PCBs can dramatically influence later behavioral function. Epidemiological studies also indicate the possible developmental neurotoxicity of PCBs accumulated in human bodies. However, the precise underlying mechanisms and which types of PCB or OH-PCB with such effects have yet to be elucidated. It is important to establish a simple, reproducible, and sensitive in vitro assay for determining the effects of PCBs and OH-PCBs on the development of the central nervous system. Recently Iwasaki et al. established a reporter assay system and disclosed that low doses of PCBs potentially interfere TH-dependent gene expressions. This is the first demonstration that PCBs and OH-PCBs directly affect TH-receptor (TR)-mediated gene expressions crucial to the brain development, through unique mechanism. We also have demonstrated TH-dependent development of Purkinje neurons in vitro using a serum-free chemically defined medium. The degree of dendritic development of Purkinje cells is TH dose-dependent and exhibits high sensitivity in the pM order. Therefore, in the present study

  17. Radiation 2006. In association with the Polymer Division, Royal Australian Chemical Institute. Incorporating the 21st AINSE Radiation Chemistry Conference and the 18th Radiation Biology Conference, conference handbook

    International Nuclear Information System (INIS)

    Lavin, M. F.; Luff, J.; Peng, Cheng; Chen, P.; Gueven, N.; Bottle, S.; Hosokawa, K.

    2006-01-01

    Full text: Ataxia-telangiectasia (A-T) is an autosomal recessive genetic disorder characterized by immunodeficiency, cancer predisposition and neurological degeneration. Cells from A-T patients are hypersensitive to radiation, display cell cycle checkpoint defects and genome instability. The gene product defective in this syndrome, ATM, is activated by double strand breaks in DNA and signals these to the DNA repair machinery and the cell cycle checkpoints via a series of phosphorylated intermediates including p53, Chk2, Nbs1 and SMC1. It has been suggested that the neurodegenerative phenotype in A-T patients arises as a consequence of oxidative stress. This is supported by observations that A-T patients have significantly reduced levels of total antioxidant capacity and that A-T cells in culture are more sensitive to oxidative stress that normal cells. We have demonstrated that in vitro survival of cerebellar Purkinje cells of Atm-mutant mice is significantly reduced compared to their wild-type littermates and most neurons from these animals have dramatically reduced dendritic branching. We also showed that in vitro administration of the antioxidant 5-carboxy-1,1,3,3-tetramethylisoindolin-2-yloxyl (CTMIO) to Atm-deficient mice reduced the rate of cell death of Purkinje cells and enhanced dendritogenesis to wild-type levels. Intraperitoneal administration of this antioxidant throughout pregnancy enhanced survival of Purkinje cell neurons from Atm-disrupted animals and protected against oxidative stress in older animals as determined by levels of nitro-tyrosinated proteins and amount of catalase activity in the cerebellum. This antioxidant, a member of the nitroxide group, is a stable, free radical, capable of scavenging reactive oxygen species and may also circumvent Fenton-derived pathways by oxidizing the metals involved. We have recently demonstrated that CTMIO correct neuro-behavioural deficits in these mice and reduces oxidative damage to Purkinje cells. We

  18. Modulation of ASIC channels in rat cerebellar purkinje neurons by ischaemia-related signals

    Science.gov (United States)

    Allen, Nicola J; Attwell, David

    2002-01-01

    Acid-sensing ion channels (ASICs), activated by a decrease of extracellular pH, are found in neurons throughout the nervous system. They have an amino acid sequence similar to that of ion channels activated by membrane stretch, and have been implicated in touch sensation. Here we characterize the pH-dependent activation of ASICs in cerebellar Purkinje cells and investigate how they are modulated by factors released in ischaemia. Lowering the external pH from 7.4 activated an inward current at −66 mV, carried largely by Na+ ions, which was half-maximal for a step to pH 6.4 and was blocked by amiloride and gadolinium. The H+-gated current desensitized within a few seconds, but approximately 30% of cells showed a sustained inward current (11% of the peak current) in response to the maintained presence of pH 6 solution. The peak H+-evoked current was potentiated by membrane stretch (which occurs in ischaemia when [K+]o rises) and by arachidonic acid (which is released when [Ca2+]i rises in ischaemia). Arachidonic acid increased to 77% the fraction of cells showing a sustained current evoked by acid pH. The ASIC currents were also potentiated by lactate (which is released when metabolism becomes anaerobic in ischaemia) and by FMRFamide (which may mimic the action of related mammalian RFamide transmitters). These data reinforce suggestions of a mechanosensory aspect to ASIC channel function, and show that the activation of ASICs reflects the integration of multiple signals which are present during ischaemia. PMID:12205186

  19. Cerebellar cortex development in the weaver condition presents regional and age-dependent abnormalities without differences in Purkinje cells neurogenesis.

    Science.gov (United States)

    Martí, Joaquín; Santa-Cruz, María C; Hervás, José P; Bayer, Shirley A; Villegas, Sandra

    2016-01-01

    Ataxias are neurological disorders associated with the degeneration of Purkinje cells (PCs). Homozygous weaver mice (wv/wv) have been proposed as a model for hereditary cerebellar ataxia because they present motor abnormalities and PC loss. To ascertain the physiopathology of the weaver condition, the development of the cerebellar cortex lobes was examined at postnatal day (P): P8, P20 and P90. Three approaches were used: 1) quantitative determination of several cerebellar features; 2) qualitative evaluation of the developmental changes occurring in the cortical lobes; and 3) autoradiographic analyses of PC generation and placement. Our results revealed a reduction in the size of the wv/wv cerebellum as a whole, confirming previous results. However, as distinguished from these reports, we observed that quantified parameters contribute differently to the abnormal growth of the wv/wv cerebellar lobes. Qualitative analysis showed anomalies in wv/wv cerebellar cytoarchitecture, depending on the age and lobe analyzed. Such abnormalities included the presence of the external granular layer after P20 and, at P90, ectopic cells located in the molecular layer following several placement patterns. Finally, we obtained autoradiographic evidence that wild-type and wv/wv PCs presented similar neurogenetic timetables, as reported. However, the innovative character of this current work lies in the fact that the neurogenetic gradients of wv/wv PCs were not modified from P8 to P90. A tendency for the accumulation of late-formed PCs in the anterior and posterior lobes was found, whereas early-generated PCs were concentrated in the central and inferior lobes. These data suggested that wv/wv PCs may migrate properly to their final destinations. The extrapolation of our results to patients affected with cerebellar ataxias suggests that all cerebellar cortex lobes are affected with several age-dependent alterations in cytoarchitectonics. We also propose that PC loss may be regionally

  20. Lactoperoxidase catalyzed radioiodination of cell surface immunoglobulin: incorporated radioactivity may not reflect relative cell surface Ig density

    International Nuclear Information System (INIS)

    Wilder, R.L.; Yuen, C.C.; Mage, R.G.

    1979-01-01

    Rabbit and mouse splenic lymphocytes were radioiodinated by the lactoperoxidase technique, extracted with non-ionic detergent, immunoprecipitated with high titered rabbit anti-kappa antisera, and compared by SDS-PAGE. Mouse sIg peaks were reproducibly larger in size than rabbit sIg peaks (often greater than 10 times). Neither differences in incorporation of label into the rabbit cell surface, nor differences in average sIg density explain this result. Total TCA-precipitable radioactivity was similar in each species. Estimation of the relative amounts of sIg in the mouse and rabbit showed similar average sIg densities. Differences in detergent solubility, proteolytic lability, or antisera used also do not adequately account for this difference. Thus, these data indicate that radioactivity incorporated after lactoperoxidase catalyzed cell surface radioiodination may not reflect cell surface Ig density. Conclusions about cell surface density based upon relative incorporation of radioactivity should be confirmed by other approaches

  1. Increasing the fidelity of noncanonical amino acid incorporation in cell-free protein synthesis.

    Science.gov (United States)

    Gan, Qinglei; Fan, Chenguang

    2017-11-01

    Cell-free protein synthesis provides a robust platform for co-translational incorporation of noncanonical amino acid (ncAA) into proteins to facilitate biological studies and biotechnological applications. Recently, eliminating the activity of release factor 1 has been shown to increase ncAA incorporation in response to amber codons. However, this approach could promote mis-incorporation of canonical amino acids by near cognate suppression. We performed a facile protocol to remove near cognate tRNA isoacceptors of the amber codon from total tRNAs, and used the phosphoserine (Sep) incorporation system as validation. By manipulating codon usage of target genes and tRNA species introduced into the cell-free protein synthesis system, we increased the fidelity of Sep incorporation at a specific position. By removing three near cognate tRNA isoacceptors of the amber stop codon [tRNA Lys , tRNA Tyr , and tRNA Gln (CUG)] from the total tRNA, the near cognate suppression decreased by 5-fold without impairing normal protein synthesis in the cell-free protein synthesis system. Mass spectrometry analyses indicated that the fidelity of ncAA incorporation was improved. Removal of near cognate tRNA isoacceptors of the amber codon could increase ncAA incorporation fidelity towards the amber stop codon in release factor deficiency systems. We provide a general strategy to improve fidelity of ncAA incorporation towards stop, quadruplet and sense codons in cell-free protein synthesis systems. This article is part of a Special Issue entitled "Biochemistry of Synthetic Biology - Recent Developments" Guest Editor: Dr. Ilka Heinemann and Dr. Patrick O'Donoghue. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Comparative sensitivity of rat cerebellar neurons to dysregulation of divalent cation homeostasis and cytotoxicity caused by methylmercury

    International Nuclear Information System (INIS)

    Edwards, Joshua R.; Marty, M. Sue; Atchison, William D.

    2005-01-01

    The objective of the present study was to determine the relative effectiveness of methylmercury (MeHg) to alter divalent cation homeostasis and cause cell death in MeHg-resistant cerebellar Purkinje and MeHg-sensitive granule neurons. Application of 0.5-5 μM MeHg to Purkinje and granule cells grown in culture caused a concentration- and time-dependent biphasic increase in fura-2 fluorescence. At 0.5 and 1 μM MeHg, the elevations of fura-2 fluorescence induced by MeHg were biphasic in both cell types, but significantly delayed in Purkinje as compared to granule cells. Application of the heavy-metal chelator, TPEN, to Purkinje cells caused a precipitous decline in a proportion of the fura-2 fluorescence signal, indicating that MeHg causes release of Ca 2+ and non-Ca 2+ divalent cations. Purkinje cells were also more resistant than granule cells to the neurotoxic effects of MeHg. At 24.5 h after-application of 5 μM MeHg, 97.7% of Purkinje cells were viable. At 3 μM MeHg there was no detectable loss of Purkinje cell viability. In contrast, only 40.6% of cerebellar granule cells were alive 24.5 h after application of 3 μM MeHg. In conclusion, Purkinje neurons in primary cultures appear to be more resistant to MeHg-induced dysregulation of divalent cation homeostasis and subsequent cell death when compared to cerebellar granule cells. There is a significant component of non-Ca 2+ divalent cation released by MeHg in Purkinje neurons

  3. A Parametric Computational Model of the Action Potential of Pacemaker Cells.

    Science.gov (United States)

    Ai, Weiwei; Patel, Nitish D; Roop, Partha S; Malik, Avinash; Andalam, Sidharta; Yip, Eugene; Allen, Nathan; Trew, Mark L

    2018-01-01

    A flexible, efficient, and verifiable pacemaker cell model is essential to the design of real-time virtual hearts that can be used for closed-loop validation of cardiac devices. A new parametric model of pacemaker action potential is developed to address this need. The action potential phases are modeled using hybrid automaton with one piecewise-linear continuous variable. The model can capture rate-dependent dynamics, such as action potential duration restitution, conduction velocity restitution, and overdrive suppression by incorporating nonlinear update functions. Simulated dynamics of the model compared well with previous models and clinical data. The results show that the parametric model can reproduce the electrophysiological dynamics of a variety of pacemaker cells, such as sinoatrial node, atrioventricular node, and the His-Purkinje system, under varying cardiac conditions. This is an important contribution toward closed-loop validation of cardiac devices using real-time heart models.

  4. Improving Efficiency of Multicrystalline Silicon and CIGS Solar Cells by Incorporating Metal Nanoparticles

    Directory of Open Access Journals (Sweden)

    Ming-Jer Jeng

    2015-10-01

    Full Text Available This work studies the use of gold (Au and silver (Ag nanoparticles in multicrystalline silicon (mc-Si and copper-indium-gallium-diselenide (CIGS solar cells. Au and Ag nanoparticles are deposited by spin-coating method, which is a simple and low cost process. The random distribution of nanoparticles by spin coating broadens the resonance wavelength of the transmittance. This broadening favors solar cell applications. Metal shadowing competes with light scattering in a manner that varies with nanoparticle concentration. Experimental results reveal that the mc-Si solar cells that incorporate Au nanoparticles outperform those with Ag nanoparticles. The incorporation of suitable concentration of Au and Ag nanoparticles into mc-Si solar cells increases their efficiency enhancement by 5.6% and 4.8%, respectively. Incorporating Au and Ag nanoparticles into CIGS solar cells improve their efficiency enhancement by 1.2% and 1.4%, respectively. The enhancement of the photocurrent in mc-Si solar cells is lower than that in CIGS solar cells, owing to their different light scattering behaviors and material absorption coefficients.

  5. Calculation of cell production from [3H]Thymidine incorporation with freshwater bacteria

    International Nuclear Information System (INIS)

    Smits, J.D.; Riemann, B.

    1988-01-01

    The conversion factor for the calculation of bacterial production from rates of [ 3 H]thymidine incorporation was examined with diluted batch cultures of freshwater bacteria. Natural bacterial assemblages were grown in aged, normal, and enriched media at 10 to 20 degree C. The generation time during 101 growth cycles covered a range from 4 to >200 h. The average conversion factor was 2.15 x 10 18 cells mol -1 of thymidine incorporated into the trichloroacetic acid (TCA) precipitate, when the generation time exceeded 20 h. At generation times of 18 cells mol -1 of thymidine incorporated into TCA precipitate. The amount of radioactivity in purified DNA increased with decreasing generation time and increasing conversion factor (calculated from the TCA precipitate), corresponding to a decrease in the percentage in protein. The conversion factors calculated from purified DNA or from the TCA precipitate gave the same variability. Conversion factors did not change significantly with the medium, but were significantly higher at 20 degree C that at 15 and 10 degree C. Results suggests that incorporation of [ 3 H]thymidine into DNA is probably limited by uptake during period with generation times of 18 cells mol -1 of thymidine incorporated is used

  6. The different electron transport of two nanotubes incorporated in working electrode of dye-sensitized solar cells

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Xiaobo, E-mail: zhangxiaobo@chnu.edu.cn [School of Physics, Huaibei Normal University, Huaibei 235000, Anhui (China); Eco-Materials and Renewable Energy Research Centre (ERERC), Nanjing University, Nanjing 210093 (China); Tian, Hanmin; Wang, Xiangyan; Xue, Guogang; Tian, Zhipeng; Zhang, Jiyuan; Yuan, Shikui [Eco-Materials and Renewable Energy Research Centre (ERERC), Nanjing University, Nanjing 210093 (China); Yu, Tao; Zou, Zhigang [Eco-Materials and Renewable Energy Research Centre (ERERC), Nanjing University, Nanjing 210093 (China); National Laboratory of Solid State Microstructures, Nanjing University, Nanjing 210093 (China)

    2013-11-25

    Highlights: •Two TiO{sub 2} nanotubes are separately incorporated in working electrode of DSSCs. •The 6-μm-tubes incorporation improves electron transport in the cell. •The 1-μm-tubes incorporation impedes electron transport in the cell. •Both 1-D electron diffusion and nanotube percolation promote electron transport. •Electron residing at the end of 1-μm-tubes maybe impedes electron transport. -- Abstract: Two different-length (6 μm and 1 μm) TiO{sub 2} nanotubes were prepared and incorporated in working electrode of dye-sensitized solar cells (DSSCs). The analyses of the electrochemical impedance spectra of cells demonstrate that, the electron transport resistance R{sub w} decreases and increases separately to 0.3 Ω in 6-μm-tubes-cell and to 15.1 Ω in 1-μm-tubes-cell comparing with that 1.4 Ω in P25-cell, reflecting the improved electron transport in 6-μm-tubes-cell and impeded electron transport in 1-μm-tubes-cell. The reason is ascribed to the different electron transport in working electrode due to the incorporation of nanotubes. For the 6-μm-tubes incorporation, both 1-D electron diffusion along nanotubes and nanotube percolation improve electron transport in working electrode, but they cannot improve electron transport for the 1-μm-tubes incorporation. On the contrary, the 1-μm-tubes incorporation may impede electron transport because of electron residing occurring seriously at the end of 1-μm-tubes. The results of this work will help to understand the specific nature of electron transport in TiO{sub 2} nanotubes in DSSCs.

  7. Hydroxyurea Treatment and Development of the Rat Cerebellum: Effects on the Neurogenetic Profiles and Settled Patterns of Purkinje Cells and Deep Cerebellar Nuclei Neurons.

    Science.gov (United States)

    Martí, Joaquín; Santa-Cruz, M C; Serra, Roger; Hervás, José P

    2016-11-01

    The current paper analyzes the development of the male and female rat cerebellum exposed to hydroxyurea (HU) (300 or 600 mg/kg) as embryo and collected at postnatal day 90. Our study reveals that the administration of this drug compromises neither the cytoarchitecture of the cerebellar cortex nor deep nuclei (DCN). However, in comparison with the saline group, we observed that several cerebellar parameters were lower in the HU injected groups. These parameters included area of the cerebellum, cerebellar cortex length, molecular layer area, Purkinje cell number, granule cell counts, internal granular layer, white matter and cerebellar nuclei areas, and number of deep cerebellar nuclei neurons. These features were larger in the rats injected with saline, smaller in those exposed to 300 mg/kg of HU and smallest in the group receiving 600 mg/kg of this agent. No sex differences in the effect of the HU were observed. In addition, we infer the neurogenetic timetables and the neurogenetic gradients of PCs and DCN neurons in rats exposed to either saline or HU as embryos. For this purpose, 5-bromo-2'-deoxyuridine was injected into pregnant rats previously administered with saline or HU. This thymidine analog was administered following a progressively delayed cumulative labeling method. The data presented here show that systematic differences exist in the pattern of neurogenesis and in the spatial location of cerebellar neurons between rats injected with saline or HU. No sex differences in the effect of the HU were observed. These findings have implications for the administration of this compound to women in gestation as the effects of HU on the development of the cerebellum might persist throughout their offsprings' life.

  8. Human cultured cells are capable to incorporate isolated plant mitochondria loaded with exogenous DNA

    Directory of Open Access Journals (Sweden)

    Laktionov P. P.

    2012-07-01

    Full Text Available Aim. To investigate the possibility of human cultured cells to incorporate isolated mitochondria together with exogenous DNA introduced into organelles. Methods. Two approaches were used for this purpose, fluorescent labelling of mitochondria and/or DNA with subsequent analysis of the cells subjected to incubation by microscopy or by quantitative PCR. Results. We have shown that human cultured cells lines, HeLa and HUVEC, are capable to uptake isolated plant mitochondria and that this process depends on the incubation time and concentration of organelles present in medium. The incorporated mitochondria can serve as vehicles to deliver exogenous DNA into human cells, this DNA is then distributed in different cell compartments. Conclusions. These results are preliminary and need further investigations, including testing the possibility of human cells to incorporate the mitochondria of human or animal origin and creating genetic construction which could provide certain selectivity or stability of the transferred exogenous DNA upon cell uptake of the mitochondria as vectors.

  9. Cell-free unnatural amino acid incorporation with alternative energy systems and linear expression templates.

    Science.gov (United States)

    Shrestha, Prashanta; Smith, Mark Thomas; Bundy, Bradley Charles

    2014-01-25

    Site-specific incorporation of unnatural amino acids (uAAs) during protein synthesis expands the proteomic code through the addition of unique residue chemistry. This field provides a unique tool to improve pharmacokinetics, cancer treatments, vaccine development, proteomics and protein engineering. The limited ability to predict the characteristics of proteins with uAA-incorporation creates a need for a low-cost system with the potential for rapid screening. Escherichia coli-based cell-free protein synthesis is a compelling platform for uAA incorporation due to the open and accessible nature of the reaction environment. However, typical cell-free systems can be expensive due to the high cost of energizing reagents. By employing alternative energy sources, we reduce the cost of uAA-incorporation in CFPS by 55%. While alternative energy systems reduce cost, the time investment to develop gene libraries can remain cumbersome. Cell-free systems allow the direct use of PCR products known as linear expression templates, thus alleviating tedious plasmid library preparations steps. We report the specific costs of CFPS with uAA incorporation, demonstrate that LETs are suitable expression templates with uAA-incorporation, and consider the substantial reduction in labor intensity using LET-based expression for CFPS uAA incorporation. Copyright © 2013 Elsevier B.V. All rights reserved.

  10. Synthesis of hydroxyeicosatetraenoic acids (HETE's) by adrenal glomerulosa cells and incorporation into cellular lipids

    International Nuclear Information System (INIS)

    Campbell, W.B.; Richards, C.F.; Brady, M.T.; Falck, J.R.

    1986-01-01

    The role of lipoxygenase metabolites of arachidonic acid (AA) in the regulation of aldosterone secretion was studied in isolated rat adrenal glomerulosa cells. Cells were incubated with 14 C-AA in the presence of angiotensin (AII). The media was extracted, metabolites isolated by HPLC, and structures of the metabolites determined by UV absorbance and mass spectrometry. The major products were 12- and 15-HETE with lesser amounts of 11- and 5-HETE. When adrenal cells were incubated with 15-, 12- or 5-HPETE or their respective HETE's (0.03-300nM), there was no significant change in basal or AII-stimulated aldosterone release. Cells were incubated with [ 3 H]-AA, -5-HETE, -15-HETE, -12-HETE or -LTB. The cellular lipids were extracted and analyzed by TLC. AA was incorporated into phospholipids (22%), cholesterol esters (50%) and triglycerides (21%). Neither the HETE's or LTB 4 were incorporated into phospholipids. 5-HETE was taken up into di- and mono-glycerides. The rates of incorporation of AA and 5-HETE were similar (+ 1/2 = 10 min). The incorporation of 5-HETE into glycerol esters did not modify the release of aldosterone by the cells. Thus, while adrenal cells synthesize HETE's, these eicosanoids do not appear to alter the synthesis of aldosterone

  11. Carbon nanotube-incorporated collagen hydrogels improve cell alignment and the performance of cardiac constructs

    Science.gov (United States)

    Sun, Hongyu; Zhou, Jing; Huang, Zhu; Qu, Linlin; Lin, Ning; Liang, Chengxiao; Dai, Ruiwu; Tang, Lijun; Tian, Fuzhou

    2017-01-01

    Carbon nanotubes (CNTs) provide an essential 2-D microenvironment for cardiomyocyte growth and function. However, it remains to be elucidated whether CNT nanostructures can promote cell–cell integrity and facilitate the formation of functional tissues in 3-D hydrogels. Here, single-walled CNTs were incorporated into collagen hydrogels to fabricate (CNT/Col) hydrogels, which improved mechanical and electrical properties. The incorporation of CNTs (up to 1 wt%) exhibited no toxicity to cardiomyocytes and enhanced cell adhesion and elongation. Through the use of immunohistochemical staining, transmission electron microscopy, and intracellular calcium-transient measurement, the incorporation of CNTs was found to improve cell alignment and assembly remarkably, which led to the formation of engineered cardiac tissues with stronger contraction potential. Importantly, cardiac tissues based on CNT/Col hydrogels were noted to have better functionality. Collectively, the incorporation of CNTs into the Col hydrogels improved cell alignment and the performance of cardiac constructs. Our study suggests that CNT/Col hydrogels offer a promising tissue scaffold for cardiac constructs, and might serve as injectable biomaterials to deliver cell or drug molecules for cardiac regeneration following myocardial infarction in the near future. PMID:28450785

  12. Incorporation of [14C]-palmitate into lipids of Brassica cells during the induction of freezing tolerance

    International Nuclear Information System (INIS)

    Lynch, D.V.; Joseph, R.A.

    1989-01-01

    Changes in plasma membrane lipid composition have been causally related to increased freezing tolerance. Studies of lipid metabolism during ABA induction of freezing tolerance in Brassica napus suspension cultures were undertaken. Cells were labeled with [ 14 C]-palmitate four days after transfer to fresh medium (control) or medium containing ABA (which increases freezing tolerance). At times between one and 20 hrs after labeling, ABA-treated cells incorporated almost twice the amount of label as controls cells. Approximately 80% of the radioactivity was associated with neutral lipids in ABA-treated cells and controls. Incorporation of label into total cellular polar lipids was 4.9 x 10 5 dpm/mg protein for control cells and 1 x 10 6 dpm/mg protein for cells transferred to medium containing ABA. Analysis of lipids following alkaline hydrolysis indicated that incorporation of [ 14 C]-palmitate into glucosylceramide of ABA-treated cells was less than 60% of control values when expressed relative to that of the total polar lipids. Incorporation into ceramides was also depressed in ABA-treated cells

  13. Enhanced incorporation of radioactive inorganic phosphate into phospholipids of HeLa cells by tumor promoters

    International Nuclear Information System (INIS)

    Nishino, H.; Fujiki, H.; Terada, M.; Sato, S.

    1983-01-01

    Teleocidin, a new tumor promoter, increased incorporation of radioactive inorganic phosphate ( 32 P/sub i/) into phospholipids in HeLa cells. This effect was detected within 1 h on incubation of the cells in medium containing teleocidin. The half-maximum effective dose of teleocidin was approximately 10 ng/ml. The main effect of teleocidin was on the incorporation of 32 P/sub i/ into the phosphatidylcholine fraction, with a lesser effect on 32 P/sub i/ incorporation into other phospholipid fractions. Increased incorporation of 32 P/sub i/ into phospholipids was also observed on incubation of the cells with 12-O-tetradecanoylphorbol-13-acetate (TPA), dihydroteleocidin B, or lyngbyatoxin A, which are all complete tumor promoters, and also with mezerein, which is an incomplete and second stage promoter. On the other hand, at concentrations of up to 1 microgram/ml, 4-O-methyl TPA and C/sub a/ 2 + ionophore A23187, which are incomplete and first stage promoters, and phorbol, which has no promoting activity in skin carcinogenesis, did not cause any increased incorporation of 32 P/sub i/ into phospholipid fractions of HeLa cells

  14. Antitumor activity of vorinostat-incorporated nanoparticles against human cholangiocarcinoma cells.

    Science.gov (United States)

    Kwak, Tae Won; Kim, Do Hyung; Jeong, Young-Il; Kang, Dae Hwan

    2015-09-26

    The aim of this study is to evaluate the anticancer activity of vorinostat-incorporated nanoparticles (vorinostat-NPs) against HuCC-T1 human cholangiocarcinoma cells. Vorinostat-NPs were fabricated by a nanoprecipitation method using poly(DL-lactide-co-glycolide)/poly(ethylene glycol) copolymer. Vorinostat-NPs exhibited spherical shapes with sizes Vorinostat-NPs have anticancer activity similar to that of vorinostat in vitro. Vorinostat-NPs as well as vorinostat itself increased acetylation of histone-H3. Furthermore, vorinostat-NPs have similar effectiveness in the suppression or expression of histone deacetylase, mutant type p53, p21, and PARP/cleaved caspase-3. However, vorinostat-NPs showed improved antitumor activity against HuCC-T1 cancer cell-bearing mice compared to vorinostat, whereas empty nanoparticles had no effect on tumor growth. Furthermore, vorinostat-NPs increased the expression of acetylated histone H3 in tumor tissue and suppressed histone deacetylase (HDAC) expression in vivo. The improved antitumor activity of vorinostat-NPs can be explained by molecular imaging studies using near-infrared (NIR) dye-incorporated nanoparticles, i.e. NIR-dye-incorporated nanoparticles were intensively accumulated in the tumor region rather than normal one. Our results demonstrate that vorinostat and vorinostat-NPs exert anticancer activity against HuCC-T1 cholangiocarcinoma cells by specific inhibition of HDAC expression. Thus, we suggest that vorinostat-NPs are a promising candidate for anticancer chemotherapy in cholangiocarcinoma. Graphical abstract Local delivery strategy of vorinostat-NPs against cholangiocarcinomas.

  15. Neuronal death and synapse elimination in the olivocerebellar system. II. Cell counts in the inferior olive of adult x-irradiated rats and weaver and reeler mutant mice

    International Nuclear Information System (INIS)

    Shojaeian, H.; Delhaye-Bouchaud, N.; Mariani, J.

    1985-01-01

    Cell death in the developing rat inferior olive precedes the regression of the polyneuronal innervation of Purkinje cells by olivary axons (i.e., climbing fibers), suggesting that the involution of the redundant olivocerebellar contacts is caused by a withdrawal of supernumerary axonal collaterals rather than by degeneration of the parent cell. However, a subsequent apparent increase of the olivary population occurs, which could eventually mask a residual presynaptic cell death taking place at the same time. Therefore, cell counts were performed in the inferior olive of adult rodents in which the multiple innervation of Purkinje cells by olivary axons is maintained, with the idea that if cell death plays a role in the regression of supernumerary climbing fibers, the number of olivary cells should be higher in these animals than in their controls. The results show that the size of the cell population in the inferior olive of weaver and reeler mutant mice and rats degranulated by early postnatal x-irradiation does not differ significantly from that of their controls. Similarly, the distribution of the cells in the four main olivary subnuclei is not modified in weaver mice and x-irradiated rats. The present data further support the assumption that the regression of the polyneuronal innervation of Purkinje cells occurs independently of cell death in the presynaptic population

  16. The ducky(2J) mutation in Cacna2d2 results in reduced spontaneous Purkinje cell activity and altered gene expression.

    Science.gov (United States)

    Donato, Roberta; Page, Karen M; Koch, Dietlind; Nieto-Rostro, Manuela; Foucault, Isabelle; Davies, Anthony; Wilkinson, Tonia; Rees, Michele; Edwards, Frances A; Dolphin, Annette C

    2006-11-29

    The mouse mutant ducky and its allele ducky(2J) represent a model for absence epilepsy characterized by spike-wave seizures and cerebellar ataxia. These mice have mutations in Cacna2d2, which encodes the alpha2delta-2 calcium channel subunit. Of relevance to the ataxic phenotype, alpha2delta-2 mRNA is strongly expressed in cerebellar Purkinje cells (PCs). The Cacna2d2(du2J) mutation results in a 2 bp deletion in the coding region and a complete loss of alpha2delta-2 protein. Here we show that du(2J)/du(2J) mice have a 30% reduction in somatic calcium current and a marked fall in the spontaneous PC firing rate at 22 degrees C, accompanied by a decrease in firing regularity, which is not affected by blocking synaptic input to PCs. At 34 degrees C, du(2J)/du(2J) PCs show no spontaneous intrinsic activity. Du(2J)/du(2J) mice also have alterations in the cerebellar expression of several genes related to PC function. At postnatal day 21, there is an elevation of tyrosine hydroxylase mRNA and a reduction in tenascin-C gene expression. Although du(2J)/+ mice have a marked reduction in alpha2delta-2 protein, they show no fall in PC somatic calcium currents or increase in cerebellar tyrosine hydroxylase gene expression. However, du(2J)/+ PCs do exhibit a significant reduction in firing rate, correlating with the reduction in alpha2delta-2. A hypothesis for future study is that effects on gene expression occur as a result of a reduction in somatic calcium currents, whereas effects on PC firing occur as a long-term result of loss of alpha2delta-2 and/or a reduction in calcium currents and calcium-dependent processes in regions other than the soma.

  17. Acute and long-term Purkinje cell loss following a single ethanol binge during the early third trimester equivalent in the rat.

    Science.gov (United States)

    Idrus, Nirelia M; Napper, Ruth M A

    2012-08-01

    In the rat, binge-like ethanol (EtOH) exposure during the early neonatal period (a developmental period equivalent to the human third trimester) can result in a permanent deficit of cerebellar Purkinje cells (Pcells). However, the consequences of a moderate binge alcohol exposure on a single day during this postnatal period have not been established. This is an issue of importance as many pregnant women binge drink periodically at social drinking levels. This study aimed to identify both the acute and long-term effects of exposure to a single alcohol binge that achieved a mean peak blood EtOH concentration of approximately 250 mg/dl during early postnatal life using a rat model of fetal alcohol spectrum disorders. Acute apoptotic Pcell death 10 hours after a moderate dose binge EtOH exposure from postnatal days (PDs) 0 to 10 was assessed using active caspase-3 immunolabeling. Acute Pcell apoptosis was quantified in cerebellar vermal lobules I-X using the physical disector method. Long-term effects were assessed at PD 60 using stereological methods to determine total Pcell numbers in the vermis, lobule III, and lobule IX, following a moderate dose binge EtOH exposure at PDs 0, 2, or 4. Acute apoptosis was induced by EtOH on PDs 1 to 8 in a time and lobular-dependent manner. For EtOH exposure on PD 2, significant long-term Pcell loss occurred in lobule III. EtOH exposure on PD 4 resulted in significant long-term Pcell loss throughout the entire vermis. These results indicate that a single, early EtOH episode of moderate dose can create significant and permanent Pcell loss in the developing cerebellum. Copyright © 2012 by the Research Society on Alcoholism.

  18. Carbon nanotube-incorporated collagen hydrogels improve cell alignment and the performance of cardiac constructs

    Directory of Open Access Journals (Sweden)

    Sun HY

    2017-04-01

    Full Text Available Hongyu Sun,* Jing Zhou,* Zhu Huang,* Linlin Qu,* Ning Lin,* Chengxiao Liang, Ruiwu Dai, Lijun Tang, Fuzhou Tian General Surgery Center, Chengdu Military General Hospital, Chengdu, China *These authors contributed equally to this work Abstract: Carbon nanotubes (CNTs provide an essential 2-D microenvironment for cardiomyocyte growth and function. However, it remains to be elucidated whether CNT nanostructures can promote cell–cell integrity and facilitate the formation of functional tissues in 3-D hydrogels. Here, single-walled CNTs were incorporated into collagen hydrogels to fabricate (CNT/Col hydrogels, which improved mechanical and electrical properties. The incorporation of CNTs (up to 1 wt% exhibited no toxicity to cardiomyocytes and enhanced cell adhesion and elongation. Through the use of immunohistochemical staining, transmission electron microscopy, and intracellular calcium-transient measurement, the incorporation of CNTs was found to improve cell alignment and assembly remarkably, which led to the formation of engineered cardiac tissues with stronger contraction potential. Importantly, cardiac tissues based on CNT/Col hydrogels were noted to have better functionality. Collectively, the incorporation of CNTs into the Col hydrogels improved cell alignment and the performance of cardiac constructs. Our study suggests that CNT/Col hydrogels offer a promising tissue scaffold for cardiac constructs, and might serve as injectable biomaterials to deliver cell or drug molecules for cardiac regeneration following myocardial infarction in the near future. Keywords: carbon nanotubes, collagen hydrogel, cardiac constructs, cell alignment, tissue functionality

  19. Studying cerebellar circuits by remote control of selected neuronal types with GABA-A receptors

    Directory of Open Access Journals (Sweden)

    William Wisden

    2009-12-01

    Full Text Available Although GABA-A receptor-mediated inhibition of cerebellar Purkinje cells by molecular layer interneurons (MLIs has been studied intensely on the cellular level, it has remained unclear how this inhibition regulates cerebellum-dependent behaviour. We have implemented two complementary approaches to investigate the function of the MLI-Purkinje cell synapse on the behavioral level. In the first approach we permanently disrupted inhibitory fast synaptic transmission at the synapse by genetically removing the postsynaptic GABA-A receptors from Purkinje cells (PC-Δγ2 mice. We found that chronic disruption of the MLI-Purkinje cell synapse strongly impaired cerebellar learning of the vestibular occular reflex (VOR, presumably by disrupting the temporal patterns of Purkinje cell activity. However, in PC-Δγ2 mice the baseline VOR reflex was only mildly affected; indeed PC-Δγ2 mice showed no ataxia or gait abnormalities suggesting that MLI control of Purkinje cell activity is either not involved in ongoing motor tasks or that the system has found a way to compensate for its loss. To investigate the latter possibility we have developed an alternative genetic technique; we made the MLI-Purkinje cell synapse selectively sensitive to rapid manipulation with the GABAA receptor modulator zolpidem (PC-γ2-swap mice. Minutes after intraperitoneal zolpidem injection, these PC-γ2-swap mice developed severe motor abnormalities, revealing a substantial contribution of the MLI-Purkinje cell synapse to real time motor control. The cell-type selective permanent knockout of synaptic GABAergic input, and the fast reversible modulation of GABAergic input at the same synapse illustrate how pursuing both strategies gives a fuller view.

  20. Zebrin II Is Expressed in Sagittal Stripes in the Cerebellum of Dragon Lizards (Ctenophorus sp.).

    Science.gov (United States)

    Wylie, Douglas R; Hoops, Daniel; Aspden, Joel W; Iwaniuk, Andrew N

    2016-01-01

    Aldolase C, also known as zebrin II (ZII), is a glycolytic enzyme that is expressed in cerebellar Purkinje cells of the vertebrate cerebellum. In both mammals and birds, ZII is expressed heterogeneously, such that there are sagittal stripes of Purkinje cells with high ZII expression (ZII+) alternating with stripes of Purkinje cells with little or no expression (ZII-). In contrast, in snakes and turtles, ZII is not expressed heterogeneously; rather all Purkinje cells are ZII+. Here, we examined the expression of ZII in the cerebellum of lizards to elucidate the evolutionary origins of ZII stripes in Sauropsida. We focused on the central netted dragon (Ctenophorus nuchalis) but also examined cerebellar ZII expression in 5 other dragon species (Ctenophorus spp.). In contrast to what has been observed in snakes and turtles, we found that in these lizards, ZII is heterogeneously expressed. In the posterior part of the cerebellum, on each side of the midline, there were 3 sagittal stripes consisting of Purkinje cells with high ZII expression (ZII+) alternating with 2 sagittal stripes with weaker ZII expression (ZIIw). More anteriorly, most of the Purkinje cells were ZII+, except laterally, where the Purkinje cells did not express ZII (ZII-). Finally, all Purkinje cells in the auricle (flocculus) were ZII-. Overall, the parasagittal heterogeneous expression of ZII in the cerebellum of lizards is similar to that in mammals and birds, and contrasts with the homogenous ZII+ expression seen in snakes and turtles. We suggest that a sagittal heterogeneous expression of ZII represents the ancestral condition in stem reptiles which was lost in snakes and turtles. © 2017 S. Karger AG, Basel.

  1. Cell Fusion along the Anterior-Posterior Neuroaxis in Mice with Experimental Autoimmune Encephalomyelitis.

    Directory of Open Access Journals (Sweden)

    Sreenivasa R Sankavaram

    Full Text Available It is well documented that bone marrow-derived cells can fuse with a diverse range of cells, including brain cells, under normal or pathological conditions. Inflammation leads to robust fusion of bone marrow-derived cells with Purkinje cells and the formation of binucleate heterokaryons in the cerebellum. Heterokaryons form through the fusion of two developmentally differential cells and as a result contain two distinct nuclei without subsequent nuclear or chromosome loss.In the brain, fusion of bone marrow-derived cells appears to be restricted to the complex and large Purkinje cells, raising the question whether the size of the recipient cell is important for cell fusion in the central nervous system. Purkinje cells are among the largest neurons in the central nervous system and accordingly can harbor two nuclei.Using a well-characterized model for heterokaryon formation in the cerebellum (experimental autoimmune encephalomyelitis - a mouse model of multiple sclerosis, we report for the first time that green fluorescent protein-labeled bone marrow-derived cells can fuse and form heterokaryons with spinal cord motor neurons. These spinal cord heterokaryons are predominantly located in or adjacent to an active or previously active inflammation site, demonstrating that inflammation and infiltration of immune cells are key for cell fusion in the central nervous system. While some motor neurons were found to contain two nuclei, co-expressing green fluorescent protein and the neuronal marker, neuron-specific nuclear protein, a number of small interneurons also co-expressed green fluorescent protein and the neuronal marker, neuron-specific nuclear protein. These small heterokaryons were scattered in the gray matter of the spinal cord.This novel finding expands the repertoire of neurons that can form heterokaryons with bone marrow-derived cells in the central nervous system, albeit in low numbers, possibly leading to a novel therapy for spinal cord

  2. LHRH inhibits [3H]thymidine incorporation by pituitary cells cultured IN VITRO

    International Nuclear Information System (INIS)

    Stepien, H.

    1981-01-01

    The effects of two synthetic neuropeptides, LHRH and neurotensin, on tritiated thymidine uptake by dispersed anterior pituitary cells were investigated. It was found that LHRH but not neurotensin (at concentrations between 10 -7 - 10 -11 M) inhibits incorporation of [ 3 H]thymidine into DNA of pituitary cell nuclei, in a dose-dependent manner. These results indicate that LHRH can regulate not only secretory activity of the gonadotrophic cells but also can be involved in the control of anterior pituitary cell replication

  3. Antitumor activity of vorinostat-incorporated nanoparticles against human cholangiocarcinoma cells

    OpenAIRE

    Kwak, Tae Won; Kim, Do Hyung; Jeong, Young-Il; Kang, Dae Hwan

    2015-01-01

    Background The aim of this study is to evaluate the anticancer activity of vorinostat-incorporated nanoparticles (vorinostat-NPs) against HuCC-T1 human cholangiocarcinoma cells. Vorinostat-NPs were fabricated by a nanoprecipitation method using poly(dl-lactide-co-glycolide)/poly(ethylene glycol) copolymer. Results Vorinostat-NPs exhibited spherical shapes with sizes

  4. Incorporation of uridine-H3 into healthy and tobacco necrosis virus-infected mesophyll cells of Chenopodium amaranticolor

    International Nuclear Information System (INIS)

    Faccioli, G.; Rubies-Autonel, C.

    1975-01-01

    Tritiated uridine was selectively incorporated into the nucleus, nucleolus and cytoplasm of actinomycin D-treated Chenopodium amaranticolor cells locally infected with a strain of tobacco necrosis virus (TNV), 3 days after inoculation. Healthy cells did not show such an incorporation. Chloroplasts, in both types of cells, were free of label. Treatment with pancreatic ribonuclease removed the label completely in the majority of nuclei and nucleoli of infected cells. Since infectivity tests showed that AMD treatment increased virus multiplication by 10-12%, it is conceivable to think that the incorporation observed was due to virus synthesis. Preliminary infectivity experiments also showed that treatment of the cells with cycloheximide inhibited virus multiplication up to 80%, while chloramphenicol increased such multiplication. Our results lead to the conclusion that nucleus, nucleolus and cytoplasm but not chloroplasts are the sites involved in the synthesis of TNV. (orig.) [de

  5. The ducky2J mutation in Cacna2d2 results in reduced spontaneous Purkinje cell activity and altered gene expression

    Science.gov (United States)

    Donato, Roberta; Page, Karen M.; Koch, Dietlind; Nieto-Rostro, Manuela; Foucault, Isabelle; Davies, Anthony; Wilkinson, Tonia; Rees, Michele; Edwards, Frances A.; Dolphin, Annette C.

    2006-01-01

    The mouse mutant ducky and its allele ducky2J represent a model for absence epilepsy characterized by spike-wave seizures, and cerebellar ataxia. These mice have mutations in Cacna2d2, which encodes the α2δ-2 calcium channel subunit. Of relevance to the ataxic phenotype, α2δ-2 mRNA is strongly expressed in cerebellar Purkinje cells (PCs). The Cacna2d2du2J mutation results in a two base-pair deletion in the coding region and a complete loss of α2δ-2 protein. Here we show that du2J/du2J mice have a 30% reduction in somatic calcium current, and a marked fall in the spontaneous PC firing rate at 22°C, accompanied by a decrease in firing regularity, which is not affected by blocking synaptic input to PCs. At 34°C du2J/du2J PCs show no spontaneous intrinsic activity. Du2J/du2J mice also have alterations in the cerebellar expression of several genes related to PC function. At P21 there is an elevation of tyrosine hydroxylase mRNA and a reduction in tenascin-C gene expression. Although du2J/+ mice have a marked reduction in α2δ-2 protein, they show no fall in PC somatic calcium currents or increase in cerebellar tryrosine hydroxylase gene expression. However, du2J/+ PCs do exhibit a significant reduction in firing rate, correlating with the reduction in α2δ-2. A hypothesis for future study is that effects on gene expression occur as a result of a reduction in somatic calcium currents, whereas effects on PC firing occur as a long-term result of loss of α2δ-2 and/or a reduction in calcium currents and calcium-dependent processes in regions other than the soma. PMID:17135419

  6. Exogenous Gene Integration for Microalgal Cell Transformation Using a Nanowire-Incorporated Microdevice.

    Science.gov (United States)

    Bae, Sunwoong; Park, Seunghye; Kim, Jung; Choi, Jong Seob; Kim, Kyung Hoon; Kwon, Donguk; Jin, EonSeon; Park, Inkyu; Kim, Do Hyun; Seo, Tae Seok

    2015-12-16

    Superior green algal cells showing high lipid production and rapid growth rate are considered as an alternative for the next generation green energy resources. To achieve the biomass based energy generation, transformed microalgae with superlative properties should be developed through genetic engineering. Contrary to the normal cells, microalgae have rigid cell walls, so that target gene delivery into cells is challengeable. In this study, we report a ZnO nanowire-incorporated microdevice for a high throughput microalgal transformation. The proposed microdevice was equipped with not only a ZnO nanowire in the microchannel for gene delivery into cells but also a pneumatic polydimethylsiloxane (PDMS) microvalve to modulate the cellular attachment and detachment from the nanowire. As a model, hygromycin B resistance gene cassette (Hyg3) was functionalized on the hydrothermally grown ZnO nanowires through a disulfide bond and released into green algal cells, Chlamydomonas reinhardtii, by reductive cleavage. During Hyg3 gene delivery, a monolithic PDMS membrane was bent down, so that algal cells were pushed down toward ZnO nanowires. The supply of vacuum in the pneumatic line made the PDMS membrane bend up, enabling the gene delivered algal cells to be recovered from the outlet of the microchannel. We successfully confirmed Hyg3 gene integrated in microalgae by amplifying the inserted gene through polymerase chain reaction (PCR) and DNA sequencing. The efficiency of the gene delivery to algal cells using the ZnO nanowire-incorporated microdevice was 6.52 × 10(4)- and 9.66 × 10(4)-fold higher than that of a traditional glass bead beating and electroporation.

  7. Quantitation of specific myeloid cells in rat bone marrow measured by in vitro /sup 35/S-sulphate incorporation

    Energy Technology Data Exchange (ETDEWEB)

    Wright, A F; Rose, M S

    1984-08-01

    A biochemical measurement which can be used for quantitation of specific early myeloid cells in rat bone marrow has been developed. This measurement consists of a rapid, simple assay for the in vitro quantitation of /sup 35/S-sulfate incorporation into rat bone marrow cells. Incubation of bone marrow cells with /sup 35/S-sulfate led to a time-dependent increase in radioactivity obtained in perchloric acid insoluble fractions of bone marrow cell suspensions. This incorporation was inhibited by cyanide and puromycin. Autoradiography has demonstrated the radiolabel to be specifically associated with immature cells of the myeloid series. The cells most active in this respect were eosinophils. When rats were treated with endotoxin, the rate of /sup 35/S-sulfate incorporation was increased. Cell number measurements, using conventional histopathology and a Coulter Counter, demonstrated that endotoxin caused an initial release of mature granulocytes from the bone marrow. The regeneration of this mature population in the marrow was rapid, and was characterized by an increase in the number of immature cells and a concomitant increase in the rate of /sup 35/S-sulfate incorporation measured in preparations of bone marrow cells in vitro. Furthermore, this response to endotoxin has demonstrated that Coulter Counting techniques can be used to distinguish specific populations of cells (e.g. mature granulocytes) within the bone marrow.

  8. Preliminary morphological and morphometric study of rat cerebellum following sodium arsenite exposure during rapid brain growth (RBG) period

    International Nuclear Information System (INIS)

    Dhar, Pushpa; Mohari, Nivedita; Mehra, Raj D.

    2007-01-01

    The effects of arsenic exposure during rapid brain growth (RBG) period were studied in rat brains with emphasis on the Purkinje cells of the cerebellum. The RBG period in rats extends from postnatal day 4 (PND 4) to postnatal day 10 (PND 10) and is reported to be highly vulnerable to environmental insults. Mother reared Wistar rat pups were administered intraperitoneal injections (i.p.) of sodium arsenite (aqueous solution) in doses of 1.0, 1.5 and 2.0 mg/kg body weight (bw) to groups II, III and IV (n = 6 animals/group) from PND 4 to 10 (sub acute). Control animals (group I) received distilled water by the same route. On PND 11, the animals were perfusion fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (PB) with pH 7.4. The cerebellum obtained from these animals was post-fixed and processed for paraffin embedding. Besides studying the morphological characteristics of Purkinje cells in cresyl violet (CV) stained paraffin sections (10 μm), morphometric analysis of Purkinje cells was carried out using Image Analysis System (Image Proplus software version 4.5) attached to Nikon Microphot-FX microscope. The results showed that on PND 11, the Purkinje cells were arranged in multiple layers extending from Purkinje cell layer (PL) to outer part of granule cell layer (GL) in experimental animals (contrary to monolayer arrangement within PL in control animals). Also, delayed maturation (well defined apical cytoplasmic cones and intense basal basophilia) was evident in Purkinje cells of experimental animals on PND 11. The mean Purkinje cell nuclear area was significantly increased in the arsenic treated animals compared to the control animals. The observations of the present study (faulty migration, delayed maturation and alteration in nuclear area measurements of Purkinje cells subsequent to arsenic exposure) thus provided the morphological evidence of structural alterations subsequent to arsenite induced developmental neurotoxicity which could be presumed to be

  9. Revised mechanism of d-alanine incorporation into cell wall polymers in Gram-positive bacteria

    Science.gov (United States)

    Reichmann, Nathalie T.; Cassona, Carolina Picarra

    2013-01-01

    Teichoic acids (TAs) are important for growth, biofilm formation, adhesion and virulence of Gram-positive bacterial pathogens. The chemical structures of the TAs vary between bacteria, though they typically consist of zwitterionic polymers that are anchored to either the peptidoglycan layer as in the case of wall teichoic acid (WTA) or the cell membrane and named lipoteichoic acid (LTA). The polymers are modified with d-alanines and a lack of this decoration leads to increased susceptibility to cationic antimicrobial peptides. Four proteins, DltA–D, are essential for the incorporation of d-alanines into cell wall polymers and it has been established that DltA transfers d-alanines in the cytoplasm of the cell onto the carrier protein DltC. However, two conflicting models have been proposed for the remainder of the mechanism. Using a cellular protein localization and membrane topology analysis, we show here that DltC does not traverse the membrane and that DltD is anchored to the outside of the cell. These data are in agreement with the originally proposed model for d-alanine incorporation through a process that has been proposed to proceed via a d-alanine undecaprenyl phosphate membrane intermediate. Furthermore, we found that WTA isolated from a Staphylococcus aureus strain lacking LTA contains only a small amount of d-alanine, indicating that LTA has a role, either direct or indirect, in the efficient d-alanine incorporation into WTA in living cells. PMID:23858088

  10. The effect of insulin on amino acid incorporation into exocrine pancreatic cells of the rat

    International Nuclear Information System (INIS)

    Kramer, M.F.; Poort, C.

    1975-01-01

    The rate of incorporation of radioactive leucine per cell in the acinar pancreatic cells of the rat increases by 50 per cent within one hour after subcutaneous administration of insulin, an effect that lasts for at least one more hour. The rate of incorporation has been measured by quantitative radioautography and by determination of the radioactivity per μg DNA in TCA-precipitable material from tissue homogenates. The capacity for amino acid (leucine and lysine) incorporation as measured by incubating pancreatic fragments in vitro is not enhanced by insulin treatment of the rat in vivo during one or more hours. Insulin was found to lower the serum concentration of most amino acids significantly, leucine by 50 per cent. The apparent effect of insulin on the incorporation of radioactive leucine in vivo can be explained by the difference in the specific radioactivity of the circulating amino acid in the treated rats as compared to the untreated ones. A change in amino acid concentration in the serum may likewise be the explanation of the decrease in amino acid incorporation rate in alloxan diabetic rats. (orig./GSE) [de

  11. Incorporation study of 18FDG through its uptake into tumour-derived cell lines

    International Nuclear Information System (INIS)

    Costa, Flavia M.; Nascimento, Leonardo T.C.; Santos, Raquel G.

    2013-01-01

    In vitro tests of radiopharmaceuticals are an important instrument to study their mechanisms of action, binding and incorporation. 18 FDG is the most used radiopharmaceutical for diagnostics positron emission tomography (PET) on oncology, on the basis of accelerated rates of absorption of glucose in cell malignancies. This radiotracer has been routinely produced at CDTN; and therefore, it was selected for preliminary assays due to its availability. Nowadays, UPPR at CDTN produces routinely 18 FDG for the local PET Centers but others PET radiopharmaceuticals are in development such as 18 F-Fluorocholine and 18 F-Fluorothymidine. According to the Brazilian Health Regulatory Agency (ANVISA) it is necessary to validate and register these new radiopharmaceuticals in order to get the approval for their commercialization. Targeting efficacy is one of the important issues to be evaluated during radiopharmaceutical validation. The aim of this study was to develop a standard protocol to determine tumor targeting efficacy of PET radiopharmaceuticals in vitro. Therefore it was developed a protocol based on the incorporation og 18 FDG through the uptake in different tumor-derived cell lines. Three variables were investigated for the standardization of the test: the number of cells to be seeded in 96-well plates, the time of incubation with the radiopharmaceutical and the radiotracer concentration. The standardized protocol was considered suitable for 18 FDG incorporation assay and showed reproductive results. The protocol developed in this work will pave the way for the in vitro studies of incorporation of the new PET radiopharmaceuticals to be produced at UPPR-CDTN, such as: 18 F-Fluorocholine and 18 F-Fluorothymidine. (author)

  12. In vitro incorporation studies of 99mTc-alendronate sodium at different bone cell lines

    International Nuclear Information System (INIS)

    Evren Gundogdu; Derya Ilem-Ozdemir; Makbule Asikoglu

    2014-01-01

    Bisphosphonates can be labeled with Technetium-99m ( 99m Tc) and are used for bone imaging because of their good localization in the skeleton and rapid clearance from soft tissues. Over the last decades bone scintigraphy has been used extensively in the evaluation of oncological patients to provide information about the sites of bone lesions, their prognosis and the effectiveness of therapy by showing the sequential changes in tracer uptake. Since the lesion visualization and lesion/bone ratio are important utilities for a bone scanning radiopharmaceutic; in this study incorporation of 99m Tc labeled alendronate sodium ( 99m Tc-ALD) was evaluated in U 2 OS (human bone osteosarcoma) and NCI-H209 (human bone carcinoma) cell lines. ALD was directly labeled by 99m Tc, radiochemical purity and stability of the complex were analyzed by radioactive thin layer chromatography and radioactive high performance liquid chromatography studies. For cell incorporation study, NCI-H209 and U 2 OS cell lines were used with standard cell culture methods. The six well plates were used for all experiments and the integrity of each cell monolayer was checked by measuring its transepithelial electrical resistance (TEER) with an epithelial voltammeter. Results confirmed that ALD was successfully radiolabeled with 99m Tc. 99m Tc-ALD incorporated with NCI-H209 and U 2 OS cells. The uptake percentages of 99m Tc-ALD in NCI-H209 and U 2 OS cell lines were found significantly different. Since 99m Tc-ALD highly uptake in cancer cell line, the results demonstrated that radiolabeled ALD may be a promising agent for bone cancer diagnosis. (author)

  13. Staphylococcus aureus Sortase A-Mediated Incorporation of Peptides: Effect of Peptide Modification on Incorporation.

    Directory of Open Access Journals (Sweden)

    Silvie Hansenová Maňásková

    Full Text Available The endogenous Staphylococcus aureus sortase A (SrtA transpeptidase covalently anchors cell wall-anchored (CWA proteins equipped with a specific recognition motif (LPXTG into the peptidoglycan layer of the staphylococcal cell wall. Previous in situ experiments have shown that SrtA is also able to incorporate exogenous, fluorescently labelled, synthetic substrates equipped with the LPXTG motif (K(FITCLPETG-amide into the bacterial cell wall, albeit at high concentrations of 500 μM to 1 mM. In the present study, we have evaluated the effect of substrate modification on the incorporation efficiency. This revealed that (i by elongation of LPETG-amide with a sequence of positively charged amino acids, derived from the C-terminal domain of physiological SrtA substrates, the incorporation efficiency was increased by 20-fold at 10 μM, 100 μM and 250 μM; (ii Substituting aspartic acid (E for methionine increased the incorporation of the resulting K(FITCLPMTG-amide approximately three times at all concentrations tested; (iii conjugation of the lipid II binding antibiotic vancomycin to K(FITCLPMTG-amide resulted in the same incorporation levels as K(FITCLPETG-amide, but much more efficient at an impressive 500-fold lower substrate concentration. These newly developed synthetic substrates can potentially find broad applications in for example the in situ imaging of bacteria; the incorporation of antibody recruiting moieties; the targeted delivery and covalent incorporation of antimicrobial compounds into the bacterial cell wall.

  14. Incorporation of UDPglucose into cell wall glucans and lipids by intact cotton fibers

    International Nuclear Information System (INIS)

    Dugger, W.M.; Palmer, R.L.

    1986-01-01

    The [ 14 C] moiety from [ 3 H]UDP[ 14 C]glucose was incorporated by intact cotton fibers into hot water soluble, acetic-nitric reagent soluble and insoluble components, and chloroform-methanol soluble lipids; the [ 3 H]UDP moiety was not incorporated. The 3 H-label can be exchanged rapidly with unlabeled substrate in a chase experiment. The cell wall apparent free space of cotton fibers was in the order of 30 picomoles per milligram of dry fibers; 25 picomoles per milligram easily exchanged and about 5 picomoles per milligram more tightly adsorbed. At 50 micromolar UDPglucose, 70% of the [ 14 C]glucose was found in the lipid fraction after both a short labeling period and chase. The percent of [ 14 C]glucose incorporated into total glucan increased within a 30-minute chase period. The data supports the concept that glucan synthesis, including cellulose, as well as the synthesis of steryl glucosides, acetylated steryl glucosides, and glucosyl-phosphoryl-polyprenol from externally supplied UDPglucose occurs at the plasma membrane-cell wall interface. The synthase enzymes for such synthesis must be part of this interfacial membrane system

  15. Enhanced incorporation of fatty acid into phosphatidyl choline that parallels histamine discharge in mast cells

    International Nuclear Information System (INIS)

    Castle, J.D.; Castle, A.M.; Ma, A.K.; Stukenbrok, H.

    1984-01-01

    Purified rat peritoneal and pleural mast cells preincubated briefly with radioactively labeled fatty acid were treated with A23187, which bypasses primary receptors in stimulating exocytosis. An enhanced incorporation of fatty acid into phosphatidyl choline (PC) that occurred in parallel with histamine release at 24-25 degrees C was observed and was initially proportional to the total amount of histamine discharged. Enhanced PC labeling and histamine secretion were also proportional at temperatures ranging from 17-37 degrees C. Both radioactive linoleic and palmitic acids were incorporated selectively at the beta-position of the glycerol backbone of PC. PC labeling by [3H]choline was not detectably different in control and stimulated cells, and phosphatidic acid did not exhibit selectively enhanced beta-acylation. Thus, the stimulated labeling in A23187-treated cells may occur secondary to the action of a phospholipase A2 that favors PC as a substrate. Other peritoneal cell types exhibit a very similar A23187-stimulated selective labeling of PC. Therefore, autoradiography has been used to provide a direct demonstration that in purified preparations, mast cells are the principal cell type engaged in A23187-elicited incorporation of fatty acid into PC. The efficacy of this approach has relied on special procedures devised to obtain significantly different autoradiographic grain densities between control and stimulated preparations that can be attributed to differences in the level of [3H]palmitate-labeled PC. Preliminary tests using compound 48/80 as a secretory stimulus for mast cells have identified a similar selectively enhanced PC labeling. In either case, however, consideration of possible relationships between PC metabolism and the secretory process are premature since they have not been tested directly

  16. Incorporation of radioactivity from [14C]lactate into the glycogen of cultured mouse astroglial cells. Evidence for gluconeogenesis in brain cells.

    Science.gov (United States)

    Dringen, R; Schmoll, D; Cesar, M; Hamprecht, B

    1993-05-01

    A pure population of astroglial cells was selected from heterogeneous astroglia-rich primary cultures in a medium containing sorbitol instead of glucose. It was shown that astroglial cells synthesize glycogen when they are returned to a glucose-containing medium, and that when [14C]lactate is also present the synthesized glycogen is radioactively labelled. Compared with the degree of incorporation of radioactivity in the presence of tritiated glucose, the incorporation of radioactivity from lactate was small but significant. After incubation of astroglial cells with radioactively labelled lactate, the glycogen was isolated and enzymatically hydrolysed to glucose, which was found to be radioactively labelled. Astrocytes are therefore able to convert lactate to glucosyl residues, a metabolic pathway known as gluconeogenesis. It is proposed that astrocytic gluconeogenesis may consume lactic acid formed in neighboring cells such as neurons, during anaerobic glycolysis at times of high energy demand.

  17. A cerebellar learning model of vestibulo-ocular reflex adaptation in wild-type and mutant mice.

    Science.gov (United States)

    Clopath, Claudia; Badura, Aleksandra; De Zeeuw, Chris I; Brunel, Nicolas

    2014-05-21

    Mechanisms of cerebellar motor learning are still poorly understood. The standard Marr-Albus-Ito theory posits that learning involves plasticity at the parallel fiber to Purkinje cell synapses under control of the climbing fiber input, which provides an error signal as in classical supervised learning paradigms. However, a growing body of evidence challenges this theory, in that additional sites of plasticity appear to contribute to motor adaptation. Here, we consider phase-reversal training of the vestibulo-ocular reflex (VOR), a simple form of motor learning for which a large body of experimental data is available in wild-type and mutant mice, in which the excitability of granule cells or inhibition of Purkinje cells was affected in a cell-specific fashion. We present novel electrophysiological recordings of Purkinje cell activity measured in naive wild-type mice subjected to this VOR adaptation task. We then introduce a minimal model that consists of learning at the parallel fibers to Purkinje cells with the help of the climbing fibers. Although the minimal model reproduces the behavior of the wild-type animals and is analytically tractable, it fails at reproducing the behavior of mutant mice and the electrophysiology data. Therefore, we build a detailed model involving plasticity at the parallel fibers to Purkinje cells' synapse guided by climbing fibers, feedforward inhibition of Purkinje cells, and plasticity at the mossy fiber to vestibular nuclei neuron synapse. The detailed model reproduces both the behavioral and electrophysiological data of both the wild-type and mutant mice and allows for experimentally testable predictions. Copyright © 2014 the authors 0270-6474/14/347203-13$15.00/0.

  18. Incorporation of Biomaterials in Multicellular Aggregates Modulates Pluripotent Stem Cell Differentiation

    Science.gov (United States)

    Bratt-Leal, Andrés M.; Carpenedo, Richard L.; Ungrin, Mark; Zandstra, Peter W.; McDevitt, Todd C.

    2010-01-01

    Biomaterials are increasingly being used to engineer the biochemical and biophysical properties of the extracellular stem cell microenvironment in order to tailor niche characteristics and direct cell phenotype. To date, stem cell-biomaterial interactions have largely been studied by introducing stem cells into artificial environments, such as 2D cell culture on biomaterial surfaces, encapsulation of cell suspensions within hydrogel materials, or cell seeding on 3D polymeric scaffolds. In this study, microparticles fabricated from different materials, such as agarose, PLGA and gelatin, were stably integrated, in a dose-dependent manner, within aggregates of pluripotent stem cells (PSCs) prior to differentiation as a means to directly examine stem cell-biomaterial interactions in 3D. Interestingly, the presence of the materials within the stem cell aggregates differentially modulated the gene and protein expression patterns of several differentiation markers without adversely affecting cell viability. Microparticle incorporation within 3D stem cell aggregates can control the spatial presentation of extracellular environmental cues (i.e. soluble factors, extracellular matrix and intercellular adhesion molecules) as a means to direct the differentiation of stem cells for tissue engineering and regenerative medicine applications. In addition, these results suggest that the physical presence of microparticles within stem cell aggregates does not compromise PSC differentiation, but in fact the choice of biomaterials can impact the propensity of stem cells to adopt particular differentiated cell phenotypes. PMID:20864164

  19. Incorporation of 35S-sulfate and 3H-glucosamine into heparan and chondroitin sulfates during the cell cycle of B16-F10 cells

    International Nuclear Information System (INIS)

    Blair, O.C.; Sartorelli, A.C.

    1984-01-01

    Changes in glycosaminoglycan composition occurring during the cell cycle were determined in B16-F10 cells sorted flow cytometrically with respect to DNA content. Incorporation of 35 S-sulfate into heparan sulfate and chondroitin sulfate of unsorted and G1,S, and G2 +M sorted cells was determined following chondroitinase ABC or nitrous acid treatment; the incorporation into surface material was measured as the difference between the radioactivity of control and trypsin-treated cells. Incorporation of 35 S-sulfate and 3 H-glucosamine into cetyl pyridinium chloride (CPC)-precipitable material was characterized before and after chondroitinase or nitrous acid treatment by Sephadex G50 chromatography. Long-term (48 h) and short-term (1 h) labeling studies demonstrate that (a) the amount of total cellular chondroitin sulfate is greater than that of heparan sulfate, with larger amounts of unsulfated heparan than chondroitin being present; (b) the rate of turnover of heparan sulfate is greater than that of chondroitin sulfate; (c) greatest short-term incorporation of 3H-glucosamine into CPC-precipitable material occurs during S phase; and (d) the rate of turnover of both heparan sulfate and chondroitin sulfate is decreased in S phase relative to G1 and G2 + M

  20. Perturbation of N-linked oligosaccharide structure results in an altered incorporation of [3H]palmitate into specific proteins in Chinese hamster ovary cells

    International Nuclear Information System (INIS)

    Wellner, R.B.; Ghosh, P.C.; Roecklein, B.; Wu, H.C.

    1987-01-01

    Increased [ 3 H]palmitate incorporation into specific cellular proteins has been reported to occur in Chinese hamster ovary and yeast mutant cells. In this paper we report studies concerning the relationship between N-linked oligosaccharide structure and [ 3 H]palmitate incorporation into proteins of Chinese hamster ovary (CHO) cells. We have compared the incorporation of [ 3 H]palmitate into proteins of wild-type and four different mutant CHO cell lines defective in various steps of N-linked protein glycosylation. Sodium dodecyl sulfate-gel electrophoretic analysis showed that three of the mutants exhibited increased [ 3 H]palmitate incorporation into several CHO cellular proteins (approximately 30,000-38,000 molecular weight) as compared to the wild-type cells. One of the affected mutants which accumulates the Man5Gn2Asn intermediate structure was examined in detail. In agreement with earlier reports, virtually all of the [ 3 H] palmitate-labeled proteins of both wild-type and mutant cell lines are membrane-bound. Pretreatment of the mutant cell line with tunicamycin blocked the increased [ 3 H]palmitate incorporation into the two specific proteins (both of approximately 30,000 molecular weight) observed in untreated cells; the decreased incorporation of [ 3 H]palmitate into the 30,000 molecular weight species was accompanied by a concomitant increase in the incorporation of [ 3 H]palmitate into two proteins of approximately 20,000 molecular weight. Pretreatment of wild-type cells with tunicamycin also caused increased [ 3 H]palmitate incorporation into the 20,000 molecular weight species

  1. Incorporation of glycine and serine into sporulating cells of Bacillus subtilis

    International Nuclear Information System (INIS)

    Mitani, Takahiko; Kadota, Hajime

    1976-01-01

    The changes during growth and sporulation in activities of cells of Bacillus subtilis to incorporate various amino acids were investigated with wild-type strain and its asporogenous mutant. In the case of wild type strain the uptake of valine, phenylalanine, and proline was largest during the logarithmic growth period. The uptake of these amino acids decreased rapidly during the early stationary phase. The uptake of valine and cysteine increased again to some extent just prior to the forespore stage. The uptake of glycine and serine, however, was largest at the forespore stage at which the formation of spore coat took place. From these observed phenomena it was assumed that the remarkable incorporation of glycine and serine into the wild type strain during sporulation was closely related to the formation of spore coat. (auth.)

  2. Radiometric study of the incorporation of labelled precursors of DNA, RNA and proteins in cell cultures inoculated with Herpes simplex virus

    International Nuclear Information System (INIS)

    Dundarov, S.; Todorov, S.; Kavaklova, L.; Tsilka, E.; Rangelova, S.; Dundarova, D.

    1976-01-01

    Results are reported of a comparative investigation of tritium-labelled thymidine, uridine and leucine incorporation in cells inoculated with HSV-1 and HSV-2. There were no essential differences in the dynamics of the biosynthesis processes in either type of herpes viruses. Viral infection led to a marked activation of DNA synthesis and progressive inhibition of overall RNA synthesis. The rate of labelled amino-acid incorporation remained almost unchanged. There was a gradual increase in 3 H-thymidine incorporation, reaching a minimum by the 12th-16th hour and a rapid decrese thereafter. After the 12th hour part of the thymidine already incorporated in DNA (the thymidine pool) starts leaving the cells as a result of increased cell membrane permeability. The rate of changes in the biosynthesis processes in HSV-inoculated cells depends on infection multiplicity. Even in high inoculation multiplicity the blockade of cell biosynthesis is incomplete which furnishes a better opportunity for HSV reproduction. (author)

  3. Ternary solution-processed organic solar cells incorporating 2D materials

    Science.gov (United States)

    Stylianakis, Minas M.; Konios, Dimitrios; Petridis, Constantinos; Kakavelakis, George; Stratakis, Emmanuel; Kymakis, Emmanuel

    2017-12-01

    Recently, the study of ternary organic solar cells (OSCs) has attracted the efforts of the scientific community, leading to significantly higher performance due to the enhanced harvesting of incoming irradiation. Here, for the first time, and in order to promote this OSC architecture, we review the progress implemented by the application of two-dimensional (2D) materials in the field of blend bulk heterojunction ternary OSCs. Power conversion efficiency (PCE) improvements of the order of 40% compared to the reference binary devices, and PCEs in excess of 8% have been reported by incorporating graphene-based or other 2D materials as a third element inside the active layer. These OSCs combine the synergetic advantages of ternary devices and the superb properties of the 2D material family. In conclusion, the incorporation of the unique properties of graphene and other 2D materials inside the active layer opens up a very promising pathway in the design and construction of high-performance, simply fabricated and low- cost photovoltaic devices.

  4. Incorporating placental tissue in cord blood banking for stem cell transplantation.

    Science.gov (United States)

    Teofili, Luciana; Silini, Antonietta R; Bianchi, Maria; Valentini, Caterina Giovanna; Parolini, Ornella

    2018-06-01

    Human term placenta is comprised of various tissues from which different cell populations can be obtained, including hematopoietic stem cells and mesenchymal stem/stromal cells (MSCs). Areas covered: This review will discuss the possibility to incorporate placental tissue cells in cord blood banking. It will discuss general features of human placenta, with a brief review of the immune cells at the fetal-maternal interface and the different cell populations isolated from placenta, with a particular focus on MSCs. It will address the question as to why placenta-derived MSCs should be banked with their hematopoietic counterparts. It will discuss clinical trials which are studying safety and efficacy of placenta tissue-derived MSCs in selected diseases, and preclinical studies which have proven their therapeutic properties in other diseases. It will discuss banking of umbilical cord blood and raise several issues for improvement, and the applications of cord blood cells in non-malignant disorders. Expert Commentary: Umbilical cord blood banking saves lives worldwide. The concomitant banking of non-hematopoietic cells from placenta, which could be applied therapeutically in the future, alone or in combination to their hematopoietic counterparts, could exploit current banking processes while laying the foundation for clinical trials exploring placenta-derived cell therapies in regenerative medicine.

  5. STD-dependent and independent encoding of input irregularity as spike rate in a computational model of a cerebellar nucleus neuron

    NARCIS (Netherlands)

    J. Luthman (Johannes); F.E. Hoebeek (Freek); R. Maex (Reinoud); N. Davey (Neil); R. Adams (Rod); C.I. de Zeeuw (Chris); V. Steuber (Volker)

    2011-01-01

    textabstractNeurons in the cerebellar nuclei (CN) receive inhibitory inputs from Purkinje cells in the cerebellar cortex and provide the major output from the cerebellum, but their computational function is not well understood. It has recently been shown that the spike activity of Purkinje cells is

  6. Normal iron absorption determined by means of whole body counting and red cell incorporation of 59Fe

    International Nuclear Information System (INIS)

    Larsen, L.; Milman, N.

    1977-01-01

    Gastrointestinal iron absorption was measured in 27 normal subjects (19 females and 8 males) by means of whole body counting. Whole body retention 14 days after oral administration of 10μCi 59 Fe together with a carrier dose of 9.9 mg Fe 2+ (as sulphate), was used as an expression of absorption. The percentage incorporation in the total erythrocyte mass of administered 59 Fe (erythrocyte incorporation) and of absorbed 59 Fe (red cell utilization) was also estimated. Geometric mean iron absorption was 8.3+-2.1 (SD% in females, 9.1+-2.2 % in males and 8.5+-2.1 % in the entire series. The difference between males and females was not significant. Erythrocyte incorporation was 7.7+-2.2 (SD) % (geometric mean) in the entire series and the correlation between iron absorption and erythrocyte incorporation was highly significant (r = 0.96,P < 0.001). Red cell utilization averaged 92.9 +- 4.0 (SEM)% (arithmetic mean) in the entire series. (author)

  7. The effects of functional magnetic nanotubes with incorporated nerve growth factor in neuronal differentiation of PC12 cells

    International Nuclear Information System (INIS)

    Xie Jining; Chen Linfeng; Varadan, Vijay K; Yancey, Justin; Srivatsan, Malathi

    2008-01-01

    In this in vitro study the efficiency of magnetic nanotubes to bind with nerve growth factor (NGF) and the ability of NGF-incorporated magnetic nanotubes to release the bound NGF are investigated using rat pheochromocytoma cells (PC12 cells). It is found that functional magnetic nanotubes with NGF incorporation enabled the differentiation of PC12 cells into neurons exhibiting growth cones and neurite outgrowth. Microscope observations show that filopodia extending from neuron growth cones were in close proximity to the NGF-incorporated magnetic nanotubes, at times appearing to extend towards or into them. These results show that magnetic nanotubes can be used as a delivery vehicle for NGF and thus may be exploited in attempts to treat neurodegenerative disorders such as Parkinson's disease with neurotrophins. Further neurite outgrowth can be controlled by manipulating magnetic nanotubes with external magnetic fields, thus helping in directed regeneration

  8. The morpho/functional discrepancy in the cerebellar cortex: Looks alone are deceptive.

    Directory of Open Access Journals (Sweden)

    Dan Rokni

    2008-12-01

    Full Text Available In a recent report we demonstrated that stimulation of cerebellar mossy fibers synchronously activates Purkinje cells that are located directly above the site of stimulation. We found that the activated Purkinje cells are arranged in a radial patch on the cerebellar surface and that this organization is independent of the integrity of the inhibitory system. This arrangement of activity is counterintuitive. The anatomical structure with the extensive parallel fiber system implies that mossy fiber stimulation will activate Purkinje cells along a beam of parallel fibers. In this short review we highlight this discrepancy between anatomical structure and functional dynamics and suggest a plausible underlying mechanism.

  9. Enhancement of the incorporation of 5-fluorodeoxyuridylate into DNA of HL-60 cells by metabolic modulations

    International Nuclear Information System (INIS)

    Tanaka, M.; Kimura, K.; Yoshida, S.

    1983-01-01

    The exposure of HL-60 human promyelocytic leukemia cells to 0.5 microM 5-fluoro-2'-[ 3 H]deoxyuridine (FdUrd) for 16 hr resulted in the incorporation of 5.14 +/- 0.31 (S.D.) X 10(-7) mol FdUrd into DNA per mol of DNA nucleotide, which corresponds to 0.146 +/- 0.082 pmol FdUrd per 10(7) cells. Pretreatment with 50 microM deoxythymidine for 24 hr led to a 2.7-fold increase in the incorporation of this analogue into newly synthesized DNA during the ensuing 16-hr exposure to 0.5 microM [ 3 H]FdUrd. Pretreatment with 0.5 microM methotrexate for 3 hr also increased the [ 3 H]FdUrd incorporation into newly synthesized DNA approximately 5-fold. The coexistence of deoxythymidine or methotrexate with [ 3 H]FdUrd, however, led to decreased incorporation of FdUrd into DNA. More than 50% of the radioactivity in DNA separated by Cs2SO4 equilibrium density gradient centrifugation was proven to be fluorodeoxyuridylate by means of its binding to Lactobacillus casei deoxythymidine monophosphate synthetase

  10. Inhibition of exogenous 3-deoxy-D-manno octulosonate incorporation into lipid A precursor of toluene-treated Salmonella typhimurium cells

    International Nuclear Information System (INIS)

    Capobianco, J.O.; Darveau, R.P.; Goldman, R.C.; Lartey, P.A.; Pernet, A.G.

    1987-01-01

    Analogs of 3-deoxy-D-manno-octulosonate (KDO) were designed to inhibit CTP:CMP-KDO cytidylyltransferase (CMP-KDO synthetase). Since these analogs lacked whole-cell antibacterial activity, a permeabilized-cell method was developed to measure intracellular compound activity directly. The method employed a mutant of Salmonella typhimurium defective in KDO-8-phosphate synthetase (kdsA), which accumulated lipid A precursor at 42 0 C. Cells permeabilized with 1% toluene were used to evaluate inhibitor effect on [ 3 H]KDO incorporation into preformed lipid A precursor. KDO incorporation proceeded through the enzymes CMP-DKO synthetase and CMP-KDO:lipid A KDO transferase. Optimum KDO incorporation occurred between pH 8 and 9 and required CTP, prior lipid A precursor accumulation, and a functional kdsB gene product, CMP-KDO synthetase. The apparent K/sub m/ for KDO in this coupled system at pH 7.6 was 1.38 mM. The reaction products isolated and characterized contained 1 and 2 KDO residues per lipid A precursor molecule. Several KDO analogs produced concentration-related reductions of DKO incorporation in toluenized cells with 50% inhibitor concentrations comparable to those obtained in purified CMP-DKO synthetase systems. Two compounds, 8-amino-2-deoxy-KDO (A-60478) and 8-aminomethyl-2-deoxy-KDO (A-60821), competitively inhibited KDO incorporation, displaying K/sub i/s of 4.2 + M for A=60478 and 2.5 + M for A-60821

  11. Selective incorporation of various C-22 polyunsaturated fatty acids in Ehrlich ascites tumor cells

    International Nuclear Information System (INIS)

    Masuzawa, Y.; Okano, S.; Waku, K.; Sprecher, H.; Lands, W.E.

    1986-01-01

    Three 14 C-labeled 22-carbon polyunsaturated fatty acids, 7,10,13,16-[ 14 C]docosatetraenoic acid (22:4(n-6)), 7,10,13,16,19-[ 14 C]docosapentaenoic acid (22:5(n-3)), and 4,7,10,13,16,19-[ 14 C]docosahexaenoic acid (22:6(n-3)), were compared with [ 3 H]arachidonic acid (20:4(n-6] and [14C]linoleic acid (18:2(n-6)) to characterize their incorporation into the lipids of Ehrlich ascites cells. The relatively rapid incorporation of the labeled 22-carbon acids into phosphatidic acid indicated that substantial amounts of these acids may be incorporated through the de novo pathway of phospholipid synthesis. In marked contrast to 20:4(n-6), the 22-carbon acids were incorporated much less into choline glycerophospholipids (CGP) and inositol glycerophospholipids (IGP). No selective preference was apparent for the (n-3) or (n-6) type of fatty acids. The amounts of the acids incorporated into diacylglycerophosphoethanolamine were in the order of: 22:6(n-3) greater than 20:4(n-6) much greater than 22:5(n-3) greater than or equal to 22:4(n-6) greater than 18:2(n-6), whereas for alkylacylglycerophosphoethanolamine they were in the order of: 22:4(n-6) greater than 22:6(n-3) greater than 22:5(n-3) much greater than 20:4(n-6) greater than 18:2(n-6). Of the mechanisms possibly responsible for the selective entry of 22-carbon acids into ethanolamine glycerophospholipids, the most reasonable explanation was that the cytidine-mediated ethanolamine phosphotransferase may have a unique double selectivity: for hexaenoic species of diacylglycerol and for 22-carbon polyunsaturated fatty acid-containing species of alkylacylglycerol. The relative distribution of fatty acids between newly incorporated and already maintained lipid classes suggested that IGP may function in Ehrlich cells as an intermediate pool for the retention of polyunsaturated fatty acids in glycerolipids

  12. Radiation-induced DNA damage in halogenated pyrimidine incorporated cells and its correlation with radiosensitivity

    International Nuclear Information System (INIS)

    Watanabe, R.; Nikjoo, H.

    2003-01-01

    Cells with DNA containing 5-halogenated pyrimidines in place of thymidine show significant reductions of slope (Do) and shoulder (Dq) of their radiation survival curves. Similar radiosensitization has also been observed in the yield of DNA strand breaks. The purpose of this study is to obtain an insight into the mechanism of cell lethality by examining the relationship between the spectrum of DNA damage and the cell survival. In this study we estimated the enhancement of strand breaks due to incorporation of halogenated pyrimidine, the complexity of DNA damage and the probability of the initial DNA damage leading to cell inactivation. Monte Carlo track structure methods were used to model and simulate the induction of strand breakage by X-rays. The increase of DNA strand break was estimated by assuming the excess strand break was caused by the highly reactive uracil radicals at the halouracil substituted sites. The assumption of the enhancement mechanism of strand breaks was examined and verified by comparison with experimental data for induction of SSB and DSB. The calculated DNA damage spectrum shows the increase in complexity of strand breaks is due to incorporation of halogenated pyrimidines. The increase in the yield of DSB and cell lethality show similar trend at various degrees of halogenated pyrimidine substitution. We asked the question whether this agreement supports the hypothesis that DSB is responsible for cell lethality? The estimated number of lethal damage from the cell survival using a linear-quadratic model is much less than the initial yield of DSB. This work examines the correlation of cell lethality as a function of frequencies of complex form of double strand breaks

  13. Temporal Sequence of Autolysis in the Cerebellar Cortex of the Mouse.

    Science.gov (United States)

    Finnie, J W; Blumbergs, P C; Manavis, J

    2016-05-01

    This study examined the temporal sequence of post-mortem changes in the cerebellar cortical granular and Purkinje cell layers of mice kept at a constant ambient temperature for up to 4 weeks. Nuclei of granule cell microneurons became pyknotic early after death, increasing progressively until, by 7 days, widespread nuclear lysis resulted in marked cellular depletion of the granular layer. Purkinje cells were relatively unaltered until about 96 h post mortem, at which time there was shrinkage and multivacuolation of the amphophilic cytoplasm, nuclear hyperchromasia and, sometimes, a perinuclear clear space. By 7 days, Purkinje cells had hypereosinophilic cytoplasm and frequent nuclear pyknosis. By 2 weeks after death, Purkinje cells showed homogenization, the cytoplasm being uniformly eosinophilic, progressing to a 'ghost-like' appearance in which the cytoplasm had pale eosinophilic staining with indistinct cell boundaries, and nuclei often absent. The results of this study could assist in differentiating post-mortem autolysis from ante-mortem lesions in the cerebellar cortex and determining the post-mortem interval. Moreover, this information could be useful when interpreting brain lesions in valuable mice found dead unexpectedly during the course of biomedical experiments. Crown Copyright © 2016. Published by Elsevier Ltd. All rights reserved.

  14. Incorporation of graphene into SnO2 photoanodes for dye-sensitized solar cells

    International Nuclear Information System (INIS)

    Batmunkh, Munkhbayar; Dadkhah, Mahnaz; Shearer, Cameron J.; Biggs, Mark J.; Shapter, Joseph G.

    2016-01-01

    Graphical abstract: Incorporation of a graphene structure into SnO 2 dye-sensitized solar cell photoanode films has been demonstrated for the first time. The use of graphene in the SnO 2 has been found to be a promising strategy to address many problems of photovoltaic cells based on SnO 2 photoanodes. - Highlights: • SnO 2 -reduced graphene oxide (RGO) hybrid is prepared using a microwave technique. • The first SnO 2 -RGO photoanode based DSSC is fabricated. • Use of RGO addresses the major shortcoming of SnO 2 when employed as a DSSC photoanode. • RGO significantly improved the electron transport rate within the DSSC devices. • Incorporation of RGO into the SnO 2 photoanode enhanced the DSSC efficiency by 91.5%. - Abstract: In dye-sensitized solar cell (DSSC) photoanodes, tin dioxide (SnO 2 ) structures present a promising alternative semiconducting oxide to the conventional titania (TiO 2 ), but they suffer from poor photovoltaic (PV) efficiency caused by insufficient dye adsorption and low energy value of the conduction band. A hybrid structure consisting of SnO 2 and reduced graphene oxide (SnO 2 -RGO) was synthesized via a microwave-assisted method and has been employed as a photoanode in DSSCs. Incorporation of RGO into the SnO 2 photoanode enhanced the power conversion efficiency of DSSC device by 91.5%, as compared to the device assembled without RGO. This efficiency improvement can be attributed to increased dye loading, enhanced electron transfer and addition of suitable energy levels in the photoanode. Finally, the use of RGO addresses the major shortcoming of SnO 2 when employed as a DSSC photoanode, namely poor dye adsorption and slow electron transfer rate.

  15. Effect of polyunsaturated fatty acids and phospholipids on [3H]-vitamin E incorporation into pulmonary artery endothelial cell membranes

    International Nuclear Information System (INIS)

    Sekharam, K.M.; Patel, J.M.; Block, E.R.

    1990-01-01

    Vitamin E, a dietary antioxidant, is presumed to be incorporated into the lipid bilayer of biological membranes to an extent proportional to the amount of polyunsaturated fatty acids or phospholipids in the membrane. In the present study we evaluated the distribution of incorporated polyunsaturated fatty acids (PUFA) and phosphatidylethanolamine (PE) in various membranes of pulmonary artery endothelial cells. We also studied whether incorporation of PUFA or PE is responsible for increased incorporation of [3H]-vitamin E into the membranes of these cells. Following a 24-hr incubation with linoleic acid (18:2), 18:2 was increased by 6.9-, 9.2-, and 13.2-fold in plasma, mitochondrial, and microsomal membranes, respectively. Incorporation of 18:2 caused significant increases in the unsaturation indexes of mitochondrial and microsomal polyunsaturated fatty acyl chains (P less than .01 versus control in both membranes). Incubation with arachidonic acid (20:4) for 24 hr resulted in 1.5-, 2.3-, and 2.4-fold increases in 20:4 in plasma, mitochondrial, and microsomal membranes, respectively. The unsaturation indexes of polyunsaturated fatty acyl chains of mitochondrial and microsomal membranes also increased (P less than .01 versus control in both membranes). Although incubations with 18:2 or 20:4 resulted in several-fold increases in membrane 18:2 or 20:4 fatty acids, incorporation of [3H]-vitamin E into these membranes was similar to that in controls. Following a 24-hr incubation with PE, membrane PE content was significantly increased, and [3H]-vitamin E incorporation was also increased to a comparable degree, i.e., plasma membrane greater than mitochondria greater than microsomes. Endogenous vitamin E content of the cells was not altered because of increased incorporation of PE and [3H]-vitamin E

  16. DNA-incorporated 125I induces more than one double-strand break per decay in mammalian cells.

    Science.gov (United States)

    Elmroth, Kecke; Stenerlöw, Bo

    2005-04-01

    The Auger-electron emitter 125I releases cascades of 20 electrons per decay that deposit a great amount of local energy, and for DNA-incorporated 125I, approximately one DNA double-strand break (DSB) is produced close to the decay site. To investigate the potential of 125I to induce additional DSBs within adjacent chromatin structures in mammalian cells, we applied DNA fragment-size analysis based on pulsed-field gel electrophoresis (PFGE) of hamster V79-379A cells exposed to DNA-incorporated 125IdU. After accumulation of decays at -70 degrees C in the presence of 10% DMSO, there was a non-random distribution of DNA fragments with an excess of fragments even higher. In contrast, using a conventional low-resolution assay without measurement of smaller DNA fragments, the yield was close to one DSB/decay. We conclude that a large fraction of the DSBs induced by DNA-incorporated 125I are nonrandomly distributed and that significantly more than one DSB/decay is induced in an intact cell. Thus, in addition to DSBs produced close to the decay site, DSBs may also be induced within neighboring chromatin fibers, releasing smaller DNA fragments that are not detected by conventional DSB assays.

  17. E-cadherin promotes incorporation of mouse epiblast stem cells into normal development.

    Directory of Open Access Journals (Sweden)

    Satoshi Ohtsuka

    Full Text Available Mouse epiblast stem cells (mEpiSCs are pluripotent stem cells derived from epiblasts of postimplantation mouse embryos. Their pluripotency is distinct from that of mouse embryonic stem cells (mESCs in several cell biological criteria. One of the distinctions is that mEpiSCs contribute either not at all or at much lower efficiency to chimeric embryos after blastocyst injection compared to mESCs. However, here we showed that mEpiSCs can be incorporated into normal development after blastocyst injection by forced expression of the E-cadherin transgene for 2 days in culture. Using this strategy, mEpiSCs gave rise to live-born chimeras from 5% of the manipulated blastocysts. There were no obvious signs of reprogramming of mEpiSCs toward the mESC-like state during the 2 days after induction of the E-cadherin transgene, suggesting that mEpiSCs possess latent ability to integrate into the normal developmental process as its origin, epiblasts.

  18. Amplification of oncogenes and integrated SV40 sequences in mammalian cells by the decay of incorporated iodine-125

    International Nuclear Information System (INIS)

    Ehrfeld, A.; Planas-Bohne, F.; Luecke-Huhle, C.

    1986-01-01

    Iodine-125, in the form of 5-[ 125 I]iododeoxyuridine (I-UdR), was incorporated into the DNA of SV40 transformed Chinese hamster embryo cells. Disintegration of the 125 I led to increased cell killing with increasing dose as measured by the colony-forming ability of single cells. The D37 (the dose at which 37% of the cells survive) amounts to 95 decays per cell, corresponding to 0.66 Gy. Variations in the copy number of specific DNA sequences was measured by using dispersed cell blotting with sensitive DNA hybridizations. A 13-fold amplification of the viral DNA sequences (SV40) and a twofold amplification of two cellular oncogenes of the ras-family (Ki-ras and Ha-ras) were found. Other cellular genes, like the alpha-actin gene, were not amplified, and no variation in gene copy number was detected after incubation of cells with cold I-UdR. We suggest the observed gene amplifications are induced by the densely ionizing radiation emitted by the decay of the incorporated 125 I atoms

  19. Modification of potentially lethal damage in irradiated Chinese hamster V79 cells after incorporation of halogenated pyrimidines

    NARCIS (Netherlands)

    Franken, N. A.; van Bree, C. V.; Kipp, J. B.; Barendsen, G. W.

    1997-01-01

    Radiosensitization of exponentially growing and plateau phase Chinese hamster V79 cells by incorporation of halogenated pyrimidines (HP) was investigated for different culture conditions that influenced repair. For this purpose cells were grown for 72 h with 0, 1, 2 and 4 microM of chloro-(CldUrd),

  20. Actions of exogenous histones and other proteins on [3H]-thymidine incorporation into DNA of Novikoff hepatoma cells

    International Nuclear Information System (INIS)

    Barra, R.; Beres, B.; Koch, M.R.; Lea, M.A.

    1976-01-01

    The effects of exogenous proteins on the incorporation of [ 3 H]-thymidine into DNA was studied in Novikoff hepatoma ascites cells incubated in Eagle's minimal essential medium. A liver cytosol fraction (8 mg protein/ml) caused approximately 80% inhibition of isotope incorporation. The inhibitory activity of cytosol fractions from Morris hepatomas 9618A 2 , 5123C and 20 were inversely related to their growth rate. Under conditions in which there appeared to be a density dependent inhibition of growth, a mean 10 to 20% stimulation of isotope incorporation was observed after addition of total calf thymus histones and individual fractions in the concentration range of 100 to 400μg/ml. In experiments with lower cell concentrations, a 60% or greater increase in [ 3 H]-thymidine incorporation could be obtained with total calf thymus histone and with Fl and arginine-rich histones from rat liver. At concentrations of 1 to 2 mg/ml, histones inhibited DNA synthesis. Bovine serum albumin had little effect on DNA synthesis. Polylysine caused an 80 to 90% inhibition at a concentration of 1 mg/ml, but stimulatory effects were detected under certain conditions at 10μg/ml. The results suggest critical dependence on the ratio of cell and exogenous protein concentration in the action of proteins on DNA synthesis. (author)

  1. Gene amplification in Chinese hamster embryo cells by the decay of incorporated iodine-125

    International Nuclear Information System (INIS)

    Luecke-Huhle, Christine; Ehrfeld, Angelika; Rau, Waltraud

    1988-01-01

    Simian Virus 40-transformed Chinese hamster embryo cells (Co631) contain 5 viral copies integrated per cell genome. These SV40 sequences were used as an endogenous indicator gene to study response of mammalian cells to radiation at gene level. Cells were internally irradiated by Auger electrons emitted by Iodine-125 which was incorporated in cell DNA in form of 5-[ 125 I] iododeoxyuridine ( 125 IdU). An increase in gene copy number was measured using dispersed cell blotting and Southern analysis in combination with highly sensitive DNA hybridization. A 13-fold amplification of the SV40 sequences and a 2-fold amplification of two cellular oncogenes of the ras family were found. Other cellular genes, like the α-actin gene, are not amplified and no variation in gene copy number was observed after incubation of cells with cold IdU. Thus, specific gene amplification seems to be the consequence of radiation-induced DNA damage and the resulting cell cycle arrest. (author)

  2. Distributed Cerebellar Motor Learning; a Spike-Timing-Dependent Plasticity Model

    Directory of Open Access Journals (Sweden)

    Niceto Rafael Luque

    2016-03-01

    Full Text Available Deep cerebellar nuclei neurons receive both inhibitory (GABAergic synaptic currents from Purkinje cells (within the cerebellar cortex and excitatory (glutamatergic synaptic currents from mossy fibres. Those two deep cerebellar nucleus inputs are thought to be also adaptive, embedding interesting properties in the framework of accurate movements. We show that distributed spike-timing-dependent plasticity mechanisms (STDP located at different cerebellar sites (parallel fibres to Purkinje cells, mossy fibres to deep cerebellar nucleus cells, and Purkinje cells to deep cerebellar nucleus cells in close-loop simulations provide an explanation for the complex learning properties of the cerebellum in motor learning. Concretely, we propose a new mechanistic cerebellar spiking model. In this new model, deep cerebellar nuclei embed a dual functionality: deep cerebellar nuclei acting as a gain adaptation mechanism and as a facilitator for the slow memory consolidation at mossy fibres to deep cerebellar nucleus synapses. Equipping the cerebellum with excitatory (e-STDP and inhibitory (i-STDP mechanisms at deep cerebellar nuclei afferents allows the accommodation of synaptic memories that were formed at parallel fibres to Purkinje cells synapses and then transferred to mossy fibres to deep cerebellar nucleus synapses. These adaptive mechanisms also contribute to modulate the deep-cerebellar-nucleus-output firing rate (output gain modulation towards optimising its working range.

  3. Evidence that Vpu modulates HIV-1 Gag-envelope interaction towards envelope incorporation and infectivity in a cell type dependent manner.

    Directory of Open Access Journals (Sweden)

    Archana Gautam

    Full Text Available The HIV-1 Vpu is required for efficient virus particle release from the plasma membrane and intracellular CD4 degradation in infected cells. In the present study, we found that the loss of virus infectivity as a result of envelope (Env incorporation defect caused by a Gag matrix (MA mutation (L30E was significantly alleviated by introducing a start codon mutation in vpu. Inactivation of Vpu partially restored the Env incorporation defect imposed by L30E substitution in MA. This effect was found to be comparable in cell types such as 293T, HeLa, NP2 and GHOST as well as in peripheral blood mononuclear cells (PBMC and monocyte-derived macrophages (MDM. However, in HeLa cells BST-2 knockdown was found to further alleviate the effect of Vpu inactivation on infectivity of L30E mutant. Our data demonstrated that the impaired infectivity of virus particles due to Env incorporation defect caused by MA mutation was modulated by start codon mutation in Vpu.

  4. Incorporation of graphene into SnO{sub 2} photoanodes for dye-sensitized solar cells

    Energy Technology Data Exchange (ETDEWEB)

    Batmunkh, Munkhbayar [School of Chemical Engineering, The University of Adelaide, Adelaide, South Australia 5005 (Australia); Centre for Nanoscale Science and Technology, School of Chemical and Physical Sciences, Flinders University, Bedford Park, Adelaide, South Australia 5042 (Australia); Dadkhah, Mahnaz; Shearer, Cameron J. [Centre for Nanoscale Science and Technology, School of Chemical and Physical Sciences, Flinders University, Bedford Park, Adelaide, South Australia 5042 (Australia); Biggs, Mark J. [School of Chemical Engineering, The University of Adelaide, Adelaide, South Australia 5005 (Australia); School of Science, Loughborough University, Loughborough, Leicestershire LE11 3TU (United Kingdom); Shapter, Joseph G., E-mail: joe.shapter@flinders.edu.au [Centre for Nanoscale Science and Technology, School of Chemical and Physical Sciences, Flinders University, Bedford Park, Adelaide, South Australia 5042 (Australia)

    2016-11-30

    Graphical abstract: Incorporation of a graphene structure into SnO{sub 2} dye-sensitized solar cell photoanode films has been demonstrated for the first time. The use of graphene in the SnO{sub 2} has been found to be a promising strategy to address many problems of photovoltaic cells based on SnO{sub 2} photoanodes. - Highlights: • SnO{sub 2}-reduced graphene oxide (RGO) hybrid is prepared using a microwave technique. • The first SnO{sub 2}-RGO photoanode based DSSC is fabricated. • Use of RGO addresses the major shortcoming of SnO{sub 2} when employed as a DSSC photoanode. • RGO significantly improved the electron transport rate within the DSSC devices. • Incorporation of RGO into the SnO{sub 2} photoanode enhanced the DSSC efficiency by 91.5%. - Abstract: In dye-sensitized solar cell (DSSC) photoanodes, tin dioxide (SnO{sub 2}) structures present a promising alternative semiconducting oxide to the conventional titania (TiO{sub 2}), but they suffer from poor photovoltaic (PV) efficiency caused by insufficient dye adsorption and low energy value of the conduction band. A hybrid structure consisting of SnO{sub 2} and reduced graphene oxide (SnO{sub 2}-RGO) was synthesized via a microwave-assisted method and has been employed as a photoanode in DSSCs. Incorporation of RGO into the SnO{sub 2} photoanode enhanced the power conversion efficiency of DSSC device by 91.5%, as compared to the device assembled without RGO. This efficiency improvement can be attributed to increased dye loading, enhanced electron transfer and addition of suitable energy levels in the photoanode. Finally, the use of RGO addresses the major shortcoming of SnO{sub 2} when employed as a DSSC photoanode, namely poor dye adsorption and slow electron transfer rate.

  5. Neuronal death and synapse elimination in the olivocerebellar system: III. Cell counts in the inferior olive of developing rats X-irradiated from birth

    International Nuclear Information System (INIS)

    Geoffroy, B.; Shojaeian, H.; Delhaye-Bouchaud, N.; Mariani, J.

    1988-01-01

    The change with age of cell number in the developing inferior olivary nucleus (ION) of the normal rat, compared to the time course of the regression of the polyneuronal innervation of Purkinje cells by olivary axons (i.e., the climbing fibers), suggests that the involution of the redundant olivocerebellar contacts is caused by a reduction of axonal branching rather than by degeneration of the parent cells, this being also suggested by the normal size of the olivary population in adult rodents whose Purkinje cells retain polyneuronal innervation. However, the similar size of the adult ION population does not necessarily imply that the development history is the same in normal and multiply innervated adult rodents. Therefore, cell counts were performed in developing rats which had been repeatedly X-irradiated from birth until postnatal day 14 and which retained polyneuronal innervation. The results show that, although less marked than during normal development, the evolution of the ION population is also characterized by a phase of cell loss followed by a slow increase. However, the number of cells in X-irradiated rats is higher than in their controls from birth to postnatal day 15 but becomes identical at 20 days and later. These data confirm that cell death in the ION does not play a major role in the shaping of olivocerebellar connections

  6. Neuropharmacologic characterization of strychnine seizure potentiation in the inferior olive lesioned rat

    International Nuclear Information System (INIS)

    Anderson, M.C.

    1988-01-01

    Cerebellar stimulation is associated with anticonvulsant activity in several animal models. There are two afferent inputs to cerebellar Purkinje cells: (1) parallel fibers, which relay mossy fiber input, from brainstem, spinal cord, cerebral cortex and cerebellum, and (2) climbing fibers, arising from the inferior olive. Both climbing and parallel fibers release excitatory amino acid neurotransmitters, which stimulate Purkinje cells and cause GABA release in the deep cerebellar nuclei. Climbing fibers also exert tonic inhibition over Purkinje cell activity by producing an absolute refractory period following stimulation, rendering Purkinje cells unresponsive to parallel fibers. Climbing fiber deafferentation by bilateral inferior olive lesions produced a specific decrease in threshold for strychnine-seizures in the rat. Inferior olive lesions produced no change in threshold to seizures induced by picrotoxin, bicuculline or pentylenetetrazole. Inferior olive lesions also produced abnormal motor behavior including, myoclonus, backward locomotion and hyperextension, which was significantly aggravated by strychnine, brucine, picrotoxin, bicuculline and pentylenetetrazole. Inferior olive lesions produced a significant increase in quisqualate sensitive [ 3 H]AMPA ((Rs)-alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid) binding to cerebellar membranes. AMPA is a glutamate analog with high affinity for quisqualate sensitive receptors

  7. Infection of neuroblastoma cells with Semliki Forest virus. Incorporation of 35S or 32P

    International Nuclear Information System (INIS)

    Anon.

    1976-01-01

    Phosphate-free medium is used for the incorporation of 32 P and methionine-free medium for 35 S-methionine labelling. After virus replication, the culture shows a clear CPE all of the cells appearing round and dead. Materials used are presented and experimental procedure is described

  8. 5-chlorodeoxycytidine sensitizes cells to x-ray and is incorporated as 5-chlorodeoxyuridine in tumor DNA

    International Nuclear Information System (INIS)

    Perez, L.M.; Greer, S.

    1985-01-01

    5-Chlorodeoxycytidine (CldC) coadministered with tetrahydrouridine (H/sub 4/U), an inhibitor of its deamination, sensitizes HEp-2 cells to X-ray and is incorporated in DNA as 5-chlorodeoxyuridine (CldU). CldC possesses a reasonable Km value (56 μM) with respect to human deoxycytidine kinase (dCK) in contrast to the low affinities of BrdC and IdC (400 and 1000μM, respectively; the Km value for dC = 2μM). Preincubation with N-phosphonacetyl-L-aspartate (PALA) and 5-fluorodeoxyuridine (FdU), inhibitors of pyrimidine biosynthesis, enhances sensitization. X-ray survival curves of HEp-2 cells treated with PALA and FdU (or FdC + H/sub 4/U) and CldC + H/sub 4/U are characterized by dose enhancement ratios of 2.5 or greater. Substantial sensitization by CldC + H/sub 4/U also occurs with Sarcoma-180 and RIF-1 cells in culture . CldC + H/sub 4/U should result in circumvention of catabolism and selective toxicity to tumors via inhibition of nucleoside reductase by CldUTP as well as selective incorporation of CldU in tumors possessing high levels of dCMP deaminase and dCK, enzymes that are markedly elevated in many human tumors. CldU, derived from CldC, is incorporated to a greater extent in the DNA of a solid tumor (S-180) than in normal tissue of the mouse. This may result in selective tumor radiosensitization

  9. Sensitivity and specificity of tritiated thymidine incorporation and ELISPOT assays in identifying antigen specific T cell immune responses

    Directory of Open Access Journals (Sweden)

    MacLeod Beth

    2007-09-01

    Full Text Available Abstract Background Standardization of cell-based immunologic monitoring is becoming increasingly important as methods for measuring cellular immunity become more complex. We assessed the ability of two commonly used cell-based assays, tritiated thymidine incorporation (proliferation and IFN-gamma ELISPOT, to predict T cell responses to HER-2/neu, tetanus toxoid (tt, and cytomegalovirus (CMV antigens. These antigens were determined to be low (HER-2/neu, moderate (tt, and robustly (CMV immunogenic proteins. Samples from 27 Stage II, III, and IV HER-2/neu positive breast cancer patients, vaccinated against the HER-2/neu protein and tt, were analyzed by tritiated thymidine incorporation and IFN-gamma ELISPOT for T cell response. Results Linear regression analysis indicates that both stimulation index (SI (p = 0.011 and IFN-gamma secreting precursor frequency (p Conclusion These data underscore the importance of taking into consideration the performance characteristics of assays used to measure T cell immunity. This consideration is particularly necessary when determining which method to utilize for assessing responses to immunotherapeutic manipulations in cancer patients.

  10. Short-term variability in bacterial abundance, cell properties, and incorporation of leucine and thymidine in subarctic sea ice

    DEFF Research Database (Denmark)

    Kaartokallio, H.; Sogaard, D. H.; Norman, L.

    2013-01-01

    Sea ice is a biome of immense size and provides a range of habitats for diverse microbial communities, many of which are adapted to living at low temperatures and high salinities in brines. We measured simultaneous incorporation of thymidine (TdR) and leucine (Leu), bacterial cell abundance...... and cell population properties (by flow cytometry) in subarctic sea ice in SW Greenland. Short-term temporal variability was moderate, and steep environmental gradients, typical for sea ice, were the main drivers of the variability in bacterial cell properties and activity. Low nucleic acid (LNA) bacteria...... and marine biofilm systems. Leu: TdR ratios were high (up to >300) in lowermost ice layers, and when compared to published respiration measurements, these results suggest non-specific Leu incorporation. There was evidence of polyhydroxyalkanoate (PHA)-containing bacteria in the sea ice, shown by brightly...

  11. Ultrarapid sonochemical synthesis of enzyme-incorporated copper nanoflowers and their application to mediatorless glucose biofuel cell

    Science.gov (United States)

    Chung, Minsoo; Nguyen, Tuan Loi; Tran, Thao Quynh Ngan; Yoon, Hyon Hee; Kim, Il Tae; Kim, Moon Il

    2018-01-01

    We have developed a mediatorless glucose biofuel cell based on hybrid nanoflowers incorporating enzymes including glucose oxidase (GOx), laccase, or catalase with copper phosphate, which were further mixed and compressed with conductive multi-walled carbon nanotube (CNT). The nanoflowers were simply synthesized within 5 min at room temperature using sonication method but yielded greatly improved stability as well as highly retained activity by the proper incorporation of enzyme molecules inside the flower-like structure. With glucose as biofuel, GOx and laccase nanoflowers were applied to form enzyme anode and cathode, respectively, and catalase nanoflowers were additionally employed to catalyze the decomposition of hydrogen peroxide, which may be deleterious for GOx, into oxygen and water. Using the enzyme nanoflowers-based biofuel cell system without any involved mediator, a high power density up to 200 μW cm-2 were obtained, which was approximately 80% to that from the biofuel cell system prepared with the corresponding free enzymes. Importantly, the enzyme nanoflowers-based biofuel cell maintained their initial power density over 90% during storage for two months at 4 °C, while most of the glucose biofuel cells in the literature present meaningful stability only in the range of one or two weeks. Based on this result, we expect that this simple but efficient strategy to prepare highly stable glucose biofuel cell using the rapidly-synthesized enzyme-inorganic hybrid nanoflowers can be readily extended to diverse applications in medical and environmental chemistry.

  12. Mosaic Expression of Thyroid Hormone Regulatory Genes Defines Cell Type-Specific Dependency in the Developing Chicken Cerebellum.

    Science.gov (United States)

    Delbaere, Joke; Van Herck, Stijn L J; Bourgeois, Nele M A; Vancamp, Pieter; Yang, Shuo; Wingate, Richard J T; Darras, Veerle M

    2016-12-01

    The cerebellum is a morphologically unique brain structure that requires thyroid hormones (THs) for the correct coordination of key cellular events driving its development. Unravelling the interplay between the multiple factors that can regulate intracellular TH levels is a key step to understanding their role in the regulation of these cellular processes. We therefore investigated the regional/cell-specific expression pattern of TH transporters and deiodinases in the cerebellum using the chicken embryo as a model. In situ hybridisation revealed expression of the TH transporters monocarboxylate transporter 8 (MCT8) and 10 (MCT10), L-type amino acid transporter 1 (LAT1) and organic anion transporting polypeptide 1C1 (OATP1C1) as well as the inactivating type 3 deiodinase (D3) in the fourth ventricle choroid plexus, suggesting a possible contribution of the resulting proteins to TH exchange and subsequent inactivation of excess hormone at the blood-cerebrospinal fluid barrier. Exclusive expression of LAT1 and the activating type 2 deiodinase (D2) mRNA was found at the level of the blood-brain barrier, suggesting a concerted function for LAT1 and D2 in the direct access of active T 3 to the developing cerebellum via the capillary endothelial cells. The presence of MCT8 mRNA in Purkinje cells and cerebellar nuclei during the first 2 weeks of embryonic development points to a potential role of this transporter in the uptake of T 3 in central neurons. At later stages, together with MCT10, detection of MCT8 signal in close association with the Purkinje cell dendritic tree suggests a role of both transporters in TH signalling during Purkinje cell synaptogenesis. MCT10 was also expressed in late-born cells in the rhombic lip lineage with a clear hybridisation signal in the outer external granular layer, indicating a potential role for MCT10 in the proliferation of granule cell precursors. By contrast, expression of D3 in the first-born rhombic lip-derived population may

  13. Improvement of minority carrier lifetime and conversion efficiency by Na incorporation in Cu2ZnSnSe4 solar cells

    Science.gov (United States)

    Tampo, Hitoshi; Kim, Kang Min; Kim, Shinho; Shibata, Hajime; Niki, Shigeru

    2017-07-01

    The effect of Na incorporation in Cu2ZnSnSe4 (CZTSe) solar cells grown by the coevaporation method was investigated via photoluminescence (PL) and time-resolved PL (TRPL) measurements as well as photovoltaic properties. The TRPL decay curves showed a monotonic increase in CZTSe lifetime from 2 to 15 ns with increasing Na incorporation, which corresponds to the increase in the correction length estimated by quantum efficiency measurements. The TRPL decay curves included two decay components, fast and slow, which were discussed and concluded as originating from the recombination at the surface and bulk of CZTSe, respectively, which is also supported by TPRL measurements with various excitation wavelengths. The lifetime of CZTSe is limited by the surface-related nonradiative recombination compared to Cu(In,Ga)Se2 devices which are fabricated with the same device structure except for the absorber, and at present, it is concluded that the surface recombination of the CZTSe limits the cell performance. In addition to the above investigations, the relationship between the CZTSe bulk lifetime and carrier concentration is discussed; deep nonradiative recombination centers in the CZTSe bulk were found to decrease by one order of magnitude with Na incorporation. The Na incorporation primarily resulted in improvement in the short circuit current density and fill factor and not in the open circuit voltage, and the results are discussed. The best performing CZTSe solar cell with Na incorporation showed a conversion efficiency of 9.57%.

  14. 2-Fluoro-L-Fucose Is a Metabolically Incorporated Inhibitor of Plant Cell Wall Polysaccharide Fucosylation

    Science.gov (United States)

    Wallace, Ian S.

    2015-01-01

    The monosaccharide L-fucose (L-Fuc) is a common component of plant cell wall polysaccharides and other plant glycans, including the hemicellulose xyloglucan, pectic rhamnogalacturonan-I (RG-I) and rhamnogalacturonan-II (RG-II), arabinogalactan proteins, and N-linked glycans. Mutations compromising the biosynthesis of many plant cell wall polysaccharides are lethal, and as a result, small molecule inhibitors of plant cell wall polysaccharide biosynthesis have been developed because these molecules can be applied at defined concentrations and developmental stages. In this study, we characterize novel small molecule inhibitors of plant fucosylation. 2-fluoro-L-fucose (2F-Fuc) analogs caused severe growth phenotypes when applied to Arabidopsis seedlings, including reduced root growth and altered root morphology. These phenotypic defects were dependent upon the L-Fuc salvage pathway enzyme L-Fucose Kinase/ GDP-L-Fucose Pyrophosphorylase (FKGP), suggesting that 2F-Fuc is metabolically converted to the sugar nucleotide GDP-2F-Fuc, which serves as the active inhibitory molecule. The L-Fuc content of cell wall matrix polysaccharides was reduced in plants treated with 2F-Fuc, suggesting that this molecule inhibits the incorporation of L-Fuc into these polysaccharides. Additionally, phenotypic defects induced by 2F-Fuc treatment could be partially relieved by the exogenous application of boric acid, suggesting that 2F-Fuc inhibits RG-II biosynthesis. Overall, the results presented here suggest that 2F-Fuc is a metabolically incorporated inhibitor of plant cellular fucosylation events, and potentially suggest that other 2-fluorinated monosaccharides could serve as useful chemical probes for the inhibition of cell wall polysaccharide biosynthesis. PMID:26414071

  15. Suppression of leukocyte inhibitory factor (LIF) production and [3H]thymidine incorporation by concanavalin A-activated mononuclear cells

    International Nuclear Information System (INIS)

    Lomnitzer, R.; Rabson, A.R.

    1979-01-01

    The capacity of human mononuclear (MN) cells pretreated with concanavalin A (Con A) to suppress the activity of fresh phytohemagglutinin (PHA)-pulsed mononuclear cells was assessed. Con A-pretreated MN cells suppressed leukocyte inhibitory factor (LIF) activity in supernatants of PHA-pulsed cell cultures and [ 3 H]thymidine incorporation by these cells. Suppression was obtained in both allogeneic and autologous systems with mitomycin-treated, irradiated, or untreated Con A-induced cells. Lymphocytes from two patients that, following treatment with Con A, did not suppress mitogen-induced proliferative response of normal cells also did not suppress LIF production

  16. Linking Essential Tremor to the Cerebellum: Neuropathological Evidence.

    Science.gov (United States)

    Louis, Elan D

    2016-06-01

    A fundamental question about essential tremor (ET) is whether its associated pathological changes and disease mechanisms are linkable to a specific brain region. To that end, recent tissue-based studies have made significant strides in elucidating changes in the ET brain. Emerging from these studies is increasing neuropathological evidence linking ET to the cerebellum. These studies have systematically identified a broad range of structural, degenerative changes in the ET cerebellum, spanning across all Purkinje cell compartments. These include the dendritic compartment (where there is an increase in number of Purkinje cell dendritic swellings, a pruning of the dendritic arbor, and a reduction in spine density), the cell body (where, aside from reductions in Purkinje cell linear density in some studies, there is an increase in the number of heterotopic Purkinje cell soma), and the axonal compartment (where a plethora of changes in axonal morphology have been observed, including an increase in the number of thickened axonal profiles, torpedoes, axonal recurrent collaterals, axonal branching, and terminal axonal sprouting). Additional changes, possibly due to secondary remodeling, have been observed in neighboring neuronal populations. These include a hypertrophy of basket cell axonal processes and changes in the distribution of climbing fiber-Purkinje cell synapses. These changes all distinguish ET from normal control brains. Initial studies further indicate that the profile (i.e., constellation) of these changes may separate ET from other diseases of the cerebellum, thereby serving as a disease signature. With the discovery of these changes, a new model of ET has arisen, which posits that it may be a neurodegenerative disorder centered in the cerebellar cortex. These newly emerging neuropathological studies pave the way for anatomically focused, hypothesis-driven, molecular mechanistic studies of disease pathogenesis.

  17. Effect of medium replenishment or composition on [3H] thymidine incorporation in uv-irradiated CHO-K1 cells

    International Nuclear Information System (INIS)

    Newman, C.N.; Miller, J.H.

    1985-03-01

    Because culture medium contains uv-absorbing material, it is usually removed just before uv-irradiation of tissue culture monolayers. However, medium removal and replenishment with fresh medium alone (sham-irradiation) causes up to a 10-fold reduction in the rate of [ 3 H]TdR incorporation in CHO-K1 cells which persists for several hours. This reduction, which is much smaller ( 3 H]TdR pulse-label in conditioned (spent) and in fresh medium; TdR in the former is converted by cells to thymine. When responses of uv-irradiated cells are normalized to responses of corresponding sham-irradiated cultures, considerable variation is observed in replicate experiments because fresh medium appears to induce transient metabolic imbalances in irradiated cells which are not readily controlled. This problem can, in part, be circumvented by replenishing treated cultures with the original spent medium; however, the presence of CdR in the growth medium still causes an anomalous 2-3-fold greater uv-induced reduction in [ 3 H]TdR incorporation than is observed in the absence of CdR. 17 refs., 3 figs., 1 tab

  18. Measurement of the capability of DNA synthesis of human fetal liver cells by the assay of 3H-TdR incorporation

    International Nuclear Information System (INIS)

    Wang Tao; Ma Xiangrui; Wang Hongyun; Cao Xia

    1987-01-01

    The fetal liver is one of the major sites of hematopoiesis during gestation. Under erythropoietin (EPO) stimulation, in erythroid precusor cells of fetal liver, proliferation and differentiation occurred and function of metabolism was enhanced. The technique of 3 H-TdR incorporation was used to measure the function of fetal liver cellular DNA synthesis. As EPO concentration at the range of approximately 20 ∼ 100 mU/ml, the counts of 3 H-TdR incorporation into fetal liver cells increased. As the concentration of EPO increased, however, its incorporation counts are lower than that in bone marrow of either the fetal or the adult. It suggested that precusors of erythrocyte of fetal liver has differentiated to later phases with the progressive accumulation of mature cells, therefore, both proliferation and function of metabolism are more or less decreased respectively. Under EPO stimulation, however, precusor of erythroid of fetal liver can greatly increase potential effects on DNA synthesis

  19. Incorporation of thymidine into onion root meristematic cell nuclei in presence of hydroxyurea and its role in recovery of mitotic activity

    International Nuclear Information System (INIS)

    Habdas, H.

    1977-01-01

    Hydroxyurea treatment of onion roots induced mitotic block which was released by transfer of bulbs to water, and also to some extent by addition of cold or 3 H-thymidine to hydroxyurea solutions. In presence of hydroxyurea there was noted very intense incorporation of 3 H-thymidine into cell nuclei, giving labelling index of 40-70%. However, all the mitotic figures appearing in presence of hydroxyurea and 3 H-thymidine were unlabelled. On the other hand, labelled mitotic figures were obtained when roots incubated with 3 H-thymidine in presence of hydroxyurea had been transferred to water. Incorporation of 3 H-uridine was unaffected by hydroxyurea. The results show that hydroxyurea arrests onion root meristematic cells, either in the S phase and the G 2 phase. Enhanced incorporation of 3 H-thymidine in the presence of hydroxyurea, and release by added thymidine of the mitotic block indicate that hydroxyurea induces in onion root meristematic cells a particular shortage of thymidylate. (author)

  20. Effect of blood serum from irradiated mice on the incorporation of DNA, RNA and protein precursor in L929 cells

    International Nuclear Information System (INIS)

    Muehlensiepen, H.; Porschen, W.; Feinendegen, L.E.

    1983-01-01

    Serum from whole-body irradiated mice inhibits incorporation of DNA precursors into DNA of L929 cells in culture in a dose-dependent way. The humoral factor interfering with the incorporation of 3 H-thymidine and 125 I-iododeoxyuridine is identical to thymidine. The degree of depression of 125 I-iododeoxyuridine-uptake is more sensitive than that of 3 H-thymidine. Irradiation of donor mice does not confer a toxic effect of blood serum on cell growth in culture. Incorporation of 3 H-leucine into protein and 3 H-cytidine into DNA and RNA is not affected by the serum of irradiated mice; there is no effect on the incorporation of 3 H-cytidine from the intracellular precursor pool into DNA or RNA either. The present findings demonstrate the specificity and high sensitivity of the assay system for measuring thymidine concentration in mouse blood serum and point to possible applications of analysing abnormalities in DNA metabolism resulting in, or from, disturbances of the thymidine reutilization pathway. (orig.) [de

  1. 2-Fluoro-L-Fucose Is a Metabolically Incorporated Inhibitor of Plant Cell Wall Polysaccharide Fucosylation.

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    Jose A Villalobos

    Full Text Available The monosaccharide L-fucose (L-Fuc is a common component of plant cell wall polysaccharides and other plant glycans, including the hemicellulose xyloglucan, pectic rhamnogalacturonan-I (RG-I and rhamnogalacturonan-II (RG-II, arabinogalactan proteins, and N-linked glycans. Mutations compromising the biosynthesis of many plant cell wall polysaccharides are lethal, and as a result, small molecule inhibitors of plant cell wall polysaccharide biosynthesis have been developed because these molecules can be applied at defined concentrations and developmental stages. In this study, we characterize novel small molecule inhibitors of plant fucosylation. 2-fluoro-L-fucose (2F-Fuc analogs caused severe growth phenotypes when applied to Arabidopsis seedlings, including reduced root growth and altered root morphology. These phenotypic defects were dependent upon the L-Fuc salvage pathway enzyme L-Fucose Kinase/ GDP-L-Fucose Pyrophosphorylase (FKGP, suggesting that 2F-Fuc is metabolically converted to the sugar nucleotide GDP-2F-Fuc, which serves as the active inhibitory molecule. The L-Fuc content of cell wall matrix polysaccharides was reduced in plants treated with 2F-Fuc, suggesting that this molecule inhibits the incorporation of L-Fuc into these polysaccharides. Additionally, phenotypic defects induced by 2F-Fuc treatment could be partially relieved by the exogenous application of boric acid, suggesting that 2F-Fuc inhibits RG-II biosynthesis. Overall, the results presented here suggest that 2F-Fuc is a metabolically incorporated inhibitor of plant cellular fucosylation events, and potentially suggest that other 2-fluorinated monosaccharides could serve as useful chemical probes for the inhibition of cell wall polysaccharide biosynthesis.

  2. Developmental expression and differentiation-related neuron-specific splicing of metastasis suppressor 1 (Mtss1 in normal and transformed cerebellar cells

    Directory of Open Access Journals (Sweden)

    Baader Stephan L

    2007-10-01

    Full Text Available Abstract Background Mtss1 encodes an actin-binding protein, dysregulated in a variety of tumors, that interacts with sonic hedgehog/Gli signaling in epidermal cells. Given the prime importance of this pathway for cerebellar development and tumorigenesis, we assessed expression of Mtss1 in the developing murine cerebellum and human medulloblastoma specimens. Results During development, Mtss1 is transiently expressed in granule cells, from the time point they cease to proliferate to their synaptic integration. It is also expressed by granule cell precursor-derived medulloblastomas. In the adult CNS, Mtss1 is found exclusively in cerebellar Purkinje cells. Neuronal differentiation is accompanied by a switch in Mtss1 splicing. Whereas immature granule cells express a Mtss1 variant observed also in peripheral tissues and comprising exon 12, this exon is replaced by a CNS-specific exon, 12a, in more mature granule cells and in adult Purkinje cells. Bioinformatic analysis of Mtss1 suggests that differential exon usage may affect interaction with Fyn and Src, two tyrosine kinases previously recognized as critical for cerebellar cell migration and histogenesis. Further, this approach led to the identification of two evolutionary conserved nuclear localization sequences. These overlap with the actin filament binding site of Mtss1, and one also harbors a potential PKA and PKC phosphorylation site. Conclusion Both the pattern of expression and splicing of Mtss1 is developmentally regulated in the murine cerebellum. These findings are discussed with a view on the potential role of Mtss1 for cytoskeletal dynamics in developing and mature cerebellar neurons.

  3. Measuring Feedforward Inhibition and Its Impact on Local Circuit Function.

    Science.gov (United States)

    Hull, Court

    2017-05-01

    This protocol describes a series of approaches to measure feedforward inhibition in acute brain slices from the cerebellar cortex. Using whole-cell voltage and current clamp recordings from Purkinje cells in conjunction with electrical stimulation of the parallel fibers, these methods demonstrate how to measure the relationship between excitation and inhibition in a feedforward circuit. This protocol also describes how to measure the impact of feedforward inhibition on Purkinje cell excitability, with an emphasis on spike timing. © 2017 Cold Spring Harbor Laboratory Press.

  4. Cerebellar abiotrophy in a family of Border Collie dogs.

    Science.gov (United States)

    Sandy, J R; Slocombe, R E; Mitten, R W; Jedwab, D

    2002-11-01

    Cerebellar abiotrophies have a nonsex-linked, autosomal, recessively inherited basis in a number of species, and lesions typically reflect profound and progressive loss of Purkinje cells. In this report, an unusual form of abiotrophy is described for two sibling Border Collies. Extensive loss of the cerebellar granular cell layer was present with relative sparing of Purkinje cells of two female pups. The biochemical basis for this form of cerebellar abiotrophy is unknown, but the lack of disease in other siblings supports an autosomal recessive mode of inheritance.

  5. Comparison of selenophene and thienothiophene incorporation into pentacyclic lactam-based conjugated polymers for organic solar cells

    KAUST Repository

    Kroon, Renee; Melianas, Armantas; Zhuang, Wenliu; Bergqvist, Jonas; Diaz De Zerio Mendaza, Amaia; Steckler, Timothy T.; Yu, Liyang; Bradley, Siobhan J.; Musumeci, Chiara; Gedefaw, Desta; Nann, Thomas; Amassian, Aram; Mü ller, Christian; Inganä s, Olle; Andersson, Mats R.

    2015-01-01

    In this work, we compare the effect of incorporating selenophene versus thienothiophene spacers into pentacyclic lactam-based conjugated polymers for organic solar cells. The two cyclic lactam-based copolymers were obtained via a new synthetic method for the lactam moiety. Selenophene incorporation results in a broader and red-shifted optical absorption while retaining a deep highest occupied molecular orbital level, whereas thienothienophene incorporation results in a blue-shifted optical absorption. Additionally, grazing-incidence wide angle X-ray scattering data indicates edge- and face-on solid state order for the selenophene-based polymer as compared to the thienothiophene-based polymer, which orders predominantly edge-on with respect to the substrate. In polymer:PCBM bulk heterojunction solar cells both materials show a similar open-circuit voltage of ∼0.80-0.84 V, however the selenophene-based polymer displays a higher fill factor of ∼0.70 vs. ∼0.65. This is due to the partial face-on backbone orientation of the selenophene-based polymer, leading to a higher hole mobility, as confirmed by single-carrier diode measurements, and a concomitantly higher fill factor. Combined with improved spectral coverage of the selenophene-based polymer, as confirmed by quantum efficiency experiments, it offers a larger short-circuit current density of ∼12 mA cm. Despite the relatively low molecular weight of both materials, a very robust power conversion efficiency ∼7% is achieved for the selenophene-based polymer, while the thienothiophene-based polymer demonstrates only a moderate maximum PCE of ∼5.5%. Hence, the favorable effects of selenophene incorporation on the photovoltaic performance of pentacyclic lactam-based conjugated polymers are clearly demonstrated.

  6. Comparison of selenophene and thienothiophene incorporation into pentacyclic lactam-based conjugated polymers for organic solar cells

    KAUST Repository

    Kroon, Renee

    2015-09-08

    In this work, we compare the effect of incorporating selenophene versus thienothiophene spacers into pentacyclic lactam-based conjugated polymers for organic solar cells. The two cyclic lactam-based copolymers were obtained via a new synthetic method for the lactam moiety. Selenophene incorporation results in a broader and red-shifted optical absorption while retaining a deep highest occupied molecular orbital level, whereas thienothienophene incorporation results in a blue-shifted optical absorption. Additionally, grazing-incidence wide angle X-ray scattering data indicates edge- and face-on solid state order for the selenophene-based polymer as compared to the thienothiophene-based polymer, which orders predominantly edge-on with respect to the substrate. In polymer:PCBM bulk heterojunction solar cells both materials show a similar open-circuit voltage of ∼0.80-0.84 V, however the selenophene-based polymer displays a higher fill factor of ∼0.70 vs. ∼0.65. This is due to the partial face-on backbone orientation of the selenophene-based polymer, leading to a higher hole mobility, as confirmed by single-carrier diode measurements, and a concomitantly higher fill factor. Combined with improved spectral coverage of the selenophene-based polymer, as confirmed by quantum efficiency experiments, it offers a larger short-circuit current density of ∼12 mA cm. Despite the relatively low molecular weight of both materials, a very robust power conversion efficiency ∼7% is achieved for the selenophene-based polymer, while the thienothiophene-based polymer demonstrates only a moderate maximum PCE of ∼5.5%. Hence, the favorable effects of selenophene incorporation on the photovoltaic performance of pentacyclic lactam-based conjugated polymers are clearly demonstrated.

  7. Concentration-Dependent Patterns of Leucine Incorporation by Coastal Picoplankton

    Science.gov (United States)

    Alonso, Cecilia; Pernthaler, Jakob

    2006-01-01

    Coastal pelagic environments are believed to feature concentration gradients of dissolved organic carbon at a microscale, and they are characterized by pronounced seasonal differences in substrate availability for the heterotrophic picoplankton. Microbial taxa that coexist in such habitats might thus differ in their ability to incorporate substrates at various concentrations. We investigated the incorporation patterns of leucine in four microbial lineages from the coastal North Sea at concentrations between 0.1 and 100 nM before and during a spring phytoplankton bloom. Community bulk incorporation rates and the fraction of leucine-incorporating cells in the different populations were analyzed. Significantly fewer bacterial cells incorporated the amino acid before (13 to 35%) than during (23 to 47%) the bloom at all but the highest concentration. The incorporation rate per active cell in the prebloom situation was constant above 0.1 nM added leucine, whereas it increased steeply with substrate concentration during the bloom. At both time points, a high proportion of members of the Roseobacter clade incorporated leucine at all concentrations (55 to 80% and 86 to 94%, respectively). In contrast, the fractions of leucine-incorporating cells increased substantially with substrate availability in bacteria from the SAR86 clade (8 to 31%) and from DE cluster 2 of the Flavobacteria-Sphingobacteria (14 to 33%). The incorporation patterns of marine Euryarchaeota were between these extremes (30 to 56% and 48 to 70%, respectively). Our results suggest that the contribution of microbial taxa to the turnover of particular substrates may be concentration dependent. This may help us to understand the specific niches of coexisting populations that appear to compete for the same resources. PMID:16517664

  8. Incorporation of Viral Glycoprotein VSV-G Improves the Delivery of DNA by Erythrocyte Ghost into Cells Refractory to Conventional Transfection.

    Science.gov (United States)

    Liu, Xin; Li, Yun-Pan; Zhong, Zhen-Min; Tan, Hui-Qi; Lin, Hao-Peng; Chen, Shao-Jun; Fu, Yu-Cai; Xu, Wen-Can; Wei, Chi-Ju

    2017-02-01

    The objective of this study was to formulate a novel gene delivery system based on the erythrocyte ghost (EG) integrated with fusogenic viral glycoprotein vesicular stomatitis virus glycoprotein G (VSV-G). VSV-G proteins were harvested as condition medium of Ad293 cells carrying a VSV-G transgene and then incorporated into EG. Plasmid DNA was condensed by various transfection reagents. A luciferase expression construct (pGL3-control) and a DsRed expression cassette (pCMV-DsRed) were used to evaluate the delivery efficiency of DNA/EG/VSV-G complexes. VSV-G proteins could be incorporated into EG in static incubation under acidic conditions as evidenced by the Western blot analysis. Condensed plasmid DNA was bound mostly to the outer surface of EG, which could be detected by electromicroscopy and measured by electrophoresis. EG/VSV-G complexes stimulated the delivery of pGL3-control into Ad293 cells significantly with the luciferase activity increased about 4-fold as compared to that of the control. The delivery of pCMV-DsRed was also enhanced with the percentage of DsRed-positive Ad293 cells increased from 55 % to about 80 %. Moreover, the transfection efficiency in 3T3, HeLa, INS-1, and bone marrow stem cell (BMSC) cells increased about 2-3-fold. Finally, confocal microscopy analysis showed that incorporation of VSV-G significantly enhanced the endocytosis of EG into target cells. In the present study, a novel type of non-viral DNA delivery vehicle consisting of EG and fusogenic VSV-G proteins was formulated, which showed superior transfection efficiency even in cells resistant to classical transfection.

  9. Ascl1 (Mash1) lineage cells contribute to discrete cell populations in CNS architecture.

    Science.gov (United States)

    Kim, Euiseok J; Battiste, James; Nakagawa, Yasushi; Johnson, Jane E

    2008-08-01

    Ascl1 (previously Mash1) is a bHLH transcription factor essential for neuronal differentiation and specification in the nervous system. Although it has been studied for its role in several neural lineages, the full complement of lineages arising from Ascl1 progenitor cells remains unknown. Using an inducible Cre-flox genetic fate-mapping strategy, Ascl1 lineages were determined throughout the brain. Ascl1 is present in proliferating progenitor cells but these cells are actively differentiating as evidenced by rapid migration out of germinal zones. Ascl1 lineage cells contribute to distinct cell types in each major brain division: the forebrain including the cerebral cortex, olfactory bulb, hippocampus, striatum, hypothalamus, and thalamic nuclei, the midbrain including superior and inferior colliculi, and the hindbrain including Purkinje and deep cerebellar nuclei cells and cells in the trigeminal sensory system. Ascl1 progenitor cells at early stages in each CNS region preferentially become neurons, and at late stages they become oligodendrocytes. In conclusion, Ascl1-expressing progenitor cells in the brain give rise to multiple, but not all, neuronal subtypes and oligodendrocytes depending on the temporal and spatial context, consistent with a broad role in neural differentiation with some subtype specification.

  10. Optimal properties of analog perceptrons with excitatory weights.

    Directory of Open Access Journals (Sweden)

    Claudia Clopath

    Full Text Available The cerebellum is a brain structure which has been traditionally devoted to supervised learning. According to this theory, plasticity at the Parallel Fiber (PF to Purkinje Cell (PC synapses is guided by the Climbing fibers (CF, which encode an 'error signal'. Purkinje cells have thus been modeled as perceptrons, learning input/output binary associations. At maximal capacity, a perceptron with excitatory weights expresses a large fraction of zero-weight synapses, in agreement with experimental findings. However, numerous experiments indicate that the firing rate of Purkinje cells varies in an analog, not binary, manner. In this paper, we study the perceptron with analog inputs and outputs. We show that the optimal input has a sparse binary distribution, in good agreement with the burst firing of the Granule cells. In addition, we show that the weight distribution consists of a large fraction of silent synapses, as in previously studied binary perceptron models, and as seen experimentally.

  11. The relationship between DNA synthesis and incorporation of (14C) lysine into different histone fractions in Ehrlich ascites tumour cells

    International Nuclear Information System (INIS)

    Malec, J.; Kornacka, L.; Wojnarowska, M.; Moscicka, M.

    1974-01-01

    The effect of inhibition of DNA synthesis by hydroxyurea on ( 14 C) lysine incorporation into the main four histone fractions in Ehrlich ascites tumor cells, was examined in vitro. The radioactivity of lysine-rich histones, especially of histone f1, was preferentially decreased. The smallest decrease was observed for histone f3. The incorporation into other cellular proteins was but slightly affected. (author)

  12. A computational model incorporating neural stem cell dynamics reproduces glioma incidence across the lifespan in the human population.

    Directory of Open Access Journals (Sweden)

    Roman Bauer

    Full Text Available Glioma is the most common form of primary brain tumor. Demographically, the risk of occurrence increases until old age. Here we present a novel computational model to reproduce the probability of glioma incidence across the lifespan. Previous mathematical models explaining glioma incidence are framed in a rather abstract way, and do not directly relate to empirical findings. To decrease this gap between theory and experimental observations, we incorporate recent data on cellular and molecular factors underlying gliomagenesis. Since evidence implicates the adult neural stem cell as the likely cell-of-origin of glioma, we have incorporated empirically-determined estimates of neural stem cell number, cell division rate, mutation rate and oncogenic potential into our model. We demonstrate that our model yields results which match actual demographic data in the human population. In particular, this model accounts for the observed peak incidence of glioma at approximately 80 years of age, without the need to assert differential susceptibility throughout the population. Overall, our model supports the hypothesis that glioma is caused by randomly-occurring oncogenic mutations within the neural stem cell population. Based on this model, we assess the influence of the (experimentally indicated decrease in the number of neural stem cells and increase of cell division rate during aging. Our model provides multiple testable predictions, and suggests that different temporal sequences of oncogenic mutations can lead to tumorigenesis. Finally, we conclude that four or five oncogenic mutations are sufficient for the formation of glioma.

  13. The cerebellum for jocks and nerds alike

    Directory of Open Access Journals (Sweden)

    Laurentiu ePopa

    2014-06-01

    Full Text Available Historically the cerebellum has been implicated in the control of movement. However, the cerebellum’s role in non-motor functions, including cognitive and emotional processes, has also received increasing attention. Starting from the premise that the uniform architecture of the cerebellum underlies a common mode of information processing, this review examines recent electrophysiological findings on the motor signals encoded in the cerebellar cortex and then relates these signals to observations in the non-motor domain. Simple spike firing of individual Purkinje cells encodes performance errors, both predicting upcoming errors as well as providing feedback about those errors. Further, this dual temporal encoding of prediction and feedback involves a change in the sign of the simple spike modulation. Therefore, Purkinje cell simple spike firing both predicts and responds to feedback about a specific parameter, consistent with computing sensory prediction errors in which the predictions about the consequences of a motor command are compared with the feedback resulting from the motor command execution. These new findings are in contrast with the historical view that complex spikes encode errors. Evaluation of the kinematic coding in the simple spike discharge shows the same dual temporal encoding, suggesting this is a common mode of signal processing in the cerebellar cortex. Decoding analyses show the considerable accuracy of the predictions provided by Purkinje cells across a range of times. Further, individual Purkinje cells encode linearly and independently a multitude of signals, both kinematic and performance errors. Therefore, the cerebellar cortex’s capacity to make associations across different sensory, motor and non-motor signals is large. The results from studying how Purkinje cells encode movement signals suggest that the cerebellar cortex circuitry can support associative learning, sequencing, working memory, and forward internal

  14. The cerebellum for jocks and nerds alike.

    Science.gov (United States)

    Popa, Laurentiu S; Hewitt, Angela L; Ebner, Timothy J

    2014-01-01

    Historically the cerebellum has been implicated in the control of movement. However, the cerebellum's role in non-motor functions, including cognitive and emotional processes, has also received increasing attention. Starting from the premise that the uniform architecture of the cerebellum underlies a common mode of information processing, this review examines recent electrophysiological findings on the motor signals encoded in the cerebellar cortex and then relates these signals to observations in the non-motor domain. Simple spike firing of individual Purkinje cells encodes performance errors, both predicting upcoming errors as well as providing feedback about those errors. Further, this dual temporal encoding of prediction and feedback involves a change in the sign of the simple spike modulation. Therefore, Purkinje cell simple spike firing both predicts and responds to feedback about a specific parameter, consistent with computing sensory prediction errors in which the predictions about the consequences of a motor command are compared with the feedback resulting from the motor command execution. These new findings are in contrast with the historical view that complex spikes encode errors. Evaluation of the kinematic coding in the simple spike discharge shows the same dual temporal encoding, suggesting this is a common mode of signal processing in the cerebellar cortex. Decoding analyses show the considerable accuracy of the predictions provided by Purkinje cells across a range of times. Further, individual Purkinje cells encode linearly and independently a multitude of signals, both kinematic and performance errors. Therefore, the cerebellar cortex's capacity to make associations across different sensory, motor and non-motor signals is large. The results from studying how Purkinje cells encode movement signals suggest that the cerebellar cortex circuitry can support associative learning, sequencing, working memory, and forward internal models in non

  15. Incorporation of functionalized gold nanoparticles into nanofibers for enhanced attachment and differentiation of mammalian cells

    Directory of Open Access Journals (Sweden)

    Jung Dongju

    2012-06-01

    Full Text Available Abstract Background Electrospun nanofibers have been widely used as substrata for mammalian cell culture owing to their structural similarity to natural extracellular matrices. Structurally consistent electrospun nanofibers can be produced with synthetic polymers but require chemical modification to graft cell-adhesive molecules to make the nanofibers functional. Development of a facile method of grafting functional molecules on the nanofibers will contribute to the production of diverse cell type-specific nanofiber substrata. Results Small molecules, peptides, and functionalized gold nanoparticles were successfully incorporated with polymethylglutarimide (PMGI nanofibers through electrospinning. The PMGI nanofibers functionalized by the grafted AuNPs, which were labeled with cell-adhesive peptides, enhanced HeLa cell attachment and potentiated cardiomyocyte differentiation of human pluripotent stem cells. Conclusions PMGI nanofibers can be functionalized simply by co-electrospinning with the grafting materials. In addition, grafting functionalized AuNPs enable high-density localization of the cell-adhesive peptides on the nanofiber. The results of the present study suggest that more cell type-specific synthetic substrata can be fabricated with molecule-doped nanofibers, in which diverse functional molecules are grafted alone or in combination with other molecules at different concentrations.

  16. A selenium-deficient Caco-2 cell model for assessing differential incorporation of chemical or food selenium into glutathione peroxidase.

    Science.gov (United States)

    Zeng, Huawei; Botnen, James H; Johnson, Luann K

    2008-01-01

    Assessing the ability of a selenium (Se) sample to induce cellular glutathione peroxidase (GPx) activity in Se-deficient animals is the most commonly used method to determine Se bioavailability. Our goal is to establish a Se-deficient cell culture model with differential incorporation of Se chemical forms into GPx, which may complement the in vivo studies. In the present study, we developed a Se-deficient Caco-2 cell model with a serum gradual reduction method. It is well recognized that selenomethionine (SeMet) is the major nutritional source of Se; therefore, SeMet, selenite, or methylselenocysteine (SeMSC) was added to cell culture media with different concentrations and treatment time points. We found that selenite and SeMSC induced GPx more rapidly than SeMet. However, SeMet was better retained as it is incorporated into proteins in place of methionine; compared with 8-, 24-, or 48-h treatment, 72-h Se treatment was a more sensitive time point to measure the potential of GPx induction in all tested concentrations. Based on induction of GPx activity, the cellular bioavailability of Se from an extract of selenobroccoli after a simulated gastrointestinal digestion was comparable with that of SeMSC and SeMet. These in vitro data are, for the first time, consistent with previous published data regarding selenite and SeMet bioavailability in animal models and Se chemical speciation studies with broccoli. Thus, Se-deficient Caco-2 cell model with differential incorporation of chemical or food forms of Se into GPx provides a new tool to study the cellular mechanisms of Se bioavailability.

  17. Effect of Rubidium Incorporation on the Structural, Electrical, and Photovoltaic Properties of Methylammonium Lead Iodide-Based Perovskite Solar Cells.

    Science.gov (United States)

    Park, Ik Jae; Seo, Seongrok; Park, Min Ah; Lee, Sangwook; Kim, Dong Hoe; Zhu, Kai; Shin, Hyunjung; Kim, Jin Young

    2017-12-06

    We report the electrical properties of rubidium-incorporated methylammonium lead iodide ((Rb x MA 1-x )PbI 3 ) films and the photovoltaic performance of (Rb x MA 1-x )PbI 3 film-based p-i-n-type perovskite solar cells (PSCs). The incorporation of a small amount of Rb + (x = 0.05) increases both the open circuit voltage (V oc ) and the short circuit photocurrent density (J sc ) of the PSCs, leading to an improved power conversion efficiency (PCE). However, a high fraction of Rb + incorporation (x = 0.1 and 0.2) decreases the J sc and thus the PCE, which is attributed to the phase segregation of the single tetragonal perovskite phase to a MA-rich tetragonal perovskite phase and a RbPbI 3 orthorhombic phase at high Rb fractions. Conductive atomic force microscopic and admittance spectroscopic analyses reveal that the single-phase (Rb 0.05 MA 0.95 )PbI 3 film has a high electrical conductivity because of a reduced deep-level trap density. We also found that Rb substitution enhances the diode characteristics of the PSC, as evidenced by the reduced reverse saturation current (J 0 ). The optimized (Rb x MA 1-x )PbI 3 PSCs exhibited a PCE of 18.8% with negligible hysteresis in the photocurrent-voltage curve. The results from this work enhance the understanding of the effect of Rb incorporation into organic-inorganic hybrid halide perovskites and enable the exploration of Rb-incorporated mixed perovskites for various applications, such as solar cells, photodetectors, and light-emitting diodes.

  18. Incorporation of podoplanin into HIV released from HEK-293T cells, but not PBMC, is required for efficient binding to the attachment factor CLEC-2

    Science.gov (United States)

    2010-01-01

    Background Platelets are associated with HIV in the blood of infected individuals and might modulate viral dissemination, particularly if the virus is directly transmitted into the bloodstream. The C-type lectin DC-SIGN and the novel HIV attachment factor CLEC-2 are expressed by platelets and facilitate HIV transmission from platelets to T-cells. Here, we studied the molecular mechanisms behind CLEC-2-mediated HIV-1 transmission. Results Binding studies with soluble proteins indicated that CLEC-2, in contrast to DC-SIGN, does not recognize the viral envelope protein, but a cellular factor expressed on kidney-derived 293T cells. Subsequent analyses revealed that the cellular mucin-like membranous glycoprotein podoplanin, a CLEC-2 ligand, was expressed on 293T cells and incorporated into virions released from these cells. Knock-down of podoplanin in 293T cells by shRNA showed that virion incorporation of podoplanin was required for efficient CLEC-2-dependent HIV-1 interactions with cell lines and platelets. Flow cytometry revealed no evidence for podoplanin expression on viable T-cells and peripheral blood mononuclear cells (PBMC). Podoplanin was also not detected on HIV-1 infected T-cells. However, apoptotic bystander cells in HIV-1 infected cultures reacted with anti-podoplanin antibodies, and similar results were obtained upon induction of apoptosis in a cell line and in PBMCs suggesting an unexpected link between apoptosis and podoplanin expression. Despite the absence of detectable podoplanin expression, HIV-1 produced in PBMC was transmitted to T-cells in a CLEC-2-dependent manner, indicating that T-cells might express an as yet unidentified CLEC-2 ligand. Conclusions Virion incorporation of podoplanin mediates CLEC-2 interactions of HIV-1 derived from 293T cells, while incorporation of a different cellular factor seems to be responsible for CLEC-2-dependent capture of PBMC-derived viruses. Furthermore, evidence was obtained that podoplanin expression is

  19. Incorporation of podoplanin into HIV released from HEK-293T cells, but not PBMC, is required for efficient binding to the attachment factor CLEC-2

    Directory of Open Access Journals (Sweden)

    Münch Jan

    2010-05-01

    Full Text Available Abstract Background Platelets are associated with HIV in the blood of infected individuals and might modulate viral dissemination, particularly if the virus is directly transmitted into the bloodstream. The C-type lectin DC-SIGN and the novel HIV attachment factor CLEC-2 are expressed by platelets and facilitate HIV transmission from platelets to T-cells. Here, we studied the molecular mechanisms behind CLEC-2-mediated HIV-1 transmission. Results Binding studies with soluble proteins indicated that CLEC-2, in contrast to DC-SIGN, does not recognize the viral envelope protein, but a cellular factor expressed on kidney-derived 293T cells. Subsequent analyses revealed that the cellular mucin-like membranous glycoprotein podoplanin, a CLEC-2 ligand, was expressed on 293T cells and incorporated into virions released from these cells. Knock-down of podoplanin in 293T cells by shRNA showed that virion incorporation of podoplanin was required for efficient CLEC-2-dependent HIV-1 interactions with cell lines and platelets. Flow cytometry revealed no evidence for podoplanin expression on viable T-cells and peripheral blood mononuclear cells (PBMC. Podoplanin was also not detected on HIV-1 infected T-cells. However, apoptotic bystander cells in HIV-1 infected cultures reacted with anti-podoplanin antibodies, and similar results were obtained upon induction of apoptosis in a cell line and in PBMCs suggesting an unexpected link between apoptosis and podoplanin expression. Despite the absence of detectable podoplanin expression, HIV-1 produced in PBMC was transmitted to T-cells in a CLEC-2-dependent manner, indicating that T-cells might express an as yet unidentified CLEC-2 ligand. Conclusions Virion incorporation of podoplanin mediates CLEC-2 interactions of HIV-1 derived from 293T cells, while incorporation of a different cellular factor seems to be responsible for CLEC-2-dependent capture of PBMC-derived viruses. Furthermore, evidence was obtained that

  20. Cell suspension culture-mediated incorporation of the rice bel gene into transgenic cotton.

    Directory of Open Access Journals (Sweden)

    Liping Ke

    Full Text Available Cotton plants engineered for resistance to the herbicides, glyphosate or glufosinate have made a considerable impact on the production of the crop worldwide. In this work, embryogenic cell cultures derived from Gossypium hirsutum L. cv Coker 312 hypocotyl callus were transformed via Agrobacterium tumefaciens with the rice cytochrome P450 gene, CYP81A6 (bel. In rice, bel has been shown to confer resistance to both bentazon and sulfanylurea herbicides. Transformed cells were selected on a liquid medium supplemented alternately or simultaneously with kanamycin (50mg/L and bentazon (4.2 µmol. A total of 17 transgenic cotton lines were recovered, based on the initial resistance to bentazon and on PCR detection of the bel transgene. Bel integration into the cotton genome was confirmed by Southern blot and expression of the transgene was verified by RT-PCR. In greenhouse and experimental plot trials, herbicide (bentazon tolerance of up to 1250 mg/L was demonstrated in the transgenic plants. Transgenic lines with a single copy of the bel gene showed normal Mendelian inheritance of the characteristic. Importantly, resistance to bentazon was shown to be stably incorporated in the T1, T2 and T3 generations of self-fertilised descendents and in plants outcrossed to another upland cotton cultivar. Engineering resistance to bentazon in cotton through the heterologous expression of bel opens the possibility of incorporating this trait into elite cultivars, a strategy that would give growers a more flexible alternative to weed management in cotton crops.

  1. Endothelium in brain: Receptors, mitogenesis, and biosynthesis in glial cells

    International Nuclear Information System (INIS)

    MacCumber, M.W.; Ross, C.A.; Snyder, S.H.

    1990-01-01

    The authors have explored the cellular loci of endothelin (ET) actions and formation in the brain, using cerebellar mutant mice was well as primary and continuous cell cultures. A glial role is favored by several observations: (1) mutant mice lacking neuronal Purkinje cells display normal ET receptor binding and enhanced stimulation by ET of inositolphospholipid turnover; (ii) in weaver mice lacking neuronal granule cells, ET stimulation of inositolphospholipid turnover is not significantly diminished; (iii) C 6 glioma cells and primary cultures of cerebellar astroglia exhibit substantial ET receptor binding and ET-induced stimulation of inositolphospholipid turnover; (iv) ET promotes mitogenesis of C 6 glioma cells and primary cerebellar astroglia; and (v) primary cultures of cerebellar astroglia contain ET mRNA. ET also appears to have a neuronal role, since it stimulates inositolphospholipid turnover in primary cultures of cerebellar granule cells, and ET binding declines in granule cell-deficient mice. Thus, ET can be produced by glia and act upon both glia and neurons in a paracrine fashion

  2. An in vitro drug sensitivity test using a higher 3H-TdR incorporation and a modified human tumor stem cell assay

    International Nuclear Information System (INIS)

    Wang Enzhong

    1991-01-01

    An in vitro drug sensitivity test was developed to evaluate the lethal effects of drugs on human pulmonary carcinoma cells (HPCC). This method was a variant and combination of Human Tumor Stem (HTSCA) and a short-term test using 3 H-TdR incorporation. It consisted of a cell containing liquid top layer and a soft agar bottom layer in 24-well microplates. The medium was RPMI 1640 supplemented with 20% malignant pleural effusion, which could enhance 3 H-TdR incorporation into malignant cells. When 50%, 40%, 30% and 30% of cell survival rate defined as sensitivity-threshold for VCR, MMC, DDP and ADM respectively, in the vitro effectiveness were close to those of clinical single-drug treatment in HPCC by Wright et al. This method was also compared with HTSCA in ten human lung cancer cell lines and four pulmonary carcinoma tissues. The agreement rates were 83% and 100% respectively. Thus we presume this system is more useful for oncological clinics than the others

  3. Effect of insulin on albumin production and incorporation of 14C-leucine into proteins in isolated parenchymal liver cells from normal rats

    DEFF Research Database (Denmark)

    Dich, J; Gluud, C N

    1975-01-01

    the immunologically determined increment in the incubation medium was 1.7 +/- 0.2 mug albumin/min per g liver wet wt. This is about 30% of the rate of production in the perfused liver. Addition of insulin (10(-6)-10(-10) M) enhanced albumin production (50-17%), and incorporation of 14C-leucine both into albumin (50......Parenchymal rat liver cells were isolated by a modification of the collagenase method of Quistorff, Bondesen and Grunnet. The cells secreted albumin into the medium and incorporated 14C-leucine both into cell proteins and proteins secreted into the medium. Albumin production measured from......-8%), secreted proteins (40-9%) and cell proteins (20-8%). Insulin does not increase the production of albumin by depleting the cells. The effect of insulin on albumin production is compatible with an effect on the rate of synthesis as the specific activity of albumin is unaffected by addition of insulin....

  4. An enzyme-linked immunosorbent assay for bromodeoxyuridine incorporation using fixed microcultures

    International Nuclear Information System (INIS)

    Muir, D.; Varon, S.; Manthorpe, M.

    1990-01-01

    We report a quantitative method by which a single microculture can be examined for cell morphology; cell number; DNA synthesis; and expression of cell antigens. This method first involves measuring by enzyme-linked immunosorbent assay (ELISA) the total bromodeoxyuridine (BrdU) incorporation into DNA by monolayer microcultures. The BrdU-ELISA measurement was followed by simultaneous immunostaining for BrdU-positive nuclei and for a cytoplasmic antigen. The method was applied to the measurement of mitogen-induced proliferation of rat sciatic nerve Schwann cell and cerebral astroglia microcultures. The ELISA measurement of BrdU incorporation compares favorably with measurements of tritiated thymidine incorporation and offers the additional advantages that the same microculture can subsequently be examined for cell number, for cell morphology, and for the percentage of cells having BrdU-labeled nuclei and other antigens

  5. An enzyme-linked immunosorbent assay for bromodeoxyuridine incorporation using fixed microcultures

    Energy Technology Data Exchange (ETDEWEB)

    Muir, D.; Varon, S.; Manthorpe, M. (Univ. of California, San Diego, La Jolla (USA))

    1990-03-01

    We report a quantitative method by which a single microculture can be examined for cell morphology; cell number; DNA synthesis; and expression of cell antigens. This method first involves measuring by enzyme-linked immunosorbent assay (ELISA) the total bromodeoxyuridine (BrdU) incorporation into DNA by monolayer microcultures. The BrdU-ELISA measurement was followed by simultaneous immunostaining for BrdU-positive nuclei and for a cytoplasmic antigen. The method was applied to the measurement of mitogen-induced proliferation of rat sciatic nerve Schwann cell and cerebral astroglia microcultures. The ELISA measurement of BrdU incorporation compares favorably with measurements of tritiated thymidine incorporation and offers the additional advantages that the same microculture can subsequently be examined for cell number, for cell morphology, and for the percentage of cells having BrdU-labeled nuclei and other antigens.

  6. Chromosome damage in Chinese hamster cells produced by 125I-UdR at the site of its incorporation

    International Nuclear Information System (INIS)

    Hughes, W.L.; Weinblatt, A.C.; Prensky, W.

    1978-01-01

    Metaphase chromosomal aberrations were produced by 125 I-labeled iododeoxyuridine ( 125 I-UdR) incorporated into Chinese hamster Don cells at the end of the S-period of the cell cycle. Chromosome damage and the number of autoradiographic silver grains were recorded for whole cells, for chromosome pairs 4 and 5 and for the X and the Y chromosomes. The X and the Y chromosomes, which label late in S, were at least twice as heavily labeled as chromosome pairs 4 and 5 - two readily recognizable autosomes of similar size. The incidence of chromosome damage was at least six times that which would have been expected from equivalent doses of X-rays and the incidence of damage was directly related to the number of silver grains over each chromosome. It is estimated that it takes four to ten disintegrations to produce a visible chromosome aberration. The finding that chromosome damage is localized at the site of the 125 I decay is most readily explained by the high flux of low energy Auger electrons occurring at the site of the decay of the incorporated 125 I atom. (Auth.)

  7. Exciton delocalization incorporated drift-diffusion model for bulk-heterojunction organic solar cells

    Science.gov (United States)

    Wang, Zi Shuai; Sha, Wei E. I.; Choy, Wallace C. H.

    2016-12-01

    Modeling the charge-generation process is highly important to understand device physics and optimize power conversion efficiency of bulk-heterojunction organic solar cells (OSCs). Free carriers are generated by both ultrafast exciton delocalization and slow exciton diffusion and dissociation at the heterojunction interface. In this work, we developed a systematic numerical simulation to describe the charge-generation process by a modified drift-diffusion model. The transport, recombination, and collection of free carriers are incorporated to fully capture the device response. The theoretical results match well with the state-of-the-art high-performance organic solar cells. It is demonstrated that the increase of exciton delocalization ratio reduces the energy loss in the exciton diffusion-dissociation process, and thus, significantly improves the device efficiency, especially for the short-circuit current. By changing the exciton delocalization ratio, OSC performances are comprehensively investigated under the conditions of short-circuit and open-circuit. Particularly, bulk recombination dependent fill factor saturation is unveiled and understood. As a fundamental electrical analysis of the delocalization mechanism, our work is important to understand and optimize the high-performance OSCs.

  8. Incorporation of cerium oxide into hydroxyapatite coating regulates osteogenic activity of mesenchymal stem cell and macrophage polarization.

    Science.gov (United States)

    Li, Kai; Shen, Qingyi; Xie, Youtao; You, Mingyu; Huang, Liping; Zheng, Xuebin

    2017-02-01

    Biomedical coatings for orthopedic implants should facilitate osseointegration and mitigate implant-induced inflammatory reactions. Cerium oxide (CeO 2 ) ceramics possess anti-oxidative properties and can be used to decrease mediators of inflammation, which makes them attractive for biomedical applications. In our work, two kinds of CeO 2 incorporated hydroxyapatite coatings (HA-10Ce and HA-30Ce) were prepared via plasma spraying technique and the effects of CeO 2 addition on the responses of bone mesenchymal stem cells (BMSCs) and RAW264.7 macrophages were investigated. An increase in CeO 2 content in the HA coatings resulted in better osteogenic behaviors of BMSCs in terms of cell proliferation, alkaline phosphatase (ALP) activity and mineralized nodule formation. RT-PCR and western blot analysis suggested that the incorporation of CeO 2 may promote the osteogenic differentiation of BMSCs through the Smad-dependent BMP signaling pathway, which activated Runx2 expression and subsequently enhanced the expression of ALP and OCN. The expression profiles of macrophages cultured on the CeO 2 modified coating revealed a tendency toward a M2 phenotype, because of an upregulation of M2 surface markers (CD163 and CD206), anti-inflammatory cytokines (TNF-α and IL-6) and osteoblastogenesis-related genes (BMP2 and TGF-β1) as well as a downregulation of M1 surface markers (CCR7 and CD11c), proinflammatory cytokines (IL-10 and IL-1ra) and reactive oxygen species production. The results suggested the regulation of BMSCs behaviors and macrophage-mediated responses at the coating's surface were associated with CeO 2 incorporation. The incorporation of CeO 2 in HA coatings can be a valuable strategy to promote osteogenic responses and reduce inflammatory reactions.

  9. Impairment of DNA synthesis in Gunn rat cerebellum.

    Science.gov (United States)

    Yamada, N; Sawasaki, Y; Nakajima, H

    1977-05-06

    Brain DNA synthesis was developmentally investigated in Gunn rat with marked cerebellar hypoplasia due to hereditary hyperbilirubinemia. In this mutant rat, the Purkinje cell was nearly selectively affected in the cerebellar cortex by bilirubin. The impaired DNA synthesis was observed in homozygous (jj) Gunn rat cerebellum, in which the DNA content and [3H]thymidine incorporation rate into DNA decreased after 10 days of age compared to those in the heterozygous (Jj)littermate. In contrast, these impairments were not found in the non-cerebellar parts of the brain and liver of jj Gunn rat. The activity of cerebellar thymidine kinase in jj Gunn rat decreased from a very early stae, being 80% of Jj rat at 6 days, and 50% at 10 days of age. The enzyme activity was not affected in the non-cerebellar parts of the brain. Although bilirubin competitively inhibited cerebellar thymidine kinase activity in vitro (15% at 10(-5) M), such bilirubin level was found to be about 1000-fold that in vivo. Moreover, photo-degradation of bilirubin in jj cerebellum exhibited no improvement in thymidine kinase activity, and the presence of an enzyme inactivator was not suggested in jj cerebellum. These results seem to indicate that the induction of thymidine kinase might be affected in jj Gunn rat cerebellum. The possibility that the impaired DNA synthesis in the external granular cells in jj cerebellum may be due to Purkinje cell damage is discussed.

  10. Changes in 2-fluoro-2-deoxy-D-glucose incorporation, hexokinase activity and lactate production by breast cancer cells responding to treatment with the anti-HER-2 antibody trastuzumab

    Energy Technology Data Exchange (ETDEWEB)

    Cheyne, Richard W. [School of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD (United Kingdom); Trembleau, Laurent; McLaughlin, Abbie [School of Natural and Computing Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD (United Kingdom); Smith, Tim A.D., E-mail: t.smith@abdn.ac.u [School of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD (United Kingdom)

    2011-04-15

    Introduction: Changes in 2-[{sup 18}F]-fluoro-2-deoxy-D-glucose (FDG) incorporation by tumors, detected using positron emission tomography, during response to chemotherapy are utilized clinically in patient management. Here, the effect of treatment with growth-inhibitory doses of the anti-human epidermal growth factor receptor-2 antibody trastuzumab (Herceptin) on the incorporation of FDG by breast tumor cells was measured along with hexokinase (HK) and glucose transport to determine the potential of FDG-positron emission tomography in predicting response to these biological anti-cancer therapies and their modulatory effects on the steps involved in FDG incorporation. Methods: The sensitivity to trastuzumab of three breast tumor cell lines, SKBr3, MDA-MB-453 and MDA-MB-468, expressing human epidermal growth factor receptor-2 at high, medium and low levels, respectively, was determined using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay over a 6-day period, and a clonogenic assay was carried out after 7- and 10-day exposures. FDG incorporation by cells treated with growth-inhibitory doses of trastuzumab was carried out after 4 h and 2, 4 and 6 days of treatment. Glucose transport (rate of uptake of the non-metabolizable analogue [{sup 3}H]O-methyl-D-glucose), HK activity and lactate production were measured on cells treated with inhibitory doses of trastuzumab for 6 days. Results: The IC{sub 50} doses for SKBr3 and MDA-MB-453 and the IC{sub 20} dose for MDA-MB-468 after 6 days of treatment with trastuzumab were 0.25, 1 and 170 {mu}g/ml, respectively. FDG incorporation by SKBr3 and MDA-MB-453 cells was found to be decreased using IC{sub 50} doses of trastuzumab for 6 days. At the IC{sub 50} doses, FDG incorporation was also decreased at 4 days and, in the case of MDA-MB-453, even after 4 h of treatment. Decreased FDG incorporation corresponded with decreased HK activity in these cells. Lactate production, previously suggested to be a

  11. SaOS-2 cell response to macro-porous boron-incorporated TiO{sub 2} coating prepared by micro-arc oxidation on titanium

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Qianli [State Key Laboratory of New Ceramics and Fine Processing, School of Materials Science and Engineering, Tsinghua University, Beijing 100084 (China); Elkhooly, Tarek A. [State Key Laboratory of New Ceramics and Fine Processing, School of Materials Science and Engineering, Tsinghua University, Beijing 100084 (China); Department of Ceramics, Inorganic Chemical Industries Division, National Research Centre, Dokki, 12622 Cairo (Egypt); Liu, Xujie [State Key Laboratory of New Ceramics and Fine Processing, School of Materials Science and Engineering, Tsinghua University, Beijing 100084 (China); Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055 (China); Zhang, Ranran; Yang, Xing; Shen, Zhijian [State Key Laboratory of New Ceramics and Fine Processing, School of Materials Science and Engineering, Tsinghua University, Beijing 100084 (China); Feng, Qingling, E-mail: biomater@mail.tsinghua.edu.cn [State Key Laboratory of New Ceramics and Fine Processing, School of Materials Science and Engineering, Tsinghua University, Beijing 100084 (China); Key Laboratory of Advanced Materials of Ministry of Education of China, School of Materials Science and Engineering, Tsinghua University, Beijing 100084 (China)

    2016-10-01

    The aims of the present study were to develop boron-incorporated TiO{sub 2} coating (B-TiO{sub 2} coating) through micro-arc oxidation (MAO) and subsequently evaluate the effect of boron incorporation on the in vitro biological performance of the coatings. The physicochemical properties of B-TiO{sub 2} coating and its response to osteoblast like cells (SaOS-2) were investigated compared to the control group without boron (TiO{sub 2} coating). The morphological and X-ray diffraction results showed that both coatings exhibited similar surface topography and phase composition, respectively. However, the incorporation of B led to an enhancement in the surface hydrophilicity of B-TiO{sub 2} coating. The spreading of SaOS-2 cells on B-TiO{sub 2} coating was faster than that on TiO{sub 2} coating. The proliferation rate of SaOS-2 cells cultured on B-TiO{sub 2} decreased after 5 days of culture compared to that on TiO{sub 2} coating. SaOS-2 cells cultured on B-TiO{sub 2} coating exhibited an enhanced alkaline phosphatase (ALP) activity, Collagen I synthesis and in vitro mineralization compared to those on TiO{sub 2} coating. The present findings suggest that B-TiO{sub 2} coating is a promising candidate surface for orthopedic implants. - Highlights: • SaOS-2 cell response to pure TiO{sub 2} and B-TiO{sub 2} coatings was investigated. • Initial cell spreading on B-TiO{sub 2} coating was accelerated compared to that on TiO{sub 2} coating. • Cell proliferation on B-TiO{sub 2} coating was inhibited compared to that on TiO{sub 2} coating. • Cell differentiation on B-TiO{sub 2} coating was enhanced compared to that on TiO{sub 2} coating.

  12. Dye-Incorporated Polynaphthalenediimide Acceptor for Additive-Free High-Performance All-Polymer Solar Cells.

    Science.gov (United States)

    Chen, Dong; Yao, Jia; Chen, Lie; Yin, Jingping; Lv, Ruizhi; Huang, Bin; Liu, Siqi; Zhang, Zhi-Guo; Yang, Chunhe; Chen, Yiwang; Li, Yongfang

    2018-04-16

    All-polymer solar cells (all-PSCs) can offer unique advantages for applications in flexible devices, and naphthalene diimide (NDI)-based polymer acceptors are the widely used polymer acceptors. However, their power conversion efficiency (PCE) still lags behind that of state-of-the-art polymer solar cells, due to low light absorption, suboptimal energy levels and the strong aggregation of the NDI-based polymer acceptor. Herein, a rhodanine-based dye molecule was introduced into the NDI-based polymer acceptor by simple random copolymerization and showed an improved light absorption coefficient, an up-shifted lowest unoccupied molecular orbital level and reduced crystallization. Consequently, additive-free all-PSCs demonstrated a high PCE of 8.13 %, which is one of the highest performance characteristics reported for all-PSCs to date. These results indicate that incorporating a dye into the n-type polymer gives insight into the precise design of high-performance polymer acceptors for all-PSCs. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. LINGO-1 and Neurodegeneration: Pathophysiologic Clues for Essential Tremor?

    Directory of Open Access Journals (Sweden)

    Zhou Zhi-dong

    2012-03-01

    Full Text Available Essential tremor (ET, one of the most common adult-onset movement disorders, has been associated with cerebellar Purkinje cell degeneration and formation of brainstem Lewy bodies. Recent findings suggest that genetic variants of the leucine-rich repeat and Ig domain containing 1 (LINGO-1 gene could be risk factors for ET. The LINGO-1 protein contains both leucine-rich repeat (LRR and immunoglobulin (Ig-like domains in its extracellular region, as well as a transmembrane domain and a short cytoplasmic tail. LINGO-1 can form a ternary complex with Nogo-66 receptor (NgR1 and p75. Binding of LINGO-1 with NgR1 can activate the NgR1 signaling pathway, leading to inhibition of oligodendrocyte differentiation and myelination in the central nervous system. LINGO-1 has also been found to bind with epidermal growth factor receptor (EGFR and induce downregulation of the activity of EGFR–PI3K–Akt signaling, which might decrease Purkinje cell survival. Therefore, it is possible that genetic variants of LINGO-1, either alone or in combination with other genetic or environmental factors, act to increase LINGO-1 expression levels in Purkinje cells and confer a risk to Purkinje cell survival in the cerebellum. Here, we provide a concise summary of the link between LINGO-1 and neurodegeneration and discuss various hypotheses as to how this could be potentially relevant to ET pathogenesis.

  14. Improved Power Conversion Efficiency of Inverted Organic Solar Cells by Incorporating Au Nanorods into Active Layer.

    Science.gov (United States)

    He, Yeyuan; Liu, Chunyu; Li, Jinfeng; Zhang, Xinyuan; Li, Zhiqi; Shen, Liang; Guo, Wenbin; Ruan, Shengping

    2015-07-29

    This Research Article describes a cooperative plasmonic effect on improving the performance of organic solar cells. When Au nanorods(NRs) are incorporated into the active layers, the designed project shows superior enhanced light absorption behavior comparing with control devices, which leads to the realization of organic solar cell with power conversion efficiency of 6.83%, accounting for 18.9% improvement. Further investigations unravel the influence of plasmonic nanostructures on light trapping, exciton generation, dissociation, and charge recombination and transport inside the thin films devices. Moreover, the introduction of high-conductivity Au NRs improves electrical conductivity of the whole device, which contributes to the enhanced fill factor.

  15. Incorporation of mesoporous silica nanoparticles into random electrospun PLGA and PLGA/gelatin nanofibrous scaffolds enhances mechanical and cell proliferation properties

    International Nuclear Information System (INIS)

    Mehrasa, Mohammad; Asadollahi, Mohammad Ali; Nasri-Nasrabadi, Bijan; Ghaedi, Kamran; Salehi, Hossein; Dolatshahi-Pirouz, Alireza; Arpanaei, Ayyoob

    2016-01-01

    Poly(lactic-co-glycolic acid) (PLGA) and PLGA/gelatin random nanofibrous scaffolds embedded with different amounts of mesoporous silica nanoparticles (MSNPs) were fabricated using electrospinning method. To evaluate the effects of nanoparticles on the scaffolds, physical, chemical, and mechanical properties as well as in vitro degradation behavior of scaffolds were investigated. The mean diameters of nanofibers were 974 ± 68 nm for the pure PLGA scaffolds vs 832 ± 70, 764 ± 80, and 486 ± 64 for the PLGA/gelatin, PLGA/10 wt% MSNPs, and the PLGA/gelatin/10 wt% MSNPs scaffolds, respectively. The results suggested that the incorporation of gelatin and MSNPs into PLGA-based scaffolds enhances the hydrophilicity of scaffolds due to an increase of hydrophilic functional groups on the surface of nanofibers. With porosity examination, it was concluded that the incorporation of MSNPs and gelatin decrease the porosity of scaffolds. Nanoparticles also improved the tensile mechanical properties of scaffolds. Using in vitro degradation analysis, it was shown that the addition of nanoparticles to the nanofibers matrix increases the weight loss percentage of PLGA-based samples, whereas it decreases the weight loss percentage in the PLGA/gelatin composites. Cultivation of rat pheochromocytoma cell line (PC12), as precursor cells of dopaminergic neural cells, on the scaffolds demonstrated that the introduction of MSNPs into PLGA and PLGA/gelatin matrix leads to improved cell attachment and proliferation and enhances cellular processes. - Highlights: • PLGA-based random nanofibers embedded with mesoporous silica nanoparticles were fabricated using electrospinning method • Incorporation of gelatin and MSNPs into PLGA-based scaffolds increased the hydrophilicity of scaffold • Addition of nanoparticles also improved the tensile mechanical properties of scaffolds • Introduction of MSNPs led to improved cell attachment and proliferation

  16. Incorporation of mesoporous silica nanoparticles into random electrospun PLGA and PLGA/gelatin nanofibrous scaffolds enhances mechanical and cell proliferation properties

    Energy Technology Data Exchange (ETDEWEB)

    Mehrasa, Mohammad [Department of Biotechnology, Faculty of Advanced Sciences and Technologies, University of Isfahan, Isfahan 81746-73441 (Iran, Islamic Republic of); Department of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology, Tehran (Iran, Islamic Republic of); Asadollahi, Mohammad Ali, E-mail: ma.asadollahi@ast.ui.ac.ir [Department of Biotechnology, Faculty of Advanced Sciences and Technologies, University of Isfahan, Isfahan 81746-73441 (Iran, Islamic Republic of); Nasri-Nasrabadi, Bijan [Department of Chemical Engineering, Isfahan University of Technology, Isfahan (Iran, Islamic Republic of); Ghaedi, Kamran [Department of Biology, Faculty of Science, University of Isfahan, Isfahan 81746-73441 (Iran, Islamic Republic of); Salehi, Hossein [Department of Anatomical Sciences, School of Medicine, Isfahan University of Medical Sciences, Isfahan (Iran, Islamic Republic of); Dolatshahi-Pirouz, Alireza [DTU Nanotech, Center for Nanomedicine and Theranostics, Technical University of Denmark (DTU), DK-2800 Kgs. Lyngby (Denmark); Arpanaei, Ayyoob, E-mail: arpanaei@yahoo.com [Department of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology, Tehran (Iran, Islamic Republic of)

    2016-09-01

    Poly(lactic-co-glycolic acid) (PLGA) and PLGA/gelatin random nanofibrous scaffolds embedded with different amounts of mesoporous silica nanoparticles (MSNPs) were fabricated using electrospinning method. To evaluate the effects of nanoparticles on the scaffolds, physical, chemical, and mechanical properties as well as in vitro degradation behavior of scaffolds were investigated. The mean diameters of nanofibers were 974 ± 68 nm for the pure PLGA scaffolds vs 832 ± 70, 764 ± 80, and 486 ± 64 for the PLGA/gelatin, PLGA/10 wt% MSNPs, and the PLGA/gelatin/10 wt% MSNPs scaffolds, respectively. The results suggested that the incorporation of gelatin and MSNPs into PLGA-based scaffolds enhances the hydrophilicity of scaffolds due to an increase of hydrophilic functional groups on the surface of nanofibers. With porosity examination, it was concluded that the incorporation of MSNPs and gelatin decrease the porosity of scaffolds. Nanoparticles also improved the tensile mechanical properties of scaffolds. Using in vitro degradation analysis, it was shown that the addition of nanoparticles to the nanofibers matrix increases the weight loss percentage of PLGA-based samples, whereas it decreases the weight loss percentage in the PLGA/gelatin composites. Cultivation of rat pheochromocytoma cell line (PC12), as precursor cells of dopaminergic neural cells, on the scaffolds demonstrated that the introduction of MSNPs into PLGA and PLGA/gelatin matrix leads to improved cell attachment and proliferation and enhances cellular processes. - Highlights: • PLGA-based random nanofibers embedded with mesoporous silica nanoparticles were fabricated using electrospinning method • Incorporation of gelatin and MSNPs into PLGA-based scaffolds increased the hydrophilicity of scaffold • Addition of nanoparticles also improved the tensile mechanical properties of scaffolds • Introduction of MSNPs led to improved cell attachment and proliferation.

  17. Simulation Methods and Validation Criteria for Modeling Cardiac Ventricular Electrophysiology.

    Directory of Open Access Journals (Sweden)

    Shankarjee Krishnamoorthi

    Full Text Available We describe a sequence of methods to produce a partial differential equation model of the electrical activation of the ventricles. In our framework, we incorporate the anatomy and cardiac microstructure obtained from magnetic resonance imaging and diffusion tensor imaging of a New Zealand White rabbit, the Purkinje structure and the Purkinje-muscle junctions, and an electrophysiologically accurate model of the ventricular myocytes and tissue, which includes transmural and apex-to-base gradients of action potential characteristics. We solve the electrophysiology governing equations using the finite element method and compute both a 6-lead precordial electrocardiogram (ECG and the activation wavefronts over time. We are particularly concerned with the validation of the various methods used in our model and, in this regard, propose a series of validation criteria that we consider essential. These include producing a physiologically accurate ECG, a correct ventricular activation sequence, and the inducibility of ventricular fibrillation. Among other components, we conclude that a Purkinje geometry with a high density of Purkinje muscle junctions covering the right and left ventricular endocardial surfaces as well as transmural and apex-to-base gradients in action potential characteristics are necessary to produce ECGs and time activation plots that agree with physiological observations.

  18. Simulation Methods and Validation Criteria for Modeling Cardiac Ventricular Electrophysiology.

    Science.gov (United States)

    Krishnamoorthi, Shankarjee; Perotti, Luigi E; Borgstrom, Nils P; Ajijola, Olujimi A; Frid, Anna; Ponnaluri, Aditya V; Weiss, James N; Qu, Zhilin; Klug, William S; Ennis, Daniel B; Garfinkel, Alan

    2014-01-01

    We describe a sequence of methods to produce a partial differential equation model of the electrical activation of the ventricles. In our framework, we incorporate the anatomy and cardiac microstructure obtained from magnetic resonance imaging and diffusion tensor imaging of a New Zealand White rabbit, the Purkinje structure and the Purkinje-muscle junctions, and an electrophysiologically accurate model of the ventricular myocytes and tissue, which includes transmural and apex-to-base gradients of action potential characteristics. We solve the electrophysiology governing equations using the finite element method and compute both a 6-lead precordial electrocardiogram (ECG) and the activation wavefronts over time. We are particularly concerned with the validation of the various methods used in our model and, in this regard, propose a series of validation criteria that we consider essential. These include producing a physiologically accurate ECG, a correct ventricular activation sequence, and the inducibility of ventricular fibrillation. Among other components, we conclude that a Purkinje geometry with a high density of Purkinje muscle junctions covering the right and left ventricular endocardial surfaces as well as transmural and apex-to-base gradients in action potential characteristics are necessary to produce ECGs and time activation plots that agree with physiological observations.

  19. Selective incorporation of 5-hydroxytryptophan into proteins in mammalian cells

    Science.gov (United States)

    Zhang, Zhiwen; Alfonta, Lital; Schultz, Peter G

    2014-02-25

    This invention provides methods and compositions for incorporation of an unnatural amino acid into a peptide using an orthogonal aminoacyl tRNA synthetase/tRNA pair. In particular, an orthogonal pair is provided to incorporate 5-hydroxy-L-tryptophan in a position encoded by an opal mutation.

  20. A rapid [3H]glucose incorporation assay for determination of lymphoid cell-mediated inhibition of Candida albicans growth

    International Nuclear Information System (INIS)

    Djeu, J.Y.; Parapanissios, A.; Halkias, D.; Friedman, H.

    1986-01-01

    [ 3 H]glucose uptake by Candida albicans after interaction with lymphoid effector cells was used to provide a quick, accurate and objective assessment of the growth inhibitory potential of lymphoid cells on candida. After 18 h coincubation of effector cells with candida, [ 3 H]glucose was added for 3 h and the amount of radiolabel incorporated into residual candida was measured. The results showed that [ 3 H]glucose uptake was proportional to the number of candida organisms left in the microwell and is dose dependent on the effector/target (E/T) ratio. At an E/T ratio of 300/1, complete inhibition of candida was seen, with significant inhibition still present at 30/1. In addition, monocytes and polymorphonuclear cells were found to be the primary cells responsible for eliminating candida. (Auth.)

  1. Differing sensitivity to fluorescent light in Chinese hamster cells containing equally incorporated quantities of BUdR versus IUdR

    International Nuclear Information System (INIS)

    Mitchell, J.B.; Morstyn, G.; Russo, A.; Kinsella, T.J.; Fornace, A. Jr.; McPherson, S.; Glatstein, E.

    1984-01-01

    Chinese hamster V79 cells that had incorporated approximately equal levels of either BUdR or IUdR into their DNA were found to be equal sensitizers to x rays. However, BUdR-substituted cells were much more sensitive to fluorescent light than IUdR-substituted cells, both on a cell survival basis and by the initial number of single strand DNA breaks induced. Since a major toxicity to the use of BUdR clinically has been light-induced skin rash, these data indicate that the use of IUdR clinically might cause less untoward toxicity but yet provide the same radiosensitization as BUdR

  2. Glucocorticoid effect on melphalan cytotoxicity, cell-cycle position, cell size, and [3H]uridine incorporation in one of three human melanoma cell lines

    International Nuclear Information System (INIS)

    Benckhuijsen, C.; Osman, A.M.; Hillebrand, M.J.; Smets, L.A.

    1987-01-01

    Three human melanoma cell lines of known content of specific glucocorticoid-binding sites were studied for colony formation after a microM dose of glucocorticoid combined with melphalan. In one of the three cell lines, M-5A, subcloned from M-5 (formerly designated RPMI 8322), the effect of combined treatment was markedly increased compared to that of melphalan even if the glucocorticoid was applied for 1 h only, 10 h before the melphalan. Semilogarithmic dose-effect plots for a reduction of final plating efficiency by glucocorticoid were curvilinear, according to a receptor-mediated process. The effects of glucocorticoid, melphalan, and their combination were linearized by bilogarithmic median-effect plotting which allowed the quantitation of a synergism which was more marked in case of glucocorticoid pretreatment, for 1 or 24 h, than on simultaneous exposure. According to sequential DNA per cell cytophotometry, melphalan abolished in M-5A a glucocorticoid-induced arrest in the G1 phase of the cell cycle. The cytotoxic synergism correlated with an apparent stimulation by glucocorticoid of the rate of acid-insoluble incorporation of [ 3 H]uridine and [ 14 C]leucine and an increase in cell size and protein content in M-5A cells but not in the other two cell lines. The way in which glucocorticoids induce an enhanced susceptibility to melphalan is not clear. Our results appear compatible with a hypothesis that chromatin in a transcriptionally activated state is more vulnerable to cytotoxic attack by an alkylating agent than under average conditions

  3. The N-Linked Outer Chain Mannans and the Dfg5p and Dcw1p Endo-α-1,6-Mannanases Are Needed for Incorporation of Candida albicans Glycoproteins into the Cell Wall

    Science.gov (United States)

    Ao, Jie; Chinnici, Jennifer L.; Maddi, Abhiram

    2015-01-01

    A biochemical pathway for the incorporation of cell wall protein into the cell wall of Neurospora crassa was recently proposed. In this pathway, the DFG-5 and DCW-1 endo-α-1,6-mannanases function to covalently cross-link cell wall protein-associated N-linked galactomannans, which are structurally related to the yeast outer chain mannans, into the cell wall glucan-chitin matrix. In this report, we demonstrate that the mannosyltransferase enzyme Och1p, which is needed for the synthesis of the N-linked outer chain mannan, is essential for the incorporation of cell wall glycoproteins into the Candida albicans cell wall. Using endoglycosidases, we show that C. albicans cell wall proteins are cross-linked into the cell wall via their N-linked outer chain mannans. We further demonstrate that the Dfg5p and Dcw1p α-1,6-mannanases are needed for the incorporation of cell wall glycoproteins into the C. albicans cell wall. Our results support the hypothesis that the Dfg5p and Dcw1p α-1,6-mannanases incorporate cell wall glycoproteins into the C. albicans cell wall by cross-linking outer chain mannans into the cell wall glucan-chitin matrix. PMID:26048011

  4. Tritiated thymidine incorporation and the growth of heterotrophic bacteria in warm core rings

    International Nuclear Information System (INIS)

    Ducklow, H.W.; Hill, S.M.

    1985-01-01

    The time-course of the incorporation rate of [methyl- 3 H]thymidine ([ 3 H]TdR) was established during 6-12 h incubations of natural bacterial populations sampled from the surface layers of warm core Gulf Stream rings. Parallel estimates of changes in cell numbers were made in order to examine the relationships between TdR incorporation and population growth for oceanic bacterial populations. Their results indicate that a conversion factor of 4 x 10 18 cells produced per mole of [ 3 H]TdR incorporated yielded estimates of bacterial production which were within a factor of 2 or 3 of production estimates derived from changes in cell numbers in seawater cultures. The authors observed a significant, direct relationship between the initial rates of TdR incorporation per cell and specific growth rates and conclude that initial short term (15-45 min) assays of TdR incorporation are a valuable tool for studying bacterial production in oceanic waters. In most incubations, the rate of TdR incorporation increased more rapidly than did cell numbers. Very large conversion factor values were derived from these data. The discrepancy between growth determined from TdR incorporation rates and total bacterial numbers in seawater cultures has not been observed in previous studies of coastal, estuarine, or lacustrine bacteria, but was a consistent feature of our studies on oceanic populations

  5. /sup 3/H-UTP incorporation in polytene chromosomes of Chironomus permeabilized cells

    Energy Technology Data Exchange (ETDEWEB)

    Diez, J L; de la Torre, C [Consejo Superior de Investigacionis Cientificas, Madrid (Spain). Inst. Biologia Celular

    1979-01-01

    A method is described that allows the cytologic detection of the transcriptional activity of endogenous RNA polymerase in fixed Chironomus polytene chromosomes. /sup 3/H-UTP is preferentially incorporated onto nucleoli and Balbiani rings where transcription is specially high. The former incorporation was sensitive to actinomycin D while incorporation on Balbiani rings was sensitive to ..cap alpha..-amanitin. The label pattern and frequency resemble those detected by /sup 3/H-uridine incorporation in the living state.

  6. Cell cycle- and chaperone-mediated regulation of H3K56ac incorporation in yeast.

    Science.gov (United States)

    Kaplan, Tommy; Liu, Chih Long; Erkmann, Judith A; Holik, John; Grunstein, Michael; Kaufman, Paul D; Friedman, Nir; Rando, Oliver J

    2008-11-01

    Acetylation of histone H3 lysine 56 is a covalent modification best known as a mark of newly replicated chromatin, but it has also been linked to replication-independent histone replacement. Here, we measured H3K56ac levels at single-nucleosome resolution in asynchronously growing yeast cultures, as well as in yeast proceeding synchronously through the cell cycle. We developed a quantitative model of H3K56ac kinetics, which shows that H3K56ac is largely explained by the genomic replication timing and the turnover rate of each nucleosome, suggesting that cell cycle profiles of H3K56ac should reveal most first-time nucleosome incorporation events. However, since the deacetylases Hst3/4 prevent use of H3K56ac as a marker for histone deposition during M phase, we also directly measured M phase histone replacement rates. We report a global decrease in turnover rates during M phase and a further specific decrease in turnover at several early origins of replication, which switch from rapidly replaced in G1 phase to stably bound during M phase. Finally, by measuring H3 replacement in yeast deleted for the H3K56 acetyltransferase Rtt109 and its two co-chaperones Asf1 and Vps75, we find evidence that Rtt109 and Asf1 preferentially enhance histone replacement at rapidly replaced nucleosomes, whereas Vps75 appears to inhibit histone turnover at those loci. These results provide a broad perspective on histone replacement/incorporation throughout the cell cycle and suggest that H3K56 acetylation provides a positive-feedback loop by which replacement of a nucleosome enhances subsequent replacement at the same location.

  7. Cell cycle- and chaperone-mediated regulation of H3K56ac incorporation in yeast.

    Directory of Open Access Journals (Sweden)

    Tommy Kaplan

    2008-11-01

    Full Text Available Acetylation of histone H3 lysine 56 is a covalent modification best known as a mark of newly replicated chromatin, but it has also been linked to replication-independent histone replacement. Here, we measured H3K56ac levels at single-nucleosome resolution in asynchronously growing yeast cultures, as well as in yeast proceeding synchronously through the cell cycle. We developed a quantitative model of H3K56ac kinetics, which shows that H3K56ac is largely explained by the genomic replication timing and the turnover rate of each nucleosome, suggesting that cell cycle profiles of H3K56ac should reveal most first-time nucleosome incorporation events. However, since the deacetylases Hst3/4 prevent use of H3K56ac as a marker for histone deposition during M phase, we also directly measured M phase histone replacement rates. We report a global decrease in turnover rates during M phase and a further specific decrease in turnover at several early origins of replication, which switch from rapidly replaced in G1 phase to stably bound during M phase. Finally, by measuring H3 replacement in yeast deleted for the H3K56 acetyltransferase Rtt109 and its two co-chaperones Asf1 and Vps75, we find evidence that Rtt109 and Asf1 preferentially enhance histone replacement at rapidly replaced nucleosomes, whereas Vps75 appears to inhibit histone turnover at those loci. These results provide a broad perspective on histone replacement/incorporation throughout the cell cycle and suggest that H3K56 acetylation provides a positive-feedback loop by which replacement of a nucleosome enhances subsequent replacement at the same location.

  8. Impact of elastin incorporation into electrochemically aligned collagen fibers on mechanical properties and smooth muscle cell phenotype.

    Science.gov (United States)

    Nguyen, Thuy-Uyen; Bashur, Chris A; Kishore, Vipuil

    2016-03-17

    Application of tissue-engineered vascular grafts (TEVGs) for the replacement of small-diameter arteries is limited due to thrombosis and intimal hyperplasia. Previous studies have attempted to address the limitations of TEVGs by developing scaffolds that mimic the composition (collagen and elastin) of native arteries to better match the mechanical properties of the graft with the native tissue. However, most existing scaffolds do not recapitulate the aligned topography of the collagen fibers found in native vessels. In the current study, based on the principles of isoelectric focusing, two different types of elastin (soluble and insoluble) were incorporated into highly oriented electrochemically aligned collagen (ELAC) fibers and the effect of elastin incorporation on the mechanical properties of the ELAC fibers and smooth muscle cell (SMC) phenotype was investigated. The results indicate that elastin incorporation significantly decreased the modulus of ELAC fibers to converge upon that of native vessels. Further, a significant increase in yield strain and decrease in Young's modulus was observed on all fibers post SMC culture compared with before the culture. Real-time polymerase chain reaction results showed a significant increase in the expression of α-smooth muscle actin and calponin on ELAC fibers with insoluble elastin, suggesting that incorporation of insoluble elastin induces a contractile phenotype in SMCs after two weeks of culture on ELAC fibers. Immunofluorescence results showed that calponin expression increased with time on all fibers. In conclusion, insoluble elastin incorporated ELAC fibers have the potential to be used for the development of functional TEVGs for the repair and replacement of small-diameter arteries.

  9. JPRS Report: Science and Technology, Central Eurasia: Life Sciences

    National Research Council Canada - National Science Library

    1993-01-01

    ... on Ultrastructure of Parallel Fiber-Purkinje Cell Synapses in Frog Cerebellum, Cloning and Expression of Human Proinsulin Gene in Bacillus Amyloliquefaciens Selected for Low Exoprotease Activity, Potato Cell...

  10. Role of Glycol Chitosan-incorporated Ursolic Acid Nanoparticles in ...

    African Journals Online (AJOL)

    Purpose: To investigate the effect of ursolic acid (UA)-incorporated glycol chitosan (GC) nanoparticles on inhibition of human osteosarcoma. Methods: U2OS and Saos-2 osteosarcoma cells were transfected with ursolic acid (UA) incorporated glycol chitosan (GC) nanoparticles. Ultraviolet (UV) spectrophotometry was used ...

  11. Effect of hydroxyurea on mitotic activity 3H-thymidine and 3H-phenylalanine incorporation in the antheridial filament cells of Chara vulgaris

    International Nuclear Information System (INIS)

    Bielecka, A.

    1979-01-01

    Hydroxyurea inhibits mitotic activity in cells of the antheridial filaments of Chara vulgaris by blocking phase S and phase G 2 . Blocking of cells in phase G 2 also occurs in the case of the root meristem cells of Helianthus annuus and Vicia faba var. minor. 3 H-thine incorporation confirmed autoradiographically the blocking of cells of the antheridial filaments in Chara vulgaris at phase S and slowing down of the rate of DNA replication. Incubation with 3 H-phenylalanine demonstrated that hydroxyurea inhibits protein synthesis. (author)

  12. Uracil incorporation in the forespore and the mother cell during spore development in Bacillus subtilis

    International Nuclear Information System (INIS)

    Ryter, A.; Whitehouse, R.

    1978-01-01

    The transcriptional activity of the two genomes of the sporangium during spore formation was determined by pulse-labeling bacteria with 3 H-uracil at different times of sporulation and preparing them for high resolution autoradiography. The quantitative analysis of autoradiographs shows that uracile incorporation in the whole sporangium decreases considerably between stages II and IV. However, the variations of the transpcriptional activity are not identical in the mother cell and in the forespore. The one of the mother cell decreases rapidly between stages II and III and then remains stable until the end of stage IV, whereas that of the forespore which is low at stage II increases as the forespore grows ovoid and then quickly diminishes. It is very weak at the beginning of stage IV and negligible at the end of this stage. (orig.) [de

  13. Solo/Trio8, a membrane-associated short isoform of Trio, modulates endosome dynamics and neurite elongation.

    Science.gov (United States)

    Sun, Ying-Jie; Nishikawa, Kaori; Yuda, Hideki; Wang, Yu-Lai; Osaka, Hitoshi; Fukazawa, Nobuna; Naito, Akira; Kudo, Yoshihisa; Wada, Keiji; Aoki, Shunsuke

    2006-09-01

    With DNA microarrays, we identified a gene, termed Solo, that is downregulated in the cerebellum of Purkinje cell degeneration mutant mice. Solo is a mouse homologue of rat Trio8-one of multiple Trio isoforms recently identified in rat brain. Solo/Trio8 contains N-terminal sec14-like and spectrin-like repeat domains followed by a single guanine nucleotide exchange factor 1 (GEF1) domain, but it lacks the C-terminal GEF2, immunoglobulin-like, and kinase domains that are typical of Trio. Solo/Trio8 is predominantly expressed in Purkinje neurons of the mouse brain, and expression begins following birth and increases during Purkinje neuron maturation. We identified a novel C-terminal membrane-anchoring domain in Solo/Trio8 that is required for enhanced green fluorescent protein-Solo/Trio8 localization to early endosomes (positive for both early-endosome antigen 1 [EEA1] and Rab5) in COS-7 cells and primary cultured neurons. Solo/Trio8 overexpression in COS-7 cells augmented the EEA1-positive early-endosome pool, and this effect was abolished via mutation and inactivation of the GEF domain or deletion of the C-terminal membrane-anchoring domain. Moreover, primary cultured neurons transfected with Solo/Trio8 showed increased neurite elongation that was dependent on these domains. These results suggest that Solo/Trio8 acts as an early-endosome-specific upstream activator of Rho family GTPases for neurite elongation of developing Purkinje neurons.

  14. Improving Mycobacterium bovis bacillus Calmette-Guèrin as a vaccine delivery vector for viral antigens by incorporation of glycolipid activators of NKT cells.

    Science.gov (United States)

    Venkataswamy, Manjunatha M; Ng, Tony W; Kharkwal, Shalu S; Carreño, Leandro J; Johnson, Alison J; Kunnath-Velayudhan, Shajo; Liu, Zheng; Bittman, Robert; Jervis, Peter J; Cox, Liam R; Besra, Gurdyal S; Wen, Xiangshu; Yuan, Weiming; Tsuji, Moriya; Li, Xiangming; Ho, David D; Chan, John; Lee, Sunhee; Frothingham, Richard; Haynes, Barton F; Panas, Michael W; Gillard, Geoffrey O; Sixsmith, Jaimie D; Korioth-Schmitz, Birgit; Schmitz, Joern E; Larsen, Michelle H; Jacobs, William R; Porcelli, Steven A

    2014-01-01

    Recombinant Mycobacterium bovis bacillus Calmette-Guèrin (rBCG) has been explored as a vector for vaccines against HIV because of its ability to induce long lasting humoral and cell mediated immune responses. To maximize the potential for rBCG vaccines to induce effective immunity against HIV, various strategies are being employed to improve its ability to prime CD8+ T cells, which play an important role in the control of HIV infections. In this study we adopted a previously described approach of incorporating glycolipids that activate CD1d-restricted natural killer T (NKT) cells to enhance priming of CD8+ T cells by rBCG strains expressing an SIV Gag antigen (rBCG-SIV gag). We found that the incorporation of the synthetic NKT activating glycolipid α-galactosylceramide (α-GC) into rBCG-SIV gag significantly enhanced CD8+ T cell responses against an immunodominant Gag epitope, compared to responses primed by unmodified rBCG-SIV gag. The abilities of structural analogues of α-GC to enhance CD8+ T cell responses to rBCG were compared in both wild type and partially humanized mice that express human CD1d molecules in place of mouse CD1d. These studies identified an α-GC analogue known as 7DW8-5, which has previously been used successfully as an adjuvant in non-human primates, as a promising compound for enhancing immunogenicity of antigens delivered by rBCG.vectors. Our findings support the incorporation of synthetic glycolipid activators of NKT cells as a novel approach to enhance the immunogenicity of rBCG-vectored antigens for induction of CD8+ T cell responses. The glycolipid adjuvant 7DW8-5 may be a promising candidate for advancing to non-human primate and human clinical studies for the development of HIV vaccines based on rBCG vectors.

  15. Improving Mycobacterium bovis bacillus Calmette-Guèrin as a vaccine delivery vector for viral antigens by incorporation of glycolipid activators of NKT cells.

    Directory of Open Access Journals (Sweden)

    Manjunatha M Venkataswamy

    Full Text Available Recombinant Mycobacterium bovis bacillus Calmette-Guèrin (rBCG has been explored as a vector for vaccines against HIV because of its ability to induce long lasting humoral and cell mediated immune responses. To maximize the potential for rBCG vaccines to induce effective immunity against HIV, various strategies are being employed to improve its ability to prime CD8+ T cells, which play an important role in the control of HIV infections. In this study we adopted a previously described approach of incorporating glycolipids that activate CD1d-restricted natural killer T (NKT cells to enhance priming of CD8+ T cells by rBCG strains expressing an SIV Gag antigen (rBCG-SIV gag. We found that the incorporation of the synthetic NKT activating glycolipid α-galactosylceramide (α-GC into rBCG-SIV gag significantly enhanced CD8+ T cell responses against an immunodominant Gag epitope, compared to responses primed by unmodified rBCG-SIV gag. The abilities of structural analogues of α-GC to enhance CD8+ T cell responses to rBCG were compared in both wild type and partially humanized mice that express human CD1d molecules in place of mouse CD1d. These studies identified an α-GC analogue known as 7DW8-5, which has previously been used successfully as an adjuvant in non-human primates, as a promising compound for enhancing immunogenicity of antigens delivered by rBCG.vectors. Our findings support the incorporation of synthetic glycolipid activators of NKT cells as a novel approach to enhance the immunogenicity of rBCG-vectored antigens for induction of CD8+ T cell responses. The glycolipid adjuvant 7DW8-5 may be a promising candidate for advancing to non-human primate and human clinical studies for the development of HIV vaccines based on rBCG vectors.

  16. Regional cerebral palmitate incorporation following transient bilateral carotid occlusion in awake gerbils

    Energy Technology Data Exchange (ETDEWEB)

    Tone, O.; Miller, J.C.; Bell, J.M.; Rapoport, S.I.

    1987-11-01

    (/sup 14/C)Palmitate was injected intravenously in awake gerbils at various times after 5 minutes of bilateral carotid artery occlusion or a sham operation. Regional rates of incorporation of plasma palmitate into the hippocampus and other regions of the anterior circulation were determined relative to the mean rate of incorporation into regions of the posterior circulation using quantitative autoradiography and a ratio method of analysis. One day after bilateral carotid occlusion, relative palmitate incorporation was elevated significantly by 16% in the CA4 pyramidal cell layer and by 20% in the dentate gyrus of the hippocampus compared with sham-operated gerbils. At 3 days, significant elevations of this magnitude were found in the CA3 and CA4 cell layers, whereas relative incorporation was reduced by 26% in the CA1 pyramidal cell layer. At 7 days, the only significant difference from control was a 15% elevated incorporation in the CA3 pyramidal cell layer. Histologic examination indicated substantial cell death in the CA1 pyramidal layer at 3 days, with extensive glial reaction and phagocytic invasion at 7 days. Our results suggest that the turnover of palmitate-containing lipids is reduced in the CA1 layer of the gerbil hippocampus but that lipid synthesis is stimulated in hippocampal regions (CA3, CA4, dentate gyrus) affected by but recovering from transient bilateral carotid occlusion.

  17. Regional cerebral palmitate incorporation following transient bilateral carotid occlusion in awake gerbils

    International Nuclear Information System (INIS)

    Tone, O.; Miller, J.C.; Bell, J.M.; Rapoport, S.I.

    1987-01-01

    [ 14 C]Palmitate was injected intravenously in awake gerbils at various times after 5 minutes of bilateral carotid artery occlusion or a sham operation. Regional rates of incorporation of plasma palmitate into the hippocampus and other regions of the anterior circulation were determined relative to the mean rate of incorporation into regions of the posterior circulation using quantitative autoradiography and a ratio method of analysis. One day after bilateral carotid occlusion, relative palmitate incorporation was elevated significantly by 16% in the CA4 pyramidal cell layer and by 20% in the dentate gyrus of the hippocampus compared with sham-operated gerbils. At 3 days, significant elevations of this magnitude were found in the CA3 and CA4 cell layers, whereas relative incorporation was reduced by 26% in the CA1 pyramidal cell layer. At 7 days, the only significant difference from control was a 15% elevated incorporation in the CA3 pyramidal cell layer. Histologic examination indicated substantial cell death in the CA1 pyramidal layer at 3 days, with extensive glial reaction and phagocytic invasion at 7 days. Our results suggest that the turnover of palmitate-containing lipids is reduced in the CA1 layer of the gerbil hippocampus but that lipid synthesis is stimulated in hippocampal regions (CA3, CA4, dentate gyrus) affected by but recovering from transient bilateral carotid occlusion

  18. PEDOT:PSS incorporated silver nanoparticles prepared by gamma radiation for the application in organic solar cells

    Directory of Open Access Journals (Sweden)

    Omayma A. Ghazy

    2015-04-01

    Full Text Available Poly(3,4-ethylenedioxythiophene:Polystyrene sulfonate (PEDOT:PSS is a dispersion used as a buffer layer on the ITO electrode in the organic solar cells. Silver nanoparticles (Ag NPs are incorporated to the dispersion using two different strategies. The first is by reduction of silver ions in the PEDOT:PSS dispersion. Chemical reduction of silver ions using sodium borohydried is compared with reduction using gamma radiation. The TEM and UV-visible spectra indicates that smaller Ag NPs are obtained for the chemical reduction method than those obtained from the radiochemical. The second strategy, is by preparing Ag NPs in polyvinyl pyrolidone (PVP solution using gamma irradiation then adding them to the PEDOT:PSS dispersion. Layers of the PEDOT:PSS incorporated different concentrations of Ag NPs (1, 2, 4, 6, 8, 10% are formed. The SEM and AFM studies of the layers morphology reveal that smooth morphology on the obtained for layers containing Ag NPs up to concentrations of 4%.

  19. Metabolism of the carbocyclic analogue of (E)-5-(2-iodovinyl)-2'-deoxyuridine in herpes simplex virus-infected cells. Incorporation of C-IVDU into DNA

    International Nuclear Information System (INIS)

    De Clercq, E.; Bernaerts, R.; Balzarini, J.; Herdewijn, P.; Verbruggen, A.

    1985-01-01

    The carbocyclic analogues of (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) and (E)-5-(2-iodovinyl)-2'-deoxyuridine (IVDU), in which the sugar moiety is replaced by a cyclopentane ring and which have been designated as C-BVDU and C-IVDU, respectively, are, like their parent compounds BVDU and IVDU, potent and selective inhibitors of herpes simplex virus type 1 (HSV-1) and, to a lesser extent, herpes simplex virus type 2 (HSV-2) replication. The authors have now synthesized the radiolabeled C-IVDU analogue, C-[ 125 I]IVDU, and determined its metabolism by HSV-infected and mock-infected Vero cells. C-[ 125 I]IVDU was effectively phosphorylated by HSV-1-infected cells and, to a lesser extent, HSV-2-infected cells. C-[ 125 I]IVDU was not phosphorylated to an appreciable extent by either mock-infected cells or cells that had been infected with a thymidine kinase-deficient mutant of HSV-1. Furthermore, C-[ 125 I]IVDU was incorporated into both viral and cellular DNA of HSV-1-infected Vero cells. This finding represents the first demonstration of the incorporation of a cyclopentylpyrimidine into DNA

  20. Neurog1 Genetic Inducible Fate Mapping (GIFM) Reveals the Existence of Complex Spatiotemporal Cyto-Architectures in the Developing Cerebellum.

    Science.gov (United States)

    Obana, Edwin A; Lundell, Travis G; Yi, Kevin J; Radomski, Kryslaine L; Zhou, Qiong; Doughty, Martin L

    2015-06-01

    Neurog1 is a pro-neural basic helix-loop-helix (bHLH) transcription factor expressed in progenitor cells located in the ventricular zone and subsequently the presumptive white matter tracts of the developing mouse cerebellum. We used genetic inducible fate mapping (GIFM) with a transgenic Neurog1-CreER allele to characterize the contributions of Neurog1 lineages to cerebellar circuit formation in mice. GIFM reveals Neurog1-expressing progenitors are fate-mapped to become Purkinje cells and all GABAergic interneuron cell types of the cerebellar cortex but not glia. The spatiotemporal sequence of GIFM is unique to each neuronal cell type. GIFM on embryonic days (E) 10.5 to E12.5 labels Purkinje cells with different medial-lateral settling patterns depending on the day of tamoxifen delivery. GIFM on E11.5 to P7 labels interneurons and the timing of tamoxifen administration correlates with the final inside-to-outside resting position of GABAergic interneurons in the cerebellar cortex. Proliferative status and long-term BrdU retention of GIFM lineages reveals Purkinje cells express Neurog1 around the time they become post-mitotic. In contrast, GIFM labels mitotic and post-mitotic interneurons. Neurog1-CreER GIFM reveals a correlation between the timing of Neurog1 expression and the spatial organization of GABAergic neurons in the cerebellar cortex with possible implications for cerebellar circuit assembly.

  1. Chlorine-Incorporation-Induced Formation of the Layered Phase for Antimony-Based Lead-Free Perovskite Solar Cells.

    Science.gov (United States)

    Jiang, Fangyuan; Yang, Dongwen; Jiang, Youyu; Liu, Tiefeng; Zhao, Xingang; Ming, Yue; Luo, Bangwu; Qin, Fei; Fan, Jiacheng; Han, Hongwei; Zhang, Lijun; Zhou, Yinhua

    2018-01-24

    The environmental toxicity of Pb in organic-inorganic hybrid perovskite solar cells remains an issue, which has triggered intense research on seeking alternative Pb-free perovskites for solar applications. Halide perovskites based on group-VA cations of Bi 3+ and Sb 3+ with the same lone-pair ns 2 state as Pb 2+ are promising candidates. Herein, through a joint experimental and theoretical study, we demonstrate that Cl-incorporated methylammonium Sb halide perovskites (CH 3 NH 3 ) 3 Sb 2 Cl X I 9-X show promise as efficient solar absorbers for Pb-free perovskite solar cells. Inclusion of methylammonium chloride into the precursor solutions suppresses the formation of the undesired zero-dimensional dimer phase and leads to the successful synthesis of high-quality perovskite films composed of the two-dimensional layered phase favored for photovoltaics. Solar cells based on the as-obtained (CH 3 NH 3 ) 3 Sb 2 Cl X I 9-X films reach a record-high power conversion efficiency over 2%. This finding offers a new perspective for the development of nontoxic and low-cost Sb-based perovskite solar cells.

  2. Synapse formation and maintenance by C1q family proteins: a new class of secreted synapse organizers.

    Science.gov (United States)

    Yuzaki, Michisuke

    2010-07-01

    Several C1q family members, especially the Cbln and C1q-like subfamilies, are highly and predominantly expressed in the central nervous system. Cbln1, a member of the Cbln subfamily, plays two unique roles at parallel fiber (PF)-Purkinje cell synapses in the cerebellum: the formation and stabilization of synaptic contact, and the control of functional synaptic plasticity by regulating the postsynaptic endocytotic pathway. The delta2 glutamate receptor (GluD2), which is predominantly expressed in Purkinje cells, plays similar critical roles in the cerebellum. In addition, viral expression of GluD2 or the application of recombinant Cbln1 induces PF-Purkinje cell synaptogenesis in vitro and in vivo. Antigen-unmasking methods were necessary to reveal the immunoreactivities for endogenous Cbln1 and GluD2 at the synaptic junction of PF synapses. We propose that Cbln1 and GluD2 are located at the synaptic cleft, where various proteins undergo intricate molecular interactions with each other, and serve as a bidirectional synaptic organizer. © The Author (2010). Journal Compilation © Federation of European Neuroscience Societies and Blackwell Publishing Ltd.

  3. Extracellular vesicle-mediated transfer of genetic information between the hematopoietic system and the brain in response to inflammation.

    Directory of Open Access Journals (Sweden)

    Kirsten Ridder

    2014-06-01

    Full Text Available Mechanisms behind how the immune system signals to the brain in response to systemic inflammation are not fully understood. Transgenic mice expressing Cre recombinase specifically in the hematopoietic lineage in a Cre reporter background display recombination and marker gene expression in Purkinje neurons. Here we show that reportergene expression in neurons is caused by intercellular transfer of functional Cre recombinase messenger RNA from immune cells into neurons in the absence of cell fusion. In vitro purified secreted extracellular vesicles (EVs from blood cells contain Cre mRNA, which induces recombination in neurons when injected into the brain. Although Cre-mediated recombination events in the brain occur very rarely in healthy animals, their number increases considerably in different injury models, particularly under inflammatory conditions, and extend beyond Purkinje neurons to other neuronal populations in cortex, hippocampus, and substantia nigra. Recombined Purkinje neurons differ in their miRNA profile from their nonrecombined counterparts, indicating physiological significance. These observations reveal the existence of a previously unrecognized mechanism to communicate RNA-based signals between the hematopoietic system and various organs, including the brain, in response to inflammation.

  4. Incorporation of metal nanoparticles into wood substrate and methods

    Science.gov (United States)

    Rector, Kirk D; Lucas, Marcel

    2015-11-04

    Metal nanoparticles were incorporated into wood. Ionic liquids were used to expand the wood cell wall structure for nanoparticle incorporation into the cell wall structure. Nanoparticles of elemental gold or silver were found to be effective surface enhanced Raman spectroscopy (SERS) imaging contrast or sensing agents. Nanoparticles of elemental iron were found to be efficient microwave absorbers and caused localized heating for disrupting the integrity of the lignocellulosic matrix. Controls suggest that the localized heating around the iron nanoparticles reduces losses of cellulose in the form of water, volatiles and CO.sub.2. The ionic liquid is needed during the incorporation process at room temperature. The use of small amounts of ionic liquid combined with the absence of an ionic liquid purification step and a lower energy and water use are expected to reduce costs in an up-scaled pretreatment process.

  5. An amino acid depleted cell-free protein synthesis system for the incorporation of non-canonical amino acid analogs into proteins.

    Science.gov (United States)

    Singh-Blom, Amrita; Hughes, Randall A; Ellington, Andrew D

    2014-05-20

    Residue-specific incorporation of non-canonical amino acids into proteins is usually performed in vivo using amino acid auxotrophic strains and replacing the natural amino acid with an unnatural amino acid analog. Herein, we present an efficient amino acid depleted cell-free protein synthesis system that can be used to study residue-specific replacement of a natural amino acid by an unnatural amino acid analog. This system combines a simple methodology and high protein expression titers with a high-efficiency analog substitution into a target protein. To demonstrate the productivity and efficacy of a cell-free synthesis system for residue-specific incorporation of unnatural amino acids in vitro, we use this system to show that 5-fluorotryptophan and 6-fluorotryptophan substituted streptavidin retain the ability to bind biotin despite protein-wide replacement of a natural amino acid for the amino acid analog. We envisage this amino acid depleted cell-free synthesis system being an economical and convenient format for the high-throughput screening of a myriad of amino acid analogs with a variety of protein targets for the study and functional characterization of proteins substituted with unnatural amino acids when compared to the currently employed in vivo methodologies. Copyright © 2014 Elsevier B.V. All rights reserved.

  6. Histomorphologic and Immunohistochemical Characterization of a Cardiac Purkinjeoma in a Bearded Seal (Erignathus barbatus

    Directory of Open Access Journals (Sweden)

    G. Krafsur

    2014-01-01

    Full Text Available The most common cardiac tumors of heart muscle are rhabdomyomas, solitary or multiple benign tumors of striated muscle origin. While cardiac rhabdomyomas are well described in human medical literature, limited information depicting the occurrence of cardiac rhabdomyomas in veterinary species exists. A case of multiple firm white nonencapsulated nodules in the heart of a bearded seal is described. Microscopic findings included cytoplasmic vacuolization with formation of spider cells, glycogen vacuoles, and striated myofibrils. These cells expressed immunoreactivity for neuron-specific enolase and protein gene product 9.5, a marker for neuronal tissue and Purkinje fiber cells. Immunoreactivity for protein gene product 9.5 along with other microscopic findings substantiates Purkinje fiber cell origin of the cardiac rhabdomyoma in the bearded seal and use of the term purkinjeoma to describe this lesion.

  7. Localizing genes to cerebellar layers by classifying ISH images.

    Directory of Open Access Journals (Sweden)

    Lior Kirsch

    Full Text Available Gene expression controls how the brain develops and functions. Understanding control processes in the brain is particularly hard since they involve numerous types of neurons and glia, and very little is known about which genes are expressed in which cells and brain layers. Here we describe an approach to detect genes whose expression is primarily localized to a specific brain layer and apply it to the mouse cerebellum. We learn typical spatial patterns of expression from a few markers that are known to be localized to specific layers, and use these patterns to predict localization for new genes. We analyze images of in-situ hybridization (ISH experiments, which we represent using histograms of local binary patterns (LBP and train image classifiers and gene classifiers for four layers of the cerebellum: the Purkinje, granular, molecular and white matter layer. On held-out data, the layer classifiers achieve accuracy above 94% (AUC by representing each image at multiple scales and by combining multiple image scores into a single gene-level decision. When applied to the full mouse genome, the classifiers predict specific layer localization for hundreds of new genes in the Purkinje and granular layers. Many genes localized to the Purkinje layer are likely to be expressed in astrocytes, and many others are involved in lipid metabolism, possibly due to the unusual size of Purkinje cells.

  8. Effect of glucagon on cyclic AMP, albumin metabolism and incorporation of 14C-leucine into proteins in isolated parenchymal rat liver cells

    DEFF Research Database (Denmark)

    Dich, J; Gluud, C N

    1976-01-01

    wet wt. This is about the rate found in the perfused liver, Glucagon (10(-8-10(-6) M) inhibited albumin secretion and the incorporation of 14C-leucine into albumin, into total proteins in the medium and into total proteins in the cell suspension. The effect of glucagon on albumin secretion...... is compatible with an effect on the rate of synthesis. A positive correlation existed between the maximal level of cyclic AMP after glucagon administration and the inhibition of both albumin secretion and the incorporation of 149leucine....

  9. Differential 3’ processing of specific transcripts expands regulatory and protein diversity across neuronal cell types

    Science.gov (United States)

    Jereb, Saša; Hwang, Hun-Way; Van Otterloo, Eric; Govek, Eve-Ellen; Fak, John J; Yuan, Yuan; Hatten, Mary E

    2018-01-01

    Alternative polyadenylation (APA) regulates mRNA translation, stability, and protein localization. However, it is unclear to what extent APA regulates these processes uniquely in specific cell types. Using a new technique, cTag-PAPERCLIP, we discovered significant differences in APA between the principal types of mouse cerebellar neurons, the Purkinje and granule cells, as well as between proliferating and differentiated granule cells. Transcripts that differed in APA in these comparisons were enriched in key neuronal functions and many differed in coding sequence in addition to 3’UTR length. We characterize Memo1, a transcript that shifted from expressing a short 3’UTR isoform to a longer one during granule cell differentiation. We show that Memo1 regulates granule cell precursor proliferation and that its long 3’UTR isoform is targeted by miR-124, contributing to its downregulation during development. Our findings provide insight into roles for APA in specific cell types and establish a platform for further functional studies. PMID:29578408

  10. Rational site-directed mutations of the LLP-1 and LLP-2 lentivirus lytic peptide domains in the intracytoplasmic tail of human immunodeficiency virus type 1 gp41 indicate common functions in cell-cell fusion but distinct roles in virion envelope incorporation.

    Science.gov (United States)

    Kalia, Vandana; Sarkar, Surojit; Gupta, Phalguni; Montelaro, Ronald C

    2003-03-01

    Two highly conserved cationic amphipathic alpha-helical motifs, designated lentivirus lytic peptides 1 and 2 (LLP-1 and LLP-2), have been characterized in the carboxyl terminus of the transmembrane (TM) envelope glycoprotein (Env) of lentiviruses. Although various properties have been attributed to these domains, their structural and functional significance is not clearly understood. To determine the specific contributions of the Env LLP domains to Env expression, processing, and incorporation and to viral replication and syncytium induction, site-directed LLP mutants of a primary dualtropic infectious human immunodeficiency virus type 1 (HIV-1) isolate (ME46) were examined. Substitutions were made for highly conserved arginine residues in either the LLP-1 or LLP-2 domain (MX1 or MX2, respectively) or in both domains (MX4). The HIV-1 mutants with altered LLP domains demonstrated distinct phenotypes. The LLP-1 mutants (MX1 and MX4) were replication defective and showed an average of 85% decrease in infectivity, which was associated with an evident decrease in gp41 incorporation into virions without a significant decrease in Env expression or processing in transfected 293T cells. In contrast, MX2 virus was replication competent and incorporated a full complement of Env into its virions, indicating a differential role for the LLP-1 domain in Env incorporation. Interestingly, the replication-competent MX2 virus was impaired in its ability to induce syncytia in T-cell lines. This defect in cell-cell fusion did not correlate with apparent defects in the levels of cell surface Env expression, oligomerization, or conformation. The lack of syncytium formation, however, correlated with a decrease of about 90% in MX2 Env fusogenicity compared to that of wild-type Env in quantitative luciferase-based cell-cell fusion assays. The LLP-1 mutant MX1 and MX4 Envs also exhibited an average of 80% decrease in fusogenicity. Altogether, these results demonstrate for the first time that

  11. Periodontal tissue regeneration with PRP incorporated gelatin hydrogel sponges

    International Nuclear Information System (INIS)

    Nakajima, Dai; Tabata, Yasuhiko; Sato, Soh

    2015-01-01

    Gelatin hydrogels have been designed and prepared for the controlled release of the transforming growth factor (TGF-b1) and the platelet-derived growth factor (PDGF-BB). PRP (Platelet rich plasma) contains many growth factors including the PDGF and TGF-b1. The objective of this study was to evaluate the regeneration of periodontal tissue following the controlled release of growth factors in PRP. For the periodontal ligament cells and osteoblast, PRP of different concentrations was added. The assessment of DNA, mitochondrial activity and ALP activity were measured. To evaluate the TGF-β1 release from PRP incorporated gelatin sponge, amounts of TGF-β1 in each supernatant sample were determined by the ELISA. Transplantation experiments to prepare a bone defect in a rat alveolar bone were an implanted gelatin sponge incorporated with different concentration PRP. In DNA assay and MTT assay, after the addition of PRP to the periodontal ligament cells and osteoblast, the cell count and mitochondrial activity had increased the most in the group with the addition of 5  ×  PRP. In the ALP assay, after the addition of PRP to the periodontal ligament cells, the cell activity had increased the most in the group with the addition of 3  ×  PRP. In the transplantation, the size of the bone regenerated in the defect with 3  ×  PRP incorporated gelatin sponge was larger than that of the other group. (paper)

  12. Periodontal tissue regeneration with PRP incorporated gelatin hydrogel sponges.

    Science.gov (United States)

    Nakajima, Dai; Tabata, Yasuhiko; Sato, Soh

    2015-10-20

    Gelatin hydrogels have been designed and prepared for the controlled release of the transforming growth factor (TGF-b1) and the platelet-derived growth factor (PDGF-BB). PRP (Platelet rich plasma) contains many growth factors including the PDGF and TGF-b1. The objective of this study was to evaluate the regeneration of periodontal tissue following the controlled release of growth factors in PRP. For the periodontal ligament cells and osteoblast, PRP of different concentrations was added. The assessment of DNA, mitochondrial activity and ALP activity were measured. To evaluate the TGF-β1 release from PRP incorporated gelatin sponge, amounts of TGF-β1 in each supernatant sample were determined by the ELISA. Transplantation experiments to prepare a bone defect in a rat alveolar bone were an implanted gelatin sponge incorporated with different concentration PRP. In DNA assay and MTT assay, after the addition of PRP to the periodontal ligament cells and osteoblast, the cell count and mitochondrial activity had increased the most in the group with the addition of 5  ×  PRP. In the ALP assay, after the addition of PRP to the periodontal ligament cells, the cell activity had increased the most in the group with the addition of 3  ×  PRP. In the transplantation, the size of the bone regenerated in the defect with 3  ×  PRP incorporated gelatin sponge was larger than that of the other group.

  13. Photovoltaic performance enhancement of dye-sensitized solar cells by incorporating poly(sodium-4-styrenesulfonate)-physisorbed MWCNTs into photoelectrode

    Energy Technology Data Exchange (ETDEWEB)

    Shih, Yen-Chen [Department of Materials Science and Engineering, National Taiwan University, Taipei 10617, Taiwan (China); Yeh, Chia-Wen [Institute of Polymer Science and Engineering, National Taiwan University, Taipei 10617, Taiwan (China); Lin, King-Fu, E-mail: kflin@ntu.edu.tw [Department of Materials Science and Engineering, National Taiwan University, Taipei 10617, Taiwan (China); Institute of Polymer Science and Engineering, National Taiwan University, Taipei 10617, Taiwan (China)

    2016-03-01

    Multi-walled carbon nanotubes (MWCNTs) with the surface physisorbed by poly(sodium-4-styrenesulfonate) (denoted as NaPSS-MWCNTs) were able to well disperse in the TiO{sub 2} paste, resulting in the increase of anatase crystalline phase and higher light transmittance after casting into film and sintering. To fabricate the photoelectrode for dye-sensitized solar cells (DSSCs), incorporating 0.03 wt% NaPSS-MWCNTs into the TiO{sub 2} mesoporous film increased the short-circuit current density (Jsc) from 18.85 ± 0.04 to 20.68 ± 0.11 mA/cm{sup 2} and power conversion efficiency (PCE) from 7.86 ± 0.05 to 8.42 ± 0.02% under AM 1.5 illumination at full sunlight. It suggested that well dispersed MWCNTs provided not only extra electron transport channels among TiO{sub 2} nanoparticles but also more light absorption of ruthenium dyes leading to higher Jsc and PCE. - Highlights: • MWCNTs physisorbed by NaPSS are able to well disperse in the TiO{sub 2} paste. • Incorporation of NaPSS-MWCNTs increases the anatase crystalline phase of TiO{sub 2}. • Incorporation of NaPSS-MWCNTs increases the light transmittance of TiO{sub 2}. • Incorporation of NaPSS-MWCNTs to the TiO{sub 2} photoanode of DSSC increases Jsc and PCE.

  14. Expression of Brain-Derived Neurotrophic Factor (BDNF Increases the Resistance of Neurons to Death in the Postresuscitation Period

    Directory of Open Access Journals (Sweden)

    I. V. Ostrova

    2015-01-01

    Full Text Available A search for substances that are able to protect brain cells from the damaging effect of hypoxia remains one of the most relevant issues in modern neurobiology and medicine. Whether neurotrophic factors, brain-derived neurotrophic factor (BDNF protein in particular, can be used to treat neurological diseases is the subject of wide speculation in the literature now. However, how the expression of this protein in the brain neurons changes after systemic circulatory arrest in the postresuscitation period remains uncertain.Objective: to estimate the level of BDNF expression in the highly ischemia-sensitive neuronal population of cerebellar Purkinje cells and the value of BDNF in the resistance of neurons to ischemia-reperfusion.Materials and methods. In mature outbred male albino rats (n=11, the heart was stopped under ether anesthesia at 12 minutes via intrathoracic ligation of the vascular fascicle, followed by revivification. A control group included pseudo-operated animals (n=11. On days 7 after revivification, a morphometric analysis of Nissl-stained paraffin sections 5—6 μm thick was used to determine the total number of Purkinje cells per 1 mm of their layer length. The expression of BDNF protein in the Purkinje cells was immunohistochemically examined by an indirect peroxidase-antiperoxidase test using primary polyclonal antibodies against BDNF. The count of Purkinje cells with different immune responses to BDNF protein was calculated. The intensity of BDNF expression was estimated from the mean optical density. Results. 12-minute systemic circulatory arrest in the rats resulted in a 12.5% reduction in the number of Purkinje cells. The immunohistochemical examination revealed a lower numbers of BDNF– neurons in the resuscitated rats. In this case, the count of BDNF+ and BDNF++ neurons corresponded to their reference level. Consequently, only BDNF-negative neurons, i.e. those that failed to express BDNF protein, died. Analysis of the

  15. Incorporation of radioactive amino acids into protein in isolated rat hepatocytes

    International Nuclear Information System (INIS)

    Seglin, P.O.

    1976-01-01

    The incorporation of radioactivity from a 14 C-labelled amino acid mixture (algal protein hydrolysate) into protein in isolated rat hepatocytes has been studied. The incorporation rate declined with increasing cell concentration, an effect which could be explained by isotope consumption, partly (and largely) by isotope dilution due to the formation of non-labelled amino acids by the cells. At a high extracellular amino acid concentration, the rate of incorporation into protein became independent of cell concentration because the isotope dilution effect was now quantitatively insignificant. The time course of protein labelling at various cell concentrations correlated better with the intracellular than with the extracellular amino acid specific activity, suggesting that amino acids for protein synthesis were taken from an intracellular pool. With increasing extracellular amino acid concentrations, both the intracellular amino acid concentration, the intracellular radioactivity and the rate of incorporation into protein increased. Protein labelling exhibited a distinct time lag at high amino acid concentrations, presumable reflecting the time-dependent expansion of the intracellular amino acid pool. The gradual increase in the rate of protein labelling could be due either to an increased intracellular specific activity, or to a real stimulation of protein synthesis by amino acids, depending on whether the total intracellular amino acid pool or just the expandable compartment is the precursor pool for protein synthesis

  16. Effect of incorporation of silver nanoparticles in PEDOT:PSS layer on performance of organic solar cell

    Science.gov (United States)

    Singh, Joginder; Nirwal, Varun Singh; Bhatnagar, P. K.; Peta, Koteswara Rao

    2018-05-01

    Solution processable organic solar cells have attracted significant interest in scientific community due to their easy processability, flexibility and eco friendly fabrication. In these organic solar cells structure, PEDOT:PSS layer has major importance as it used as hole transporting layer. In the present work, we have analyzed the effect of incorporation of silver nanoparticles (AgNPs) in PEDOT:PSS layer for P3HT:PCBM based organic solar cells. The presence of Ag nanoparticles in PEDOT:PSS film is confirmed by atomic force microscopy (AFM) images. It has been observed that PEDOT:PSS layer with AgNPs has ˜5.4% more transmittance than PEDOT:PSS layer in most of the visible region, which helps in reaching more light on active layer. Finally, solar cell with structure ITO/PEDOT:PSS:AgNPs/Al is fabricated and J-V characteristics are plotted under illumination. It is observed that there is a significant (˜10%) enhancement in short circuit current and slight increment in open circuit voltage with addition of AgNPs in PEDOT:PSS layer. The calculated value of power conversion efficiency (PCE) of fabricated device without AgNPs in PEDOT:PSS was 1.67%, which increased to 2.02% after addition of AgNPs in PEDOT:PSS layer.

  17. Use of digitonin permeabilization for characterization of calcium incorporated into. beta. cells in response to glucose

    Energy Technology Data Exchange (ETDEWEB)

    Wesslen, N [Uppsala Univ. (Sweden)

    1985-01-01

    ..beta.. cell-rich pancreatic islets were used to characterize /sup 45/Ca uptake into pools with different sensitivities to permeabilization with digitonin. The plasma and secretory granule membranes were among the membranes most sensitive to digitonin treatment whereas the mitochondria were more resistant. Most of the /sup 45/Ca incorporated in response to glucose was found in a fraction released after exposure to a high concentration of digitonin. The results suggest that glucose promotes active sequestration of calcium in mitochondria and that the uptake of /sup 45/Ca in the secretory granules in secondary to a raised cytosolic Ca/sup 2 +/ activity.

  18. Preparation, Characterization and Tests of Incorporation in Stem Cells of Superparamagnetic Iron Oxide

    International Nuclear Information System (INIS)

    Haddad, P S; Britos, T N; Li, L M; Li, L D S

    2015-01-01

    Superparamagnetic iron oxide nanoparticles (SPIONs) have been produced and used as contrast-enhancing agents in magnetic resonance imaging (MRI) for diagnostic use in a wide range of maladies including cardiovascular, neurological disorders, and cancer. The reasons why these SPIONs are attractive for medical purposes are based on their important and unique features. The large surface area of the nanoparticles and their manipulation through an external magnetic field are features that allow their use for carrying a large number of molecules such as biomolecules or drugs. In this scenario, the present work reports on the synthesis and characterization of SPIONs and in vitro MRI experiments to increase their capacity as probes for MRI applications on stem cells therapy. Initially, the SPIONs were prepared through the co-precipitation method using ferrous and ferric chlorides in acidic solution. The SPIONs were coated with two thiolmolecules such as mercaptosuccinic acid (MSA) and cysteine (Cys) (molar ratio SPIONs:ligand = 1:20), leading to the formation of a stable aqueous dispersion of thiolated nanoparticles (SH-SPIONs). The SH-SPIONs were characterized by Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), transmission electron microscopy (TEM), and vibrating sample magnetometry (VSM). The results showed that the SH-SPIONs have a mean diameter of 14 nm and display superparamagnetic behavior at room temperature. Preliminary tests of incorporation of SH-SPIONs were evaluated stem cells. The results showed that the thiolated nanoparticles have no toxic effects for stem cells and successfully internalized and enhance the contrast in MRI. (paper)

  19. THE INCORPORATION OF RADIOACTIVITY FROM GLYCINE-C$sup 14$ BY MAMMALIAN SPERMATOZOA

    Energy Technology Data Exchange (ETDEWEB)

    Graves, C. N.

    1962-05-15

    The metabolic pathways of glycine incorporation were investigated by biochemical and radibautographic methods. Results show that glycine is utilized hy bovine spermatoza and is incorporated into all fractions of the sperm cell. Incorporation into the nucleic acid fraction and especially into thymine indicates that there is a turnover in the desoxyribenucleic acid during storage of bovine spermatoza. (C.H.)

  20. Incorporation of chimeric HIV-SIV-Env and modified HIV-Env proteins into HIV pseudovirions

    International Nuclear Information System (INIS)

    Devitt, Gerard; Emerson, Vanessa; Holtkotte, Denise; Pfeiffer, Tanya; Pisch, Thorsten; Bosch, Valerie

    2007-01-01

    Low level incorporation of the viral glycoprotein (Env) into human immunodeficiency virus (HIV) particles is a major drawback for vaccine strategies against HIV/AIDS in which HIV particles are used as immunogen. Within this study, we have examined two strategies aimed at achieving higher levels of Env incorporation into non-infectious pseudovirions (PVs). First, we have generated chimeric HIV/SIV Env proteins containing the truncated C-terminal tail region of simian immunodeficiency virus (SIV)mac239-Env767 stop , which mediates strongly increased incorporation of SIV-Env into SIV particles. In a second strategy, we have employed a truncated HIV-Env protein (Env-Tr752 N750K ) which we have previously demonstrated to be incorporated into HIV virions, generated in infected T-cells, to a higher level than that of Wt-HIV-Env. Although the chimeric HIV/SIV Env proteins were expressed at the cell surface and induced increased levels of cell-cell fusion in comparison to Wt-HIV-Env, they did not exhibit increased incorporation into either HIV-PVs or SIV-PVs. Only Env-Tr752 N750K exhibited significantly higher (threefold) levels of incorporation into HIV-PVs, an improvement, which, although not dramatic, is worthwhile for the large-scale preparation of non-infectious PVs for vaccine studies aimed at inducing Env humoral responses

  1. Flow cytometric analysis of RNA synthesis by detection of bromouridine incorporation

    DEFF Research Database (Denmark)

    Larsen, J K; Jensen, Peter Østrup; Larsen, J

    2001-01-01

    RNA synthesis has traditionally been investigated by a laborious and time-consuming radiographic method involving incorporation of tritiated uridine. Now a faster non-radioactive alternative has emerged, based on immunocytochemical detection. This method utilizes the brominated RNA precursor...... bromouridine, which is taken into a cell, phosphorylated, and incorporated into nascent RNA. The BrU-substituted RNA is detected by permeabilizing the cells and staining with certain anti-BrdU antibodies. This dynamic approach yields information complementing that provided by cellular RNA content analysis...

  2. Randomized Controlled Trial Examining the Effects of Fish Oil and Multivitamin Supplementation on the Incorporation of n-3 and n-6 Fatty Acids into Red Blood Cells

    Directory of Open Access Journals (Sweden)

    Andrew Pipingas

    2014-05-01

    Full Text Available The present randomized, placebo-controlled, double-blind, parallel-groups clinical trial examined the effects of fish oil and multivitamin supplementation on the incorporation of n-3 and n-6 fatty acids into red blood cells. Healthy adult humans (n = 160 were randomized to receive 6 g of fish oil, 6 g of fish oil plus a multivitamin, 3 g of fish oil plus a multivitamin or a placebo daily for 16 weeks. Treatment with 6 g of fish oil, with or without a daily multivitamin, led to higher eicosapentaenoic acid (EPA composition at endpoint. Docosahexaenoic acid (DHA composition was unchanged following treatment. The long chain LC n-3 PUFA index was only higher, compared to placebo, in the group receiving the combination of 6 g of fish oil and the multivitamin. Analysis by gender revealed that all treatments increased EPA incorporation in females while, in males, EPA was only significantly increased by the 6 g fish oil multivitamin combination. There was considerable individual variability in the red blood cell incorporation of EPA and DHA at endpoint. Gender contributed to a large proportion of this variability with females generally showing higher LC n-3 PUFA composition at endpoint. In conclusion, the incorporation of LC n-3 PUFA into red blood cells was influenced by dosage, the concurrent intake of vitamin/minerals and gender.

  3. Influence of radiosterilized cells on cells L1210 growth

    International Nuclear Information System (INIS)

    Malaise, E.P.; Decheva-Ninova, Z.; Tubiana, M.

    1975-01-01

    The effect of cells sterilized by acute X-irradiation is investigated on the growth of L 1210 cells. For this purpose young male mice DBA 2 are injected intraperitoneally or hypodermically with suspension of either live cells or live and sterile cells. The effect is considered according to survival time of treated animals and the number of leukemic cells examined in dynamics after their intraperitoneal incorporation or according to tumor size after their hypodermical incorporation. In both cases the incorporation of sterile cells has an inhibitory effect - life duration of treated mice is increased. This common effect disappears if animals are previously irradiated with 350 R. The sterile cells have also a local stimulating effect when incorporated hypodermically - time for their duplication is reduced from 15,8 to 13,7 hours. This stimulation is much more expressed when the recipients are previously irradiated - the time for tumor cells duplication being 12,2 hours. Direct stimulating effect of sterilized cells is not established when they are intraperitoneally incorporated. (author)

  4. A Microdosimetric-Kinetic Model of Cell Killing by Irradiation from Permanently Incorporated Radionuclides.

    Science.gov (United States)

    Hawkins, Roland B

    2018-01-01

    An expression for the surviving fraction of a replicating population of cells exposed to permanently incorporated radionuclide is derived from the microdosimetric-kinetic model. It includes dependency on total implant dose, linear energy transfer (LET), decay rate of the radionuclide, the repair rate of potentially lethal lesions in DNA and the volume doubling time of the target population. This is used to obtain an expression for the biologically effective dose ( BED α / β ) based on the minimum survival achieved by the implant that is equivalent to, and can be compared and combined with, the BED α / β calculated for a fractionated course of radiation treatment. Approximate relationships are presented that are useful in the calculation of BED α / β for alpha- or beta-emitting radionuclides with half-life significantly greater than, or nearly equal to, the approximately 1-h repair half-life of radiation-induced potentially lethal lesions.

  5. Study of the Mg incorporation in CdTe for developing wide band gap Cd1−xMgxTe thin films for possible use as top-cell absorber in a tandem solar cell

    International Nuclear Information System (INIS)

    Martínez, Omar S.; Millán, Aduljay Remolina; Huerta, L.; Santana, G.; Mathews, N.R.; Ramon-Garcia, M.L.; Morales, Erik R.; Mathew, X.

    2012-01-01

    Highlights: ► Thin films of Cd 1−x Mg x Te with high spatial uniformity and band gap in the range of 1.6–1.96 eV were deposited by vacuum co-evaporation of CdTe and Mg. ► Obtained Cd 1−x Mg x Te films have the structural characteristics of the CdTe, evidence of the change in atomic scattering due to incorporation of Mg was observed. ► XRD and XPS data confirmed the incorporation of Mg in the lattice of CdTe. ► SEM images revealed the impact of Mg incorporation on the morphology of the films, the changes in grain size and grain morphology are noticeable. - Abstract: Thin films of Cd 1−x Mg x Te with band gap in the range of 1.6–1.96 eV were deposited by vacuum co-evaporation of CdTe and Mg on glass substrates heated at 300 °C. Different experimental techniques such as XRD, UV–vis spectroscopy, SEM, and XPS were used to study the effect of Mg incorporation into the lattice of CdTe. The band gap of the films showed a clear tendency to increase as the Mg content in the film is increased. The Cd 1−x Mg x Te films maintain all the structural characteristics of the CdTe, however, diminishing of intensity for the XRD patterns is observed due to both change in preferential orientation and change in atomic scattering due to the incorporation of Mg. SEM images showed significant evidences of morphological changes due to the presence of Mg. XRD, UV–vis spectroscopy, and XPS data confirmed the incorporation of Mg in the lattice of CdTe. The significant increase in band gap of CdTe due to incorporation of Mg suggests that the Cd 1−x Mg x Te thin film is a candidate material to use as absorber layer in the top-cell of a tandem solar cell.

  6. Biochemical properties of Hemigraphis alternata incorporated chitosan hydrogel scaffold.

    Science.gov (United States)

    Annapoorna, M; Sudheesh Kumar, P T; Lakshman, Lakshmi R; Lakshmanan, Vinoth-Kumar; Nair, Shantikumar V; Jayakumar, R

    2013-02-15

    In this work, Hemigraphis alternata extract incorporated chitosan scaffold was synthesized and characterized for wound healing. The antibacterial activity of Hemigraphis incorporated chitosan scaffold (HIC) against Escherichia coli and Staphylococcus aureus was evaluated which showed a reduction in total colony forming units by 45-folds toward E. coli and 25-fold against S. aureus respectively. Cell viability studies using Human Dermal Fibroblast cells (HDF) showed 90% viability even at 48 h when compared to the chitosan control. The herbal scaffold made from chitosan was highly haemostatic and antibacterial. The obtained results were in support that the herbal scaffold can be effectively applied for infectious wounds. Copyright © 2012 Elsevier Ltd. All rights reserved.

  7. Thymidine uptake, thymidine incorporation, and thymidine kinase activity in marine bacterium isolates

    International Nuclear Information System (INIS)

    Jeffrey, W.H.; Paul, J.H.

    1990-01-01

    One assumption made in bacterial production estimates from [ 3 H]thymidine incorporation is that all heterotrophic bacteria can incorporate exogenous thymidine into DNA. Heterotrophic marine bacterium isolates from Tampa Bay, Fla., Chesapeake Bay, Md., and a coral surface microlayer were examined for thymidine uptake (transport), thymidine incorporation, the presence of thymidine kinase genes, and thymidine kinase enzyme activity. Of the 41 isolates tested, 37 were capable of thymidine incorporation into DNA. The four organisms that could not incorporate thymidine also transported the thymidine poorly and lacked thymidine kinase activity. Attempts to detect thymidine kinase genes in the marine isolates by molecular probing with gene probes made from Escherichia coli and herpes simplex virus thymidine kinase genes proved unsuccessful. To determine if the inability to incorporate thymidine was due to the lack of thymidine kinase, one organism, Vibro sp. strain DI9, was transformed with a plasmid (pGQ3) that contained an E. coli thymidine kinase gene. Although enzyme assays indicated high levels of thymidine kinase activity in transformants, these cells still failed to incorporate exogenous thymidine into DNA or to transport thymidine into cells. These results indicate that the inability of certain marine bacteria to incorporate thymidine may not be solely due to the lack of thymidine kinase activity but may also be due to the absence of thymidine transport systems

  8. HERC 1 ubiquitin ligase mutation affects neocortical, CA3 hippocampal and spinal cord projection neurons. An ultrastructural study

    Directory of Open Access Journals (Sweden)

    Rocío eRuiz

    2016-04-01

    Full Text Available The spontaneous mutation tambaleante is caused by the Gly483Glu substitution in the highly conserved N terminal RCC1-like domain of the HERC1 protein, which leads to the increase of mutated protein levels responsible for cerebellar Purkinje cell death by autophagy. Until now, Purkinje cells have been the only central nervous neurons reported as being targeted by the mutation, and their degeneration elicits an ataxic syndrome in adult mutant mice. However, the ultrastructural analysis performed here demonstrates that signs of autophagy, such as autophagosomes, lysosomes, and altered mitochondria, are present in neocortical pyramidal, CA3 hippocampal pyramidal, and spinal cord motor neurons. The main difference is that the reduction in the number of neurons affected in the tambaleante mutation in the neocortex, the hippocampus, and the spinal cord is not so evident as the dramatic loss of cerebellar Purkinje cells. Interestingly, signs of autophagy are absent in both interneurons and neuroglia cells. Affected neurons have in common that they are projection neurons which receive strong and varied synaptic inputs, and possess the highest degree of neuronal activity. Therefore, because the integrity of the ubiquitin-proteasome system is essential for protein degradation and, hence, for normal protein turnover, it could be hypothesized that the deleterious effects of the misrouting of these pathways would depend directly on the neuronal activity.

  9. Cerebellar Fastigial Nucleus Electrical Stimulation Alleviates Depressive-Like Behaviors in Post-Stroke Depression Rat Model and Potential Mechanisms

    Directory of Open Access Journals (Sweden)

    Lei Zhang

    2017-03-01

    Full Text Available Objective: To identify the molecular mechanism of post-stroke depression (PSD, and observe the therapeutic effects of cerebellar fastigial nucleus electrical stimulation (FNS on the behaviors and regional cerebral blood flow (rCBF in a PSD rat model. Methods: Healthy SD rats were randomly divided into four groups (sham, stroke, post-stroke depress and FNS group. Sham group (n = 6 underwent sham operation. The other three groups (n = 6*3 underwent MCAO. Rats were examined twice a week in open filed test. Moreover, neuroprotective effect on cerebellar Purkinje cells and expression of cytokines in hippocampal tissue were examined. Results: The PSD group showed a significant weight loss, decreased consumption of sucrose water, reduced rearing and locomotor activities. The FNS significantly alleviates the body weight loss and sucrose preference, locomotor and rearing activities. The bilateral rCBF was also restored after FNS treatment. Moreover, FNS improved the neuroprotection via suppressing apoptosis of cerebellar Purkinje cells. And the inflammatory cytokines mRNA level in hippocampus was significantly decreased. Conclusion: FNS treatment alleviates depressive-like behaviors and rCBF in PSD rats model, which could be attributed to its ability to protect cerebellar Purkinje cells and decrease the mRNA level of inflammatory cytokines.

  10. Epigallocatechin gallate incorporation into lignin enhances the alkaline delignification and enzymatic saccharification of cell walls

    Directory of Open Access Journals (Sweden)

    Elumalai Sasikumar

    2012-08-01

    Full Text Available Abstract Background Lignin is an integral component of the plant cell wall matrix but impedes the conversion of biomass into biofuels. The plasticity of lignin biosynthesis should permit the inclusion of new compatible phenolic monomers such as flavonoids into cell wall lignins that are consequently less recalcitrant to biomass processing. In the present study, epigallocatechin gallate (EGCG was evaluated as a potential lignin bioengineering target for rendering biomass more amenable to processing for biofuel production. Results In vitro peroxidase-catalyzed polymerization experiments revealed that both gallate and pyrogallyl (B-ring moieties in EGCG underwent radical cross-coupling with monolignols mainly by β–O–4-type cross-coupling, producing benzodioxane units following rearomatization reactions. Biomimetic lignification of maize cell walls with a 3:1 molar ratio of monolignols and EGCG permitted extensive alkaline delignification of cell walls (72 to 92% that far exceeded that for lignified controls (44 to 62%. Alkali-insoluble residues from EGCG-lignified walls yielded up to 34% more glucose and total sugars following enzymatic saccharification than lignified controls. Conclusions It was found that EGCG readily copolymerized with monolignols to become integrally cross-coupled into cell wall lignins, where it greatly enhanced alkaline delignification and subsequent enzymatic saccharification. Improved delignification may be attributed to internal trapping of quinone-methide intermediates to prevent benzyl ether cross-linking of lignin to structural polysaccharides during lignification, and to the cleavage of ester intra-unit linkages within EGCG during pretreatment. Overall, our results suggest that apoplastic deposition of EGCG for incorporation into lignin would be a promising plant genetic engineering target for improving the delignification and saccharification of biomass crops.

  11. Radiation dose to trabecular bone marrow stem cells from 3H, 14C and selected α-emitters incorporated in a bone remodeling compartment

    International Nuclear Information System (INIS)

    Nie Huiling; Richardson, Richard B

    2009-01-01

    A Monte Carlo simulation of repeated cubic units representing trabecular bone cavities in adult bone was employed to determine absorbed dose fractions evaluated for 3 H, 14 C and a set of α-emitters incorporated within a bone remodeling compartment (BRC). The BRC consists of a well-oxygenated vascular microenvironment located within a canopy of bone-lining cells. The International Commission on Radiological Protection (ICRP) considers that an important target for radiation-induced bone cancer is the endosteum marrow layer adjacent to bone surface where quiescent bone stem cells reside. It is proposed that the active stem cells and progenitor cells located above the BRC canopy, the 'BRC stem cell niche', is a more important radiation-induced cancer target volume. Simulation results from a static model, where no remodeling occurs, indicate that the mean dose from bone and bone surface to the 50 μm quiescent bone stem cell niche, the current ICRP target, was substantially lower (two to three times lower) than that to the narrower and hypoxic 10 μm endosteum for 3 H, 14 C and α-particles with energy range 0.5-10 MeV. The results from a dynamic model indicate that the temporal α-radiation dose to active stem/progenitor cells located in the BRC stem cell niche from the material incorporated in and buried by forming bone was 9- to 111-fold greater than the dose to the quiescent bone stem cell niche. This work indicates that the remodeling portion of the bone surface, rather than the quiescent (endosteal) surface, has the greatest risk of radiation-induced bone cancer, particularly from short-range radiation, due to the elevated dose and the radiosensitizing oxygen effect.

  12. The organization of plasticity in the cerebellar cortex: from synapses to control.

    Science.gov (United States)

    D'Angelo, Egidio

    2014-01-01

    The cerebellum is thought to play a critical role in procedural learning, but the relationship between this function and the underlying cellular and synaptic mechanisms remains largely speculative. At present, at least nine forms of long-term synaptic and nonsynaptic plasticity (some of which are bidirectional) have been reported in the cerebellar cortex and deep cerebellar nuclei. These include long-term potentiation (LTP) and long-term depression at the mossy fiber-granule cell synapse, at the synapses formed by parallel fibers, climbing fibers, and molecular layer interneurons on Purkinje cells, and at the synapses formed by mossy fibers and Purkinje cells on deep cerebellar nuclear cells, as well as LTP of intrinsic excitability in granule cells, Purkinje cells, and deep cerebellar nuclear cells. It is suggested that the complex properties of cerebellar learning would emerge from the distribution of plasticity in the network and from its dynamic remodeling during the different phases of learning. Intrinsic and extrinsic factors may hold the key to explain how the different forms of plasticity cooperate to select specific transmission channels and to regulate the signal-to-noise ratio through the cerebellar cortex. These factors include regulation of neuronal excitation by local inhibitory networks, engagement of specific molecular mechanisms by spike bursts and theta-frequency oscillations, and gating by external neuromodulators. Therefore, a new and more complex view of cerebellar plasticity is emerging with respect to that predicted by the original "Motor Learning Theory," opening issues that will require experimental and computational testing. © 2014 Elsevier B.V. All rights reserved.

  13. Sites of sulfate incorporation into mammotrophs and somatotrophs of the rat pituitary as determined by quantitative electron microscopic autoradiography

    International Nuclear Information System (INIS)

    Rosenzweig, L.J.; Farquhar, M.G.

    1980-01-01

    Dispersed pituitary cells were labeled with [ 35 S]sulfate followed by a chase incubation in order to study sulfate incorporation and transport in anterior pituitary cells. The initial site of incorporation of sulfate, the kinetics of sulfate transport, and the intracellular localization of incorporated sulfate were studied by quantitative electron microscope autoradiography. Analysis of autoradiograms from estrogen-treated female rats revealed that all granulated cell types incorporate sulfate. The labeling index of the various cell types was greatest for mammotrophs, slightly less for corticotrophs, gonadotrophs, and thyrotrophs and least for somatotrophs. These results indicate the [ 35 S]sulfate is initially incorporated into the Golgi complex of all interior pituitary cell types. The majority of the sulfate-labeled macromolecules are then packaged into immature secretion granules in the Golgi region, which become mature granules. In addition, a considerable amount (approx. 30% in mammotrophs) of the radioactivity remains associated within the Golgi region for up to 2 h post pulse. The incorporation of sulfate into the Golgi complex and its transfer to secretory granule membranes and/or contents thus appears to be a general property of anterior pituitary cells

  14. Phosphatidylserine biosynthesis in cultured Chinese hamster ovary cells. I. Inhibition of de novo phosphatidylserine biosynthesis by exogenous phosphatidylserine and its efficient incorporation

    International Nuclear Information System (INIS)

    Nishijima, M.; Kuge, O.; Akamatsu, Y.

    1986-01-01

    The effect of phosphatidylserine exogenously added to the medium on de novo biosynthesis of phosphatidylserine was investigated in cultured Chinese hamster ovary cells. When cells were cultured for several generations in medium supplemented with phosphatidylserine and 32 Pi, the incorporation of 32 Pi into cellular phosphatidylserine was remarkably inhibited, the degree of inhibition being dependent upon the concentration of added phosphatidylserine. 32 Pi uptake into cellular phosphatidylethanolamine was also partly reduced by the addition of exogenous phosphatidylserine, consistent with the idea that phosphatidylethanolamine is biosynthesized via decarboxylation of phosphatidylserine. However, incorporation of 32 Pi into phosphatidylcholine, sphingomyelin, and phosphatidylinositol was not significantly affected. In contrast, the addition of either phosphatidylcholine, sphingomyelin, phosphatidylethanolamine, or phosphatidylinositol to the medium did not inhibit endogenous biosynthesis of the corresponding phospholipid. Radiochemical and chemical analyses of the cellular phospholipid composition revealed that phosphatidylserine in cells grown with 80 microM phosphatidylserine was almost entirely derived from the added phospholipid. Phosphatidylserine uptake was also directly determined by using [ 3 H]serine-labeled phospholipid. Pulse and pulse-chase experiments with L-[U- 14 C] serine showed that when cells were cultured with 80 microM phosphatidylserine, the rate of synthesis of phosphatidylserine was reduced 3-5-fold. Enzyme assaying of extracts prepared from cells grown with and without phosphatidylserine indicated that the inhibition of de novo phosphatidylserine biosynthesis by the added phosphatidylserine appeared not to be caused by a reduction in the level of the enzyme involved in the base-exchange reaction between phospholipids and serine

  15. The role of p38 in mitochondrial respiration in male and female mice.

    Science.gov (United States)

    Ju, Xiaohua; Wen, Yi; Metzger, Daniel; Jung, Marianna

    2013-06-07

    p38 is a mitogen-activated protein kinase and mediates cell growth, cell differentiation, and synaptic plasticity. The aim of this study is to determine the extent to which p38 plays a role in maintaining mitochondrial respiration in male and female mice under a normal condition. To achieve this aim, we have generated transgenic mice that lack p38 in cerebellar Purkinje neurons by crossing Pcp2 (Purkinje cell protein 2)-Cre mice with p38(loxP/loxP) mice. Mitochondria from cerebellum were then isolated from the transgenic and wild-type mice to measure mitochondrial respiration using XF24 respirometer. The mRNA and protein expression of cytochrome c oxidase (COX) in cerebellum were also measured using RT-PCR and immunoblot methods. Separately, HT22 cells were used to determine the involvement of 17β-estradiol (E2) and COX in mitochondrial respiration. The genetic knockout of p38 in Purkinje neurons suppressed the mitochondrial respiration only in male mice and increased COX expression only in female mice. The inhibition of COX by sodium azide (SA) sharply suppressed mitochondrial respiration of HT22 cells in a manner that was protected by E2. These data suggest that p38 is required for the mitochondrial respiration of male mice. When p38 is below a normal level, females may maintain mitochondrial respiration through COX up-regulation. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  16. Incorporation of graphene into SnO2 photoanodes for dye-sensitized solar cells

    Science.gov (United States)

    Batmunkh, Munkhbayar; Dadkhah, Mahnaz; Shearer, Cameron J.; Biggs, Mark J.; Shapter, Joseph G.

    2016-11-01

    In dye-sensitized solar cell (DSSC) photoanodes, tin dioxide (SnO2) structures present a promising alternative semiconducting oxide to the conventional titania (TiO2), but they suffer from poor photovoltaic (PV) efficiency caused by insufficient dye adsorption and low energy value of the conduction band. A hybrid structure consisting of SnO2 and reduced graphene oxide (SnO2-RGO) was synthesized via a microwave-assisted method and has been employed as a photoanode in DSSCs. Incorporation of RGO into the SnO2 photoanode enhanced the power conversion efficiency of DSSC device by 91.5%, as compared to the device assembled without RGO. This efficiency improvement can be attributed to increased dye loading, enhanced electron transfer and addition of suitable energy levels in the photoanode. Finally, the use of RGO addresses the major shortcoming of SnO2 when employed as a DSSC photoanode, namely poor dye adsorption and slow electron transfer rate.

  17. Paroxysmal atrioventricular block: Electrophysiological mechanism of phase 4 conduction block in the His-Purkinje system: A comparison with phase 3 block.

    Science.gov (United States)

    Shenasa, Mohammad; Josephson, Mark E; Wit, Andrew L

    2017-11-01

    Paroxysmal atrioventricular (A-V) block is relatively rare, and due to its transient nature, it is often under recognized. It is often triggered by atrial, junctional, or ventricular premature beats, and occurs in the presence of a diseased His-Purkinje system (HPS). Here, we present a 45-year-old white male who was admitted for observation due to recurrent syncope and near-syncope, who had paroxysmal A-V block. The likely cellular electrophysiological mechanisms(s) of paroxysmal A-V block and its differential diagnosis and management are discussed. Continuous electrocardiographic monitoring was done while the patient was in the cardiac unit. Multiple episodes of paroxysmal A-V block were documented in this case. All episodes were initiated and terminated with atrial/junctional premature beats. The patient underwent permanent pacemaker implantation and has remained asymptomatic since then. Paroxysmal A-V block is rare and often causes syncope or near-syncope. Permanent pacemaker implantation is indicated according to the current guidelines. Paroxysmal A-V block occurs in the setting of diseased HPS and is bradycardia-dependent. The detailed electrophysiological mechanisms, which involve phase 4 diastolic depolarization, and differential diagnosis are discussed. © 2017 Wiley Periodicals, Inc.

  18. Enhanced conversion efficiency of dye-sensitized solar cells using a CNT-incorporated TiO2 slurry-based photoanode

    OpenAIRE

    Jiaoping Cai; Zexiang Chen; Jun Li; Yan Wang; Dong Xiang; Jijun Zhang; Hai Li

    2015-01-01

    A new titanium dioxide (TiO2) slurry formulation is herein reported for the fabrication of TiO2 photoanode for use in dye-sensitized solar cells (DSSCs). The prepared TiO2 photoanode featured a highly uniform mesoporous structure with well-dispersed TiO2 nanoparticles. The energy conversion efficiency of the resulting TiO2 slurry-based DSSC was ∼63% higher than that achieved by a DSSC prepared using a commercial TiO2 slurry. Subsequently, the incorporation of acid-treated multi-walled carbon ...

  19. The feasibility of incorporating Vpx into lentiviral gene therapy vectors

    Directory of Open Access Journals (Sweden)

    Samantha A McAllery

    2016-01-01

    Full Text Available While current antiretroviral therapy has significantly improved, challenges still remain in life-long targeting of HIV-1 reservoirs. Lentiviral gene therapy has the potential to deliver protective genes into the HIV-1 reservoir. However, inefficient reverse transcription (RT occurs in HIV-1 reservoirs during lentiviral gene delivery. The viral protein Vpx is capable of increasing lentiviral RT by antagonizing the restriction factor SAMHD1. Incorporating Vpx into lentiviral vectors could substantially increase gene delivery into the HIV-1 reservoir. The feasibility of this Vpx approach was tested in resting cell models utilizing macrophages and dendritic cells. Our results showed Vpx exposure led to increased permissiveness of cells over a period that exceeded 2 weeks. Consequently, significant lower potency of HIV-1 antiretrovirals inhibiting RT and integration was observed. When Vpx was incorporated with anti-HIV-1 genes inhibiting either pre-RT or post-RT stages of the viral life-cycle, transduction levels significantly increased. However, a stronger antiviral effect was only observed with constructs that inhibit pre-RT stages of the viral life cycle. In conclusion this study demonstrates a way to overcome the major delivery obstacle of gene delivery into HIV-1 reservoir cell types. Importantly, incorporating Vpx with pre-RT anti-HIV-1 genes, demonstrated the greatest protection against HIV-1 infection.

  20. ZnO nanoparticle incorporated nanostructured metallic titanium for increased mesenchymal stem cell response and antibacterial activity

    Science.gov (United States)

    Elizabeth, Elmy; Baranwal, Gaurav; Krishnan, Amit G.; Menon, Deepthy; Nair, Manitha

    2014-03-01

    Recent trends in titanium implants are towards the development of nanoscale topographies that mimic the nanoscale properties of bone tissue. Although the nanosurface promotes the integration of osteoblast cells, infection related problems can also occur, leading to implant failure. Therefore it is imperative to reduce bacterial adhesion on an implant surface, either with or without the use of drugs/antibacterial agents. Herein, we have investigated two different aspects of Ti surfaces in inhibiting bacterial adhesion and concurrently promoting mammalian cell adhesion. These include (i) the type of nanoscale topography (Titania nanotube (TNT) and Titania nanoleaf (TNL)) and (ii) the presence of an antibacterial agent like zinc oxide nanoparticles (ZnOnp) on Ti nanosurfaces. To address this, periodically arranged TNT (80-120 nm) and non-periodically arranged TNL surfaces were generated by the anodization and hydrothermal techniques respectively, and incorporated with ZnOnp of different concentrations (375 μM, 750 μM, 1.125 mM and 1.5 mM). Interestingly, TNL surfaces decreased the adherence of staphylococcus aureus while increasing the adhesion and viability of human osteosarcoma MG63 cell line and human mesenchymal stem cells, even in the absence of ZnOnp. In contrast, TNT surfaces exhibited an increased bacterial and mammalian cell adhesion. The influence of ZnOnp on these surfaces in altering the bacterial and cell adhesion was found to be concentration dependent, with an optimal range of 375-750 μM. Above 750 μM, although bacterial adhesion was reduced, cellular viability was considerably affected. Thus our study helps us to infer that nanoscale topography by itself or its combination with an optimal concentration of antibacterial ZnOnp would provide a differential cell behavior and thereby a desirable biological response, facilitating the long term success of an implant.

  1. Single and double strand breaks induced by 3H incorporated in DNA of cultured human kidney cells

    International Nuclear Information System (INIS)

    Tisljar-Lentulis, G.; Henneberg, P.; Mielke, T.; Feinendegen, L.E.

    1978-01-01

    In the course of the investigations of the biological effects of radionuclides incorporated in DNA single (SSB) and double strand breaks (DSB) caused tritium-decay were measured and compared with respective data resulting from 125 I. Tritium bound to thymidine and iododeoxyuridine seems to be more effective than tritium bound to other DNA-precursors. On the basis of decay, methyl- 3 H thymidine appears to be more effective with regard to the production of strand breaks than 3 H in position 6 of the pyrimidine ring. Based on the numbers of strand-breaks per rad, position 6 is more effective in accordance with data obtained by F. Krasin et al. The ratio of SSBs to DSBs per tritium decay appears to be approximately 8 in mammlian cells. Not only SSBs but also DSBs induced by 3 H in mammalian cells are reapairable. (orig./AJ) [de

  2. In Vivo Assessment of Bone Regeneration in Alginate/Bone ECM Hydrogels with Incorporated Skeletal Stem Cells and Single Growth Factors

    Science.gov (United States)

    Gothard, David; Smith, Emma L.; Kanczler, Janos M.; Black, Cameron R.; Wells, Julia A.; Roberts, Carol A.; White, Lisa J.; Qutachi, Omar; Peto, Heather; Rashidi, Hassan; Rojo, Luis; Stevens, Molly M.; El Haj, Alicia J.; Rose, Felicity R. A. J.; Shakesheff, Kevin M.; Oreffo, Richard O. C.

    2015-01-01

    The current study has investigated the use of decellularised, demineralised bone extracellular matrix (ECM) hydrogel constructs for in vivo tissue mineralisation and bone formation. Stro-1-enriched human bone marrow stromal cells were incorporated together with select growth factors including VEGF, TGF-β3, BMP-2, PTHrP and VitD3, to augment bone formation, and mixed with alginate for structural support. Growth factors were delivered through fast (non-osteogenic factors) and slow (osteogenic factors) release PLGA microparticles. Constructs of 5 mm length were implanted in vivo for 28 days within mice. Dense tissue assessed by micro-CT correlated with histologically assessed mineralised bone formation in all constructs. Exogenous growth factor addition did not enhance bone formation further compared to alginate/bone ECM (ALG/ECM) hydrogels alone. UV irradiation reduced bone formation through degradation of intrinsic growth factors within the bone ECM component and possibly also ECM cross-linking. BMP-2 and VitD3 rescued osteogenic induction. ALG/ECM hydrogels appeared highly osteoinductive and delivery of angiogenic or chondrogenic growth factors led to altered bone formation. All constructs demonstrated extensive host tissue invasion and vascularisation aiding integration and implant longevity. The proposed hydrogel system functioned without the need for growth factor incorporation or an exogenous inducible cell source. Optimal growth factor concentrations and spatiotemporal release profiles require further assessment, as the bone ECM component may suffer batch variability between donor materials. In summary, ALG/ECM hydrogels provide a versatile biomaterial scaffold for utilisation within regenerative medicine which may be tailored, ultimately, to form the tissue of choice through incorporation of select growth factors. PMID:26675008

  3. In vitro method determing sensitivity of anticancer agents by incorporation of radioactive precursors

    International Nuclear Information System (INIS)

    Sakakibara, Satoshi

    1983-01-01

    A new sensitivity test of anticancer agents was developed to measure the lethal effects of cancer cells by the incorporation of radioactive precursors. The thousand cancer cells were cultured in a microplate in the presence of anticancer agents. These cells were exposed to radioactive precursors. Two or three days later, the cancer cells were harvested on a glass fiver filter by a multiple automatic cell-harvester and the incorporation of precursors was counted by a liquid scintillation counter. In this study, the in vivo results of drug testing in animal model systems were compared with drug sensitivities. Mice inoculated Ehrlich ascites cells were treated with various kinds of anticancer drugs. The development of the cells was compatible with the result of the sensitivity test. The growths of Lauson and ME-180 cells derived from human cancers implanted subcutaneously to nude mice were also well correlated with this sensitivity test. (author)

  4. Enhancing the performance of dye-sensitized solar cells by incorporating nanomica in gel electrolytes☆

    KAUST Repository

    Lai, Yi-Hsuan

    2010-04-01

    Gel-type dye-sensitized solar cells (DSSCs) were fabricated with 5.0 wt% polyvinyidene fluoride-co-hexafluoro propylene (PVDF-HFP) in methoxy propionitrile (MPN) as gel polymer electrolyte (GPE), 1-butyl-3-methylimidazolium iodide (BMII)/iodine (I2) as redox couple, 4-tertiary butyl pyridine (TBP) and guanidine thiocyanate as additives. The incorporation of alkyl-modified nanomica (AMNM) in the PVDF-HFP gel electrolytes caused the reduction of crystallization of PVDF-HFP, which was confirmed by X-ray diffraction (XRD) analysis. The short-circuit current density (JSC) of the cell increased due to the decrease of diffusion resistance, as judged by the electrochemical impedance spectra (EIS) analysis, while the open-circuit voltage (VOC) remained almost the same. As the loading of AMNM in the PVDF-HFP gel electrolyte was increased to 3.0 wt%, the JSC and power conversion efficiency (η) of the cells increased from 8.3 to 13.6 mA/cm2 and 3.5% to 5.7%, respectively. However, the JSC decreased as the loading of AMNM exceeded 3.0 wt%. At higher AMNM loadings, nanomica acted as a barrier interface between the electrolyte and the dye molecules to hinder electron transfer, and thus reducing the cell\\'s photocurrent density. Furthermore, the DSSCs fabricated by dispersing polymethyl methacrylate (PMMA) microspheres in the TiO2 electrode with the GPE containing 3.0 wt% AMNM improved the η to 6.70%. The TiO2 films would exhibit larger porosity by blending with PMMA, leading the penetration of GPEs into the porous TiO2 easier, thus improving the contact between the dye-adsorbed TiO2 surfaces and the GPEs, as characterized by EIS. Moreover, the η of gel-type DSSCs with a 25 μm thickness of surlyn reached 7.96% as compared with 6.70% for the DSSCs with a 60 μm surlyn. © 2009 Elsevier B.V. All rights reserved.

  5. Cytoplasmic location of α1A voltage-gated calcium channel C-terminal fragment (Cav2.1-CTF aggregate is sufficient to cause cell death.

    Directory of Open Access Journals (Sweden)

    Makoto Takahashi

    Full Text Available The human α1A voltage-dependent calcium channel (Cav2.1 is a pore-forming essential subunit embedded in the plasma membrane. Its cytoplasmic carboxyl(C-tail contains a small poly-glutamine (Q tract, whose length is normally 4∼19 Q, but when expanded up to 20∼33Q, the tract causes an autosomal-dominant neurodegenerative disorder, spinocerebellar ataxia type 6 (SCA6. A recent study has shown that a 75-kDa C-terminal fragment (CTF containing the polyQ tract remains soluble in normal brains, but becomes insoluble mainly in the cytoplasm with additional localization to the nuclei of human SCA6 Purkinje cells. However, the mechanism by which the CTF aggregation leads to neurodegeneration is completely elusive, particularly whether the CTF exerts more toxicity in the nucleus or in the cytoplasm. We tagged recombinant (rCTF with either nuclear-localization or nuclear-export signal, created doxycyclin-inducible rat pheochromocytoma (PC12 cell lines, and found that the CTF is more toxic in the cytoplasm than in the nucleus, the observations being more obvious with Q28 (disease range than with Q13 (normal-length. Surprisingly, the CTF aggregates co-localized both with cAMP response element-binding protein (CREB and phosphorylated-CREB (p-CREB in the cytoplasm, and Western blot analysis showed that the quantity of CREB and p-CREB were both decreased in the nucleus when the rCTF formed aggregates in the cytoplasm. In human brains, polyQ aggregates also co-localized with CREB in the cytoplasm of SCA6 Purkinje cells, but not in other conditions. Collectively, the cytoplasmic Cav2.1-CTF aggregates are sufficient to cause cell death, and one of the pathogenic mechanisms may be abnormal CREB trafficking in the cytoplasm and reduced CREB and p-CREB levels in the nuclei.

  6. Cytoplasmic Location of α1A Voltage-Gated Calcium Channel C-Terminal Fragment (Cav2.1-CTF) Aggregate Is Sufficient to Cause Cell Death

    Science.gov (United States)

    Takahashi, Makoto; Obayashi, Masato; Ishiguro, Taro; Sato, Nozomu; Niimi, Yusuke; Ozaki, Kokoro; Mogushi, Kaoru; Mahmut, Yasen; Tanaka, Hiroshi; Tsuruta, Fuminori; Dolmetsch, Ricardo; Yamada, Mitsunori; Takahashi, Hitoshi; Kato, Takeo; Mori, Osamu; Eishi, Yoshinobu; Mizusawa, Hidehiro; Ishikawa, Kinya

    2013-01-01

    The human α1A voltage-dependent calcium channel (Cav2.1) is a pore-forming essential subunit embedded in the plasma membrane. Its cytoplasmic carboxyl(C)-tail contains a small poly-glutamine (Q) tract, whose length is normally 4∼19 Q, but when expanded up to 20∼33Q, the tract causes an autosomal-dominant neurodegenerative disorder, spinocerebellar ataxia type 6 (SCA6). A recent study has shown that a 75-kDa C-terminal fragment (CTF) containing the polyQ tract remains soluble in normal brains, but becomes insoluble mainly in the cytoplasm with additional localization to the nuclei of human SCA6 Purkinje cells. However, the mechanism by which the CTF aggregation leads to neurodegeneration is completely elusive, particularly whether the CTF exerts more toxicity in the nucleus or in the cytoplasm. We tagged recombinant (r)CTF with either nuclear-localization or nuclear-export signal, created doxycyclin-inducible rat pheochromocytoma (PC12) cell lines, and found that the CTF is more toxic in the cytoplasm than in the nucleus, the observations being more obvious with Q28 (disease range) than with Q13 (normal-length). Surprisingly, the CTF aggregates co-localized both with cAMP response element-binding protein (CREB) and phosphorylated-CREB (p-CREB) in the cytoplasm, and Western blot analysis showed that the quantity of CREB and p-CREB were both decreased in the nucleus when the rCTF formed aggregates in the cytoplasm. In human brains, polyQ aggregates also co-localized with CREB in the cytoplasm of SCA6 Purkinje cells, but not in other conditions. Collectively, the cytoplasmic Cav2.1-CTF aggregates are sufficient to cause cell death, and one of the pathogenic mechanisms may be abnormal CREB trafficking in the cytoplasm and reduced CREB and p-CREB levels in the nuclei. PMID:23505410

  7. Centrioles regulate centrosome size by controlling the rate of Cnn incorporation into the PCM.

    Science.gov (United States)

    Conduit, Paul T; Brunk, Kathrin; Dobbelaere, Jeroen; Dix, Carly I; Lucas, Eliana P; Raff, Jordan W

    2010-12-21

    centrosomes are major microtubule organizing centers in animal cells, and they comprise a pair of centrioles surrounded by an amorphous pericentriolar material (PCM). Centrosome size is tightly regulated during the cell cycle, and it has recently been shown that the two centrosomes in certain stem cells are often asymmetric in size. There is compelling evidence that centrioles influence centrosome size, but how centrosome size is set remains mysterious. we show that the conserved Drosophila PCM protein Cnn exhibits an unusual dynamic behavior, because Cnn molecules only incorporate into the PCM closest to the centrioles and then spread outward through the rest of the PCM. Cnn incorporation into the PCM is driven by an interaction with the conserved centriolar proteins Asl (Cep152 in humans) and DSpd-2 (Cep192 in humans). The rate of Cnn incorporation into the PCM is tightly regulated during the cell cycle, and this rate influences the amount of Cnn in the PCM, which in turn is an important determinant of overall centrosome size. Intriguingly, daughter centrioles in syncytial embryos only start to incorporate Cnn as they disengage from their mothers; this generates a centrosome size asymmetry, with mother centrioles always initially organizing more Cnn than their daughters. centrioles can control the amount of PCM they organize by regulating the rate of Cnn incorporation into the PCM. This mechanism can explain how centrosome size is regulated during the cell cycle and also allows mother and daughter centrioles to set centrosome size independently of one another.

  8. The effect of incorporation of SDF-1alpha into PLGA scaffolds on stem cell recruitment and the inflammatory response.

    Science.gov (United States)

    Thevenot, Paul T; Nair, Ashwin M; Shen, Jinhui; Lotfi, Parisa; Ko, Cheng-Yu; Tang, Liping

    2010-05-01

    Despite significant advances in the understanding of tissue responses to biomaterials, most implants are still plagued by inflammatory responses which can lead to fibrotic encapsulation. This is of dire consequence in tissue engineering, where seeded cells and bioactive components are separated from the native tissue, limiting the regenerative potential of the design. Additionally, these interactions prevent desired tissue integration and angiogenesis, preventing functionality of the design. Recent evidence supports that mesenchymal stem cells (MSC) and hematopoietic stem cells (HSC) can have beneficial effects which alter the inflammatory responses and improve healing. The purpose of this study was to examine whether stem cells could be targeted to the site of biomaterial implantation and whether increasing local stem cell responses could improve the tissue response to PLGA scaffold implants. Through incorporation of SDF-1alpha through factor adsorption and mini-osmotic pump delivery, the host-derived stem cell response can be improved resulting in 3X increase in stem cell populations at the interface for up to 2 weeks. These interactions were found to significantly alter the acute mast cell responses, reducing the number of mast cells and degranulated mast cells near the scaffold implants. This led to subsequent downstream reduction in the inflammatory cell responses, and through altered mast cell activation and stem cell participation, increased angiogenesis and decreased fibrotic responses to the scaffold implants. These results support that enhanced recruitment of autologous stem cells can improve the tissue responses to biomaterial implants through modifying/bypassing inflammatory cell responses and jumpstarting stem cell participation in healing at the implant interface. Copyright 2010 Elsevier Ltd. All rights reserved.

  9. Kolaviron and vitamin E ameliorate hematotoxicity and oxidative stress in brains of prepubertal rats treated with an anticonvulsant phenytoin.

    Science.gov (United States)

    Owoeye, Olatunde; Adedara, Isaac A; Bakare, Oluwafemi S; Adeyemo, Oluwatobi A; Egun, Christa; Farombi, Ebenezer O

    2014-06-01

    Phenytoin (PHT), an anticonvulsant agent, widely used for the treatment of epilepsy has been reported to exhibit toxic side effects. The present study investigated the protective effects of kolaviron and vitamin E on hematotoxicity and neurotoxicity induced by phenytoin, in prepubertal male rats. The animals were treated with PHT (75 mg/kg) separately or in combination with either kolaviron (200 mg/kg) or vitamin E (500 mg/kg) for 14 days. Phenytoin treatment significantly decreased the hemoglobin, white blood cells, lymphocytes and mean corpuscular volume levels without affecting red blood cell, packed cell volume, neutrophils, mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration when compared with the control rats. There was a significant increase in lipid peroxidation and hydrogen peroxide levels with marked depletion in antioxidant status in brains of PHT-treated rats when compared with the control. Although PHT treatment had no effect on the granular layer, widest diameter of Purkinje cells and Purkinje layer of the cerebellum, it significantly reduced its molecular layer and the density of Purkinje cell. Administration of PHT significantly reduced the densities of the granule cells of the dentate gyrus and the pyramidal neurons of the cornu ammonis of hippocampus proper. Co-treatment with kolaviron and vitamin E effectively reversed the PHT-mediated alterations in the hematology, brain antioxidant status and histomorphometry when compared to PHT only. Taken together, the present data indicate the abilities of kolaviron and vitamin E to ameliorate phenytoin-induced hematotoxicity and oxidative stress in brains of rats.

  10. Genetic incorporation of recycled unnatural amino acids.

    Science.gov (United States)

    Ko, Wooseok; Kim, Sanggil; Jo, Kyubong; Lee, Hyun Soo

    2016-02-01

    The genetic incorporation of unnatural amino acids (UAAs) into proteins has been a useful tool for protein engineering. However, most UAAs are expensive, and the method requires a high concentration of UAAs, which has been a drawback of the technology, especially for large-scale applications. To address this problem, a method to recycle cultured UAAs was developed. The method is based on recycling a culture medium containing the UAA, in which some of essential nutrients were resupplemented after each culture cycle, and induction of protein expression was controlled with glucose. Under optimal conditions, five UAAs were recycled for up to seven rounds of expression without a decrease in expression level, cell density, or incorporation fidelity. This method can generally be applied to other UAAs; therefore, it is useful for reducing the cost of UAAs for genetic incorporation and helpful for expanding the use of the technology to industrial applications.

  11. Enhancement in irradiated mononuclear cells in culture of mitogen-induced incorporation of [3H]thymidine by homologous conditioned medium

    International Nuclear Information System (INIS)

    Sandru, G.; Greiner, R.

    1994-01-01

    Incorporation of [ 3 H]thymidine in irradiated peripheral blood mononuclear cell cultures irradiated in vitro was stimulated significantly by either concanavalin A or phytohemagglutinin only in the presence of homologous conditioned medium. Production of this activity by mononuclear cells was enhanced by irradiation and/or pulsed exposure to puromycin but was abolished by actinomycin D. Addition of anti-interleukin 1 or anti-interleukin 2 monoclonal antibodies to the conditioned medium before assay did not influence the stimulatory action. A similar significant stimulation of mononuclear cell cultures irradiated with 6 Gy by concanavalin A was obtained when purified preparations of homologous conditioned medium were used in the assay. Purification was done by ultrafiltration and concentration, heparin agarose chromatography, ammonium sulfate precipitation, concanavalin A agarose chromatography, DEAE-ion exchange chromatography and HPLC gel filtration chromatography. With SDS-PAGE and silver staining, the active HPLC fraction gave one band of 50 kDa, suggesting that this protein is responsible for the co-stimulatory effect of homologous conditioned medium for both mitogen-induced irradiated and nonirradiated mononuclear cell cultures. 42 refs., 9 figs., 3 tabs

  12. Effects of piracetam on the incorporation of 32P into the phospholipids of neurons and glial cells isolated from rabbit cerebral cortex

    International Nuclear Information System (INIS)

    Woelk, H.

    1979-01-01

    In the search for the biochemical basis of the action of Piracetam, the effects of this encephalotropic substance on the neuronal and glial phospholipid metabolism was investigated. Piracetam increases the incorporation of 32 P into phosphatidylinositol and phosphatidyl choline of both glia and neuronal cell bodies. When taking the important role of phosphatidylinoitol in the processes of synaptic transmission and axonal conduction into consideration, the data obtained in the present work suggest that piracetam may stimulate excitatory neurons and may be involved in the process of synaptic transmission. The stimulatory effect of piracetam on the incorporation of 32 P into phosphatidylinositol and phosphatidyl choline appears to be mediated by norepinephrine or another neurotransmitter. (orig.) [de

  13. Computational optogenetics: empirically-derived voltage- and light-sensitive channelrhodopsin-2 model.

    Directory of Open Access Journals (Sweden)

    John C Williams

    Full Text Available Channelrhodospin-2 (ChR2, a light-sensitive ion channel, and its variants have emerged as new excitatory optogenetic tools not only in neuroscience, but also in other areas, including cardiac electrophysiology. An accurate quantitative model of ChR2 is necessary for in silico prediction of the response to optical stimulation in realistic tissue/organ settings. Such a model can guide the rational design of new ion channel functionality tailored to different cell types/tissues. Focusing on one of the most widely used ChR2 mutants (H134R with enhanced current, we collected a comprehensive experimental data set of the response of this ion channel to different irradiances and voltages, and used these data to develop a model of ChR2 with empirically-derived voltage- and irradiance- dependence, where parameters were fine-tuned via simulated annealing optimization. This ChR2 model offers: 1 accurate inward rectification in the current-voltage response across irradiances; 2 empirically-derived voltage- and light-dependent kinetics (activation, deactivation and recovery from inactivation; and 3 accurate amplitude and morphology of the response across voltage and irradiance settings. Temperature-scaling factors (Q10 were derived and model kinetics was adjusted to physiological temperatures. Using optical action potential clamp, we experimentally validated model-predicted ChR2 behavior in guinea pig ventricular myocytes. The model was then incorporated in a variety of cardiac myocytes, including human ventricular, atrial and Purkinje cell models. We demonstrate the ability of ChR2 to trigger action potentials in human cardiomyocytes at relatively low light levels, as well as the differential response of these cells to light, with the Purkinje cells being most easily excitable and ventricular cells requiring the highest irradiance at all pulse durations. This new experimentally-validated ChR2 model will facilitate virtual experimentation in neural and

  14. Novel experimental results in human cardiac electrophysiology: measurement of the Purkinje fibre action potential from the undiseased human heart.

    Science.gov (United States)

    Nagy, Norbert; Szél, Tamás; Jost, Norbert; Tóth, András; Gy Papp, Julius; Varró, András

    2015-09-01

    Data obtained from canine cardiac electrophysiology studies are often extrapolated to the human heart. However, it has been previously demonstrated that because of the lower density of its K(+) currents, the human ventricular action potential has a less extensive repolarization reserve. Since the relevance of canine data to the human heart has not yet been fully clarified, the aim of the present study was to determine for the first time the action potentials of undiseased human Purkinje fibres (PFs) and to compare them directly with those of dog PFs. All measurements were performed at 37 °C using the conventional microelectrode technique. At a stimulation rate of 1 Hz, the plateau potential of human PFs is more positive (8.0 ± 1.8 vs 8.6 ± 3.4 mV, n = 7), while the amplitude of the spike is less pronounced. The maximal rate of depolarization is significantly lower in human PKs than in canine PFs (406.7 ± 62 vs 643 ± 36 V/s, respectively, n = 7). We assume that the appreciable difference in the protein expression profiles of the 2 species may underlie these important disparities. Therefore, caution is advised when canine PF data are extrapolated to humans, and further experiments are required to investigate the characteristics of human PF repolarization and its possible role in arrhythmogenesis.

  15. Activation patterns of Purkinje fibers during long-duration ventricular fibrillation in an isolated canine heart model.

    Science.gov (United States)

    Tabereaux, Paul B; Walcott, Greg P; Rogers, Jack M; Kim, Jong; Dosdall, Derek J; Robertson, Peter G; Killingsworth, Cheryl R; Smith, William M; Ideker, Raymond E

    2007-09-04

    The roles of Purkinje fibers (PFs) and focal wave fronts, if any, in the maintenance of ventricular fibrillation (VF) are unknown. If PFs are involved in VF maintenance, it should be possible to map wave fronts propagating from PFs into the working ventricular myocardium during VF. If wave fronts ever arise focally during VF, it should be possible to map them appearing de novo. Six canine hearts were isolated, and the left main coronary artery was cannulated and perfused. The left ventricular cavity was exposed, which allowed direct endocardial mapping of the anterior papillary muscle insertion. Nonperfused VF was induced, and 6 segments of data, each 5 seconds long, were analyzed during 10 minutes of VF. During 36 segments of data that were analyzed, 1018 PF or focal wave fronts of activation were identified. In 534 wave fronts, activation was mapped propagating from working ventricular myocardium to PF. In 142 wave fronts, activation was mapped propagating from PF to working ventricular myocardium. In 342 wave fronts, activation was mapped arising focally. More than 1 of these 3 patterns could occur in the same wave front. PFs are highly active throughout the first 10 minutes of VF. In addition to retrograde propagation from the working ventricular myocardium to PFs, antegrade propagation occurs from PFs to working ventricular myocardium, which suggests PFs are important in VF maintenance. Prior plunge needle recordings in dogs indicate activation propagates from the endocardium toward the epicardium after 1 minute of VF, which suggests that focal sites on the endocardium may represent foci and not breakthrough. If so, in addition to reentry, abnormal automaticity or triggered activity may also occur during VF.

  16. Evidence of a genetic instability induced by the incorporation of a DNA precursor marked with tritium

    International Nuclear Information System (INIS)

    Saintigny, Y.; Laurent, D.; Lahayel, J.B.; Roche, St.; Meynard, D.; Lopez, B.S.

    2009-01-01

    The authors report a molecular geno-toxicology investigation which allowed molecular events induced par intracellular incorporation of tritium to be studied, and the genetic instability resulting from a chronic exposure even at low dose to be analysed. For this purpose, they developed cell models (hamster tumorous cells and human fibroblasts) in which they know how to incorporate given quantities of marked nucleotides in the DNA. They show that the incorporation of tritium, even with doses which are said to be non toxic, causes a prolonged exposure of the cell to a genotoxic stress, and maybe a genetic instability due to a too great number of recombination events

  17. Permeability changes and incorporation of labelled thymidine into DNA and whole cells of the fibroblast culture of Chinese hamsters affected by MEA and low temperature

    International Nuclear Information System (INIS)

    Ermekova, V.M.; Kondakova, N.V.; Levitman, M.Kh.; Saugabaeva, K.M.; Ehjdus, L.Kh.

    1976-01-01

    Action of MEA and low temperature (20degC) on the incorporation of labelled thymidine into DNA and whole cells of the fibroblast culture of chinese hamsters has been studied. It has been found that each of the above-mentioned factors equally decreases the label uptake into the cell and DNA. It is concluded that MEA and low temperature do not substantially influence the rate of DNA synthesis

  18. Cerebellar nuclei neurons show only small excitatory responses to optogenetic olivary stimulation in transgenic mice: in vivo and in vitro studies

    Directory of Open Access Journals (Sweden)

    Huo eLu

    2016-03-01

    Full Text Available To study the olivary input to the cerebellar nuclei (CN we used optogenetic stimulation in transgenic mice expressing channelrhodopsin-2 (ChR2 in olivary neurons. We obtained in vivo extracellular Purkinje cell (PC and CN recordings in anesthetized mice while stimulating the contralateral inferior olive (IO with a blue laser (single pulse, 10 - 50 ms duration. Peri-stimulus histograms were constructed to show the spike rate changes after optical stimulation. Among 29 CN neurons recorded, 15 showed a decrease in spike rate of variable strength and duration, and only 1 showed a transient spiking response. These results suggest that direct olivary input to CN neurons is usually overridden by stronger Purkinje cell inhibition triggered by climbing fiber responses. To further investigate the direct input from the climbing fiber collaterals we also conducted whole cell recordings in brain slices, where we used local stimulation with blue light. Due to the expression of ChR2 in Purkinje cell axons as well as the IO in our transgenic line, strong inhibitory responses could be readily triggered with optical stimulation (13 of 15 neurons. After blocking this inhibition with GABAzine, only in 5 of 13 CN neurons weak excitatory responses were revealed. Therefore our in vitro results support the in vivo findings that the excitatory input to CN neurons from climbing fiber collaterals in adult mice is masked by the inhibition under normal conditions.

  19. Garcinia kola aqueous suspension prevents cerebellar neurodegeneration in long-term diabetic rat - a type 1 diabetes mellitus model.

    Science.gov (United States)

    Farahna, Mohammed; Seke Etet, Paul F; Osman, Sayed Y; Yurt, Kıymet K; Amir, Naheed; Vecchio, Lorella; Aydin, Isınsu; Aldebasi, Yousef H; Sheikh, Azimullah; Chijuka, John C; Kaplan, Süleyman; Adem, Abdu

    2017-01-04

    The development of compounds able to improve metabolic syndrome and mitigate complications caused by inappropriate glycemic control in type 1 diabetes mellitus is challenging. The medicinal plant with established hypoglycemic properties Garcinia kola Heckel might have the potential to mitigate diabetes mellitus metabolic syndrome and complications. We have investigated the neuroprotective properties of a suspension of G. kola seeds in long-term type 1 diabetes mellitus rat model. Wistar rats, made diabetic by single injection of streptozotocin were monitored for 8 months. Then, they were administered with distilled water or G. kola oral aqueous suspension daily for 30 days. Body weight and glycemia were determined before and after treatment. After sacrifice, cerebella were dissected out and processed for stereological quantification of Purkinje cells. Histopathological and immunohistochemical analyses of markers of neuroinflammation and neurodegeneration were performed. Purkinje cell counts were significantly increased, and histopathological signs of apoptosis and neuroinflammation decreased, in diabetic animals treated with G. kola compared to diabetic rats given distilled water. Glycemia was also markedly improved and body weight restored to non-diabetic control values, following G. kola treatment. These results suggest that G. kola treatment improved the general condition of long-term diabetic rats and protected Purkinje cells partly by improving the systemic glycemia and mitigating neuroinflammation. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  20. Hypericum perforatum incorporated chitosan films as potential bioactive wound dressing material.

    Science.gov (United States)

    Güneş, Seda; Tıhmınlıoğlu, Funda

    2017-09-01

    Recent studies in wound dressing applications offer new therapies and promote wound healing process. The aim of this study was to develop Hypericum perforatum (St John's Wort) oil incorporated chitosan films for wound dressing applications. H. perforatum oil as a potential therapeutic agent was encapsulated in chitosan film to achieve a better wound dressing material. Oil incorporated chitosan films were successfully prepared by solvent casting method in different oil concentrations (0.25-1.5%v/v). Water vapor permeability (WVP), mechanical test, swelling behavior and surface hydrophobicity were performed in order to characterize the prepared films. Antimicrobial test was performed by disc diffusion method and the growth inhibition effects of the films including different amount of H. perforatum oil were investigated on Escherichia coli and Staphylococcus aureus. WVP increased with oil incorporation and the highest value was obtained for 0.25% oil concentration.The highest strain value was obtained in 0.25% oil content films although tensile stress decreased with increasing oil content. H. perforatum oil incorporated films had antimicrobial effect on both microorganisms. Chitosan based films had no cytotoxic effects on NIH3T3fibroblast cells and provided a good surface for cell attachment and proliferation. The results showed that the H. perforatum incorporated chitosan films seems to be a potential and novel biomaterial for wound healing applications. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Targeted transgene insertion into the CHO cell genome using Cre recombinase-incorporating integrase-defective retroviral vectors.

    Science.gov (United States)

    Kawabe, Yoshinori; Shimomura, Takuya; Huang, Shuohao; Imanishi, Suguru; Ito, Akira; Kamihira, Masamichi

    2016-07-01

    Retroviral vectors have served as efficient gene delivery tools in various biotechnology fields. However, viral DNA is randomly inserted into the genome, which can cause problems, such as insertional mutagenesis and gene silencing. Previously, we reported a site-specific gene integration system, in which a transgene is integrated into a predetermined chromosomal locus of Chinese hamster ovary (CHO) cells using integrase-defective retroviral vectors (IDRVs) and Cre recombinase. In this system, a Cre expression plasmid is transfected into founder cells before retroviral transduction. In practical applications of site-specific gene modification such as for hard-to-transfect cells or for in vivo gene delivery, both the transgene and the Cre protein into retroviral virions should be encapsulate. Here, we generated novel hybrid IDRVs in which viral genome and enzymatically active Cre can be delivered (Cre-IDRVs). Cre-IDRVs encoding marker genes, neomycin resistance and enhanced green fluorescent protein (EGFP), flanked by wild-type and mutated loxP sites were produced using an expression plasmid for a chimeric protein of Cre and retroviral gag-pol. After analyzing the incorporation of the Cre protein into retroviral virions by Western blotting, the Cre-IDRV was infected into founder CHO cells, in which marker genes (hygromycin resistance and red fluorescent protein) flanked with corresponding loxP sites are introduced into the genome. G418-resistant colonies expressing GFP appeared and the site-specific integration of the transgene into the expected chromosomal site was confirmed by PCR and sequencing of amplicons. Moreover, when Cre-IDRV carried a gene expression unit for a recombinant antibody, the recombinant cells in which the antibody expression cassette was integrated in a site-specific manner were generated and the cells produced the recombinant antibody. This method may provide a promising tool to perform site-specific gene modification according to Cre

  2. ZnO nanoparticle incorporated nanostructured metallic titanium for increased mesenchymal stem cell response and antibacterial activity

    International Nuclear Information System (INIS)

    Elizabeth, Elmy; Baranwal, Gaurav; Krishnan, Amit G; Menon, Deepthy; Nair, Manitha

    2014-01-01

    Recent trends in titanium implants are towards the development of nanoscale topographies that mimic the nanoscale properties of bone tissue. Although the nanosurface promotes the integration of osteoblast cells, infection related problems can also occur, leading to implant failure. Therefore it is imperative to reduce bacterial adhesion on an implant surface, either with or without the use of drugs/antibacterial agents. Herein, we have investigated two different aspects of Ti surfaces in inhibiting bacterial adhesion and concurrently promoting mammalian cell adhesion. These include (i) the type of nanoscale topography (Titania nanotube (TNT) and Titania nanoleaf (TNL)) and (ii) the presence of an antibacterial agent like zinc oxide nanoparticles (ZnOnp) on Ti nanosurfaces. To address this, periodically arranged TNT (80–120 nm) and non-periodically arranged TNL surfaces were generated by the anodization and hydrothermal techniques respectively, and incorporated with ZnOnp of different concentrations (375 μM, 750 μM, 1.125 mM and 1.5 mM). Interestingly, TNL surfaces decreased the adherence of staphylococcus aureus while increasing the adhesion and viability of human osteosarcoma MG63 cell line and human mesenchymal stem cells, even in the absence of ZnOnp. In contrast, TNT surfaces exhibited an increased bacterial and mammalian cell adhesion. The influence of ZnOnp on these surfaces in altering the bacterial and cell adhesion was found to be concentration dependent, with an optimal range of 375−750 μM. Above 750 μM, although bacterial adhesion was reduced, cellular viability was considerably affected. Thus our study helps us to infer that nanoscale topography by itself or its combination with an optimal concentration of antibacterial ZnOnp would provide a differential cell behavior and thereby a desirable biological response, facilitating the long term success of an implant. (paper)

  3. In vitro evaluation of Spirulina platensis extract incorporated skin cream with its wound healing and antioxidant activities.

    Science.gov (United States)

    Gunes, Seda; Tamburaci, Sedef; Dalay, Meltem Conk; Deliloglu Gurhan, Ismet

    2017-12-01

    Algae have gained importance in cosmeceutical product development due to their beneficial effects on skin health and therapeutical value with bioactive compounds. Spirulina platensis Parachas (Phormidiaceae) is renowned as a potential source of high-value chemicals and recently used in skincare products. This study develops and evaluates skin creams incorporated with bioactive S. platensis extract. Spirulina platensis was cultivated, the aqueous crude extract was prepared and in vitro cytotoxicity of S. platensis extract in the range of 0.001-1% concentrations for 1, 3 and 7 d on HS2 keratinocyte cells was determined. Crude extracts were incorporated in skin cream formulation at 0.01% (w/w) concentration and in vitro wound healing and genotoxicity studies were performed. Immunohistochemical staining was performed to determine the collagen activity. 0.1% S. platensis extract exhibited higher proliferation activity compared with the control group with 198% of cell viability after 3 d. Skin cream including 1.125% S. platensis crude extract showed enhanced wound healing effect on HS2 keratinocyte cell line and the highest HS2 cell viability % was obtained with this concentration. The micronucleus (MN) assay results indicated that S. platensis extract incorporated creams had no genotoxic effect on human peripheral blood cells. Immunohistochemical analysis showed that collagen 1 immunoreactivity was improved by increased extract concentration and it was strongly positive in cells treated with 1.125% extract incorporated skin cream. The cell viability, wound healing activity and genotoxicity results showed that S. platensis incorporated skin cream could be of potential value in cosmeceutical and biomedical applications.

  4. Targeting p38 Mitogen-Activated Protein Kinase Signaling Restores Subventricular Zone Neural Stem Cells and Corrects Neuromotor Deficits in Atm Knockout Mouse

    Science.gov (United States)

    Kim, Jeesun

    2012-01-01

    Ataxia-telangiectasia (A-T) is a progressive degenerative disorder that results in major neurological disability. In A-T patients, necropsy has revealed atrophy of cerebellar cortical layers along with Purkinje and granular cell loss. We have previously identified an oxidative stress-mediated increase in phospho-p38 mitogen-activated protein kinase (MAPK) and the resultant downregulation of Bmi-1 and upregulation of p21 as key components of the mechanism causing defective proliferation of neural stem cells (NSCs) isolated from the subventricular zone (SVZ) of Atm−/− mice. However, the in vivo aspect of alteration in SVZ tissue and the functional significance of p38MAPK activation in NSCs for neuropathogenesis of ATM deficiency remain unknown. Here we show that the NSC population was abnormally decreased in the SVZ of 3-month-old Atm−/− mice; this decrease was accompanied by p38MAPK activation. However, after a 2-month treatment with the p38MAPK inhibitor SB203580, starting at 1 month old, Atm−/− mice showed restoration of normal levels of Bmi-1 and p21 with the rescue of NSC population in the SVZ. In addition, treated Atm−/− mice exhibited more Purkinje cells in the cerebellum. Most importantly, motor coordination of Atm−/− mice was significantly improved in the treatment group. Our results show for the first time in vivo evidence of depleted NSCs in the SVZ of Atm−/− mice and also demonstrate that pharmacologic inhibition of p38MAPK signaling has the potential to treat neurological defects of A-T. This study provides a promising approach targeting the oxidative stress-dependent p38 signaling pathway not only for A-T but also for other neurodegenerative disorders. PMID:23197859

  5. Enhancing the performance of dye-sensitized solar cells by incorporating nanomica in gel electrolytes☆

    KAUST Repository

    Lai, Yi-Hsuan; Lin, Chia-Yu; Chen, Jian-Ging; Wang, Chun-Chieh; Huang, Kuan-Chieh; Liu, Ken-Yen; Lin, King-Fu; Lin, Jiang-Jen; Ho, Kuo-Chuan

    2010-01-01

    Gel-type dye-sensitized solar cells (DSSCs) were fabricated with 5.0 wt% polyvinyidene fluoride-co-hexafluoro propylene (PVDF-HFP) in methoxy propionitrile (MPN) as gel polymer electrolyte (GPE), 1-butyl-3-methylimidazolium iodide (BMII)/iodine (I2) as redox couple, 4-tertiary butyl pyridine (TBP) and guanidine thiocyanate as additives. The incorporation of alkyl-modified nanomica (AMNM) in the PVDF-HFP gel electrolytes caused the reduction of crystallization of PVDF-HFP, which was confirmed by X-ray diffraction (XRD) analysis. The short-circuit current density (JSC) of the cell increased due to the decrease of diffusion resistance, as judged by the electrochemical impedance spectra (EIS) analysis, while the open-circuit voltage (VOC) remained almost the same. As the loading of AMNM in the PVDF-HFP gel electrolyte was increased to 3.0 wt%, the JSC and power conversion efficiency (η) of the cells increased from 8.3 to 13.6 mA/cm2 and 3.5% to 5.7%, respectively. However, the JSC decreased as the loading of AMNM exceeded 3.0 wt%. At higher AMNM loadings, nanomica acted as a barrier interface between the electrolyte and the dye molecules to hinder electron transfer, and thus reducing the cell's photocurrent density. Furthermore, the DSSCs fabricated by dispersing polymethyl methacrylate (PMMA) microspheres in the TiO2 electrode with the GPE containing 3.0 wt% AMNM improved the η to 6.70%. The TiO2 films would exhibit larger porosity by blending with PMMA, leading the penetration of GPEs into the porous TiO2 easier, thus improving the contact between the dye-adsorbed TiO2 surfaces and the GPEs, as characterized by EIS. Moreover, the η of gel-type DSSCs with a 25 μm thickness of surlyn reached 7.96% as compared with 6.70% for the DSSCs with a 60 μm surlyn. © 2009 Elsevier B.V. All rights reserved.

  6. Ataxin-1 with an expanded glutamine tract alters nuclear matrix-associated structures

    DEFF Research Database (Denmark)

    Skinner, P J; Koshy, B T; Cummings, C J

    1997-01-01

    a similar pattern of nuclear localization; with expanded ataxin-1 occurring in larger structures that are fewer in number than those of normal ataxin-1. Colocalization studies show that mutant ataxin-1 causes a specific redistribution of the nuclear matrix-associated domain containing promyelocytic...... the subcellular localization of wild-type human ataxin-1 (the protein encoded by the SCA1 gene) and mutant ataxin-1 in the Purkinje cells of transgenic mice. We found that ataxin-1 localizes to the nuclei of cerebellar Purkinje cells. Normal ataxin-1 localizes to several nuclear structures approximately 0.......5 microm across, whereas the expanded ataxin-1 localizes to a single approximately 2-microm structure, before the onset of ataxia. Mutant ataxin-1 localizes to a single nuclear structure in affected neurons of SCA1 patients. Similarly, COS-1 cells transfected with wild-type or mutant ataxin-1 show...

  7. Incorporation of zinc into the coccoliths of the microalga Emiliania huxleyi.

    Science.gov (United States)

    Santomauro, Giulia; Sun, Wei-Lin; Brümmer, Franz; Bill, Joachim

    2016-04-01

    The coccolithophore Emiliania huxleyi is covered with elaborated calcite plates, the so-called coccoliths, which are produced inside the cells. We investigated the incorporation of zinc into the coccoliths of E. huxleyi by applying different zinc and calcium amounts via the culture media and subsequently analyzing the zinc content in the cells and the Zn/Ca ratio of the coccoliths. To investigate the Zn/Ca ratio of coccoliths built in the manipulated media, the algae have first to be decalcified, i.e. coccolith free. We used a newly developed decalcification method to obtain 'naked' cells for cultivation. E. huxleyi proliferated and produced new coccoliths in all media with manipulated Zn/Ca ratios. The cells and the newly built coccoliths were investigated regarding their zinc content and their Zn/Ca ratio, respectively. High zinc amounts were taken up by the algae. The Zn/Ca ratio of the coccoliths was positively correlated to the Zn/Ca ratio of the applied media. The unique feature of the coccoliths was maintained also at high Zn/Ca ratios. We suggest the following pathway of the zinc ions into the coccoliths: first, the zinc ions are bound to the cell surface, followed by their transportation into the cytoplasm. Obviously, the zinc ions are removed afterwards into the coccolith vesicle, where the zinc is incorporated into the calcite coccoliths which are then extruded. The incorporation of toxic zinc ions into the coccoliths possibly due to a new function of the coccoliths as detoxification sites is discussed.

  8. Data on cell growth inhibition induced by anti-VEGF siRNA delivered by Stealth liposomes incorporating G2 PAMAM-cholesterol versus Metafectene® as a function of exposure time and siRNA concentration

    Directory of Open Access Journals (Sweden)

    Nasim Golkar

    2016-09-01

    Full Text Available In this data article, carboxyfluorescein-loaded liposomes were prepared and purified from free carboxyfluorescein using gel filtration chromatography in the first part. In the next part, following preparation of anti-VEGF siRNA loaded liposomes incorporating hydrophobically modified G2 PAMAM dendrimer (G2-Chol40% (Golkar et al., 2016 [1], the cell growth inhibition induced by the formulations (siRNA/Metafectene complexes and siRNA loaded liposomes incorporating hydrophobic G2 was evaluated at two exposure times through MTT assay in a breast cancer cell (SKBR-3 and compared by two-way ANOVA. Keywords: Anti-VEGF siRNA, Cell growth inhibition, Polyamidoaminedendrimer, Liposome

  9. 3D Culture for Self-Formation of the Cerebellum from Human Pluripotent Stem Cells Through Induction of the Isthmic Organizer.

    Science.gov (United States)

    Muguruma, Keiko

    2017-01-01

    Pluripotent stem cells (PSCs) possess self-organizing abilities in 3D culture. This property has been demonstrated in recent studies, including the generation of various neuroectodermal and endodermal tissues. For example, PSCs are able to differentiate into specific type of neural tissues, such as the neocortex and the optic cup, in response to local positional information brought about by signals during embryogenesis. In contrast, the generation of cerebellar tissue from PSCs requires a secondary induction by a signaling center, called the isthmic organizer, which first appears in the cell aggregate in 3D culture. Such developmental complexity of cerebellum has hampered establishment of effective differentiation culture system from PSCs, thus far.We recently reported that cerebellar neurons are generated from human PSCs (hPSCs). In this chapter, we describe an efficient protocol for differentiation of 3D cerebellar neuroepithelium from hPSCs. We also describe the protocols for further differentiation into specific neurons in the cerebellar cortex, such as Purkinje cells and the granule cells.

  10. Study of the Mg incorporation in CdTe for developing wide band gap Cd{sub 1-x}Mg{sub x}Te thin films for possible use as top-cell absorber in a tandem solar cell

    Energy Technology Data Exchange (ETDEWEB)

    Martinez, Omar S. [Centro de Investigacion en Energia, Universidad Nacional Autonoma de Mexico, 62580 Temixco, Morelos (Mexico); Universidad Politecnica del Estado de Guerrero, Comunidad de Puente Campuzano, C.P. 40325 Taxco de Alarcon, Guerrero (Mexico); Millan, Aduljay Remolina [Centro de Investigacion en Energia, Universidad Nacional Autonoma de Mexico, 62580 Temixco, Morelos (Mexico); Huerta, L.; Santana, G. [Instituto de Investigaciones en Materiales, Universidad Nacional Autonoma de Mexico. C.P 04510 Mexico D.F. (Mexico); Mathews, N.R.; Ramon-Garcia, M.L.; Morales, Erik R. [Centro de Investigacion en Energia, Universidad Nacional Autonoma de Mexico, 62580 Temixco, Morelos (Mexico); Mathew, X., E-mail: xm@cie.unam.mx [Centro de Investigacion en Energia, Universidad Nacional Autonoma de Mexico, 62580 Temixco, Morelos (Mexico)

    2012-02-15

    Highlights: Black-Right-Pointing-Pointer Thin films of Cd{sub 1-x}Mg{sub x}Te with high spatial uniformity and band gap in the range of 1.6-1.96 eV were deposited by vacuum co-evaporation of CdTe and Mg. Black-Right-Pointing-Pointer Obtained Cd{sub 1-x}Mg{sub x}Te films have the structural characteristics of the CdTe, evidence of the change in atomic scattering due to incorporation of Mg was observed. Black-Right-Pointing-Pointer XRD and XPS data confirmed the incorporation of Mg in the lattice of CdTe. Black-Right-Pointing-Pointer SEM images revealed the impact of Mg incorporation on the morphology of the films, the changes in grain size and grain morphology are noticeable. - Abstract: Thin films of Cd{sub 1-x}Mg{sub x}Te with band gap in the range of 1.6-1.96 eV were deposited by vacuum co-evaporation of CdTe and Mg on glass substrates heated at 300 Degree-Sign C. Different experimental techniques such as XRD, UV-vis spectroscopy, SEM, and XPS were used to study the effect of Mg incorporation into the lattice of CdTe. The band gap of the films showed a clear tendency to increase as the Mg content in the film is increased. The Cd{sub 1-x}Mg{sub x}Te films maintain all the structural characteristics of the CdTe, however, diminishing of intensity for the XRD patterns is observed due to both change in preferential orientation and change in atomic scattering due to the incorporation of Mg. SEM images showed significant evidences of morphological changes due to the presence of Mg. XRD, UV-vis spectroscopy, and XPS data confirmed the incorporation of Mg in the lattice of CdTe. The significant increase in band gap of CdTe due to incorporation of Mg suggests that the Cd{sub 1-x}Mg{sub x}Te thin film is a candidate material to use as absorber layer in the top-cell of a tandem solar cell.

  11. Altered cerebellar development in nuclear receptor TAK1/ TR4 null mice is associated with deficits in GLAST(+) glia, alterations in social behavior, motor learning, startle reactivity, and microglia.

    Science.gov (United States)

    Kim, Yong-Sik; Harry, G Jean; Kang, Hong Soon; Goulding, David; Wine, Rob N; Kissling, Grace E; Liao, Grace; Jetten, Anton M

    2010-09-01

    Previously, deficiency in the expression of the nuclear orphan receptor TAK1 was found to be associated with delayed cerebellar granule cell migration and Purkinje cell maturation with a permanent deficit in foliation of lobules VI–VII, suggesting a role for TAK1 in cerebellum development. In this study, we confirm that TAK1-deficient (TAK1(−/−)) mice have a smaller cerebellum and exhibit a disruption of lobules VI–VII. We extended these studies and show that at postnatal day 7, TAK1(−/−) mice exhibit a delay in monolayer maturation of dysmorphic calbindin 28K-positive Purkinje cells. The astrocyte-specific glutamate transporter (GLAST) was expressed within Bergmann fibers and internal granule cell layer at significantly lower levels in the cerebellum of TAK1(−/−) mice. At PND21, Golgi-positive Purkinje cells in TAK1(−/−) mice displayed a smaller soma (18%) and shorter distance to first branch point (35%). Neuronal death was not observed in TAK1(−/−) mice at PND21; however, activated microglia were present in the cerebellum, suggestive of earlier cell death. These structural deficits in the cerebellum were not sufficient to alter motor strength, coordination, or activity levels; however, deficits in acoustic startle response, prepulse startle inhibition, and social interactions were observed. Reactions to a novel environment were inhibited in a light/dark chamber, open-field, and home-cage running wheel. TAK1(−/−) mice displayed a plateau in performance on the running wheel, suggesting a deficit in learning to coordinate performance on a motor task. These data indicate that TAK1 is an important transcriptional modulator of cerebellar development and neurodevelopmentally regulated behavior.

  12. Fabrication and Characterization of Chitosan Nanoparticle-Incorporated Quaternized Poly(Vinyl Alcohol) Composite Membranes as Solid Electrolytes for Direct Methanol Alkaline Fuel Cells

    International Nuclear Information System (INIS)

    Li, Pin-Chieh; Liao, Guan–Ming; Kumar, S. Rajesh; Shih, Chao-Ming; Yang, Chun-Chen; Wang, Da-Ming; Lue, Shingjiang Jessie

    2016-01-01

    Highlights: • Preparation of chitosan nanoparticles from bulk to enhance the degree of deacetylation. • The incorporation of chitosan nanoparticles into a QPVA matrix to form a nanocomposite membrane. • The nanocomposite constructed into thin-film membranes using the solution casting method. • To improve permeability, glutaraldehyde was cross-linked with the nanocomposite membranes. • A direct methanol alkaline fuel cell was studied at different temperatures. - Abstract: In this study, we designed a method for the preparation of chitosan nanoparticles incorporated into a quaternized poly(vinyl alcohol) (QPVA) matrix for direct methanol alkaline fuel cells (DMAFCs). The structural and morphological properties of the prepared nanocomposites were studied using X-ray diffraction (XRD), field-emission scanning electron microscopy (FE-SEM), transmission electron microscope (TEM) and dynamic laser-light scattering (DLS). The crystallinity of the nanocomposite solid electrolytes containing 0 and 10% chitosan nanoparticles were investigated using differential scanning calorimetry (DSC). The electrochemical measurement of resulting nanocomposite membranes were analyzed according to the following parameters: methanol permeability, liquid uptakes, ionic conductivity and cell performances. The composite membranes with 10% chitosan nanoparticles in a QPVA matrix (CQPVA) show suppressed methanol permeability and higher ionic conductivity than pristine QPVA. In addition, the glutaraldehyde cross-linked nanocomposite film exhibited improvement on the methanol barrier property at 80 °C. The peak power density of the DMAFCs reached 67 mW cm −2 when fed into 1 M of methanol in 6 M of KOH.

  13. Evidence of a genetic instability induced by the incorporation of a DNA precursor marked with tritium; Mise en evidence d'une instabilite genetique induite par l'incorporation d'un precurseur de L'ADN marque au tritium

    Energy Technology Data Exchange (ETDEWEB)

    Saintigny, Y.; Laurent, D.; Lahayel, J.B. [CEA Fontenay-aux-Roses, IRCM-LRTS, U967 - CEA/INSERM/Universites Paris 7 and Paris-11, 92 (France); Roche, St.; Meynard, D.; Lopez, B.S. [CEA Fontenay-aux-Roses, LMR - UMR 217 - CEA/CNRS, Institut de Radiobiologie Cellulaire et moleculaire, Direction des Sciences du Vivant, 92 (France)

    2009-07-01

    The authors report a molecular geno-toxicology investigation which allowed molecular events induced par intracellular incorporation of tritium to be studied, and the genetic instability resulting from a chronic exposure even at low dose to be analysed. For this purpose, they developed cell models (hamster tumorous cells and human fibroblasts) in which they know how to incorporate given quantities of marked nucleotides in the DNA. They show that the incorporation of tritium, even with doses which are said to be non toxic, causes a prolonged exposure of the cell to a genotoxic stress, and maybe a genetic instability due to a too great number of recombination events

  14. Competitor analogs for defined T cell antigens: peptides incorporating a putative binding motif and polyproline or polyglycine spacers.

    Science.gov (United States)

    Maryanski, J L; Verdini, A S; Weber, P C; Salemme, F R; Corradin, G

    1990-01-12

    We describe a new approach for modeling antigenic peptides recognized by T cells. Peptide A24 170-182 can compete with other antigenic peptides that are recognized by H-2kd-restricted cytolytic T cells, presumably by binding to the Kd molecule. By comparing substituted A24 peptides as competitors in a functional competition assay, the A24 residues Tyr-171, Thr-178, and Leu-179 were identified as possible contact residues for Kd. A highly active competitor peptide analog was synthesized in which Tyr was separated from the Thr-Leu pair by a pentaproline spacer. The choice of proline allowed the prediction of a probable conformation for the analog when bound to the Kd molecule. The simplest conformation of the A24 peptide that allows the same spacing and orientation of the motif as in the analog would be a nearly extended polypeptide chain incorporating a single 3(10) helical turn or similar structural kink.

  15. Amo 1618 effects on incorporation of 14C-MVA and 14C-acetate into sterols in Nicotiana and Digitalis seedlings and cell-free preparations from Nicotiana

    International Nuclear Information System (INIS)

    Douglas, T.J.; Paleg, L.G.

    1978-01-01

    Incorporation of radioactivity from acetate-[ 14 C] and MVA-[ 14 C] into sterols and sterol precursors in tobacco was inhibited by Amo 1618; differing patterns of accumulation were obtained with the two precursors, suggesting more than one point of inhibition. This was borne out with cell-free preparations with which it was demonstrated that both HMG-CoA reductase and squalene-2,3-epoxide cyclase were inhibited, the latter more strongly than the former. GLC analysis of gross sterol and hydrocarbon fractions confirmed previous indications that incorporation of radioactivity into individual sterols was inhibited by Amo 1618. Finally, incorporation of MVA-[ 14 C] into sterols and sterol precursors of Digitalis was significantly altered by the retardant, thus expanding the generality of the relationship between sterol (particularly 4-desmethylsterol) biosynthesis inhibition and retardant effect. (author)

  16. PIXE maps of intracellular element distribution in cerebellar neurons

    Czech Academy of Sciences Publication Activity Database

    Kranda, Karel; Havránek, Vladimír; Purkrtová, Z.; Vožeh, F.; Hájková, L.

    2012-01-01

    Roč. 22, 1-2 (2012), s. 65-72 ISSN 0129-0835 R&D Projects: GA ČR GA309/09/1189 Institutional support: RVO:61389005 Keywords : MicroPIXE * 2D-mapping * cerebellum * mutant mice * Purkinje cells * cell death * metan concentration Subject RIV: BG - Nuclear, Atomic and Molecular Physics, Colliders

  17. Cerebellar Circuit Mechanisms Which Accompany Coordinated Limb Trajectory Patterns in the Rat: Use of a Model of Spontaneous Changes

    National Research Council Canada - National Science Library

    Smith, Sherry

    1997-01-01

    ...) and its target, Purkinje cells in the paravermal cerebellum. In many cases, simultaneous recordings were carried out from as many as 48 neurons in both areas during tredmill locomotion tasks used to evaluate concomitant sensorimotor performance...

  18. Stimulation of neural differentiation in human bone marrow mesenchymal stem cells by extremely low-frequency electromagnetic fields incorporated with MNPs.

    Science.gov (United States)

    Choi, Yun-Kyong; Lee, Dong Heon; Seo, Young-Kwon; Jung, Hyun; Park, Jung-Keug; Cho, Hyunjin

    2014-10-01

    Human bone marrow-derived mesenchymal stem cells (hBM-MSCs) have been investigated as a new cell-therapeutic solution due to their capacity that could differentiate into neural-like cells. Extremely low-frequency electromagnetic fields (ELF-EMFs) therapy has emerged as a novel technique, using mechanical stimulus to differentiate hBM-MSCs and significantly enhance neuronal differentiation to affect cellular and molecular reactions. Magnetic iron oxide (Fe3O4) nanoparticles (MNPs) have recently achieved widespread use for biomedical applications and polyethylene glycol (PEG)-labeled nanoparticles are used to increase their circulation time, aqueous solubility, biocompatibility, and nonspecific cellular uptake as well as to decrease immunogenicity. Many studies have used MNP-labeled cells for differentiation, but there have been no reports of MNP-labeled neural differentiation combined with EMFs. In this study, synthesized PEG-phospholipid encapsulated magnetite (Fe3O4) nanoparticles are used on hBM-MSCs to improve their intracellular uptake. The PEGylated nanoparticles were exposed to the cells under 50 Hz of EMFs to improve neural differentiation. First, we measured cell viability and intracellular iron content in hBM-MSCs after treatment with MNPs. Analysis was conducted by RT-PCR, and immunohistological analysis using neural cell type-specific genes and antibodies after exposure to 50 Hz electromagnetic fields. These results suggest that electromagnetic fields enhance neural differentiation in hBM-MSCs incorporated with MNPs and would be an effective method for differentiating neural cells.

  19. Silver nanoparticles-incorporated Nb2O5 surface passivation layer for efficiency enhancement in dye-sensitized solar cells.

    Science.gov (United States)

    Suresh, S; Unni, Gautam E; Satyanarayana, M; Sreekumaran Nair, A; Mahadevan Pillai, V P

    2018-08-15

    Guiding and capturing photons at the nanoscale by means of metal nanoparticles and interfacial engineering for preventing back-electron transfer are well documented techniques for performance enhancement in excitonic solar cells. Drifting from the conventional route, we propose a simple one-step process to integrate both metal nanoparticles and surface passivation layer in the porous photoanode matrix of a dye-sensitized solar cell. Silver nanoparticles and Nb 2 O 5 surface passivation layer are simultaneously deposited on the surface of a highly porous nanocrystalline TiO 2 photoanode, facilitating an absorption enhancement in the 465 nm and 570 nm wavelength region and a reduction in back-electron transfer in the fabricated dye-sensitized solar cells together. The TiO 2 photoanodes were prepared by spray pyrolysis deposition method from a colloidal solution of TiO 2 nanoparticles. An impressive 43% enhancement in device performance was accomplished in photoanodes having an Ag-incorporated Nb 2 O 5 passivation layer as against a cell without Ag nanoparticles. By introducing this idea, we were able to record two benefits - the metal nanoparticles function as the absorption enhancement agent, and the Nb 2 O 5 layer as surface passivation for TiO 2 nanoparticles and as an energy barrier layer for preventing back-electron transfer - in a single step. Copyright © 2018 Elsevier Inc. All rights reserved.

  20. Cell-type-specific expression of NFIX in the developing and adult cerebellum.

    Science.gov (United States)

    Fraser, James; Essebier, Alexandra; Gronostajski, Richard M; Boden, Mikael; Wainwright, Brandon J; Harvey, Tracey J; Piper, Michael

    2017-07-01

    Transcription factors from the nuclear factor one (NFI) family have been shown to play a central role in regulating neural progenitor cell differentiation within the embryonic and post-natal brain. NFIA and NFIB, for instance, promote the differentiation and functional maturation of granule neurons within the cerebellum. Mice lacking Nfix exhibit delays in the development of neuronal and glial lineages within the cerebellum, but the cell-type-specific expression of this transcription factor remains undefined. Here, we examined the expression of NFIX, together with various cell-type-specific markers, within the developing and adult cerebellum using both chromogenic immunohistochemistry and co-immunofluorescence labelling and confocal microscopy. In embryos, NFIX was expressed by progenitor cells within the rhombic lip and ventricular zone. After birth, progenitor cells within the external granule layer, as well as migrating and mature granule neurons, expressed NFIX. Within the adult cerebellum, NFIX displayed a broad expression profile, and was evident within granule cells, Bergmann glia, and interneurons, but not within Purkinje neurons. Furthermore, transcriptomic profiling of cerebellar granule neuron progenitor cells showed that multiple splice variants of Nfix are expressed within this germinal zone of the post-natal brain. Collectively, these data suggest that NFIX plays a role in regulating progenitor cell biology within the embryonic and post-natal cerebellum, as well as an ongoing role within multiple neuronal and glial populations within the adult cerebellum.

  1. Somatostatin reduces 3H-thymidine incorporation and c-myc, but not thyroglobulin ribonucleic acid levels in human thyroid follicular cells in vitro

    International Nuclear Information System (INIS)

    degli Uberti, E.C.; Hanau, S.; Rossi, R.; Piva, R.; Margutti, A.; Trasforini, G.; Pansini, G.; del Senno, L.

    1991-01-01

    The action of somatostatin (SRIH) on 3 H-thymidine (thy) incorporation and on c-myc and thyroglobulin RNA levels in a suspension of follicles from normal and goitrous human thyroid was examined. SRIH, at 10 - 7 M concentration, inhibited basal thy incorporation (maximally by 4 h lasting for up 24 h), which effect was greater in goiter than in normal thyroid and was also detected in growing adherent epithelial cells. Moreover, in a follicle suspension SRIH prevented TSH-stimulated thy incorporation, both in normal and in goitrous thyroid. Basal expression of c-myc RNA was not affected by SRIH in either tissue, whereas the TSH-stimulated c-myc RNA level was significantly reduced in goiter. No effect of SRIH was observed on basal or TSH-stimulated thyroglobulin RNA levels. SRIH did not alter basal cAMP concentrations in normal or goitrous follicles, but it significantly reduced TSH-stimulated cAMP accumulation both in normal thyroid and in goiter. Overall, our data indicate a direct inhibitory action of SRIH on growth, but not on differentiation, of human thyroid, probably by a mechanism not entirely cAMP dependent

  2. Cerebellar neurodegeneration in the absence of microRNAs

    Science.gov (United States)

    Schaefer, Anne; O'Carroll, Dónal; Tan, Chan Lek; Hillman, Dean; Sugimori, Mutsuyuki; Llinas, Rodolfo; Greengard, Paul

    2007-01-01

    Genome-encoded microRNAs (miRNAs) are potent regulators of gene expression. The significance of miRNAs in various biological processes has been suggested by studies showing an important role of these small RNAs in regulation of cell differentiation. However, the role of miRNAs in regulation of differentiated cell physiology is not well established. Mature neurons express a large number of distinct miRNAs, but the role of miRNAs in postmitotic neurons has not been examined. Here, we provide evidence for an essential role of miRNAs in survival of differentiated neurons. We show that conditional Purkinje cell–specific ablation of the key miRNA-generating enzyme Dicer leads to Purkinje cell death. Deficiency in Dicer is associated with progressive loss of miRNAs, followed by cerebellar degeneration and development of ataxia. The progressive neurodegeneration in the absence of Dicer raises the possibility of an involvement of miRNAs in neurodegenerative disorders. PMID:17606634

  3. Yeast cells lacking all known ceramide synthases continue to make complex sphingolipids and to incorporate ceramides into glycosylphosphatidylinositol (GPI) anchors

    DEFF Research Database (Denmark)

    Vionnet, Christine; Roubaty, Carole; Ejsing, Christer S.

    2010-01-01

    In yeast, the inositolphosphorylceramides mostly contain C26:0 fatty acids. Inositolphosphorylceramides were considered to be important for viability, since the inositolphosphorylceramide synthase AUR1 is essential. Yet, lcb1 cells, unable to make sphingoid bases and inositolphosphorylceramides......, are viable if they harbor SLC1-1, a gain of function mutation in the 1-acyl-glycerol-3-phosphate acyltransferase SLC1. SLC1-1 allows to incorporate C26:0 fatty acids into phosphatidylinositol (PI), thus generating PIii, an abnormal, C26-containing PI, presumably acting as surrogate...

  4. behavioural, biochemical and neurocytoarchitechural impact of ...

    African Journals Online (AJOL)

    2017-01-20

    Jan 20, 2017 ... dimensions, 100cm wide, 100cm long, and 50cm .... eyes color in the short term. Although .... coordination and memory function (Callaghan et al., 2006 .... of cerebellar Purkinje cells modulated by sensory stimulation. Nature ...

  5. The role of Abcb5 alleles in susceptibility to haloperidol-induced toxicity in mice and humans.

    KAUST Repository

    Zheng, Ming; Zhang, Haili; Dill, David L; Clark, J David; Tu, Susan; Yablonovitch, Arielle L; Tan, Meng How; Zhang, Rui; Rujescu, Dan; Wu, Manhong; Tessarollo, Lino; Vieira, Wilfred; Gottesman, Michael M; Deng, Suhua; Eberlin, Livia S; Zare, Richard N; Billard, Jean-Martin; Gillet, Jean-Pierre; Li, Jin Billy; Peltz, Gary

    2015-01-01

    that genetic variation within an ABC-drug efflux transporter (Abcb5) affected susceptibility to HIT. In situ hybridization results reveal that Abcb5 is expressed in brain capillaries, and by cerebellar Purkinje cells. We also analyzed chromosome substitution

  6. Incorporating pushing in exclusion-process models of cell migration.

    Science.gov (United States)

    Yates, Christian A; Parker, Andrew; Baker, Ruth E

    2015-05-01

    The macroscale movement behavior of a wide range of isolated migrating cells has been well characterized experimentally. Recently, attention has turned to understanding the behavior of cells in crowded environments. In such scenarios it is possible for cells to interact, inducing neighboring cells to move in order to make room for their own movements or progeny. Although the behavior of interacting cells has been modeled extensively through volume-exclusion processes, few models, thus far, have explicitly accounted for the ability of cells to actively displace each other in order to create space for themselves. In this work we consider both on- and off-lattice volume-exclusion position-jump processes in which cells are explicitly allowed to induce movements in their near neighbors in order to create space for themselves to move or proliferate into. We refer to this behavior as pushing. From these simple individual-level representations we derive continuum partial differential equations for the average occupancy of the domain. We find that, for limited amounts of pushing, comparison between the averaged individual-level simulations and the population-level model is nearly as good as in the scenario without pushing. Interestingly, we find that, in the on-lattice case, the diffusion coefficient of the population-level model is increased by pushing, whereas, for the particular off-lattice model that we investigate, the diffusion coefficient is reduced. We conclude, therefore, that it is important to consider carefully the appropriate individual-level model to use when representing complex cell-cell interactions such as pushing.

  7. β-Catenin is critical for cerebellar foliation and lamination.

    Directory of Open Access Journals (Sweden)

    Jing Wen

    Full Text Available The cerebellum has a conserved foliation pattern and a well-organized layered structure. The process of foliation and lamination begins around birth. β-catenin is a downstream molecule of Wnt signaling pathway, which plays a critical role in tissue organization. Lack of β-catenin at early embryonic stages leads to either prenatal or neonatal death, therefore it has been difficult to resolve its role in cerebellar foliation and lamination. Here we used GFAP-Cre to ablate β-catenin in neuronal cells of the cerebellum after embryonic day 12.5, and found an unexpected role of β-catenin in determination of the foliation pattern. In the mutant mice, the positions of fissure formation were changed, and the meninges were improperly incorporated into fissures. At later stages, some lobules were formed by Purkinje cells remaining in deep regions of the cerebellum and the laminar structure was dramatically altered. Our results suggest that β-catenin is critical for cerebellar foliation and lamination. We also found a non cell-autonomous role of β-catenin in some developmental properties of major cerebellar cell types during specific stages.

  8. Collagen/silk fibroin composite scaffold incorporated with PLGA microsphere for cartilage repair

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Jianhua; Yang, Qiu; Cheng, Niangmei [Institute of Biomedical and Pharmaceutical Technology, Fuzhou University, Fuzhou 350002 (China); Tao, Xiaojun [Department of Pharmacy, School of Medicine, Hunan Normal University, Changsha, 410013, Hunan (China); Zhang, Zhihua; Sun, Xiaomin [Institute of Biomedical and Pharmaceutical Technology, Fuzhou University, Fuzhou 350002 (China); Zhang, Qiqing, E-mail: zhangqiq@126.com [Institute of Biomedical and Pharmaceutical Technology, Fuzhou University, Fuzhou 350002 (China); Key Laboratory of Biomedical Materials of Tianjin, Institute of Biomedical Engineering, Chinese Academy of Medical Science & Peking Union Medical College, Tianjin 300192 (China)

    2016-04-01

    For cartilage repair, ideal scaffolds should mimic natural extracellular matrix (ECM) exhibiting excellent characteristics, such as biocompatibility, suitable porosity, and good cell affinity. This study aimed to prepare a collagen/silk fibroin composite scaffold incorporated with poly-lactic-co-glycolic acid (PLGA) microsphere that can be applied in repairing cartilage. To obtain optimum conditions for manufacturing a composite scaffold, a scaffold composed of different collagen-to-silk fibroin ratios was evaluated by determining porosity, water absorption, loss rate in hot water, and cell proliferation. Results suggested that the optimal ratio of collagen and silk fibroin composite scaffold was 7:3. The microstructure and morphological characteristics of the obtained scaffold were also examined through scanning electron microscopy and Fourier transform infrared spectroscopy. The results of in vitro fluorescence staining of bone marrow stromal cells revealed that collagen/silk fibroin composite scaffold enhanced cell proliferation without eliciting side effects. The prepared composite scaffold incorporated with PLGA microsphere was implanted in fully thick articular cartilage defects in rabbits. Collagen/silk fibroin composite scaffold with PLGA microspheres could enhance articular cartilage regeneration and integration between the repaired cartilage and the surrounding cartilage. Therefore, this composite will be a promising material for cartilage repair and regeneration. - Highlights: • Collagen/silk fibroin composite scaffold incorporated with PLGA microsphere proposed for cartilage repair was created. • In vivo, scaffold could enhance cartilage regeneration and integration between the repaired and surrounding cartilage. • In vitro, scaffold exhibits excellent characteristics, such as, improved porosity water absorption and good cell affinity.

  9. Incorporation of thymidine into onion root meristematic cell nuclei in presence of hydroxyurea and its role in recovery of mitotic activity

    OpenAIRE

    H. Habdas

    2015-01-01

    Hydroxyurea treatment of onion roots induced mitotic block which was released by transfer of bulbs to water, and also to some extent by addition of cold or 3H-thymidine to hydroxyurea solutions. In presence of hydroxyurea there was noted very intense incorporation of 3H-thymidine into cell nuclei, giving labelling index of 40-70%. However, all the mitotic figures appearing in presence of hydroxyurea and 3H-thymidine were unlabelled. On the other hand, labelled mitotic figures were obtained wh...

  10. Altered subcellular localization of ornithine decarboxylase in Alzheimer's disease brain

    International Nuclear Information System (INIS)

    Nilsson, Tatjana; Bogdanovic, Nenad; Volkman, Inga; Winblad, Bengt; Folkesson, Ronnie; Benedikz, Eirikur

    2006-01-01

    The amyloid precursor protein can through ligand-mimicking induce expression of ornithine decarboxylase (ODC), the initial and rate-limiting enzyme in polyamine biosynthesis. We report here the regional distribution and cellular localization of ODC immunoreactivity in Alzheimer's disease (AD) brains. In frontal cortex and hippocampus of control cases, the most pronounced ODC immunoreactivity was found in the nucleus. In possible and definite AD the immunoreactivity had shifted to the cytoplasm. In cerebellum of control cases, ODC staining was found in a small portion of Purkinje cells, mostly in the nucleus. In AD, both possible and definite, the number of stained Purkinje cells increased significantly and immunoreactivity was shifted to the cytoplasm, even though it was still prominent in the nucleus. In conclusion, our study reveals an early shift of the ODC immunoreactivity in AD from the nuclear compartment towards the cytoplasm

  11. Questioning the cerebellar doctrine.

    Science.gov (United States)

    Galliano, Elisa; De Zeeuw, Chris I

    2014-01-01

    The basic principles of cerebellar function were originally described by Flourens, Cajal, and Marr/Albus/Ito, and they constitute the pillars of what can be considered to be the classic cerebellar doctrine. In their concepts, the main cerebellar function is to control motor behavior, Purkinje cells are the only cortical neuron receiving and integrating inputs from climbing fiber and mossy-parallel fiber pathways, and plastic modification at the parallel fiber synapses onto Purkinje cells constitutes the substrate of motor learning. Yet, because of recent technical advances and new angles of investigation, all pillars of the cerebellar doctrine now face regular re-examination. In this review, after summarizing the classic concepts and recent disputes, we attempt to synthesize an integrated view and propose a revisited version of the cerebellar doctrine. © 2014 Elsevier B.V. All rights reserved.

  12. Visualization of the African swine fever virus infection in living cells by incorporation into the virus particle of green fluorescent protein-p54 membrane protein chimera

    International Nuclear Information System (INIS)

    Hernaez, Bruno; Escribano, Jose M.; Alonso, Covadonga

    2006-01-01

    Many stages of African swine fever virus infection have not yet been studied in detail. To track the behavior of African swine fever virus (ASFV) in the infected cells in real time, we produced an infectious recombinant ASFV (B54GFP-2) that expresses and incorporates into the virus particle a chimera of the p54 envelope protein fused to the enhanced green fluorescent protein (EGFP). The incorporation of the fusion protein into the virus particle was confirmed immunologically and it was determined that p54-EGFP was fully functional by confirmation that the recombinant virus made normal-sized plaques and presented similar growth curves to the wild-type virus. The tagged virus was visualized as individual fluorescent particles during the first stages of infection and allowed to visualize the infection progression in living cells through the viral life cycle by confocal microscopy. In this work, diverse potential applications of B54GFP-2 to study different aspects of ASFV infection are shown. By using this recombinant virus it was possible to determine the trajectory and speed of intracellular virus movement. Additionally, we have been able to visualize for first time the ASFV factory formation dynamics and the cytophatic effect of the virus in live infected cells. Finally, we have analyzed virus progression along the infection cycle and infected cell death as time-lapse animations

  13. Influence of incorporated bromodeoxyuridine on the induction of chromosomal alterations by ionizing radiation and long-wave UV in CHO cells.

    Science.gov (United States)

    Zwanenburg, T S; van Zeeland, A A; Natarajan, A T

    1985-01-01

    Incorporation of BrdUrd into nuclear DNA sensitizes CHO cells (1) to the induction of chromosomal aberrations by X-rays and 0.5 MeV neutrons and (2) to induction of chromosomal aberrations and SCEs by lw-UV. We have attempted to establish a correlation between induced chromosomal alterations and induced single- or double-strand breaks in DNA. The data show that while DSBs correlate very well with X-ray-induced aberrations, no clear correlation could be established between lw-UV induced SSBs (including alkali-labile sites) and chromosomal alterations. In addition the effect of 3-aminobenzamide (3AB) on the induction of chromosomal aberrations and SCEs induced by lw-UV has been determined. It is shown that 3AB is without any effect when lw-UV-irradiated cells are posttreated with this inhibitor. The significance of these results is discussed.

  14. Incorporation of [1-C14] Isopentenyl Pyrophosphate into Carotenoids and Homo carotenoids using a Cell-free Preparation of Micrococcus Luteus

    International Nuclear Information System (INIS)

    Al-Wandawi, H.

    1998-01-01

    The early steps up to the formation of acyclic unsaturated carotenes (e.g.,phytoene to lycopene) are presumed to be common to the biosynthesis of all carotenoids with 40 or more carbon atoms, nevertheless, no direct evidence so far available to confirm this for homo carotenoids (c 45 and c 50 carotenoids). In the present study, an active cell-free preparation was obtained from diphenylamine-inhibited cells of Micrococcus Iuteus and found to be capable to incorporate radioactivity from Isopentenyl pyrophosphate (labelled with C-14)into carotenoids and homo carotenoids, providing for the first time a direct evidence which suggests that both carotenoids and homo carotenoids are sharing the same biological origin. Furthermore, the technique developed in this study may be considered as a valuable method for preparation of biological-active labelled compounds which may have some advantages over conventional chemical syntheses methods

  15. Age-related changes of structures in cerebellar cortex of cat

    Indian Academy of Sciences (India)

    Madhu

    ness of the cerebellar cortex as well as loss of neurons, and hypertrophy and ... Purkinje cells. (PCs) in old cats showed much fewer NF-IR dendrites than those in young adults. ... diminution in motor control and motor learning) underlying.

  16. Angiogenin activates phospholipase C and elicits a rapid incorporation of fatty acid into cholesterol esters in vascular smooth muscle cells

    International Nuclear Information System (INIS)

    Moore, F.; Riordan, J.F.

    1990-01-01

    Angiogenin activates the phosphoinositide-specific phospholipase C (PLC) in cultured rat aortic smooth muscle cells to yield a transient (30 s) peak of 1,2-diacylglycerol (DG) and inositol trisphosphate. Within 1 min, the DG level falls below that of the control and remains so for at least 20 min. A transient increase in monoacylglycerol indicates that depletion of DG may be the consequence of hydrolysis by DG lipase. In addition to these changes in second messengers, a rapid increase in incorporating of radiolabeled tracer into cellular cholesterol esters is observed. Stimulated cholesterol ester labeling is inhibited by preincubation with either the DG lipase inhibitor RHC 80267 or the acyl coenzyme A:cholesterol acyltransferase inhibitor Sandoz 58035. Cells prelabeled with [ 3 H]arachidonate show a sustained increase in labeling of cholesterol esters following exposure to angiogenin. In contrast, cells prelabeled with [ 3 H]oleate show only a transient elevation that returns to the basal level by 5 min. This suggests initial cholesterol esterification by oleate followed by arachidonate that is released by stimulation of the PLC/DG lipase pathway

  17. The Biomineralization of a Bioactive Glass-Incorporated Light-Curable Pulp Capping Material Using Human Dental Pulp Stem Cells

    Directory of Open Access Journals (Sweden)

    Soo-Kyung Jun

    2017-01-01

    Full Text Available The aim of this study was to investigate the biomineralization of a newly introduced bioactive glass-incorporated light-curable pulp capping material using human dental pulp stem cells (hDPSCs. The product (Bioactive® [BA] was compared with a conventional calcium hydroxide-incorporated (Dycal [DC] and a light-curable (Theracal® [TC] counterpart. Eluates from set specimens were used for investigating the cytotoxicity and biomineralization ability, determined by alkaline phosphatase (ALP activity and alizarin red staining (ARS. Cations and hydroxide ions in the extracts were measured. An hDPSC viability of less than 70% was observed with 50% diluted extract in all groups and with 25% diluted extract in the DC. Culturing with 12.5% diluted BA extract statistically lowered ALP activity and biomineralization compared to DC (p0.05. Ca (~110 ppm and hydroxide ions (pH 11 were only detected in DC and TC. Ionic supplement-added BA, which contained similar ion concentrations as TC, showed similar ARS mineralization compared to TC. In conclusion, the BA was similar to, yet more cytotoxic to hDPSCs than, its DC and TC. The BA was considered to stimulate biomineralization similar to DC and TC only when it released a similar amount of Ca and hydroxide ions.

  18. Enhanced conversion efficiency of dye-sensitized solar cells using a CNT-incorporated TiO2 slurry-based photoanode

    Directory of Open Access Journals (Sweden)

    Jiaoping Cai

    2015-02-01

    Full Text Available A new titanium dioxide (TiO2 slurry formulation is herein reported for the fabrication of TiO2 photoanode for use in dye-sensitized solar cells (DSSCs. The prepared TiO2 photoanode featured a highly uniform mesoporous structure with well-dispersed TiO2 nanoparticles. The energy conversion efficiency of the resulting TiO2 slurry-based DSSC was ∼63% higher than that achieved by a DSSC prepared using a commercial TiO2 slurry. Subsequently, the incorporation of acid-treated multi-walled carbon nanotubes (CNTs into the TiO2 slurry was examined. More specifically, the effect of varying the concentration of the CNTs in this slurry on the performance of the resulting DSSCs was studied. The chemical state of the CNTs-incorporated TiO2 photoanode was investigated by Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy. A high energy conversion efficiency of 6.23% was obtained at an optimum CNT concentration of ∼0.06 wt.%. The obtained efficiency corresponds to a 63% enhancement when compared with that obtained from a DSSC based on a commercial TiO2 slurry. The higher efficiency was attributed to the improvement in the collection and transport of excited electrons in the presence of the CNTs.

  19. Enhanced conversion efficiency of dye-sensitized solar cells using a CNT-incorporated TiO2 slurry-based photoanode

    Science.gov (United States)

    Cai, Jiaoping; Chen, Zexiang; Li, Jun; Wang, Yan; Xiang, Dong; Zhang, Jijun; Li, Hai

    2015-02-01

    A new titanium dioxide (TiO2) slurry formulation is herein reported for the fabrication of TiO2 photoanode for use in dye-sensitized solar cells (DSSCs). The prepared TiO2 photoanode featured a highly uniform mesoporous structure with well-dispersed TiO2 nanoparticles. The energy conversion efficiency of the resulting TiO2 slurry-based DSSC was ˜63% higher than that achieved by a DSSC prepared using a commercial TiO2 slurry. Subsequently, the incorporation of acid-treated multi-walled carbon nanotubes (CNTs) into the TiO2 slurry was examined. More specifically, the effect of varying the concentration of the CNTs in this slurry on the performance of the resulting DSSCs was studied. The chemical state of the CNTs-incorporated TiO2 photoanode was investigated by Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy. A high energy conversion efficiency of 6.23% was obtained at an optimum CNT concentration of ˜0.06 wt.%. The obtained efficiency corresponds to a 63% enhancement when compared with that obtained from a DSSC based on a commercial TiO2 slurry. The higher efficiency was attributed to the improvement in the collection and transport of excited electrons in the presence of the CNTs.

  20. Enhancing the performance of dye-sensitized solar cells by incorporating nanosilicate platelets in gel electrolyte

    KAUST Repository

    Lai, Yi-Hsuan

    2009-10-01

    Two kinds of gel-type dye-sensitized solar cells (DSSCs), composed of two types of electrolytes, were constructed and the respective cell performance was evaluated in this study. One electrolyte, TEOS-Triton X-100 gel, was based on a hybrid organic/inorganic gel electrolyte made by the sol-gel method and the other was based on poly(vinyidene fluoride-co-hexafluoro propylene) (PVDF-HFP) copolymer. TEOS-Triton X-100 gel was based on the reticulate structure of silica, formed by hydrolysis, and condensation of tetraethoxysilane (TEOS), while its organic subphase was a mixture of surfactant (Triton X-100) and ionic liquid electrolytes. Both DSSC gel-type electrolytes were composed of iodine, 1-propy-3-methyl-imidazolium iodide, and 3-methoxypropionitrile to create the redox couple of I3 -/I-. Based on the results obtained from the I-V characteristics, it was found that the optimal iodine concentrations for the TEOS-Triton X-100 gel electrolyte and PVDF-HFP gel electrolyte are 0.05 M and 0.1 M, respectively. Although the increase in the iodine concentration could enhance the short-circuit current density (JSC), a further increase in the iodine concentration would reduce the JSC due to increased dark current. Therefore, the concentration of I2 is a significant factor in determining the performance of DSSCs. In order to enhance cell performance, the addition of nanosilicate platelets (NSPs) in the above-mentioned gel electrolytes was investigated. By incorporating NSP-Triton X-100 into the electrolytes, the JSC of the cells increased due to the decrease of diffusion resistance, while the open circuit voltage (VOC) remained almost the same. As the loading of the NSP-Triton X-100 in the TEOS-Triton X-100 gel electrolyte increased to 0.5 wt%, the JSC and the conversion efficiency increased from 8.5 to 12 mA/cm2 and from 3.6% to 4.7%, respectively. However, the JSC decreased as the loading of NSP-Triton X-100 exceeded 0.5 wt%. At higher NSP-Triton X-100 loading, NSPs acted as

  1. Hydrogen exchange during cell-free incorporation of deuterated amino acids and an approach to its inhibition

    Energy Technology Data Exchange (ETDEWEB)

    Tonelli, Marco; Singarapu, Kiran K. [University of Wisconsin-Madison, National Magnetic Resonance Facility at Madison (NMRFAM), Department of Biochemistry (United States); Makino, Shin-ichi; Sahu, Sarata C.; Matsubara, Yuko [University of Wisconsin-Madison, Center for Eukaryotic Structural Genomics (CESG), Department of Biochemistry (United States); Endo, Yaeta [Ehime University, Cell-Free Science and Technology Research Center (Japan); Kainosho, Masatsune [Tokyo Metropolitan University, Center for Priority Areas (Japan); Markley, John L., E-mail: markley@nmrfam.wisc.edu [University of Wisconsin-Madison, National Magnetic Resonance Facility at Madison (NMRFAM), Department of Biochemistry (United States)

    2011-12-15

    Perdeuteration, selective deuteration, and stereo array isotope labeling (SAIL) are valuable strategies for NMR studies of larger proteins and membrane proteins. To minimize scrambling of the label, it is best to use cell-free methods to prepare selectively labeled proteins. However, when proteins are prepared from deuterated amino acids by cell-free translation in H{sub 2}O, exchange reactions can lead to contamination of {sup 2}H sites by {sup 1}H from the solvent. Examination of a sample of SAIL-chlorella ubiquitin prepared by Escherichia coli cell-free synthesis revealed that exchange had occurred at several residues (mainly at Gly, Ala, Asp, Asn, Glu, and Gln). We present results from a study aimed at identifying the exchanging sites and level of exchange and at testing a strategy for minimizing {sup 1}H contamination during wheat germ cell-free translation of proteins produced from deuterated amino acids by adding known inhibitors of transaminases (1 mM aminooxyacetic acid) and glutamate synthetase (0.1 mM l-methionine sulfoximine). By using a wheat germ cell-free expression system, we produced [U-{sup 2}H, {sup 15}N]-chlorella ubiquitin without and with added inhibitors, and [U-{sup 15}N]-chlorella ubiquitin as a reference to determine the extent of deuterium incorporation. We also prepared a sample of [U-{sup 13}C, {sup 15}N]-chlorella ubiquitin, for use in assigning the sites of exchange. The added inhibitors did not reduce the protein yield and were successful in blocking hydrogen exchange at C{sup {alpha}} sites, with the exception of Gly, and at C{sup {beta}} sites of Ala. We discovered, in addition, that partial exchange occurred with or without the inhibitors at certain side-chain methyl and methylene groups: Asn-H{sup {beta}}, Asp-H{sup {beta}}, Gln-H{sup {gamma}}, Glu-H{sup {gamma}}, and Lys-H{sup {epsilon}}. The side-chain labeling pattern, in particular the mixed chiral labeling resulting from partial exchange at certain sites, should be of

  2. Hard X-ray submicrometer tomography of human brain tissue at Diamond Light Source

    Science.gov (United States)

    Khimchenko, A.; Bikis, C.; Schulz, G.; Zdora, M.-C.; Zanette, I.; Vila-Comamala, J.; Schweighauser, G.; Hench, J.; Hieber, S. E.; Deyhle, H.; Thalmann, P.; Müller, B.

    2017-06-01

    There is a lack of the necessary methodology for three-dimensional (3D) investigation of soft tissues with cellular resolution without staining or tissue transformation. Synchrotron radiation based hard X-ray in-line phase contrast tomography using single-distance phase reconstruction (SDPR) provides high spatial resolution and density contrast for the visualization of individual cells using a standard specimen preparation and data reconstruction. In this study, we demonstrate the 3D characterization of a formalin-fixed paraffin-embedded (FFPE) human cerebellum specimen by SDPR at the Diamond-Manchester Imaging Branchline I13-2 (Diamond Light Source, UK) at pixel sizes down to 0.45 μm. The approach enables visualization of cerebellar layers (Stratum moleculare and Stratum granulosum), the 3D characterization of individual cells (Purkinje, stellate and granule cells) and can even resolve some subcellular structures (nucleus and nucleolus of Purkinje cells). The tomographic results are qualitatively compared to hematoxylin and eosin (H&E) stained histological sections. We demonstrate the potential benefits of hard X-ray microtomography for the investigations of biological tissues in comparison to conventional histology.

  3. Hard X-ray submicrometer tomography of human brain tissue at Diamond Light Source

    International Nuclear Information System (INIS)

    Khimchenko, A; Bikis, C; Schulz, G; Hieber, S E; Deyhle, H; Thalmann, P; Müller, B; Zdora, M-C; Zanette, I; Vila-Comamala, J; Schweighauser, G; Hench, J

    2017-01-01

    There is a lack of the necessary methodology for three-dimensional (3D) investigation of soft tissues with cellular resolution without staining or tissue transformation. Synchrotron radiation based hard X-ray in-line phase contrast tomography using single-distance phase reconstruction (SDPR) provides high spatial resolution and density contrast for the visualization of individual cells using a standard specimen preparation and data reconstruction. In this study, we demonstrate the 3D characterization of a formalin-fixed paraffin-embedded (FFPE) human cerebellum specimen by SDPR at the Diamond-Manchester Imaging Branchline I13-2 (Diamond Light Source, UK) at pixel sizes down to 0.45 μm. The approach enables visualization of cerebellar layers ( Stratum moleculare and Stratum granulosum ), the 3D characterization of individual cells (Purkinje, stellate and granule cells) and can even resolve some subcellular structures (nucleus and nucleolus of Purkinje cells). The tomographic results are qualitatively compared to hematoxylin and eosin (H and E) stained histological sections. We demonstrate the potential benefits of hard X-ray microtomography for the investigations of biological tissues in comparison to conventional histology. (paper)

  4. Post-mortem 3H-thymidine incorporation in human epidermis and oral mucosa

    International Nuclear Information System (INIS)

    Schellmann, B.

    1981-01-01

    Using the 3H-thymidine labelling method, the authors studied post-mortem incorporation activity in the epidermis and oral mucosa of corpses which were stored with their clothes on under conditions of normal room temperature (+20 0 ) and of cooling (+4 0 C). Samples were taken in the form of skin punches at 2 h or 4 h intervals, respec.. Using histo-autoradiograms, the incorporation of 3H-thymidine in dependence from the time interval between the points of time of death and sampling were determined in situe and given as the ratio of labelled cells of the germinative layer per 100 μm length of basement membrane. A linear drop of post-mortem thymidine incorporation rates in epidermis and oral mucosa was found in human corpse skin correlating with increasing temporal distance from the point of time of death. Incorporation rates in the oral mucosa were markedly higher (by a factor of 3 to 5) than those of the epidermis which agrees well with in vivo conditions. No labelling of cell nuclei, i.e. no synthetic activity of the germinative layer, could be detected in the epidermis 35-40 h after individual death at the latest (in the oral mucosa after 45-50 h). However, clear incorporation activities could be observed in the germinative layer of epidermis and oral mucosa after more than 4 d in the case of storage at +4 0 C. (orig./MG) [de

  5. Manipulation of Cell Cycle and Chromatin Configuration by Means of Cell-Penetrating Geminin.

    Directory of Open Access Journals (Sweden)

    Yoshinori Ohno

    Full Text Available Geminin regulates chromatin remodeling and DNA replication licensing which play an important role in regulating cellular proliferation and differentiation. Transcription of the Geminin gene is regulated via an E2F-responsive region, while the protein is being closely regulated by the ubiquitin-proteasome system. Our objective was to directly transduce Geminin protein into cells. Recombinant cell-penetrating Geminin (CP-Geminin was generated by fusing Geminin with a membrane translocating motif from FGF4 and was efficiently incorporated into NIH 3T3 cells and mouse embryonic fibroblasts. The withdrawal study indicated that incorporated CP-Geminin was quickly reduced after removal from medium. We confirmed CP-Geminin was imported into the nucleus after incorporation and also that the incorporated CP-Geminin directly interacted with Cdt1 or Brahma/Brg1 as the same manner as Geminin. We further demonstrated that incorporated CP-Geminin suppressed S-phase progression of the cell cycle and reduced nuclease accessibility in the chromatin, probably through suppression of chromatin remodeling, indicating that CP-Geminin constitutes a novel tool for controlling chromatin configuration and the cell cycle. Since Geminin has been shown to be involved in regulation of stem cells and cancer cells, CP-Geminin is expected to be useful for elucidating the role of Geminin in stem cells and cancer cells, and for manipulating their activity.

  6. Antibacterial and cytocompatible nanotextured Ti surface incorporating silver via single step hydrothermal processing

    Energy Technology Data Exchange (ETDEWEB)

    Mohandas, Anu; Krishnan, Amit G.; Biswas, Raja; Menon, Deepthy, E-mail: deepthymenon@aims.amrita.edu; Nair, Manitha B., E-mail: manithanair@aims.amrita.edu

    2017-06-01

    Nanosurface modification of Titanium (Ti) implants and prosthesis is proved to enhance osseointegration at the tissue–implant interface. However, many of these products lack adequate antibacterial capability, which leads to implant loosening. As a curative strategy, in this study, nanotextured Ti substrates embedded with silver nanoparticles were developed through a single step hydrothermal processing in an alkaline medium containing silver nitrate at different concentrations (15, 30 and 75 μM). Scanning electron micrographs revealed a non-periodically oriented nanoleafy structure on Ti (TNL) decorated with Ag nanoparticles (nanoAg), which was verified by XPS, XRD and EDS analysis. This TNLAg substrate proved to be mechanically stable upon nanoindentation and nanoscratch tests. Silver ions at detectable levels were released for a period of ~ 28 days only from substrates incorporating higher nanoAg content. The samples demonstrated antibacterial activity towards both Escherichia coli and Staphylococcus aureus, with a more favorable response to the former. Simultaneously, Ti substrates incorporating nanoAg at all concentrations supported the viability, proliferation and osteogenic differentiation of mesenchymal stem cells. Overall, nanoAg incorporation into surface modified Ti via a simple one-step thermochemical method is a favorable strategy for producing implants with dual characteristics of antibacterial activity and cell compatibility. - Highlights: • Nanosilver was incorporated within Ti nanoleafy topography by simple one-step thermochemical method • The nanosurface demonstrated antibacterial activity against gram positive and gram negative bacteria • The nanosurface promoted the viability, proliferation and osteogenic differentiation of mesenchymal stem cells.

  7. Effects of Incorporating Carboxymethyl Chitosan into PMMA Bone Cement Containing Methotrexate.

    Directory of Open Access Journals (Sweden)

    Bo-Ming Liu

    Full Text Available Treatment of bone metastases usually includes surgical resection with local filling of methotrexate (MTX in polymethyl methacrylate (PMMA cement. We investigated whether incorporating carboxymethyl chitosan (CMCS in MTX-PMMA cement might overcome disadvantages associated with MTX. To determine the optimal CMCS+MTX concentration to suppress the viability of cancer cells, an integrated microfluidic chip culturing highly metastatic lung cancer cells (H460 was employed. The mechanical properties, microstructure, and MTX release of (CMCS+MTX-PMMA cement were evaluated respectively by universal mechanical testing machine, scanning electron microscopy (SEM, and incubation in simulated body fluid with subsequent HPLC-MS. Implants of MTX-PMMA and (CMCS+MTX-PMMA cement were evaluated in vivo in guinea pig femurs over time using spiral computed tomography with three-dimensional image reconstruction, and SEM at 6 months. Viability of H460 cells was significantly lowest after treatment with 57 μg/mL CMCS + 21 μg/mL MTX, which was thus used in subsequent experiments. Incorporation of 1.6% (w/w CMCS to MTX-PMMA significantly increased the bending modulus, bending strength, and compressive strength by 5, 2.8, and 5.2%, respectively, confirmed by improved microstructural homogeneity. Incorporation of CMCS delayed the time-to-plateau of MTX release by 2 days, but increased the fraction released at the plateau from 3.24% (MTX-PMMA to 5.34%. Relative to the controls, the (CMCS+MTX-PMMA implants integrated better with the host bone. SEM revealed pores in the cement of the (CMCS+MTX-PMMA implants that were not obvious in the controls. In conclusion, incorporation of CMCS in MTX-PMMA appears a feasible and effective modification for improving the anti-tumor properties of MTX-PMMA cement.

  8. Should mediastinoscopy actually be incorporated into the FDG PET strategy for patients with non-small cell lung carcinoma?

    International Nuclear Information System (INIS)

    Hayashi, Katsumi; Abe, Katsumi; Yano, Fuzuki; Watanabe, Sadahiro; Iwasaki, Yoshie; Kosuda, Shigeru

    2005-01-01

    Incorporating mediastinoscopy (MS) into the PET-based strategy for non-small cell lung carcinoma (NSCLC) patients might be cost-effective because MS can allow unnecessary thoracotomies to be avoided. The objective of our study was to assess the cost-effectiveness of incorporating MS into a PET strategy for NSCLC patients. To determine life expectancy (LE), quality adjusted life years (QALY), and the incremental cost-effectiveness ratio (ICER), a decision-tree sensitivity analysis was designed for histopathologically confirmed NSCLC patients with M0 disease, based on the three competing strategies of chest CT only vs. PET+CT vs. PET+CT+MS. A simulation of 1,000 NSCLC patients was created using baselines of other relevant variables in regard to sensitivity, specificity, mortality, LE, utilities and cost from published data. One-way sensitivity analyses were performed to determine the influences of mediastinal metastasis prevalence on LE, QALY and ICER. The LE and QALY per patient in the CT only strategy, PET+CT strategy and PET+CT+MS strategy were 4.79 and 4.35, 5.33 and 4.93 and 5.68 and 5.33 years, respectively, with a 20% prevalence of mediastinal metastasis. The ICERs were 906.6 yen x 10 3 (US$7,555)/QALY/patient at a 20% mediastinal metastasis prevalence, and 2,194 yen x 10 3 (US$18,282)/QALY/patient at a 50% prevalence, but exceeded 5,280 yen x 10 3 (US$44,000)/QALY/patient at 80%. Our study quantitatively showed the CT+PET+MS strategy in place of the PET+CT strategy in managing NSCLC patients to be cost-effective. MS should be incorporated into the PET+CT strategy for NSCLC patients except in those highly suspected of having mediastinal disease on chest CT or PET. (author)

  9. Colloidal graphene quantum dots incorporated with a Cobalt electrolyte in a dye sensitized solar cell

    Science.gov (United States)

    Lim, Hyuna

    The utilization of sun light as a renewable energy source has been pursued for a long time, but the ultimate goal of developing inexpensive and highly efficient photovoltaic devices remains elusive. To address this problem, colloidal graphene quantum dots (GQDs) were synthesized and used as a new sensitizer in dye sensitized solar cells (DSCs). Not only do the GQDs have a well-defined structure, but their large absorptivity, tunable bandgap, and size- and functional group-dependent redox potentials make them promising candidates for photovoltaic applications. Because volatile organic solvents in electrolyte solutions hinder long-term use and mass production of DSC devices, imidazolium based ionic liquids (ILs) were investigated. Cobalt-bipyridine complexes were successfully synthesized and characterized for use as new redox shuttles in DSCs. In the tested DSCs, J-V (current density-voltage) curves illustrate that the short circuit current and fill factor decrease significantly as the active area in the TiO2 photo anode increases. Dark current measurement indicated that the diode factor is bigger than one, which is different from the conventional p-n junction type solar cells, due to the high efficiency of photoelectron injection. The variation of the diode factor in dark and in light would show various types of recombination behaviors in DSCs. The performance of the DSC stained by GQDs incorporated with the cobalt redox couple was tested, but further study to improve the efficiency and to understand photochemical reaction in the DSCs is needed.

  10. Phospholipid synthesis in the squid giant axon: incorporation of lipid precursors

    Energy Technology Data Exchange (ETDEWEB)

    Gould, R.M.; Pant, H.; Gainer, H.; Tytell, M.

    1983-05-01

    The squid giant axon and extruded axoplasm from the giant axon were used to study the capacity of axoplasm for phospholipid synthesis. Extruded axoplasm, suspended in chemically defined media, catalyzed the synthesis of phospholipids from all of the precursors tested. /sup 32/P-Labeled inorganic phosphate and gamma-labeled ATP were actively incorporated into phosphatidylinositol phosphate, while (2-/sup 3/H)myo-inositol and L-(/sup 3/H(G))serine were actively incorporated into phosphatidylinositol and phosphatidylserine, respectively. Though less well utilized. (2-/sup 3/H)glycerol was incorporated into phosphatidic acid, phosphatidylinositol, and triglyceride, and methyl-3H)choline and (1-/sup 3/H)ethanolamine were incorporated into phosphatidylcholine and phosphatidylethanolamine, respectively. Isolated squid giant axons were incubated in artificial seawater containing the above precursors. The axoplasm was extruded following the incubations. Although most of the product lipids were recovered in the sheath (composed of cortical axoplasm, axolemma, and surrounding satellite cells), significant amounts (4-20%) were present in the extruded axoplasm. With tritiated choline and myo-inositol, the major labeled phospholipids found in both the extruded axoplasm and the sheath were phosphatidylcholine and phosphatidylinositol, respectively. With both glycerol and phosphate, phosphatidylethanolamine was a major labeled lipid in both axoplasm and sheath. These findings demonstrate that all classes of phospholipids are formed by endogenous synthetic enzymes in axoplasm. In addition, we feel that the different patterns of incorporation by intact axons and extruded axoplasm indicate that surrounding sheath cells contribute lipids to axoplasm. A comprehensive picture of axonal lipid metabolism should include axoplasmic synthesis and glial-axon transfer as pathways complementing the axonal transport of perikaryally formed lipids.

  11. Comparative study of human-induced pluripotent stem cells derived from bone marrow cells, hair keratinocytes, and skin fibroblasts.

    Science.gov (United States)

    Streckfuss-Bömeke, Katrin; Wolf, Frieder; Azizian, Azadeh; Stauske, Michael; Tiburcy, Malte; Wagner, Stefan; Hübscher, Daniela; Dressel, Ralf; Chen, Simin; Jende, Jörg; Wulf, Gerald; Lorenz, Verena; Schön, Michael P; Maier, Lars S; Zimmermann, Wolfram H; Hasenfuss, Gerd; Guan, Kaomei

    2013-09-01

    Induced pluripotent stem cells (iPSCs) provide a unique opportunity for the generation of patient-specific cells for use in disease modelling, drug screening, and regenerative medicine. The aim of this study was to compare human-induced pluripotent stem cells (hiPSCs) derived from different somatic cell sources regarding their generation efficiency and cardiac differentiation potential, and functionalities of cardiomyocytes. We generated hiPSCs from hair keratinocytes, bone marrow mesenchymal stem cells (MSCs), and skin fibroblasts by using two different virus systems. We show that MSCs and fibroblasts are more easily reprogrammed than keratinocytes. This corresponds to higher methylation levels of minimal promoter regions of the OCT4 and NANOG genes in keratinocytes than in MSCs and fibroblasts. The success rate and reprogramming efficiency was significantly higher by using the STEMCCA system than the OSNL system. All analysed hiPSCs are pluripotent and show phenotypical characteristics similar to human embryonic stem cells. We studied the cardiac differentiation efficiency of generated hiPSC lines (n = 24) and found that MSC-derived hiPSCs exhibited a significantly higher efficiency to spontaneously differentiate into beating cardiomyocytes when compared with keratinocyte-, and fibroblast-derived hiPSCs. There was no significant difference in the functionalities of the cardiomyocytes derived from hiPSCs with different origins, showing the presence of pacemaker-, atrial-, ventricular- and Purkinje-like cardiomyocytes, and exhibiting rhythmic Ca2+ transients and Ca2+ sparks in hiPSC-derived cardiomyocytes. Furthermore, spontaneously and synchronously beating and force-developing engineered heart tissues were generated. Human-induced pluripotent stem cells can be reprogrammed from all three somatic cell types, but with different efficiency. All analysed iPSCs can differentiate into cardiomyocytes, and the functionalities of cardiomyocytes derived from different cell

  12. Cell cycle synchronization and BrdU incorporation as a tool to study the possible selective elimination of ErbB1 gene in the micronuclei in A549 cells

    International Nuclear Information System (INIS)

    Lauand, C.; Niero, E.L.; Dias, V.M.; Machado-Santelli, G.M.

    2015-01-01

    Lung cancer often exhibits molecular changes, such as the overexpression of the ErbB1 gene that encodes epidermal growth factor receptor (EGFR). ErbB1 amplification and mutation are associated with tumor aggressiveness and low response to therapy. The aim of the present study was to design a schedule to synchronize the cell cycle of A549 cell line (a non-small cell lung cancer) and to analyze the possible association between the micronuclei (MNs) and the extrusion of ErbB1 gene extra-copies. After double blocking, by the process of fetal bovine serum deprivation and vincristine treatment, MNs formation was monitored with 5-bromo-2-deoxyuridine (BrdU) incorporation, which is an S-phase marker. Statistical analyses allowed us to infer that MNs may arise both in mitosis as well as in interphase. The MNs were able to replicate their DNA and this process seemed to be non-synchronous with the main cell nuclei. The presence of ErbB1 gene in the MNs was evaluated by fluorescent in situ hybridization (FISH). ErbB1 sequences were detected in the MNs, but a relation between the MNs formation and extrusion of amplified ErbB1could not be established. The present study sought to elucidate the meaning of MNs formation and its association with the elimination of oncogenes or other amplified sequences from the tumor cells

  13. Cell cycle synchronization and BrdU incorporation as a tool to study the possible selective elimination of ErbB1 gene in the micronuclei in A549 cells

    Energy Technology Data Exchange (ETDEWEB)

    Lauand, C.; Niero, E.L.; Dias, V.M.; Machado-Santelli, G.M. [Departamento de Biologia Celular e do Desenvolvimento, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, SP (Brazil)

    2015-03-06

    Lung cancer often exhibits molecular changes, such as the overexpression of the ErbB1 gene that encodes epidermal growth factor receptor (EGFR). ErbB1 amplification and mutation are associated with tumor aggressiveness and low response to therapy. The aim of the present study was to design a schedule to synchronize the cell cycle of A549 cell line (a non-small cell lung cancer) and to analyze the possible association between the micronuclei (MNs) and the extrusion of ErbB1 gene extra-copies. After double blocking, by the process of fetal bovine serum deprivation and vincristine treatment, MNs formation was monitored with 5-bromo-2-deoxyuridine (BrdU) incorporation, which is an S-phase marker. Statistical analyses allowed us to infer that MNs may arise both in mitosis as well as in interphase. The MNs were able to replicate their DNA and this process seemed to be non-synchronous with the main cell nuclei. The presence of ErbB1 gene in the MNs was evaluated by fluorescent in situ hybridization (FISH). ErbB1 sequences were detected in the MNs, but a relation between the MNs formation and extrusion of amplified ErbB1could not be established. The present study sought to elucidate the meaning of MNs formation and its association with the elimination of oncogenes or other amplified sequences from the tumor cells.

  14. A single episode of neonatal seizures alters the cerebellum of immature rats

    Czech Academy of Sciences Publication Activity Database

    Lomoio, S.; Necchi, D.; Mareš, Vladislav; Scherini, E.

    2011-01-01

    Roč. 93, č. 1 (2011), s. 17-24 ISSN 0920-1211 Institutional research plan: CEZ:AV0Z50110509 Keywords : metrazol seizures * cerebellum * Purkinje cells * GluR2/3 * GLT1 Subject RIV: FH - Neurology Impact factor: 2.290, year: 2011

  15. m-AAA proteases, mitochondrial calcium homeostasis and neurodegeneration.

    Science.gov (United States)

    Patron, Maria; Sprenger, Hans-Georg; Langer, Thomas

    2018-03-01

    The function of mitochondria depends on ubiquitously expressed and evolutionary conserved m-AAA proteases in the inner membrane. These ATP-dependent peptidases form hexameric complexes built up of homologous subunits. AFG3L2 subunits assemble either into homo-oligomeric isoenzymes or with SPG7 (paraplegin) subunits into hetero-oligomeric proteolytic complexes. Mutations in AFG3L2 are associated with dominant spinocerebellar ataxia (SCA28) characterized by the loss of Purkinje cells, whereas mutations in SPG7 cause a recessive form of hereditary spastic paraplegia (HSP7) with motor neurons of the cortico-spinal tract being predominantly affected. Pleiotropic functions have been assigned to m-AAA proteases, which act as quality control and regulatory enzymes in mitochondria. Loss of m-AAA proteases affects mitochondrial protein synthesis and respiration and leads to mitochondrial fragmentation and deficiencies in the axonal transport of mitochondria. Moreover m-AAA proteases regulate the assembly of the mitochondrial calcium uniporter (MCU) complex. Impaired degradation of the MCU subunit EMRE in AFG3L2-deficient mitochondria results in the formation of deregulated MCU complexes, increased mitochondrial calcium uptake and increased vulnerability of neurons for calcium-induced cell death. A reduction of calcium influx into the cytosol of Purkinje cells rescues ataxia in an AFG3L2-deficient mouse model. In this review, we discuss the relationship between the m-AAA protease and mitochondrial calcium homeostasis and its relevance for neurodegeneration and describe a novel mouse model lacking MCU specifically in Purkinje cells. Our results pledge for a novel view on m-AAA proteases that integrates their pleiotropic functions in mitochondria to explain the pathogenesis of associated neurodegenerative disorders.

  16. Changes in the rat lung after exposure to radon and its progeny: Effects on incorporation of bromodeoxyuridine in epithelial cells and on the incidence of nuclear aberrations in Alveolar macrophages

    International Nuclear Information System (INIS)

    Taya, A.; Morgan, A.; Baker, S.T.; Humphreys, J.A.H.; Collier, C.G.; Bisson, M.

    1994-01-01

    The aim of this study was to investigate some responses of cells in the rat respiratory tract as a function of time after inhalation exposure to various levels of radon and its progeny. Rats were exposed to a constant concentration of radon and its progeny to give cumulative exposure levels of 120, 225, 440 and 990 working level months (WLM). An additional unexposed group of rats served as controls. The end points selected for investigation were (a) the incorporation of bromodeoxyuridine (BrdU) in epithelial cells of the conducting airways and of the alveolar region of the respiratory tract and (b) the incidence of alveolar macrophages with nuclear aberrations. After exposure, the incidence of epithelial cells incorporating BrdU-the labeling index-increased in all regions of the respiratory tract examined, but the increase occurred later in alveolar than in airway epithelial cells. The highest labeling index was found in bronchial epithelial cells, which probably received the highest radiation dose. After an initial induction period, the incidence of alveolar macrophages with nuclear aberrations also increased. The possibility of using the labeling index of alveolar and airway epithelial cells, and/or the incidence of nuclear aberrations in alveolar macrophages, to estimate the radiation dose to various regions of the respiratory tract after exposure of rats to radon and its progeny is discussed. 22 refs., 3 figs., 1 tab

  17. The C1q complement family of synaptic organizers: not just complementary.

    Science.gov (United States)

    Yuzaki, Michisuke

    2017-08-01

    Molecules that regulate formation, differentiation, and maintenance of synapses are called synaptic organizers. Recently, various 'C1q family' proteins have been shown to be released from neurons, and serve as a new class of synaptic organizers. Cbln1 and C1ql1 proteins regulate the formation and maintenance of parallel fiber-Purkinje cell and climbing fiber-Purkinje cell synapses, respectively, in the cerebellum. Cbln1 also modulates the function of postsynaptic delta2 glutamate receptors to regulate synaptic plasticity. C1ql2 and C1ql3, released from mossy fibers, determine the synaptic localization of postsynaptic kainate receptors in the hippocampus. C1ql3 also regulates the formation of synapses between the basolateral amygdala and the prefrontal cortex. These findings indicate the diverse functions of C1q family proteins in various brain regions. Copyright © 2017 Elsevier Ltd. All rights reserved.

  18. Impaired Cerebellar Maturation, Growth Restriction, and Circulating Insulin-Like Growth Factor 1 in Preterm Rabbit Pups

    Science.gov (United States)

    Sveinsdóttir, Kristbjörg; Länsberg, John-Kalle; Sveinsdóttir, Snjólaug; Garwicz, Martin; Ohlsson, Lennart; Hellström, Ann; Smith, Lois; Gram, Magnus; Ley, David

    2018-01-01

    Cerebellar growth is impeded following very preterm birth in human infants and the observed reduction in cerebellar volume is associated with neurodevelopmental impairment. Decreased levels of circulating insulin-like growth factor 1 (IGF-1) are associated with decreased cerebellar volume. The relationship between preterm birth, circulating IGF-1, and key cell populations supporting cerebellar proliferation is unknown. The aim of this study was to evaluate the effect of preterm birth on postnatal growth, circulating IGF-1, and cerebellar maturation in a preterm rabbit pup model. Preterm rabbit pups (PT) were delivered by cesarean section at day 29 of gestation, cared for in closed incubators with humidified air, and gavage fed with formula. Control term pups (T) delivered by spontaneous vaginal delivery at day 32 of gestation were housed and fed by their lactating doe. In vivo perfusion-fixation for immunohistochemical evaluation of cerebellar proliferation, cell maturation, and apoptosis was performed at repeated time points in PT and T pups. Results show that the mean weight of the pups and circulating IGF-1 protein levels were lower in the PT group at all time points (p staining at P0 (p = 0.003), P2 (p = 0.004), and P5 (p = 0.04) in the PT group compared to in the T group. Staining for sonic hedgehog was positive in neuronal EGL progenitors and Purkinje cells at early time points but was restricted to a well-defined Purkinje cell monolayer at later time points. Preterm birth in rabbit pups is associated with lower circulating levels of IGF-1, decreased postnatal growth, and decreased cerebellar EGL proliferation and Purkinje cell maturation. The preterm rabbit pup model exhibits important characteristics of human preterm birth, and may thus be suitable for the evaluation of interventions aiming to modify growth and cerebellar development in the preterm population. PMID:28972955

  19. Cerebellum neurotransmission during postnatal development: [Pt(O,O'-acac)(γ-acac)(DMS)] vs cisplatin and neurotoxicity.

    Science.gov (United States)

    Piccolini, Valeria Maria; Esposito, Alessandra; Dal Bo, Veronica; Insolia, Violetta; Bottone, Maria Grazia; De Pascali, Sandra Angelica; Fanizzi, Francesco Paolo; Bernocchi, Graziella

    2015-02-01

    Several chemotherapeutic drugs are known to cause neurotoxicity. Platinum-based agents in use or in clinical trials display neurotoxic potential accompanied by neurological complications; recent studies have identified a large number of behavioural issues in paediatric oncology patients. To understand the toxicity of platinum drugs at the molecular and cellular levels, this study compares the possible cytotoxic effects of an older platinum compound, cisplatin and a new platinum compound, [Pt(O,O'-acac)(γ-acac)(DMS)], on the CNS of postnatally developing rats, which is much more vulnerable to injury than the CNS of adult rats. Since several drugs interact with neurotransmitters during neuronal maturation, we performed immunostainings with antibodies raised against markers of glutamate and GABA, the major neurotransmitters in the cerebellum. After a single injection of cisplatin at postnatal day 10 (PD10), the labelling of Purkinje cells with the neurotransmitter markers evidenced alterations between PD11 and PD30, i.e. atrophy of the dendrite tree, changes in the distribution of synaptic contacts of parallel and climbing fibres, delay in the elimination of transient synapses on cell soma and severely impaired pinceau formation at the axon hillock. After treatment with [Pt(O,O'-acac)(γ-acac)(DMS)], the sole relevant change concerned the timing of climbing fibres elimination; the transient synapses disappearance on the Purkinje cell soma was delayed in some cells; instead, the growth of Purkinje cell dendrite tree was normal as was the formation of inhibitory synaptic contacts on these neurons. These findings add new evidence not only on the lower neurotoxicity of [Pt(O,O'-acac)(γ-acac)(DMS)] vs cisplatin but also on the involvement of neurotransmitters and relative synaptic connections in the maturation of central nerve tissue. Copyright © 2014 ISDN. Published by Elsevier Ltd. All rights reserved.

  20. Autoradiographic demonstration of 3H-estradiol and 3H-cholesterol incorporation in hamster gonads

    International Nuclear Information System (INIS)

    Angelova, P.; Martinova, J.; Kyncheva, L.; Baleva-Ivanova, K.

    1989-01-01

    Male and female hamster gonads were investigated on day 14 of pregnancy, at birth, on days 7, 18 and 25 after birth and at sexual maturity. [2,4,6,7 3 H]-estradiol -17β, specific activity 110 Ci.mmol -1 and [1α, 2α - 3 H] - cholesterol specific activity 44 Ci.mmol -1 have been used for labelling. On embrional day 14 the histological image has been similar to that in the neonatal gonads - diffusive labelling includding germ, satellite and Leyding cells in fetal ovaries and testes. On the 7th postnatal day in the ovary a formation of primary follicles began in the deeper layers of gonads and an incorporation of the labelled substances in the germ and prefollicular cells in both ovary and testis have been observed. On the 18th postnatal day growing follicles have been seen in the ovary and labelling have been noticed in the oocytes and follicular cells. In the prepubertal testis the meiolic process has started, spermatocytes have been found and an incorporation of the radioactive substances in germ, Sertoli and Leydig cells has been established. In the ovaries of both 25th day old hamsters and adult animals multi-layered and preovulatory follicles have been seen. Sertoli cells, spermatogonia, spermatocytes and spertamids in the seminiferons tubules have been observed. The incorporation of 3 H-estradiol and 3 H cholesterol in both germ and Sertoli cells has been found. A presence has been observed of specific estradiol receptors in all three main cell types of fetal and developing gonads: germ, satellite and intertitial cells. The presence of estradiol receptors in developing hamster gonads has indicated a participation of steroids in the process of development and differentiation of male and female gonads

  1. Syntheses and characterization of liposome-incorporated adamantyl aminoguanidines.

    Science.gov (United States)

    Šekutor, Marina; Štimac, Adela; Mlinarić-Majerski, Kata; Frkanec, Ruža

    2014-08-21

    A series of mono and bis-aminoguanidinium adamantane derivatives has been synthesized and incorporated into liposomes. They combine two biomedically significant molecules, the adamantane moiety and the guanidinium group. The adamantane moiety possesses the membrane compatible features while the cationic guanidinium subunit was recognized as a favourable structural feature for binding to complementary molecules comprising phosphate groups. The liposome formulations of adamantyl aminoguanidines were characterized and it was shown that the entrapment efficiency of the examined compounds is significant. In addition, it was demonstrated that liposomes with incorporated adamantyl aminoguanidines effectively recognized the complementary liposomes via the phosphate group. These results indicate that adamantane derivatives bearing guanidinium groups might be versatile tools for biomedical application, from studies of molecular recognition processes to usage in drug formulation and cell targeting.

  2. Synbiotic yogurt-ice cream produced via incorporation of microencapsulated lactobacillus acidophilus (la-5) and fructooligosaccharide.

    Science.gov (United States)

    Ahmadi, Abbas; Milani, Elnaz; Madadlou, Ashkan; Mortazavi, Seyed Ali; Mokarram, Reza Rezaei; Salarbashi, Davoud

    2014-08-01

    Yogurt-ice cream is a nutritious product with a refreshing taste and durability profoundly longer than that of yogurt. The probiotic Lactobacillus acidophilus (La-5) cells either in free or encapsulated form were incorporated into yog-ice cream and their survivability were studied. Fructooligosaccharide (FOS) as a prebiotic compound at three levels (0, 4 & 8 % w/w) was added to yogurt-ice cream mix and its effects on some chemical properties, overrun and firmness of product were evaluated. The higher the incorporated FOS concentration, the lower were the pH value and higher the total solid content of treatments. FOS incorporation (8 %) significantly increased the overrun of treatments and reduced their firmness. The viable counts of free probiotics decreased from ~9.55 to ~7.3 log cfu/g after 60 days of frozen storage while that of encapsulated cells merely decreased less than 1 log cycle. Encapsulation with alginate microbeads protected the probiotic cells against injuries in the freezing stage as well as, during frozen storage.

  3. Enhancing the performance of dye-sensitized solar cells by incorporating nanosilicate platelets in gel electrolyte

    KAUST Repository

    Lai, Yi-Hsuan; Chiu, Chih-Wei; Chen, Jian-Ging; Wang, Chun-Chieh; Lin, Jiang-Jen; Lin, King-Fu; Ho, Kuo-Chuan

    2009-01-01

    Two kinds of gel-type dye-sensitized solar cells (DSSCs), composed of two types of electrolytes, were constructed and the respective cell performance was evaluated in this study. One electrolyte, TEOS-Triton X-100 gel, was based on a hybrid organic/inorganic gel electrolyte made by the sol-gel method and the other was based on poly(vinyidene fluoride-co-hexafluoro propylene) (PVDF-HFP) copolymer. TEOS-Triton X-100 gel was based on the reticulate structure of silica, formed by hydrolysis, and condensation of tetraethoxysilane (TEOS), while its organic subphase was a mixture of surfactant (Triton X-100) and ionic liquid electrolytes. Both DSSC gel-type electrolytes were composed of iodine, 1-propy-3-methyl-imidazolium iodide, and 3-methoxypropionitrile to create the redox couple of I3 -/I-. Based on the results obtained from the I-V characteristics, it was found that the optimal iodine concentrations for the TEOS-Triton X-100 gel electrolyte and PVDF-HFP gel electrolyte are 0.05 M and 0.1 M, respectively. Although the increase in the iodine concentration could enhance the short-circuit current density (JSC), a further increase in the iodine concentration would reduce the JSC due to increased dark current. Therefore, the concentration of I2 is a significant factor in determining the performance of DSSCs. In order to enhance cell performance, the addition of nanosilicate platelets (NSPs) in the above-mentioned gel electrolytes was investigated. By incorporating NSP-Triton X-100 into the electrolytes, the JSC of the cells increased due to the decrease of diffusion resistance, while the open circuit voltage (VOC) remained almost the same. As the loading of the NSP-Triton X-100 in the TEOS-Triton X-100 gel electrolyte increased to 0.5 wt%, the JSC and the conversion efficiency increased from 8.5 to 12 mA/cm2 and from 3.6% to 4.7%, respectively. However, the JSC decreased as the loading of NSP-Triton X-100 exceeded 0.5 wt%. At higher NSP-Triton X-100 loading, NSPs acted as

  4. In Vivo Evidence for Lysosome Depletion and Impaired Autophagic Clearance in Hereditary Spastic Paraplegia Type SPG11.

    Directory of Open Access Journals (Sweden)

    Rita-Eva Varga

    2015-08-01

    Full Text Available Hereditary spastic paraplegia (HSP is characterized by a dying back degeneration of corticospinal axons which leads to progressive weakness and spasticity of the legs. SPG11 is the most common autosomal-recessive form of HSPs and is caused by mutations in SPG11. A recent in vitro study suggested that Spatacsin, the respective gene product, is needed for the recycling of lysosomes from autolysosomes, a process known as autophagic lysosome reformation. The relevance of this observation for hereditary spastic paraplegia, however, has remained unclear. Here, we report that disruption of Spatacsin in mice indeed causes hereditary spastic paraplegia-like phenotypes with loss of cortical neurons and Purkinje cells. Degenerating neurons accumulate autofluorescent material, which stains for the lysosomal protein Lamp1 and for p62, a marker of substrate destined to be degraded by autophagy, and hence appears to be related to autolysosomes. Supporting a more generalized defect of autophagy, levels of lipidated LC3 are increased in Spatacsin knockout mouse embryonic fibrobasts (MEFs. Though distinct parameters of lysosomal function like processing of cathepsin D and lysosomal pH are preserved, lysosome numbers are reduced in knockout MEFs and the recovery of lysosomes during sustained starvation impaired consistent with a defect of autophagic lysosome reformation. Because lysosomes are reduced in cortical neurons and Purkinje cells in vivo, we propose that the decreased number of lysosomes available for fusion with autophagosomes impairs autolysosomal clearance, results in the accumulation of undegraded material and finally causes death of particularly sensitive neurons like cortical motoneurons and Purkinje cells in knockout mice.

  5. Disruption of the LTD dialogue between the cerebellum and the cortex in Angelman syndrome model: a timing hypothesis

    Directory of Open Access Journals (Sweden)

    Guy eCheron

    2014-11-01

    Full Text Available Angelman syndrome is a genetic neurodevelopmental disorder in which cerebellar functioning impairment has been documented despite the absence of gross structural abnormalities. Characteristically, a spontaneous 160 Hz oscillation emerges in the Purkinje cells network of the Ube3am-/p+ Angelman mouse model. This abnormal oscillation is induced by enhanced Purkinje cell rhythmicity and hypersynchrony along the parallel fiber beam. We present a pathophysiological hypothesis for the neurophysiology underlying major aspects of the clinical phenotype of Angelman syndrome, including cognitive, language and motor deficits, involving long-range connection between the cerebellar and the cortical networks. This hypothesis states that the alteration of the cerebellar rhythmic activity impinges cerebellar long-term depression (LTD plasticity, which in turn alters the LTD plasticity in the cerebral cortex. This hypothesis was based on preliminary experiments using electrical stimulation of the whiskers pad performed in alert mice showing that after a 8 Hz LTD-inducing protocol, the cerebellar LTD accompanied by a delayed response in the wild type mice is missing in Ube3am-/p+ mice and that the LTD induced in the barrel cortex following the same peripheral stimulation in wild mice is reversed into a LTP in the Ube3am-/p+ mice. The control exerted by the cerebellum on the excitation vs inhibition balance in the cerebral cortex and possible role played by the timing plasticity of the Purkinje cell LTD on the spike–timing dependent plasticity (STDP of the pyramidal neurons are discussed in the context of the present hypothesis.

  6. Effects of perinatal hypo- and hyperthyroidism on the levels of nerve growth factor and its low-affinity receptor in cerebellum.

    Science.gov (United States)

    Figueiredo, B C; Otten, U; Strauss, S; Volk, B; Maysinger, D

    1993-04-16

    Deficits or excesses of thyroid hormones during critical periods of mammalian cerebellar development can lead to profound biochemical and morphological abnormalities in this system. The goal of this study was to investigate the effects of perinatal hypo- and hyperthyroidism on the ontogeny of nerve growth factor (NGF) and its low-affinity receptor (p75NGFR) in the rat cerebellum. The concentration of NGF and of p75NGFR immunoreactivity (IR) were measured, several days after birth, in cerebella of rats which had received propylthiouracil (PTU) or thyroxine. NGF concentration was markedly enhanced only on postnatal day 2 (P2) in hyperthyroid rats, whereas in hypothyroid (PTU-treated) rats NGF values were similar to age-matched controls. These observations suggest that thyroid hormone affects NGF synthesis during early periods of cerebellar development. In Purkinje cells of control animals, p75NGFR IR peaked at P10. In hypothyroid rats, the expression of p75NGFR was retarded, peaking at P15, whereas in hyperthyroid rats it was advanced, peaking at P8. The increased p75NGFR IR found in Purkinje cell bodies and the delayed disappearance of p75NGFR IR from the external granular layer of hypothyroid rats suggest different roles for thyroid hormone in the developing cerebellum. We conclude that p75NGFR and NGF are independently regulated by thyroid hormone during critical periods of cerebellar development. The effect of thyroid hormone deficiency on p75NGFR content in Purkinje cells may involve complex mechanisms such as impaired efficiency of axonal transport.

  7. P(MMA-EMA Random Copolymer Electrolytes Incorporating Sodium Iodide for Potential Application in a Dye-Sensitized Solar Cell

    Directory of Open Access Journals (Sweden)

    Nurul Akmaliah Dzulkurnain

    2015-02-01

    Full Text Available Polymer electrolytes based on 90 wt% of methyl methacrylate and 10 wt% of ethyl methacrylate (90MMA-co-10EMA incorporating different weight ratios of sodium iodide were prepared using the solution casting method. The complexation between salt and copolymer host has been investigated using Fourier transform infrared spectroscopy. The ionic conductivity and thermal stability of the electrolytes were measured using impedance spectroscopy and differential scanning calorimetry, respectively. Scanning electron microscopy was used to study the morphology of the polymer electrolytes. The ionic conductivity and glass transition temperature increased up to 20 wt% of sodium iodide (5.19 × 10−6 S·cm−1 and decreased with the further addition of salt concentration, because of the crosslinked effect. The morphology behavior of the highest conducting sample also showed smaller pores compared to the other concentration. The total ionic transference number proved that this system was mainly due to ions, and the electrochemical stability window was up to 2.5 V, which is suitable for a dye-sensitized solar cell application. This sample was then tested in a dye-sensitized solar cell and exhibited an efficiency of 0.62%.

  8. Rapid, High-Throughput, and Direct Molecular Beacon Delivery to Human Cancer Cells Using a Nanowire-Incorporated and Pneumatic Pressure-Driven Microdevice.

    Science.gov (United States)

    Kim, Kyung Hoon; Kim, Jung; Choi, Jong Seob; Bae, Sunwoong; Kwon, Donguk; Park, Inkyu; Kim, Do Hyun; Seo, Tae Seok

    2015-12-01

    Tracking and monitoring the intracellular behavior of mRNA is of paramount importance for understanding real-time gene expression in cell biology. To detect specific mRNA sequences, molecular beacons (MBs) have been widely employed as sensing probes. Although numerous strategies for MB delivery into the target cells have been reported, many issues such as the cytotoxicity of the carriers, dependence on the random probability of MB transfer, and critical cellular damage still need to be overcome. Herein, we have developed a nanowire-incorporated and pneumatic pressure-driven microdevice for rapid, high-throughput, and direct MB delivery to human breast cancer MCF-7 cells to monitor survivin mRNA expression. The proposed microdevice is composed of three layers: a pump-associated glass manifold layer, a monolithic polydimethylsiloxane (PDMS) membrane, and a ZnO nanowire-patterned microchannel layer. The MB is immobilized on the ZnO nanowires by disulfide bonding, and the glass manifold and PDMS membrane serve as a microvalve, so that the cellular attachment and detachment on the MB-coated nanowire array can be manipulated. The combination of the nanowire-mediated MB delivery and the microvalve function enable the transfer of MB into the cells in a controllable way with high cell viability and to detect survivin mRNA expression quantitatively after docetaxel treatment. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. The conducting tissue in the adult chicken atria. A histological and immunohistochemical analysis

    NARCIS (Netherlands)

    de Groot, I. J.; Hardy, G. P.; Sanders, E.; Los, J. A.; Moorman, A. F.

    1985-01-01

    A three-dimensional reconstruction from serial sections of adult chicken heart was made to verify whether Purkinje cells, that can be recognized by a number of well-known histological criteria, form specialized tracts in the adult chicken atria. This reconstruction revealed a loosely arranged

  10. A cerebellar learning model of vestibulo-ocular reflex adaptation in wild-type and mutant mice

    NARCIS (Netherlands)

    Clopath, Claudia; Badura, Aleksandra; De Zeeuw, Chris I; Brunel, Nicolas

    2014-01-01

    Mechanisms of cerebellar motor learning are still poorly understood. The standard Marr-Albus-Ito theory posits that learning involves plasticity at the parallel fiber to Purkinje cell synapses under control of the climbing fiber input, which provides an error signal as in classical supervised

  11. Switch in the expression of mGlu1 and mGlu5 metabotropic glutamate receptors in the cerebellum of mice developing experimental autoimmune encephalomyelitis and in autoptic cerebellar samples from patients with multiple sclerosis

    NARCIS (Netherlands)

    Fazio, F.; Notartomaso, S.; Aronica, E.; Storto, M.; Battaglia, G.; Vieira, E.; Gatti, S.; Bruno, V.; Biagioni, F.; Gradini, R.; Nicoletti, F.; Di Marco, R.

    2008-01-01

    Recent evidence suggests that changes in the expression of membrane receptors/ion channels in cerebellar Purkinje cells contribute to the onset of cerebellar motor symptoms in patients with multiple sclerosis (MS). We examined the expression of group-I metabotropic glutamate receptors (mGlu1 and

  12. An Investigation into the Effects of Peptide Neurotransmitters and Intracellular Second Messengers in Rat Central Neurons in Culture.

    Science.gov (United States)

    1988-02-04

    Purkinje neurons. 3. Neuromodulation of synaptic efficacy in an invertebrate preparation that may be a useful model system for the actions of histamine in...neurotransmitters, neuromodulators , affect brain function. Nerve cells are the functional units of the brain, and changes in neuronal activity are ultimately

  13. Behavioral Analysis and Rescue of a Novel Cerebellar Mouse Model of Tuberous Sclerosis Complex

    Science.gov (United States)

    2012-05-01

    including: Purkinje cell loss, general cerebellar hypoplasia, vermal hypoplasia and hyperplasia, reduced gray matter, GABA dysfunction, and decreased...Lond B Biol Sci. 287, 167-201. Cappon, D., 1953. Clinical manifestations of autism and schizophrenia in childhood. Can Med Assoc J. 69, 44-9. Chan

  14. Intrinsic electrical properties of mammalian neurons and CNS function: a historical perspective

    OpenAIRE

    Llinás, Rodolfo R.

    2014-01-01

    This brief review summarizes work done in mammalian neuroscience concerning the intrinsic electrophysiological properties of four neuronal types; Cerebellar Purkinje cells, inferior olivary cells, thalamic cells, and some cortical interneurons. It is a personal perspective addressing an interesting time in neuroscience when the reflex view of brain function, as the paradigm to understand global neuroscience, began to be modified towards one in which sensory input modulates rather than dictate...

  15. Determination of rumen microbial growth in vitro form 32P-labelled phosphate incorporation

    International Nuclear Information System (INIS)

    Nevel, C.J. Van; Demeyer, D.I.

    1977-01-01

    The extracellular phosphate pool in incubations of rumen fluid or washed cell suspensions of mixed rumen bacteria (WCS) was labelled with 32 P. From the constant extracellular phosphate pool specific activity and the amount of radioactivity incorporated during incubation, the amount of P incorporated in the microbial fraction was calculated. From the value for nitrogen: P determined in microbial matter, the amount of N incorporated was calculated as a measure of microbial growth. Incorporation of soluble non-protein-N in incubations devoid of substrate protein was 50 and 80% of the values obtained using isotope method for rumen fluid and WCS respectively. Incorporation of 32 P in P-containing microbial components (mainly nucleic acids) was compared with net synthesis of these components in incubations of WCS. When N incorporation, calculated from results obtained using isotope method in incubations with rumen fluid, was compared with the amount of carbohydrate substrate fermented and the type of fermentation, values between 18.3 and 44.6 g N incorporated kg of organic matter fermented were obtained. The use of isotopes for determination of rumen microbial growth in vitro is critically discussed. (author)

  16. Method Development for Efficient Incorporation of Unnatural Amino Acids

    KAUST Repository

    Harris, Paul D.

    2014-04-01

    The synthesis of proteins bearing unnatural amino acids has the potential to enhance and elucidate many processes in biochemistry and molecular biology. There are two primary methods for site specific unnatural amino acid incorporation, both of which use the cell’s native protein translating machinery: in vitro chemical acylation of suppressor tRNAs and the use of orthogonal amino acyl tRNA synthetases. Total chemical synthesis is theoretically possible, but current methods severely limit the maximum size of the product protein. In vivo orthogonal synthetase methods suffer from the high cost of the unnatural amino acid. In this thesis I sought to address this limitation by increasing cell density, first in shake flasks and then in a bioreactor in order to increase the yield of protein per amount of unnatural amino acid used. In a parallel project, I used the in vitro chemical acylation system to incorporate several unnatural amino acids, key among them the fluorophore BODIPYFL, with the aim of producing site specifically fluorescently labeled protein for single molecule FRET studies. I demonstrated successful incorporation of these amino acids into the trial protein GFP, although incorporation was not demonstrated in the final target, FEN1. This also served to confirm the effectiveness of a new procedure developed for chemical acylation.

  17. Method Development for Efficient Incorporation of Unnatural Amino Acids

    KAUST Repository

    Harris, Paul D.

    2014-01-01

    The synthesis of proteins bearing unnatural amino acids has the potential to enhance and elucidate many processes in biochemistry and molecular biology. There are two primary methods for site specific unnatural amino acid incorporation, both of which use the cell’s native protein translating machinery: in vitro chemical acylation of suppressor tRNAs and the use of orthogonal amino acyl tRNA synthetases. Total chemical synthesis is theoretically possible, but current methods severely limit the maximum size of the product protein. In vivo orthogonal synthetase methods suffer from the high cost of the unnatural amino acid. In this thesis I sought to address this limitation by increasing cell density, first in shake flasks and then in a bioreactor in order to increase the yield of protein per amount of unnatural amino acid used. In a parallel project, I used the in vitro chemical acylation system to incorporate several unnatural amino acids, key among them the fluorophore BODIPYFL, with the aim of producing site specifically fluorescently labeled protein for single molecule FRET studies. I demonstrated successful incorporation of these amino acids into the trial protein GFP, although incorporation was not demonstrated in the final target, FEN1. This also served to confirm the effectiveness of a new procedure developed for chemical acylation.

  18. Cadmium incorporation by the marine copepod Pseudodiaptomus coronatus

    International Nuclear Information System (INIS)

    Sick, L.V.; Baptist, G.J.

    1979-01-01

    Pseudodiaptomus coronatus, after exposure to phytoplankton and cadmium in concentrations like those in temperate, coastal environments, incorporated 109 Cd at higher rates from ambient water than from phytoplankton food. When ambient stable cadmium concentrations were increased from 0.03 to 1.00 μg.liter -1 , cadmium concentration by phytoplankton cells increased and the rate of cell ingestion by P. coronatus decreased. This inverse relation between the accumulation of cadmium in phytoplankton cells and the animal's ingestion rate resulted in relatively small net increases in the cadmium accumulated from phytoplankton by copepods. Rates of stable cadmium ingestion for P. coronatus ranged from 0.18 to 0.38 ng.mg animal dry wt -1 .h -1 , depending on the initial algal cell density and the ambient cadmium concentration. For cadmium concentrations of 0.03 to 1.00 μg.liter -1 , percentage assimilation efficiencies ranged from 13.20 to 68.40. Both rates of cadmium ingestion and assimilation efficiencies were higher than published values for carnivorous zooplankton

  19. Secretin Modulates the Postnatal Development of Mouse Cerebellar Cortex Via PKA- and ERK-dependent Pathways

    Directory of Open Access Journals (Sweden)

    Lei Wang

    2017-11-01

    Full Text Available Postnatal development of the cerebellum is critical for its intact function such as motor coordination and has been implicated in the pathogenesis of psychiatric disorders. We previously reported that deprivation of secretin (SCT from cerebellar Purkinje neurons impaired motor coordination and motor learning function, while leaving the potential role of SCT in cerebellar development to be determined. SCT and its receptor (SCTR were constitutively expressed in the postnatal cerebellum in a temporal and cell-specific manner. Using a SCT knockout mouse model, we provided direct evidence showing altered developmental patterns of Purkinje cells (PCs and granular cells (GCs. SCT deprivation reduced the PC density, impaired the PC dendritic formation, induced accelerated GC migration and potentiated cerebellar apoptosis. Furthermore, our results indicated the involvement of protein kinase A (PKA and extracellular signal regulated kinase (ERK signaling pathways in SCT-mediated protective effects against neuronal apoptosis. Results of this study illustrated a novel function of SCT in the postnatal development of cerebellum, emphasizing the necessary role of SCT in cerebellar-related functions.

  20. Cerebellum-from J. E. Purkyně up to Contemporary Research.

    Science.gov (United States)

    Vožeh, František

    2017-06-01

    Jan. Evangelista Purkyně, the most famous among Czech physiologists, was the first who identified and described the largest nerve cells in the cerebellum. The most distinguished researchers of the nervous system then recommended naming these neurons Purkinje cells in his honor. Through experiments by Purkinje and his followers, the function of the cerebellum was properly attributed to the precision of motor movements and skills. This traditional concept was valid until early 1990s, when it was readjusted and replenished with new and important findings. It was discovered that the cerebellar cortex contains more neurons than the cerebral cortex and shortly thereafter was gradually revealed that such enormous numbers of neural cells are not without impact on brain functions. It was shown that the cerebellum, in addition to its traditional role, also participates in higher nervous activity. These new findings were obtained thanks to the introduction of modern methods of examination into the clinical praxis, and experimental procedures using animal models of cerebellar disorders described in this work.

  1. Differential responses of neuronal and spermatogenic cells to the doppel cytotoxicity.

    Directory of Open Access Journals (Sweden)

    Kefeng Qin

    Full Text Available Although structurally and biochemically similar to the cellular prion (PrP(C, doppel (Dpl is unique in its biological functions. There are no reports about any neurodegenerative diseases induced by Dpl. However the artificial expression of Dpl in the PrP-deficient mouse brain causes ataxia with Purkinje cell death. Abundant Dpl proteins have been found in testis and depletion of the Dpl gene (Prnd causes male infertility. Therefore, we hypothesize different regulations of Prnd in the nerve and male productive systems. In this study, by electrophoretic mobility shift assays we have determined that two different sets of transcription factors are involved in regulation of the Prnd promoter in mouse neuronal N2a and GC-1 spermatogenic (spg cells, i.e., upstream stimulatory factors (USF in both cells, Brn-3 and Sp1 in GC-1 spg cells, and Sp3 in N2a cells, leading to the expression of Dpl in GC-1 spg but not in N2a cells. We have further defined that, in N2a cells, Dpl induces oxidative stress and apoptosis, which stimulate ataxia-telangiectasia mutated (ATM-modulating bindings of transcription factors, p53 and p21, to Prnp promoter, resulting the PrP(C elevation for counteraction of the Dpl cytotoxicity; in contrast, in GC-1 spg cells, phosphorylation of p21 and N-terminal truncated PrP may play roles in the control of Dpl-induced apoptosis, which may benefit the physiological function of Dpl in the male reproduction system.

  2. Exogenous incorporation of neugc-rich mucin augments n-glycolyl sialic acid content and promotes malignant phenotype in mouse tumor cell lines

    Directory of Open Access Journals (Sweden)

    Alonso Daniel F

    2009-12-01

    Full Text Available Abstract Background Carbohydrates embedded in the plasma membrane are one of the main actors involved in the communication of cells with the microenvironment. Neuraminic sialic acids are glycocalyx sugars that play important roles in the modulation of malignant cell behaviour. N-glycolylneuraminic acid (NeuGc is synthesized by the cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH, an enzyme expressed in all mammals except humans. In mice, this sugar is synthesized in several somatic tissues. Methods We used the B16 melanoma and F3II mammary carcinoma mouse tumor cell lines. By CMAH directed RT-PCR and NeuGc detection with the specific anti-NeuGc-GM3 antibody 14F7 we evaluated enzyme and ganglioside expression in tumor cells, respectively. Expression of NeuGc-GM3 ganglioside was reached by in vitro incubation with NeuGc-rich bovine submaxillary mucin and evaluated by slot-blot and immunohistochemistry assays using the 14F7 antibody. Tumor cells treated with mucin or purified NeuGc were injected s.c. and i.v. in syngeneic mice to evaluate tumor and metastatic growth. Results In the present work we demonstrated the absence of expression of CMAH enzyme in B16 melanoma and F3II mammary carcinoma cells. In vitro incubation of these NeuGc-negative cells with NeuGc-rich mucin increased the presence of NeuGc in cell membranes for at least 48-72 h, as a component of the GM3 ganglioside. Preincubation with NeuGc-rich mucin reduced tumor latency and increased the metastatic potential of tumor cells in syngeneic animals. Similar results were obtained when cells were incubated with purified NeuGc alone. Conclusion Our results indicate that B16 and F3II mouse tumor cell lines do not express NeuGc in cell membranes but they are able to incorporate NeuGc from an exogenous source, contributing to the malignant phenotype of melanoma and mammary carcinoma cells.

  3. The endozepine ODN stimulates [3H]thymidine incorporation in cultured rat astrocytes

    International Nuclear Information System (INIS)

    Gandolfo, P.; Patte, C.; Thoumas, J.L.; Leprince, J.; Vaudry, H.; Tonon, M.C.

    1999-01-01

    High concentrations of diazepam-binding inhibitor (DBI) mRNA have been detected in astrocytoma, suggesting that DBI-derived peptides may play a role in glial cell proliferation. In the present study, we have investigated the effect of a processing product of DBI, the octadecaneuropeptide ODN, on DNA synthesis in cultured rat astrocytes. At very low concentrations (10 -14 to 10 -11 M), ODN caused a dose-dependent increase of [ 3 H]thymidine incorporation. At higher doses (10 -10 to 10 -5 M), the effect of ODN gradually declined. The central-type benzodiazepine receptor antagonist flumazenil (10 -6 M) completely suppressed the stimulatory action of ODN whereas the peripheral-type benzodiazepine receptor ligand, PK11195 (10 -6 M) had no effect. The ODN-induced stimulation of [ 3 H]thymidine incorporation was mimicked by methyl 6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate (DMCM). The GABA A receptor antagonist bicuculline (10 -4 M) suppressed the effect of both ODN and DMCM on DNA synthesis. Exposure of cultured astrocytes to the specific GABA A agonist 3APS (10 -10 to 10 -4 M) also induced a dose-related increase of [ 3 H]thymidine incorporation. The present study indicates that ODN, acting through central-type benzodiazepine receptors associated with the GABA A receptor complex, stimulates DNA synthesis in rat glial cells. These data provide evidence for an autocrine role of endozepines in the control of glial cell proliferation. (Copyright (c) 1999 Elsevier Science B.V., Amsterdam. All rights reserved.)

  4. Structural incorporation of MgCl2 into ice VII at room temperature

    Science.gov (United States)

    Watanabe, Mao; Komatsu, Kazuki; Noritake, Fumiya; Kagi, Hiroyuki

    2017-05-01

    Raman spectra and X-ray diffraction patterns were obtained from 1:100 and 1:200 \\text{MgCl}2:\\text{H}2\\text{O} solutions (in molar ratio) at pressures up to 6 GPa using diamond anvil cells (DACs) and compared with those of pure water. The O-H stretching band from ice VII crystallized from the 1:200 solution was approximately 10 cm-1 higher than that of pure ice VII. The phase boundaries between ice VII and VIII crystallized from the MgCl2 solutions at 4 GPa were 2 K lower than those of pure ice VII and VIII. These observations indicate that ice VII incorporates MgCl2 into its structure. The unit cell volumes of ice VII crystallized from pure water and the two solutions coincided with each other within the experimental error, and salt incorporation was not detectable from the cell volume. Possible configurations of ion substitution and excess volume of ice VIII were simulated on the basis of density functional theory (DFT) calculations.

  5. Cerebellar Codings for Control of Compensatory Eye Movements

    NARCIS (Netherlands)

    M. Schonewille (Martijn)

    2008-01-01

    textabstractThis thesis focuses on the control of the cerebellum on motor behaviour, and more specifically on the role of the cerebellar Purkinje cells in exerting this control. As the cerebellum is an online control system, we look at both motor performance and learning, trying to identify

  6. Questioning the cerebellar doctrine

    NARCIS (Netherlands)

    Galliano, Elisa; De Zeeuw, Chris I

    2014-01-01

    The basic principles of cerebellar function were originally described by Flourens, Cajal, and Marr/Albus/Ito, and they constitute the pillars of what can be considered to be the classic cerebellar doctrine. In their concepts, the main cerebellar function is to control motor behavior, Purkinje cells

  7. Evolving Models of Pavlovian Conditioning: Cerebellar Cortical Dynamics in Awake Behaving Mice

    Directory of Open Access Journals (Sweden)

    Michiel M. ten Brinke

    2015-12-01

    Full Text Available Three decades of electrophysiological research on cerebellar cortical activity underlying Pavlovian conditioning have expanded our understanding of motor learning in the brain. Purkinje cell simple spike suppression is considered to be crucial in the expression of conditional blink responses (CRs. However, trial-by-trial quantification of this link in awake behaving animals is lacking, and current hypotheses regarding the underlying plasticity mechanisms have diverged from the classical parallel fiber one to the Purkinje cell synapse LTD hypothesis. Here, we establish that acquired simple spike suppression, acquired conditioned stimulus (CS-related complex spike responses, and molecular layer interneuron (MLI activity predict the expression of CRs on a trial-by-trial basis using awake behaving mice. Additionally, we show that two independent transgenic mouse mutants with impaired MLI function exhibit motor learning deficits. Our findings suggest multiple cerebellar cortical plasticity mechanisms underlying simple spike suppression, and they implicate the broader involvement of the olivocerebellar module within the interstimulus interval.

  8. Altered Cerebellar Organization and Function in Monoamine Oxidase A Hypomorphic Mice

    Science.gov (United States)

    Alzghoul, Loai; Bortolato, Marco; Delis, Foteini; Thanos, Panayotis K.; Darling, Ryan D.; Godar, Sean C; Zhang, Junlin; Grant, Samuel; Wang, Gene-Jack; Simpson, Kimberly L.; Chen, Kevin; Volkow, Nora D.; Lin, Rick C.S.; Shih, Jean C.

    2012-01-01

    Monoamine oxidase A (MAO-A) is the key enzyme for the degradation of brain serotonin (5-hydroxytryptamine, 5-HT), norepinephrine (NE) and dopamine (DA). We recently generated and characterized a novel line of MAO-A hypormorphic mice (MAO-ANeo), featuring elevated monoamine levels, social deficits and perseverative behaviors as well as morphological changes in the basolateral amygdala and orbitofrontal cortex. Here we showed that MAO-ANeo mice displayed deficits in motor control, manifested as subtle disturbances in gait, motor coordination, and balance. Furthermore, magnetic resonance imaging of the cerebellum revealed morphological changes and a moderate reduction in the cerebellar size of MAO- ANeo mice compared to wild type (WT) mice. Histological and immunohistochemical analyses using calbindin-D-28k (CB) expression of Purkinje cells revealed abnormal cerebellar foliation with vermal hypoplasia and decreased in Purkinje cell count and their dendritic density in MAO- ANeo mice compared to WT. Our current findings suggest that congenitally low MAO-A activity leads to abnormal development of the cerebellum. PMID:22971542

  9. Sensitivity difference between anterior and posterior lobes of rat cerebellum to prenatal exposure to 2.5 Gy X-irradiation. A histological study

    International Nuclear Information System (INIS)

    Darmanto, W.; Hayasaka, Shizu; Takagishi, Yoshiko; Aolad, H.M.; Inouye, Minoru

    1997-01-01

    We investigated the histological differences in abnormality between anterior lobes (vermian lobules II, III and culmen IV, V) and posterior lobes (lobules IX, X) of the rat cerebellum following prenatal exposure to X-irradiation. Pregnant rats were exposed to 2.5 Gy X-irradiation at gestation day-21 (GD-21), and pups were sacrificed from birth through 15 days of age. Their cerebella were examined histologically and immunohistochemically for glial fibrillary acidic protein in Bergmann fibers. Extensive cell death was found in the external granular layer (EGL) of the cerebellum on the day of birth. In the anterior lobes, the number of cell deaths was higher than in the posterior lobes. During 5 days after birth, the recovery of EGL was earlier in the posterior lobes than in the anterior lobes. Seven days after birth, Bergmann fibers were more irregular in the anterior lobes than in the posterior lobes. The number of Purkinje cells in ectopic locations was higher in the anterior lobes than in the posterior lobes. The EGL and migrating Purkinje cells showed different responses to X-irradiation in the anterior than in the posterior lobes of the cerebellum. (author)

  10. Dosimetry techniques for applications of incorporated radionuclides

    International Nuclear Information System (INIS)

    Howell, R.W.; Rao, D.V.; Haydock, C.

    1989-01-01

    Beta particle emitters are attracting attention as the radiolabels of choice for therapeutic radiopharmaceutical. Their use in cancer therapy has drawn attention to a variety of problems in estimating the absorbed dose to primary tumors and metastases from incorporated Β-emitters. Experimental evidence indicates that the distribution of radiopharmaceutical, such as radiolabeled monoclonal antibodies, is highly nonuniform in tumor tissue. Three levels of nonuniformity may be noted: (1) inhomogeneity at the macroscopic level due to poor penetration of the radiopharmaceutical into the tumor, (2) microscopic inhomogeneity due to large variations in the number of binding sites on the tumor cells, and (3) nonuniformity at the subcellular level. Conventional application of the MIRD Schema for calculating absorbed doses from incorporated radionuclides may be inadequate under these circumstances since this approach assumes that the, distribution of radioactivity in the organ is uniform. The conventional dosimetry may be modified to handle inhomogeneous activity distributions by dividing the tumor into a number of subregions. At the macroscopic level a spherical tumor may be broken up into a group of concentric annular regions of tissue. At the microscopic level the tumor or metastasis may be considered as a multicellular cluster which in essence divides the tumor into many subtumors of cellular dimensions. Finally, at the subcellular level, a cancer cell may be viewed as consisting of several compartments: the cell membrane, cytoplasm, and nucleus. In each case absorbed fractions, and therefore the total absorbed doses, may be calculated for the various subregions of the tumor using standard MIRD procedures. Using macroscopic and multicellular dosimetry models, the relative importance of these various levels of inhomogeneity in radionuclide distribution is examined. A dosimetry model which accounts for the possible time dependence of the tumor mass is formulated

  11. 5-aminolevulinic acid-incorporated nanoparticles of methoxy poly(ethylene glycol-chitosan copolymer for photodynamic therapy

    Directory of Open Access Journals (Sweden)

    Chung CW

    2013-02-01

    Full Text Available Chung-Wook Chung,1,* Kyu-Don Chung,2,* Young-Il Jeong,1 Dae Hwan Kang,1 1National Research and Development Center for Hepatobiliary Disease, Pusan National University Yangsan Hospital, Gyeongnam, Republic of Korea; 2Department of Anesthesiology and Pain Medicine, College of Medicine, The Catholic University, Seoul, Republic of Korea*These authors contributed equally to this workPurpose: The aim of this study was to make 5-aminolevulinic acid (5-ALA-incorporated nanoparticles using methoxy polyethylene glycol/chitosan (PEG-Chito copolymer for application in photodynamic therapy for colon cancer cells.Methods: 5-ALA-incorporated (PEG-Chito-5-ALA nanoparticles were prepared by ion complex formation between 5-ALA and chitosan. Protoporphyrin IX accumulation in the tumor cells and phototoxicity induced by PEG-Chito-5-ALA nanoparticles were assessed using CT26 cells in vitro.Results: PEG-Chito-5-ALA nanoparticles have spherical shapes with sizes diameters 200 nm. More specifically, microscopic observation revealed a core-shell structure of PEG-Chito-5-ALA nanoparticles. 1H NMR spectra showed that 5-ALA was incorporated in the core of the nanoparticles. In the absence of light irradiation, all components such as 5-ALA, empty nanoparticles, and PEG-Chito-5-ALA nanoparticles did not affect the viability of cells. However, 5-ALA or PEG-Chito-5-ALA nanoparticles induced tumor cell death under light irradiation, and the viability of tumor cells was dose-dependently decreased according to the increase in irradiation time. In particular, PEG-Chito-5-ALA nanoparticles induced increased phototoxicity and higher protoporphyrin IX accumulation into the tumor cells than did 5-ALA alone. Furthermore, PEG-Chito-5-ALA nanoparticles accelerated apoptosis/necrosis of tumor cells, compared to 5-ALA alone.Conclusion: PEG-Chito-5-ALA nanoparticles showed superior delivery capacity of 5-ALA and phototoxicity against tumor cells. These results show that PEG-Chito-5-ALA

  12. Beneficial effects of amino acid-functionalized graphene nanosheets incorporated in the photoanode material of dye-sensitized solar cells: A practical and theoretical study

    International Nuclear Information System (INIS)

    Taki, Mahmood; Rezaei, Behzad; Fani, Najmeh; Borandeh, Sedigheh; Abdolmaleki, Amir; Ensafi, Ali A.

    2017-01-01

    Highlights: • Electrochemical properties of GO was improved with incorporated amino acids. • Functionalized amino acids could act as a reducing agent for GO. • Aromatic amino acids-GO had improved photovoltaic properties over aliphatic ones. • Aromatic ring of tyrosine was aligned in parallel to the GO sheet. • A bang gap for GO was emerged with functionalizing with amino acids. - Abstract: In this research, covalently functionalized graphene oxide (GO) with some biocompatible amino acids were incorporated to the TiO_2 film and employed as the photoanodes of dye-sensitized solar cells (DSSCs). Electrochemical analysis of the amino acids-functionalized graphene oxide (AFGs) confirmed that the attached amino acids could be acted as a reducing agent of the GO. The photovoltaic performance of the assembled DSSCs under illumination of simulated AM 1.5 sunlight (100 mW cm"−"2) showed an enhancement of about 4.1 and 1.8 fold for the solar cell assembled with the tyrosine-functionalized GO in relation to the control solar cells constructed with GO-TiO_2 composite and blank TiO_2 film, respectively. These results were in accordance with electron life time and transport time resulted from the open circuit voltage decay (OCVD), electrochemical impedance spectroscopy (EIS) and intensity modulated photocurrent spectroscopy (IMPS) analysis. The density functional theory (DFT) calculations exhibited a proper spacial arrangement for the tyrosine-GO structure that could improve electron transfer between the adjucent GO sheets. Density of electronic states (DOS) exhibited a gap between the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) energy levels for the simulated AFG structures. This effect could facilitate the light adsorption process in near-IR region.

  13. Beneficial effects of amino acid-functionalized graphene nanosheets incorporated in the photoanode material of dye-sensitized solar cells: A practical and theoretical study

    Energy Technology Data Exchange (ETDEWEB)

    Taki, Mahmood [Department of Chemistry, Isfahan University of Technology, Isfahan 84156-83111, Islamic Republic of Iran (Iran, Islamic Republic of); Rezaei, Behzad, E-mail: rezaei@cc.iut.ac.ir [Department of Chemistry, Isfahan University of Technology, Isfahan 84156-83111, Islamic Republic of Iran (Iran, Islamic Republic of); Fani, Najmeh [Department of Chemistry, University of Isfahan, Isfahan 81746-73441, Islamic Republic of Iran (Iran, Islamic Republic of); Borandeh, Sedigheh [Center for Nanotechnology in Drug Delivery, Shiraz University of Medical Science, Shiraz 71345, Islamic Republic of Iran (Iran, Islamic Republic of); Abdolmaleki, Amir; Ensafi, Ali A. [Department of Chemistry, Isfahan University of Technology, Isfahan 84156-83111, Islamic Republic of Iran (Iran, Islamic Republic of)

    2017-05-01

    Highlights: • Electrochemical properties of GO was improved with incorporated amino acids. • Functionalized amino acids could act as a reducing agent for GO. • Aromatic amino acids-GO had improved photovoltaic properties over aliphatic ones. • Aromatic ring of tyrosine was aligned in parallel to the GO sheet. • A bang gap for GO was emerged with functionalizing with amino acids. - Abstract: In this research, covalently functionalized graphene oxide (GO) with some biocompatible amino acids were incorporated to the TiO{sub 2} film and employed as the photoanodes of dye-sensitized solar cells (DSSCs). Electrochemical analysis of the amino acids-functionalized graphene oxide (AFGs) confirmed that the attached amino acids could be acted as a reducing agent of the GO. The photovoltaic performance of the assembled DSSCs under illumination of simulated AM 1.5 sunlight (100 mW cm{sup −2}) showed an enhancement of about 4.1 and 1.8 fold for the solar cell assembled with the tyrosine-functionalized GO in relation to the control solar cells constructed with GO-TiO{sub 2} composite and blank TiO{sub 2} film, respectively. These results were in accordance with electron life time and transport time resulted from the open circuit voltage decay (OCVD), electrochemical impedance spectroscopy (EIS) and intensity modulated photocurrent spectroscopy (IMPS) analysis. The density functional theory (DFT) calculations exhibited a proper spacial arrangement for the tyrosine-GO structure that could improve electron transfer between the adjucent GO sheets. Density of electronic states (DOS) exhibited a gap between the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) energy levels for the simulated AFG structures. This effect could facilitate the light adsorption process in near-IR region.

  14. The effects of acute morphine treatment on the incorporation of [3H]L-lysine by normal and regenerating facial nucleus neurons

    International Nuclear Information System (INIS)

    Sinatra, R.S.; Ford, D.H.; Rhines, R.K.

    1979-01-01

    The effect of morphine on the incorporation of [ 3 H]L-lysine into proteins of facial nucleus neurons was examined by light microscopic radioautography. Silver grains present within various compartments of normal and regenerating (3-, 7-. 14- and 21 days post-axotomy) neurons from saline-treated Wistar rats were compared with the amount present in similar cells from animals receiving 40 mg/kg morphine sulfate i.v. At 14- and 21-days post-axotomy, regenerating neurons were larger and the grain count in the emulsion over these cells was greater than that observed in normal (unoperated) neurons. In normal facial neurons, the accumulation of lysine into the nucleus and nucleolus was significantly lower 60 min after morphine adminstration. However, morphine's inhibition of lysine incorporation was even more pronounced in regenerating neurons. In these cells, nuclear lysine uptake was depressed at 3 and 7 days, while maximum inhibition of cytoplasmic incorporation occurred at 14-days post-axotomy. Morphine adminstration decreased nucleolar lysine incorporation at all survival intervals. (Auth.)

  15. Orthogonal translation components for the in vivo incorporation of unnatural amino acids

    Science.gov (United States)

    Schultz, Peter G.; Xie, Jianming; Zeng, Huaqiang

    2012-07-10

    The invention relates to orthogonal pairs of tRNAs and aminoacyl-tRNA synthetases that can incorporate unnatural amino acids into proteins produced in eubacterial host cells such as E. coli, or in a eukaryotic host such as a yeast cell. The invention provides, for example but not limited to, novel orthogonal synthetases, methods for identifying and making the novel synthetases, methods for producing proteins containing unnatural amino acids, and translation systems.

  16. Design of a novel integration-deficient lentivector technology that incorporates genetic and posttranslational elements to target human dendritic cells.

    Science.gov (United States)

    Tareen, Semih U; Kelley-Clarke, Brenna; Nicolai, Christopher J; Cassiano, Linda A; Nelson, Lisa T; Slough, Megan M; Vin, Chintan D; Odegard, Jared M; Sloan, Derek D; Van Hoeven, Neal; Allen, James M; Dubensky, Thomas W; Robbins, Scott H

    2014-03-01

    As sentinels of the immune system, dendritic cells (DCs) play an essential role in regulating cellular immune responses. One of the main challenges of developing DC-targeted therapies includes the delivery of antigen to DCs in order to promote the activation of antigen-specific effector CD8 T cells. With the goal of creating antigen-directed immunotherapeutics that can be safely administered directly to patients, Immune Design has developed a platform of novel integration-deficient lentiviral vectors that target and deliver antigen-encoding nucleic acids to human DCs. This platform, termed ID-VP02, utilizes a novel genetic variant of a Sindbis virus envelope glycoprotein with posttranslational carbohydrate modifications in combination with Vpx, a SIVmac viral accessory protein, to achieve efficient targeting and transduction of human DCs. In addition, ID-VP02 incorporates safety features in its design that include two redundant mechanisms to render ID-VP02 integration-deficient. Here, we describe the characteristics that allow ID-VP02 to specifically transduce human DCs, and the advances that ID-VP02 brings to conventional third-generation lentiviral vector design as well as demonstrate upstream production yields that will enable manufacturing feasibility studies to be conducted.

  17. effect of o,p'-DDD on the in vivo incorporation of 3H-thymidine into DNA

    International Nuclear Information System (INIS)

    Lund, B.-O.; Brandt, I.; Busk, L.; Hellmann, B.

    1990-01-01

    The effects of o,p'-DDD on the DNA synthesis in the C57Bl mouse lung and liver were studied. As determined by 3 H-thymidine incorporation into DNA, a selective increase in the lung DNA synthesis (+59%) was observed 2 day after a single intraperiotoneal injection of 100 mg/kg o,p'-DDD. Microautoradiography showed that incorporated 3 H-thymidine was confined to a restricted number of heavily labelled cells, presumably proliferating type II cells. At the most, a 9 times higher rate of cell proliferation was observed in the lung 4 days after an intraperitoneal injection of 500 mg/kg o,p'-DDD. Using mouse lung or liver S-9 as activating system, no mutagenic activity of o,p'-DDD was detected in the Ames test. The induced cell proliferation may indicate a tissue-selective promotor activity of o,p'-DDD in the mouse lung. (author)

  18. Nucleotide pools dictate the identity and frequency of ribonucleotide incorporation in mitochondrial DNA.

    Directory of Open Access Journals (Sweden)

    Anna-Karin Berglund

    2017-02-01

    Full Text Available Previous work has demonstrated the presence of ribonucleotides in human mitochondrial DNA (mtDNA and in the present study we use a genome-wide approach to precisely map the location of these. We find that ribonucleotides are distributed evenly between the heavy- and light-strand of mtDNA. The relative levels of incorporated ribonucleotides reflect that DNA polymerase γ discriminates the four ribonucleotides differentially during DNA synthesis. The observed pattern is also dependent on the mitochondrial deoxyribonucleotide (dNTP pools and disease-causing mutations that change these pools alter both the absolute and relative levels of incorporated ribonucleotides. Our analyses strongly suggest that DNA polymerase γ-dependent incorporation is the main source of ribonucleotides in mtDNA and argues against the existence of a mitochondrial ribonucleotide excision repair pathway in human cells. Furthermore, we clearly demonstrate that when dNTP pools are limiting, ribonucleotides serve as a source of building blocks to maintain DNA replication. Increased levels of embedded ribonucleotides in patient cells with disturbed nucleotide pools may contribute to a pathogenic mechanism that affects mtDNA stability and impair new rounds of mtDNA replication.

  19. Nucleotide pools dictate the identity and frequency of ribonucleotide incorporation in mitochondrial DNA.

    Science.gov (United States)

    Berglund, Anna-Karin; Navarrete, Clara; Engqvist, Martin K M; Hoberg, Emily; Szilagyi, Zsolt; Taylor, Robert W; Gustafsson, Claes M; Falkenberg, Maria; Clausen, Anders R

    2017-02-01

    Previous work has demonstrated the presence of ribonucleotides in human mitochondrial DNA (mtDNA) and in the present study we use a genome-wide approach to precisely map the location of these. We find that ribonucleotides are distributed evenly between the heavy- and light-strand of mtDNA. The relative levels of incorporated ribonucleotides reflect that DNA polymerase γ discriminates the four ribonucleotides differentially during DNA synthesis. The observed pattern is also dependent on the mitochondrial deoxyribonucleotide (dNTP) pools and disease-causing mutations that change these pools alter both the absolute and relative levels of incorporated ribonucleotides. Our analyses strongly suggest that DNA polymerase γ-dependent incorporation is the main source of ribonucleotides in mtDNA and argues against the existence of a mitochondrial ribonucleotide excision repair pathway in human cells. Furthermore, we clearly demonstrate that when dNTP pools are limiting, ribonucleotides serve as a source of building blocks to maintain DNA replication. Increased levels of embedded ribonucleotides in patient cells with disturbed nucleotide pools may contribute to a pathogenic mechanism that affects mtDNA stability and impair new rounds of mtDNA replication.

  20. Supplementation with a fish oil-enriched, high-protein medical food leads to rapid incorporation of EPA into white blood cells and modulates immune responses within one week in healthy men and women.

    Science.gov (United States)

    Faber, Joyce; Berkhout, Marloes; Vos, Arjan P; Sijben, John W C; Calder, Philip C; Garssen, Johan; van Helvoort, Ardy

    2011-05-01

    Immune modulatory effects of EPA and DHA are well described. However, these fatty acids must be effectively incorporated into cell membrane phospholipids to modify cell function. To address the absence of human data regarding short-term incorporation, the present study investigated the incorporation of EPA and DHA into white blood cells (WBC) at different time points during 1 wk of supplementation with a medical food, which is high in protein and leucine and enriched with fish oil and specific oligosaccharides. Additionally, the effects on ex vivo immune function were determined. In a single-arm, open label study, 12 healthy men and women consumed 2 × 200 mL of medical food providing 2.4 g EPA, 1.2 g DHA, 39.7 g protein (including 4.4 g L-leucine), and 5.6 g oligosaccharides daily. Blood samples were taken at d 0 (baseline), 1, 2, 4, and 7. Within 1 d of nutritional intervention, the percentage of EPA in phospholipids of WBC increased from 0.5% at baseline to 1.3% (P blood cultures was significantly increased within 1 wk. Nutritional supplementation with a fish oil-enriched medical food significantly increased the percentage of EPA in phospholipids of WBC within 1 wk. Simultaneously, ex vivo immune responsiveness to LPS increased significantly. These results hold promise for novel applications such as fast-acting nutritional interventions in cancer patients, which should be investigated in future studies.

  1. Autoradiographic demonstration of sup 3 H-estradiol and sup 3 H-cholesterol incorporation in hamster gonads

    Energy Technology Data Exchange (ETDEWEB)

    Angelova, P; Martinova, J; Kyncheva, L; Baleva-Ivanova, K [Bylgarska Akademiya na Naukite, Sofia (Bulgaria). Inst. po Morfologiya

    1989-01-01

    Male and female hamster gonads were investigated on day 14 of pregnancy, at birth, on days 7, 18 and 25 after birth and at sexual maturity. (2,4,6,7 {sup 3}H)-estradiol -17{beta}, specific activity 110 Ci.mmol{sup -1} and (1{alpha}, 2{alpha} -{sup 3}H) - cholesterol specific activity 44 Ci.mmol{sup -1} have been used for labelling. On embrional day 14 the histological image has been similar to that in the neonatal gonads - diffusive labelling includding germ, satellite and Leyding cells in fetal ovaries and testes. On the 7th postnatal day in the ovary a formation of primary follicles began in the deeper layers of gonads and an incorporation of the labelled substances in the germ and prefollicular cells in both ovary and testis have been observed. On the 18th postnatal day growing follicles have been seen in the ovary and labelling have been noticed in the oocytes and follicular cells. In the prepubertal testis the meiolic process has started, spermatocytes have been found and an incorporation of the radioactive substances in germ, Sertoli and Leydig cells has been established. In the ovaries of both 25th day old hamsters and adult animals multi-layered and preovulatory follicles have been seen. Sertoli cells, spermatogonia, spermatocytes and spertamids in the seminiferons tubules have been observed. The incorporation of {sup 3}H-estradiol and {sup 3}H cholesterol in both germ and Sertoli cells has been found. A presence has been observed of specific estradiol receptors in all three main cell types of fetal and developing gonads: germ, satellite and intertitial cells. The presence of estradiol receptors in developing hamster gonads has indicated a participation of steroids in the process of development and differentiation of male and female gonads.

  2. Abnormal secretion or extracellular matrix incorporation of fibrillin by dermal fibroblasts from patients with thoracic aortic aneurysms

    Energy Technology Data Exchange (ETDEWEB)

    Milewicz, D.; Cao, S.; Cosselli, J. [Univ. of Texas Medical School, Houston, TX (United States)

    1994-09-01

    Abnormal synthesis, secretion, and extracellular matrix incorporation of fibrillin is observed in the majority of fibroblast cell strains obtained from individuals with the Marfan syndrome (>85%). These fibrillin protein abnormalities are due to mutations in the FBN1 gene. We have screened fibroblast cell strains from patients with thoracic aortic aneurysms (TAA) without skeletal or ocular features of the Marfan syndrome for defects in fibrillin synthesis or processing. Dermal fibroblasts obtained from biopsies were pulse labeled with [{sup 35}S]cysteine for 30 minutes and then chased for 0, 4, and 20 hours. The media, cell lysate and extracellular matrix were harvested separately, then analyzed by SDS-PAGE. We selected fibroblasts from 17 TAA patients to study based on the development of a TAA at a young age or a family history of TAAs. Cells from 3 patients synthesized and secreted fibrillin normally, but did not incorporate the fibrillin in the extracellular matrix. None of the cell strains were found to have diminished synthesis of fibrillin when compared with control cells. We were unable to detect abnormalities in the synthesis, secretion, or matrix incorporation of fibrillin by cells from 9 of the 17 patients. These results indicate that fibrillin protein defects are found in a significant number of patients with TAAs who are young or have a family history of TAAs. Analysis of the FBN1 gene for mutations in these patients with fibrillin protein defects will determine if the observed protein abnormalities are the result of FBN1 gene mutations.

  3. Studies on lipids and fatty acids in rats with streptozotocin-induced insulin deficiency II. Incorporation of 1-(14)C-sodium acetate into lipids and fatty acids of liver slices and whole blood cells

    OpenAIRE

    三宅,寛治

    1988-01-01

    In order to study the lipid and fatty acid metabolism in the insulin deficient state, the in vitro incorporation of 1-(14)C-sodium acetate into major lipid fractions and fatty acids of liver slices and whole blood cells was determined. Rats were studied one week, one month and three months after insulin deficiency was induced by administration of streptozotocin.The net incorporation of (14)C into lipid fractions and total fatty acids of liver slices significantly decreased after one week. On ...

  4. Effects of titanium surface anodization with CaP incorporation on human osteoblastic response

    Science.gov (United States)

    OLIVEIRA, Natássia Cristina Martins; MOURA, Camilla Christian Gomes; ZANETTA-BARBOSA, Darceny; MENDONÇA, Daniela Baccelli Silveira; MENDONÇA, Gustavo; DECHICHI, Paula

    2015-01-01

    In this study we investigated whether anodization with calcium phosphate (CaP) incorporation (Vulcano®) enhances growth factors secretion, osteoblast-specific gene expression, and cell viability, when compared to acid etched surfaces (Porous®) and machined surfaces (Screw®) after 3 and 7 days. Results showed significant cell viability for Porous and Vulcano at day 7, when compared with Screw (p=0.005). At the same time point, significant differences regarding runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP) and bone sialoprotein (BSP) expression were found for all surfaces (p0.05). Although no significant correlation was found for growth factors secretion and Runx2 expression, a significant positive correlation between this gene and ALP/BSP expression showed that their strong association is independent on the type of surface. The incorporation of CaP affected the biological parameters evaluated similar to surfaces just acid etched. The results presented here support the observations that roughness also may play an important role in determining cell response. PMID:23498218

  5. HIV-1 Envelope Glycoprotein Trafficking through the Endosomal Recycling Compartment Is Required for Particle Incorporation.

    Science.gov (United States)

    Kirschman, Junghwa; Qi, Mingli; Ding, Lingmei; Hammonds, Jason; Dienger-Stambaugh, Krista; Wang, Jaang-Jiun; Lapierre, Lynne A; Goldenring, James R; Spearman, Paul

    2018-03-01

    The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) encodes specific trafficking signals within its long cytoplasmic tail (CT) that regulate incorporation into HIV-1 particles. Rab11-family interacting protein 1C (FIP1C) and Rab14 are host trafficking factors required for Env particle incorporation, suggesting that Env undergoes sorting from the endosomal recycling compartment (ERC) to the site of particle assembly on the plasma membrane. We disrupted outward sorting from the ERC by expressing a C-terminal fragment of FIP1C (FIP1C 560-649 ) and examined the consequences on Env trafficking and incorporation into particles. FIP1C 560-649 reduced cell surface levels of Env and prevented its incorporation into HIV-1 particles. Remarkably, Env was trapped in an exaggerated perinuclear ERC in a CT-dependent manner. Mutation of either the Yxxϕ endocytic motif or the YW 795 motif in the CT prevented Env trapping in the ERC and restored incorporation into particles. In contrast, simian immunodeficiency virus SIVmac239 Env was not retained in the ERC, while substitution of the HIV-1 CT for the SIV CT resulted in SIV Env retention in this compartment. These results provide the first direct evidence that Env traffics through the ERC and support a model whereby HIV-1 Env is specifically targeted to the ERC prior to FIP1C- and CT-dependent outward sorting to the particle assembly site on the plasma membrane. IMPORTANCE The HIV envelope protein is an essential component of the viral particle. While many aspects of envelope protein structure and function have been established, the pathway it follows in the cell prior to reaching the site of particle assembly is not well understood. The envelope protein has a very long cytoplasmic tail that interacts with the host cell trafficking machinery. Here, we utilized a truncated form of the trafficking adaptor FIP1C protein to arrest the intracellular transport of the envelope protein, demonstrating that it becomes

  6. p38α phosphorylates serine 258 within the cytoplasmic domain of tissue factor and prevents its incorporation into cell-derived microparticles.

    Science.gov (United States)

    Ettelaie, Camille; Elkeeb, Azza M; Maraveyas, Anthony; Collier, Mary Elizabeth W

    2013-03-01

    We previously showed that the phosphorylation of Ser253 within the cytoplasmic domain of human tissue factor (TF) initiates the incorporation and release of this protein into cell-derived microparticles. Furthermore, subsequent phosphorylation of Ser258 terminates this process. However, the identity of the kinase responsible for the phosphorylation of Ser258 and mode of action of this enzyme remain unknown. In this study, p38α was identified as the proline-directed kinase capable of phosphorylating Ser258 specifically, and without any detectable activity towards Ser253. Furthermore, using synthetic peptides, it was shown that the Km for the reaction decreased by approximately 10 fold on substitution of Ser253 with phospho-Ser253. Either inhibition of p38 using SB202190 or knockdown of p38α expression in coronary artery endothelial cells overexpressing wild-type TF, resulted in decreased phosphorylation of Ser258, following activation of cells with PAR2-agonist peptide (PAR2-AP). In agreement with our previous data, inhibition of phosphorylation of this residue maintained the release of TF. Activation of PAR2 in cells transfected to overexpress TF, resulted in two separate peaks of p38 activity at approximately 40 and 120 min post-activation. Furthermore, overexpression of Ala253-substituted TF enhanced the second p38 activation peak. However, the second peak was absent in cells devoid of TF or in cells overexpressing the Asp253-substituted TF. Our data clearly identifies p38α as a kinase capable of phosphorylating Ser258 within the cytoplasmic domain of TF. Moreover, it appears that the presence of TF within the cells regulates the late activation of p38 and consequently the termination of TF release into microparticles. Copyright © 2012 Elsevier B.V. All rights reserved.

  7. Histomorphometric studies on the effect of cyanide consumption of ...

    African Journals Online (AJOL)

    The density and size of the Purkinje cells were the same in both the control and experimental groups (P>0.05). Conclusion: Maternal consumption of 500 ppm cyanide in rats does not significantly affect light microscopic prenatal cerebellar development, but causes mild changes in the post-natal life. Maternal cyanide ...

  8. Energy-neutral sustainable nutrient recovery incorporated with the wastewater purification process in an enlarged microbial nutrient recovery cell

    Science.gov (United States)

    Sun, Dongya; Gao, Yifan; Hou, Dianxun; Zuo, Kuichang; Chen, Xi; Liang, Peng; Zhang, Xiaoyuan; Ren, Zhiyong Jason; Huang, Xia

    2018-04-01

    Recovery of nutrient resources from the wastewater is now an inevitable strategy to maintain the supply of both nutrient and water for our huge population. While the intensive energy consumption in conventional nutrient recovery technologies still remained as the bottleneck towards the sustainable nutrient recycle. This study proposed an enlarged microbial nutrient recovery cell (EMNRC) which was powered by the energy contained in wastewater and achieved multi-cycle nutrient recovery incorporated with in situ wastewater treatment. With the optimal recovery solution of 3 g/L NaCl and the optimal volume ratio of wastewater to recovery solution of 10:1, >89% of phosphorus and >62% of ammonium nitrogen were recovered into struvite. An extremely low water input ratio of water. It was proved the EMNRC system was a promising technology which could utilize the chemical energy contained in wastewater itself and energy-neutrally recover nutrient during the continuous wastewater purification process.

  9. Leucine incorporation by aerobic anoxygenic phototrophic bacteria in the Delaware estuary

    Science.gov (United States)

    Stegman, Monica R; Cottrell, Matthew T; Kirchman, David L

    2014-01-01

    Aerobic anoxygenic phototrophic (AAP) bacteria are well known to be abundant in estuaries, coastal regions and in the open ocean, but little is known about their activity in any aquatic ecosystem. To explore the activity of AAP bacteria in the Delaware estuary and coastal waters, single-cell 3H-leucine incorporation by these bacteria was examined with a new approach that combines infrared epifluorescence microscopy and microautoradiography. The approach was used on samples from the Delaware coast from August through December and on transects through the Delaware estuary in August and November 2011. The percent of active AAP bacteria was up to twofold higher than the percentage of active cells in the rest of the bacterial community in the estuary. Likewise, the silver grain area around active AAP bacteria in microautoradiography preparations was larger than the area around cells in the rest of the bacterial community, indicating higher rates of leucine consumption by AAP bacteria. The cell size of AAP bacteria was 50% bigger than the size of other bacteria, about the same difference on average as measured for activity. The abundance of AAP bacteria was negatively correlated and their activity positively correlated with light availability in the water column, although light did not affect 3H-leucine incorporation in light–dark experiments. Our results suggest that AAP bacteria are bigger and more active than other bacteria, and likely contribute more to organic carbon fluxes than indicated by their abundance. PMID:24824666

  10. Leucine incorporation by aerobic anoxygenic phototrophic bacteria in the Delaware estuary.

    Science.gov (United States)

    Stegman, Monica R; Cottrell, Matthew T; Kirchman, David L

    2014-11-01

    Aerobic anoxygenic phototrophic (AAP) bacteria are well known to be abundant in estuaries, coastal regions and in the open ocean, but little is known about their activity in any aquatic ecosystem. To explore the activity of AAP bacteria in the Delaware estuary and coastal waters, single-cell (3)H-leucine incorporation by these bacteria was examined with a new approach that combines infrared epifluorescence microscopy and microautoradiography. The approach was used on samples from the Delaware coast from August through December and on transects through the Delaware estuary in August and November 2011. The percent of active AAP bacteria was up to twofold higher than the percentage of active cells in the rest of the bacterial community in the estuary. Likewise, the silver grain area around active AAP bacteria in microautoradiography preparations was larger than the area around cells in the rest of the bacterial community, indicating higher rates of leucine consumption by AAP bacteria. The cell size of AAP bacteria was 50% bigger than the size of other bacteria, about the same difference on average as measured for activity. The abundance of AAP bacteria was negatively correlated and their activity positively correlated with light availability in the water column, although light did not affect (3)H-leucine incorporation in light-dark experiments. Our results suggest that AAP bacteria are bigger and more active than other bacteria, and likely contribute more to organic carbon fluxes than indicated by their abundance.

  11. Computational cell quantification in the human brain tissues based on hard x-ray phase-contrast tomograms

    Science.gov (United States)

    Hieber, Simone E.; Bikis, Christos; Khimchenko, Anna; Schulz, Georg; Deyhle, Hans; Thalmann, Peter; Chicherova, Natalia; Rack, Alexander; Zdora, Marie-Christine; Zanette, Irene; Schweighauser, Gabriel; Hench, Jürgen; Müller, Bert

    2016-10-01

    Cell visualization and counting plays a crucial role in biological and medical research including the study of neurodegenerative diseases. The neuronal cell loss is typically determined to measure the extent of the disease. Its characterization is challenging because the cell density and size already differs by more than three orders of magnitude in a healthy cerebellum. Cell visualization is commonly performed by histology and fluorescence microscopy. These techniques are limited to resolve complex microstructures in the third dimension. Phase- contrast tomography has been proven to provide sufficient contrast in the three-dimensional imaging of soft tissue down to the cell level and, therefore, offers the basis for the three-dimensional segmentation. Within this context, a human cerebellum sample was embedded in paraffin and measured in local phase-contrast mode at the beamline ID19 (ESRF, Grenoble, France) and the Diamond Manchester Imaging Branchline I13-2 (Diamond Light Source, Didcot, UK). After the application of Frangi-based filtering the data showed sufficient contrast to automatically identify the Purkinje cells and to quantify their density to 177 cells per mm3 within the volume of interest. Moreover, brain layers were segmented in a region of interest based on edge detection. Subsequently performed histological analysis validated the presence of the cells, which required a mapping from the two- dimensional histological slices to the three-dimensional tomogram. The methodology can also be applied to further tissue types and shows potential for the computational tissue analysis in health and disease.

  12. Microfluidic platform for efficient Nanodisc assembly, membrane protein incorporation, and purification.

    Science.gov (United States)

    Wade, James H; Jones, Joshua D; Lenov, Ivan L; Riordan, Colleen M; Sligar, Stephen G; Bailey, Ryan C

    2017-08-22

    The characterization of integral membrane proteins presents numerous analytical challenges on account of their poor activity under non-native conditions, limited solubility in aqueous solutions, and low expression in most cell culture systems. Nanodiscs are synthetic model membrane constructs that offer many advantages for studying membrane protein function by offering a native-like phospholipid bilayer environment. The successful incorporation of membrane proteins within Nanodiscs requires experimental optimization of conditions. Standard protocols for Nanodisc formation can require large amounts of time and input material, limiting the facile screening of formation conditions. Capitalizing on the miniaturization and efficient mass transport inherent to microfluidics, we have developed a microfluidic platform for efficient Nanodisc assembly and purification, and demonstrated the ability to incorporate functional membrane proteins into the resulting Nanodiscs. In addition to working with reduced sample volumes, this platform simplifies membrane protein incorporation from a multi-stage protocol requiring several hours or days into a single platform that outputs purified Nanodiscs in less than one hour. To demonstrate the utility of this platform, we incorporated Cytochrome P450 into Nanodiscs of variable size and lipid composition, and present spectroscopic evidence for the functional active site of the membrane protein. This platform is a promising new tool for membrane protein biology and biochemistry that enables tremendous versatility for optimizing the incorporation of membrane proteins using microfluidic gradients to screen across diverse formation conditions.

  13. Kinetics of /sup 3/H-thymidine incorporation into nuclei of cells of the regenerating liver of rats exposed to various doses of x rays

    Energy Technology Data Exchange (ETDEWEB)

    Aksenovich, T I; Polishchuk, A M [AN SSSR, Novosibirsk. Inst. Tsitologii i Genetiki

    1976-01-01

    Action of x radiation on the incorporation of /sup 3/H-thymidine into nuclei of cells of the regenerating liver of rats has been studied. A whole-body exposure of rats to 150 to 300 R has been found to inhibit /sup 3/H-thymidine uptake into DNA of S-cells of the regenerating liver, and this effect can be attributed to the inhibition of DNA synthesis rather than to changes in the concentration of a label in the intracellular pool of DNA precursors. In addition to the DNA synthesis decrease, inhibition of the label uptake into the pool is observed after doses of 600 to 1200 R. On the basis of the data obtained, a hypothesis is proposed that explains the mechanism of inhibition of DNA synthesis under the action of radiation.

  14. Investigating the prevalence of queuine in Escherichia coli RNA via incorporation of the tritium-labeled precursor, preQ1

    International Nuclear Information System (INIS)

    Brooks, Allen F.; Vélez-Martínez, Carol S.; Hollis Showalter, H.D.; Garcia, George A.

    2012-01-01

    Highlights: ► Escherichia coli cells deficient in preQ 1 biosynthesis can utilize exogenous preQ 1 from growth media. ► Exogenous radiolabeled preQ 1 is incorporated into cellular RNA in both wild-type and preQ 1 deficient cells. ► Radiolabeled preQ 1 is incorporated into RNAs of differing sizes larger than tRNA. ► Analysis of isolated ribosomes shows that radiolabeled preQ 1 is incorporated into ribosomal RNA. ► The conclusion of this study is that queuine is incorporated into many other RNAs in addition to the known tRNAs. -- Abstract: There are over 100 modified bases that occur in RNA with the majority found in transfer RNA. It has been widely believed that the queuine modification is limited to four transfer RNA species in vivo. However, given the vast amount of the human genome (60–70%) that is transcribed into non-coding RNA (Mattick ), probing the presence of modified bases in these RNAs is of fundamental importance. The mechanism of incorporation of queuine, via transglycosylation, makes this uniquely poised to probe base modification in RNA. Results of incubations of Escherichia coli cell cultures with [ 3 H] preQ 1 (a queuine precursor in eubacteria) clearly demonstrate preQ 1 incorporation into a number of RNA species of various sizes larger than transfer RNA. Specifically, significant levels of preQ 1 incorporation into ribosomal RNA are observed. The modification of other large RNAs was also observed. These results confirm that non-coding RNAs contain modified bases and lead to the supposition that these modifications are necessary to control non-coding RNA structure and function as has been shown for transfer RNA.

  15. Incorporating an Electrode Modification Layer with a Vertical Phase Separated Photoactive Layer for Efficient and Stable Inverted Nonfullerene Polymer Solar Cells.

    Science.gov (United States)

    Shi, Zhenzhen; Liu, Hao; Wang, Yaping; Li, Jinyan; Bai, Yiming; Wang, Fuzhi; Bian, Xingming; Hayat, Tasawar; Alsaedi, Ahmed; Tan, Zhan'ao

    2017-12-20

    For bulk heterojunction polymer solar cells (PSCs), the donors and acceptors featuring specific phase separation and concentration distribution within the electron donor/acceptor blends crucially affect the exciton dissociation and charge transportation. Herein, efficient and stable nonfullerene inverted PSCs incorporating a phase separated photoactive layer and a titanium chelate electrode modification layer are demonstrated. Water contact angle (WCA), scanning kelvin probe microscopy (SKPM), and atomic force microscopy (AFM) techniques are implemented to characterize the morphology of photoactive layers. Compared with the control conventional device, the short-circuit current density (J sc ) is enhanced from 14.74 to 17.45 mAcm -2 . The power conversion efficiency (PCE) for the inverted PSCs with a titanium (diisopropoxide)-bis-(2,4-pentanedionate) (TIPD) layer increases from 9.67% to 11.69% benefiting from the declined exciton recombination and fairly enhanced charge transportation. Furthermore, the nonencapsulated inverted device with a TIPD layer demonstrates the best long-term stability, 85% of initial PCE remaining and an almost undecayed open-circuit voltage (V oc ) after 1440 h. Our results reveal that the titanium chelate is an excellent electrode modification layer to incorporate with a vertical phase separated photoactive layer for producing high-efficiency and high-stability inverted nonfullerene PSCs.

  16. Engineering the bone-ligament interface using polyethylene glycol diacrylate incorporated with hydroxyapatite.

    Science.gov (United States)

    Paxton, Jennifer Z; Donnelly, Kenneth; Keatch, Robert P; Baar, Keith

    2009-06-01

    Ligaments and tendons have previously been tissue engineered. However, without the bone attachment, implantation of a tissue-engineered ligament would require it to be sutured to the remnant of the injured native tissue. Due to slow repair and remodeling, this would result in a chronically weak tissue that may never return to preinjury function. In contrast, orthopaedic autograft reconstruction of the ligament often uses a bone-to-bone technique for optimal repair. Since bone-to-bone repairs heal better than other methods, implantation of an artificial ligament should also occur from bone-to-bone. The aim of this study was to investigate the use of a poly(ethylene glycol) diacrylate (PEGDA) hydrogel incorporated with hydroxyapatite (HA) and the cell-adhesion peptide RGD (Arg-Gly-Asp) as a material for creating an in vitro tissue interface to engineer intact ligaments (i.e., bone-ligament-bone). Incorporation of HA into PEG hydrogels reduced the swelling ratio but increased mechanical strength and stiffness of the hydrogels. Further, HA addition increased the capacity for cell growth and interface formation. RGD incorporation increased the swelling ratio but decreased mechanical strength and stiffness of the material. Optimum levels of cell attachment were met using a combination of both HA and RGD, but this material had no better mechanical properties than PEG alone. Although adherence of the hydrogels containing HA was achieved, failure occurs at about 4 days with 5% HA. Increasing the proportion of HA improved interface formation; however, with high levels of HA, the PEG HA composite became brittle. This data suggests that HA, by itself or with other materials, might be well suited for engineering the ligament-bone interface.

  17. Effect of SkQ1 Antioxidant on Structural and Functional Conditions of The Brain in PostResuscitation Period

    Directory of Open Access Journals (Sweden)

    M. L. Lovat

    2016-01-01

    Full Text Available The aim was to assess the efficacy of mitochondriatargeted antioxidant SkQ1 in prevention of structural and functional abnormalities of brain postresuscitation after cardiac arrest.Materials and methods. Adult male Wistar rats (n=19 underwent cardiac arrest for 7 minutes followed by resuscitation. Nine rats were administered with 500 nmol/kg SkQ1 per os with water for 2 weeks (1 week before and 1 week after resuscitation. A control group consisted of shamoperated animals (n=10. At days 4—6 post operation locomotor activity and anxiety («elevated plus maze» test and sensorimotor function of limbs («beam walking» test were examined. Total numbers of neurons per 1 mm of their layer length in vulnerable neuronal populations (cerebellar Purkinje cells and piramidal neurons of hippocampus fields CA1 and CA4 were estimated by histological analysis of the specimens stained with cresyl violet on day 7 postresuscitation. To identify possible mechanisms of SkQ1 action, the immunohistochemical study of a glialderived neurotrophic factor (GDNF expression in piramidal neurons of hippocampus was performed by indirect peroxidaseantiperoxidase method and antiGDNF primary polyclonal antibodies.Results. Ischemiareperfusion resulted in neuronal loss in all studied brain areas followed by reduction in locomotor activity and development of sensorimotor deficit. SkQ1 prevented development of postresuscitative locomotor and sensorimotor irregularities, significantly reduced Purkinje cells loss, prevented death of piramidal neurons in hippocampal field CA4, but not in CA1. Data demonstrated, that iIn Purkinje cells from resuscitated rats treated with SkQ1 there was a significant increase in number of GDNFpositive neurons, which were more resistant to ischemia (transition of GDNFnegative cells toward the category of cells actively expressing this factor that promoted their survival postresuscitation.Conclusion. Data confirm the positive effects of SkQ1

  18. Opsoclonus-myoclonus syndrome: Correlation of radiographic and pathological observations

    International Nuclear Information System (INIS)

    Tuchman, R.F.; Alvarez, L.A.

    1989-01-01

    We report a case of a child with opsoclonus-myoclonus syndrome. Neuroradiological studies indicated a lesion in the cerebellar vermis. A cerebellar biopsy revealed changes consisting of Purkinje and granular cell loss with gliosis. This case report documents the correlation of radiologic and pathological findings in a patient with opsoclonus-myoclonus syndrome. (orig.)

  19. Extensive Lesions of Cholinergic Basal Forebrain Neurons Do Not Impair Spatial Working Memory

    Science.gov (United States)

    Vuckovich, Joseph A.; Semel, Mara E.; Baxter, Mark G.

    2004-01-01

    A recent study suggests that lesions to all major areas of the cholinergic basal forebrain in the rat (medial septum, horizontal limb of the diagonal band of Broca, and nucleus basalis magnocellularis) impair a spatial working memory task. However, this experiment used a surgical technique that may have damaged cerebellar Purkinje cells. The…

  20. [Incorporation monitoring of employees of a radioiodine therapy ward. Is incorporation monitoring required for routine?].

    Science.gov (United States)

    Happel, C; Kratzel, U; Selkinski, I; Bockisch, B; Etzel, M; Korkusuz, H; Sauter, B; Staudt, J; von Müller, F; Grünwald, F; Kranert, W T

    2013-01-01

    Aim of the study was to determine the annual incorporation of staff on a radioiodine therapy ward and the resulting annual effective dose (aed). Following the German incorporation guideline (gig), incorporation monitoring is not necessary for potential aed below 0.5 mSv/a. For aed > 0.5 mSv/a adherence to the 1 mSv dose limit must be verified. For doses > 1 mSv/a incorporation has to be monitored by the authority. Furthermore, the (131)I incorporation factor from the gig should be verified. To determine the actual work related incorporation, the (131)I activity concentration in urine samples (collection over 24 h) of 14 employees of different professions were examined over a period of 27 months. Measured activity concentrations were related to the individual time of exposure. A constant activity supply for at least three days was assumed. The mean annual effective doses were 2.4 · 10⁻¹ mSv/a (nursing staff; n = 3), 5.6 · 10⁻² mSv/a (cleaning staff; n = 2), 2.8 · 10⁻³ mSv/a (technical staff; n = 2) and 5.2 · 10⁻³ mSv/a (physicians; n = 7). All aed were below the dose limits of the gig. The calculated mean incorporation factors ranged from 3.0 · 10⁻⁸ for the nursing staff to 3.6 · 10⁻¹⁰ for the technical staff (cleaning staff: 7 · 10⁻⁹; physicians: 6.5 · 10⁻¹⁰) and were therefore well below the (131)I incorporation factor defined by the gig. To estimate the aed caused by incorporation of (131)I it has to be subdivided for the different requirements in the diverse fields of activity of the employees. Regarding those who spend most of their time nearby the patient an incorporation monitoring by the authority might be required. The (131)I incorporation factor from the guideline (10⁻⁶) can be reduced by a factor of 10. For (99m)Tc and (18)F an incorporation factor of 10⁻⁷ is accepted.

  1. High-pass filtering and dynamic gain regulation enhance vertical bursts transmission along the mossy fiber pathway of cerebellum

    Directory of Open Access Journals (Sweden)

    Jonathan Mapelli

    2010-05-01

    Full Text Available Signal elaboration in the cerebellum mossy fiber input pathway presents controversial aspects, especially concerning gain regulation and the spot-like (rather than beam-like appearance of granular-to-molecular layer transmission. By using voltage-sensitive dye (VSD imaging in rat cerebellar slices (Mapelli et al., 2010, we found that mossy fiber bursts optimally excited the granular layer above ~50 Hz and the overlaying molecular layer above ~100 Hz, thus generating a cascade of high-pass filters. NMDA receptors enhanced transmission in the granular, while GABA-A receptors depressed transmission in both the granular and molecular layer. Burst transmission gain was controlled through a dynamic frequency-dependent involvement of these receptors. Moreover, while high-frequency transmission was enhanced along vertical lines connecting the granular to molecular layer, no high-frequency enhancement was observed along the parallel fiber axis in the molecular layer. This was probably due to the stronger effect of Purkinje cell GABA-A receptor-mediated inhibition occurring along the parallel fibers than along the granule cell axon ascending branch. The consequent amplification of burst responses along vertical transmission lines could explain the spot-like activation of Purkinje cells observed following punctuate stimulation in vivo .

  2. HIV-1 incorporates and proteolytically processes human NDR1 and NDR2 serine-threonine kinases

    International Nuclear Information System (INIS)

    Devroe, Eric; Silver, Pamela A.; Engelman, Alan

    2005-01-01

    Mammalian genomes encode two related serine-threonine kinases, nuclear Dbf2 related (NDR)1 and NDR2, which are homologous to the Saccharomyces cerevisiae Dbf2 kinase. Recently, a yeast genetic screen implicated the Dbf2 kinase in Ty1 retrotransposition. Since several virion-incorporated kinases regulate the infectivity of human immunodeficiency virus type 1 (HIV-1), we speculated that the human NDR1 and NDR2 kinases might play a role in the HIV-1 life cycle. Here we show that the NDR1 and NDR2 kinases were incorporated into HIV-1 particles. Furthermore, NDR1 and NDR2 were cleaved by the HIV-1 protease (PR), both within virions and within producer cells. Truncation at the PR cleavage site altered NDR2 subcellular localization and inhibited NDR1 and NDR2 enzymatic activity. These studies identify two new virion-associated host cell enzymes and suggest a novel mechanism by which HIV-1 alters the intracellular environment of human cells

  3. Reduction of inflammatory responses and enhancement of extracellular matrix formation by vanillin-incorporated poly(lactic-co-glycolic acid) scaffolds.

    Science.gov (United States)

    Lee, Yujung; Kwon, Jeongil; Khang, Gilson; Lee, Dongwon

    2012-10-01

    Vanillin is one of the major components of vanilla, a commonly used flavoring agent and preservative and is known to exert potent antioxidant and anti-inflammatory activities. In this work, vanillin-incorporated poly(lactic-co-glycolic acid) (PLGA) films and scaffolds were fabricated to evaluate the effects of vanillin on the inflammatory responses and extracellular matrix (ECM) formation in vitro and in vivo. The incorporation of vanillin to PLGA films induced hydrophilic nature, resulting in the higher cell attachment and proliferation than the pure PLGA film. Vanillin also reduced the generation of reactive oxygen species (ROS) in cells cultured on the pure PLGA film and significantly inhibited the PLGA-induced inflammatory responses in vivo, evidenced by the reduced accumulation of inflammatory cells and thinner fibrous capsules. The effects of vanillin on the ECM formation were evaluated using annulus fibrous (AF) cell-seeded porous PLGA/vanillin scaffolds. PLGA/vanillin scaffolds elicited the more production of glycosaminoglycan and collagen than the pure PLGA scaffold, in a concentration-dependent manner. Based on the low level of inflammatory responses and enhanced ECM formation, vanillin-incorporated PLGA constructs make them promising candidates in the future biomedical applications.

  4. Reduction of Inflammatory Responses and Enhancement of Extracellular Matrix Formation by Vanillin-Incorporated Poly(Lactic-co-Glycolic Acid) Scaffolds

    Science.gov (United States)

    Lee, Yujung; Kwon, Jeongil; Khang, Gilson

    2012-01-01

    Vanillin is one of the major components of vanilla, a commonly used flavoring agent and preservative and is known to exert potent antioxidant and anti-inflammatory activities. In this work, vanillin-incorporated poly(lactic-co-glycolic acid) (PLGA) films and scaffolds were fabricated to evaluate the effects of vanillin on the inflammatory responses and extracellular matrix (ECM) formation in vitro and in vivo. The incorporation of vanillin to PLGA films induced hydrophilic nature, resulting in the higher cell attachment and proliferation than the pure PLGA film. Vanillin also reduced the generation of reactive oxygen species (ROS) in cells cultured on the pure PLGA film and significantly inhibited the PLGA-induced inflammatory responses in vivo, evidenced by the reduced accumulation of inflammatory cells and thinner fibrous capsules. The effects of vanillin on the ECM formation were evaluated using annulus fibrous (AF) cell-seeded porous PLGA/vanillin scaffolds. PLGA/vanillin scaffolds elicited the more production of glycosaminoglycan and collagen than the pure PLGA scaffold, in a concentration-dependent manner. Based on the low level of inflammatory responses and enhanced ECM formation, vanillin-incorporated PLGA constructs make them promising candidates in the future biomedical applications. PMID:22551555

  5. The endozepine ODN stimulates [{sup 3}H]thymidine incorporation in cultured rat astrocytes

    Energy Technology Data Exchange (ETDEWEB)

    Gandolfo, P.; Patte, C.; Thoumas, J.L.; Leprince, J.; Vaudry, H.; Tonon, M.C. [European Institute for Peptide Research (IFRMP no. 23), Laboratory of Cellular and Molecular Neuroendocrinology, INSERM U 413, UA CNRS, University of Rouen, 76821 Mont-Saint-Aignan (France)

    1999-05-15

    High concentrations of diazepam-binding inhibitor (DBI) mRNA have been detected in astrocytoma, suggesting that DBI-derived peptides may play a role in glial cell proliferation. In the present study, we have investigated the effect of a processing product of DBI, the octadecaneuropeptide ODN, on DNA synthesis in cultured rat astrocytes. At very low concentrations (10{sup -14} to 10{sup -11} M), ODN caused a dose-dependent increase of [{sup 3}H]thymidine incorporation. At higher doses (10{sup -10} to 10{sup -5} M), the effect of ODN gradually declined. The central-type benzodiazepine receptor antagonist flumazenil (10{sup -6} M) completely suppressed the stimulatory action of ODN whereas the peripheral-type benzodiazepine receptor ligand, PK11195 (10{sup -6} M) had no effect. The ODN-induced stimulation of [{sup 3}H]thymidine incorporation was mimicked by methyl 6,7-dimethoxy-4-ethyl-{beta}-carboline-3-carboxylate (DMCM). The GABA{sub A} receptor antagonist bicuculline (10{sup -4} M) suppressed the effect of both ODN and DMCM on DNA synthesis. Exposure of cultured astrocytes to the specific GABA{sub A} agonist 3APS (10{sup -10} to 10{sup -4} M) also induced a dose-related increase of [{sup 3}H]thymidine incorporation. The present study indicates that ODN, acting through central-type benzodiazepine receptors associated with the GABA{sub A} receptor complex, stimulates DNA synthesis in rat glial cells. These data provide evidence for an autocrine role of endozepines in the control of glial cell proliferation. (Copyright (c) 1999 Elsevier Science B.V., Amsterdam. All rights reserved.)

  6. In silico investigation of the short QT syndrome, using human ventricle models incorporating electromechanical coupling

    Directory of Open Access Journals (Sweden)

    Ismail eAdeniran

    2013-07-01

    Full Text Available Introduction Genetic forms of the Short QT Syndrome (SQTS arise due to cardiac ion channel mutations leading to accelerated ventricular repolarisation, arrhythmias and sudden cardiac death. Results from experimental and simulation studies suggest that changes to refractoriness and tissue vulnerability produce a substrate favourable to re-entry. Potential electromechanical consequences of the SQTS are less well understood. The aim of this study was to utilize electromechanically coupled human ventricle models to explore electromechanical consequences of the SQTS. Methods and results: The Rice et al. mechanical model was coupled to the ten Tusscher et al. ventricular cell model. Previously validated K+ channel formulations for SQT variants 1 and 3 were incorporated. Functional effects of the SQTS mutations on transients, sarcomere length shortening and contractile force at the single cell level were evaluated with and without the consideration of stretch activated channel current (Isac. Without Isac, the SQTS mutations produced dramatic reductions in the amplitude of transients, sarcomere length shortening and contractile force. When Isac was incorporated, there was a considerable attenuation of the effects of SQTS-associated action potential shortening on Ca2+ transients, sarcomere shortening and contractile force. Single cell models were then incorporated into 3D human ventricular tissue models. The timing of maximum deformation was delayed in the SQTS setting compared to control. Conclusion: The incorporation of Isac appears to be an important consideration in modelling functional effects of SQT 1 and 3 mutations on cardiac electro-mechanical coupling. Whilst there is little evidence of profoundly impaired cardiac contractile function in SQTS patients, our 3D simulations correlate qualitatively with reported evidence for dissociation between ventricular repolarization and the end of mechanical systole.

  7. Incorporating genomic, transcriptomic and clinical data: a prognostic and stem cell-like MYC and PRC imbalance in high-risk neuroblastoma.

    Science.gov (United States)

    Yang, Xinan Holly; Tang, Fangming; Shin, Jisu; Cunningham, John M

    2017-10-03

    the PRC2-mediated tumor cell growth and differentiation in neuroblastoma, which may exert oncogenic effects together with MYC regulation. Our results propose a prognostic effect of imbalanced MYC and PRC2 moderations in pediatric HR-NB for the first time. This study demonstrates an incorporation of genomic landscapes and transcriptomic profiles into the hypothesis-driven precision prognosis and biomarker discovery. The application of this approach to neuroblastoma, as well as other cancer more broadly, could contribute to reduced relapse and mortality rates in the long term.

  8. Experimental analysis of embryogenesis of cerebellum in rat. II. Morphogenetic malformations following x-ray irradiation on day 18 of gestation

    International Nuclear Information System (INIS)

    Das, G.D.

    1977-01-01

    Rat embryos of 18 days gestation were exposed in utero to 170 R of x-ray irradiation. Embryos were collected six hours, 1, 2, and 3 days after irradiation, and animals of 2-, 6-, 15- and 30-day-old postnatal age were sacrificed. Six hours after irradiation pyknosis of cells was noticed in the external granular layer along the posterior aspect of the cerebellum. Neuroblasts, destined to differentiate into Purkinje cells, were found arrested in their migratory path. During subsequent periods of embryogenesis the external granular layer was found recovered, and clustering of the neuroblasts were disorganized and fragmented. This abnormal clustering of neuroblasts was permanent, and the external granular layer followed the same abnormal pattern in its growth. During postnatal development the internal granular layer also was found to follow the abnormal pattern of Purkinje cell layer. Those abnormal developmental events were seen to lead to malformed folia in the anterior regions of the cerebellum. In addition to it the cerebellum of x-ray irradiated animals appeared smaller than the normal. Issues having a bearing on the differential radiosensitivity of different cells, factors determining the small size of the cerebellum, and cellular events determining the morphogenetic malformations are discussed

  9. Incorporation of parallel electrospun fibers for improved topographical guidance in 3D nerve guides

    International Nuclear Information System (INIS)

    Jeffries, Eric M; Wang Yadong

    2013-01-01

    Three dimensional (3D) conduits facilitate nerve regeneration. Parallel microfibers have been shown to guide axon extension and Schwann cell migration on flat sheets via topographical cues. However, incorporation of aligned microfibers into 3D conduits to accelerate nerve regeneration has proven challenging. We report an electrospinning technique to incorporate parallel microfibers into 3D constructs at high surface areas while retaining an open architecture. The nerve guide consists of many microchannels lined with a thin layer of longitudinally-aligned microfibers. This design aims to maximize benefits of topographical cues without inhibiting cellular infiltration. We support this hypothesis by demonstrating efficient cell infiltration in vitro. Additionally, this new technique reduces wall thickness compared to our previous design, providing a greater total area for tissue growth. This approach results in an architecture that very closely mimics the structure of decellularized nerve but with larger microchannel diameters to encourage cell infiltration. We believe that reproducing the native architecture is the first step toward matching autograph efficacy. Furthermore, this design can be combined with other biochemical cues to promote nerve regeneration. (paper)

  10. Methylation of deoxycytidine incorporated by excision-repair synthesis of DNA

    International Nuclear Information System (INIS)

    Kastan, M.B.; Gowans, B.J.; Lieberman, M.W.

    1982-01-01

    Methylation of deoxycytidine incorporated by DNA excision-repair was studied in human diploid fibroblasts following damage with ultraviolet radiation, N-methyl-N-nitrosourea, or N-acetoxy-2-acetylaminofluorene. In confluent, nondividing cells, methylation in repair patches induced by all three agents is slow and incomplete. Whereas after DNA replication in logarithmic-phase cultures a steady state level of 3.4% 5-methylcytosine is reached in less than 2 hr after cells are labeled with 6- 3H-deoxycytidine, following ultraviolet-stimulated repair synthesis in confluent cells it takes about 3 days to reach a level of approximately 2.0% 5-methylcytosine in the repair patch. In cells from cultures in logarithmic-phase growth, 5-methylcytosine formation in ultraviolet-induced repair patches occurs faster and to a greater extent, reaching a level of approximately 2.7% in 10-20 hr. Preexisting hypomethylated repair patches in confluent cells are methylated further when the cells are stimulated to divide; however, the repair patch may still not be fully methylated before cell division occurs. Thus DNA damage and repair may lead to heritable loss of methylation at some sites

  11. Anatomical evidence for brainstem circuits mediating feeding motor programs in the leopard frog, Rana pipiens.

    Science.gov (United States)

    Anderson, C W

    2001-09-01

    Using injections of small molecular weight fluorescein dextran amines, combined with activity-dependent uptake of sulforhodamine 101 (SR101), brainstem circuits presumed to be involved in feeding motor output were investigated. As has been shown previously in other studies, projections to the cerebellar nuclei were identified from the cerebellar cortex, the trigeminal motor nucleus, and the vestibular nuclei. Results presented here suggest an additional pathway from the hypoglossal motor nuclei to the cerebellar nucleus as well as an afferent projection from the peripheral hypoglossal nerve to the Purkinje cell layer of the cerebellar cortex. Injections in the cerebellar cortex combined with retrograde labeling of the peripheral hypoglossal nerve demonstrate anatomical convergence at the level of the medial reticular formation. This suggests a possible integrative region for afferent feedback from the hypoglossal nerve and information through the Purkinje cell layer of the cerebellar cortex. The activity-dependent uptake of SR101 additionally suggests a reciprocal, polysynaptic pathway between this same area of the medial reticular formation and the trigeminal motor nuclei. The trigeminal motor neurons innervate the m adductor mandibulae, the primary mouth-closing muscle. The SR101 uptake clearly labeled the ventrolateral hypoglossal nuclei, the medial reticular formation, and the Purkinje cell layer of the cerebellar cortex. Unlike retrograde labeling of the peripheral hypoglossal nerve, stimulating the hypoglossal nerve while SR101 was bath-applied labeled trigeminal motor neurons. This, combined with the dextran labeling, suggests a reciprocal connection between the trigeminal motor nuclei and the cerebellar nuclei, as well as the medulla. Taken together, these data are important for understanding the neurophysiological pathways used to coordinate the proper timing of an extremely rapid, goal-directed movement and may prove useful for elucidating some of the

  12. Spinocerebellar Ataxia Type 6 Protein Aggregates Cause Deficits in Motor Learning and Cerebellar Plasticity

    NARCIS (Netherlands)

    Mark, Melanie D; Krause, Martin; Boele, Henk-Jan; Kruse, Wolfgang; Pollok, Stefan; Kuner, Thomas; Dalkara, Deniz; Koekkoek, Sebastiaan; De Zeeuw, Chris I; Herlitze, Stefan

    2015-01-01

    Spinocerebellar ataxia type 6 (SCA6) is linked to poly-glutamine (polyQ) within the C terminus (CT) of the pore-forming subunits of P/Q-type Ca(2+) channels (Cav2.1) and is characterized by CT protein aggregates found in cerebellar Purkinje cells (PCs). One hypothesis regarding SCA6 disease is that

  13. Caveats in transneuronal tracing with unmodified rabies virus: an evaluation of aberrant results using a nearly perfect tracing technique

    Directory of Open Access Journals (Sweden)

    Tom J.H. Ruigrok

    2016-07-01

    Full Text Available Apart from the genetically engineered, modified, strains of rabies virus (RABV, unmodified ‘fixed’ virus strains of RABV, such as the ‘French’ subtype of CVS11, are used to examine synaptically connected networks in the brain. This technique has been shown to have all the prerequisite characteristics for ideal tracing as it does not metabolically affect infected neurons within the time span of the experiment, it is transferred transneuronally in one direction only and to all types of neurons presynaptic to the infected neuron, number of transneuronal steps can be precisely controlled by survival time and it is easily detectable with a sensitive technique.Here, using the ‘French’ CVS 11 subtype of RABV in Wistar rats, we show that some of these characteristics may not be as perfect as previously indicated. Using injection of RABV in hind limb muscles, we show that RABV-infected spinal motoneurons may already show lysis 1 or 2 days after infection. Using longer survival times we were able to establish that Purkinje cells may succumb approximately 3 days after infection. In addition, some neurons seem to resist infection, as we noted that the number of RABV-infected inferior olivary neurons did not progress in the same rate as other infected neurons. Furthermore, in our hands, we noted that infection of Purkinje cells did not result in expected transneuronal labeling of cell types that are presynaptic to Purkinje cells such as molecular layer interneurons and granule cells. However, these cell types were readily infected when RABV was injected directly in the cerebellar cortex. Conversely, neurons in the cerebellar nuclei that project to the inferior olive did not take up RABV when this was injected in the inferior olive, whereas these cells could be infected with RABV via a transneuronal route. These results suggest that viral entry from the extracellular space depends on other factors or mechanisms than those used for retrograde

  14. Aquaporin-11: A channel protein lacking apparent transport function expressed in brain

    Directory of Open Access Journals (Sweden)

    Tsunenari Takashi

    2006-05-01

    Full Text Available Abstract Background The aquaporins are a family of integral membrane proteins composed of two subfamilies: the orthodox aquaporins, which transport only water, and the aquaglyceroporins, which transport glycerol, urea, or other small solutes. Two recently described aquaporins, numbers 11 and 12, appear to be more distantly related to the other mammalian aquaporins and aquaglyceroporins. Results We report on the characterization of Aquaporin-11 (AQP11. AQP11 RNA and protein is found in multiple rat tissues, including kidney, liver, testes and brain. AQP11 has a unique distribution in brain, appearing in Purkinje cell dendrites, hippocampal neurons of CA1 and CA2, and cerebral cortical neurons. Immunofluorescent staining of Purkinje cells indicates that AQP11 is intracellular. Unlike other aquaporins, Xenopus oocytes expressing AQP11 in the plasma membrane failed to transport water, glycerol, urea, or ions. Conclusion AQP11 is functionally distinct from other proteins of the aquaporin superfamily and could represent a new aquaporin subfamily. Further studies are necessary to elucidate the role of AQP11 in the brain.

  15. Understanding the Process of Envelope Glycoprotein Incorporation into Virions in Simian and Feline Immunodeficiency Viruses

    Directory of Open Access Journals (Sweden)

    José L. Affranchino

    2014-01-01

    Full Text Available The lentiviral envelope glycoproteins (Env mediate virus entry by interacting with specific receptors present at the cell surface, thereby determining viral tropism and pathogenesis. Therefore, Env incorporation into the virions formed by assembly of the viral Gag polyprotein at the plasma membrane of the infected cells is a key step in the replication cycle of lentiviruses. Besides being useful models of human immunodeficiency virus (HIV infections in humans and valuable tools for developing AIDS therapies and vaccines, simian and feline immunodeficiency viruses (SIV and FIV, respectively are relevant animal retroviruses; the study of which provides important information on how lentiviral replication strategies have evolved. In this review, we discuss the molecular mechanisms underlying the incorporation of the SIV and FIV Env glycoproteins into viral particles.

  16. Effect of epileptogenic agents on the incorporation of /sup 3/H-glycine into proteins in the cat's cerebral cortex

    Energy Technology Data Exchange (ETDEWEB)

    Rojik, I.; Feher, O.

    1982-06-01

    Filter paper strips soaked in /sup 3/H-glycine solution were applied to acoustic cortex of cats, anaesthetized with Nembutal and pretreated with epileptogenic agents (Metrazol, G-penicillin, and 3-amino-pyridine) and cycloheximide. The untreated contralateral hemisphere served as control. After 1 h incubation, both cortical samples were excised simultaneously and fixed in Bouin solution for autoradiography. Incorporation was blocked by cycloheximide. There was no glycine incorporation on the penicillin-treated side, while pyramidal cells were intensively labelled in layers II-V of the mirror focus. 3-Aminopyridine produced the same result. Metrazol as convulsant proved to be far weaker than the previous two. The intensity of incorporation was significantly more intensive in the mirror focus than in the primary one. Penicillin and 3-aminopyridine, while provoking cortical seizures, seem to inhibit glycine incorporation into a neuron-specific, function-dependent protein contained by the labelled cells in the autoradiogram.

  17. Ameliorative effect of Pimpinella anisum oil on immunohistochemical and ultrastuctural changes of cerebellum of albino rats induced by aspartame.

    Science.gov (United States)

    Abdul-Hamid, Manal; Gallaly, Sanaa Rida

    2014-05-01

    The study aims to investigate the protective effect of Pimpinella anisum oil on aspartame (ASP) which resulted in cerebellar changes. The rats were divided into four equal groups: Group 1: (control group): served as control animals. Group 2: control P. anisum oil received .5 mL/kg/d/b wt. once daily. Group 3 (ASP group): received daily 250 mg/kg/b wt. of ASP dissolved in distilled water and given orally to the animals by intra-gastric tube for 2 months. Group 4: received .5 mL/kg/b wt. of prophylactic P. anisum oil once daily, followed by ASP after 2 h for 2 months. The histopathological approach revealed marked changes in the Purkinje cells, myleinated nerve fibers and granular cells of ASP-treated animals. Some of these cells appeared with deeply stained cytoplasm. Ultrastructural examination showed Purkinje cells with dilated rough endoplasmic reticulum and condensed mitochondria. Granular cells appeared with less c nuclei and surrounded by dissolution of most Mossy rosettes structures. Most myelinated nerve fibers showed thickening of myelinated sheath and others showed splitting of their myelin sheath. The histopathological, immunohistochemical and ultrastructural alterations were much less observed in concomitant use of P. anisum oil with ASP. Cerebellar cortex is considered target areas of ASP neurotoxicity, while P. anisum oil, when used in combination with ASP displays a protective action against neurotoxicity.

  18. Gold nanorod-incorporated gelatin-based conductive hydrogels for engineering cardiac tissue constructs.

    Science.gov (United States)

    Navaei, Ali; Saini, Harpinder; Christenson, Wayne; Sullivan, Ryan Tanner; Ros, Robert; Nikkhah, Mehdi

    2016-09-01

    The development of advanced biomaterials is a crucial step to enhance the efficacy of tissue engineering strategies for treatment of myocardial infarction. Specific characteristics of biomaterials including electrical conductivity, mechanical robustness and structural integrity need to be further enhanced to promote the functionalities of cardiac cells. In this work, we fabricated UV-crosslinkable gold nanorod (GNR)-incorporated gelatin methacrylate (GelMA) hybrid hydrogels with enhanced material and biological properties for cardiac tissue engineering. Embedded GNRs promoted electrical conductivity and mechanical stiffness of the hydrogel matrix. Cardiomyocytes seeded on GelMA-GNR hybrid hydrogels exhibited excellent cell retention, viability, and metabolic activity. The increased cell adhesion resulted in abundance of locally organized F-actin fibers, leading to the formation of an integrated tissue layer on the GNR-embedded hydrogels. Immunostained images of integrin β-1 confirmed improved cell-matrix interaction on the hybrid hydrogels. Notably, homogeneous distribution of cardiac specific markers (sarcomeric α-actinin and connexin 43), were observed on GelMA-GNR hydrogels as a function of GNRs concentration. Furthermore, the GelMA-GNR hybrids supported synchronous tissue-level beating of cardiomyocytes. Similar observations were also noted by, calcium transient assay that demonstrated the rhythmic contraction of the cardiomyocytes on GelMA-GNR hydrogels as compared to pure GelMA. Thus, the findings of this study clearly demonstrated that functional cardiac patches with superior electrical and mechanical properties can be developed using nanoengineered GelMA-GNR hybrid hydrogels. In this work, we developed gold nanorod (GNR) incorporated gelatin-based hydrogels with suitable electrical conductivity and mechanical stiffness for engineering functional cardiac tissue constructs (e.g. cardiac patches). The synthesized conductive hybrid hydrogels properly

  19. Incorporation of glucose carbons into rat lung lipids after exposure to 0.6 ppm ozone

    International Nuclear Information System (INIS)

    Bassett, D.J.; Rabinowitz, J.L.

    1985-01-01

    Continuous exposure to low concentrations of ozone has previously been associated with proliferation of lung alveolar type II epithelial cells. In this study, 14 C incorporation into tissue lipids was determined in isolated rat lungs by perfusion with [U- 14 C]glucose, at a time of maximal hyperplasia brought about by 3 days continuous exposure to 0.6 ppm ozone. Ozone exposed lungs exhibited increased rates of glycolytic energy production, indicated by an 89% increase in 3 H 2 O generation on perfusion with [5-3H]glucose. Ozone exposure resulted in enhanced 14 C incorporations into glyceride-glycerol and fatty acid moieties of lung lipids of 95 and 180%, respectively, with a greater proportion of label being recovered in shorter chain fatty acids. Although increased labeling was observed in both neutral and phospholipids, the pattern of 14 C recovery suggested a relative increased glucose carbon incorporation into lung free fatty acids, phosphatidic acid, and such membrane associated lipids as phosphatidylinositol and those containing sphingosine. These results are consistent with the needs of a dividing cell population for enhanced energy production and synthesis of new lipids

  20. Fuel cell subassemblies incorporating subgasketed thrifted membranes

    Science.gov (United States)

    Iverson, Eric J.; Pierpont, Daniel M.; Yandrasits, Michael A.; Hamrock, Steven J.; Obradovich, Stephan J.; Peterson, Donald G.

    2016-03-01

    A fuel cell roll good subassembly is described that includes a plurality of individual electrolyte membranes. One or more first subgaskets are attached to the individual electrolyte membranes. Each of the first subgaskets has at least one aperture and the first subgaskets are arranged so the center regions of the individual electrolyte membranes are exposed through the apertures of the first subgaskets. A second subgasket comprises a web having a plurality of apertures. The second subgasket web is attached to the one or more first subgaskets so the center regions of the individual electrolyte membranes are exposed through the apertures of the second subgasket web. The second subgasket web may have little or no adhesive on the subgasket surface facing the electrolyte membrane.

  1. Microbial turnover and incorporation of organic compounds in oil sand mining reclamation sites

    Science.gov (United States)

    Lappé, M.; Kallmeyer, J.

    2013-12-01

    Microorganisms play an important role in the development of new soils and in the reclamation of disturbed landscapes. Especially in hydrocarbon-contaminated soils their ability to degrade organic matter and pollutants makes them essential to re-establish full ecosystem functionality. Microbes are also involved in the mobilization of nutrients for plant growth and in the production of greenhouse gases. Reclamation sites from oil sand mining activities in Alberta, Canada, contain residual bitumen as well as other hydrocarbons. So, these areas provide a great opportunity to study microbial degradation of residual contaminants from oil sand. To get an impression of degradation rates as well as metabolic pathways, incubation experiments were performed in the lab. We measured microbial turnover (catabolic metabolism) and incorporation (anabolic metabolism) rates of different common organic compounds in samples from differently treated reclamation sites - with plant cover and without plant cover. About 10 g of sample material was suspended in 10 mL of a solution that mimics the in-situ concentration of dissolved ions. Radioactively labelled 14C-acetate was added as a common substrate, whereas 14C-naphthenic acid was chosen to investigate the microbial community's capability to utilize a typical hydrocarbon pollutant in oil sand tailings as a nutrient source. To test for the influence of fertilizers on microbial activity, phosphate, nitrate and potassium were added to some samples in different combinations. Incubations were run over two different time periods (7 and 14 days). At the end of each incubation experiment, the amount of produced 14CO2, 14C incorporated into the cells and the remaining unreacted 14C in the slurry were measured. First results show that most of the added 14C-acetate is used for respiration as it is mostly released as 14CO2. In upper soil layers only about 3% of 14C is incorporated into cells, whereas in deeper horizons with lower cell abundances

  2. Physico-chemical and Biological Evaluation of Flavonols: Fisetin, Quercetin and Kaempferol Alone and Incorporated in beta Cyclodextrins.

    Science.gov (United States)

    Corina, Danciu; Bojin, Florina; Ambrus, Rita; Muntean, Delia; Soica, Codruta; Paunescu, Virgil; Cristea, Mirabela; Pinzaru, Iulia; Dehelean, Cristina

    2017-01-01

    Fisetin,quercetin and kaempferol are among the important representatives of flavonols, biological active phytocomounds, with low water solubility. To evaluate the antimicrobial effect, respectively the antiproliferative and pro apoptotic activity on the B164A5 murine melanoma cell line of pure flavonols and their beta cyclodextrins complexes. Incorporation of fisetin, quercetin and kaempferol in beta cyclodextrins was proved by scanning electron microscopy (SEM), differencial scanning calorimetry (DSC) and X-ray powder diffraction (XRPD). Pure compounds and their complexes were tested for antiproliferative (MTT) and pro-apoptotic activity (Annexin V-PI) on the B164A5 murine melanoma cell line and for the antimicrobial properties (Disk Diffusion Method) on the selected strains. The phytocompounds presented in a different manner in vitro chemopreventive activity against B164A5 murine melanoma cell line and weak antimicrobial effect. The three flavonols: fisetin, quercetin and kaempferol were successfully incorporated in beta-cyclodextrin (BCD) and hydroxylpropyl-beta-cyclodextrin (HPBCD). Incorporation in beta cyclodextrins had a mix effect on the biological activity conducing to decrease, increase or consistent effect compared to pure phytocompound, depending on the screened process and on the chosen combination. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  3. The Incorporation of Chromium in Rice Straw Fermented with Ganoderma lucidum

    Directory of Open Access Journals (Sweden)

    F. Agustin

    2013-04-01

    Full Text Available The research was designed to evaluate chromium incorporation by Ganoderma lucidum in rice straw substrate supplemented with chromium chloride. Treatments were combination of Cr level (0, 500, 1000, 1500, 2000, 25000, and 3000 ppm and fermentation time (0, 2, 3, and 4 wk. The treatments were arranged in a factorial 7 x 4 and allocated in completely randomized design with three replications. G. lucidum was grown in potato dextrose agar (PDA medium for 10 days and was inoculated to the substrate which have been sterilized and mixed with CrCl3.6H2O. The moisture of substrate was maintained at 70%. Fiber and protein components of growth media of G. lucidum was determined and analyzed for their Cr content. The finding result showed that addition of Cr up to 3000 ppm into the substrate stimulated the G. lucidum growth. Chromium was incorporated into the fiber and protein components of the growth substrate of G. lucidum during fermentation. Incorporation of Cr into the protein of substrate containing 3000 ppm Cr was highest when fermented for 4 wk. Protein component of substrate contained 9.29% Cr while in NDF and ADF was 27.20% and 10.55% Cr, respectively. It is concluded that Cr was incorporated into the G. lucidum cells during fermentation.

  4. Incorporation of Novel Nanostructured Materials into Solar Cells and Nanoelectronic Devices

    Energy Technology Data Exchange (ETDEWEB)

    Rodriguez, Rene; Pak, Joshua; Holland, Andrew; Hunt, Alan; Bitterwolf, Thomas; Qiang, You; Bergman, Leah; Berven, Christine; Punnoose, Alex; Tenne, Dmitri

    2011-11-11

    Each of the investigators on this project has had significant accomplishments toward the production of semiconductor nanoparticles, particles, and thin films and attempts to incorporate these materials into photovoltaics or sensors; to use them for improving fluorescence diagnostics; or to employ them as cancer fighting agents. The synthesis and characterization of the nanomaterials, and more recently the device construction and testing of these materials, have been the subject of several publications and presentations by team members. During the course of the investigations, several students were fully involved as part of their graduate and undergraduate training. The nature of these projects in material development dictates that the students have gained significant experience in a diverse array of material-related topics.

  5. Incorporation of antibacterial agent derived deep eutectic solvent into an active dental composite.

    Science.gov (United States)

    Wang, Jing; Dong, Xiaoqing; Yu, Qingsong; Baker, Sheila N; Li, Hao; Larm, Nathaniel E; Baker, Gary A; Chen, Liang; Tan, Jingwen; Chen, Meng

    2017-12-01

    To incorporate an antibacterial agent derived deep eutectic solvent (DES) into a dental resin composite, and investigate the resulting mechanical properties and antibacterial effects. The DES was derived from benzalkonium chloride (BC) and acrylic acid (AA) and was incorporated into the dental resin composite through rapid mixing. A three-point bending test was employed to measure the flexural strength of the composite. An agar diffusion test was used to investigate antibacterial activity. Artificial (accelerated) aging was undertaken by immersing the composites in buffer solutions at an elevated temperature for up to 4 weeks. UV-vis spectrophotometry and NMR analysis were conducted to study BC release from the composite. Finally, the biocompatibility of the composite materials was evaluated using osteoblast cell culture for 7 days. Results were compared to those of a control composite which contained no BC. The DES-incorporated composite (DES-C) displayed higher flexural strength than a similar BC-incorporated composite BC (BC-C) for the same level of BC. The inclusion of BC conferred antibacterial activity to both BC-containing composites, although BC-C produced larger inhibition halos than DES-C at the same loading of BC. Control composites which contained no BC showed negligible antibacterial activity. After artificial aging, the DES-C composite showed better maintenance of the mechanical properties of the control compared with BC-C, although a decrease was observed during the three-point bending test, particularly upon storage at elevated temperatures. No BC release was detected in the aged solutions of DES-C, whereas the BC-C showed a linear increase in BC release with storage time. Significantly, cell viability results indicated that DES-C has better biocompatibility than BC-C. The incorporation of a BC-based DES into a dental resin composite provides a new strategy to develop antibacterial dental materials with better biocompatibility and longer effective

  6. Adhesion of Candida albicans to Vanillin Incorporated Self-Curing Orthodontic PMMA Resin.

    Science.gov (United States)

    Zam, K.; Sawaengkit, P.; Thaweboon, S.; Thaweboon, B.

    2018-02-01

    It has been observed that there is an increase in Candida carriers during the treatment with orthodontic removable appliance. Vanillin is flavouring agent, which is known to have antioxidant and antimicrobial properties. The aim of this study was to evaluate the effect of vanillin incorporated PMMA on adhesion of Candida albicans. A total of 36 orthodontic self-curing PMMA resin samples were fabricated. The samples were divided into 3 groups depending on percentage of vanillin incorporated (0.1%, 0.5% and PMMA without vanillin as control). PMMA samples were coated with saliva. The adhesion assay was performed with C. albicans (ATCC 10231). The adherent yeast cells were stained with crystal violet and counted under microscope by random selection of 3 fields at 10X magnification. The statistical analyses performed by Kruskal Wallis and Mann Whitney non-parametric test. It was found that the PMMA resin samples with vanillin incorporation significantly reduced the adhesion of C. albicans as compared to the control group. This study indicates that vanillin incorporated resin can impede the adhesion of C. albicans to about 45 - 56 %. With further testing and development, vanillin can be employed as an antifungal agent to prevent adhesion of C. albicans to orthodontic self-curing PMMA resin.

  7. Tub-Tag Labeling; Chemoenzymatic Incorporation of Unnatural Amino Acids.

    Science.gov (United States)

    Helma, Jonas; Leonhardt, Heinrich; Hackenberger, Christian P R; Schumacher, Dominik

    2018-01-01

    Tub-tag labeling is a chemoenzymatic method that enables the site-specific labeling of proteins. Here, the natural enzyme tubulin tyrosine ligase incorporates noncanonical tyrosine derivatives to the terminal carboxylic acid of proteins containing a 14-amino acid recognition sequence called Tub-tag. The tyrosine derivative carries a unique chemical reporter allowing for a subsequent bioorthogonal modification of proteins with a great variety of probes. Here, we describe the Tub-tag protein modification protocol in detail and explain its utilization to generate labeled proteins for advanced applications in cell biology, imaging, and diagnostics.

  8. Scheduling a Single Mobile Robot Incorporated into Production Environment

    DEFF Research Database (Denmark)

    Dang, Vinh Quang; Nielsen, Izabela Ewa; Steger-Jensen, Kenn

    2013-01-01

    to the challenges of issues such as energy conservation and pollution preventions. Facing the central tension between manufacturing and environmental drivers is difficult, but critical to develop new technologies, particularly mobile robots, that can be incorporated into production to achieve holistic solutions....... This chapter deals with the problem of finding optimal operating sequence in a manufacturing cell of a mobile robot with manipulation arm that feeds materials to feeders. The “Bartender Concept” is discussed to show the cooperation between the mobile robot and industrial environment. The performance criterion...

  9. Direct imaging of glycans in Arabidopsis roots via click labeling of metabolically incorporated azido-monosaccharides.

    Science.gov (United States)

    Hoogenboom, Jorin; Berghuis, Nathalja; Cramer, Dario; Geurts, Rene; Zuilhof, Han; Wennekes, Tom

    2016-10-10

    Carbohydrates, also called glycans, play a crucial but not fully understood role in plant health and development. The non-template driven formation of glycans makes it impossible to image them in vivo with genetically encoded fluorescent tags and related molecular biology approaches. A solution to this problem is the use of tailor-made glycan analogs that are metabolically incorporated by the plant into its glycans. These metabolically incorporated probes can be visualized, but techniques documented so far use toxic copper-catalyzed labeling. To further expand our knowledge of plant glycobiology by direct imaging of its glycans via this method, there is need for novel click-compatible glycan analogs for plants that can be bioorthogonally labelled via copper-free techniques. Arabidopsis seedlings were incubated with azido-containing monosaccharide analogs of N-acetylglucosamine, N-acetylgalactosamine, L-fucose, and L-arabinofuranose. These azido-monosaccharides were metabolically incorporated in plant cell wall glycans of Arabidopsis seedlings. Control experiments indicated active metabolic incorporation of the azido-monosaccharide analogs into glycans rather than through non-specific absorption of the glycan analogs onto the plant cell wall. Successful copper-free labeling reactions were performed, namely an inverse-electron demand Diels-Alder cycloaddition reaction using an incorporated N-acetylglucosamine analog, and a strain-promoted azide-alkyne click reaction. All evaluated azido-monosaccharide analogs were observed to be non-toxic at the used concentrations under normal growth conditions. Our results for the metabolic incorporation and fluorescent labeling of these azido-monosaccharide analogs expand the possibilities for studying plant glycans by direct imaging. Overall we successfully evaluated five azido-monosaccharide analogs for their ability to be metabolically incorporated in Arabidopsis roots and their imaging after fluorescent labeling. This expands

  10. Incorporation of ophiobolin a into novel chemoembolization particles for cancer cell treatment.

    Science.gov (United States)

    Morrison, Rachel; Gardiner, Chris; Evidente, Antonio; Kiss, Robert; Townley, Helen

    2014-10-01

    To design and synthesize chemoembolization particles for the delivery of Ophiobolin A (OphA), a promising fungal-derived chemotherapeutic, directly at the tumour location. To investigate cell death mechanism of OphA on a Rhabdomyosarcoma cancer (RD) cell line. Rhabdomyosarcoma is the most common soft tissue sarcoma in children; with a 5-year survival rate of between 30 and 65%. Multimodal chemoembolization particles were prepared by sintering mesoporous silica nanoparticles, prepared by the sol-gel method, onto the surface of polystyrene microspheres, prepared by suspension copolymerisation. The chemoembolization particles were subsequently loaded with OphA. The effects of OphA in vitro were characterised by flow cytometry and nanoparticle tracking analysis (NanoSight). High loading of OphA onto the chemoembolization particles was achieved. The subsequent release of OphA onto RD cells in culture showed a 70% reduction in cell viability. OphA caused RD cells to round up and their membrane to bleb and caused cell death via apoptosis. OphA caused both an increase in the number of microvesicles produced and an increase in DNA content within these microvesicles. The prepared chemoembolization particles showed good efficacy against RD cells in culture.

  11. Ionic liquid pretreatment of poplar wood at room temperature: swelling and incorporation of nanoparticles.

    Science.gov (United States)

    Lucas, Marcel; Macdonald, Brian A; Wagner, Gregory L; Joyce, Stephen A; Rector, Kirk D

    2010-08-01

    Lignocellulosic biomass offers economic and environmental advantages over corn starch for biofuels production. However, its fractionation currently requires energy-intensive pretreatments, due to the lignin chemical resistance and complex cell wall structure. Recently, ionic liquids have been used to dissolve biomass at high temperatures. In this study, thin sections of poplar wood were swollen by ionic liquid (1-ethyl-3-methylimidazolium acetate) pretreatment at room temperature. The samples contract when rinsed with deionized water. The controlled expansion and contraction of the wood structure can be used to incorporate enzymes and catalysts deep into the wood structure for improved pretreatments and accelerated cellulose hydrolysis. As a proof of concept, silver and gold nanoparticles of diameters ranging from 20 to 100 nm were incorporated at depths up to 4 mum. Confocal surface-enhanced Raman images at different depths show that a significant number of nanoparticles were incorporated into the pretreated sample, and they remained on the samples after rinsing. Quantitative X-ray fluorescence microanalyses indicate that the majority of nanoparticle incorporation occurs after an ionic liquid pretreatment of less than 1 h. In addition to improved pretreatments, the incorporation of materials and chemicals into wood and paper products enables isotope tracing, development of new sensing, and imaging capabilities.

  12. Numeral Incorporation in Japanese Sign Language

    Science.gov (United States)

    Ktejik, Mish

    2013-01-01

    This article explores the morphological process of numeral incorporation in Japanese Sign Language. Numeral incorporation is defined and the available research on numeral incorporation in signed language is discussed. The numeral signs in Japanese Sign Language are then introduced and followed by an explanation of the numeral morphemes which are…

  13. Kaempferol nanoparticles achieve strong and selective inhibition of ovarian cancer cell viability

    Science.gov (United States)

    Luo, Haitao; Jiang, Bingbing; Li, Bingyun; Li, Zhaoliang; Jiang, Bing-Hua; Chen, Yi Charlie

    2012-01-01

    Ovarian cancer is one of the leading causes of cancer death for women throughout the Western world. Kaempferol, a natural flavonoid, has shown promise in the chemoprevention of ovarian cancer. A common concern about using dietary supplements for chemoprevention is their bioavailability. Nanoparticles have shown promise in increasing the bioavailability of some chemicals. Here we developed five different types of nanoparticles incorporating kaempferol and tested their efficacy in the inhibition of viability of cancerous and normal ovarian cells. We found that positively charged nanoparticle formulations did not lead to a significant reduction in cancer cell viability, whereas nonionic polymeric nanoparticles resulted in enhanced reduction of cancer cell viability. Among the nonionic polymeric nanoparticles, poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) nanoparticles incorporating kaempferol led to significant reduction in cell viability of both cancerous and normal cells. Poly(DL-lactic acid-co-glycolic acid) (PLGA) nanoparticles incorporating kaempferol resulted in enhanced reduction of cancer cell viability together with no significant reduction in cell viability of normal cells compared with kaempferol alone. Therefore, both PEO-PPO-PEO and PLGA nanoparticle formulations were effective in reducing cancer cell viability, while PLGA nanoparticles incorporating kaempferol had selective toxicity against cancer cells and normal cells. A PLGA nanoparticle formulation could be advantageous in the prevention and treatment of ovarian cancers. On the other hand, PEO-PPO-PEO nanoparticles incorporating kaempferol were more effective inhibitors of cancer cells, but they also significantly reduced the viability of normal cells. PEO-PPO-PEO nanoparticles incorporating kaempferol may be suitable as a cancer-targeting strategy, which could limit the effects of the nanoparticles on normal cells while retaining their potency against cancer cells. We

  14. High frequency switched-mode stimulation can evoke postsynaptic responses in cerebellar principal neurons

    Directory of Open Access Journals (Sweden)

    Marijn Van Dongen

    2015-03-01

    Full Text Available This paper investigates the efficacy of high frequency switched-mode neural stimulation. Instead of using a constant stimulation amplitude, the stimulus is switched on and off repeatedly with a high frequency (up to 100kHz duty cycled signal. By means of tissue modeling that includes the dynamic properties of both the tissue material as well as the axon membrane, it is first shown that switched-mode stimulation depolarizes the cell membrane in a similar way as classical constant amplitude stimulation.These findings are subsequently verified using in vitro experiments in which the response of a Purkinje cell is measured due to a stimulation signal in the molecular layer of the cerebellum of a mouse. For this purpose a stimulator circuit is developed that is able to produce a monophasic high frequency switched-mode stimulation signal. The results confirm the modeling by showing that switched-mode stimulation is able to induce similar responses in the Purkinje cell as classical stimulation using a constant current source. This conclusion opens up possibilities for novel stimulation designs that can improve the performance of the stimulator circuitry. Care has to be taken to avoid losses in the system due to the higher operating frequency.

  15. Lack of connexin43-mediated Bergmann glial gap junctional coupling does not affect cerebellar long-term depression, motor coordination, or eyeblink conditioning

    Directory of Open Access Journals (Sweden)

    Mika Tanaka

    2008-04-01

    Full Text Available Bergmann glial cells are specialized astrocytes in the cerebellum. In the mature cerebellar molecular layer, Bergmann glial processes are closely associated with Purkinje cells, enclosing Purkinje cell dendritic synapses with a glial sheath. There is intensive gap junctional coupling between Bergmann glial processes, but their significance in cerebellar functions is not known. Connexin43 (Cx43, a major component of astrocytic gap junction channels, is abundantly expressed in Bergmann glial cells. To examine the role of Cx43-mediated gap junctions between Bergmann glial cells in cerebellar functions, we generated Cx43 conditional knockout mice with the S100b-Cre transgenic line (Cx43fl/fl:S100b-Cre, which exhibited a significant loss of Cx43 in the Bergmann glial cells and astrocytes in the cerebellum with a postnatal onset. The Cx43fl/fl:S100b-Cre mice had normal cerebellar architecture. Although gap junctional coupling between the Bergmann glial cells measured by spreading of microinjected Lucifer yellow was virtually abolished in Cx43fl/fl:S100b-Cre mice, electrophysiologic analysis revealed that cerebellar long-term depression could be induced and maintained normally in thier cerebellar slices. In addition, at the behavioral level, Cx43fl/fl:S100b-Cre mice had normal motor coordination in the rotarod task and normal conditioned eyelid response. Our findings suggest that Cx43-mediated gap junctional coupling between Bergmann glial cells is not necessary for the neuron-glia interactions required for cerebellum-dependent motor coordination and motor learning.

  16. Nitric oxide-induced calcium release: activation of type 1 ryanodine receptor by endogenous nitric oxide.

    Science.gov (United States)

    Kakizawa, Sho; Yamazawa, Toshiko; Iino, Masamitsu

    2013-01-01

    Ryanodine receptors (RyRs), located in the sarcoplasmic/endoplasmic reticulum (SR/ER) membrane, are required for intracellular Ca2+ release that is involved in a wide range of cellular functions. In addition to Ca2+-induced Ca2+ release in cardiac cells and voltage-induced Ca2+ release in skeletal muscle cells, we recently identified another mode of intracellular Ca2+ mobilization mediated by RyR, i.e., nitric oxide-induced Ca2+ release (NICR), in cerebellar Purkinje cells. NICR is evoked by neuronal activity, is dependent on S-nitrosylation of type 1 RyR (RyR1) and is involved in the induction of long-term potentiation (LTP) of cerebellar synapses. In this addendum, we examined whether peroxynitrite, which is produced by the reaction of nitric oxide with superoxide, may also have an effect on the Ca2+ release via RyR1 and the cerebellar LTP. We found that scavengers of peroxynitrite have no significant effect either on the Ca2+ release via RyR1 or on the cerebellar LTP. We also found that an application of a high concentration of peroxynitrite does not reproduce neuronal activity-dependent Ca2+ release in Purkinje cells. These results support that NICR is induced by endogenous nitric oxide produced by neuronal activity through S-nitrosylation of RyR1.

  17. Anoctamin Calcium-Activated Chloride Channels May Modulate Inhibitory Transmission in the Cerebellar Cortex.

    Directory of Open Access Journals (Sweden)

    Weiping Zhang

    Full Text Available Calcium-activated chloride channels of the anoctamin (alias TMEM16 protein family fulfill critical functions in epithelial fluid transport, smooth muscle contraction and sensory signal processing. Little is known, however, about their contribution to information processing in the central nervous system. Here we examined the recent finding that a calcium-dependent chloride conductance impacts on GABAergic synaptic inhibition in Purkinje cells of the cerebellum. We asked whether anoctamin channels may underlie this chloride conductance. We identified two anoctamin channel proteins, ANO1 and ANO2, in the cerebellar cortex. ANO1 was expressed in inhibitory interneurons of the molecular layer and the granule cell layer. Both channels were expressed in Purkinje cells but, while ANO1 appeared to be retained in the cell body, ANO2 was targeted to the dendritic tree. Functional studies confirmed that ANO2 was involved in a calcium-dependent mode of ionic plasticity that reduces the efficacy of GABAergic synapses. ANO2 channels attenuated GABAergic transmission by increasing the postsynaptic chloride concentration, hence reducing the driving force for chloride influx. Our data suggest that ANO2 channels are involved in a Ca2+-dependent regulation of synaptic weight in GABAergic inhibition. Thus, in balance with the chloride extrusion mechanism via the co-transporter KCC2, ANO2 appears to regulate ionic plasticity in the cerebellum.

  18. Assessment of 3H-TdR incorporation for the viability of bone marrow hematopoietic stem cells after freezing and thawing

    International Nuclear Information System (INIS)

    Zhang Hongquan; Shen Baijun; Fu Zengju

    1993-01-01

    The authors have studied 3 H-TdR incorporation rates of bone marrow MNC at different concentrations and incubation periods. The rates are compared with the viability of MNC preserved at different range of time and temperatures by means of 3 H-TdR incorporation combined with Trypan Blue and CFU-GM culture methods. The result suggested that 3 H-TdR incorporation was a sensitive and reliable assess to evaluate the viability of bone marrow MNC after cryopreservation and thawing

  19. Hydrogen Incorporation during Aluminium Anodisation on Silicon Wafer Surfaces

    International Nuclear Information System (INIS)

    Lu, Pei Hsuan Doris; Strutzberg, Hartmuth; Wenham, Stuart; Lennon, Alison

    2014-01-01

    Hydrogen can act to reduce recombination at silicon surfaces for solar cell devices and consequently the ability of dielectric layers to provide a source of hydrogen for this purpose is of interest. However, due to the ubiquitous nature of hydrogen and its mobility, direct measurements of hydrogen incorporation in dielectric layers are challenging. In this paper, we report the use of secondary ion mass spectrometry measurements to show that deuterium from an electrolyte can be incorporated in an anodic aluminium oxide (AAO) layer and be introduced into an underlying amorphous silicon layer during anodisation of aluminium on silicon wafers. After annealing at 400 °C, the concentration of deuterium in the AAO was reduced by a factor of two, as the deuterium was re-distributed to the interface between the amorphous silicon and AAO and to the amorphous silicon. The assumption that hydrogen, from an aqueous electrolyte, could be similarly incorporated in AAO, is supported by the observation that the hydrogen content in the underlying amorphous silicon was increased by a factor of ∼ 3 after anodisation. Evidence for hydrogen being introduced into crystalline silicon after aluminium anodisation was provided by electrochemical capacitance voltage measurements indicating boron electrical deactivation in the underlying crystalline silicon. If introduced hydrogen can electrically deactivate dopant atoms at the surface, then it is reasonable to assume that it could also deactivate recombination-active states at the crystalline silicon interface therefore enabling higher minority carrier lifetimes in the silicon wafer

  20. Effects of hyaluronic acid- chitosan-gelatin complex on the apoptosis and cell cycle of L929 cells

    Institute of Scientific and Technical Information of China (English)

    MAO Jinshu; WANG Xianghui; CUI Yuanlu; YAO Kangde

    2003-01-01

    With the development in the field of tissue engineering, the interaction between biomaterials and cells has been deeply studied. Viewing the cells seeded on the surface of materials as an organic whole, cell cycle and apoptosis are analyzed to deepen the study of cell compatibility on biomaterials, while cellproliferation and differentiation are studied at the same time. In this paper, hyaluronic acid is incorporated into the chitosan-gelatin system. Propidium iodide (PI) was used in cell cycle analysis and the double-staining of cells with annexin-V and PI was applied in cell apoptosis analysis. The results show that incorporated hyaluronic acid shortens the adaptation period of cells on the material surface, and then cells enter the normal cell cycle quickly. In addition, added hyaluronic acid inhibits cell apoptosis triggered by the membranes. Therefore,hyaluronic acid improves the cell compatibility of chitosan-gelatin system and benefits the design of biomimetic materials.

  1. Preparation and evaluation of unilamellar liposomes incorporating boron-containing derivatives of cholesterol

    International Nuclear Information System (INIS)

    Feakes, D.A.; Tate, C.C.; Stefanutti, S.J.

    2000-01-01

    The application of boron neutron capture therapy is dependent on the identification and preparation of boron-containing compounds that can be delivered and retained by the tumor cells. Unilamellar liposomes have been investigated as potential tumor-specific delivery vehicles for boron-containing compounds that have no inherent tumor specificity. A series of carborane-containing derivatives of cholesterol have been prepared and incorporated into the bilayer of unilamellar liposomes. The cholesterol derivatives vary in the linker moiety (ester and ether), the chain length between the cholesterol and the carborane substituent, and the identity of the carborane group itself (closo- and nido-). The ability of the boron-containing derivatives of cholesterol to be incorporated into the bilayer of the unilamellar liposomes and the stability of the resulting liposome formulations will be presented. (author)

  2. Postresuscitative Changes of Brain-Derived Neurotrophic Factor (BDNF Protein Expression: Association With Neuronal Death

    Directory of Open Access Journals (Sweden)

    M. Sh. Avrushchenko

    2017-01-01

    Full Text Available Aim of the study: to evaluate expression level of BDNF and its association with the postresuscitative neuronal death in highly hypoxia-sensitive brain regions.Materials and methods. Cardiac arrest in adult albino male rats was evoked by intrathoracic clamping of supracardiac bundle of vessels for 10 min. Pyramidal neurons of the hippocampus and Purkinje cells of the cerebellum were analyzed at various time points after resuscitation (days 1, 4, 7, 14. Shame-operated rats served as controls. The expression of BDNF protein was immunohistochemically determined. The BDNF expression level was determined by evalution on the base of the average optical density. The number of neurons with different BDNF expression levels and the total number of neurons per 1 mm of the layer length were computed. Image analysis systems (Intel personal computer, Olympus BX-41 microscope, ImageScopeM, ImageJ 1,48v and MS Excel 2007 software packages were used in the study. Data statistical processing was performed with the aid of Statistica 7.0 program and Kolmogorov-Smirnov λ-test, Mann-Whitney U-test and Student's t-test.Results. The dynamics of postresuscitative shifts of BDNF immunoreactivity in neuronal populations of hippocampal pyramidal cells and cerebellar Purkinje cells was established. It was shown that the level of BDNF expression within the two neuronal populations decreased, that was accompanied by neuronal death. In the Purkinje cell population the neuronal death occurred by the 4th day after resuscitation, while in the hippocampus, it occurs only by the 7th day. Notably, only BDNF-negative neurons or neurons with low level of BDNF expression died in both neuronal populations.Conclusion. The results of the study indicate the existence of an interrelation between the shifts in BDNF expression and the postresuscitative neuronal death. It was shown that only the cells with none or poor BDNF expression underwent death in highly hypoxia-sensitive neuronal

  3. Incorporation of a prolyl hydroxylase inhibitor into scaffolds: a strategy for stimulating vascularization.

    Science.gov (United States)

    Sham, Adeline; Martinez, Eliana C; Beyer, Sebastian; Trau, Dieter W; Raghunath, Michael

    2015-03-01

    Clinical applications of tissue engineering are constrained by the ability of the implanted construct to invoke vascularization in adequate extent and velocity. To overcome the current limitations presented by local delivery of single angiogenic factors, we explored the incorporation of prolyl hydroxylase inhibitors (PHIs) into scaffolds as an alternative vascularization strategy. PHIs are small molecule drugs that can stabilize the alpha subunit of hypoxia-inducible factor-1 (HIF-1), a key transcription factor that regulates a variety of angiogenic mechanisms. In this study, we conjugated the PHI pyridine-2,4-dicarboxylic acid (PDCA) through amide bonds to a gelatin sponge (Gelfoam(®)). Fibroblasts cultured on PDCA-Gelfoam were able to infiltrate and proliferate in these scaffolds while secreting significantly more vascular endothelial growth factor than cells grown on Gelfoam without PDCA. Reporter cells expressing green fluorescent protein-tagged HIF-1α exhibited dose-dependent stabilization of this angiogenic transcription factor when growing within PDCA-Gelfoam constructs. Subsequently, we implanted PDCA-Gelfoam scaffolds into the perirenal fat tissue of Sprague Dawley rats for 8 days. Immunostaining of explants revealed that the PDCA-Gelfoam scaffolds were amply infiltrated by cells and promoted vascular ingrowth in a dose-dependent manner. Thus, the incorporation of PHIs into scaffolds appears to be a feasible strategy for improving vascularization in regenerative medicine applications.

  4. The Incorporation of Strontium to Improve Bone-Regeneration Ability of Mesoporous Bioactive Glasses

    Directory of Open Access Journals (Sweden)

    Sonia Fiorilli

    2018-04-01

    Full Text Available Over the recent years, mesoporous bioactive glasses (MBGs gained interest as bone regeneration systems, due to their excellent bioactivity and ability to release therapeutic molecules. In order to improve the bone regeneration ability of MBGs, the incorporation of Sr2+ ions, due to its recognized pro-osteogenenic potential, represents a very promising strategy. In this study, MBGs based on the SiO2–CaO system and containing different percentages (2 and 4 mol % of strontium were prepared by two synthesis methods, in the form of microspheres and nanoparticles. Sr-containing MBGs were characterized by FE-SEM, XRD and N2 adsorption/desorption analysis. The in vitro bioactivity in SBF resulted excellent. The assessment of fibroblast cell (line L929 viability showed that Sr-containing MBGs were biocompatible both in form of micro- and nanoparticles. The osteogenic response of osteoblast-like SAOS-2 cells was investigated by analysing the expression of GAPDH, COL1a1, RANKL, SPARC, OPG and ALPL genes, as cell differentiation markers. The results indicate that the incorporation of Sr into MBG is beneficial for bone regeneration as promotes a pro-osteogenic effect, paving the way to the design of advanced devices enabled by these nanocarriers also in combination with drug release, for the treatment of bone pathologies, particularly in patients with osteoporosis.

  5. Functional analysis of protein N-myristoylation: Metabolic labeling studies using three oxygen-substituted analogs of myristic acid and cultured mammalian cells provide evidence for protein-sequence-specific incorporation and analog-specific redistribution

    International Nuclear Information System (INIS)

    Johnson, D.R.; Heuckeroth, R.O.; Gordon, J.I.; Cox, A.D.; Solski, P.A.; Buss, J.E.; Devadas, B.; Adams, S.P.; Leimgruber, R.M.

    1990-01-01

    Covalent attachment of myristic acid (C14:0) to the NH 2 -terminal glycine residue of a number of cellular, viral, and oncogene-encoded proteins is essential for full expression of their biological function. Substitution of oxygen for methylene groups in this fatty acid does not produce a significant change in chain length or stereochemistry but does result in a reduction in hydrophobicity. These heteroatom-containing analogs serve as alternative substrates for mammalian myristoyl-CoA: protein N-myristoyltransferase and offer the opportunity to explore structure/function relationships of myristate in N-myristoyltransferase proteins. The authors have synthesized three tritiated analogs of myristate with oxygen substituted for methylene groups at C6, C11, and C13. Metabolic labeling studies were performed with these compounds and (i) a murine myocyte cell line (BC 3 H1), (ii) a rat fibroblast cell that produces p60 v-src (3Xsrc), or (iii) NIH 3T3 cells that have been engineered to express a fusion protein consisting of an 11-residue myristoylation signal from the Rasheed sarcoma virus (RaSV) gag protein linked to c-Ha-ras with a Cys → Ser-186 mutation. Two-dimensional gel electrophoresis of membrane and soluble fractions prepared from cell lysates revealed different patterns of incorporation of the analogs into cellular N-myristoyl proteins. The demonstration that these analogs differ in the extent to which they are incorporated and in their ability to cause redistribution of any single protein suggests that they may also have sufficient selectivity to be of potential therapeutic value

  6. Deficient incorporation of spike protein into virions contributes to the lack of infectivity following establishment of a persistent, non-productive infection in oligodendroglial cell culture by murine coronavirus

    International Nuclear Information System (INIS)

    Liu Yin; Herbst, Werner; Cao Jianzhong; Zhang Xuming

    2011-01-01

    Infection of mouse oligodendrocytes with a recombinant mouse hepatitis virus (MHV) expressing a green fluorescence protein facilitated specific selection of virus-infected cells and subsequent establishment of persistence. Interestingly, while viral genomic RNAs persisted in infected cells over 14 subsequent passages with concomitant synthesis of viral subgenomic mRNAs and structural proteins, no infectious virus was isolated beyond passage 2. Further biochemical and electron microscopic analyses revealed that virions, while assembled, contained little spike in the envelope, indicating that lack of infectivity during persistence was likely due to deficiency in spike incorporation. This type of non-lytic, non-productive persistence in oligodendrocytes is unique among animal viruses and resembles MHV persistence previously observed in the mouse central nervous system. Thus, establishment of such a culture system that can recapitulate the in vivo phenomenon will provide a powerful approach for elucidating the mechanisms of coronavirus persistence in glial cells at the cellular and molecular levels.

  7. Leucine incorporation by aerobic anoxygenic phototrophic bacteria in the Delaware estuary

    OpenAIRE

    Stegman, Monica R; Cottrell, Matthew T; Kirchman, David L

    2014-01-01

    Aerobic anoxygenic phototrophic (AAP) bacteria are well known to be abundant in estuaries, coastal regions and in the open ocean, but little is known about their activity in any aquatic ecosystem. To explore the activity of AAP bacteria in the Delaware estuary and coastal waters, single-cell 3H-leucine incorporation by these bacteria was examined with a new approach that combines infrared epifluorescence microscopy and microautoradiography. The approach was used on samples from the Delaware c...

  8. Transmutation of americium and curium incorporated in zirconia-based host materials

    International Nuclear Information System (INIS)

    Raison, P.E.

    2001-01-01

    Presented are studies involving the incorporation of americium and curium in zirconia-based materials. First explored was the pseudo ternary system AmO 2 -ZrO 2 -Y 2 O 3 . It was determined that selected Y-CSZ materials can incorporate significant quantities of americium oxide and remain cubic single-phase. The cell parameters of these fluorite-type products were established to be linear with the AmO 2 content. The Cm 2 O 3 -ZrO 2 system was also investigated. It was found that at 25 mol% of CmO 1.5 , the Cm(III) stabilized zirconia in its cubic form (a = 5.21 ±0.01 Angstrom). At higher and lower concentrations, diphasic materials were encountered. At 50 mol% of CmO 1.5 , a pyrochlore oxide - Cm 2 Zr 2 O 7 - is formed (a = 10.63 ±0.02 Angstrom). (author)

  9. Human cathepsin L rescues the neurodegeneration and lethality incathepsin B/L double deficient mice

    Energy Technology Data Exchange (ETDEWEB)

    Sevenich, Lisa; Pennacchio, Len A.; Peters, Christoph; Reinheckel, Thomas

    2006-01-09

    Cathepsin B (CTSB) and cathepsin L (CTSL) are two widelyexpressed cysteine proteases thought to predominantly reside withinlysosomes. Functional analysis of CTSL in humans is complicated by theexistence of two CTSL-like homologues (CTSL and CTSL2), in contrast tomice which contain only one CTSL enzyme. Thus transgenic expression ofhuman CTSL in CTSL deficient mice provides an opportunity to study the invivo functions of this human protease without interference by its highlyrelated homologue. While mice with single gene deficiencies for murineCTSB or CTSL survive without apparent neuromuscular impairment, murineCTSB/CTSL double deficient mice display degeneration of cerebellarPurkinje cells and neurons of the cerebral cortex, resulting in severehypotrophy, motility defects, and lethality during their third to fourthweek of life. Here we show that expression of human CTSL through agenomic transgene results in widespread expression of human CTSL in themouse which is capable of rescuing the lethality found in CTSB/CTSLdouble-deficient animals. Human CTSL is expressed in the brain of thesecompound mutants predominantly in neurons of the cerebral cortex and inPurkinje cells of the cerebellum, where it appears to prevent neuronalcell death.

  10. Effects of turtle oil on insulin sensitivity and glucose metabolism in insulin resistant cell model

    International Nuclear Information System (INIS)

    Bai Jing; Tian Yaping; Guo Duo

    2007-01-01

    To evaluate the effects of turtle oil on insulin sensitivity and glucose metabolism in an insulin-resistant (IR) cell model which was established by the way of high concentration of insulin induction with HepG 2 cell in vitro culture. The IR cells were treated by turtle oil, the glucose consumption and 3 H-D-glucose incorporation rate in IR cells were detected by the way of glucose oxidase and 3 H-D-glucose incorporation assay respectively. The state of cell proliferation was tested by MTT method. The results showed that the incorporation rate of 3 H-D-glucose in IR cells was significantly lower than that in the control cells(P 3 H-D-glucose incorporation rate in either IR cells or control cells was increased with the increase of insulin concentration. Moreover, the 3 H-D-glucose incorporation rate of IR cells increased slower than that of control cells. The MTT assay showed that turtle oil can promote the proliferation of IR cell and control cell. The glucose uptake and glucose consumption in IR cell which treated with turtle oil was significantly increase than that in the control cells (P<0.05). Turtle oil can improve the insulin sensitivity and glucose metabolism in the IR cell model. (authors)

  11. Strontium incorporation to optimize the antibacterial and biological characteristics of silver-substituted hydroxyapatite coating

    Energy Technology Data Exchange (ETDEWEB)

    Geng, Zhen; Cui, Zhenduo [School of Materials Science and Engineering, Tianjin University, Tianjin 300072 (China); Li, Zhaoyang, E-mail: zyli@tju.edu.cn [School of Materials Science and Engineering, Tianjin University, Tianjin 300072 (China); Tianjin Key Laboratory of Composite and Functional Materials, Tianjin 300072 (China); Zhu, Shengli [School of Materials Science and Engineering, Tianjin University, Tianjin 300072 (China); Tianjin Key Laboratory of Composite and Functional Materials, Tianjin 300072 (China); Liang, Yanqin [School of Materials Science and Engineering, Tianjin University, Tianjin 300072 (China); Liu, Yunde; Li, Xue; He, Xin; Yu, Xiaoxu; Wang, Renfeng [School of Laboratory Medicine, Tianjin Medical University, Tianjin 300072 (China); Yang, Xianjin, E-mail: xjyang@tju.edu.cn [School of Materials Science and Engineering, Tianjin University, Tianjin 300072 (China); Tianjin Key Laboratory of Composite and Functional Materials, Tianjin 300072 (China)

    2016-01-01

    Infection in primary total joint prostheses is attracting considerable attention. In this study, silver (Ag) was incorporated into hydroxyapatite (HA) using a hydrothermal method in order to improve its antimicrobial properties. Strontium (Sr) was added as a second binary element to improve the biocompatibility. The substituted HA samples were fixed on titanium (Ti) substrates by dopamine-assisted immobilization in order to evaluate their antibacterial and biological properties. The results showed that Ag and Sr were successfully incorporated into HA without affecting their crystallinity. Further, the antibacterial tests showed that all the Ag-substituted samples had good anti-bacterial properties against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). Despite their good antibacterial ability, the Ag-substituted samples showed evidence of cytotoxicity on MG63 cells, characterized by low cell density and poor spreadability. The addition of Sr to the Ag-substituted samples considerably reduced the cytotoxicity of Ag. Although the viability of the cells grown on the surfaces of co-substituted HA was not as high as that of the cells grown on the HA surfaces, it is believed that excellent antibacterial properties and good biological activity can be achieved by balancing the dosage of Sr and Ag. - Highlights: • Ag- and Sr-substituted HA was prepared by hydrothermal method. • Ag- and Sr-substituted HA coating was deposited on dopamine functionalized titanium. • Ag-substituted HA biofilm showed a remarkable antibacterial activity. • Sr could offset the side effects of Ag.

  12. Tritium ingestion as organically bound tritium (OBT) - incorporation in different organs of pregnant and non-pregnant rats

    International Nuclear Information System (INIS)

    Bhatia, A.L.; Pollaris, K.; Vandecasteele, C.M.; Kowalska, M.

    1998-01-01

    For a better understanding of the hazard of tritium, its bound form in the food constituents (organically bound tritium (OBT)) has not been investigated though study on tritiated water are many. Hence an evaluation of the uptake of tritium incorporated in basic constituents of food viz, proteins, carbohydrates and lipids is warranted. Present study cells with the incorporated three organically bound tritium components separated from tritiated milk powder (casein, butter and lactose). This is further compared in the organs of pregnant (after parturition) and non-pregnant rats

  13. Intrinsic electrical properties of mammalian neurons and CNS function: a historical perspective

    Science.gov (United States)

    Llinás, Rodolfo R.

    2014-01-01

    This brief review summarizes work done in mammalian neuroscience concerning the intrinsic electrophysiological properties of four neuronal types; Cerebellar Purkinje cells, inferior olivary cells, thalamic cells, and some cortical interneurons. It is a personal perspective addressing an interesting time in neuroscience when the reflex view of brain function, as the paradigm to understand global neuroscience, began to be modified toward one in which sensory input modulates rather than dictates brain function. The perspective of the paper is not a comprehensive description of the intrinsic electrical properties of all nerve cells but rather addresses a set of cell types that provide indicative examples of mechanisms that modulate brain function. PMID:25408634

  14. INTRINSIC ELECTRICAL PROPERTIES OF MAMMALIAN NEURONS AND CNS FUNCTION: A HISTORICAL PERSPECTIVE

    Directory of Open Access Journals (Sweden)

    Rodolfo R Llinas

    2014-11-01

    Full Text Available This brief review summarizes work done in mammalian neuroscience concerning the intrinsic electrophysiological properties of four neuronal types; Cerebellar Purkinje cells, inferior olivary cells, thalamic cells, and some cortical interneurons. It is a personal perspective addressing an interesting time in neuroscience when the reflex view of brain function, as the paradigm to understand global neuroscience, began to be modified towards one in which sensory input modulates rather than dictates brain function. The perspective of the paper is not a comprehensive description of the intrinsic electrical properties of all nerve cells but rather addresses a set of cell types that provide indicative examples of mechanisms that modulate brain function.

  15. Incorporation of bone marrow cells in pancreatic pseudoislets improves posttransplant vascularization and endocrine function.

    Directory of Open Access Journals (Sweden)

    Christine Wittig

    Full Text Available Failure of revascularization is known to be the major reason for the poor outcome of pancreatic islet transplantation. In this study, we analyzed whether pseudoislets composed of islet cells and bone marrow cells can improve vascularization and function of islet transplants. Pancreatic islets isolated from Syrian golden hamsters were dispersed into single cells for the generation of pseudoislets containing 4×10(3 cells. To create bone marrow cell-enriched pseudoislets 2×10(3 islet cells were co-cultured with 2×10(3 bone marrow cells. Pseudoislets and bone marrow cell-enriched pseudoislets were transplanted syngeneically into skinfold chambers to study graft vascularization by intravital fluorescence microscopy. Native islet transplants served as controls. Bone marrow cell-enriched pseudoislets showed a significantly improved vascularization compared to native islets and pseudoislets. Moreover, bone marrow cell-enriched pseudoislets but not pseudoislets normalized blood glucose levels after transplantation of 1000 islet equivalents under the kidney capsule of streptozotocin-induced diabetic animals, although the bone marrow cell-enriched pseudoislets contained only 50% of islet cells compared to pseudoislets and native islets. Fluorescence microscopy of bone marrow cell-enriched pseudoislets composed of bone marrow cells from GFP-expressing mice showed a distinct fraction of cells expressing both GFP and insulin, indicating a differentiation of bone marrow-derived cells to an insulin-producing cell-type. Thus, enrichment of pseudoislets by bone marrow cells enhances vascularization after transplantation and increases the amount of insulin-producing tissue. Accordingly, bone marrow cell-enriched pseudoislets may represent a novel approach to increase the success rate of islet transplantation.

  16. Translation system engineering in Escherichia coli enhances non-canonical amino acid incorporation into proteins.

    Science.gov (United States)

    Gan, Rui; Perez, Jessica G; Carlson, Erik D; Ntai, Ioanna; Isaacs, Farren J; Kelleher, Neil L; Jewett, Michael C

    2017-05-01

    The ability to site-specifically incorporate non-canonical amino acids (ncAAs) into proteins has made possible the study of protein structure and function in fundamentally new ways, as well as the bio synthesis of unnatural polymers. However, the task of site-specifically incorporating multiple ncAAs into proteins with high purity and yield continues to present a challenge. At the heart of this challenge lies the lower efficiency of engineered orthogonal translation system components compared to their natural counterparts (e.g., translation elements that specifically use a ncAA and do not interact with the cell's natural translation apparatus). Here, we show that evolving and tuning expression levels of multiple components of an engineered translation system together as a whole enhances ncAA incorporation efficiency. Specifically, we increase protein yield when incorporating multiple p-azido-phenylalanine(pAzF) residues into proteins by (i) evolving the Methanocaldococcus jannaschii p-azido-phenylalanyl-tRNA synthetase anti-codon binding domain, (ii) evolving the elongation factor Tu amino acid-binding pocket, and (iii) tuning the expression of evolved translation machinery components in a single vector. Use of the evolved translation machinery in a genomically recoded organism lacking release factor one enabled enhanced multi-site ncAA incorporation into proteins. We anticipate that our approach to orthogonal translation system development will accelerate and expand our ability to site-specifically incorporate multiple ncAAs into proteins and biopolymers, advancing new horizons for synthetic and chemical biotechnology. Biotechnol. Bioeng. 2017;114: 1074-1086. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  17. The Incorporation of Ribonucleotides Induces Structural and Conformational Changes in DNA.

    Science.gov (United States)

    Meroni, Alice; Mentegari, Elisa; Crespan, Emmanuele; Muzi-Falconi, Marco; Lazzaro, Federico; Podestà, Alessandro

    2017-10-03

    Ribonucleotide incorporation is the most common error occurring during DNA replication. Cells have hence developed mechanisms to remove ribonucleotides from the genome and restore its integrity. Indeed, the persistence of ribonucleotides into DNA leads to severe consequences, such as genome instability and replication stress. Thus, it becomes important to understand the effects of ribonucleotides incorporation, starting from their impact on DNA structure and conformation. Here we present a systematic study of the effects of ribonucleotide incorporation into DNA molecules. We have developed, to our knowledge, a new method to efficiently synthesize long DNA molecules (hundreds of basepairs) containing ribonucleotides, which is based on a modified protocol for the polymerase chain reaction. By means of atomic force microscopy, we could therefore investigate the changes, upon ribonucleotide incorporation, of the structural and conformational properties of numerous DNA populations at the single-molecule level. Specifically, we characterized the scaling of the contour length with the number of basepairs and the scaling of the end-to-end distance with the curvilinear distance, the bending angle distribution, and the persistence length. Our results revealed that ribonucleotides affect DNA structure and conformation on scales that go well beyond the typical dimension of the single ribonucleotide. In particular, the presence of ribonucleotides induces a systematic shortening of the molecules, together with a decrease of the persistence length. Such structural changes are also likely to occur in vivo, where they could directly affect the downstream DNA transactions, as well as interfere with protein binding and recognition. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  18. Deuterium incorporation into Escherichia-coli proteins

    DEFF Research Database (Denmark)

    Lederer, H.; May, R. P.; Kjems, Jørgen

    1986-01-01

    Neutron small-angle scattering studies of single protein subunits in a protein-DNA complex require the adjustment of the neutron scattering-length densities of protein and DNA, which is attainable by specific deuteration of the protein. The neutron scattering densities of unlabelled DNA and DNA......-dependent RNA polymerase of Escherichia coli match when RNA polymerase is isolated from cells grown in a medium containing 46% D2O and unlabelled glucose as carbon source. Their contrasts vanish simultaneously in a dialysis buffer containing 65% D2O. An expression was evaluated which allows the calculation...... of the degree of deuteration and match point of any E. coli protein from the D2O content of the growth medium, taking the 2H incorporation into RNA polymerase amino acids to be representative for all amino acids in E. coli proteins. The small-angle scattering results, on which the calculation of the degree...

  19. Memory window engineering of Ta2O5-x oxide-based resistive switches via incorporation of various insulating frames

    Science.gov (United States)

    Lee, Ah Rahm; Baek, Gwang Ho; Kim, Tae Yoon; Ko, Won Bae; Yang, Seung Mo; Kim, Jongmin; Im, Hyun Sik; Hong, Jin Pyo

    2016-07-01

    Three-dimensional (3D) stackable memory frames, including nano-scaled crossbar arrays, are one of the most reliable building blocks to meet the demand of high-density non-volatile memory electronics. However, their utilization has the disadvantage of introducing issues related to sneak paths, which can negatively impact device performance. We address the enhancement of complementary resistive switching (CRS) features via the incorporation of insulating frames as a generic approach to extend their use; here, a Pt/Ta2O5-x/Ta/Ta2O5-x/Pt frame is chosen as the basic CRS cell. The incorporation of Ta/Ta2O5-x/Ta or Pt/amorphous TaN/Pt insulting frames into the basic CRS cell ensures the appreciably advanced memory features of CRS cells including higher on/off ratios, improved read margins, and increased selectivity without reliability degradation. Experimental observations identified that a suitable insulating frame is crucial for adjusting the abrupt reset events of the switching element, thereby facilitating the enhanced electrical characteristics of CRS cells that are suitable for practical applications.

  20. Microsystem to evaluate the incorporation of 3H-uridine in macrophage RNA

    International Nuclear Information System (INIS)

    Varesio, L.; Naglich, J.; Brunda, M.J.; Taramelli, D.; Eva, A.

    1981-01-01

    A method is described for the evaluation of the total 3H-uridine incorporated by macrophages in vitro into trichloroacetic acid (TCA)-precipitable material. The technique is based upon solubilization of the macrophage monolayers by guanidine-HC1, followed by TCA precipitation. The recovery of RNA into the precipitate and the reproducibility of the results were strictly dependent on the use of filtered reagents and on incubation of the TCA precipitate for 2 or more hours at 4 degree C before harvesting. Treatment with quanidine-HC1 did not affect the recovery of labeled RNA. Moreover, we observed that radioactive precipitate had the characteristics of RNA, since its recovery was sensitive to the addition of unlabeled uridine in the culture medium and to the treatment of the macrophages with inhibitors of RNA synthesis, but not of protein synthesis. Tritiated uridine incorporation in microcultures of macrophages can be assessed with this technique, by processing the cells directly in the wells. The main advantages of this procedure are: 1) the radioactivity can be measured by semiautomatic cell harvesters, 2) a small number of macrophages are required, and 3) many samples can be processed simultaneously. Overall, the technique is simple, rapid, and could be successfully adapted to study other metabolic pathways

  1. Construction and Characterization of Insect Cell-Derived Influenza VLP: Cell Binding, Fusion, and EGFP Incorporation

    Directory of Open Access Journals (Sweden)

    Yi-Shin Pan

    2010-01-01

    Full Text Available We have constructed virus-like particles (VLPs harboring hemagglutinin (HA, neuraminidase (NA, matrix protein 1 (M1 ,and proton channel protein (M2 using baculovirus as a vector in the SF9 insect cell. The size of the expressed VLP was estimated to be ~100 nm by light scattering experiment and transmission electron microscopy. Recognition of HA on the VLP surface by the HA2-specific monoclonal antibody IIF4 at acidic pH, as probed by surface plasmon resonance, indicated the pH-induced structural rearrangement of HA. Uptake of the particle by A549 mediated by HA-sialylose receptor interaction was visualized by the fluorescent-labeled VLP. The HA-promoted cell-virus fusion activity was illustrated by fluorescence imaging on the Jurkat cells incubated with rhodamine-loaded VLP performed at fusogenic pH. Furthermore, the green fluorescence protein (GFP was fused to NA to produce VLP with a pH-sensitive probe, expanding the use of VLP as an antigen carrier and a tool for viral tracking.

  2. High Efficiency Semi-Fuel Cell Incorporating an Ion Exchange Membrane

    National Research Council Canada - National Science Library

    Medeiros, Maria G; Dow, Eric G; Bessette, Russell R; Yan, Susan G; Dischert, Dwayne W

    2004-01-01

    It is a general purpose and object of the present invention to eliminate the parasitic direct reaction of the catholyte with the metal anode in a semi-fuel cell, thereby improving the overall energy...

  3. Effects of [3H]UdR on the cell-cycle progression of L1210 cells

    International Nuclear Information System (INIS)

    Darzynkiewicz, Z.; Carter, S.; Kimmel, M.

    1984-01-01

    Tritium-labelled uridine (( 3 H)UdR)perturbs progression of L1210 cells through the mitotic cycle. A slowdown of G 2 cells is observed 2 hr after addition of 0.5-5.0 μci/ml of ( 3 H)UdR into cultures. At 2.5-5.0 μCi/ml of ( 3 H)UdR a slowdown of cell progression through S is also apparent. Additionally, there is an increase in the number of cells with DNA values higher than 4C in cultures growing in the presence of ( 3 H)UdR for 8-24 hr. A pulse of ( 3 H)UdR of 2 hr duration labels predominantly (95%) cellular RNA. The first cell-cycle effects (G 2 slowdown) are observed when the amount of the incorporated ( 3 H)UdR is such that, on average there are fewer than thirty-six ( 3 H) decays per cell which corresponds to approximately 12-19 rads. The S-phase slowdown is seen at a dose of incorporated ( 3 H)UdR twice as high as that inducing G 2 effects. The specific localization of ( 3 H)UdR in nucleoli, peripheral nucleoplasm and in cytoplasm, as well as differences in the kinetics of the incorporation in relation to phases of the cell cycle are discussed. Mathematical modelling of the cell-cycle effects of ( 3 H)UdR is provided. (author)

  4. Host thin films incorporating nanoparticles

    Science.gov (United States)

    Qureshi, Uzma

    The focus of this research project was the investigation of the functional properties of thin films that incorporate a secondary nanoparticulate phase. In particular to assess if the secondary nanoparticulate material enhanced a functional property of the coating on glass. In order to achieve this, new thin film deposition methods were developed, namely use of nanopowder precursors, an aerosol assisted transport technique and an aerosol into atmospheric pressure chemical vapour deposition system. Aerosol assisted chemical vapour deposition (AACVD) was used to deposit 8 series of thin films on glass. Five different nanoparticles silver, gold, ceria, tungsten oxide and zinc oxide were tested and shown to successfully deposit thin films incorporating nanoparticles within a host matrix. Silver nanoparticles were synthesised and doped within a titania film by AACVD. This improved solar control properties. A unique aerosol assisted chemical vapour deposition (AACVD) into atmospheric pressure chemical vapour deposition (APCVD) system was used to deposit films of Au nanoparticles and thin films of gold nanoparticles incorporated within a host titania matrix. Incorporation of high refractive index contrast metal oxide particles within a host film altered the film colour. The key goal was to test the potential of nanopowder forms and transfer the suspended nanopowder via an aerosol to a substrate in order to deposit a thin film. Discrete tungsten oxide nanoparticles or ceria nanoparticles within a titanium dioxide thin film enhanced the self-cleaning and photo-induced super-hydrophilicity. The nanopowder precursor study was extended by deposition of zinc oxide thin films incorporating Au nanoparticles and also ZnO films deposited from a ZnO nanopowder precursor. Incorporation of Au nanoparticles within a VO: host matrix improved the thermochromic response, optical and colour properties. Composite VC/TiC and Au nanoparticle/V02/Ti02 thin films displayed three useful

  5. In Vitro Biocompatibility of Endodontic Sealers Incorporating Antibacterial Nanoparticles

    Directory of Open Access Journals (Sweden)

    Itzhak Abramovitz

    2012-01-01

    Full Text Available The main cause of endodontic disease is bacteria. Disinfection is presently achieved by cleaning the root canal system prior to obturation. Following setting, root canal filling is devoid of any antibacterial effect. Endodontic sealers with antimicrobial properties yet biocompatible may enhance root canal therapy. For this purpose, quaternized polyethylenimine nanoparticles which are antibacterial polymers, biocompatible, nonvolatile, and stable may be used. The aim of the present study was to examine the impact of added QPEI on the cytotoxicity of AH Plus, Epiphany, and GuttaFlow endodontic sealers. The effect of these sealers on the proliferation of RAW 264.7 macrophage and L-929 fibroblast cell lines and on the production of TNFα from macrophages was examined. Cell vitality was evaluated using a colorimetric XTT assay. The presence of cytokines was determined by two-site ELISA. Results show that QPEI at 1% concentration does not impair the basic properties of the examined sealers in both macrophages and fibroblast cell lines. Incorporation of 1% QPEI into the sealers did not impair their biocompatibility. QPEI is a potential clinical candidate to improve antibacterial activity of sealers without increasing cytotoxicity.

  6. Resistance of tumor cells to hydrogen peroxide as a factor of selection of highly metastatic cell variants in vivo

    International Nuclear Information System (INIS)

    Deichman, G.I.; Vendrov, E.L.

    1986-01-01

    The authors investigate the theory that tumor cells which catabolize H 2 O 2 more actively may possibly have selected advantages in vivo (of different origin). The authors tested the sensitivity to H 2 O 2 of parental cells of strain STHE (which did not progress in vivo) and 17 of its daughter variants isolated from lung tissue of experimental animals at different stages of formation of metastases (before and after their formation) and differing in metastatic activity. Intact cells were placed into test tube No. 6 of each series. After 30 min of exposure at 20 0 C, cells treated with H 2 O 2 and intact cells were washed out by centrifugation, and resuspended in nutritive medium containing 10% bovine serum and 3 H-thymidine, and each sample was diffused at a volume of 2.0 ml of cell suspension in each of three scintillation vials. The proliferative pool of cells in each sample was determined according to incorporation (for 22 h at 37 0 C) of 3 H-thymidine into cell nuclei. Data concerning incorporation of 3 H-thymidine was expressed for each sample of cells in percentages in relation to corresponding intact control (incorporation of label in a control culture of intact cells was taken as 100%). Each cell variant was investigated repeatedly in two-three more experiments

  7. Sensibilization of escherichia coli cells by cholesterol incorporated into their membrane

    International Nuclear Information System (INIS)

    Breslev, S.E.; Rozenberg, O.A.; Noskin, L.A.; Stepanova, I.M.; Beketova, A.G.; Loshakova, L.V.; Kovaleva, I.G.

    1984-01-01

    It has been established earlier that a level of cell radiosensitivity is defined by membrane viscosity changing in a wide temperature range. Therefore in epsilon coli cells of a natural type lethal doses of gamma rays are increased approximately a 3.5 times at 45 deg C, as compared to 4 deg C. Cholesterol changing a phase state of membrane lipids was used as a modifying factor. Liposomes were used with the goal of effective bacteria transfer to a membrane. It is established that liposomes without cholesterol do not affect their radioresistance and an increase of its content leads to resistance decrease. The effect is attained only at a sufficient long time of incubation of cells with liposomes (10-16 h). At 4 deg C lipids of E. coli membrane are in a solid-crystalline state independently on pholesterol presence, because of this, radiosensitivity does not change. Temperature increase up to 45 deg C transfer a part of lipids to a liquid-crystalline state, thus decreasing membrane viscosity. In this case cholesterol manifests itself. The authors explain viscosity increase with a violation in functioning of those enzyme systems, which activity is connected with membrane structural state, including enzymes of DNA repair. The authors assume that the radiosensibilization effect of cholesterol introduction into a bacterial membrane in high-temperature cell irradiation is explained by this phenomenon

  8. Rubrocerebellar Feedback Loop Isolates the Interposed Nucleus as an Independent Processor of Corollary Discharge Information in Mice.

    Science.gov (United States)

    Beitzel, Christy S; Houck, Brenda D; Lewis, Samantha M; Person, Abigail L

    2017-10-18

    Understanding cerebellar contributions to motor coordination requires deeper insight into how the output structures of the cerebellum, the cerebellar nuclei, integrate their inputs and influence downstream motor pathways. The magnocellular red nucleus (RNm), a brainstem premotor structure, is a major target of the interposed nucleus (IN), and has also been described in previous studies to send feedback collaterals to the cerebellum. Because such a pathway is in a key position to provide motor efferent information to the cerebellum, satisfying predictions about the use of corollary discharge in cerebellar computations, we studied it in mice of both sexes. Using anterograde viral tracing, we show that innervation of cerebellum by rubrospinal neuron collaterals is remarkably selective for the IN compared with the cerebellar cortex. Optogenetic activation of the pathway in acute mouse brain slices drove IN activity despite small amplitude synaptic currents, suggesting an active role in IN information processing. Monosynaptic transsynaptic rabies tracing indicated the pathway contacts multiple cell types within the IN. By contrast, IN inputs to the RNm targeted a region that lacked inhibitory neurons. Optogenetic drive of IN inputs to the RNm revealed strong, direct excitation but no inhibition of RNm neurons. Together, these data indicate that the cerebellar nuclei are under afferent control independent of the cerebellar cortex, potentially diversifying its roles in motor control. SIGNIFICANCE STATEMENT The common assumption that all cerebellar mossy fibers uniformly collateralize to the cerebellar nuclei and cortex underlies classic models of convergent Purkinje influence on cerebellar output. Specifically, mossy fibers are thought to both directly excite nuclear neurons and drive polysynaptic feedforward inhibition via Purkinje neurons, setting up a fundamental computational unit. Here we present data that challenge this rule. A dedicated cerebellar nuclear afferent

  9. The long adventurous journey of rhombic lip cells in jawed vertebrates: a comparative developmental analysis

    Directory of Open Access Journals (Sweden)

    Mario F Wullimann

    2011-04-01

    Full Text Available This review summarizes vertebrate rhombic lip and early cerebellar development covering classic approaches up to modern developmental genetics which identifies the relevant differential gene expression domains and their progeny. Most of this information is derived from amniotes. However, progress in anamniotes, particularly in the zebrafish, has recently been made. The current picture suggests that rhombic lip and cerebellar development in jawed vertebrates (gnathostomes share many characteristics. Regarding cerebellar development, these include a ptf1a expressing ventral cerebellar proliferation (VCP giving rise to Purkinje cells and other inhibitory cerebellar cell types, and an atoh1 expressing upper rhombic lip giving rise to an external granular layer (EGL, i.e., excitatory granule cells and an early ventral migration into the anterior rhombencephalon (cholinergic nuclei. As for the lower rhombic lip (LRL, gnathostome commonalities likely include the formation of precerebellar nuclei (mossy fiber origins and partially primary auditory nuclei (likely convergently evolved from the atoh1 expressing dorsal zone. The fate of the ptf1a expressing ventral LRL zone which gives rise to (excitatory cells of the inferior olive (climbing fiber origin and (inhibitory cells of cochlear nuclei in amniotes, has not been determined in anamniotes. Special for the zebrafish in comparison to amniotes is the predominant origin of anamniote excitatory deep cerebellar nuclei homologues (i.e., eurydendroid cells from ptf1a expressing VCP cells, the sequential activity of various atoh1 paralogues and the incomplete coverage of the subpial cerebellar plate with proliferative EGL cells. Nevertheless, the conclusion that a rhombic lip and its major derivatives evolved with gnathostome vertebrates only and are thus not an ancestral craniate character complex is supported by the absence of a cerebellum (and likely absence of its afferent and efferent nuclei in jawless

  10. Expression pattern of the thrombopoietin receptor (Mpl) in the murine central nervous system.

    Science.gov (United States)

    Ivanova, Anna; Wuerfel, Jens; Zhang, Juan; Hoffmann, Olaf; Ballmaier, Matthias; Dame, Christof

    2010-07-28

    Thrombopoietin (Thpo) and its receptor (Mpl), which regulate megakaryopoiesis, are expressed in the central nervous system (CNS), where Thpo is thought to exert pro-apoptotic effects on newly generated neurons. Mpl expression has been analysed in brain tissue on transcript level and in cultured primary rat neurons and astrocytes on protein level. Herein, we analysed Mpl expression in the developing and adult murine CNS by immunohistochemistry and investigated the brain of mice with homozygous Mpl deficiency (Mpl-/-) by MRI. Mpl was not detectable at developmental stages E12 to E15 in any resident cells of the CNS. From E18 onwards, robust Mpl expression was found in various brain areas, including cerebral cortex, olfactory bulb, thalamus, hypothalamus, medulla, pons, and the grey matter of spinal cord. However, major developmental changes became obvious: In the subventricular zone of the cerebral cortex Mpl expression occurred only during late gestation, while in the hippocampus Mpl expression was detectable for first time at stage P4. In the white matter of the cerebellum Mpl expression was restricted to the perinatal period. In the adult cerebellum, Mpl expression switched to Purkinje cell. The majority of other Mpl-positive cells were NeuN-positive neurons. None of the cells could be double-labelled with astrocyte marker GFAP. Mpl-/- mice showed no gross abnormalities of the brain. Our data locate Mpl expression to neurons at different subdivisions of the spinal cord, rhombencephalon, midbrain and prosencephalon. Besides neuronal cells Mpl protein is also expressed in Purkinje cells of the adult cerebellum.

  11. Photosensitive semiconducting polymer-incorporated nanofibers for promoting the regeneration of skin wound

    Energy Technology Data Exchange (ETDEWEB)

    Jin, Guorui [Institute of Materials Research and Engineering, A*STAR, 2 Fusionopolis Way, Innovis, #08-03, 138634 (Singapore); The Key Laboratory of Biomedical Information Engineering of Ministry of Education, Xi' an Jiaotong University, School of Life Science and Technology, Xi' an 710049 (China); Li, Jun [Institute of Materials Research and Engineering, A*STAR, 2 Fusionopolis Way, Innovis, #08-03, 138634 (Singapore); Department of Chemistry and Centre for Plastic Electronics, Imperial College London, London SW7 2AZ (United Kingdom); Li, Kai, E-mail: kai_li_cn@hotmail.com [Institute of Materials Research and Engineering, A*STAR, 2 Fusionopolis Way, Innovis, #08-03, 138634 (Singapore); Department of Radiology and Molecular Imaging Program at Stanford (MIPS), Stanford School of Medicine, Stanford, 94305 (United States)

    2017-01-01

    Photosensitive semiconducting polymer (SP) combined with light stimulation has shown the capability in promoting the proliferation of human dermal fibroblasts (HDFs). However, the high cytotoxicity of the used SP hindered its further application in bioactive scaffolds. In this contribution, we designed and synthesized a SP, poly (N,N-bis(2-octyldodecyl)-3,6-di(thiophen-2-yl)-2,5-dihydropyrrolo[3,4-c] pyrrole-1,4-dione-alt-thieno[3,2-b]thiophene) (PDBTT) with low cytotoxicity and strong absorbance in red and near-infrared region (600–1200 nm). The photosensitive SP was then applied in electrospun poly(ε-caprolactone) (PCL) nanofibrous scaffold and evaluated its proliferative effect on HDFs under the illumination from red light-emitting diode (LED) with high tissue penetration. After 9 days of continuous stimulation, the hybrid electrospun PCL/PDBTT nanofibers with low cytotoxicity showed excellent support for HDFs adhesion, proliferation and collagen secretion than neat PCL nanofibers and HDFs on the stimulated PCL/PDBTT nanofibers gained typical spindle morphology, indicating the well cell spreading on the stimulated PCL/PDBTT nanofibers. The incorporation of functional materials within synthetic biomaterials could be a novel way in improving the performance of engineered tissue constructs by providing multiple cues (e.g. electrical stimulation) to the attached cells. - Highlights: • A photosensitive semiconducting polymer (SP) was applied in electrospun nanofibrous scaffold. • The SP-incorporated scaffold could promote cell proliferation upon light stimulation. • The designed photosensitive SP could be applied as functional material with low cost and high durability in skin tissue engineering.

  12. 17 CFR 230.411 - Incorporation by reference.

    Science.gov (United States)

    2010-04-01

    ... following provisions: (1) Non-financial information may be incorporated by reference to any document; (2) Financial information may be incorporated by reference to any document, provided any financial statement so incorporated meets the requirements of the forms on which the statement is filed. Financial statements or other...

  13. Transmutation of americium and curium incorporated in zirconia-based host materials

    Energy Technology Data Exchange (ETDEWEB)

    Raison, P.E. [CEA Cadarache, 13 - Saint-Paul-lez-Durance (France). Dept. d' Etudes des Combustibles; Haire, R.G. [Oak Ridge National Lab., TN (United States)

    2001-07-01

    Presented are studies involving the incorporation of americium and curium in zirconia-based materials. First explored was the pseudo ternary system AmO{sub 2}-ZrO{sub 2}-Y{sub 2}O{sub 3}. It was determined that selected Y-CSZ materials can incorporate significant quantities of americium oxide and remain cubic single-phase. The cell parameters of these fluorite-type products were established to be linear with the AmO{sub 2} content. The Cm{sub 2}O{sub 3}-ZrO{sub 2} system was also investigated. It was found that at 25 mol% of CmO{sub 1.5}, the Cm(III) stabilized zirconia in its cubic form (a = 5.21 {+-}0.01 Angstrom). At higher and lower concentrations, diphasic materials were encountered. At 50 mol% of CmO{sub 1.5}, a pyrochlore oxide - Cm{sub 2}Zr{sub 2}O{sub 7} - is formed (a = 10.63 {+-}0.02 Angstrom). (author)

  14. Preparation of Lipid Nano emulsions Incorporating Curcumin for Cancer Therapy

    International Nuclear Information System (INIS)

    Anuchapreeda, S.; Anuchapreeda, S.; Fukumori, Y.; Ichikawa, H.; Okonogi, S.

    2012-01-01

    The aim of this study was to develop a new formulation of a curcumin lipid nano emulsion having the smallest particle size, the highest loading, and a good physical stability for cancer chemotherapy. Curcumin lipid nano emulsions were prepared by a modified thin-film hydration method followed by sonication. Soybean oil, hydrogenated L-α-phosphatidylcholine from egg yolk, and co surfactants were used to formulate the emulsions. The resultant nano emulsions showed mean particle diameter of 47-55 nm, could incorporate 23-28 mg curcumin per 30 mL, and were stable in particle size for 60 days at 4 degree C. The cytotoxicity studies of curucumin solution and curcumin-loaded nano emulsion using B16F10 and leukemic cell lines showed IC 50 values ranging from 3.5 to 30.1 and 22.2 to 53.7μM, respectively. These results demonstrated the successful incorporation of curcumin into lipid nano emulsion particles with small particle size, high loading capacity, good physical stability, and preserved cytotoxicity

  15. Myeloperoxidase-catalyzed incorporation of amines into proteins: role of hypochlorous acid and dichloramines.

    Science.gov (United States)

    Thomas, E L; Jefferson, M M; Grisham, M B

    1982-11-23

    obtained by reacting RNCl2 with polyhistidine or polytyrosine, and to a lesser extent with polylysine at high pH, but not with other poly(amino acids). Precipitable derivatives were also obtained by incubating MPO-containing extracts from leukocyte granules with hydrogen peroxide, Cl-, and labeled amines. The extracts were found to have a high content of substances with primary amino groups, which competed for incorporation. The results account for oxidative incorporation of amines into proteins in leukocytes and provide evidence that HOCl and nitrogen-chlorine (N-Cl) derivatives are formed in these cells. The characteristics of the incorporation reaction suggest that it would not contribute significantly to the antimicrobial activity of myeloperoxidase (MPO). Nevertheless, the reaction may provide a sensitive method for studying MPO action in vivo.

  16. Rabbit uterine epithelial cells: Co-culture with spermatozoa

    International Nuclear Information System (INIS)

    Boice, M.L.

    1988-01-01

    A primary culture of rabbit uterine epithelial cells was established and their effects on sperm function were examined in vitro. Epithelial cells were isolated from uteri of estrous rabbits and cultured on floating collagen gels in phenol red-free medium supplemented with 5% fetal bovine serum. Light microscopy and keratin staining showed that the epithelial cell population established in culture had morphological characteristics similar to that seen in the intact endometrium. Cells were cultured with 3 H-leucine and uptake of label by cells and its incorporation into cellular and secretory proteins determined. When compared to cells cultured for 24-48 h, incorporation of label into cellular protein was lower at 72-96 h, but secretion increased. Estradiol 17-β did not affect label uptake or incorporation, but did enhance proliferation of cells as judged by total DNA content of the cell population. Analysis of proteins in media by sodium dodecyl sulfate polyacrylamide gel electrophoresis and fluorography suggested that epithelial and stromal cells synthesis proteins that may be secretory in nature during 72-96 h culture. Twenty-nine to thirty-one h after initiation of epithelial cultures, 1-2 x 10 6 sperm were co-incubated with cells and sperm viability, motility, loss of acrosome and fertilizing ability determined

  17. VEGF-incorporated biomimetic poly(lactide-co-glycolide) sintered microsphere scaffolds for bone tissue engineering.

    Science.gov (United States)

    Jabbarzadeh, Ehsan; Deng, Meng; Lv, Qing; Jiang, Tao; Khan, Yusuf M; Nair, Lakshmi S; Laurencin, Cato T

    2012-11-01

    Regenerative engineering approaches utilizing biomimetic synthetic scaffolds provide alternative strategies to repair and restore damaged bone. The efficacy of the scaffolds for functional bone regeneration critically depends on their ability to induce and support vascular infiltration. In the present study, three-dimensional (3D) biomimetic poly(lactide-co-glycolide) (PLAGA) sintered microsphere scaffolds were developed by sintering together PLAGA microspheres followed by nucleation of minerals in a simulated body fluid. Further, the angiogenic potential of vascular endothelial growth factor (VEGF)-incorporated mineralized PLAGA scaffolds were examined by monitoring the growth and phenotypic expression of endothelial cells on scaffolds. Scanning electron microscopy micrographs confirmed the growth of bone-like mineral layers on the surface of microspheres. The mineralized PLAGA scaffolds possessed interconnectivity and a compressive modulus of 402 ± 61 MPa and compressive strength of 14.6 ± 2.9 MPa. Mineralized scaffolds supported the attachment and growth and normal phenotypic expression of endothelial cells. Further, precipitation of apatite layer on PLAGA scaffolds resulted in an enhanced VEGF adsorption and prolonged release compared to nonmineralized PLAGA and, thus, a significant increase in endothelial cell proliferation. Together, these results demonstrated the potential of VEGF-incorporated biomimetic PLAGA sintered microsphere scaffolds for bone tissue engineering as they possess the combined effects of osteointegrativity and angiogenesis. Copyright © 2012 Wiley Periodicals, Inc.

  18. Effects of (/sup 3/H)UdR on the cell-cycle progression of L1210 cells

    Energy Technology Data Exchange (ETDEWEB)

    Darzynkiewicz, Z.; Carter, S.; Kimmel, M. (Memorial Sloan-Kettering Cancer Center, New York (USA))

    1984-11-01

    Tritium-labelled uridine ((/sup 3/H)UdR)perturbs progression of L1210 cells through the mitotic cycle. A slowdown of G/sub 2/ cells is observed 2 hr after addition of 0.5-5.0 ..mu..ci/ml of (/sup 3/H)UdR into cultures. At 2.5-5.0 ..mu..Ci/ml of (/sup 3/H)UdR a slowdown of cell progression through S is also apparent. Additionally, there is an increase in the number of cells with DNA values higher than 4C in cultures growing in the presence of (/sup 3/H)UdR for 8-24 hr. A pulse of (/sup 3/H)UdR of 2 hr duration labels predominantly (95%) cellular RNA. The first cell-cycle effects (G/sub 2/ slowdown) are observed when the amount of the incorporated (/sup 3/H)UdR is such that, on average there are fewer than thirty-six (/sup 3/H) decays per cell which corresponds to approximately 12-19 rads. The S-phase slowdown is seen at a dose of incorporated (/sup 3/H)UdR twice as high as that inducing G/sub 2/ effects. The specific localization of (/sup 3/H)UdR in nucleoli, peripheral nucleoplasm and in cytoplasm, as well as differences in the kinetics of the incorporation in relation to phases of the cell cycle are discussed. Mathematical modelling of the cell-cycle effects of (/sup 3/H)UdR is provided.

  19. The Effect of UV Aging on Antimicrobial and Mechanical Properties of PLA Films with Incorporated Zinc Oxide Nanoparticles.

    Science.gov (United States)

    Mizielińska, Małgorzata; Kowalska, Urszula; Jarosz, Michał; Sumińska, Patrycja; Landercy, Nicolas; Duquesne, Emmanuel

    2018-04-18

    The aim of this study was to examine the influence of accelerated UV-aging on the activity against chosen microorganisms and the mechanical properties of poly-lactic acid (PLA) films enhanced with ZnO nanoparticles. The pure PLA films and tri-layered PLAZnO1%/PLA/PLAZnO1% films of 150 µm thickness were extruded. The samples were treated with UV-A and Q-SUN irradiation. After irradiation the antimicrobial activity and mechanical properties of the films were analyzed. The results of the study demonstrated that PLA films did not inhibit the growth of Staphylococcus aureus , Bacillus cereus , Escherichia coli , Bacillus atrophaeus , and Candida albicans cells. PLA films with incorporated zinc oxide nanoparticles decreased the number of analyzed microorganisms. Accelerated UV aging had no negative effect on the activity of the film containing nano-ZnO against Gram-positive bacteria, but it influenced the activity against Gram-negative cells and C. albicans . Q-SUN irradiation decreased the antimicrobial effect of films with incorporated nanoparticles against B. cereus . UV-A and Q-UV irradiation did not influence the mechanical properties of PLA films containing incorporated ZnO nanoparticles.

  20. Au nanorods-incorporated plasmonic-enhanced inverted organic solar cells

    Science.gov (United States)

    Peng, Ling; Mei, Yang; Chen, Shu-Fen; Zhang, Yu-Pei; Hao, Jing-Yu; Deng, Ling-Ling; Huang, Wei

    2015-11-01

    The effect of Au nanorods (NRs) on optical-to-electric conversion efficiency is investigated in inverted polymer solar cells, in which Au NRs are sandwiched between two layers of ZnO. Accompanied by the optimization of thickness of ZnO covered on Au NRs, a high-power conversion efficiency of 3.60% and an enhanced short-circuit current density (JSC) of 10.87 mA/cm2 are achieved in the poly(3-hexylthiophene): [6,6]-phenyl-C61-butyric acid methyl ester (P3HT:PC60BM)-based inverted cell and the power conversion efficiency (PCE) is enhanced by 19.6% compared with the control device. The detailed analyses of the light absorption characteristics, the simulated scattering induced by Au NRs, and the electromagnetic field around Au NRs show that the absorption improvement in the photoactive layer due to the light scattering from the longitudinal axis and the near-field increase around Au NRs induced by localized surface plasmon resonance plays a key role in enhancing the performances. Project supported by the Ministry of Science and Technology, China (Grant No. 2012CB933301), the National Natural Science Foundation of China (Grant Nos. 61274065, 51173081, 61136003, BZ2010043, 51372119, and 51172110), and the Priority Academic Program Development of Jiangsu Provincial Higher Education Institutions and Synergetic Innovation Center for Organic Electronics and Information Displays, China.