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Sample records for cultured primary motor

  1. Motor sequence learning and motor adaptation in primary cervical dystonia.

    PubMed

    Katschnig-Winter, Petra; Schwingenschuh, Petra; Davare, Marco; Sadnicka, Anna; Schmidt, Reinhold; Rothwell, John C; Bhatia, Kailash P; Edwards, Mark J

    2014-06-01

    Motor sequence learning and motor adaptation rely on overlapping circuits predominantly involving the basal ganglia and cerebellum. Given the importance of these brain regions to the pathophysiology of primary dystonia, and the previous finding of abnormal motor sequence learning in DYT1 gene carriers, we explored motor sequence learning and motor adaptation in patients with primary cervical dystonia. We recruited 12 patients with cervical dystonia and 11 healthy controls matched for age. Subjects used a joystick to move a cursor from a central starting point to radial targets as fast and accurately as possible. Using this device, we recorded baseline motor performance, motor sequence learning and a visuomotor adaptation task. Patients with cervical dystonia had a significantly higher peak velocity than controls. Baseline performance with random target presentation was otherwise normal. Patients and controls had similar levels of motor sequence learning and motor adaptation. Our patients had significantly higher peak velocity compared to controls, with similar movement times, implying a different performance strategy. The preservation of motor sequence learning in cervical dystonia patients contrasts with the previously observed deficit seen in patients with DYT1 gene mutations, supporting the hypothesis of differing pathophysiology in different forms of primary dystonia. Normal motor adaptation is an interesting finding. With our paradigm we did not find evidence that the previously documented cerebellar abnormalities in cervical dystonia have a behavioral correlate, and thus could be compensatory or reflect "contamination" rather than being directly pathological.

  2. Primary face motor area as the motor representation of articulation.

    PubMed

    Terao, Yasuo; Ugawa, Yoshikazu; Yamamoto, Tomotaka; Sakurai, Yasuhisa; Masumoto, Tomohiko; Abe, Osamu; Masutani, Yoshitaka; Aoki, Shigeki; Tsuji, Shoji

    2007-04-01

    No clinical data have yet been presented to show that a lesion localized to the primary motor area (M1) can cause severe transient impairment of articulation, although a motor representation for articulation has been suggested to exist within M1. Here we describe three cases of patients who developed severe dysarthria, temporarily mimicking speech arrest or aphemia, due to a localized brain lesion near the left face representation of the human primary motor cortex (face-M1). Speech was slow, effortful, lacking normal prosody, and more affected than expected from the degree of facial or tongue palsy. There was a mild deficit in tongue movements in the sagittal plane that impaired palatolingual contact and rapid tongue movements. The speech disturbance was limited to verbal output, without aphasia or orofacial apraxia. Overlay of magnetic resonance images revealed a localized cortical region near face-M1, which displayed high intensity on diffusion weighted images, while the main portion of the corticobulbar fibers arising from the lower third of the motor cortex was preserved. The cases suggest the existence of a localized brain region specialized for articulation near face-M1. Cortico-cortical fibers connecting face-M1 with the lower premotor areas including Broca's area may also be important for articulatory control. PMID:17380243

  3. Postnatal development of the motor representation in primary motor cortex.

    PubMed

    Chakrabarty, S; Martin, J H

    2000-11-01

    The purpose of this study was to examine when the muscles and joints of the forelimb become represented in primary motor cortex (M1) during postnatal life and how local representation patterns change. We examined these questions in cats that were anesthetized (45-90 days, n = 14; adults, n = 3) and awake (n = 4; 52-86 days). We used intracortical microstimulation (45 ms duration train, 330 Hz, 0.2-ms balanced biphasic pulses, with a leading cathodic pulse; up to 100 microA). In young animals (less than day 70), we also used stimulus trains and pulses that could produce greater temporal summation (up to 200-ms train duration, down to 143-Hz stimulus frequency, up to 0.8-ms pulse width). Anesthetized animals were areflexic, and muscle tone was similar to that of the awake cats (i.e., relaxed, not weight or load bearing, with minimal resistance to passive stretch). We monitored the kinematic effects of microstimulation and changes in electromyographic (EMG) activity in forelimb muscles. There was an age-dependent reduction in the number of sites where microstimulation did not produce a motor effect (i.e., ineffective sites), from 95% in animals younger than 60 days to 33% between 81 and 90 days. In adults, 24% of sites were ineffective. Median current thresholds for evoking movements dropped from 79 microA in animals younger than day 60 to 38 and 28 microA in day 81-90 animals and adults, respectively. There was a proximal-to-distal development of the somatotopic organization of the motor map. Stimulation at the majority of sites in animals younger than day 71 produced shoulder and elbow movement. Wrist sites were first present by day 71, and digit sites by day 81. Sites at which multiple responses were evoked, between 1.0 and 1.5 times threshold, were present after day 71, and increased with age. A higher percentage of distal joints were co-represented with other joints, rather than being represented alone. We found that effective sites initially were scattered and

  4. Three Dimensional Primary Hepatocyte Culture

    NASA Technical Reports Server (NTRS)

    Yoffe, Boris

    1998-01-01

    Our results demonstrated for the first time the feasibility of culturing PHH in microgravity bioreactors that exceeded the longest period obtained using other methods. Within the first week of culture, isolated hepatocytes started to form aggregates, which continuously increased in size (up to 1 cm) and macroscopically appeared as a multidimensional tissue-like assembly. To improve oxygenation and nutrition within the spheroids we performed experiments with the biodegradable nonwoven fiber-based polymers made from PolyGlycolic Acid (PGA). It has been shown that PGA scaffolds stimulate isolated cells to regenerate tissue with defined sizes and shapes and are currently being studied for various tissue-engineering applications. Our data demonstrated that culturing hepatocytes in the presence of PGA scaffolds resulted in more efficient cell assembly and formations of larger cell spheroids (up to 3 cm in length, see figure). The histology of cell aggregates cultured with PGA showed polymer fibers with attached hepatocytes. We initiated experiments to co-culture primary human hepatocytes with human microvascular endothelial cells in the bioreactor. The presence of endothelial cells in co-cultures were established by immunohistochemistry using anti-CD34 monoclonal Ab. Our preliminary data demonstrated that cultures of purified hepatocytes with human microvascular endothelial cells exhibited better growth and expressed higher levels of albumin MRNA for a longer period of time than cultures of ppfified, primary human hepatocytes cultured alone. We also evaluated microsomal deethylation activity of hepatocytes cultured in the presence of endothelial cells.In summary, we have established liver cell culture, which mimicked the structure and function of the parent tissue.

  5. Primary Culture of Mouse Dopaminergic Neurons

    PubMed Central

    Gaven, Florence; Marin, Philippe; Claeysen, Sylvie

    2014-01-01

    Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions such as motor control, motivation, and working memory. Nigrostriatal dopaminergic neurons selectively degenerate in Parkinson's disease (PD). This progressive neuronal loss is unequivocally associated with the motors symptoms of the pathology (bradykinesia, resting tremor, and muscular rigidity). The main agent responsible of dopaminergic neuron degeneration is still unknown. However, these neurons appear to be extremely vulnerable in diverse conditions. Primary cultures constitute one of the most relevant models to investigate properties and characteristics of dopaminergic neurons. These cultures can be submitted to various stress agents that mimic PD pathology and to neuroprotective compounds in order to stop or slow down neuronal degeneration. The numerous transgenic mouse models of PD that have been generated during the last decade further increased the interest of researchers for dopaminergic neuron cultures. Here, the video protocol focuses on the delicate dissection of embryonic mouse brains. Precise excision of ventral mesencephalon is crucial to obtain neuronal cultures sufficiently rich in dopaminergic cells to allow subsequent studies. This protocol can be realized with embryonic transgenic mice and is suitable for immunofluorescence staining, quantitative PCR, second messenger quantification, or neuronal death/survival assessment. PMID:25226064

  6. Repetitive Transcranial Magnetic Stimulation to the Primary Motor Cortex Interferes with Motor Learning by Observing

    ERIC Educational Resources Information Center

    Brown, Liana E.; Wilson, Elizabeth T.; Gribble, Paul L.

    2009-01-01

    Neural representations of novel motor skills can be acquired through visual observation. We used repetitive transcranial magnetic stimulation (rTMS) to test the idea that this "motor learning by observing" is based on engagement of neural processes for learning in the primary motor cortex (M1). Human subjects who observed another person learning…

  7. WEIRD Walking: Cross-Cultural Research on Motor Development

    PubMed Central

    Karasik, Lana B.; Adolph, Karen E.; Tamis-LeMonda, Catherine S.; Bornstein, Marc H.

    2011-01-01

    Motor development—traditionally studied in WEIRD populations—falls victim to assumptions of universality similar to other domains described by Henrich et al. (current issue). However, cross-cultural research illustrates the extraordinary diversity that is normal in motor skill acquisition. Indeed, motor development provides an important domain for evaluating cultural challenges to a general behavioral science. PMID:20546664

  8. Primary Motor Cortex Involvement in Initial Learning during Visuomotor Adaptation

    ERIC Educational Resources Information Center

    Riek, Stephan; Hinder, Mark R.; Carson, Richard G.

    2012-01-01

    Human motor behaviour is continually modified on the basis of errors between desired and actual movement outcomes. It is emerging that the role played by the primary motor cortex (M1) in this process is contingent upon a variety of factors, including the nature of the task being performed, and the stage of learning. Here we used repetitive TMS to…

  9. Different Modulation of Common Motor Information in Rat Primary and Secondary Motor Cortices

    PubMed Central

    Saiki, Akiko; Kimura, Rie; Samura, Toshikazu; Fujiwara-Tsukamoto, Yoko; Sakai, Yutaka; Isomura, Yoshikazu

    2014-01-01

    Rodents have primary and secondary motor cortices that are involved in the execution of voluntary movements via their direct and parallel projections to the spinal cord. However, it is unclear whether the rodent secondary motor cortex has any motor function distinct from the primary motor cortex to properly control voluntary movements. In the present study, we quantitatively examined neuronal activity in the caudal forelimb area (CFA) of the primary motor cortex and rostral forelimb area (RFA) of the secondary motor cortex in head-fixed rats performing forelimb movements (pushing, holding, and pulling a lever). We found virtually no major differences between CFA and RFA neurons, regardless of neuron subtypes, not only in their basal spiking properties but also in the time-course, amplitude, and direction preference of their functional activation for simple forelimb movements. However, the RFA neurons, as compared with the CFA neurons, showed obviously a greater susceptibility of their functional activation to an alteration in a behavioral situation, a 'rewarding' response that leads to reward or a 'consummatory' response that follows reward water, which might be accompanied by some internal adaptations without affecting the motor outputs. Our results suggest that, although the CFA and RFA neurons commonly process fundamental motor information to properly control forelimb movements, the RFA neurons may be functionally differentiated to integrate motor information with internal state information for an adaptation to goal-directed behaviors. PMID:24893154

  10. Motor Speech Disorders Associated with Primary Progressive Aphasia

    PubMed Central

    Duffy, Joseph R.; Strand, Edythe A.; Josephs, Keith A.

    2014-01-01

    Background Primary progressive aphasia (PPA) and conditions that overlap with it can be accompanied by motor speech disorders. Recognition and understanding of motor speech disorders can contribute to a fuller clinical understanding of PPA and its management as well as its localization and underlying pathology. Aims To review the types of motor speech disorders that may occur with PPA, its primary variants, and its overlap syndromes (progressive supranuclear palsy syndrome, corticobasal syndrome, motor neuron disease), as well as with primary progressive apraxia of speech. Main Contribution The review should assist clinicians' and researchers' understanding of the relationship between motor speech disorders and PPA and its major variants. It also highlights the importance of recognizing neurodegenerative apraxia of speech as a condition that can occur with little or no evidence of aphasia. Conclusion Motor speech disorders can occur with PPA. Their recognition can contribute to clinical diagnosis and management of PPA and to understanding and predicting the localization and pathology associated with PPA variants and conditions that can overlap with them. PMID:25309017

  11. The current status of primary hepatocyte culture

    PubMed Central

    Mitaka, Toshihiro

    1998-01-01

    Recently, there have been significant advances toward the development of culture conditions that promote proliferation of primary rodent hepatocytes. There are two major methods for the multiplication of hepatocytes in vitro: one is the use of nicotinamide, the other is the use of a nutrient-rich medium. In the medium containing a high concentration of nicotinamide and a growth factor, primary hepatocytes can proliferate well. In this culture condition small mononucleate cells, which are named small hepatocytes, appear and form colonies. Small hepatocytes have a high potential to proliferate while maintaining hepatic characteristics, and can differentiate into mature ones. On the other hand, combining the nutrient-rich medium with 2% DMSO, the proliferated hepatocytes can recover the hepatic differentiated functions and maintain them for a long time. In this review I describe the culture conditions for the proliferation and differentiation of primary hepatocytes and discuss the small hepatocytes, especially their roles in liver growth. PMID:10319020

  12. Concurrent TMS to the primary motor cortex augments slow motor learning.

    PubMed

    Narayana, Shalini; Zhang, Wei; Rogers, William; Strickland, Casey; Franklin, Crystal; Lancaster, Jack L; Fox, Peter T

    2014-01-15

    Transcranial magnetic stimulation (TMS) has shown promise as a treatment tool, with one FDA approved use. While TMS alone is able to up- (or down-) regulate a targeted neural system, we argue that TMS applied as an adjuvant is more effective for repetitive physical, behavioral and cognitive therapies, that is, therapies which are designed to alter the network properties of neural systems through Hebbian learning. We tested this hypothesis in the context of a slow motor learning paradigm. Healthy right-handed individuals were assigned to receive 5 Hz TMS (TMS group) or sham TMS (sham group) to the right primary motor cortex (M1) as they performed daily motor practice of a digit sequence task with their non-dominant hand for 4 weeks. Resting cerebral blood flow (CBF) was measured by H2(15)O PET at baseline and after 4 weeks of practice. Sequence performance was measured daily as the number of correct sequences performed, and modeled using a hyperbolic function. Sequence performance increased significantly at 4 weeks relative to baseline in both groups. The TMS group had a significant additional improvement in performance, specifically, in the rate of skill acquisition. In both groups, an improvement in sequence timing and transfer of skills to non-trained motor domains was also found. Compared to the sham group, the TMS group demonstrated increases in resting CBF specifically in regions known to mediate skill learning namely, the M1, cingulate cortex, putamen, hippocampus, and cerebellum. These results indicate that TMS applied concomitantly augments behavioral effects of motor practice, with corresponding neural plasticity in motor sequence learning network. These findings are the first demonstration of the behavioral and neural enhancing effects of TMS on slow motor practice and have direct application in neurorehabilitation where TMS could be applied in conjunction with physical therapy.

  13. Concurrent TMS to the primary motor cortex augments slow motor learning

    PubMed Central

    Narayana, Shalini; Zhang, Wei; Rogers, William; Strickland, Casey; Franklin, Crystal; Lancaster, Jack L.; Fox, Peter T.

    2013-01-01

    Transcranial magnetic stimulation (TMS) has shown promise as a treatment tool, with one FDA approved use. While TMS alone is able to up- (or down-) regulate a targeted neural system, we argue that TMS applied as an adjuvant is more effective for repetitive physical, behavioral and cognitive therapies, that is, therapies which are designed to alter the network properties of neural systems through Hebbian learning. We tested this hypothesis in the context of a slow motor learning paradigm. Healthy right-handed individuals were assigned to receive 5 Hz TMS (TMS group) or sham TMS (sham group) to the right primary motor cortex (M1) as they performed daily motor practice of a digit sequence task with their non-dominant hand for 4 weeks. Resting cerebral blood flow (CBF) was measured by H215O PET at baseline and after 4 weeks of practice. Sequence performance was measured daily as the number of correct sequences performed, and modeled using a hyperbolic function. Sequence performance increased significantly at 4 weeks relative to baseline in both groups. The TMS group had a significant additional improvement in performance, specifically, in the rate of skill acquisition. In both groups, an improvement in sequence timing and transfer of skills to non-trained motor domains was also found. Compared to the sham group, the TMS group demonstrated increases in resting CBF specifically in regions known to mediate skill learning namely, the M1, cingulate cortex, putamen, hippocampus, and cerebellum. These results indicate that TMS applied concomitantly augments behavioral effects of motor practice, with corresponding neural plasticity in motor sequence learning network. These findings are the first demonstration of the behavioral and neural enhancing effects of TMS on slow motor practice and have direct application in neurorehabilitation where TMS could be applied in conjunction with physical therapy. PMID:23867557

  14. Effect of tactile stimulation on primary motor cortex excitability during action observation combined with motor imagery.

    PubMed

    Tanaka, Megumi; Kubota, Shinji; Onmyoji, Yusuke; Hirano, Masato; Uehara, Kazumasa; Morishita, Takuya; Funase, Kozo

    2015-07-23

    We aimed to investigate the effects of the tactile stimulation to an observer's fingertips at the moment that they saw an object being pinched by another person on the excitability of observer's primary motor cortex (M1) using transcranial magnetic stimulation (TMS). In addition, the above effects were also examined during action observation combined with the motor imagery. Motor evoked potentials (MEP) were evoked from the subjects' right first dorsal interosseous (FDI) and abductor digiti minimi (ADM) muscles. Electrical stimulation (ES) inducing tactile sensation was delivered to the subjects' first and second fingertips at the moment of pinching action performed by another person. Although neither the ES nor action observation alone had significant effects on the MEP amplitude of the FDI or ADM, the FDI MEP amplitude which acts as the prime mover during pinching was reduced when ES and action observation were combined; however, no such changes were seen in the ADM. Conversely, that reduced FDI MEP amplitude was increased during the motor imagery. These results indicated that the M1 excitability during the action observation of pinching action combined with motor imagery could be enhanced by the tactile stimulation delivered to the observer's fingertips at the moment corresponding to the pinching being observed.

  15. Glycosphingolipid patterns in primary mouse kidney cultures

    SciTech Connect

    Lyerla, T.A.; Gross, S.K.; McCluer, R.H.

    1986-12-01

    Primary kidney cultures from C57BL/6J mice, 6 weeks of age or older, were produced using D-valine medium to select for epithelial cell growth. After allowing the cells to attach and proliferate for 1 week following plating, medium was changed once per week. Cells formed nearly confluent monolayers during the second week of culture. The cultured cells contained all of the glycosphingolipids seen in the adult kidney, analyzed by high performance liquid chromatography as their perbenzoyl derivatives. Glucosylceramide, however, was highly predominant in the cultured cells, whereas dihexosyl- and trihexosylceramides predominate in the intact kidney. Sex differences in glycolipid contents found in the intact kidney were also apparent in these cultured cells: The concentration of neutral glycolipids, in general, was higher in male cells than in those derived from females, and the male-specific glycolipid nonhydroxy fatty acid digalactosylceramide was high in male cells but very low in female cells. Neutral glycosphingolipids were labeled in 2-week-old cultures using (/sup 3/H)palmitate. The (/sup 3/H)palmitate was incorporated into all of the glycolipids within 2 hr of labeling. Hence, adult mouse kidney cells in D-valine medium retain their differentiated characteristics for a sufficient period of time to allow investigation of glycolipid syntheses in monolayer cultures of epithelial cells derived from this organ.

  16. Molluscan cells in culture: primary cell cultures and cell lines

    PubMed Central

    Yoshino, T. P.; Bickham, U.; Bayne, C. J.

    2013-01-01

    In vitro cell culture systems from molluscs have significantly contributed to our basic understanding of complex physiological processes occurring within or between tissue-specific cells, yielding information unattainable using intact animal models. In vitro cultures of neuronal cells from gastropods show how simplified cell models can inform our understanding of complex networks in intact organisms. Primary cell cultures from marine and freshwater bivalve and gastropod species are used as biomonitors for environmental contaminants, as models for gene transfer technologies, and for studies of innate immunity and neoplastic disease. Despite efforts to isolate proliferative cell lines from molluscs, the snail Biomphalaria glabrata Say, 1818 embryonic (Bge) cell line is the only existing cell line originating from any molluscan species. Taking an organ systems approach, this review summarizes efforts to establish molluscan cell cultures and describes the varied applications of primary cell cultures in research. Because of the unique status of the Bge cell line, an account is presented of the establishment of this cell line, and of how these cells have contributed to our understanding of snail host-parasite interactions. Finally, we detail the difficulties commonly encountered in efforts to establish cell lines from molluscs and discuss how these difficulties might be overcome. PMID:24198436

  17. Redundant information encoding in primary motor cortex during natural and prosthetic motor control.

    PubMed

    So, Kelvin; Ganguly, Karunesh; Jimenez, Jessica; Gastpar, Michael C; Carmena, Jose M

    2012-06-01

    Redundant encoding of information facilitates reliable distributed information processing. To explore this hypothesis in the motor system, we applied concepts from information theory to quantify the redundancy of movement-related information encoded in the macaque primary motor cortex (M1) during natural and neuroprosthetic control. Two macaque monkeys were trained to perform a delay center-out reaching task controlling a computer cursor under natural arm movement (manual control, 'MC'), and using a brain-machine interface (BMI) via volitional control of neural ensemble activity (brain control, 'BC'). During MC, we found neurons in contralateral M1 to contain higher and more redundant information about target direction than ipsilateral M1 neurons, consistent with the laterality of movement control. During BC, we found that the M1 neurons directly incorporated into the BMI ('direct' neurons) contained the highest and most redundant target information compared to neurons that were not incorporated into the BMI ('indirect' neurons). This effect was even more significant when comparing to M1 neurons of the opposite hemisphere. Interestingly, when we retrained the BMI to use ipsilateral M1 activity, we found that these neurons were more redundant and contained higher information than contralateral M1 neurons, even though ensembles from this hemisphere were previously less redundant during natural arm movement. These results indicate that ensembles most associated to movement contain highest redundancy and information encoding, which suggests a role for redundancy in proficient natural and prosthetic motor control.

  18. 9 CFR 3.138 - Primary conveyances (motor vehicle, rail, air, and marine).

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Primary conveyances (motor vehicle, rail, air, and marine). 3.138 Section 3.138 Animals and Animal Products ANIMAL AND PLANT HEALTH... (motor vehicle, rail, air, and marine). (a) The animal cargo space of primary conveyances used...

  19. Development in primary cell culture of demosponges.

    PubMed

    De Rosa, Salvatore; De Caro, Salvatore; Iodice, Carmine; Tommonaro, Giuseppina; Stefanov, Kamen; Popov, Simeon

    2003-01-23

    We have established primary cell culture of the marine demosponge Dysidea avara and Suberites domuncula. Microbial contamination was controlled by the use of a pool of antibiotics confirming the goodness of this procedure. Effect of pH, temperature and light was studied to establish the better growth conditions. The comparison of lipid composition of sponge and cells suggested a series of experiments to optimise the medium. A glucose dose-dependent experiment showed that the ideal glucose concentration is 1 g l(-1). Supplementing the medium with unsaturated fatty acid and retinol, no promotion of growth was observed, but the compounds were totally metabolised by cells. Increments from 70 to 160% in the number of cells were observed, supplementing the medium with different concentration of cholesterol. These results suggest that the analysis of the chemical composition of sponge and cells give indication on the composition of the nutrient media.

  20. Predicting Organizational Commitment from Organizational Culture in Turkish Primary Schools

    ERIC Educational Resources Information Center

    Ipek, Cemalettin

    2010-01-01

    This study aims to describe organizational culture and commitment and to predict organizational commitment from organizational culture in Turkish primary schools. Organizational Culture Scale (Ipek "1999") and Organizational Commitment Scale (Balay "2000") were used in the data gathering process. The data were collected from 415 primary teachers…

  1. Improvement in precision grip force control with self-modulation of primary motor cortex during motor imagery

    PubMed Central

    Blefari, Maria L.; Sulzer, James; Hepp-Reymond, Marie-Claude; Kollias, Spyros; Gassert, Roger

    2015-01-01

    Motor imagery (MI) has shown effectiveness in enhancing motor performance. This may be due to the common neural mechanisms underlying MI and motor execution (ME). The main region of the ME network, the primary motor cortex (M1), has been consistently linked to motor performance. However, the activation of M1 during motor imagery is controversial, which may account for inconsistent rehabilitation therapy outcomes using MI. Here, we examined the relationship between contralateral M1 (cM1) activation during MI and changes in sensorimotor performance. To aid cM1 activity modulation during MI, we used real-time fMRI neurofeedback-guided MI based on cM1 hand area blood oxygen level dependent (BOLD) signal in healthy subjects, performing kinesthetic MI of pinching. We used multiple regression analysis to examine the correlation between cM1 BOLD signal and changes in motor performance during an isometric pinching task of those subjects who were able to activate cM1 during motor imagery. Activities in premotor and parietal regions were used as covariates. We found that cM1 activity was positively correlated to improvements in accuracy as well as overall performance improvements, whereas other regions in the sensorimotor network were not. The association between cM1 activation during MI with performance changes indicates that subjects with stronger cM1 activation during MI may benefit more from MI training, with implications toward targeted neurotherapy. PMID:25762907

  2. Motor profile of Portuguese preschool children on the Peabody Developmental Motor Scales-2: a cross-cultural study.

    PubMed

    Saraiva, Linda; Rodrigues, Luís P; Cordovil, Rita; Barreiros, João

    2013-06-01

    This study was designed to examine the cultural sensitivity of the PDMS-2 for Portuguese preschool children aged 36-71 months. A total of 540 children (255 males and 285 females) from 15 public preschools of Viana do Castelo, Portugal, were assessed. Age and gender effects in motor performance were examined. Results indicated that PDMS-2 is valid instrument to differentiate Portuguese age groups. Girls presented higher scores than boys in the Grasping and Visuo-motor integration subtests and lower scores in the Object Manipulation subtest. Portuguese preschoolers performed above US norms on Grasping, Visual-motor integration, and Stationary subtests, and bellow on Locomotion and Object Manipulation subtests. Overall, Portuguese children showed better results on the Fine Motor Quotient comparing to the Gross Motor Quotient. These results underline different motor development profiles between Portuguese and American children. PMID:23584176

  3. Action Verbs and the Primary Motor Cortex: A Comparative TMS Study of Silent Reading, Frequency Judgments, and Motor Imagery

    ERIC Educational Resources Information Center

    Tomasino, Barbara; Fink, Gereon R.; Sparing, Roland; Dafotakis, Manuel; Weiss, Peter H.

    2008-01-01

    Single pulse transcranial magnetic stimulation (TMS) was applied to the hand area of the left primary motor cortex or, as a control, to the vertex (STIMULATION: TMS[subscript M1] vs. TMS[subscript vertex]) while right-handed volunteers silently read verbs related to hand actions. We examined three different tasks and time points for stimulation…

  4. The non-motor syndrome of primary dystonia: clinical and pathophysiological implications

    PubMed Central

    Stamelou, Maria; Edwards, Mark J.; Hallett, Mark

    2012-01-01

    Dystonia is typically considered a movement disorder characterized by motor manifestations, primarily involuntary muscle contractions causing twisting movements and abnormal postures. However, growing evidence indicates an important non-motor component to primary dystonia, including abnormalities in sensory and perceptual functions, as well as neuropsychiatric, cognitive and sleep domains. Here, we review this evidence and discuss its clinical and pathophysiological implications. PMID:21933808

  5. Ventral Premotor to Primary Motor Cortical Interactions during Noxious and Naturalistic Action Observation

    ERIC Educational Resources Information Center

    Lago, Angel; Koch, Giacomo; Cheeran, Binith; Marquez, Gonzalo; Sanchez, Jose Andres; Ezquerro, Milagros; Giraldez, Manolo; Fernandez-del-Olmo, Miguel

    2010-01-01

    Within the motor system, cortical areas such as the primary motor cortex (M1) and the ventral premotor cortex (PMv), are thought to be activated during the observation of actions performed by others. However, it is not known how the connections between these areas become active during action observation or whether these connections are modulated…

  6. Form and Function in Motor Mimicry: Topographic Evidence that the Primary Function is Communicative.

    ERIC Educational Resources Information Center

    Bavelas, Janet Beavin; And Others

    1988-01-01

    Proposes that motor mimicry functions as a nonverbal, analogic, relationship message about similarity between observer and other, and that this message is encoded according to Gestalt principles of form. Concludes that the primary function of motor mimicry must be communicative and that any relationship to vicarious processes is secondary. (RAE)

  7. Photoacoustic imaging of functional domains in primary motor cortex in rhesus macaques

    NASA Astrophysics Data System (ADS)

    Jo, Janggun; Zhang, Hongyu; Cheney, Paul; Yang, Xinmai

    2012-02-01

    Functional detection in primate brains has particular advantages because of the similarity between non-human primate brain and human brain and the potential for relevance to a wide range of conditions such as stroke and Parkinson's disease. In this research, we used photoacoustic imaging (PAI) technique to detect functional changes in primary motor cortex of awake rhesus monkeys. We observed strong increases in photoacoustic signal amplitude during both passive and active forelimb movement, which indicates an increase in total hemoglobin concentration resulting from activation of primary motor cortex. Further, with PAI approach, we were able to obtain depthresolved functional information from primary motor cortex. The results show that PAI can reliably detect primary motor cortex activation associated with forelimb movement in rhesus macaques with a minimal-invasive approach.

  8. Primary Teacher Identity, Commitment and Career in Performative School Cultures

    ERIC Educational Resources Information Center

    Troman, Geoff

    2008-01-01

    The research reported here maps changes in primary teachers' identity, commitment and perspectives and subjective experiences of occupational career in the context of performative primary school cultures. The research aimed to provide in-depth knowledge of performative school culture and teachers' subjective experiences in their work of teaching.…

  9. Examining School Culture in Flemish and Chinese Primary Schools

    ERIC Educational Resources Information Center

    Zhu, Chang; Devos, Geert; Tondeur, Jo

    2014-01-01

    The aim of this research is to gain understanding about school culture characteristics of primary schools in the Flemish and Chinese context. The study was carried out in Flanders (Belgium) and China, involving a total of 44 Flemish schools and 40 Chinese schools. The School Culture Scales were used to measure five school culture dimensions with…

  10. Teaching Cultural History from Primary Events

    ERIC Educational Resources Information Center

    Carson, Robert N.

    2004-01-01

    This article explores the relationship between specific cultural events such as Galileo's work with the pendulum and a curriculum design that seeks to establish in skeletal form a comprehensive epic narrative about the co-evolution of cultural systems and human consciousness. The article explores some of the challenges and some of the strategies…

  11. Zonisamide Enhances Neurite Elongation of Primary Motor Neurons and Facilitates Peripheral Nerve Regeneration In Vitro and in a Mouse Model

    PubMed Central

    Yagi, Hideki; Ohkawara, Bisei; Nakashima, Hiroaki; Ito, Kenyu; Tsushima, Mikito; Ishii, Hisao; Noto, Kimitoshi; Ohta, Kyotaro; Masuda, Akio; Imagama, Shiro; Ishiguro, Naoki; Ohno, Kinji

    2015-01-01

    No clinically applicable drug is currently available to enhance neurite elongation after nerve injury. To identify a clinically applicable drug, we screened pre-approved drugs for neurite elongation in the motor neuron-like NSC34 cells. We found that zonisamide, an anti-epileptic and anti-Parkinson’s disease drug, promoted neurite elongation in cultured primary motor neurons and NSC34 cells in a concentration-dependent manner. The neurite-scratch assay revealed that zonisamide enhanced neurite regeneration. Zonisamide was also protective against oxidative stress-induced cell death of primary motor neurons. Zonisamide induced mRNA expression of nerve growth factors (BDNF, NGF, and neurotrophin-4/5), and their receptors (tropomyosin receptor kinase A and B). In a mouse model of sciatic nerve autograft, intragastric administration of zonisamide for 1 week increased the size of axons distal to the transected site 3.9-fold. Zonisamide also improved the sciatic function index, a marker for motor function of hindlimbs after sciatic nerve autograft, from 6 weeks after surgery. At 8 weeks after surgery, zonisamide was protective against denervation-induced muscle degeneration in tibialis anterior, and increased gene expression of Chrne, Colq, and Rapsn, which are specifically expressed at the neuromuscular junction. We propose that zonisamide is a potential therapeutic agent for peripheral nerve injuries as well as for neuropathies due to other etiologies. PMID:26571146

  12. tDCS-induced alterations in GABA concentration within primary motor cortex predict motor learning and motor memory: a 7 T magnetic resonance spectroscopy study.

    PubMed

    Kim, Soyoung; Stephenson, Mary C; Morris, Peter G; Jackson, Stephen R

    2014-10-01

    Transcranial direct current stimulation (tDCS) is a non-invasive brain stimulation technique that alters cortical excitability in a polarity specific manner and has been shown to influence learning and memory. tDCS may have both on-line and after-effects on learning and memory, and the latter are thought to be based upon tDCS-induced alterations in neurochemistry and synaptic function. We used ultra-high-field (7 T) magnetic resonance spectroscopy (MRS), together with a robotic force adaptation and de-adaptation task, to investigate whether tDCS-induced alterations in GABA and Glutamate within motor cortex predict motor learning and memory. Note that adaptation to a robot-induced force field has long been considered to be a form of model-based learning that is closely associated with the computation and 'supervised' learning of internal 'forward' models within the cerebellum. Importantly, previous studies have shown that on-line tDCS to the cerebellum, but not to motor cortex, enhances model-based motor learning. Here we demonstrate that anodal tDCS delivered to the hand area of the left primary motor cortex induces a significant reduction in GABA concentration. This effect was specific to GABA, localised to the left motor cortex, and was polarity specific insofar as it was not observed following either cathodal or sham stimulation. Importantly, we show that the magnitude of tDCS-induced alterations in GABA concentration within motor cortex predicts individual differences in both motor learning and motor memory on the robotic force adaptation and de-adaptation task.

  13. 9 CFR 3.114 - Primary conveyances (motor vehicle, rail, air and marine).

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... conveyances (motor vehicle, rail, air and marine). (a) The animal cargo space of primary conveyances used in... safety and comfort of the marine mammals contained within at all times. All primary conveyances used must... animal cargo space must be constructed and maintained in a manner that will prevent the ingress of...

  14. Motor Training Promotes Both Synaptic and Intrinsic Plasticity of Layer II/III Pyramidal Neurons in the Primary Motor Cortex

    PubMed Central

    Kida, Hiroyuki; Tsuda, Yasumasa; Ito, Nana; Yamamoto, Yui; Owada, Yuji; Kamiya, Yoshinori; Mitsushima, Dai

    2016-01-01

    Motor skill training induces structural plasticity at dendritic spines in the primary motor cortex (M1). To further analyze both synaptic and intrinsic plasticity in the layer II/III area of M1, we subjected rats to a rotor rod test and then prepared acute brain slices. Motor skill consistently improved within 2 days of training. Voltage clamp analysis showed significantly higher α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/N-methyl-d-aspartate (AMPA/NMDA) ratios and miniature EPSC amplitudes in 1-day trained rats compared with untrained rats, suggesting increased postsynaptic AMPA receptors in the early phase of motor learning. Compared with untrained controls, 2-days trained rats showed significantly higher miniature EPSC amplitude and frequency. Paired-pulse analysis further demonstrated lower rates in 2-days trained rats, suggesting increased presynaptic glutamate release during the late phase of learning. One-day trained rats showed decreased miniature IPSC frequency and increased paired-pulse analysis of evoked IPSC, suggesting a transient decrease in presynaptic γ-aminobutyric acid (GABA) release. Moreover, current clamp analysis revealed lower resting membrane potential, higher spike threshold, and deeper afterhyperpolarization in 1-day trained rats—while 2-days trained rats showed higher membrane potential, suggesting dynamic changes in intrinsic properties. Our present results indicate dynamic changes in glutamatergic, GABAergic, and intrinsic plasticity in M1 layer II/III neurons after the motor training. PMID:27193420

  15. Establishment and characterization of Xenopus oviduct cells in primary culture

    SciTech Connect

    Marsh, J.; Tata, J.R. )

    1987-11-01

    Based on previously established procedure of Xenopus hepatocytes, the authors describe tubular oviduct cells in primary culture which continue to secrete substantial quantities of egg jelly for several days, as can be visualized microscopically. Freshly isolated cells exhibited a culture shock response, from which they recovered by the third day in culture. This recovery was characterized by (a) the diminished synthesis of heat shock proteins hsp 70 and hsp 85, (b) the cessation of the drop in number of estrogen receptor, and (c) the enhanced rate of synthesis of cellular and secreted proteins. The oviduct estrogen receptor had the same characteristics as those in other estrogen target tissues and was present in the same amount as in adult female Xenopus hepatocytes. The successful establishment and characterization of primary cultures of both liver and oviduct cells now fulfill the conditions required for investigating the basis for tissue specificity of regulation by estrogen of Xenopus egg protein gene expression in primary cell culture.

  16. Action verbs and the primary motor cortex: a comparative TMS study of silent reading, frequency judgments, and motor imagery.

    PubMed

    Tomasino, Barbara; Fink, Gereon R; Sparing, Roland; Dafotakis, Manuel; Weiss, Peter H

    2008-01-01

    Single pulse transcranial magnetic stimulation (TMS) was applied to the hand area of the left primary motor cortex or, as a control, to the vertex (STIMULATION: TMS(M1) vs. TMS(vertex)) while right-handed volunteers silently read verbs related to hand actions. We examined three different tasks and time points for stimulation within the same experiment: subjects indicated with their left foot when they (i) had finished reading, (ii) had judged whether the corresponding movement involved a hand rotation after simulating the hand movement, and (iii) had judged whether they would frequently encounter the action verb in a newspaper (TASK: silent reading, motor imagery, and frequency judgment). Response times were compared between TMS(M1) and TMS(vertex), both applied at different time points after stimulus onset (DELAY: 150, 300, 450, 600, and 750 ms). TMS(M1) differentially modulated task performance: there was a significant facilitatory effect of TMS(M1) for the imagery task only (about 88 ms), with subjects responding about 10% faster (compared to TMS(vertex)). In contrast, response times for silent reading and frequency judgments were unaffected by TMS(M1). No differential effect of the time point of TMS(M1) was observed. The differential effect of TMS(M1) when subjects performed a motor imagery task (relative to performing silent reading or frequency judgments with the same set of verbs) suggests that the primary motor cortex is critically involved in processing action verbs only when subjects are simulating the corresponding movement. This task-dependent effect of hand motor cortex TMS on the processing of hand-related action verbs is discussed with respect to the notion of embodied cognition and the associationist theory. PMID:18328510

  17. The Relationship between Social and Motor Cognition in Primary School Age-Children

    PubMed Central

    Kenny, Lorcan; Hill, Elisabeth; Hamilton, Antonia F. de C.

    2016-01-01

    There is increased interest in the relationship between motor skills and social skills in child development, with evidence that the mechanisms underlying these behaviors may be linked. We took a cognitive approach to this problem, and examined the relationship between four specific cognitive domains: theory of mind, motor skill, action understanding, and imitation. Neuroimaging and adult research suggest that action understanding and imitation are closely linked, but are somewhat independent of theory of mind and low-level motor control. Here, we test if a similar pattern is shown in child development. A sample of 101 primary school aged children with a wide ability range completed tests of IQ (Raven’s matrices), theory of mind, motor skill, action understanding, and imitation. Parents reported on their children’s social, motor and attention performance as well as developmental concerns. The results showed that action understanding and imitation correlate, with the latter having a weak link to motor control. Theory of mind was independent of the other tasks. These results imply that independent cognitive processes for social interaction (theory of mind) and for motor control can be identified in primary school age children, and challenge approaches that link all these domains together. PMID:26941685

  18. Absent movement-related cortical potentials in children with primary motor stereotypies.

    PubMed

    Houdayer, Elise; Walthall, Jessica; Belluscio, Beth A; Vorbach, Sherry; Singer, Harvey S; Hallett, Mark

    2014-08-01

    The underlying pathophysiologic mechanism for complex motor stereotypies in children is unknown, with hypotheses ranging from an arousal to a motor control disorder. Movement-related cortical potentials (MRCPs), representing the activation of cerebral areas involved in the generation of movements, precede and accompany self-initiated voluntary movements. The goal of this study was to compare cerebral activity associated with stereotypies to that seen with voluntary movements in children with primary complex motor stereotypies. Electroencephalographic (EEG) activity synchronized with video recording was recorded in 10 children diagnosed with primary motor stereotypies and 7 controls. EEG activity related to stereotypies and self-paced arm movements were analyzed for presence or absence of early or late MRCP, a steep negativity beginning about 1 second before the onset of a voluntary movement. Early MRCPs preceded self-paced arm movements in 8 of 10 children with motor stereotypies and in 6 of 7 controls. Observed MRCPs did not differ between groups. No MRCP was identified before the appearance of a complex motor stereotypy. Unlike voluntary movements, stereotypies are not preceded by MRCPs. This indicates that premotor areas are likely not involved in the preparation of these complex movements and suggests that stereotypies are initiated by mechanisms different from voluntary movements. Further studies are required to determine the site of the motor control abnormality within cortico-striatal-thalamo-cortical pathways and to identify whether similar findings would be found in children with secondary stereotypies.

  19. N-cadherin regulates primary motor axons growth and branching during zebrafish embryonic development

    PubMed Central

    Brusés, Juan L

    2013-01-01

    N-cadherin is a classical type I cadherin that contributes to the formation of neural circuits by regulating growth cone migration and the formation of synaptic contacts. This study analyzed the role of N-cadherin in primary motor axons growth during development of the zebrafish (Danio rerio) embryo. After exiting the spinal cord, primary motor axons migrate ventrally through a common pathway and form the first neuromuscular junction with the muscle pioneer cells located at the horizontal myoseptum, which serves as a choice point for cell-type specific pathway selection. Analysis of N-cadherin mutants (cdh2hi3644Tg) and embryos injected with N-cadherin antisense morpholinos showed primary motor axons extending aberrant axonal branches at the choice point in ~40% of the somitic hemisegments, and an ~150% increase in the number of branches per axon length within the ventral myotome. Analysis of individual axons trajectories showed that the caudal (CaP) and rostral (RoP) motor neurons axons formed aberrant branches at the choice point which abnormally extended in the rostrocaudal axis and ventrally to the horizontal myoseptum. Expression of a dominant-interfering N-cadherin cytoplasmic domain in primary motor neurons caused some axons to abnormally stall at the horizontal myoseptum and to impair their migration into the ventral myotome. However, in N-cadherin depleted embryos the majority of primary motor axons innervated their appropriate myotomal territories indicating that N-cadherin regulates motor axon growth and branching without severely affecting the mechanisms that control axonal target selection. PMID:21452216

  20. Intersections between interprofessional practice, cultural competency and primary healthcare.

    PubMed

    Oelke, Nelly D; Thurston, Wilfreda E; Arthur, Nancy

    2013-09-01

    The concepts of interprofessional collaborative practice (IPCP), cultural competency and primary healthcare (PHC) appear to be linked in theory and practice. This discussion article provides arguments explicating the potential linkages between IPCP and cultural competency. We argue that cultural competency is an important component of IPCP both for relationships with patients and/or communities in which providers work and between team members. Organizational structures also play an important role in facilitating IPCP and cultural competency. The integration of both IPCP and cultural competency has the potential to enhance positive health outcomes. Furthermore, we argue IPCP and cultural competency have important implications for PHC service design, given interprofessional teams are a key component of PHC systems. Linking these concepts in providing PHC services can be essential for impacting outcomes at all levels of primary healthcare, including patient, provider and systems. PMID:23683058

  1. Motor Profile of Portuguese Preschool Children on the Peabody Developmental Motor Scales-2: A Cross-Cultural Study

    ERIC Educational Resources Information Center

    Saraiva, Linda; Rodrigues, Luis P.; Cordovil, Rita; Barreiros, Joao

    2013-01-01

    This study was designed to examine the cultural sensitivity of the PDMS-2 for Portuguese preschool children aged 36-71 months. A total of 540 children (255 males and 285 females) from 15 public preschools of Viana do Castelo, Portugal, were assessed. Age and gender effects in motor performance were examined. Results indicated that PDMS-2 is valid…

  2. Event-related desynchronization reflects downregulation of intracortical inhibition in human primary motor cortex.

    PubMed

    Takemi, Mitsuaki; Masakado, Yoshihisa; Liu, Meigen; Ushiba, Junichi

    2013-09-01

    There is increasing interest in electroencephalogram (EEG)-based brain-computer interface (BCI) as a tool for rehabilitation of upper limb motor functions in hemiplegic stroke patients. This type of BCI often exploits mu and beta oscillations in EEG recorded over the sensorimotor areas, and their event-related desynchronization (ERD) following motor imagery is believed to represent increased sensorimotor cortex excitability. However, it remains unclear whether the sensorimotor cortex excitability is actually correlated with ERD. Thus we assessed the association of ERD with primary motor cortex (M1) excitability during motor imagery of right wrist movement. M1 excitability was tested by motor evoked potentials (MEPs), short-interval intracortical inhibition (SICI), and intracortical facilitation (ICF) with transcranial magnetic stimulation (TMS). Twenty healthy participants were recruited. The participants performed 7 s of rest followed by 5 s of motor imagery and received online visual feedback of the ERD magnitude of the contralateral hand M1 while performing the motor imagery task. TMS was applied to the right hand M1 when ERD exceeded predetermined thresholds during motor imagery. MEP amplitudes, SICI, and ICF were recorded from the agonist muscle of the imagined hand movement. Results showed that the large ERD during wrist motor imagery was associated with significantly increased MEP amplitudes and reduced SICI but no significant changes in ICF. Thus ERD magnitude during wrist motor imagery represents M1 excitability. This study provides electrophysiological evidence that a motor imagery task involving ERD may induce changes in corticospinal excitability similar to changes accompanying actual movements.

  3. Effects of an Exhaustive Exercise on Motor Skill Learning and on the Excitability of Primary Motor Cortex and Supplementary Motor Area

    PubMed Central

    Coco, Marinella; Perciavalle, Vincenzo; Cavallari, Paolo; Perciavalle, Valentina

    2016-01-01

    Abstract We examined, on 28 healthy adult subjects, the possible correlations of an exhaustive exercise, and the consequent high blood lactate levels, on immediate (explicit) and delayed (implicit) motor execution of sequential finger movements (cognitive task). Moreover, we determined with transcranial magnetic stimulation whether changes in motor performance are associated with variations in excitability of primary motor area (M1) and supplementary motor area (SMA). We observed that, after an acute exhaustive exercise, the large increase of blood lactate is associated with a significant worsening of both explicit and implicit sequential visuomotor task paradigms, without gender differences. We also found that, at the end of the exhaustive exercise, there is a change of excitability in both M1 and SMA. In particular, the excitability of M1 was increased whereas that of SMA decreased and, also in this case, without gender differences. These results support the idea that an increase of blood lactate after an exhaustive exercise appears to have a protective effect at level of primary cortical areas (as M1), although at the expense of efficiency of adjacent cortical regions (as SMA). PMID:26986109

  4. Effects of an Exhaustive Exercise on Motor Skill Learning and on the Excitability of Primary Motor Cortex and Supplementary Motor Area.

    PubMed

    Coco, Marinella; Perciavalle, Vincenzo; Cavallari, Paolo; Perciavalle, Valentina

    2016-03-01

    We examined, on 28 healthy adult subjects, the possible correlations of an exhaustive exercise, and the consequent high blood lactate levels, on immediate (explicit) and delayed (implicit) motor execution of sequential finger movements (cognitive task). Moreover, we determined with transcranial magnetic stimulation whether changes in motor performance are associated with variations in excitability of primary motor area (M1) and supplementary motor area (SMA). We observed that, after an acute exhaustive exercise, the large increase of blood lactate is associated with a significant worsening of both explicit and implicit sequential visuomotor task paradigms, without gender differences. We also found that, at the end of the exhaustive exercise, there is a change of excitability in both M1 and SMA. In particular, the excitability of M1 was increased whereas that of SMA decreased and, also in this case, without gender differences. These results support the idea that an increase of blood lactate after an exhaustive exercise appears to have a protective effect at level of primary cortical areas (as M1), although at the expense of efficiency of adjacent cortical regions (as SMA).

  5. Effects of an Exhaustive Exercise on Motor Skill Learning and on the Excitability of Primary Motor Cortex and Supplementary Motor Area.

    PubMed

    Coco, Marinella; Perciavalle, Vincenzo; Cavallari, Paolo; Perciavalle, Valentina

    2016-03-01

    We examined, on 28 healthy adult subjects, the possible correlations of an exhaustive exercise, and the consequent high blood lactate levels, on immediate (explicit) and delayed (implicit) motor execution of sequential finger movements (cognitive task). Moreover, we determined with transcranial magnetic stimulation whether changes in motor performance are associated with variations in excitability of primary motor area (M1) and supplementary motor area (SMA). We observed that, after an acute exhaustive exercise, the large increase of blood lactate is associated with a significant worsening of both explicit and implicit sequential visuomotor task paradigms, without gender differences. We also found that, at the end of the exhaustive exercise, there is a change of excitability in both M1 and SMA. In particular, the excitability of M1 was increased whereas that of SMA decreased and, also in this case, without gender differences. These results support the idea that an increase of blood lactate after an exhaustive exercise appears to have a protective effect at level of primary cortical areas (as M1), although at the expense of efficiency of adjacent cortical regions (as SMA). PMID:26986109

  6. Matrix Metalloproteinases in Primary Culture of Cardiomyocytes.

    PubMed

    Bildyug, N B; Voronkina, I V; Smagina, L V; Yudintseva, N M; Pinaev, G P

    2015-10-01

    The highly organized contractile apparatus of cardiomyocytes in heart tissue allows for their continuous contractility, whereas extracellular matrix components are synthesized and spatially organized by fibroblasts and endothelial cells. However, reorganization of the cardiomyocyte contractile apparatus occurs upon their 2D cultivation, which is accompanied by transient loss of their contractility and acquired capability of extracellular matrix synthesis (Bildyug, N. B., and Pinaev, G. P. (2013) Tsitologiya, 55, 713-724). In this study, matrix metalloproteinases were investigated at different times of cardiomyocyte 2D cultivation and 3D cultivation in collagen gels. It was found that cardiomyocytes in 2D culture synthesize matrix metalloproteinases MMP-2 and MMP-9, wherein their amount varies with the cultivation time. The peak MMP-9 amount is at early cultivation time, when the reorganization of cardiomyocyte contractile apparatus occurs, and the MMP-2 peak precedes the recovery of the initial organization of their contractile apparatus. Upon cardiomyocyte cultivation in 3D collagen gels, in which case their contractile apparatus does not rearrange, a steady small amount of MMP-2 and MMP-9 is observed. These data indicate that the cardiomyocyte contractile apparatus reorganization in culture is associated with synthesis and spatial organization of their own extracellular matrix.

  7. A digital microfluidic platform for primary cell culture and analysis.

    PubMed

    Srigunapalan, Suthan; Eydelnant, Irwin A; Simmons, Craig A; Wheeler, Aaron R

    2012-01-21

    Digital microfluidics (DMF) is a technology that facilitates electrostatic manipulation of discrete nano- and micro-litre droplets across an array of electrodes, which provides the advantages of single sample addressability, automation, and parallelization. There has been considerable interest in recent years in using DMF for cell culture and analysis, but previous studies have used immortalized cell lines. We report here the first digital microfluidic method for primary cell culture and analysis. A new mode of "upside-down" cell culture was implemented by patterning the top plate of a device using a fluorocarbon liftoff technique. This method was useful for culturing three different primary cell types for up to one week, as well as implementing a fixation, permeabilization, and staining procedure for F-actin and nuclei. A multistep assay for monocyte adhesion to endothelial cells (ECs) was performed to evaluate functionality in DMF-cultured primary cells and to demonstrate co-culture using a DMF platform. Monocytes were observed to adhere in significantly greater numbers to ECs exposed to tumor necrosis factor (TNF)-α than those that were not, confirming that ECs cultured in this format maintain in vivo-like properties. The ability to manipulate, maintain, and assay primary cells demonstrates a useful application for DMF in studies involving precious samples of cells from small animals or human patients.

  8. The effects of acute aerobic exercise on the primary motor cortex.

    PubMed

    Singh, Amaya M; Staines, W Richard

    2015-01-01

    The effect of aerobic exercise on primary motor cortical excitability is a relevant area of interest for both motor learning and motor rehabilitation. Transient excitability changes that may follow an exercise session are a necessary precursor to more lasting neuroplastic changes. While the number of studies is limited, research suggests that a session of aerobic exercise can create an ideal environment for the early induction of plasticity. Potential mechanisms include the upregulation of neurotransmitter activity, altered cerebral metabolism and cortisol levels, and increases in brain-derived neurotrophic factor. While there is considerable evidence that chronic physical activity positively impacts brain health and function, studies examining cortical excitability changes and motor performance after a single session of exercise are lacking. Further research is required to determine the clinical utility and feasibility of aerobic exercise.

  9. Primary motor cortex reports efferent control of vibrissa motion on multiple time scales

    PubMed Central

    Hill, Daniel N.; Curtis, John C.; Moore, Jeffrey D.; Kleinfeld, David

    2011-01-01

    Exploratory whisking in rat is an example of self-generated movement on multiple time scales, from slow variations in the envelope of whisking to the rapid sequence of muscle contractions during a single whisk cycle. We find that, as a population, spike trains of single units in primary vibrissa motor cortex report the absolute angle of vibrissa position. This representation persists after sensory nerve transection, indicating an efferent source. About two-thirds of the units are modulated by slow variations in the envelope of whisking while relatively few units report rapid changes in position within the whisk cycle. The combined results from this study and past measurements, which show that primary sensory cortex codes the whisking envelope as a motor copy signal, imply that signals present in both sensory and motor cortices are necessary to compute coordinates based on vibrissa touch. PMID:22017992

  10. 9 CFR 3.114 - Primary conveyances (motor vehicle, rail, air and marine).

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... conveyances (motor vehicle, rail, air and marine). (a) The animal cargo space of primary conveyances used in... animal cargo space must be constructed and maintained in a manner that will prevent the ingress of engine... mammals must only be placed in animal cargo spaces that have a supply of air sufficient for each...

  11. 9 CFR 3.88 - Primary conveyances (motor vehicle, rail, air, and marine).

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... conveyances (motor vehicle, rail, air, and marine). (a) The animal cargo space of primary conveyances used to... cargo space must have a supply of air that is sufficient for the normal breathing of all the animals... the animal cargo space in a manner that provides protection from the elements and that allows...

  12. 9 CFR 3.88 - Primary conveyances (motor vehicle, rail, air, and marine).

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... conveyances (motor vehicle, rail, air, and marine). (a) The animal cargo space of primary conveyances used to... cargo space must have a supply of air that is sufficient for the normal breathing of all the animals... the animal cargo space in a manner that provides protection from the elements and that allows...

  13. 9 CFR 3.15 - Primary conveyances (motor vehicle, rail, air, and marine).

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... conveyances (motor vehicle, rail, air, and marine). (a) The animal cargo space of primary conveyances used to transport dogs and cats must be designed, constructed, and maintained in a manner that at all times protects... cargo space must have a supply of air that is sufficient for the normal breathing of all the...

  14. 9 CFR 3.37 - Primary conveyances (motor vehicle, rail, air, and marine).

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... the safety and comfort of the live guinea pigs and hamsters at all times. (b) The animal cargo space... conveyances (motor vehicle, rail, air, and marine). (a) The animal cargo space of primary conveyances used in... be placed in an animal cargo space that does not have a supply of air sufficient for normal...

  15. 9 CFR 3.138 - Primary conveyances (motor vehicle, rail, air, and marine).

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... comfort of the live animals contained therein at all times. (b) The animal cargo space shall be... (motor vehicle, rail, air, and marine). (a) The animal cargo space of primary conveyances used in... cargo space that does not have a supply of air sufficient for normal breathing for each live...

  16. 9 CFR 3.37 - Primary conveyances (motor vehicle, rail, air, and marine).

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... the safety and comfort of the live guinea pigs and hamsters at all times. (b) The animal cargo space... conveyances (motor vehicle, rail, air, and marine). (a) The animal cargo space of primary conveyances used in... be placed in an animal cargo space that does not have a supply of air sufficient for normal...

  17. 9 CFR 3.62 - Primary conveyances (motor vehicle, rail, air, and marine).

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... comfort of the rabbits contained therein at all times. (b) The animal cargo space shall be constructed and... (motor vehicle, rail, air, and marine). (a) The animal cargo space of primary conveyances used in... during transportation in commerce. (c) No live rabbit shall be placed in an animal cargo space that...

  18. 9 CFR 3.88 - Primary conveyances (motor vehicle, rail, air, and marine).

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... conveyances (motor vehicle, rail, air, and marine). (a) The animal cargo space of primary conveyances used to transport nonhuman primates must be designed, constructed, and maintained in a manner that at all times... cargo space must have a supply of air that is sufficient for the normal breathing of all the...

  19. 9 CFR 3.62 - Primary conveyances (motor vehicle, rail, air, and marine).

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... comfort of the rabbits contained therein at all times. (b) The animal cargo space shall be constructed and... (motor vehicle, rail, air, and marine). (a) The animal cargo space of primary conveyances used in... during transportation in commerce. (c) No live rabbit shall be placed in an animal cargo space that...

  20. 9 CFR 3.88 - Primary conveyances (motor vehicle, rail, air, and marine).

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... conveyances (motor vehicle, rail, air, and marine). (a) The animal cargo space of primary conveyances used to... cargo space must have a supply of air that is sufficient for the normal breathing of all the animals... the animal cargo space in a manner that provides protection from the elements and that allows...

  1. 9 CFR 3.114 - Primary conveyances (motor vehicle, rail, air and marine).

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... conveyances (motor vehicle, rail, air and marine). (a) The animal cargo space of primary conveyances used in... animal cargo space must be constructed and maintained in a manner that will prevent the ingress of engine... mammals must only be placed in animal cargo spaces that have a supply of air sufficient for each...

  2. 9 CFR 3.88 - Primary conveyances (motor vehicle, rail, air, and marine).

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... conveyances (motor vehicle, rail, air, and marine). (a) The animal cargo space of primary conveyances used to... cargo space must have a supply of air that is sufficient for the normal breathing of all the animals... the animal cargo space in a manner that provides protection from the elements and that allows...

  3. Enhancing Access to Primary Cultural Heritage Materials of India

    NASA Astrophysics Data System (ADS)

    Scharf, Peter M.; Hyman, Malcolm

    This chapter is about enhancing access to primary cultural heritage materials of India housed in academic libraries by integrating them with machine-readable texts, lexical resources, and linguistic software in a digital library. Integrating primary cultural materials with a digital library can enable broad use of Indic collections for research and education. For the purposes of illustrating this procedure, we outline here the development of a prototype using the collections of Sanskrit manuscripts in the libraries at Brown University and the University of Pennsylvania and integrating them with The Sanskrit Library. The result is extendable to collections of Indic materials throughout the world and can serve as a model for digitization projects of cultural materials in other major culture-bearing languages such as Greek, Latin, Arabic, Persian, and Chinese.

  4. Laminin is produced by early rat astrocytes in primary culture

    PubMed Central

    1983-01-01

    The production of laminin by early rat astrocytes in primary culture was investigated by double immunofluorescence staining for laminin and the glial fibrillary acidic protein (GFAP), a defined astrocyte marker. In early cultures (3 d in vitro; 3 DIV) cytoplasmic laminin was detected in all the GFAP-positive cells which formed the major population (80%) of the nonneuronal cells present in cultures from 20- 21-d embryonic, newborn, or 5-d-old rat brains. Monensin treatment (10 microM, 4 h) resulted in accumulation of laminin in the Golgi region, located using labeled wheat germ agglutinin. Laminin started gradually to disappear from the cells with the time in culture, was absent in star-shaped, apparently mature astrocytes, but remained as pericellular matrix deposits. The disappearance of cellular laminin was dependent on the age of the animal and the time in culture so that it started earlier in cultures from 5-d-old rat brains (5 DIV) and approximately following the in vivo age difference in cultures from newborn (12 DIV) and embryonic (14 DIV) rat brains. Our results indicate that laminin is a protein of early astrocytes and also deposited by them in primary culture, thus suggesting a role for this glycoprotein in the development of the central nervous system. PMID:6339524

  5. A gait paradigm reveals different patterns of abnormal cerebellar motor learning in primary focal dystonias.

    PubMed

    Hoffland, B S; Veugen, L C; Janssen, M M H P; Pasman, J W; Weerdesteyn, V; van de Warrenburg, B P

    2014-12-01

    Accumulating evidence points to a role of the cerebellum in the pathophysiology of primary dystonia. The aim of this study was to investigate whether the abnormalities of cerebellar motor learning in primary dystonia are solely detectable in more pure forms of cerebellum-dependent associative motor learning paradigms, or whether these are also present in other motor learning paradigms that rely heavily on the cerebellum but in addition require a more widespread sensorimotor network. Twenty-six patients with various forms of focal dystonia and 10 age-matched healthy controls participated in a motor learning paradigm on a split-belt treadmill. By using reflective markers, three-dimensional kinematics were recorded using a 6-camera motion analysis system. Adaptation walking parameters were analyzed offline, comparing the different dystonia groups and healthy controls. Patients with blepharospasm and writer's cramp were significantly impaired on various adaptation walking parameters. Whereas results of cervical dystonia patients did not differ from healthy controls in terms of adaptation walking parameters, differences in parameters of normal gait were found. We have here demonstrated abnormal sensorimotor adaptation with the split-belt paradigm in patients with blepharospasm and writer's cramp. This reinforces the current concept of cerebellar dysfunction in primary dystonia, and that this extends beyond more pure forms of cerebellum-dependent associative motor learning paradigms. However, the finding of normal adaptation in cervical dystonia patients indicates that the pattern of cerebellar dysfunction may be slightly different for the various forms of primary focal dystonia, suggesting that actual cerebellar pathology may not be a primary driving force in dystonia.

  6. Coculture of Primary Motor Neurons and Schwann Cells as a Model for In Vitro Myelination

    PubMed Central

    Hyung, Sujin; Yoon Lee, Bo; Park, Jong-Chul; Kim, Jinseok; Hur, Eun-Mi; Francis Suh, Jun-Kyo

    2015-01-01

    A culture system that can recapitulate myelination in vitro will not only help us better understand the mechanism of myelination and demyelination, but also find out possible therapeutic interventions for treating demyelinating diseases. Here, we introduce a simple and reproducible myelination culture system using mouse motor neurons (MNs) and Schwann cells (SCs). Dissociated motor neurons are plated on a feeder layer of SCs, which interact with and wrap around the axons of MNs as they differentiate in culture. In our MN-SC coculture system, MNs survived over 3 weeks and extended long axons. Both viability and axon growth of MNs in the coculture were markedly enhanced as compared to those of MN monoculture. Co-labeling of myelin basic proteins (MBPs) and neuronal microtubules revealed that SC formed myelin sheaths by wrapping around the axons of MNs. Furthermore, using the coculture system we found that treatment of an antioxidant substance coenzyme Q10 (Co-Q10) markedly facilitated myelination. PMID:26456300

  7. Coculture of Primary Motor Neurons and Schwann Cells as a Model for In Vitro Myelination.

    PubMed

    Hyung, Sujin; Yoon Lee, Bo; Park, Jong-Chul; Kim, Jinseok; Hur, Eun-Mi; Francis Suh, Jun-Kyo

    2015-10-12

    A culture system that can recapitulate myelination in vitro will not only help us better understand the mechanism of myelination and demyelination, but also find out possible therapeutic interventions for treating demyelinating diseases. Here, we introduce a simple and reproducible myelination culture system using mouse motor neurons (MNs) and Schwann cells (SCs). Dissociated motor neurons are plated on a feeder layer of SCs, which interact with and wrap around the axons of MNs as they differentiate in culture. In our MN-SC coculture system, MNs survived over 3 weeks and extended long axons. Both viability and axon growth of MNs in the coculture were markedly enhanced as compared to those of MN monoculture. Co-labeling of myelin basic proteins (MBPs) and neuronal microtubules revealed that SC formed myelin sheaths by wrapping around the axons of MNs. Furthermore, using the coculture system we found that treatment of an antioxidant substance coenzyme Q10 (Co-Q10) markedly facilitated myelination.

  8. Multiparameter analysis of primary epithelial cultures grown on cyclopore membranes.

    PubMed

    De Boer, W I; Rebel, J M; Vermey, M; Thijssen, C D; Van der Kwast, T H

    1994-02-01

    The use of porous membranes as culture support for epithelial cells has previously been shown to cause functional differentiation of these cells mimicking an in vivo condition, in contrast to culture on plastic. The different materials of which the membranes are made also have different properties, such as transparency, rigidity, and retention of molecules. Cyclopore membranes (polyethylene terephtalate) are permeable, transparent, rigid, and have low protein retention. In this study we examined the applicability of assessing multiple parameters on a single culture of primary epithelial cells on a Cyclopore membrane. Cultures of transitional epithelial cells on these membranes differentiate into an organoid-like epithelium. We were able to perform morphometric analysis during and after cell culture and to quantitate proliferation and differentiation by double immunoenzymatic staining. On these cultures, quantitative radiochemical analysis could also be achieved, retaining the morphology and the immunohistochemical staining. Cross-sections of paraffin-embedded and plastic-embedded cultures were analyzed qualitatively by light and transmission electron microscopy, respectively. Finally, cytokeratins in these cultures could also be visualized by immunofluorescence analysis. This suitability for simultaneous assessment of both qualitative and quantitative parameters on a single cell culture grown on a Cyclopore membrane reduces the need of biological materials and may lead to better insight into physiological processes. PMID:7507144

  9. Organizational culture, job satisfaction, and clinician turnover in primary care.

    PubMed

    Hall, Charles B; Brazil, Kevin; Wakefield, Dorothy; Lerer, Trudy; Tennen, Howard

    2010-04-01

    The purpose of this study is to examine how organizational culture and job satisfaction affect clinician turnover in primary care pediatric practices. One hundred thirty clinicians from 36 primary care pediatric practices completed the Primary Care Organizational Questionnaire (PCOQ), which evaluates interactions among members of the practice and job-related attributes measuring 8 organizational factors, along with a separate 3-item instrument measuring job satisfaction. Random effects logistic models were used to assess the associations between job satisfaction, the organizational factors from the PCOQ, and clinician turnover over the subsequent year. All 8 measured organizational factors from the PCOQ, particularly perceived effectiveness, were associated with job satisfaction. Five of the 8 organizational factors were also associated with clinician turnover. The effects of the organizational factors on turnover were substantially reduced in a model that included job satisfaction; only 1 organizational factor, communication between clinicians and nonclinicians, remained significant (P = .05). This suggests that organizational culture affects subsequent clinician turnover primarily through its effect on job satisfaction. Organizational culture, in particular perceived effectiveness and communication, affects job satisfaction, which in turn affects clinician turnover in primary care pediatric practices. Strategies to improve job satisfaction through changes in organizational culture could potentially reduce clinician turnover. PMID:23804066

  10. Creativity and Performativity Policies in Primary School Cultures

    ERIC Educational Resources Information Center

    Troman, Geoff; Jeffrey, Bob; Raggl, Andrea

    2007-01-01

    Cultures of performativity in English primary schools refer to systems and relationships of: target-setting; Ofsted inspections; school league tables constructed from pupil test scores; performance management; performance related pay; threshold assessment; and advanced skills teachers. Systems which demand that teachers "perform" and in which…

  11. Where Cultural Games Count: The Voices of Primary Classroom Teachers

    ERIC Educational Resources Information Center

    Nabie, Michael Johnson

    2015-01-01

    This study explored Ghanaian primary school teachers' values and challenges of integrating cultural games in teaching mathematics. Using an In-depth conversational interview, ten (10) certificated teachers' voices on the values and challenges of integrating games were examined. Thematic data analysis was applied to the qualitative data from the…

  12. Acetaminophen metabolism, cytotoxicity, and genotoxicity in rat primary hepatocyte cultures

    SciTech Connect

    Milam, K.M.; Byard, J.L.

    1985-06-30

    Acetaminophen (APAP) metabolism, cytotoxicity, and genotoxicity were measured in primary cultures of rat hepatocytes. Although 3 mM APAP caused a slight increase in cellular release of lactate dehydrogenase into the culture medium, cellular glutathione concentration (an index of APAP metabolism) was reduced by 50%. APAP at 7 mM was significantly more toxic to these hepatocytes and had a similar but more marked effect on glutathione concentrations. In spite of its cytotoxicity, neither dose of APAP stimulated DNA repair synthesis when monitored by the rate of incorporation of (/sup 3/H)thymidine into DNA following exposure to APAP. Thus, although APAP has been shown to be both hepato- and nephrotoxic in several in vivo and in vitro systems, the reactive toxic metabolite of APAP is not genotoxic in rat primary hepatocyte cultures.

  13. [Participation of the primary motor cortex in programming of muscle activity during catching of falling object].

    PubMed

    Kazennikov, O V; Lipshits, M I

    2011-01-01

    Object fell into the cup that sitting subject held between thumb and index fingers. Transcranial magnetic stimulation (TMS) of the primary motor cortex was performed early before and during anticipatory grip force increasing. Comparison of current EMG activity of adductor pollicis brevis and first dorsal interosseous muscles and responses of these muscles on TMS showed that responses were increased before the raising of muscle activity. From the other side only slight augmentation of responses was observed during subsequent strong muscle activation. It is assumed that the increasing of the TMS responses that occurred before the initiation of muscle activity reflects the enhancement ofthe motor cortex excitability associated to specific processes related to the motor cortex participation in programming of the muscles activities. PMID:22117465

  14. Speed of processing in the primary motor cortex: a continuous theta burst stimulation study.

    PubMed

    Lakhani, Bimal; Bolton, David A E; Miyasike-Dasilva, Veronica; Vette, Albert H; McIlroy, William E

    2014-03-15

    'Temporally urgent' reactions are extremely rapid, spatially precise movements that are evoked following discrete stimuli. The involvement of primary motor cortex (M1) and its relationship to stimulus intensity in such reactions is not well understood. Continuous theta burst stimulation (cTBS) suppresses focal regions of the cortex and can assess the involvement of motor cortex in speed of processing. The primary objective of this study was to explore the involvement of M1 in speed of processing with respect to stimulus intensity. Thirteen healthy young adults participated in this experiment. Behavioral testing consisted of a simple button press using the index finger following median nerve stimulation of the opposite limb, at either high or low stimulus intensity. Reaction time was measured by the onset of electromyographic activity from the first dorsal interosseous (FDI) muscle of each limb. Participants completed a 30 min bout of behavioral testing prior to, and 15 min following, the delivery of cTBS to the motor cortical representation of the right FDI. The effect of cTBS on motor cortex was measured by recording the average of 30 motor evoked potentials (MEPs) just prior to, and 5 min following, cTBS. Paired t-tests revealed that, of thirteen participants, five demonstrated a significant attenuation, three demonstrated a significant facilitation and five demonstrated no significant change in MEP amplitude following cTBS. Of the group that demonstrated attenuated MEPs, there was a biologically significant interaction between stimulus intensity and effect of cTBS on reaction time and amplitude of muscle activation. This study demonstrates the variability of potential outcomes associated with the use of cTBS and further study on the mechanisms that underscore the methodology is required. Importantly, changes in motor cortical excitability may be an important determinant of speed of processing following high intensity stimulation.

  15. Bringing transcranial mapping into shape: Sulcus-aligned mapping captures motor somatotopy in human primary motor hand area.

    PubMed

    Raffin, Estelle; Pellegrino, Giovanni; Di Lazzaro, Vincenzo; Thielscher, Axel; Siebner, Hartwig Roman

    2015-10-15

    Motor representations express some degree of somatotopy in human primary motor hand area (M1HAND), but within-M1HAND corticomotor somatotopy has been difficult to study with transcranial magnetic stimulation (TMS). Here we introduce a "linear" TMS mapping approach based on the individual shape of the central sulcus to obtain mediolateral corticomotor excitability profiles of the abductor digiti minimi (ADM) and first dorsal interosseus (FDI) muscles. In thirteen young volunteers, we used stereotactic neuronavigation to stimulate the right M1HAND with a small eight-shaped coil at 120% of FDI resting motor threshold. We pseudorandomly stimulated six targets located on a straight mediolateral line corresponding to the overall orientation of the central sulcus with a fixed coil orientation of 45° to the mid-sagittal line (STRAIGHT-450FIX) or seven targets in the posterior part of the crown of the central sulcus following the bending of the central sulcus (CURVED). CURVED mapping employed a fixed (CURVED-450FIX) or flexible coil orientation producing always a current perpendicular to the sulcal wall (CURVED-900FLEX). During relaxation, CURVED but not STRAIGHT mapping revealed distinct corticomotor excitability peaks in M1HAND with the excitability maximum of ADM located medially to the FDI maximum. This mediolateral somatotopy was still present during tonic contraction of the ADM or FDI. During ADM contraction, cross-correlation between the spatial excitability profiles of ADM and FDI was lowest for CURVED-900FLEX. Together, the results show that within-M1HAND somatotopy can be readily probed with linear TMS mapping aligned to the sulcal shape. Sulcus-aligned linear mapping will benefit non-invasive studies of representational plasticity in human M1HAND.

  16. Bringing transcranial mapping into shape: Sulcus-aligned mapping captures motor somatotopy in human primary motor hand area.

    PubMed

    Raffin, Estelle; Pellegrino, Giovanni; Di Lazzaro, Vincenzo; Thielscher, Axel; Siebner, Hartwig Roman

    2015-10-15

    Motor representations express some degree of somatotopy in human primary motor hand area (M1HAND), but within-M1HAND corticomotor somatotopy has been difficult to study with transcranial magnetic stimulation (TMS). Here we introduce a "linear" TMS mapping approach based on the individual shape of the central sulcus to obtain mediolateral corticomotor excitability profiles of the abductor digiti minimi (ADM) and first dorsal interosseus (FDI) muscles. In thirteen young volunteers, we used stereotactic neuronavigation to stimulate the right M1HAND with a small eight-shaped coil at 120% of FDI resting motor threshold. We pseudorandomly stimulated six targets located on a straight mediolateral line corresponding to the overall orientation of the central sulcus with a fixed coil orientation of 45° to the mid-sagittal line (STRAIGHT-450FIX) or seven targets in the posterior part of the crown of the central sulcus following the bending of the central sulcus (CURVED). CURVED mapping employed a fixed (CURVED-450FIX) or flexible coil orientation producing always a current perpendicular to the sulcal wall (CURVED-900FLEX). During relaxation, CURVED but not STRAIGHT mapping revealed distinct corticomotor excitability peaks in M1HAND with the excitability maximum of ADM located medially to the FDI maximum. This mediolateral somatotopy was still present during tonic contraction of the ADM or FDI. During ADM contraction, cross-correlation between the spatial excitability profiles of ADM and FDI was lowest for CURVED-900FLEX. Together, the results show that within-M1HAND somatotopy can be readily probed with linear TMS mapping aligned to the sulcal shape. Sulcus-aligned linear mapping will benefit non-invasive studies of representational plasticity in human M1HAND. PMID:26188259

  17. Functional connectivity of primary motor cortex is dependent on genetic burden in prodromal Huntington disease.

    PubMed

    Koenig, Katherine A; Lowe, Mark J; Harrington, Deborah L; Lin, Jian; Durgerian, Sally; Mourany, Lyla; Paulsen, Jane S; Rao, Stephen M

    2014-09-01

    Subtle changes in motor function have been observed in individuals with prodromal Huntington disease (prHD), but the underlying neural mechanisms are not well understood nor is the cumulative effect of the disease (disease burden) on functional connectivity. The present study examined the resting-state functional magnetic resonance imaging (rs-fMRI) connectivity of the primary motor cortex (M1) in 16 gene-negative (NEG) controls and 48 gene-positive prHD participants with various levels of disease burden. The results showed that the strength of the left M1 connectivity with the ipsilateral M1 and somatosensory areas decreased as disease burden increased and correlated with motor symptoms. Weakened M1 connectivity within the motor areas was also associated with abnormalities in long-range connections that evolved with disease burden. In this study, M1 connectivity was decreased with visual centers (bilateral cuneus), but increased with a hub of the default mode network (DMN; posterior cingulate cortex). Changes in connectivity measures were associated with worse performance on measures of cognitive-motor functioning. Short- and long-range functional connectivity disturbances were also associated with volume loss in the basal ganglia, suggesting that weakened M1 connectivity is partly a manifestation of striatal atrophy. Altogether, the results indicate that the prodromal phase of HD is associated with abnormal interhemispheric interactions among motor areas and disturbances in the connectivity of M1 with visual centers and the DMN. These changes may, respectively, contribute to increased motor symptoms, visuomotor integration problems, and deficits in the executive control of movement as individuals approach a manifest diagnosis.

  18. Primary Motor Cortex Representation of Handgrip Muscles in Patients with Leprosy

    PubMed Central

    Rangel, Maria Luíza Sales; Sanchez, Tiago Arruda; Moreira, Filipe Azaline; Hoefle, Sebastian; Souto, Inaiacy Bittencourt; da Cunha, Antônio José Ledo Alves

    2015-01-01

    Background Leprosy is an endemic infectious disease caused by Mycobacterium leprae that predominantly attacks the skin and peripheral nerves, leading to progressive impairment of motor, sensory and autonomic function. Little is known about how this peripheral neuropathy affects corticospinal excitability of handgrip muscles. Our purpose was to explore the motor cortex organization after progressive peripheral nerve injury and upper-limb dysfunction induced by leprosy using noninvasive transcranial magnetic stimulation (TMS). Methods In a cross-sectional study design, we mapped bilaterally in the primary motor cortex (M1) the representations of the hand flexor digitorum superficialis (FDS), as well as of the intrinsic hand muscles abductor pollicis brevis (APB), first dorsal interosseous (FDI) and abductor digiti minimi (ADM). All participants underwent clinical assessment, handgrip dynamometry and motor and sensory nerve conduction exams 30 days before mapping. Wilcoxon signed rank and Mann-Whitney tests were performed with an alpha-value of p<0.05. Findings Dynamometry performance of the patients’ most affected hand (MAH), was worse than that of the less affected hand (LAH) and of healthy controls participants (p = 0.031), confirming handgrip impairment. Motor threshold (MT) of the FDS muscle was higher in both hemispheres in patients as compared to controls, and lower in the hemisphere contralateral to the MAH when compared to that of the LAH. Moreover, motor evoked potential (MEP) amplitudes collected in the FDS of the MAH were higher in comparison to those of controls. Strikingly, MEPs in the intrinsic hand muscle FDI had lower amplitudes in the hemisphere contralateral to MAH as compared to those of the LAH and the control group. Taken together, these results are suggestive of a more robust representation of an extrinsic hand flexor and impaired intrinsic hand muscle function in the hemisphere contralateral to the MAH due to leprosy. Conclusion Decreased

  19. Transcranial direct current stimulation of the primary motor cortex improves word-retrieval in older adults

    PubMed Central

    Meinzer, Marcus; Lindenberg, Robert; Sieg, Mira M.; Nachtigall, Laura; Ulm, Lena; Flöel, Agnes

    2014-01-01

    Language facilitation by transcranial direct current stimulation (tDCS) in healthy individuals has generated hope that tDCS may also allow improving language impairment after stroke (aphasia). However, current stimulation protocols have yielded variable results and may require identification of residual language cortex using functional magnetic resonance imaging (fMRI), which complicates incorporation into clinical practice. Based on previous behavioral studies that demonstrated improved language processing by motor system pre-activation, the present study assessed whether tDCS administered to the primary motor cortex (M1) can enhance language functions. This proof-of-concept study employed a sham-tDCS controlled, cross-over, within-subject design and assessed the impact of unilateral excitatory (anodal) and bihemispheric (dual) tDCS in 18 healthy older adults during semantic word-retrieval and motor speech tasks. Simultaneous fMRI scrutinized the neural mechanisms underlying tDCS effects. Both active tDCS conditions significantly improved word-retrieval compared to sham-tDCS. The direct comparison of activity elicited by word-retrieval vs. motor-speech trials revealed bilateral frontal activity increases during both anodal- and dual-tDCS compared to sham-tDCS. This effect was driven by more pronounced deactivation of frontal regions during the motor-speech task, while activity during word-retrieval trials was unaffected by the stimulation. No effects were found in M1 and secondary motor regions. Our results show that tDCS administered to M1 can improve word-retrieval in healthy individuals, thereby providing a rationale to explore whether M1-tDCS may offer a novel approach to improve language functions in aphasia. Functional magnetic resonance imaging revealed neural facilitation specifically during motor speech trials, which may have reduced switching costs between the overlapping neural systems for lexical retrieval and speech processing, thereby resulting in

  20. Impaired off-line motor skills consolidation in young primary insomniacs.

    PubMed

    Cellini, Nicola; de Zambotti, Massimiliano; Covassin, Naima; Sarlo, Michela; Stegagno, Luciano

    2014-10-01

    Compelling evidence indicates that sleep can facilitate the off-line consolidation of declarative, perceptual, emotional and procedural memories. Here we assessed the sleep-related off-line consolidation of motor skills in 13 young primary insomniacs (23.31±2.5 yrs) compared to 13 healthy sleepers (24.31±1.6 yrs) using the sequential finger tapping task. During a training session insomniacs performed less correct sequences than controls. However, both groups exhibited similar on-line motor learning in the pre-sleep evening session. After a night of sleep, healthy controls improved their performance, indicating an overnight effect of sleep on motor skills consolidation. In contrast, insomniacs failed to exhibit a sleep-related enhancement in memory performance indicating impairment in the off-line motor skills consolidation process. Our results suggest that young adults with insomnia experience impaired off-line memory consolidation which seems not to be associated with reduced ability to acquire new motor information.

  1. Stimulus-response profile during single-pulse transcranial magnetic stimulation to the primary motor cortex.

    PubMed

    Hanakawa, Takashi; Mima, Tatsuya; Matsumoto, Riki; Abe, Mitsunari; Inouchi, Morito; Urayama, Shin-Ichi; Anami, Kimitaka; Honda, Manabu; Fukuyama, Hidenao

    2009-11-01

    We examined the stimulus-response profile during single-pulse transcranial magnetic stimulation (TMS) by measuring motor-evoked potentials (MEPs) with electromyographic monitoring and hemodynamic responses with functional magnetic resonance imaging (fMRI) at 3 Tesla. In 16 healthy subjects, single TMS pulses were irregularly delivered to the left primary motor cortex at a mean frequency of 0.15 Hz with a wide range of stimulus intensities. The measurement of MEP proved a typical relationship between stimulus intensity and MEP amplitude in the concurrent TMS-fMRI environment. In the population-level analysis of the suprathreshold stimulation conditions, significant increases in hemodynamic responses were detected in the motor/somatosensory network, reflecting both direct and remote effects of TMS, and also the auditory/cognitive areas, perhaps related to detection of clicks. The stimulus-response profile showed both linear and nonlinear components in the direct and remote motor/somatosensory network. A detailed analysis suggested that the nonlinear components of the motor/somatosensory network activity might be induced by nonlinear recruitment of neurons in addition to sensory afferents resulting from movement. These findings expand our basic knowledge of the quantitative relationship between TMS-induced neural activations and hemodynamic signals measured by neuroimaging techniques.

  2. Altered structural and functional connectivity between the bilateral primary motor cortex in unilateral subcortical stroke

    PubMed Central

    Zhang, Yong; Li, Kuang-Shi; Ning, Yan-Zhe; Fu, Cai-Hong; Liu, Hong-Wei; Han, Xiao; Cui, Fang-Yuan; Ren, Yi; Zou, Yi-Huai

    2016-01-01

    Abstract A large number of functional imaging studies have focused on the understanding of motor-related neural activities after ischemic stroke. However, the knowledge is still limited in the structural and functional changes of the interhemispheric connections of the bilateral primary motor cortices (M1s) and their potential influence on motor function recovery following stroke. Twenty-four stroke patients with right hemispheric subcortical infarcts and 25 control subjects were recruited to undergo multimodal magnetic resonance imaging examinations. Structural impairments between the bilateral M1s were measured by fractional anisotropy. Functional changes of the bilateral M1s were assessed via M1-M1 resting-state functional connectivity. Task-evoked activation analysis was applied to identify the roles of the bilateral hemispheres in motor function recovery. Compared with control subjects, unilateral subcortical stroke patients revealed significantly decreased fractional anisotropy and functional connectivity between the bilateral M1s. Stroke patients also revealed higher activations in multiple brain regions in both hemispheres and that more regions were located in the contralesional hemisphere. This study increased our understanding of the structural and functional alterations between the bilateral M1s that occur in unilateral subcortical stroke and provided further evidence for the compensatory role played by the contralesional hemisphere for these alterations during motor function recovery. PMID:27495109

  3. Oncogenic transformation of diverse gastrointestinal tissues in primary organoid culture

    PubMed Central

    Li, Xingnan; Nadauld, Lincoln; Ootani, Akifumi; Corney, David C.; Pai, Reetesh K.; Gevaert, Olivier; Cantrell, Michael A.; Rack, Paul G.; Neal, James T.; Chan, Carol W-M.; Yeung, Trevor; Gong, Xue; Yuan, Jenny; Wilhelmy, Julie; Robine, Sylvie; Attardi, Laura D.; Plevritis, Sylvia K.; Hung, Kenneth E.; Chen, Chang-Zheng; Ji, Hanlee P.; Kuo, Calvin J.

    2014-01-01

    The application of primary organoid cultures containing epithelial and mesenchymal elements to cancer modeling holds promise for combining the accurate multilineage differentiation and physiology of in vivo systems with the facile in vitro manipulation of transformed cell lines. Here, a single air-liquid interface culture method was used without modification to engineer oncogenic mutations into primary epithelial/mesenchymal organoids from mouse colon, stomach and pancreas. Pancreatic and gastric organoids exhibited dysplasia upon KrasG12D expression and/or p53 loss, and readily generated adenocarcinoma upon in vivo transplantation. In contrast, primary colon organoids required combinatorial Apc, p53, KrasG12D and Smad4 mutations for progressive transformation to invasive adenocarcinoma-like histology in vitro and tumorigenicity in vivo, recapitulating multi-hit models of colorectal cancer (CRC), and versus more promiscuous transformation of small intestinal organoids. Colon organoid culture functionally validated the microRNA miR-483 as a dominant driver oncogene at the Insulin-like growth factor-2 (IGF2) 11p15.5 CRC amplicon, inducing dysplasia in vitro and tumorigenicity in vivo. These studies demonstrate the general utility of a highly tractable primary organoid system for cancer modeling and driver oncogene validation in diverse gastrointestinal tissues. PMID:24859528

  4. Oncogenic transformation of diverse gastrointestinal tissues in primary organoid culture.

    PubMed

    Li, Xingnan; Nadauld, Lincoln; Ootani, Akifumi; Corney, David C; Pai, Reetesh K; Gevaert, Olivier; Cantrell, Michael A; Rack, Paul G; Neal, James T; Chan, Carol W-M; Yeung, Trevor; Gong, Xue; Yuan, Jenny; Wilhelmy, Julie; Robine, Sylvie; Attardi, Laura D; Plevritis, Sylvia K; Hung, Kenneth E; Chen, Chang-Zheng; Ji, Hanlee P; Kuo, Calvin J

    2014-07-01

    The application of primary organoid cultures containing epithelial and mesenchymal elements to cancer modeling holds promise for combining the accurate multilineage differentiation and physiology of in vivo systems with the facile in vitro manipulation of transformed cell lines. Here we used a single air-liquid interface culture method without modification to engineer oncogenic mutations into primary epithelial and mesenchymal organoids from mouse colon, stomach and pancreas. Pancreatic and gastric organoids exhibited dysplasia as a result of expression of Kras carrying the G12D mutation (Kras(G12D)), p53 loss or both and readily generated adenocarcinoma after in vivo transplantation. In contrast, primary colon organoids required combinatorial Apc, p53, Kras(G12D) and Smad4 mutations for progressive transformation to invasive adenocarcinoma-like histology in vitro and tumorigenicity in vivo, recapitulating multi-hit models of colorectal cancer (CRC), as compared to the more promiscuous transformation of small intestinal organoids. Colon organoid culture functionally validated the microRNA miR-483 as a dominant driver oncogene at the IGF2 (insulin-like growth factor-2) 11p15.5 CRC amplicon, inducing dysplasia in vitro and tumorigenicity in vivo. These studies demonstrate the general utility of a highly tractable primary organoid system for cancer modeling and driver oncogene validation in diverse gastrointestinal tissues.

  5. CLINICAL, ENDOSCOPIC AND MANOMETRIC FEATURES OF THE PRIMARY MOTOR DISORDERS OF THE ESOPHAGUS

    PubMed Central

    MARTINEZ, Júlio César; LIMA, Gustavo Rosa de Almeida; SILVA, Diego Henrique; DUARTE, Alexandre Ferreira; NOVO, Neil Ferreira; da SILVA, Ernesto Carlos; PINTO, Pérsio Campos Correia; MAIA, Alexandre Moreira

    2015-01-01

    Background Significant incidence, diagnostic difficulties, clinical relevance and therapeutic efficacy associated with the small number of publications on the primary esophageal motor disorders, motivated the present study. Aim To determine the manometric prevalence of these disorders and correlate them to the endoscopic and clinical findings. Methods A retrospective study of 2614 patients, being 1529 (58.49%) women and 1085 (41.51%) men. From 299 manometric examinations diagnosed with primary esophageal motor disorder, were sought-clinical data (heartburn, regurgitation, dysphagia, odynophagia, non-cardiac chest pain, pharyngeal globe and extra-esophageal symptoms) and/or endoscopic (hiatal hernia, erosive esophagitis, food waste) that motivated the performance of manometry. Results Were found 49 cases of achalasia, 73 diffuse spasm, 89 nutcracker esophagus, 82 ineffective esophageal motility, and six lower esophageal sphincter hypertension. In relation to the correlations, it was observed that in 119 patients clinical conditions were associated with dysphagia, found in achalasia more than in other conditions; in relationship between endoscopic findings and clinical conditions there was no statistical significance between data. Conclusions The clinical and endoscopic findings have little value in the characterization of the primary motor disorders of the esophagus, showing even more the need for manometry, particularly in the preoperative period of gastroesophageal reflux disease. PMID:25861066

  6. Phosphorylation of Dpsyl2 (CRMP2) and Dpsyl3 (CRMP4) is required for positioning of caudal primary motor neurons in the zebrafish spinal cord.

    PubMed

    Morimura, Rii; Nozawa, Keisuke; Tanaka, Hideomi; Ohshima, Toshio

    2013-12-01

    Dpysls (CRMPs) that were initially identified as mediator proteins of Semaphorin3a (Sema3a) signaling are involved in neuronal polarity and axon elongation in cultured neurons. Previous studies have shown that knockdown of neuropilin1a, one of the sema3a receptors, exhibited ectopic primary motor neurons (PMNs) outside of the spinal cord in zebrafish. However, downstream molecules of sema3a signaling involved in the positioning of motor neurons are largely unknown. Here, we addressed the role of Dpysl2 (CRMP2) and Dpysl3 (CRMP4) in the positioning of PMNs in the zebrafish spinal cord. We found that the knockdown of dpysls by antisense morpholino oligonucleotides (AMO) causes abnormal positioning of caudal primary (CaP) motor neurons outside the spinal cord. The knockdown of cdk5 and dyrk2 by AMO also caused similar phenotype in the positioning of CaP motor neurons, and this phenotype was rescued by co-injection of phosphorylation-mimic type dpysl2 mRNA. These results suggest that the phosphorylation of Dpysl2 and Dpysl3 by Cdk5 and Dyrk2 is required for correct positioning of CaP motor neurons in the zebrafish spinal cord.

  7. Epigenetic Modifications as Antidedifferentiation Strategy for Primary Hepatocytes in Culture.

    PubMed

    Bolleyn, Jennifer; Fraczek, Joanna; Rogiers, Vera; Vanhaecke, Tamara

    2015-01-01

    A well-known problem of cultured primary hepatocytes is their rapid dedifferentiation. During the last years, several strategies to counteract this phenomenon have been developed, of which changing the in vitro environment is the most popular one. However, mimicking the in vivo setting in vitro by adding soluble media additives or the restoration of both cell-cell and cell-extracellular matrix contacts is not sufficient and only delays the dedifferentiation process instead of counteracting it. In this chapter, new strategies to prevent the deterioration of the liver-specific phenotype of primary hepatocytes in culture by targeting the (epi)genetic mechanisms that drive hepatocellular gene expression are described. PMID:26272144

  8. Microbiologic and clinical value of primary broth cultures of wound specimens collected with swabs.

    PubMed Central

    Silletti, R P; Ailey, E; Sun, S; Tang, D

    1997-01-01

    In order to assess the microbiologic and clinical value of primary broth culture of wound specimens collected with swabs and submitted to the laboratory in transport medium, we compared the results of primary agar culture with the results of a corresponding primary broth culture for 344 aerobic specimens and 176 anaerobic specimens. While 8.7% (45 of 520) of the specimens yielded organisms from the primary broth culture that were not recovered from the corresponding primary agar culture, only 5.0% (26 of 520) of the specimens yielded organisms from the primary broth culture other than Staphylococcus epidermidis, viridans group streptococci, and Corynebacterium spp. Moreover, the primary broth culture of only 0.6% (3 of 520) of the specimens yielded organisms not recovered from the primary agar culture that caused a change in the therapy of the patient. Our conclusion is that primary broth cultures are unnecessary for the processing of wound specimens properly collected with swabs. PMID:9230370

  9. Cross-cultural analysis of the motor development of Brazilian, Greek and Canadian infants assessed with the Alberta Infant Motor Scale

    PubMed Central

    Saccani, Raquel; Valentini, Nadia Cristina

    2013-01-01

    OBJECTIVE: To compare the motor development of infants from three population samples (Brazil, Canada and Greece), to investigate differences in the percentile curves of motor development in these samples, and to investigate the prevalence of motor delays in Brazilian children. METHODS: Observational, descriptive and cross-sectional study with 795 Brazilian infants from zero to 18 months of age, assessed by the Alberta Infant Motor Scale (AIMS) at day care centers, nurseries, basic health units and at home. The Brazilian infants' motor scores were compared to the results of two population samples from Greece (424 infants) and Canada (2,400 infants). Descriptive statistics was used, with one-sample t-test and binomial tests, being significant p≤0.05. RESULTS: 65.4% of Brazilian children showed typical motor development, although with lower mean scores. In the beginning of the second year of life, the differences in the motor development among Brazilian, Canadian and Greek infants were milder; at 15 months of age, the motor development became similar in the three groups. A non-linear motor development trend was observed. CONCLUSIONS: The lowest motor percentiles of the Brazilian sample emphasized the need for national norms in order to correctly categorize the infant motor development. The different ways of motor development may be a consequence of cultural differences in infant care. PMID:24142318

  10. Spatiotemporal relations of primary sensorimotor and secondary motor activation patterns mapped by NIR imaging

    PubMed Central

    Khan, Bilal; Chand, Pankaj; Alexandrakis, George

    2011-01-01

    Functional near infrared (fNIR) imaging was used to identify spatiotemporal relations between spatially distinct cortical regions activated during various hand and arm motion protocols. Imaging was performed over a field of view (FOV, 12 x 8.4 cm) including the secondary motor, primary sensorimotor, and the posterior parietal cortices over a single brain hemisphere. This is a more extended FOV than typically used in current fNIR studies. Three subjects performed four motor tasks that induced activation over this extended FOV. The tasks included card flipping (pronation and supination) that, to our knowledge, has not been performed in previous functional magnetic resonance imaging (fMRI) or fNIR studies. An earlier rise and a longer duration of the hemodynamic activation response were found in tasks requiring increased physical or mental effort. Additionally, analysis of activation images by cluster component analysis (CCA) demonstrated that cortical regions can be grouped into clusters, which can be adjacent or distant from each other, that have similar temporal activation patterns depending on whether the performed motor task is guided by visual or tactile feedback. These analyses highlight the future potential of fNIR imaging to tackle clinically relevant questions regarding the spatiotemporal relations between different sensorimotor cortex regions, e.g. ones involved in the rehabilitation response to motor impairments. PMID:22162826

  11. [Primary motor cortex as one of the levels of the construction of movements].

    PubMed

    Pavlova, O G

    2014-01-01

    The data obtained during the recent decades led to revision of the dominant in neurophysiology view of primary motor cortex as "the cord area" which transfers the motor commands to the spinal cord. Contrary to this point of view, it was shown that MI primates neurons participate in all stages of organization of motor behavior and that the final postures of complex coordinated movements are represented in the MI map. Characteristics of movements controlled by MI revealed by currently available methods were predicted and explained by N.A. Bernstein about 70 years ago. According to his concept, there are some levels of the construction of movements that exist in the central nervous system. Area 4 (i.e. MI), which is one of them, appeared on the definite stage of evolution for resolving the particular movement tasks. In support of this conception we are showing that: 1) MI controls the movements that differ from the movements of other levels by their characteristics (the mode of operating and the sense content); 2) some voluntary movements can be executed without participation of MI; 3) different motor areas of the cortex are coupled with different aspects of movement behavior.

  12. Mirror illusion reduces motor cortical inhibition in the ipsilateral primary motor cortex during forceful unilateral muscle contractions.

    PubMed

    Zult, Tjerk; Goodall, Stuart; Thomas, Kevin; Hortobágyi, Tibor; Howatson, Glyn

    2015-04-01

    Forceful, unilateral contractions modulate corticomotor paths targeting the resting, contralateral hand. However, it is unknown whether mirror-viewing of a slowly moving but forcefully contracting hand would additionally affect these paths. Here we examined corticospinal excitability and short-interval intracortical inhibition (SICI) of the right-ipsilateral primary motor cortex (M1) in healthy young adults under no-mirror and mirror conditions at rest and during right wrist flexion at 60% maximal voluntary contraction (MVC). During the no-mirror conditions neither hand was visible, whereas in the mirror conditions participants looked at the right hand's reflection in the mirror. Corticospinal excitability increased during contractions in the left flexor carpi radialis (FCR) (contraction 0.41 mV vs. rest 0.21 mV) and extensor carpi radialis (ECR) (contraction 0.56 mV vs. rest 0.39 mV), but there was no mirror effect (FCR: P = 0.743, ηp (2) = 0.005; ECR: P = 0.712, ηp (2) = 0.005). However, mirror-viewing of the contracting and moving wrist attenuated SICI relative to test pulse in the left FCR by ∼9% compared with the other conditions (P < 0.05, d ≥ 0.62). Electromyographic activity in the resting left hand prior to stimulation was not affected by the mirror (FCR: P = 0.255, ηp (2) = 0.049; ECR: P = 0.343, ηp (2) = 0.035) but increased twofold during contractions. Thus viewing the moving hand in the mirror and not just the mirror image of the nonmoving hand seems to affect motor cortical inhibitory networks in the M1 associated with the mirror image. Future studies should determine whether the use of a mirror could increase interlimb transfer produced by cross-education, especially in patient groups with unilateral orthopedic and neurological conditions.

  13. Mirror illusion reduces motor cortical inhibition in the ipsilateral primary motor cortex during forceful unilateral muscle contractions.

    PubMed

    Zult, Tjerk; Goodall, Stuart; Thomas, Kevin; Hortobágyi, Tibor; Howatson, Glyn

    2015-04-01

    Forceful, unilateral contractions modulate corticomotor paths targeting the resting, contralateral hand. However, it is unknown whether mirror-viewing of a slowly moving but forcefully contracting hand would additionally affect these paths. Here we examined corticospinal excitability and short-interval intracortical inhibition (SICI) of the right-ipsilateral primary motor cortex (M1) in healthy young adults under no-mirror and mirror conditions at rest and during right wrist flexion at 60% maximal voluntary contraction (MVC). During the no-mirror conditions neither hand was visible, whereas in the mirror conditions participants looked at the right hand's reflection in the mirror. Corticospinal excitability increased during contractions in the left flexor carpi radialis (FCR) (contraction 0.41 mV vs. rest 0.21 mV) and extensor carpi radialis (ECR) (contraction 0.56 mV vs. rest 0.39 mV), but there was no mirror effect (FCR: P = 0.743, ηp (2) = 0.005; ECR: P = 0.712, ηp (2) = 0.005). However, mirror-viewing of the contracting and moving wrist attenuated SICI relative to test pulse in the left FCR by ∼9% compared with the other conditions (P < 0.05, d ≥ 0.62). Electromyographic activity in the resting left hand prior to stimulation was not affected by the mirror (FCR: P = 0.255, ηp (2) = 0.049; ECR: P = 0.343, ηp (2) = 0.035) but increased twofold during contractions. Thus viewing the moving hand in the mirror and not just the mirror image of the nonmoving hand seems to affect motor cortical inhibitory networks in the M1 associated with the mirror image. Future studies should determine whether the use of a mirror could increase interlimb transfer produced by cross-education, especially in patient groups with unilateral orthopedic and neurological conditions. PMID:25632077

  14. Organizational culture in the primary healthcare setting of Cyprus

    PubMed Central

    2013-01-01

    Background The concept of organizational culture is important in understanding the behaviour of individuals in organizations as they manage external demands and internal social changes. Cyprus healthcare system is under restructuring and soon a new healthcare scheme will be implemented starting at the Primary Healthcare (PHC) level. The aim of the study was to investigate the underlying culture encountered in the PHC setting of Cyprus and to identify possible differences in desired and prevailing cultures among healthcare professionals. Methods The population of the study included all general practitioners (GPs) and nursing staff working at the 42 PHC centres throughout the island. The shortened version of the Organizational Culture Profile questionnaire comprising 28 statements on organizational values was used in the study. The instrument was already translated and validated in Greek and cross-cultural adaptation was performed. Participants were required to indicate the organization’s characteristic cultural values orientation along a five-point Likert scale ranging from “Very Much = 1” to “Not at all= 5”. Statistical analysis was performed using SPSS 16.0. Student t-test was used to compare means between two groups of variables whereas for more than two groups analysis of variance (ANOVA) was applied. Results From the total of 306 healthcare professionals, 223 participated in the study (72.9%). The majority of participants were women (75.3%) and mean age was 42.6 ± 10.7 years. Culture dimension “performance orientation” was the desired culture among healthcare professionals (mean: 1.39 ± 0.45). “Supportiveness” and “social responsibility” were the main cultures encountered in PHC (means: 2.37 ± 0.80, 2.38 ± 0.83). Statistical significant differences were identified between desired and prevailing cultures for all culture dimensions (p= 0.000). Conclusions This was the first study performed in Cyprus assessing organizational culture in

  15. Young patients with focal seizures may have the primary motor area for the hand in the postcentral gyrus

    PubMed Central

    Haseeb, Ateeq; Asano, Eishi; Juhász, Csaba; Shah, Aashit; Sood, Sandeep; Chugani, Harry T.

    2007-01-01

    Summary Objective: We determined whether the primary motor hand area was most frequently located in the precentral gyrus in young patients with intractable focal seizures. Methods: Sixty-five patients with focal seizures aged between 5 months and 20 years who underwent a two-stage epilepsy surgery using chronic subdural-EEG monitoring were studied. Pairs of subdural electrodes were electrically stimulated, and the brain region with contralateral hand movement induced by the lowest-intense stimulus was defined as the primary motor hand area. Results: Contralateral hand movement was induced without afterdischarges in 50 children but not in the remaining 15 children. The unpaired t-test revealed that failure to induce contralateral hand motor movement was associated with younger age of subjects. Among the 50 patients with a positive motor response, the primary motor hand area was confined to the precentral gyrus in 9 patients, confined to the postcentral gyrus in 24, and located in both the pre- and post-central gyri in the remaining 17. The McNemar's test revealed that the observed frequency of 24 patients showing the primary motor hand area confined to the postcentral gyrus was larger than chance frequency. Logistic regression analysis failed to demonstrate that the observation of the primary motor hand area confined to the postcentral gyrus was associated with the age, the presence of dysplastic lesion or the seizure onset involving the frontal lobe. Conclusion: Our study failed to support the traditionally-accepted notion that the primary motor hand area is most frequently located in the precentral gyrus but rather demonstrated that a substantial proportion of young patients had the primary motor hand area in the postcentral gyrus. PMID:17723289

  16. Continuous Force Decoding from Local Field Potentials of the Primary Motor Cortex in Freely Moving Rats

    PubMed Central

    Khorasani, Abed; Heydari Beni, Nargess; Shalchyan, Vahid; Daliri, Mohammad Reza

    2016-01-01

    Local field potential (LFP) signals recorded by intracortical microelectrodes implanted in primary motor cortex can be used as a high informative input for decoding of motor functions. Recent studies show that different kinematic parameters such as position and velocity can be inferred from multiple LFP signals as precisely as spiking activities, however, continuous decoding of the force magnitude from the LFP signals in freely moving animals has remained an open problem. Here, we trained three rats to press a force sensor for getting a drop of water as a reward. A 16-channel micro-wire array was implanted in the primary motor cortex of each trained rat, and obtained LFP signals were used for decoding of the continuous values recorded by the force sensor. Average coefficient of correlation and the coefficient of determination between decoded and actual force signals were r = 0.66 and R2 = 0.42, respectively. We found that LFP signal on gamma frequency bands (30–120 Hz) had the most contribution in the trained decoding model. This study suggests the feasibility of using low number of LFP channels for the continuous force decoding in freely moving animals resembling BMI systems in real life applications. PMID:27767063

  17. Genomics and proteomics analysis of cultured primary rat hepatocytes.

    PubMed

    Beigel, Juergen; Fella, Kerstin; Kramer, Peter-Juergen; Kroeger, Michaela; Hewitt, Philip

    2008-02-01

    The use of animal models in pharmaceutical research is a costly and sometimes misleading method of generating toxicity data and hence predicting human safety. Therefore, in vitro test systems, such as primary rat hepatocytes, and the developing genomics and proteomics technologies, are playing an increasingly important role in toxicological research. Gene and protein expression analysis were investigated in a time series (up to 5 days) of primary rat hepatocytes cultured on collagen coated dishes. Especially after 24h, a significant down-regulation of many important Phase I and Phase II enzymes (e.g., cytochrome P450's, glutathione-S-transferases, sulfotransferases) involved in xenobiotic metabolism, and antioxidative enzymes (e.g., catalase, superoxide dismutase, glutathione peroxidase) was observed. Acute-phase-response enzymes were frequently up-regulated (e.g., LPS binding protein, alpha-2-macro-globulin, ferritin, serine proteinase inhibitor B, haptoglobin), which is likely to be a result of cellular stress caused by the cell isolation procedure (perfusion) itself. A parallel observation was the increased expression of several structural genes (e.g., beta-actin, alpha-tubulin, vimentin), possibly caused by other proliferating cell types in the culture, such as fibroblasts or alternatively by hepatocyte dedifferentiation. In conclusion, the careful interpretation of data derived from this in vitro system indicates that primary hepatocytes can be successfully used for short-term toxicity studies up to 24h. However, culturing conditions need to be further optimized to reduce the massive changes of gene and protein expression of long-term cultured hepatocytes to allow practical applications as a long-term toxicity test system.

  18. Experience in primary culture of human peritoneal mesothelial cell.

    PubMed

    Chen, Kuo-Su; Chen, Wen-Shiang

    2012-08-31

    To compare the growth condition between different sources and different culture environments, mesothelial cells were isolated from omentum and peritoneal dialysate effluent (PDE), seeded at different densities (5 × 10⁵, 1 × 10⁵, 5 × 10⁴, 1 × 10⁴, 5 × 10³, 1 × 10³ and 5 × 10² cells/cm², respectively), supported with different fetal calf serum (FCS) concentrations (3%, 6%, 10% and 15%) and grown in dishes with and without gelatin pre-coating. Growth condition was evaluated by simple morphological observation. Cells phenotype was examined by immunofluorescent staining. The results showed that omentum-derived mesothelial cells generally showed a uniform growth pattern with good quality. Alternatively, there was a wide patient-to-patient variation in PDE-derived culture. Heterogeneous colonies composed of a mixture of large, small or abortive mesothelial colonies as well as fibroblastoid colonies were frequently observed. A minimum seeding density of 5 × 10³ cells/cm² is required for the omentum-derived mesothelial cells to grow to confluent monolayer (1-5 × 10⁴ cells/cm² for initial culture from fresh PDE). Appropriate seeding density is always associated with successful culture in omentumbased culture, but not in PDE-based culture. Mesothelial cells could grow to confluency regardless of FCS concentration and gelatin pre-coating. However, growth rate was slower in lower FCS concentrations and on dishes without gelatin coating. Most cells in culture expressed cytokeratin and vimentin, but not VWF. Alpha-smooth muscle actin frequently appeared in cytokeratin+ mesothelial cells, especially in higher FCS concentrations and in PDE-derived culture. Our data demonstrate that PDE, in contrast to omentum, provides a source of mesothelial cells with poor and unstable quality for primary culture. Healthy cell quality and sufficient seeding density seem to be the most important factors for successful culture of mesothelial cells. The frequent occurrence

  19. Effects of Ranolazine on Astrocytes and Neurons in Primary Culture

    PubMed Central

    Aldasoro, Martin; Guerra-Ojeda, Sol; Aguirre-Rueda, Diana; Mauricio, Mª Dolores; Vila, Jose Mª; Marchio, Patricia; Iradi, Antonio; Aldasoro, Constanza; Jorda, Adrian; Obrador, Elena; Valles, Soraya L.

    2016-01-01

    Ranolazine (Rn) is an antianginal agent used for the treatment of chronic angina pectoris when angina is not adequately controlled by other drugs. Rn also acts in the central nervous system and it has been proposed for the treatment of pain and epileptic disorders. Under the hypothesis that ranolazine could act as a neuroprotective drug, we studied its effects on astrocytes and neurons in primary culture. We incubated rat astrocytes and neurons in primary cultures for 24 hours with Rn (10−7, 10−6 and 10−5 M). Cell viability and proliferation were measured using trypan blue exclusion assay, MTT conversion assay and LDH release assay. Apoptosis was determined by Caspase 3 activity assay. The effects of Rn on pro-inflammatory mediators IL-β and TNF-α was determined by ELISA technique, and protein expression levels of Smac/Diablo, PPAR-γ, Mn-SOD and Cu/Zn-SOD by western blot technique. In cultured astrocytes, Rn significantly increased cell viability and proliferation at any concentration tested, and decreased LDH leakage, Smac/Diablo expression and Caspase 3 activity indicating less cell death. Rn also increased anti-inflammatory PPAR-γ protein expression and reduced pro-inflammatory proteins IL-1 β and TNFα levels. Furthermore, antioxidant proteins Cu/Zn-SOD and Mn-SOD significantly increased after Rn addition in cultured astrocytes. Conversely, Rn did not exert any effect on cultured neurons. In conclusion, Rn could act as a neuroprotective drug in the central nervous system by promoting astrocyte viability, preventing necrosis and apoptosis, inhibiting inflammatory phenomena and inducing anti-inflammatory and antioxidant agents. PMID:26950436

  20. Effects of Ranolazine on Astrocytes and Neurons in Primary Culture.

    PubMed

    Aldasoro, Martin; Guerra-Ojeda, Sol; Aguirre-Rueda, Diana; Mauricio, M Dolores; Vila, Jose M; Marchio, Patricia; Iradi, Antonio; Aldasoro, Constanza; Jorda, Adrian; Obrador, Elena; Valles, Soraya L

    2016-01-01

    Ranolazine (Rn) is an antianginal agent used for the treatment of chronic angina pectoris when angina is not adequately controlled by other drugs. Rn also acts in the central nervous system and it has been proposed for the treatment of pain and epileptic disorders. Under the hypothesis that ranolazine could act as a neuroprotective drug, we studied its effects on astrocytes and neurons in primary culture. We incubated rat astrocytes and neurons in primary cultures for 24 hours with Rn (10-7, 10-6 and 10-5 M). Cell viability and proliferation were measured using trypan blue exclusion assay, MTT conversion assay and LDH release assay. Apoptosis was determined by Caspase 3 activity assay. The effects of Rn on pro-inflammatory mediators IL-β and TNF-α was determined by ELISA technique, and protein expression levels of Smac/Diablo, PPAR-γ, Mn-SOD and Cu/Zn-SOD by western blot technique. In cultured astrocytes, Rn significantly increased cell viability and proliferation at any concentration tested, and decreased LDH leakage, Smac/Diablo expression and Caspase 3 activity indicating less cell death. Rn also increased anti-inflammatory PPAR-γ protein expression and reduced pro-inflammatory proteins IL-1 β and TNFα levels. Furthermore, antioxidant proteins Cu/Zn-SOD and Mn-SOD significantly increased after Rn addition in cultured astrocytes. Conversely, Rn did not exert any effect on cultured neurons. In conclusion, Rn could act as a neuroprotective drug in the central nervous system by promoting astrocyte viability, preventing necrosis and apoptosis, inhibiting inflammatory phenomena and inducing anti-inflammatory and antioxidant agents. PMID:26950436

  1. Stimulation over primary motor cortex during action observation impairs effector recognition.

    PubMed

    Naish, Katherine R; Barnes, Brittany; Obhi, Sukhvinder S

    2016-04-01

    Recent work suggests that motor cortical processing during action observation plays a role in later recognition of the object involved in the action. Here, we investigated whether recognition of the effector making an action is also impaired when transcranial magnetic stimulation (TMS) - thought to interfere with normal cortical activity - is applied over the primary motor cortex (M1) during action observation. In two experiments, single-pulse TMS was delivered over the hand area of M1 while participants watched short clips of hand actions. Participants were then asked whether an image (experiment 1) or a video (experiment 2) of a hand presented later in the trial was the same or different to the hand in the preceding video. In Experiment 1, we found that participants' ability to recognise static images of hands was significantly impaired when TMS was delivered over M1 during action observation, compared to when no TMS was delivered, or when stimulation was applied over the vertex. Conversely, stimulation over M1 did not affect recognition of dot configurations, or recognition of hands that were previously presented as static images (rather than action movie clips) with no object. In Experiment 2, we found that effector recognition was impaired when stimulation was applied part way through (300ms) and at the end (500ms) of the action observation period, indicating that 200ms of action-viewing following stimulation was not long enough to form a new representation that could be used for later recognition. The findings of both experiments suggest that interfering with cortical motor activity during action observation impairs subsequent recognition of the effector involved in the action, which complements previous findings of motor system involvement in object memory. This work provides some of the first evidence that motor processing during action observation is involved in forming representations of the effector that are useful beyond the action observation period

  2. Fine Motor Skills Predict Maths Ability Better than They Predict Reading Ability in the Early Primary School Years.

    PubMed

    Pitchford, Nicola J; Papini, Chiara; Outhwaite, Laura A; Gulliford, Anthea

    2016-01-01

    Fine motor skills have long been recognized as an important foundation for development in other domains. However, more precise insights into the role of fine motor skills, and their relationships to other skills in mediating early educational achievements, are needed to support the development of optimal educational interventions. We explored concurrent relationships between two components of fine motor skills, Fine Motor Precision and Fine Motor Integration, and early reading and maths development in two studies with primary school children of low-to-mid socio-economic status in the UK. Two key findings were revealed. First, despite being in the first 2 years of primary school education, significantly better performance was found in reading compared to maths across both studies. This may reflect the protective effects of recent national-level interventions to promote early literacy skills in young children in the UK that have not been similarly promoted for maths. Second, fine motor skills were a better predictor of early maths ability than they were of early reading ability. Hierarchical multiple regression revealed that fine motor skills did not significantly predict reading ability when verbal short-term memory was taken into account. In contrast, Fine Motor Integration remained a significant predictor of maths ability, even after the influence of non-verbal IQ had been accounted for. These results suggest that fine motor skills should have a pivotal role in educational interventions designed to support the development of early mathematical skills. PMID:27303342

  3. Fine Motor Skills Predict Maths Ability Better than They Predict Reading Ability in the Early Primary School Years.

    PubMed

    Pitchford, Nicola J; Papini, Chiara; Outhwaite, Laura A; Gulliford, Anthea

    2016-01-01

    Fine motor skills have long been recognized as an important foundation for development in other domains. However, more precise insights into the role of fine motor skills, and their relationships to other skills in mediating early educational achievements, are needed to support the development of optimal educational interventions. We explored concurrent relationships between two components of fine motor skills, Fine Motor Precision and Fine Motor Integration, and early reading and maths development in two studies with primary school children of low-to-mid socio-economic status in the UK. Two key findings were revealed. First, despite being in the first 2 years of primary school education, significantly better performance was found in reading compared to maths across both studies. This may reflect the protective effects of recent national-level interventions to promote early literacy skills in young children in the UK that have not been similarly promoted for maths. Second, fine motor skills were a better predictor of early maths ability than they were of early reading ability. Hierarchical multiple regression revealed that fine motor skills did not significantly predict reading ability when verbal short-term memory was taken into account. In contrast, Fine Motor Integration remained a significant predictor of maths ability, even after the influence of non-verbal IQ had been accounted for. These results suggest that fine motor skills should have a pivotal role in educational interventions designed to support the development of early mathematical skills.

  4. Fine Motor Skills Predict Maths Ability Better than They Predict Reading Ability in the Early Primary School Years

    PubMed Central

    Pitchford, Nicola J.; Papini, Chiara; Outhwaite, Laura A.; Gulliford, Anthea

    2016-01-01

    Fine motor skills have long been recognized as an important foundation for development in other domains. However, more precise insights into the role of fine motor skills, and their relationships to other skills in mediating early educational achievements, are needed to support the development of optimal educational interventions. We explored concurrent relationships between two components of fine motor skills, Fine Motor Precision and Fine Motor Integration, and early reading and maths development in two studies with primary school children of low-to-mid socio-economic status in the UK. Two key findings were revealed. First, despite being in the first 2 years of primary school education, significantly better performance was found in reading compared to maths across both studies. This may reflect the protective effects of recent national-level interventions to promote early literacy skills in young children in the UK that have not been similarly promoted for maths. Second, fine motor skills were a better predictor of early maths ability than they were of early reading ability. Hierarchical multiple regression revealed that fine motor skills did not significantly predict reading ability when verbal short-term memory was taken into account. In contrast, Fine Motor Integration remained a significant predictor of maths ability, even after the influence of non-verbal IQ had been accounted for. These results suggest that fine motor skills should have a pivotal role in educational interventions designed to support the development of early mathematical skills. PMID:27303342

  5. Hemispheric asymmetry in cerebrovascular reactivity of the human primary motor cortex: an in vivo study at 7 T.

    PubMed

    Driver, Ian D; Andoh, Jamila; Blockley, Nicholas P; Francis, Susan T; Gowland, Penny A; Paus, Tomáš

    2015-05-01

    Current functional MRI (fMRI) approaches assess underlying neuronal activity through monitoring the related local variations in cerebral blood oxygenation, blood volume and blood flow. This vascular response is likely to vary across brain regions and across individuals, depending on the composition of the local vascular bed and on the vascular capacity to dilate. The most widely used technique uses the blood oxygen level dependent (BOLD) fMRI signal, which arises from a complex combination of all of these factors. The model of handedness provides a case where one brain region (dominant motor cortex) is known to have a stronger BOLD response over another (non-dominant motor cortex) during hand motor task performance. We predict that this is accompanied by a higher vascular reactivity in the dominant motor cortex, when compared with the non-dominant motor cortex. Precise measurement of end-tidal CO2 and a novel sinusoidal CO2 respiratory challenge were combined with the high sensitivity and finer spatial resolution available for fMRI at 7 T to measure BOLD cerebrovascular reactivity (CVR) in eight healthy male participants. BOLD CVR was compared between the left (dominant) and right (non-dominant) primary motor cortices of right-handed adults. Hemispheric asymmetry in vascular reactivity was predicted and observed in the primary motor cortex (left CVR = 0.60 ± 0.15%/mm Hg; right CVR = 0.47 ± 0.08%/mm Hg; left CVR > right CVR, P = 0.04), the first reported evidence of such a vascular difference. These findings demonstrate a cerebral vascular asymmetry between the left and right primary motor cortex. The origin of this asymmetry largely arises from the contribution of large draining veins. This work has implications for future motor laterality studies that use BOLD, and it is also suggestive of a vascular plasticity in the human primary motor cortex. PMID:25788020

  6. Primary culture of embryonic rat olfactory receptor neurons.

    PubMed

    Micholt, Evelien; Jans, Danny; Callewaert, Geert; Bartic, Carmen; Lammertyn, Jeroen; Nicolai, Bart

    2012-12-01

    Embryonic cells are very robust in surviving dissection and culturing protocols and easily adapt to their in vitro environment. Despite these advantages, research in the olfactory field on cultured embryonic olfactory neurons is sparse. In this study, two primary rat olfactory explant cultures of different embryonic d (E17 and E20) were established, comprising epithelium and bulb. The functionality of these neurons was tested by measuring intracellular calcium responses to cAMP-inducing agents forskolin (FSK) and 3-isobutyl-1-methylxanthine (IBMX) with fluorescence microscopy. For E17, the responsive cell fraction increased over time, from an initial 3% at the 1 d in vitro (DIV) to a maximum of 19% at 11 DIV. The response of E20 neurons fluctuated over time around a more or less stable 13%. A logistic regression analysis indicated a significant difference between both embryonic d in the response to FSK + IBMX. In addition, of these functional neurons, 23.3% of E17 and 54.3% of E20 cultures were responsive to the odorant isoamyl acetate. PMID:23150136

  7. Insulin Cannot Induce Adipogenic Differentiation in Primary Cardiac Cultures.

    PubMed

    Parameswaran, Sreejit; Sharma, Rajendra K

    2016-09-01

    Cardiac tissue contains a heterogeneous population of cardiomyocytes and nonmyocyte population especially fibroblasts. Fibroblast differentiation into adipogenic lineage is important for fat accumulation around the heart which is important in cardiac pathology. The differentiation in fibroblast has been observed both spontaneously and due to increased insulin stimulation. The present study aims to observe the effect of insulin in adipogenic differentiation of cardiac cells present in primary murine cardiomyocyte cultures. Oil Red O (ORO) staining has been used for observing the lipid accumulations formed due to adipogenic differentiation in murine cardiomyocyte cultures. The accumulated lipids were quantified by ORO assay and normalized using protein estimation. The lipid accumulation in cardiac cultures did not increase in presence of insulin. However, addition of other growth factors like insulin-like growth factor 1 and epidermal growth factor promoted adipogenic differentiation even in the presence of insulin and other inhibitory molecules such as vitamins. Lipid accumulation also increased in cells grown in media without insulin after an initial exposure to insulin-containing growth media. The current study adds to the existing knowledge that the insulin by itself cannot induce adipogenic induction in the cardiac cultures. The data have significance in the understanding of cardiovascular health especially in diabetic patients. PMID:27574386

  8. Geniposide prevents rotenone-induced apoptosis in primary cultured neurons

    PubMed Central

    Li, Lin; Zhao, Juan; Liu, Ke; Li, Guang-lai; Han, Yan-qing; Liu, Yue-ze

    2015-01-01

    Geniposide, a monomer extracted from gardenia and widely used in Chinese medicine, is a novel agonist at the glucagon-like peptide-1 receptor. This receptor is involved in neuroprotection. In the present study, we sought to identify an anti-apoptotic mechanism for the treatment of neurodegenerative diseases. Primary cultured neurons were treated with different concentrations of rotenone for 48 hours. Morphological observation, cell counting kit-8 assay, lactate dehydrogenase detection and western blot assay demonstrated that 0.5 nM rotenone increased lactate dehydrogenase release, decreased the expression of procaspase-3 and Bcl-2, and increased cleaved caspase-3 expression in normal neurons. All these effects were prevented by geniposide. Our results indicate that geniposide diminished rotenone-induced injury in primary neurons by suppressing apoptosis. This may be one of the molecular mechanisms underlying the efficacy of geniposide in the treatment of neurodegenerative diseases. PMID:26692859

  9. Cross-cultural comparison of motor competence in children from Australia and Belgium.

    PubMed

    Bardid, Farid; Rudd, James R; Lenoir, Matthieu; Polman, Remco; Barnett, Lisa M

    2015-01-01

    Motor competence in childhood is an important determinant of physical activity and physical fitness in later life. However, childhood competence levels in many countries are lower than desired. Due to the many different motor skill instruments in use, children's motor competence across countries is rarely compared. The purpose of this study was to evaluate the motor competence of children from Australia and Belgium using the Körperkoordinationstest für Kinder (KTK). The sample consisted of 244 (43.4% boys) Belgian children and 252 (50.0% boys) Australian children, aged 6-8 years. A MANCOVA for the motor scores showed a significant country effect. Belgian children scored higher on jumping sideways, moving sideways and hopping for height but not for balancing backwards. Moreover, a Chi squared test revealed significant differences between the Belgian and Australian score distribution with 21.3% Belgian and 39.3% Australian children scoring "below average." The very low levels reported by Australian children may be the result of cultural differences in physical activity contexts such as physical education and active transport. When compared to normed scores, both samples scored significantly worse than children 40 years ago. The decline in children's motor competence is a global issue, largely influenced by increasing sedentary behavior and a decline in physical activity.

  10. Cell culture models using rat primary alveolar type I cells

    PubMed Central

    Downs, Charles A.; Montgomery, David W.; Merkle, Carrie J.

    2011-01-01

    There is a lack of cell culture models using primary alveolar type I (AT I) cells. The purpose of this study was to develop cell culture models using rat AT I cells and microvascular endothelial cells from the lung (MVECL). Two types of model systems were developed: single and co-culture systems; additionally a 3-dimensional model system was developed. Pure AT I cell (96.3 ±2.7%) and MVECL (97.9 ±1.1 %) preparations were used. AT I cell morphology, mitochondrial number and distribution, actin filament arrangement and number of apoptotic cells at confluence, and telomere attrition were characterized. AT I cells maintained their morphometric characteristics through at least population doubling (PD) 35, while demonstrating telomere attrition through at least PD 100. Furthermore, AT I cells maintained the expression of their specific markers, T1α and AQ-5, through PD 42. For the co-cultures, AT I cells were grown on the top and MVECL were grown on the bottom of fibronectin coated 24 well Transwell Fluroblok™ filter inserts. Neither cell type transmigrated the 1 micron pores. Additionally AT I cells were grown in a thick layer of Matrigel® to create a 3-dimensional model in which primary AT I cells form ring-like structures that resemble an alveolus. The development of these model systems offers the opportunities to investigate AT I cell cells and their interactions with MVECL in response to pharmacological interventions and in the processes of disease, repair and regeneration. PMID:21624488

  11. Effect of primary culture medium type for culture of canine fibroblasts on production of cloned dogs.

    PubMed

    Kim, Geon A; Oh, Hyun Ju; Kim, Min Jung; Jo, Young Kwang; Choi, Jin; Kim, Jin Wook; Lee, Tae Hee; Lee, Byeong Chun

    2015-09-01

    Fibroblasts are common source of donor cells for SCNT. It is suggested that donor cells' microenvironment, including the primary culture, affects development of reconstructed embryos. To prove this, canine embryos were cloned with fibroblasts that were cultured in two different primary media (RCMEp vs. Dulbecco's modified Eagle's medium [DMEM]) and in vivo developments were compared with relative amount of stemness, reprogramming, apoptosis gene transcripts, and telomerase activity. Donor cells cultured in RCMEp contained a significantly higher amount of SOX2, NANOG, DPPA2, REXO1, HDAC, DNMT1, MECP2 and telomerase activity than those cultured in DMEM (P < 0.05). In vivo developmental potential of cloned embryos with donor cells cultured in RCMEp had a higher birth rate than that of embryos derived from DMEM (P < 0.05). The culture medium can induce changes in gene expression of donor cells and telomerase activity, and these alterations can also affect in vivo developmental competence of the cloned embryos.

  12. Primary motor cortex underlies multi-joint integration for fast feedback control.

    PubMed

    Pruszynski, J Andrew; Kurtzer, Isaac; Nashed, Joseph Y; Omrani, Mohsen; Brouwer, Brenda; Scott, Stephen H

    2011-10-20

    A basic difficulty for the nervous system is integrating locally ambiguous sensory information to form accurate perceptions about the outside world. This local-to-global problem is also fundamental to motor control of the arm, because complex mechanical interactions between shoulder and elbow allow a particular amount of motion at one joint to arise from an infinite combination of shoulder and elbow torques. Here we show, in humans and rhesus monkeys, that a transcortical pathway through primary motor cortex (M1) resolves this ambiguity during fast feedback control. We demonstrate that single M1 neurons of behaving monkeys can integrate shoulder and elbow motion information into motor commands that appropriately counter the underlying torque within about 50 milliseconds of a mechanical perturbation. Moreover, we reveal a causal link between M1 processing and multi-joint integration in humans by showing that shoulder muscle responses occurring ∼50 milliseconds after pure elbow displacement can be potentiated by transcranial magnetic stimulation. Taken together, our results show that transcortical processing through M1 permits feedback responses to express a level of sophistication that rivals voluntary control; this provides neurophysiological support for influential theories positing that voluntary movement is generated by the intelligent manipulation of sensory feedback. PMID:21964335

  13. Perturbation-evoked responses in primary motor cortex are modulated by behavioral context.

    PubMed

    Omrani, Mohsen; Pruszynski, J Andrew; Murnaghan, Chantelle D; Scott, Stephen H

    2014-12-01

    Corrective responses to external perturbations are sensitive to the behavioral task being performed. It is believed that primary motor cortex (M1) forms part of a transcortical pathway that contributes to this sensitivity. Previous work has identified two distinct phases in the perturbation response of M1 neurons, an initial response starting ∼20 ms after perturbation onset that does not depend on the intended motor action and a task-dependent response that begins ∼40 ms after perturbation onset. However, this invariant initial response may reflect ongoing postural control or a task-independent response to the perturbation. The present study tested these two possibilities by examining if being engaged in an ongoing postural task before perturbation onset modulated the initial perturbation response in M1. Specifically, mechanical perturbations were applied to the shoulder and/or elbow while the monkey maintained its hand at a central target or when it was watching a movie and not required to respond to the perturbation. As expected, corrective movements, muscle stretch responses, and M1 population activity in the late perturbation epoch were all significantly diminished in the movie task. Strikingly, initial perturbation responses (<40 ms postperturbation) remained the same across tasks, suggesting that the initial phase of M1 activity constitutes a task-independent response that is sensitive to the properties of the mechanical perturbation but not the goal of the ongoing motor task.

  14. Supplementary motor area and primary auditory cortex activation in an expert break-dancer during the kinesthetic motor imagery of dance to music.

    PubMed

    Olshansky, Michael P; Bar, Rachel J; Fogarty, Mary; DeSouza, Joseph F X

    2015-01-01

    The current study used functional magnetic resonance imaging to examine the neural activity of an expert dancer with 35 years of break-dancing experience during the kinesthetic motor imagery (KMI) of dance accompanied by highly familiar and unfamiliar music. The goal of this study was to examine the effect of musical familiarity on neural activity underlying KMI within a highly experienced dancer. In order to investigate this in both primary sensory and motor planning cortical areas, we examined the effects of music familiarity on the primary auditory cortex [Heschl's gyrus (HG)] and the supplementary motor area (SMA). Our findings reveal reduced HG activity and greater SMA activity during imagined dance to familiar music compared to unfamiliar music. We propose that one's internal representations of dance moves are influenced by auditory stimuli and may be specific to a dance style and the music accompanying it.

  15. Linoleate impairs collagen synthesis in primary cultures of avian chondrocytes.

    PubMed

    Watkins, B A; Xu, H; Turek, J J

    1996-06-01

    The effects of supplemental fatty acids, vitamin E (VIT E), and iron-induced oxidative stress on collagen synthesis, cellular injury, and lipid peroxidation were evaluated in primary cultures of avian epiphyseal chondrocytes. The treatments included oleic and linoleic acids (O or 50 microM) complexed with BSA and dl-alpha-tocopheryl acetate (VIT E at 0 or 100 microM). After 14 days of preculture, the chondrocytes were enriched with fatty acids for 8 days then cultured with VIT E for 2 days. The chondrocytes were then treated with ferrous sulfate (O or 20 microM) for 24 hr to induce oxidative stress. Collagen synthesis was the lowest and the activity of lactate dehydrogenase (LDH) was the highest in chondrocyte cultures treated with 50 microM linoleic acid and 0 VIT E. In contrast, VIT E supplemented at 100 microM partially restored collagen synthesis in the chondrocytes enriched with linoleic acid and lowered LDH activity in the media. The iron oxidative inducer significantly increased the values of thiobarbituric acid-reactive substances (TBARS) in the culture medium. The data showed that linoleic acid impaired chondrocyte cell function and caused cellular injury but that VIT E reversed these effects. Results from a previous study demonstrated that VIT E stimulated bone formation in chicks fed unsaturated fat, and the present findings in cultures of epiphyseal chondrocytes suggest that VIT E is important for chondrocyte function in the presence of polyunsaturated fatty acids. VIT E appears to be beneficial for growth cartilage biology and in optimizing bone growth.

  16. Zinc Modulates Nanosilver-Induced Toxicity in Primary Neuronal Cultures.

    PubMed

    Ziemińska, Elżbieta; Strużyńska, Lidia

    2016-02-01

    Silver nanoparticles (NAg) have recently become one of the most commonly used nanomaterials. Since the ability of nanosilver to enter the brain has been confirmed, there has been a need to investigate mechanisms of its neurotoxicity. We previously showed that primary neuronal cultures treated with nanosilver undergo destabilization of calcium homeostasis via a mechanism involving glutamatergic NMDA receptors. Considering the fact that zinc interacts with these receptors, the aim of the present study was to examine the role of zinc in mechanisms of neuronal cell death in primary cultures. In cells treated with nanosilver, we noted an imbalance between extracellular and intracellular zinc levels. Thus, the influence of zinc deficiency and supplementation on nanosilver-evoked cytotoxicity was investigated by treatment with TPEN (a chelator of zinc ions), or ZnCl(2), respectively. Elimination of zinc leads to complete death of nanosilver-treated CGCs. In contrast, supplementation with ZnCl(2) increases viability of CGCs in a dose-dependent manner. Addition of zinc provided protection against the extra/intracellular calcium imbalance in a manner similar to MK-801, an antagonist of NMDA receptors. Zinc chelation by TPEN decreases the mitochondrial potential and dramatically increases the rate of production of reactive oxygen species. Our results indicate that zinc supplementation positively influences nanosilver-evoked changes in CGCs. This is presumed to be due to an inhibitory effect on NMDA-sensitive calcium channels.

  17. The Effect of Visual and Auditory Enhancements on Excitability of the Primary Motor Cortex during Motor Imagery: A Pilot Study

    ERIC Educational Resources Information Center

    Ikeda, Kohei; Higashi, Toshio; Sugawara, Kenichi; Tomori, Kounosuke; Kinoshita, Hiroshi; Kasai, Tatsuya

    2012-01-01

    The effect of visual and auditory enhancements of finger movement on corticospinal excitability during motor imagery (MI) was investigated using the transcranial magnetic stimulation technique. Motor-evoked potentials were elicited from the abductor digit minimi muscle during MI with auditory, visual and, auditory and visual information, and no…

  18. Modulation of Cortical Inhibitory Circuits after Cathodal Transcranial Direct Current Stimulation over the Primary Motor Cortex

    PubMed Central

    Sasaki, Ryoki; Miyaguchi, Shota; Kotan, Shinichi; Kojima, Sho; Kirimoto, Hikari; Onishi, Hideaki

    2016-01-01

    Here, we aimed to evaluate whether cathodal transcranial direct current stimulation (tDCS) over the primary motor cortex (M1) and primary somatosensory cortex (S1) can modulate cortical inhibitory circuits. Sixteen healthy subjects participated in this study. Cathodal tDCS was positioned over the left M1 (M1 cathodal) or left S1 (S1 cathodal) with an intensity of 1 mA for 10 min. Sham tDCS was applied for 10 min over the left M1 (sham). Motor evoked potentials (MEPs) elicited by transcranial magnetic stimulation (TMS) were recorded from the right abductor pollicis brevis (APB) muscle before the intervention (pre) and 10 and 30 min after the intervention (post 1 and post 2, respectively). Cortical inhibitory circuits were evaluated using short-interval intracortical inhibition (SICI) and short-latency afferent inhibition (SAI). M1 cathodal decreased single-pulse MEP amplitudes at post 1 and decreased SAI at post 1 and post 2; however, SICI did not exhibit any change. S1 cathodal and sham did not show any changes in MEP amplitudes at any of the three time points. These results demonstrated that cathodal tDCS over the M1 not only decreases the M1 excitability but also affects the cortical inhibitory circuits related to SAI. PMID:26869909

  19. Automatic detection and quantitative analysis of cells in the mouse primary motor cortex

    NASA Astrophysics Data System (ADS)

    Meng, Yunlong; He, Yong; Wu, Jingpeng; Chen, Shangbin; Li, Anan; Gong, Hui

    2014-09-01

    Neuronal cells play very important role on metabolism regulation and mechanism control, so cell number is a fundamental determinant of brain function. Combined suitable cell-labeling approaches with recently proposed three-dimensional optical imaging techniques, whole mouse brain coronal sections can be acquired with 1-μm voxel resolution. We have developed a completely automatic pipeline to perform cell centroids detection, and provided three-dimensional quantitative information of cells in the primary motor cortex of C57BL/6 mouse. It involves four principal steps: i) preprocessing; ii) image binarization; iii) cell centroids extraction and contour segmentation; iv) laminar density estimation. Investigations on the presented method reveal promising detection accuracy in terms of recall and precision, with average recall rate 92.1% and average precision rate 86.2%. We also analyze laminar density distribution of cells from pial surface to corpus callosum from the output vectorizations of detected cell centroids in mouse primary motor cortex, and find significant cellular density distribution variations in different layers. This automatic cell centroids detection approach will be beneficial for fast cell-counting and accurate density estimation, as time-consuming and error-prone manual identification is avoided.

  20. Accumulation of silver nanoparticles by cultured primary brain astrocytes

    NASA Astrophysics Data System (ADS)

    Luther, Eva M.; Koehler, Yvonne; Diendorf, Joerg; Epple, Matthias; Dringen, Ralf

    2011-09-01

    Silver nanoparticles (AgNP) are components of various food industry products and are frequently used for medical equipment and materials. Although such particles enter the vertebrate brain, little is known on their biocompatibility for brain cells. To study the consequences of an AgNP exposure of brain cells we have treated astrocyte-rich primary cultures with polyvinylpyrrolidone (PVP)-coated AgNP. The incubation of cultured astrocytes with micromolar concentrations of AgNP for up to 24 h resulted in a time- and concentration-dependent accumulation of silver, but did not compromise the cell viability nor lower the cellular glutathione content. In contrast, the incubation of astrocytes for 4 h with identical amounts of silver as AgNO3 already severely compromised the cell viability and completely deprived the cells of glutathione. The accumulation of AgNP by astrocytes was proportional to the concentration of AgNP applied and significantly lowered by about 30% in the presence of the endocytosis inhibitors chloroquine or amiloride. Incubation at 4 °C reduced the accumulation of AgNP by 80% compared to the values obtained for cells that had been exposed to AgNP at 37 °C. These data demonstrate that viable cultured brain astrocytes efficiently accumulate PVP-coated AgNP in a temperature-dependent process that most likely involves endocytotic pathways.

  1. Optimal 3-D culture of primary articular chondrocytes for use in the Rotating Wall Vessel Bioreactor

    PubMed Central

    Mellor, Liliana F.; Baker, Travis L.; Brown, Raquel J.; Catlin, Lindsey W.; Oxford, Julia Thom

    2014-01-01

    INTRODUCTION Reliable culturing methods for primary articular chondrocytes are essential to study the effects of loading and unloading on joint tissue at the cellular level. Due to the limited proliferation capacity of primary chondrocytes and their tendency to dedifferentiate in conventional culture conditions, long-term culturing conditions of primary chondrocytes can be challenging. The goal of this study was to develop a suspension culturing technique that not only would retain the cellular morphology but also maintain gene expression characteristics of primary articular chondrocytes. METHODS Three-dimensional culturing methods were compared and optimized for primary articular chondrocytes in the rotating wall vessel bioreactor, which changes the mechanical culture conditions to provide a form of suspension culture optimized for low shear and turbulence. We performed gene expression analysis and morphological characterization of cells cultured in alginate beads, Cytopore-2 microcarriers, primary monolayer culture, and passaged monolayer cultures using reverse transcription-PCR and laser scanning confocal microscopy. RESULTS Primary chondrocytes grown on Cytopore-2 microcarriers maintained the phenotypical morphology and gene expression pattern observed in primary bovine articular chondrocytes, and retained these characteristics for up to 9 days. DISCUSSION Our results provide a novel and alternative culturing technique for primary chondrocytes suitable for studies that require suspension such as those using the rotating wall vessel bioreactor. In addition, we provide an alternative culturing technique for primary chondrocytes that can impact future mechanistic studies of osteoarthritis progression, treatments for cartilage damage and repair, and cartilage tissue engineering. PMID:25199120

  2. The Non-motor Features of Essential Tremor: A Primary Disease Feature or Just a Secondary Phenomenon?

    PubMed Central

    Jhunjhunwala, Ketan; Pal, Pramod K.

    2014-01-01

    Essential tremor (ET) is a pathologically heterogeneous neurodegenerative disorder with both motor and increasingly recognized non-motor features. It is debated whether the non-motor manifestations in ET result from widespread neurodegeneration or are merely secondary to impaired motor functions and decreased quality of life due to tremor. It is important to review these features to determine how to best treat the non-motor symptoms of patients and to understand the basic pathophysiology of the disease and develop appropriate pharmacotherapies. In this review, retrospective and prospective clinical studies were critically analyzed to identify possible correlations between the severities of non-motor features and tremor. We speculated that if such a correlation existed, the non-motor features were likely to be secondary to tremor. According to the current literature, the deficits in executive function, attention, concentration, and memory often observed in ET are likely to be a primary manifestation of the disease. It has also been documented that patients with ET often exhibit characteristic personality traits. However, it remains to be determined whether the other non-motor features often seen in ET, such as anxiety, depression, and sleep disturbances are primary or secondary to motor manifestations of ET and subsequent poor quality of life. Finally, there is evidence that patients with ET can also have impaired color vision, disturbances of olfaction, and hearing impairments, though there are few studies in these areas. Further investigations of large cohorts of patients with ET are required to understand the prevalence, nature, and true significance of the non-motor features in ET. PMID:25120945

  3. The effects of phthalate esters on fibroblasts in primary culture.

    PubMed

    Teranishi, H; Kasuya, M

    1980-06-01

    The toxicity of butylbenzyl phthalate(BLP), di-n-heptyl phthalate (DNHP) and n-butyl lauryl phthalate (BLP) to fibroblasts from newborn rat cerebellum in primary culture was significant at concentrations of 7.0, 2.7, and 5.0 x 10(-4) M, respectively. The toxicity of di-methoxyethyl phthalate(DMEP), butyl phthalyl butyl glycolate(BPBG), di-n-octyl phthalate(DNOP), and di-(2-ethylhexyl) phthalate(DEHP) was not significant. Phthalic acid and potassium hydrogen phthalate (K-phthalate) were the least toxic to fibroblasts. Comparison of the toxicity to fibroblasts of five phthalate esters of normal series showed that dimethyl phthalate(DMP) < diethyl phthalate(DEP) < di-n-butyl phthalate(DNBP) > DNHP > DNOP.

  4. Parallel Cortical Networks Formed by Modular Organization of Primary Motor Cortex Outputs.

    PubMed

    Hamadjida, Adjia; Dea, Melvin; Deffeyes, Joan; Quessy, Stephan; Dancause, Numa

    2016-07-11

    In primates, the refinement of motor behaviors, in particular hand use, is associated with the establishment of more direct projections from primary motor cortex (M1) onto cervical motoneurons [1, 2] and the appearance of additional premotor and sensory cortical areas [3]. All of these areas have reciprocal connections with M1 [4-7]. Thus, during the evolution of the sensorimotor network, the number of interlocutors with which M1 interacts has tremendously increased. It is not clear how these additional interconnections are organized in relation to one another within the hand representation of M1. This is important because the organization of connections between M1 and phylogenetically newer and specialized cortical areas is likely to be key to the increased repertoire of hand movements in primates. In cebus monkeys, we used injections of retrograde tracers into the hand representation of different cortical areas of the sensorimotor network (ventral and dorsal premotor areas [PMv and PMd], supplementary motor area [SMA], and posterior parietal cortex [area 5]), and we analyzed the pattern of labeled neurons within the hand representation of M1. Instead of being uniformly dispersed across M1, neurons sending projections to each distant cortical area were largely segregated in different subregions of M1. These data support the view that primates split the cortical real estate of M1 into modules, each preferentially interconnected with a particular cortical area within the sensorimotor network. This modular organization could sustain parallel processing of interactions with multiple specialized cortical areas to increase the behavioral repertoire of the hand. PMID:27322001

  5. Bilateral tDCS on Primary Motor Cortex: Effects on Fast Arm Reaching Tasks

    PubMed Central

    Arias, Pablo; Corral-Bergantiños, Yoanna; Robles-García, Verónica; Madrid, Antonio; Oliviero, Antonio; Cudeiro, Javier

    2016-01-01

    Background The effects produced by transcranial direct current stimulation (tDCS) applied to the motor system have been widely studied in the past, chiefly focused on primary motor cortex (M1) excitability. However, the effects on functional tasks are less well documented. Objective This study aims to evaluate the effect of tDCS-M1 on goal-oriented actions (i.e., arm-reaching movements; ARM), in a reaction-time protocol. Methods 13 healthy subjects executed dominant ARM as fast as possible to one of two targets in front of them while surface EMG was recorded. Participants performed three different sessions. In each session they first executed ARM (Pre), then received tDCS, and finally executed Post, similar to Pre. Subjects received three different types of tDCS, one per session: In one session the anode was on right-M1 (AR), and the cathode on the left-M1 (CL), thus termed AR-CL; AL-CR reversed the montage; and Sham session was applied likewise. Real stimulation was 1mA-10min while subjects at rest. Three different variables and their coefficients of variation (CV) were analyzed: Premotor times (PMT), reaction-times (RT) and movement-times (MT). Results triceps-PMT were significantly increased at Post-Sham, suggesting fatigue. Results obtained with real tDCS were not different depending on the montage used, in both cases PMT were significantly reduced in all recorded muscles. RT and MT did not change for real or sham stimulation. RT-CV and PMT-CV were reduced after all stimulation protocols. Conclusion tDCS reduces premotor time and fatigability during the execution of fast motor tasks. Possible underlying mechanisms are discussed. PMID:27490752

  6. Parallel Cortical Networks Formed by Modular Organization of Primary Motor Cortex Outputs.

    PubMed

    Hamadjida, Adjia; Dea, Melvin; Deffeyes, Joan; Quessy, Stephan; Dancause, Numa

    2016-07-11

    In primates, the refinement of motor behaviors, in particular hand use, is associated with the establishment of more direct projections from primary motor cortex (M1) onto cervical motoneurons [1, 2] and the appearance of additional premotor and sensory cortical areas [3]. All of these areas have reciprocal connections with M1 [4-7]. Thus, during the evolution of the sensorimotor network, the number of interlocutors with which M1 interacts has tremendously increased. It is not clear how these additional interconnections are organized in relation to one another within the hand representation of M1. This is important because the organization of connections between M1 and phylogenetically newer and specialized cortical areas is likely to be key to the increased repertoire of hand movements in primates. In cebus monkeys, we used injections of retrograde tracers into the hand representation of different cortical areas of the sensorimotor network (ventral and dorsal premotor areas [PMv and PMd], supplementary motor area [SMA], and posterior parietal cortex [area 5]), and we analyzed the pattern of labeled neurons within the hand representation of M1. Instead of being uniformly dispersed across M1, neurons sending projections to each distant cortical area were largely segregated in different subregions of M1. These data support the view that primates split the cortical real estate of M1 into modules, each preferentially interconnected with a particular cortical area within the sensorimotor network. This modular organization could sustain parallel processing of interactions with multiple specialized cortical areas to increase the behavioral repertoire of the hand.

  7. Regulation of human renin expression in chorion cell primary cultures

    SciTech Connect

    Duncan, K.G.; Haidar, M.A.; Baxter, J.D.; Reudelhuber, T.L. )

    1990-10-01

    The human renin gene is expressed in the kidney, placenta, and several other sites. The release of renin or its precursor, prorenin, can be affected by several regulatory agents. In this study, primary cultures of human placental cells were used to examine the regulation of prorenin release and renin mRNA levels and of the transfected human renin promoter linked to chloramphenicol acetyltransferase reporter sequences. Treatment of the cultures with a calcium ionophore alone, calcium ionophore plus forskolin (that activates adenylate cyclase), or forskolin plus a phorbol ester increased prorenin release and renin mRNA levels 1.3{endash} to 6{endash}fold, but several classes of steroids did not affect prorenin secretion or renin RNA levels. These results suggest that (i) the first 584 base pairs of the renin gene 5'{endash}flanking DNA do not contain functional glucocorticoid or estrogen response elements, (ii) placental prorenin release and renin mRNA are regulated by calcium ion and by the combinations of cAMP with either C kinase or calcium ion, and (iii) the first 100 base pairs of the human renin 5'{endash}flanking DNA direct accurate initiation of transcription and can be regulated by cAMP. Thus, some control of renin release in the placenta (and by inference in other tissues) occurs via transcriptional influences on its promoter.

  8. Reduced functional connectivity within the primary motor cortex of patients with brachial plexus injury.

    PubMed

    Fraiman, D; Miranda, M F; Erthal, F; Buur, P F; Elschot, M; Souza, L; Rombouts, S A R B; Schimmelpenninck, C A; Norris, D G; Malessy, M J A; Galves, A; Vargas, C D

    2016-01-01

    This study aims at the effects of traumatic brachial plexus lesion with root avulsions (BPA) upon the organization of the primary motor cortex (M1). Nine right-handed patients with a right BPA in whom an intercostal to musculocutaneous (ICN-MC) nerve transfer was performed had post-operative resting state fMRI scanning. The analysis of empirical functional correlations between neighboring voxels revealed faster correlation decay as a function of distance in the M1 region corresponding to the arm in BPA patients as compared to the control group. No differences between the two groups were found in the face area. We also investigated whether such larger decay in patients could be attributed to a gray matter diminution in M1. Structural imaging analysis showed no difference in gray matter density between groups. Our findings suggest that the faster decay in neighboring functional correlations without significant gray matter diminution in BPA patients could be related to a reduced activity in intrinsic horizontal connections in M1 responsible for upper limb motor synergies. PMID:27547727

  9. Cell and neuron densities in the primary motor cortex of primates

    PubMed Central

    Young, Nicole A.; Collins, Christine E.; Kaas, Jon H.

    2013-01-01

    Cell and neuron densities vary across the cortical sheet in a predictable manner across different primate species (Collins et al., 2010b). Primary motor cortex, M1, is characterized by lower neuron densities relative to other cortical areas. M1 contains a motor representation map of contralateral body parts from tail to tongue in a mediolateral sequence. Different functional movement representations within M1 likely require specialized microcircuitry for control of different body parts, and these differences in circuitry may be reflected by variation in cell and neuron densities. Here we determined cell and neuron densities for multiple sub-regions of M1 in six primate species, using the semi-automated flow fractionator method. The results verify previous reports of lower overall neuron densities in M1 compared to other parts of cortex in the six primate species examined. The most lateral regions of M1 that correspond to face and hand movement representations, are more neuron dense relative to medial locations in M1, which suggests differences in cortical circuitry within movement zones. PMID:23450743

  10. Fast and Slow Oscillations in Human Primary Motor Cortex Predict Oncoming Behaviorally Relevant Cues

    PubMed Central

    Saleh, Maryam; Reimer, Jacob; Penn, Richard; Ojakangas, Catherine L.; Hatsopoulos, Nicholas G.

    2011-01-01

    SUMMARY Beta oscillations (12-30Hz) in local field potentials are prevalent in the motor system, yet their functional role within the context of planning a movement is still debated. In this study, a human participant implanted with a multi-electrode array in the hand area of primary motor cortex (MI) was instructed to plan a movement using either the second or fourth of five sequentially presented instruction cues. The beta amplitude increased from the start of the trial until the informative (second or fourth) cue, and was diminished afterwards. Moreover, the beta amplitude peaked just prior to each instruction cue and the delta frequency (0.5-1.5Hz) entrained to the interval between the cues - but only until the informative cue. This result suggests that the beta amplitude and delta phase in MI reflect the subject’s engagement with the rhythmically-presented cues and work together to enhance sensitivity to predictable and task-relevant visual cues. PMID:20188651

  11. Voltage-sensitive dye imaging of primary motor cortex activity produced by ventral tegmental area stimulation.

    PubMed

    Kunori, Nobuo; Kajiwara, Riichi; Takashima, Ichiro

    2014-06-25

    The primary motor cortex (M1) receives dopaminergic projections from the ventral tegmental area (VTA) through the mesocortical dopamine pathway. However, few studies have focused on changes in M1 neuronal activity caused by VTA activation. To address this issue, we used voltage-sensitive dye imaging (VSD) to reveal the spatiotemporal dynamics of M1 activity induced by single-pulse stimulation of VTA in anesthetized rats. VSD imaging showed that brief electrical stimulation of unilateral VTA elicited a short-latency excitatory-inhibitory sequence of neuronal activity not only in the ipsilateral but also in the contralateral M1. The contralateral M1 response was not affected by pharmacological blockade of ipsilateral M1 activity, but it was completely abolished by corpus callosum transection. Although the VTA-evoked neuronal activity extended throughout the entire M1, we found the most prominent activity in the forelimb area of M1. The 6-OHDA-lesioned VTA failed to evoke M1 activity. Furthermore, both excitatory and inhibitory intact VTA-induced activity was entirely extinguished by blocking glutamate receptors in the target M1. When intracortical microstimulation of M1 was paired with VTA stimulation, the evoked forelimb muscle activity was facilitated or inhibited, depending on the interval between the two stimuli. These findings suggest that VTA neurons directly modulate the excitability of M1 neurons via fast glutamate signaling and, consequently, may control the last cortical stage of motor command processing. PMID:24966388

  12. Reduced functional connectivity within the primary motor cortex of patients with brachial plexus injury.

    PubMed

    Fraiman, D; Miranda, M F; Erthal, F; Buur, P F; Elschot, M; Souza, L; Rombouts, S A R B; Schimmelpenninck, C A; Norris, D G; Malessy, M J A; Galves, A; Vargas, C D

    2016-01-01

    This study aims at the effects of traumatic brachial plexus lesion with root avulsions (BPA) upon the organization of the primary motor cortex (M1). Nine right-handed patients with a right BPA in whom an intercostal to musculocutaneous (ICN-MC) nerve transfer was performed had post-operative resting state fMRI scanning. The analysis of empirical functional correlations between neighboring voxels revealed faster correlation decay as a function of distance in the M1 region corresponding to the arm in BPA patients as compared to the control group. No differences between the two groups were found in the face area. We also investigated whether such larger decay in patients could be attributed to a gray matter diminution in M1. Structural imaging analysis showed no difference in gray matter density between groups. Our findings suggest that the faster decay in neighboring functional correlations without significant gray matter diminution in BPA patients could be related to a reduced activity in intrinsic horizontal connections in M1 responsible for upper limb motor synergies.

  13. Political, cultural and economic foundations of primary care in Europe.

    PubMed

    Kringos, Dionne S; Boerma, Wienke G W; van der Zee, Jouke; Groenewegen, Peter P

    2013-12-01

    This article explores various contributing factors to explain differences in the strength of the primary care (PC) structure and services delivery across Europe. Data on the strength of primary care in 31 European countries in 2009/10 were used. The results showed that the national political agenda, economy, prevailing values, and type of healthcare system are all important factors that influence the development of strong PC. Wealthier countries are associated with a weaker PC structure and lower PC accessibility, while Eastern European countries seemed to have used their growth in national income to strengthen the accessibility and continuity of PC. Countries governed by left-wing governments are associated with a stronger PC structure, accessibility and coordination of PC. Countries with a social-security based system are associated with a lower accessibility and continuity of PC; the opposite is true for transitional systems. Cultural values seemed to affect all aspects of PC. It can be concluded that strengthening PC means mobilising multiple leverage points, policy options, and political will in line with prevailing values in a country. PMID:24355465

  14. Predicting hand orientation in reach-to-grasp tasks using neural activities from primary motor cortex.

    PubMed

    Zhang, Peng; Ma, Xuan; Huang, Hailong; He, Jiping

    2014-01-01

    Hand orientation is an important control parameter during reach-to-grasp task. In this paper, we presented a study for predicting hand orientation of non-human primate by decoding neural activities from primary motor cortex (M1). A non-human primate subject was guided to do reaching and grasping tasks meanwhile neural activities were acquired by chronically implanted microelectrode arrays. A Support Vector Machines (SVMs) classifier has been trained for predicting three different hand orientations using these M1 neural activities. Different number of neurons were selected and analyzed; the classifying accuracy was 94.1% with 2 neurons and was 100% with 8 neurons. Data from highly event related neuron units contribute a lot to the accuracy of hand orientation prediction. These results indicate that three different hand orientations can be predicted accurately and effectively before the actual movements occurring with a small number of related neurons in M1.

  15. Preparation of Rodent Primary Cultures for Neuron–Glia, Mixed Glia, Enriched Microglia, and Reconstituted Cultures with Microglia

    PubMed Central

    Chen, Shih-Heng; Oyarzabal, Esteban A.; Hong, Jau-Shyong

    2016-01-01

    Microglia, neurons, and macroglia (astrocytes and oligodendrocytes) are the major cell types in the central nervous system. In the past decades, primary microglia-enriched cultures have been widely used to study the biological functions of microglia in vitro. In order to study the interactions between microglia and other brain cells, neuron–glia, neuron–microglia, and mixed glia cultures were developed. The aim of this chapter is to provide basic and adaptable protocols for the preparation of these microglia-containing primary cultures from rodent. Meanwhile, we also want to provide a collection of tips from our collective experiences doing primary brain cell cultures. PMID:23813383

  16. Laterality of movement-related activity reflects transformation of coordinates in ventral premotor cortex and primary motor cortex of monkeys.

    PubMed

    Kurata, Kiyoshi

    2007-10-01

    The ventral premotor cortex (PMv) and the primary motor cortex (MI) of monkeys participate in various sensorimotor integrations, such as the transformation of coordinates from visual to motor space, because the areas contain movement-related neuronal activity reflecting either visual or motor space. In addition to relationship to visual and motor space, laterality of the activity could indicate stages in the visuomotor transformation. Thus we examined laterality and relationship to visual and motor space of movement-related neuronal activity in the PMv and MI of monkeys performing a fast-reaching task with the left or right arm, toward targets with visual and motor coordinates that had been dissociated by shift prisms. We determined laterality of each activity quantitatively and classified it into four types: activity that consistently depended on target locations in either head-centered visual coordinates (V-type) or motor coordinates (M-type) and those that had either differential or nondifferential activity for both coordinates (B- and N-types). A majority of M-type neurons in the areas had preferences for reaching movements with the arm contralateral to the hemisphere where neuronal activity was recorded. In contrast, most of the V-type neurons were recorded in the PMv and exhibited less laterality than the M-type. The B- and N-types were recorded in the PMv and MI and exhibited intermediate properties between the V- and M-types when laterality and correlations to visual and motor space of them were jointly examined. These results suggest that the cortical motor areas contribute to the transformation of coordinates to generate final motor commands.

  17. Interspecies differences in metabolism of arsenic by cultured primary hepatocytes

    SciTech Connect

    Drobna, Zuzana; Walton, Felecia S.; Harmon, Anne W.; Thomas, David J.; Styblo, Miroslav

    2010-05-15

    Biomethylation is the major pathway for the metabolism of inorganic arsenic (iAs) in many mammalian species, including the human. However, significant interspecies differences have been reported in the rate of in vivo metabolism of iAs and in yields of iAs metabolites found in urine. Liver is considered the primary site for the methylation of iAs and arsenic (+3 oxidation state) methyltransferase (As3mt) is the key enzyme in this pathway. Thus, the As3mt-catalyzed methylation of iAs in the liver determines in part the rate and the pattern of iAs metabolism in various species. We examined kinetics and concentration-response patterns for iAs methylation by cultured primary hepatocytes derived from human, rat, mice, dog, rabbit, and rhesus monkey. Hepatocytes were exposed to [{sup 73}As]arsenite (iAs{sup III}; 0.3, 0.9, 3.0, 9.0 or 30 nmol As/mg protein) for 24 h and radiolabeled metabolites were analyzed in cells and culture media. Hepatocytes from all six species methylated iAs{sup III} to methylarsenic (MAs) and dimethylarsenic (DMAs). Notably, dog, rat and monkey hepatocytes were considerably more efficient methylators of iAs{sup III} than mouse, rabbit or human hepatocytes. The low efficiency of mouse, rabbit and human hepatocytes to methylate iAs{sup III} was associated with inhibition of DMAs production by moderate concentrations of iAs{sup III} and with retention of iAs and MAs in cells. No significant correlations were found between the rate of iAs methylation and the thioredoxin reductase activity or glutathione concentration, two factors that modulate the activity of recombinant As3mt. No associations between the rates of iAs methylation and As3mt protein structures were found for the six species examined. Immunoblot analyses indicate that the superior arsenic methylation capacities of dog, rat and monkey hepatocytes examined in this study may be associated with a higher As3mt expression. However, factors other than As3mt expression may also contribute to

  18. Reference gene for primary culture of prostate cancer cells.

    PubMed

    Souza, Aline Francielle Damo; Brum, Ilma Simoni; Neto, Brasil Silva; Berger, Milton; Branchini, Gisele

    2013-04-01

    Selection of reference genes to normalize mRNA levels between samples is critical for gene expression studies because their expression can vary depending on the tissues or cells used and the experimental conditions. We performed ten cell cultures from samples of prostate cancer. Cells were divided into three groups: control (with no transfection protocol), cells transfected with siRNA specific to knockdown the androgen receptor and cells transfected with inespecific siRNAs. After 24 h, mRNA was extracted and gene expression was analyzed by Real-time qPCR. Nine candidates to reference genes for gene expression studies in this model were analyzed (aminolevulinate, delta-, synthase 1 (ALAS1); beta-actin (ACTB); beta-2-microglobulin (B2M); glyceraldehyde-3-phosphate dehydrogenase (GAPDH); hypoxanthine phosphoribosyltransferase 1 (HPRT1); succinate dehydrogenase complex, subunit A, flavoprotein (Fp) (SDHA); TATA box binding protein (TBP); ubiquitin C (UBC); tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide (YWHAZ)). Expression stability was calculated NormFinder algorithm to find the most stable genes. NormFinder calculated SDHA as the most stable gene and the gene with the lowest intergroup and intragroup variation, and indicated GAPDH and SDHA as the best combination of two genes for the purpose of normalization. Androgen receptor mRNA expression was evaluated after normalization by each candidate gene and showed statistical difference in the transfected group compared to control group only when normalized by combination of GAPDH and SDHA. Based on the algorithm analysis, the combination of SDHA and GAPDH should be used to normalize target genes mRNA levels in primary culture of prostate cancer cells submitted to transfection with siRNAs.

  19. Accumulation of pyrethroid compounds in primary cultures of rat cortical neurons

    EPA Science Inventory

    Recent studies have demonstrated that lipophilic compounds (e.g. methylmercury, polychlorinated biphenyls (PCBs) and polybrominated diphenylethers (PBDEs)) rapidly accumulate in cells in culture to concentrations much higher than in the surrounding media. Primary cultures of neur...

  20. Primary motor cortex activity reduction under the regulation of SMA by real-time fMRI

    NASA Astrophysics Data System (ADS)

    Guo, Jia; Zhao, Xiaojie; Li, Yi; Yao, Li; Chen, Kewei

    2012-03-01

    Real-time fMRI (rtfMRI) is a new technology which allows human subjects to observe and control their own BOLD signal change from one or more localized brain regions during scanning. Current rtfMRI-neurofeedback studies mainly focused on the target region itself without considering other related regions influenced by the real-time feedback. However, there always exits important directional influence between many of cooperative regions. On the other hand, rtfMRI based on motor imagery mainly aimed at somatomotor cortex or primary motor area, whereas supplement motor area (SMA) was a relatively more integrated and pivotal region. In this study, we investigated whether the activities of SMA can be controlled utilizing different motor imagery strategies, and whether there exists any possible impact on an unregulated but related region, primary motor cortex (M1). SMA was first localized using overt finger tapping task, the activities of SMA were feedback to subjects visually on line during each of two subsequent imagery motor movement sessions. All thirteen healthy participants were found to be able to successfully control their SMA activities by self-fit imagery strategies which involved no actual motor movements. The activation of right M1 was also found to be significantly reduced in both intensity and extent with the neurofeedback process targeted at SMA, suggestive that not only the part of motor cortex activities were influenced under the regulation of a key region SMA, but also the increased difference between SMA and M1 might reflect the potential learning effect.

  1. Central Motor Conduction Studies and Diagnostic Magnetic Resonance Imaging in Children with Severe Primary and Secondary Dystonia

    ERIC Educational Resources Information Center

    McClelland, Verity; Mills, Kerry; Siddiqui, Ata; Selway, Richard; Lin, Jean-Pierre

    2011-01-01

    Aim: Dystonia in childhood has many causes. Imaging may suggest corticospinal tract dysfunction with or without coexistent basal ganglia damage. There are very few published neurophysiological studies on children with dystonia; one previous study has focused on primary dystonia. We investigated central motor conduction in 62 children (34 males, 28…

  2. Preparing Pre-Service Primary School Teachers to Assess Fundamental Motor Skills: Two Skills and Two Approaches

    ERIC Educational Resources Information Center

    Haynes, John; Miller, Judith

    2015-01-01

    Background: Pre-service teacher education (PSTE) programmes for generalist primary school teachers have limited time allocated to Physical Education, Health and Personal Development. In practice, teachers in schools are required to assess motor skills despite the fact that their training provides minimal preparation. This necessitates creative…

  3. HTS compatible assay for antioxidative agents using primary cultured hepatocytes.

    PubMed

    Gaunitz, Frank; Heise, Kerstin

    2003-06-01

    We have used primary cultured rat hepatocytes to establish a system that is compatible with HTS for screening substance libraries for biologically active compounds. The hepatocytes were treated with t-BHP to induce oxidative stress, leading to the formation ROS. The involvement of ROS in oxidative stress and pathological alterations has been of major interest in recent years, and there is great demand to identify new compounds with antioxidant potential. In most HTS programs each compound is tested in duplicate, and may only be tested once. Because of this it is important to develop assays that can identify candidate compounds accurately and with high confidence. Using newly available cell-based assay systems, we have developed a system that can detect active compounds (hits) with a high degree of confidence. As an example of an agent that can be detected from a substance library, we analyzed the effect of fisetin as an antioxidative compound using this system. All measurements were performed using the newly developed and highly versatile Multilabel-Reader Mithras LB 940 (Berthold Technologies, Bad Wildbad, Germany). The data presented show that all Z' factors determined were highly reliable. Although the protocol is primarily designed to screen for substances with antioxidative potential, it can easily be adapted to screen for other biologically active substances.

  4. Direction of Movement Is Encoded in the Human Primary Motor Cortex

    PubMed Central

    Toxopeus, Carolien M.; de Jong, Bauke M.; Valsan, Gopal; Conway, Bernard A.; Leenders, Klaus L.; Maurits, Natasha M.

    2011-01-01

    The present study investigated how direction of hand movement, which is a well-described parameter in cerebral organization of motor control, is incorporated in the somatotopic representation of the manual effector system in the human primary motor cortex (M1). Using functional magnetic resonance imaging (fMRI) and a manual step-tracking task we found that activation patterns related to movement in different directions were spatially disjoint within the representation area of the hand on M1. Foci of activation related to specific movement directions were segregated within the M1 hand area; activation related to direction 0° (right) was located most laterally/superficially, whereas directions 180° (left) and 270° (down) elicited activation more medially within the hand area. Activation related to direction 90° was located between the other directions. Moreover, by investigating differences between activations related to movement along the horizontal (0°+180°) and vertical (90°+270°) axis, we found that activation related to the horizontal axis was located more anterolaterally/dorsally in M1 than for the vertical axis, supporting that activations related to individual movement directions are direction- and not muscle related. Our results of spatially segregated direction-related activations in M1 are in accordance with findings of recent fMRI studies on neural encoding of direction in human M1. Our results thus provide further evidence for a direct link between direction as an organizational principle in sensorimotor transformation and movement execution coded by effector representations in M1. PMID:22110768

  5. Direction of movement is encoded in the human primary motor cortex.

    PubMed

    Toxopeus, Carolien M; de Jong, Bauke M; Valsan, Gopal; Conway, Bernard A; Leenders, Klaus L; Maurits, Natasha M

    2011-01-01

    The present study investigated how direction of hand movement, which is a well-described parameter in cerebral organization of motor control, is incorporated in the somatotopic representation of the manual effector system in the human primary motor cortex (M1). Using functional magnetic resonance imaging (fMRI) and a manual step-tracking task we found that activation patterns related to movement in different directions were spatially disjoint within the representation area of the hand on M1. Foci of activation related to specific movement directions were segregated within the M1 hand area; activation related to direction 0° (right) was located most laterally/superficially, whereas directions 180° (left) and 270° (down) elicited activation more medially within the hand area. Activation related to direction 90° was located between the other directions. Moreover, by investigating differences between activations related to movement along the horizontal (0°+180°) and vertical (90°+270°) axis, we found that activation related to the horizontal axis was located more anterolaterally/dorsally in M1 than for the vertical axis, supporting that activations related to individual movement directions are direction- and not muscle related. Our results of spatially segregated direction-related activations in M1 are in accordance with findings of recent fMRI studies on neural encoding of direction in human M1. Our results thus provide further evidence for a direct link between direction as an organizational principle in sensorimotor transformation and movement execution coded by effector representations in M1.

  6. Energy-dependent volume regulation in primary cultured cerebral astrocytes.

    PubMed

    Olson, J E; Sankar, R; Holtzman, D; James, A; Fleischhacker, D

    1986-08-01

    Cell volume regulation and energy metabolism were studied in primary cultured cerebral astrocytes during exposure to media of altered osmolarity. Cells suspended in medium containing 1/2 the normal concentration of NaCl (hypoosmotic) swell immediately to a volume 40-50% larger than cells suspended in isoosmotic medium. The cell volume in hypoosmotic medium then decreases over 30 min to a volume approximately 25% larger than cells in isoosmotic medium. In hyperosmotic medium (containing twice the normal concentration of NaCl), astrocytes shrink by 29%. Little volume change occurs following this initial shrinkage. Cells resuspended in isoosmotic medium after a 30 min incubation in hypoosmotic medium shrink immediately to a volume 10% less than the volume of cells incubated continuously in isoosmotic medium. Thus, the regulatory volume decrease (RVD) in hypoosmotic medium involves a net reduction of intracellular osmoles. The RVD is partially blocked by inhibitors of mitochondrial electron transport but is unaffected by an inhibitor of glycolysis or by an uncoupler of oxidative phosphorylation. Inhibition of RVD by these metabolic agents is correlated with decreased cellular ATP levels. Ouabain, added immediately after hypoosmotic induced swelling, completely inhibits RVD, but does not alter cell volume if added after RVD has taken place. Ouabain also inhibits cell respiration 27% more in hypoosmotic medium than in isoosmotic medium indicating that the (Na,K)-ATPase-coupled ion pump is more active in the hypoosmotic medium. These data suggest that the cell volume response of astrocytes in hypoosmotic medium involves the net movement of osmoles by a mechanism dependent on cellular energy and tightly coupled to the (Na,K)-ATPase ion pump. This process may be important in the energy-dependent osmoregulation in the brain, a critical role attributed to the astrocyte in vivo. PMID:3015986

  7. Neurochemical Analysis of Primary Motor Cortex in Chronic Low Back Pain

    PubMed Central

    Sharma, Neena K.; Brooks, William M.; Popescu, Anda E.; VanDillen, Linda; George, Steven Z.; McCarson, Kenneth E.; Gajewski, Byron J.; Gorman, Patrick; Cirstea, Carmen M.

    2012-01-01

    The involvement of the primary motor cortex (M1) in chronic low back pain (LBP) is a relatively new concept. Decreased M1 excitability and an analgesic effect after M1 stimulation have been recently reported. However, the neurochemical changes underlying these functional M1 changes are unknown. The current study investigated whether neurochemicals specific to neurons and glial cells in both right and left M1 are altered. N-Acetylaspartate (NAA) and myo-inositol (mI) were measured with proton magnetic resonance spectroscopy in 19 subjects with chronic LBP and 14 healthy controls. We also examined correlations among neurochemicals within and between M1 and relationships between neurochemical concentrations and clinical features of pain. Right M1 NAA was lower in subjects with LBP compared to controls (p = 0.008). Left M1 NAA and mI were not significantly different between LBP and control groups. Correlations between neurochemical concentrations across M1s were different between groups (p = 0.008). There were no significant correlations between M1 neurochemicals and pain characteristics. These findings provide preliminary evidence of neuronal depression and altered neuronal-glial interactions across M1 in chronic LBP. PMID:23766894

  8. Prediction of hand trajectory from electrocorticography signals in primary motor cortex.

    PubMed

    Chen, Chao; Shin, Duk; Watanabe, Hidenori; Nakanishi, Yasuhiko; Kambara, Hiroyuki; Yoshimura, Natsue; Nambu, Atsushi; Isa, Tadashi; Nishimura, Yukio; Koike, Yasuharu

    2013-01-01

    Due to their potential as a control modality in brain-machine interfaces, electrocorticography (ECoG) has received much focus in recent years. Studies using ECoG have come out with success in such endeavors as classification of arm movements and natural grasp types, regression of arm trajectories in two and three dimensions, estimation of muscle activity time series and so on. However, there still remains considerable work to be done before a high performance ECoG-based neural prosthetic can be realized. In this study, we proposed an algorithm to decode hand trajectory from 15 and 32 channel ECoG signals recorded from primary motor cortex (M1) in two primates. To determine the most effective areas for prediction, we applied two electrode selection methods, one based on position relative to the central sulcus (CS) and another based on the electrodes' individual prediction performance. The best coefficients of determination for decoding hand trajectory in the two monkeys were 0.4815 ± 0.0167 and 0.7780 ± 0.0164. Performance results from individual ECoG electrodes showed that those with higher performance were concentrated at the lateral areas and areas close to the CS. The results of prediction according with different numbers of electrodes based on proposed methods were also shown and discussed. These results also suggest that superior decoding performance can be achieved from a group of effective ECoG signals rather than an entire ECoG array.

  9. Lamina-dependent calibrated BOLD response in human primary motor cortex.

    PubMed

    Guidi, Maria; Huber, Laurentius; Lampe, Leonie; Gauthier, Claudine J; Möller, Harald E

    2016-11-01

    Disentangling neural activity at different cortical depths during a functional task has recently generated growing interest, since this would allow to separate feedforward and feedback activity. The majority of layer-dependent studies have, so far, relied on gradient-recalled echo (GRE) blood-oxygenation-level dependent (BOLD) acquisitions, which are weighted towards the large draining veins at the cortical surface. The current study aims to obtain quantitative brain activity responses in the primary motor cortex on a laminar scale without the contamination due to accompanying secondary vascular effects. Evoked oxidative metabolism was evaluated using the Davis model, to investigate its applicability, advantages, and limits in lamina-dependent fMRI. Average values for the calibration parameter, M, and for changes in the cerebral metabolic rate of oxygen consumption (CMRO2) during a unilateral finger-tapping task were (11±2)% and (30±7)%, respectively, with distinct variation features across the cortical depth. The results presented here showed an uncoupling between BOLD-based functional magnetic resonance imaging (fMRI) and metabolic changes across cortical depth, while the tight coupling between CMRO2 and CBV was conserved across cortical layers. We conclude that the Davis model can help to obtain estimates of lamina-dependent metabolic changes without contamination from large draining veins, with high consistency and reproducibility across participants.

  10. Lamina-dependent calibrated BOLD response in human primary motor cortex.

    PubMed

    Guidi, Maria; Huber, Laurentius; Lampe, Leonie; Gauthier, Claudine J; Möller, Harald E

    2016-11-01

    Disentangling neural activity at different cortical depths during a functional task has recently generated growing interest, since this would allow to separate feedforward and feedback activity. The majority of layer-dependent studies have, so far, relied on gradient-recalled echo (GRE) blood-oxygenation-level dependent (BOLD) acquisitions, which are weighted towards the large draining veins at the cortical surface. The current study aims to obtain quantitative brain activity responses in the primary motor cortex on a laminar scale without the contamination due to accompanying secondary vascular effects. Evoked oxidative metabolism was evaluated using the Davis model, to investigate its applicability, advantages, and limits in lamina-dependent fMRI. Average values for the calibration parameter, M, and for changes in the cerebral metabolic rate of oxygen consumption (CMRO2) during a unilateral finger-tapping task were (11±2)% and (30±7)%, respectively, with distinct variation features across the cortical depth. The results presented here showed an uncoupling between BOLD-based functional magnetic resonance imaging (fMRI) and metabolic changes across cortical depth, while the tight coupling between CMRO2 and CBV was conserved across cortical layers. We conclude that the Davis model can help to obtain estimates of lamina-dependent metabolic changes without contamination from large draining veins, with high consistency and reproducibility across participants. PMID:27364473

  11. Biological factors and age-dependence of primary motor cortex experimental plasticity.

    PubMed

    Polimanti, Renato; Simonelli, Ilaria; Zappasodi, Filippo; Ventriglia, Mariacarla; Pellicciari, Maria Concetta; Benussi, Luisa; Squitti, Rosanna; Rossini, Paolo Maria; Tecchio, Franca

    2016-02-01

    To evaluate whether the age-dependence of brain plasticity correlates with the levels of proteins involved in hormone and brain functions we executed a paired associative stimulation (PAS) protocol and blood tests. We measured the PAS-induced plasticity in the primary motor cortex. Blood levels of the brain-derived neurotrophic factor (BDNF), estradiol, the insulin-like growth factor (IGF)-1, the insulin-like growth factor binding protein (IGFBP)-3, progesterone, sex hormone-binding globulin (SHBG), testosterone, and the transforming growth factor beta 1 (TGF-β1) were determined in 15 healthy men and 20 healthy women. We observed an age-related reduction of PAS-induced plasticity in females that it is not present in males. In females, PAS-induced plasticity displayed a correlation with testosterone (p = 0.006) that became a trend after the adjustment for the age effect (p = 0.078). In males, IGF-1 showed a nominally significant correlation with the PAS-induced plasticity (p = 0.043). In conclusion, we observed that hormone blood levels (testosterone in females and IGF-1 in males) may be involved in the age-dependence of brain plasticity.

  12. Primary motor cortex of the parkinsonian monkey: altered neuronal responses to muscle stretch

    PubMed Central

    Pasquereau, Benjamin; Turner, Robert S.

    2013-01-01

    Exaggeration of the long-latency stretch reflex (LLSR) is a characteristic neurophysiologic feature of Parkinson's disease (PD) that contributes to parkinsonian rigidity. To explore one frequently-hypothesized mechanism, we studied the effects of fast muscle stretches on neuronal activity in the macaque primary motor cortex (M1) before and after the induction of parkinsonism by unilateral administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). We compared results from the general population of M1 neurons and two antidromically-identified subpopulations: distant-projecting pyramidal-tract type neurons (PTNs) and intra-telecenphalic-type corticostriatal neurons (CSNs). Rapid rotations of elbow or wrist joints evoked short-latency responses in 62% of arm-related M1 neurons. As in PD, the late electromyographic responses that constitute the LLSR were enhanced following MPTP. This was accompanied by a shortening of M1 neuronal response latencies and a degradation of directional selectivity, but surprisingly, no increase in single unit response magnitudes. The results suggest that parkinsonism alters the timing and specificity of M1 responses to muscle stretch. Observation of an exaggerated LLSR with no change in the magnitude of proprioceptive responses in M1 is consistent with the idea that the increase in LLSR gain that contributes to parkinsonian rigidity is localized to the spinal cord. PMID:24324412

  13. Patterns of cortical input to the primary motor area in the marmoset monkey.

    PubMed

    Burman, Kathleen J; Bakola, Sophia; Richardson, Karyn E; Reser, David H; Rosa, Marcello G P

    2014-03-01

    In primates the primary motor cortex (M1) forms a topographic map of the body, whereby neurons in the medial part of this area control movements involving trunk and hindlimb muscles, those in the intermediate part control movements involving forelimb muscles, and those in the lateral part control movements of facial and other head muscles. This topography is accompanied by changes in cytoarchitectural characteristics, raising the question of whether the anatomical connections also vary between different parts of M1. To address this issue, we compared the patterns of cortical afferents revealed by retrograde tracer injections in different locations within M1 of marmoset monkeys. We found that the entire extent of this area is unified by projections from the dorsocaudal and medial subdivisions of premotor cortex (areas 6DC and 6M), from somatosensory areas 3a, 3b, 1/2, and S2, and from posterior parietal area PE. While cingulate areas projected to all subdivisions, they preferentially targeted the medial part of M1. Conversely, the ventral premotor areas were preferentially connected with the lateral part of M1. Smaller but consistent inputs originated in frontal area 6DR, ventral posterior parietal cortex, the retroinsular cortex, and area TPt. Connections with intraparietal, prefrontal, and temporal areas were very sparse, and variable. Our results demonstrate that M1 is unified by a consistent pattern of major connections, but also shows regional variations in terms of minor inputs. These differences likely reflect requirements for control of voluntary movement involving different body parts. PMID:23939531

  14. Prediction of Hand Trajectory from Electrocorticography Signals in Primary Motor Cortex

    PubMed Central

    Nakanishi, Yasuhiko; Kambara, Hiroyuki; Yoshimura, Natsue; Nambu, Atsushi; Isa, Tadashi; Nishimura, Yukio; Koike, Yasuharu

    2013-01-01

    Due to their potential as a control modality in brain-machine interfaces, electrocorticography (ECoG) has received much focus in recent years. Studies using ECoG have come out with success in such endeavors as classification of arm movements and natural grasp types, regression of arm trajectories in two and three dimensions, estimation of muscle activity time series and so on. However, there still remains considerable work to be done before a high performance ECoG-based neural prosthetic can be realized. In this study, we proposed an algorithm to decode hand trajectory from 15 and 32 channel ECoG signals recorded from primary motor cortex (M1) in two primates. To determine the most effective areas for prediction, we applied two electrode selection methods, one based on position relative to the central sulcus (CS) and another based on the electrodes' individual prediction performance. The best coefficients of determination for decoding hand trajectory in the two monkeys were 0.4815±0.0167 and 0.7780±0.0164. Performance results from individual ECoG electrodes showed that those with higher performance were concentrated at the lateral areas and areas close to the CS. The results of prediction according with different numbers of electrodes based on proposed methods were also shown and discussed. These results also suggest that superior decoding performance can be achieved from a group of effective ECoG signals rather than an entire ECoG array. PMID:24386223

  15. Patient Safety Culture in Nephrology Nurse Practice Settings: Results by Primary Work Unit, Organizational Work Setting, and Primary Role.

    PubMed

    Ulrich, Beth; Kear, Tamara

    2015-01-01

    Patient safety culture is critical to the achievement of patient safety. In 2014, a landmark national study was conducted to investigate patient safety culture in nephrology nurse practice settings. In this secondary analysis of data from that study, we report the status of patient safety culture by primary work unit (chronic hemodialysis unit, acute hemodialysis unit, peritoneal dialysis unit) and organizational work setting (for-profit organization, not-for-profit organization), and compare the perceptions of direct care nurses and managers/administrators on components of patient safety culture.

  16. Impaired cholesterol esterification in primary brain cultures of the lysosomal cholesterol storage disorder (LCSD) mouse mutant

    SciTech Connect

    Patel, S.C.; Suresh, S.; Weintroub, H.; Brady, R.O.; Pentchev, P.G.

    1987-02-27

    Esterification of cholesterol was investigated in primary neuroglial cultures obtained from newborn lysosomal cholesterol storage disorder (LCSD) mouse mutants. An impairment in /sup 3/H-oleic acid incorporation into cholesteryl esters was demonstrated in cultures of homozygous LCSD brain. Primary cultures derived from other phenotypically normal pups of the carrier breeders esterified cholesterol at normal levels or at levels which were intermediary between normal and deficient indicating a phenotypic expression of the LCSD heterozygote genotype. These observations on LCSD mutant brain cells indicate that the defect in cholesterol esterification is closely related to the primary genetic defect and is expressed in neuroglial cells in culture.

  17. Transient inhibition of primary motor cortex suppresses hand muscle responses during a reactive reach to grasp.

    PubMed

    Bolton, David A E; Patel, Rupesh; Staines, W Richard; McIlroy, William E

    2011-10-24

    Rapid balance reactions such as compensatory reach to grasp represent important response strategies following unexpected loss of balance. While it has been assumed that early corrective actions arise from subcortical networks, recent research has prompted speculation about the potential role of cortical involvement. With reach to grasp reactions there is evidence of parallels in the control of perturbation-evoked reaching versus rapid voluntary reaching. However, the potential role of cortical involvement in such rapid balance reactions remains speculative. To test if cortical motor regions are involved we used continuous theta burst stimulation (cTBS) to temporarily suppress the hand area of primary motor cortex (M1) in participants involved in two reaching conditions: (1) rapid compensatory perturbation-evoked reach to grasp and (2) voluntary reach to grasp in response to an auditory cue. We hypothesized that following cTBS to the left M1 hand area we would find diminished EMG responses in the reaching (right) hand for both compensatory and voluntary movements. To isolate balance reactions to the upper limb participants were seated in an elevated tilt-chair with a stable handle positioned in front of their right shoulder. The chair was held vertical by a magnet and triggered to fall backward randomly. To regain balance, participants were instructed to reach for the handle as quickly as possible with the right hand upon chair release. Intermixed with perturbation trials, participants were also required to reach for the same handle but in response to an auditory tone. Muscle activity was recorded from several muscles of the right arm/hand using electromyography. As expected, movement time and muscle onsets were much faster following perturbation versus auditory-cued reaching. The novel finding from our study was the reduced amplitude of hand muscle activity post-cTBS for both perturbation-cued and auditory-cued reaches. Moreover, this reduction was specific to the

  18. Hand Preference for Tool-Use in Capuchin Monkeys (Cebus apella) is Associated with Asymmetry of the Primary Motor Cortex

    PubMed Central

    Phillips, Kimberley A.; Thompson, Claudia R.

    2012-01-01

    Skilled motor actions are associated with handedness and neuroanatomical specializations in humans. Recent reports have documented similar neuroanatomical asymmetries and their relationship to hand preference in some nonhuman primate species, including chimpanzees and capuchin monkeys. We investigated whether capuchins displayed significant hand preferences for a tool use task and whether such preferences were associated with motor-processing regions of the brain. Handedness data on a dipping tool-use task and high-resolution 3T MRI scans were collected from 15 monkeys. Capuchins displayed a significant group-level left-hand preference for this type of tool use, and handedness was associated with asymmetry of the primary motor cortex. Left-hand preferent individuals displayed a deeper central sulcus in the right hemisphere. Our results suggest that capuchins show an underlying right-hemisphere bias for skilled movement. PMID:22987442

  19. Nogo receptor 1 is expressed in both primary cultured glial cells and neurons

    PubMed Central

    Ukai, Junichi; Imagama, Shiro; Ohgomori, Tomohiro; Ito, Zenya; Ando, Kei; Ishiguro, Naoki; Kadomatsu, Kenji

    2016-01-01

    ABSTRACT Nogo receptor (NgR) is common in myelin-derived molecules, i.e., Nogo, MAG, and OMgp, and plays important roles in both axon fasciculation and the inhibition of axonal regeneration. In contrast to NgR’s roles in neurons, its roles in glial cells have been poorly explored. Here, we found a dynamic regulation of NgR1 expression during development and neuronal injury. NgR1 mRNA was consistently expressed in the brain from embryonic day 18 to postnatal day 25. In contrast, its expression significantly decreased in the spinal cord during development. Primary cultured neurons, microglia, and astrocytes expressed NgR1. Interestingly, a contusion injury in the spinal cord led to elevated NgR1 mRNA expression at the injury site, but not in the motor cortex, 14 days after injury. Consistent with this, astrocyte activation by TGFβ1 increased NgR1 expression, while microglia activation rather decreased NgR1 expression. These results collectively suggest that NgR1 expression is enhanced in a milieu of neural injury. Our findings may provide insight into the roles of NgR1 in glial cells. PMID:27578914

  20. Nogo receptor 1 is expressed in both primary cultured glial cells and neurons.

    PubMed

    Ukai, Junichi; Imagama, Shiro; Ohgomori, Tomohiro; Ito, Zenya; Ando, Kei; Ishiguro, Naoki; Kadomatsu, Kenji

    2016-08-01

    Nogo receptor (NgR) is common in myelin-derived molecules, i.e., Nogo, MAG, and OMgp, and plays important roles in both axon fasciculation and the inhibition of axonal regeneration. In contrast to NgR's roles in neurons, its roles in glial cells have been poorly explored. Here, we found a dynamic regulation of NgR1 expression during development and neuronal injury. NgR1 mRNA was consistently expressed in the brain from embryonic day 18 to postnatal day 25. In contrast, its expression significantly decreased in the spinal cord during development. Primary cultured neurons, microglia, and astrocytes expressed NgR1. Interestingly, a contusion injury in the spinal cord led to elevated NgR1 mRNA expression at the injury site, but not in the motor cortex, 14 days after injury. Consistent with this, astrocyte activation by TGFβ1 increased NgR1 expression, while microglia activation rather decreased NgR1 expression. These results collectively suggest that NgR1 expression is enhanced in a milieu of neural injury. Our findings may provide insight into the roles of NgR1 in glial cells. PMID:27578914

  1. Isolation and Transfection of Primary Culture Bovine Retinal Pericytes.

    PubMed

    Primo, Vincent A; Arboleda-Velasquez, Joseph F

    2016-01-01

    This protocol describes an enzymatic approach for isolating homogeneous cultures of pericytes from retinas of bovine source. In summary, retinas are dissected, washed, digested, filtered, cultured in specific media to select for pericytes, and finally expanded for a low passage culture of about 14 million bovine retinal pericytes (BRP) within 4-6 weeks. This protocol also describes a liposomal-based technique for transfection of BRPs. PMID:27172949

  2. Effect of Organizational Culture on Patient Access, Care Continuity, and Experience of Primary Care.

    PubMed

    Hung, Dorothy; Chung, Sukyung; Martinez, Meghan; Tai-Seale, Ming

    2016-01-01

    This study examined relationships between organizational culture and patient-centered outcomes in primary care. Generalized least squares regression was used to analyze patient access, care continuity, and reported experiences of care among 357 physicians in 41 primary care departments. Compared with a "Group-oriented" culture, a "Rational" culture type was associated with longer appointment wait times, and both "Hierarchical" and "Developmental" culture types were associated with less care continuity, but better patient experiences with care. Understanding the unique effects of organizational culture can enhance the delivery of more patient-centered care.

  3. Effect of Organizational Culture on Patient Access, Care Continuity, and Experience of Primary Care.

    PubMed

    Hung, Dorothy; Chung, Sukyung; Martinez, Meghan; Tai-Seale, Ming

    2016-01-01

    This study examined relationships between organizational culture and patient-centered outcomes in primary care. Generalized least squares regression was used to analyze patient access, care continuity, and reported experiences of care among 357 physicians in 41 primary care departments. Compared with a "Group-oriented" culture, a "Rational" culture type was associated with longer appointment wait times, and both "Hierarchical" and "Developmental" culture types were associated with less care continuity, but better patient experiences with care. Understanding the unique effects of organizational culture can enhance the delivery of more patient-centered care. PMID:27232685

  4. Determinants of cardiomyocyte development in long-term primary culture.

    PubMed

    Piper, H M; Jacobson, S L; Schwartz, P

    1988-09-01

    The influence of cell attachment to substrates and of medium composition on development of cardiomyocytes from adult rats in cultures up to 9 days old was investigated. Cardiomyocytes prevented from attaching to a culture substratum deteriorated within 3 days in medium 199 (M199) with or without fetal calf serum (FCS). Rapid attachment during the first 4 h after plating could be attained equally well on FCS or laminin coated surfaces. In M199 without FCS, attached cardiomyocytes on FCS coated dishes were able to retain their overall elongated morphology, but the number of cells remaining attached constantly decreased during the first 9 days in serum free culture. Attached on laminin the rate of loss from serum free cultures was lower. In the presence of 20% FCS, attached cardiomyocytes spread extensively after day 3, both on FCS and on laminin coated dishes. In serum containing media many cells pass through a spherical intermediate state before spreading extensively. Almost all cardiomyocytes cultured with 20% FCS on untreated tissue culture plastic gradually become spherical before attaching. With 20% FCS in culture media, the number of cells remaining in culture after 9 days was similar whether cells were rapidly attached to FCS treated or laminin coated substrata, or were plated on culture plastic, i.e., 52, 63, and 45% of the maximal number attached on day 1. By day 9 in all three culture types cells were spread and were beating spontaneously. These results indicate that adult cardiomyocytes do not establish in a stable morphological state in long-term cultures, in other than a surface attached spread cell form. For this stability the presence of yet unidentified components of fetal calf serum is required. PMID:3230587

  5. Establishment of Asian citrus psllid (Diaphorina citri) primary cultures

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A new cell line was developed from the Asian citrus psyllid (AsCP), Diaphorina citri (Hemiptera: Psyllidae), as a novel approach to culture the bacteria associated with huanglongbing disease (HLB), also known as citrus greening disease. Methods to culture the phloem-inhabiting bacterium Candidatus L...

  6. Culturally Responsive Dance Pedagogy in the Primary Classroom

    ERIC Educational Resources Information Center

    Melchior, Elizabeth

    2011-01-01

    Dance has an important place in multicultural education and the development of culturally responsive pedagogy. Through dance, children can explore and express their own and others' cultures and share their stories in ways other than the spoken and written word. This paper presents a case study concerning a professional development programme in…

  7. [Hygienic assessment of organization of motor activity in primary class pupils of full-day schools].

    PubMed

    Khramtsov, P I; Bakanov, I M

    2009-01-01

    The impact of a routine with the traditional organization of motor activity, a more extensive motor regimen with additional lessons of physical training, eurhythmics, and swimming, as well as a motor regimen of prophylactic and health-improving orientation of the Health School, which is at the most integrated into an educational process, on exercise performance, lung capacity, carpal muscle strength, physical fitness, and nonspecific resistance was studied in 156 first-to-second-form pupils at a two-year follow-up. The traditional motor regimen was found to fail to significantly increase functional parameters. Higher increment rates of the study parameters were observed with the extensive motor regimen. The Health School motor regimen providing a uniform motor activity distribution in the first and second half of a day and a predominance of a dynamic component over a statistical one was also favorable to the maintenance of increment rates of the parameters at 2 years of the follow-up, which implies the developing nature of this regimen. Hygienic recommendations to optimize the traditional motor regimen were worked out for full-day school pupils.

  8. Sensory-Motor Interactions for Vocal Pitch Monitoring in Non-Primary Human Auditory Cortex

    PubMed Central

    Larson, Charles R.; Jackson, Adam W.; Chen, Fangxiang; Hansen, Daniel R.; Oya, Hiroyuki; Kawasaki, Hiroto; Howard, Matthew A.

    2013-01-01

    The neural mechanisms underlying processing of auditory feedback during self-vocalization are poorly understood. One technique used to study the role of auditory feedback involves shifting the pitch of the feedback that a speaker receives, known as pitch-shifted feedback. We utilized a pitch shift self-vocalization and playback paradigm to investigate the underlying neural mechanisms of audio-vocal interaction. High-resolution electrocorticography (ECoG) signals were recorded directly from auditory cortex of 10 human subjects while they vocalized and received brief downward (−100 cents) pitch perturbations in their voice auditory feedback (speaking task). ECoG was also recorded when subjects passively listened to playback of their own pitch-shifted vocalizations. Feedback pitch perturbations elicited average evoked potential (AEP) and event-related band power (ERBP) responses, primarily in the high gamma (70–150 Hz) range, in focal areas of non-primary auditory cortex on superior temporal gyrus (STG). The AEPs and high gamma responses were both modulated by speaking compared with playback in a subset of STG contacts. From these contacts, a majority showed significant enhancement of high gamma power and AEP responses during speaking while the remaining contacts showed attenuated response amplitudes. The speaking-induced enhancement effect suggests that engaging the vocal motor system can modulate auditory cortical processing of self-produced sounds in such a way as to increase neural sensitivity for feedback pitch error detection. It is likely that mechanisms such as efference copies may be involved in this process, and modulation of AEP and high gamma responses imply that such modulatory effects may affect different cortical generators within distinctive functional networks that drive voice production and control. PMID:23577157

  9. Cultural democracy: the way forward for primary care of hard to reach New Zealanders.

    PubMed

    Finau, Sitaleki A; Finau, Eseta

    2007-09-01

    The use of cultural democracy, the freedom to practice one's culture without fear, as a framework for primary care service provision is essential for improved health service in a multi cultural society like New Zealand. It is an effective approach to attaining health equity for all. Many successful health ventures are ethnic specific and have gone past cultural competency to the practice of cultural democracy. That is, the services are freely taking on the realities of clients without and malice from those of other ethnicities. In New Zealand the scientific health service to improve the health of a multi cultural society are available but there is a need to improve access and utilization by hard to reach New Zealanders. This paper discusses cultural democracy and provide example of how successful health ventures that had embraced cultural democracy were implemented. It suggests that cultural democracy will provide the intellectual impetus and robust philosophy for moving from equality to equity in health service access and utilization. This paper would provide a way forward to improved primary care utilization, efficiency, effectiveness and equitable access especially for the hard to reach populations. use the realities of Pacificans in New Zealand illustrate the use of cultural democracy, and thus equity to address the "inverse care law" of New Zealand. The desire is for primary care providers to take cognizance and use cultural democracy and equity as the basis for the design and practice of primary health care for the hard to reach New Zealanders.

  10. Synaptogenesis of hippocampal neurons in primary cell culture.

    PubMed

    Grabrucker, Andreas; Vaida, Bianca; Bockmann, Jürgen; Boeckers, Tobias M

    2009-12-01

    Hippocampal neurons in dissociated cell culture are one of the most extensively used model systems in the field of molecular and cellular neurobiology. Only limited data are however available on the normal time frame of synaptogenesis, synapse number and ultrastructure of excitatory synapses during early development in culture. Therefore, we analyzed the synaptic ultrastructure and morphology and the localization of presynaptic (Bassoon) and postsynaptic (ProSAP1/Shank2) marker proteins in cultures established from rat embryos at embryonic day 19, after 3, 7, 10, 14, and 21 days in culture. First excitatory synapses were identified at day 7 with a clearly defined postsynaptic density and presynaptically localized synaptic vesicles. Mature synapses on dendritic spines were seen from day 10 onward, and the number of synapses steeply increased in the third week. Fenestrated or multiple synapses were found after 14 or 21 days, respectively. So-called dense-core vesicles, responsible for the transport of proteins to the active zone of the presynaptic specialization, were seen on cultivation day 3 and 7 and could be detected in axons and especially in the presynaptic subcompartments. The expression and localization of the presynaptic protein Bassoon and of the postsynaptic molecule ProSAP1/Shank2 was found to correlate nicely with the ultrastructural results. This regular pattern of development and maturation of excitatory synapses in hippocampal culture starting from day 7 in culture should ease the comparison of synapse number and morphology of synaptic contacts in this widely used model system.

  11. The preparation of primary hematopoietic cell cultures from murine bone marrow for electroporation.

    PubMed

    Kroeger, Kelly; Collins, Michelle; Ugozzoli, Luis

    2009-01-01

    It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed. PMID:19229174

  12. Complex Organization of Human Primary Motor Cortex: A High-Resolution fMRI Study

    PubMed Central

    Meier, Jeffrey D.; Aflalo, Tyson N.; Kastner, Sabine; Graziano, Michael S. A.

    2008-01-01

    A traditional view of the human motor cortex is that it contains an overlapping sequence of body part representations from the tongue in a ventral location to the foot in a dorsal location. In this study, high-resolution functional MRI (1.5 × 1.5 × 2 mm) was used to examine the somatotopic map in the lateral motor cortex of humans, to determine whether it followed the traditional somatotopic order or whether it contained any violations of that somatotopic order. The arm and hand representation had a complex organization in which the arm was relatively emphasized in two areas: one dorsal and the other ventral to a region that emphasized the fingers. This violation of a traditional somatotopic order suggests that the motor cortex is not merely a map of the body but is topographically shaped by other influences, perhaps including correlations in the use of body parts in the motor repertoire. PMID:18684903

  13. Intravenous immunoglobulin treatment preserves and protects primary rat hippocampal neurons and primary human brain cultures against oxidative insults.

    PubMed

    Lahiri, Debomoy K; Ray, Balmiki

    2014-01-01

    Alzheimer's disease (AD) is characterized by deleterious accumulation of amyloid-β (Aβ) peptide into senile plaque, neurofibrillary tangles formed from hyperphosphorylated tau protein, and loss of cholinergic synapses in the cerebral cortex. The deposition of Aβ-loaded plaques results in microglial activation and subsequent production of reactive oxygen species (ROS), including free radicals. Neurons in aging and AD brains are particularly vulnerable to ROS and other toxic stimuli. Therefore, agents that decrease the vulnerability of neurons against ROS may provide therapeutic values for the treatment or prevention of AD. In the present study, our goal was to test whether intravenous immunoglobulin (IVIG) treatment could preserve as well as protect neurons from oxidative damage. We report that treatment with IVIG protects neuronal viability and synaptic proteins in primary rat hippocampal neurons. Further, we demonstrate the tolerability of IVIG treatment in the primary human fetal mixed brain cultures. Indeed, a high dose (20 mg/ml) of IVIG treatment was well-tolerated by primary human brain cultures that exhibit a normal neuronal phenotype. We also observed a potent neuropreservatory effect of IVIG against ROS-mediated oxidative insults in these human fetal brain cultures. These results indicate that IVIG treatment has great potential to preserve and protect primary human neuronal-enriched cultures and to potentially rescue dying neurons from oxidative insults. Therefore, our findings suggest that IVIG treatment may represent an important therapeutic agent for clinical trials designed to prevent and delay the onset of neurodegeneration as well as AD pathology. PMID:25115544

  14. Characterization of motor units in behaving adult mice shows a wide primary range.

    PubMed

    Ritter, Laura K; Tresch, Matthew C; Heckman, C J; Manuel, Marin; Tysseling, Vicki M

    2014-08-01

    The mouse is essential for genetic studies of motor function in both normal and pathological states. Thus it is important to consider whether the structure of motor output from the mouse is in fact analogous to that recorded in other animals. There is a striking difference in the basic electrical properties of mouse motoneurons compared with those in rats, cats, and humans. The firing evoked by injected currents produces a unique frequency-current (F-I) function that emphasizes recruitment of motor units at their maximum force. These F-I functions, however, were measured in anesthetized preparations that lacked two key components of normal synaptic input: high levels of synaptic noise and neuromodulatory inputs. Recent studies suggest that the alterations in the F-I function due to these two components are essential for recreating firing behavior of motor units in human subjects. In this study we provide the first data on firing patterns of motor units in the awake mouse, focusing on steady output in quiet stance. The resulting firing patterns did not match the predictions from the mouse F-I behaviors but instead revealed rate modulation across a remarkably wide range (10-60 Hz). The low end of the firing range may be due to changes in the F-I relation induced by synaptic noise and neuromodulatory inputs. The high end of the range may indicate that, unlike other species, quiet standing in the mouse involves recruitment of relatively fast-twitch motor units. PMID:24805075

  15. Role of primary afferents in the developmental regulation of motor axon synapse numbers on Renshaw cells.

    PubMed

    Siembab, Valerie C; Gomez-Perez, Laura; Rotterman, Travis M; Shneider, Neil A; Alvarez, Francisco J

    2016-06-15

    Motor function in mammalian species depends on the maturation of spinal circuits formed by a large variety of interneurons that regulate motoneuron firing and motor output. Interneuron activity is in turn modulated by the organization of their synaptic inputs, but the principles governing the development of specific synaptic architectures unique to each premotor interneuron are unknown. For example, Renshaw cells receive, at least in the neonate, convergent inputs from sensory afferents (likely Ia) and motor axons, raising the question of whether they interact during Renshaw cell development. In other well-studied neurons, such as Purkinje cells, heterosynaptic competition between inputs from different sources shapes synaptic organization. To examine the possibility that sensory afferents modulate synaptic maturation on developing Renshaw cells, we used three animal models in which afferent inputs in the ventral horn are dramatically reduced (ER81(-/-) knockout), weakened (Egr3(-/-) knockout), or strengthened (mlcNT3(+/-) transgenic). We demonstrate that increasing the strength of sensory inputs on Renshaw cells prevents their deselection and reduces motor axon synaptic density, and, in contrast, absent or diminished sensory afferent inputs correlate with increased densities of motor axons synapses. No effects were observed on other glutamatergic inputs. We conclude that the early strength of Ia synapses influences their maintenance or weakening during later development and that heterosynaptic influences from sensory synapses during early development regulates the density and organization of motor inputs on mature Renshaw cells. PMID:26660356

  16. Vitamin D and Caudal Primary Motor Cortex: A Magnetic Resonance Spectroscopy Study

    PubMed Central

    Annweiler, Cedric; Beauchet, Olivier; Bartha, Robert; Hachinski, Vladimir; Montero-Odasso, Manuel

    2014-01-01

    Background Vitamin D is involved in brain physiology and lower-extremity function. We investigated spectroscopy in a cohort of older adults to explore the hypothesis that lower vitamin D status was associated with impaired neuronal function in caudal primary motor cortex (cPMC) measured by proton magnetic resonance spectroscopic imaging. Methods Twenty Caucasian community-dwellers (mean±standard deviation, 74.6±6.2 years; 35.0% female) from the ‘Gait and Brain Study’ were included in this analysis. Ratio of N-acetyl-aspartate to creatine (NAA/Cr), a marker of neuronal function, was calculated in cPMC. Participants were categorized according to mean NAA/Cr. Lower vitamin D status was defined as serum 25-hydroxyvitamin D (25OHD) concentration <75 nmol/L. Age, gender, number of comorbidities, vascular risk, cognition, gait performance, vitamin D supplements, undernourishment, cPMC thickness, white matter hyperintensities grade, serum parathyroid hormone concentration, and season of evaluation were used as potential confounders. Results Compared to participants with high NAA/Cr (n = 11), those with low NAA/Cr (i.e., reduced neuronal function) had lower serum 25OHD concentration (P = 0.044) and more frequently lower vitamin D status (P = 0.038). Lower vitamin D status was cross-sectionally associated with a decrease in NAA/Cr after adjustment for clinical characteristics (β = −0.41, P = 0.047), neuroimaging measures (β = −0.47, P = 0.032) and serum measures (β = −0.45, P = 0.046). Conclusions Lower vitamin D status was associated with reduced neuronal function in cPMC. These novel findings need to be replicated in larger and preferably longitudinal cohorts. They contribute to explain the pathophysiology of gait disorders in older adults with lower vitamin D status, and provide a scientific base for vitamin D replacement trials. PMID:24498072

  17. Competition with Primary Sensory Afferents Drives Remodeling of Corticospinal Axons in Mature Spinal Motor Circuits

    PubMed Central

    Jiang, Yu-Qiu; Zaaimi, Boubker

    2016-01-01

    Injury to the mature motor system drives significant spontaneous axonal sprouting instead of axon regeneration. Knowing the circuit-level determinants of axonal sprouting is important for repairing motor circuits after injury to achieve functional rehabilitation. Competitive interactions are known to shape corticospinal tract axon outgrowth and withdrawal during development. Whether and how competition contributes to reorganization of mature spinal motor circuits is unclear. To study this question, we examined plastic changes in corticospinal axons in response to two complementary proprioceptive afferent manipulations: (1) enhancing proprioceptive afferents activity by electrical stimulation; or (2) diminishing their input by dorsal rootlet rhizotomy. Experiments were conducted in adult rats. Electrical stimulation produced proprioceptive afferent sprouting that was accompanied by significant corticospinal axon withdrawal and a decrease in corticospinal connections on cholinergic interneurons in the medial intermediate zone and C boutons on motoneurons. In contrast, dorsal rootlet rhizotomy led to a significant increase in corticospinal connections, including those on cholinergic interneurons; C bouton density increased correspondingly. Motor cortex-evoked muscle potentials showed parallel changes to those of corticospinal axons, suggesting that reciprocal corticospinal axon changes are functional. Using the two complementary models, we showed that competitive interactions between proprioceptive and corticospinal axons are an important determinant in the organization of mature corticospinal axons and spinal motor circuits. The activity- and synaptic space-dependent properties of the competition enables prediction of the remodeling of spared corticospinal connection and spinal motor circuits after injury and informs the target-specific control of corticospinal connections to promote functional recovery. SIGNIFICANCE STATEMENT Neuroplasticity is limited in maturity

  18. Pyrethroid insecticide accumulation in primary cultures of cortical neurons in vitro

    EPA Science Inventory

    Primary cultures of neurons have been widely utilized to study the actions of pyrethroids and other neurotoxicants, with the presumption that the media concentration accurately reflects the dose received by the cells. However, recent studies have demonstrated that lipophilic comp...

  19. Correction: Understanding metal homeostasis in primary cultured neurons. Studies using single neuron subcellular and quantitative metallomics.

    PubMed

    Colvin, Robert A; Lai, Barry; Holmes, William R; Lee, Daewoo

    2015-09-01

    Correction for 'Understanding metal homeostasis in primary cultured neurons. Studies using single neuron subcellular and quantitative metallomics' by Robert A. Colvin et al., Metallomics, 2015, 7, 1111-1123.

  20. Multisynaptic projections from the ventrolateral prefrontal cortex to hand and mouth representations of the monkey primary motor cortex.

    PubMed

    Miyachi, Shigehiro; Hirata, Yoshihiro; Inoue, Ken-ichi; Lu, Xiaofeng; Nambu, Atsushi; Takada, Masahiko

    2013-07-01

    Different sectors of the prefrontal cortex have distinct neuronal connections with higher-order sensory areas and/or limbic structures and are related to diverse aspects of cognitive functions, such as visual working memory and reward-based decision-making. Recent studies have revealed that the prefrontal cortex (PF), especially the lateral PF, is also involved in motor control. Hence, different sectors of the PF may contribute to motor behaviors with distinct body parts. To test this hypothesis anatomically, we examined the patterns of multisynaptic projections from the PF to regions of the primary motor cortex (MI) that represent the arm, hand, and mouth, using retrograde transsynaptic transport of rabies virus. Four days after rabies injections into the hand or mouth region, particularly dense neuron labeling was observed in the ventrolateral PF, including the convexity part of ventral area 46. After the rabies injections into the mouth region, another dense cluster of labeled neurons was seen in the orbitofrontal cortex (area 13). By contrast, rabies labeling of PF neurons was rather sparse in the arm-injection cases. The present results suggest that the PF-MI multisynaptic projections may be organized such that the MI hand and mouth regions preferentially receive cognitive information for execution of elaborate motor actions. PMID:23664864

  1. Isolation, culture and characterization of primary mouse RPE cells.

    PubMed

    Fernandez-Godino, Rosario; Garland, Donita L; Pierce, Eric A

    2016-07-01

    Mouse models are powerful tools for the study of ocular diseases. Alterations in the morphology and function of the retinal pigment epithelium (RPE) are common features shared by many ocular disorders. We report a detailed protocol to collect, seed, culture and characterize RPE cells from mice. We describe a reproducible method that we previously developed to collect and culture murine RPE cells on Transwells as functional polarized monolayers. The collection of RPE cells takes ∼3 h, and the cultures mimic in vivo RPE cell features within 1 week. This protocol also describes methods to characterize the cells on Transwells within 1-2 weeks by transmission and scanning electron microscopy (TEM and SEM, respectively), immunostaining of vibratome sections and flat mounts, and measurement of transepithelial electrical resistance. The RPE cell cultures are suitable to study the biology of the RPE from wild-type and genetically modified strains of mice between the ages of 10 d and 12 months. The RPE cells can also be manipulated to investigate molecular mechanisms underlying the RPE pathology in the numerous mouse models of ocular disorders. Furthermore, modeling the RPE pathology in vitro represents a new approach to testing drugs that will help accelerate the development of therapies for vision-threatening disorders such as macular degeneration (MD). PMID:27281648

  2. Effects of wound dressings on cultured primary keratinocytes.

    PubMed

    Esteban-Vives, Roger; Young, Matthew T; Ziembicki, Jenny; Corcos, Alain; Gerlach, Jörg C

    2016-02-01

    Autologous cell-spray grafting of non-cultured epidermal cells is an innovative approach for the treatment of severe second-degree burns. After treatment, wounds are covered with dressings that are widely used in wound care management; however, little is known about the effects of wound dressings on individually isolated cells. The sprayed cells have to actively attach, spread, proliferate, and migrate in the wound for successful re-epithelialization, during the healing process. It is expected that exposure to wound dressing material might interfere with cell survival, attachment, and expansion. Two experiments were performed to determine whether some dressing materials have a negative impact during the early phases of wound healing. In one experiment, freshly isolated cells were seeded and cultured for one week in combination with eight different wound dressings used during burn care. Cells, which were seeded and cultured with samples of Adaptic(®), Xeroform(®), EZ Derm(®), and Mepilex(®) did not attach, nor did they survive during the first week. Mepitel(®), N-Terface(®), Polyskin(®), and Biobrane(®) dressing samples had no negative effect on cell attachment and cell growth when compared to the controls. In a second experiment, the same dressings were exposed to pre-cultured cells in order to exclude the effects of attachment and spreading. The results confirm the above findings. This study could be of interest for establishing skin cell grafting therapies in burn medicine and also for wound care in general.

  3. Homophobia, Transphobia and Culture: Deconstructing Heteronormativity in English Primary Schools

    ERIC Educational Resources Information Center

    DePalma, Renee; Jennett, Mark

    2010-01-01

    This article presents some of the advances in legal support for addressing homophobia and transphobia in school settings and provides a critique of school-based policies that focus on these phenomena as particular incidents involving bullies and victims. Defining heteronormativity as a cultural phenomenon underpinning recognisable acts of…

  4. Polygonal networks, "geodomes", of adult rat hepatocytes in primary culture.

    PubMed

    Mochizuki, Y; Furukawa, K; Mitaka, T; Yokoi, T; Kodama, T

    1988-01-01

    Polygonal networks, "geodomes", in cultured hepatocytes of adult rats were examined by both light and electron microscopy. On light microscopical examinations of specimens stained with Coomassie blue after the treatment with Triton X-100, the networks were detected 5 days after culture, which consisted of triangles arranged mainly in hexagonal patterns. They surrounded main cell body, looking like a headband, or were occasionally situated over nuclei, looking like a geodesic dome. Scanning electron microscopical observations after Triton treatment revealed that these structures were located underneath surface membrane. Transmission electron microscopical investigations revealed that the connecting fibers of networks consisted of microfilaments which radiated in a compact bundle from electron-dense vertices. PMID:3396075

  5. Effects of wound dressings on cultured primary keratinocytes.

    PubMed

    Esteban-Vives, Roger; Young, Matthew T; Ziembicki, Jenny; Corcos, Alain; Gerlach, Jörg C

    2016-02-01

    Autologous cell-spray grafting of non-cultured epidermal cells is an innovative approach for the treatment of severe second-degree burns. After treatment, wounds are covered with dressings that are widely used in wound care management; however, little is known about the effects of wound dressings on individually isolated cells. The sprayed cells have to actively attach, spread, proliferate, and migrate in the wound for successful re-epithelialization, during the healing process. It is expected that exposure to wound dressing material might interfere with cell survival, attachment, and expansion. Two experiments were performed to determine whether some dressing materials have a negative impact during the early phases of wound healing. In one experiment, freshly isolated cells were seeded and cultured for one week in combination with eight different wound dressings used during burn care. Cells, which were seeded and cultured with samples of Adaptic(®), Xeroform(®), EZ Derm(®), and Mepilex(®) did not attach, nor did they survive during the first week. Mepitel(®), N-Terface(®), Polyskin(®), and Biobrane(®) dressing samples had no negative effect on cell attachment and cell growth when compared to the controls. In a second experiment, the same dressings were exposed to pre-cultured cells in order to exclude the effects of attachment and spreading. The results confirm the above findings. This study could be of interest for establishing skin cell grafting therapies in burn medicine and also for wound care in general. PMID:26678326

  6. Creative Partnerships? Cultural Policy and Inclusive Arts Practice in One Primary School

    ERIC Educational Resources Information Center

    Hall, Christine; Thomson, Pat

    2007-01-01

    This article traces the "cultural turn" in UK educational policy through an analysis of the Creative Partnerships policy (New Labour's "flagship programme in the cultural education field") and a consideration of an arts project funded under this initiative in one primary school. It argues that current educational policy foregrounds the economic…

  7. English and French Pedagogical Cultures: Convergence and Divergence in Cameroonian Primary School Teachers' Discourse

    ERIC Educational Resources Information Center

    Esch, Edith

    2012-01-01

    This article approaches the phenomenon of the continuing influence of French and English pedagogical cultures in Africa relying on post-modern notions of time and space. It reports on a project carried out in Cameroon where both cultures are in contact and where the teachers from two primary schools were observed and interviewed over a period of…

  8. A qualitative study of the cultural changes in primary care organisations needed to implement clinical governance.

    PubMed Central

    Marshall, Martin; Sheaff, Rod; Rogers, Anne; Campbell, Stephen; Halliwell, Shirley; Pickard, Susan; Sibbald, Bonnie; Roland, Martin

    2002-01-01

    BACKGROUND: It is commony claimed that changing the culture of health organisations is a fundamental prerequisite for improving the National Health Service (NHS). Little is currently known about the nature or importance of culture and cultural change in primary care groups and trusts (PCG/Ts) or their constituent general practices. AIMS: To investigate the importance of culture and cultural change for the implementation of clinical governance in general practice by PCG/Ts, to identify perceived desirable and undesirable cultural attributes of general practice, and to describe potential facilitators and barriers to changing culture. DESIGN: Qualitative: case studies using data derived from semi-structured interviews and review of documentary evidence. SETTING: Fifty senior non-clinical and clinical managers from 12 purposely sampled PCGs or trusts in England. RESULTS: Senior primary care managers regard culture and cultural change as fundamental aspects of clinical governance. The most important desirable cultural traits were the value placed on a commitment to public accountability by the practices, their willingness to work together and learn from each other, and the ability to be self-critical and learn from mistakes. The main barriers to cultural change were the high level of autonomy of practices and the perceived pressure to deliver rapid measurable changes in general practice. CONCLUSIONS: The culture of general practice is perceived to be an important component of health system reform and quality improvement. This study develops our understanding of a changing organisational culture in primary care; however, further work is required to determine whether culture is a useful practical lever for initiating or managing improvement. PMID:12171222

  9. Meaningful Cultural Learning by Imitative Participation: The Case of Abstract Thinking in Primary School

    ERIC Educational Resources Information Center

    van Oers, Bert

    2012-01-01

    The article describes a theory-driven approach to meaningful learning in primary schools, based on the Vygotskian cultural-historical theory of human development and learning. This approach is elaborated into an educational concept called "developmental education" that is implemented in the Netherlands in many primary schools. In this approach,…

  10. Exploring the Effects of Classroom Culture on Primary Pre-Service Teachers' Professional Development

    ERIC Educational Resources Information Center

    Altun, Taner

    2013-01-01

    This study aims to examine primary student teachers' (PSTs) perceptions about the effects of pre-formed classroom culture on their professional development. In the study, a mixed method approach was used. The study group consisted of 4th year student teachers who attend a primary teacher education program leading to a B.Ed. degree at the…

  11. Turkish Primary School Teachers' Perceptions of School Culture Regarding ICT Integration

    ERIC Educational Resources Information Center

    Tezci, Erdogan

    2011-01-01

    The current study aimed at identifying Turkish primary school teachers' perceptions of school culture regarding ICT integration in education. In addition, the current study was designed to investigate factors that might influence their perceptions. The participants were 1540 primary school teachers. The findings revealed that the teachers'…

  12. The Effect of Organizational Trust on the Culture of Teacher Leadership in Primary Schools

    ERIC Educational Resources Information Center

    Demir, Kamile

    2015-01-01

    The purpose of this research is to examine the effect of the level of trust of primary school teachers towards their organization in relation to their perceptions of the school having a culture of teacher leadership. Participants of the study consisted of 378 teachers working in Burdur public primary schools. The data collection tool used two…

  13. The acquisition of skilled motor performance: Fast and slow experience-driven changes in primary motor cortex

    PubMed Central

    Karni, Avi; Meyer, Gundela; Rey-Hipolito, Christine; Jezzard, Peter; Adams, Michelle M.; Turner, Robert; Ungerleider, Leslie G.

    1998-01-01

    Behavioral and neurophysiological studies suggest that skill learning can be mediated by discrete, experience-driven changes within specific neural representations subserving the performance of the trained task. We have shown that a few minutes of daily practice on a sequential finger opposition task induced large, incremental performance gains over a few weeks of training. These gains did not generalize to the contralateral hand nor to a matched sequence of identical component movements, suggesting that a lateralized representation of the learned sequence of movements evolved through practice. This interpretation was supported by functional MRI data showing that a more extensive representation of the trained sequence emerged in primary motor cortex after 3 weeks of training. The imaging data, however, also indicated important changes occurring in primary motor cortex during the initial scanning sessions, which we proposed may reflect the setting up of a task-specific motor processing routine. Here we provide behavioral and functional MRI data on experience-dependent changes induced by a limited amount of repetitions within the first imaging session. We show that this limited training experience can be sufficient to trigger performance gains that require time to become evident. We propose that skilled motor performance is acquired in several stages: “fast” learning, an initial, within-session improvement phase, followed by a period of consolidation of several hours duration, and then “slow” learning, consisting of delayed, incremental gains in performance emerging after continued practice. This time course may reflect basic mechanisms of neuronal plasticity in the adult brain that subserve the acquisition and retention of many different skills. PMID:9448252

  14. Dual-hemisphere transcranial direct current stimulation over primary motor cortex enhances consolidation of a ballistic thumb movement.

    PubMed

    Koyama, Soichiro; Tanaka, Satoshi; Tanabe, Shigeo; Sadato, Norihiro

    2015-02-19

    Transcranial direct current stimulation (tDCS) is a noninvasive technique that modulates motor performance and learning. Previous studies have shown that tDCS over the primary motor cortex (M1) can facilitate consolidation of various motor skills. However, the effect of tDCS on consolidation of newly learned ballistic movements remains unknown. The present study tested the hypothesis that tDCS over M1 enhances consolidation of ballistic thumb movements in healthy adults. Twenty-eight healthy subjects participated in an experiment with a single-blind, sham-controlled, between-group design. Fourteen subjects practiced a ballistic movement with their left thumb during dual-hemisphere tDCS. Subjects received 1mA anodal tDCS over the contralateral M1 and 1mA cathodal tDCS over the ipsilateral M1 for 25min during the training session. The remaining 14 subjects underwent identical training sessions, except that dual-hemisphere tDCS was applied for only the first 15s (sham group). All subjects performed the task again at 1h and 24h later. Primary measurements examined improvement in peak acceleration of the ballistic thumb movement at 1h and 24h after stimulation. Improved peak acceleration was significantly greater in the tDCS group (144.2±15.1%) than in the sham group (98.7±9.1%) (P<0.05) at 24h, but not 1h, after stimulation. Thus, dual-hemisphere tDCS over M1 enhanced consolidation of ballistic thumb movement in healthy adults. Dual-hemisphere tDCS over M1 may be useful to improve elemental motor behaviors, such as ballistic movements, in patients with subcortical strokes.

  15. Ganoderma Lucidum polysaccharides protect against MPP+ and rotenone-induced apoptosis in primary dopaminergic cell cultures through inhibiting oxidative stress

    PubMed Central

    Guo, Shan-Shan; Cui, Xiao-Lan; Rausch, Wolf-Dieter

    2016-01-01

    Oxidative stress plays a pivotal role in the progressive neurodegeneration in Parkinson’s disease (PD) which is responsible for disabling motor abnormalities in more than 6.5 million people worldwide. Polysaccharides are the main active constituents from Ganoderma lucidum which is characterized with anti-oxidant, antitumor and immunostimulant properties. In the present study, primary dopaminergic cell cultures prepared from embryonic mouse mesencephala were used to investigate the neuroprotective effects and the potential mechanisms of Ganoderma lucidum polysaccharides (GLP) on the degeneration of dopaminergic neurons induced by the neurotoxins methyl-4-phenylpyridine (MPP+) and rotenone. Results revealed that GLP can protect dopamine neurons against MPP+ and rotenone at the concentrations of 100, 50 and 25 μg/ml in primary mesencephalic cultures in a dose-dependent manner. Interestingly, either with or without neurotoxin treatment, GLP treatment elevated the survival of THir neurons, and increased the length of neurites of dopaminergic neurons. The Trolox equivalent anti-oxidant capacity (TEAC) of GLP was determined to be 199.53 μmol Trolox/g extract, and the decrease of mitochondrial complex I activity induced by MPP+ and rotenone was elevated by GLP treatment (100, 50, 25 and 12.5 μg/ml) in a dose dependent manner. Furthermore, GLP dramatically decreased the relative number of apoptotic cells and increased the declining mitochondrial membrane potential (ΔΨm) induced by MPP+ and rotenone in a dose-dependent manner. In addition, GLP treatment reduced the ROS formation induced by MPP+ and rotenone at the concentrations of 100, 50 and 25 μg/ml in a dose-dependent manner. Our study indicates that GLP possesses neuroprotective properties against MPP+ and rotenone neurotoxicity through suppressing oxidative stress in primary mesencephalic dopaminergic cell culture owning to its antioxidant activities. PMID:27335703

  16. Ganoderma Lucidum polysaccharides protect against MPP(+) and rotenone-induced apoptosis in primary dopaminergic cell cultures through inhibiting oxidative stress.

    PubMed

    Guo, Shan-Shan; Cui, Xiao-Lan; Rausch, Wolf-Dieter

    2016-01-01

    Oxidative stress plays a pivotal role in the progressive neurodegeneration in Parkinson's disease (PD) which is responsible for disabling motor abnormalities in more than 6.5 million people worldwide. Polysaccharides are the main active constituents from Ganoderma lucidum which is characterized with anti-oxidant, antitumor and immunostimulant properties. In the present study, primary dopaminergic cell cultures prepared from embryonic mouse mesencephala were used to investigate the neuroprotective effects and the potential mechanisms of Ganoderma lucidum polysaccharides (GLP) on the degeneration of dopaminergic neurons induced by the neurotoxins methyl-4-phenylpyridine (MPP(+)) and rotenone. Results revealed that GLP can protect dopamine neurons against MPP(+) and rotenone at the concentrations of 100, 50 and 25 μg/ml in primary mesencephalic cultures in a dose-dependent manner. Interestingly, either with or without neurotoxin treatment, GLP treatment elevated the survival of THir neurons, and increased the length of neurites of dopaminergic neurons. The Trolox equivalent anti-oxidant capacity (TEAC) of GLP was determined to be 199.53 μmol Trolox/g extract, and the decrease of mitochondrial complex I activity induced by MPP(+) and rotenone was elevated by GLP treatment (100, 50, 25 and 12.5 μg/ml) in a dose dependent manner. Furthermore, GLP dramatically decreased the relative number of apoptotic cells and increased the declining mitochondrial membrane potential (ΔΨm) induced by MPP(+) and rotenone in a dose-dependent manner. In addition, GLP treatment reduced the ROS formation induced by MPP(+) and rotenone at the concentrations of 100, 50 and 25 μg/ml in a dose-dependent manner. Our study indicates that GLP possesses neuroprotective properties against MPP(+) and rotenone neurotoxicity through suppressing oxidative stress in primary mesencephalic dopaminergic cell culture owning to its antioxidant activities. PMID:27335703

  17. Ganoderma Lucidum polysaccharides protect against MPP(+) and rotenone-induced apoptosis in primary dopaminergic cell cultures through inhibiting oxidative stress.

    PubMed

    Guo, Shan-Shan; Cui, Xiao-Lan; Rausch, Wolf-Dieter

    2016-01-01

    Oxidative stress plays a pivotal role in the progressive neurodegeneration in Parkinson's disease (PD) which is responsible for disabling motor abnormalities in more than 6.5 million people worldwide. Polysaccharides are the main active constituents from Ganoderma lucidum which is characterized with anti-oxidant, antitumor and immunostimulant properties. In the present study, primary dopaminergic cell cultures prepared from embryonic mouse mesencephala were used to investigate the neuroprotective effects and the potential mechanisms of Ganoderma lucidum polysaccharides (GLP) on the degeneration of dopaminergic neurons induced by the neurotoxins methyl-4-phenylpyridine (MPP(+)) and rotenone. Results revealed that GLP can protect dopamine neurons against MPP(+) and rotenone at the concentrations of 100, 50 and 25 μg/ml in primary mesencephalic cultures in a dose-dependent manner. Interestingly, either with or without neurotoxin treatment, GLP treatment elevated the survival of THir neurons, and increased the length of neurites of dopaminergic neurons. The Trolox equivalent anti-oxidant capacity (TEAC) of GLP was determined to be 199.53 μmol Trolox/g extract, and the decrease of mitochondrial complex I activity induced by MPP(+) and rotenone was elevated by GLP treatment (100, 50, 25 and 12.5 μg/ml) in a dose dependent manner. Furthermore, GLP dramatically decreased the relative number of apoptotic cells and increased the declining mitochondrial membrane potential (ΔΨm) induced by MPP(+) and rotenone in a dose-dependent manner. In addition, GLP treatment reduced the ROS formation induced by MPP(+) and rotenone at the concentrations of 100, 50 and 25 μg/ml in a dose-dependent manner. Our study indicates that GLP possesses neuroprotective properties against MPP(+) and rotenone neurotoxicity through suppressing oxidative stress in primary mesencephalic dopaminergic cell culture owning to its antioxidant activities.

  18. Trout gill cells in primary culture on solid and permeable supports.

    PubMed

    Leguen, I; Cauty, C; Odjo, N; Corlu, A; Prunet, P

    2007-12-01

    Trout gill cells in primary culture on solid and permeable supports were compared. Cultures were carried out by directly seeding cells on each support after gill dissociation. Most of the cell types present in culture were similar, regardless of culture support (pavement cells, mucous cells (3-4%), but no mitochondria-rich cells). However, insertion of mucous cells in cultured epithelium on permeable support presented a morphology more similar to gills in situ. Gene expression of ion transporters and hormonal receptors indicated similar mRNA levels in both systems. Cortisol inhibited cell proliferation on both supports and maintained or increased the total cell number on solid and permeable membranes, respectively. This inhibition of mitosis associated with an increase or maintenance of total gill cells suggests that cortisol reduced cell degeneration. In the presence of cortisol, transepithelial resistance of cultured gill cells on permeable membranes was increased and maintained for a longer time in culture. In conclusion, gill cells in primary culture on permeable support present: (i) a morphology more similar to epithelium in situ; and (ii) specific responses to cortisol treatment. New findings and differences with previous studies on primary cultures of trout gill cells on permeable membrane are discussed.

  19. Organisation and function of the primary motor cortex in chronic pain: protocol for a systematic review and meta-analysis

    PubMed Central

    Chang, Wei-Ju; O'Connell, Neil E; Burns, Emma; Chipchase, Lucy S; Liston, Matthew B

    2015-01-01

    Introduction Primary motor cortical (M1) adaptation in the form of altered organisation and function is hypothesised to underpin motor dysfunction observed in chronic pain. The aim of this review is to assess the evidence for altered M1 organisation and function in chronic pain. Methods and analysis Systematic review and meta-analysis. We will search electronic databases with predetermined search terms to identify relevant studies and evaluate the studies for inclusion and risks of bias. Two independent reviewers will extract data. Any disagreement will be resolved through a third reviewer. Cross-sectional or prospective studies published in English before May 2015 that investigate M1 organisation and function in chronic pain will be included if they meet the eligibility criteria. Primary outcomes will include M1 cortical excitability, spatial cortical representation, the function of inhibitory and facilitatory intracortical networks, cortical reactivity and cortical glucose metabolism. Clinical measures such as pain and disability will be included where the correlation with the primary outcomes of M1 organisation and function were investigated in the included studies. Ethics and dissemination This systematic review does not require ethical approval. The results of this review will be submitted for peer-reviewed publication regardless of outcome and will be presented at relevant conferences. Trial registration number Our systematic review protocol was registered with the International Prospective Register of Systematic Reviews (PROSPERO; registration number CRD42015014823). PMID:26621512

  20. Prevalence of Persistent Primary Reflexes and Motor Problems in Children with Reading Difficulties

    ERIC Educational Resources Information Center

    McPhillips, M.; Sheehy, N.

    2004-01-01

    It has been shown that some children with reading difficulties have underlying developmental delay and that this may be related to the persistence of primary reflexes. This study investigated the prevalence of persistent primary reflexes in the ordinary primary school population and how this related to other cognitive and social factors. Three…

  1. A Microfluidic Interface for the Culture and Sampling of Adiponectin from Primary Adipocytes

    PubMed Central

    Godwin, Leah A.; Brooks, Jessica C.; Hoepfner, Lauren D.; Wanders, Desiree; Judd, Robert L.; Easley, Christopher J.

    2014-01-01

    Secreted from adipose tissue, adiponectin is a vital endocrine hormone that acts in glucose metabolism, thereby establishing its crucial role in diabetes, obesity, and other metabolic disease states. Insulin exposure to primary adipocytes cultured in static conditions has been shown to stimulate adiponectin secretion. However, conventional, static methodology for culturing and stimulating adipocytes falls short of truly mimicking physiological environments. Along with decreases in experimental costs and sample volume, and increased temporal resolution, microfluidic platforms permit small-volume flowing cell culture systems, which more accurately represent the constant flow conditions through vasculature in vivo. Here, we have integrated a customized primary tissue culture reservoir into a passively operated microfluidic device made of polydimethylsiloxane (PDMS). Fabrication of the reservoir was accomplished through unique PDMS “landscaping” above sampling channels, with a design strategy targeted to primary adipocytes to overcome issues of positive cell buoyancy. This reservoir allowed three-dimensional culture of primary murine adipocytes, accurate control over stimulants via constant perfusion, and sampling of adipokine secretion during various treatments. As the first report of primary adipocyte culture and sampling within microfluidic systems, this work sets the stage for future studies in adipokine secretion dynamics. PMID:25423362

  2. 10 CFR 830 Major Modification Determination for Replacement of ATR Primary Coolant Pumps and Motors

    SciTech Connect

    Noel Duckwitz

    2011-05-01

    The continued safe and reliable operation of the ATR is critical to the Department of Energy (DOE) Office of Nuclear Energy (NE) mission. While ATR is safely fulfilling current mission requirements, a variety of aging and obsolescence issues challenge ATR engineering and maintenance personnel’s capability to sustain ATR over the long term. First documented in a series of independent assessments, beginning with an OA Environmental Safety and Health Assessment conducted in 2003, the issues were validated in a detailed Material Condition Assessment (MCA) conducted as a part of the ATR Life Extension Program in 2007.Accordingly, near term replacement of aging and obsolescent original ATR equipment has become important to ensure ATR capability in support of NE’s long term national missions. To that end, a mission needs statement has been prepared for a non-major system acquisition which is comprised of three interdependent subprojects. The first project will replace the existent diesel-electrical bus (E-3), switchgear, and the 50-year-old obsolescent marine diesels with commercial power that is backed with safety related emergency diesel generators, switchgear, and uninterruptible power supply (UPS). The second project, the subject of this major modification determination, will replace the four, obsolete, original primary coolant pumps (PCPs) and motors. Completion of this and the two other age-related projects (replacement of the ATR diesel bus [E-3] and switchgear and replacement of the existent emergency firewater injection system) will resolve major age-related operational issues plus make a significant contribution in sustaining the ATR safety and reliability profile. The major modification criteria evaluation of the project pre-conceptual design identified several issues that lead to the conclusion that the project is a major modification: 1. Evaluation Criteria #3 (Change of existing process). The proposed strategy for equipping the replacement PCPs with VFDs

  3. A primary culture system of mouse thick ascending limb cells with preserved function and uromodulin processing.

    PubMed

    Glaudemans, Bob; Terryn, Sara; Gölz, Nadine; Brunati, Martina; Cattaneo, Angela; Bachi, Angela; Al-Qusairi, Lama; Ziegler, Urs; Staub, Olivier; Rampoldi, Luca; Devuyst, Olivier

    2014-02-01

    The epithelial cells lining the thick ascending limb (TAL) of the loop of Henle perform essential transport processes and secrete uromodulin, the most abundant protein in normal urine. The lack of differentiated cell culture systems has hampered studies of TAL functions. Here, we report a method to generate differentiated primary cultures of TAL cells, developed from microdissected tubules obtained in mouse kidneys. The TAL tubules cultured on permeable filters formed polarized confluent monolayers in ∼12 days. The TAL cells remain differentiated and express functional markers such as uromodulin, NKCC2, and ROMK at the apical membrane. Electrophysiological measurements on primary TAL monolayers showed a lumen-positive transepithelial potential (+9.4 ± 0.8 mV/cm(2)) and transepithelial resistance similar to that recorded in vivo. The transepithelial potential is abolished by apical bumetanide and in primary cultures obtained from ROMK knockout mice. The processing, maturation and apical secretion of uromodulin by primary TAL cells is identical to that observed in vivo. The primary TAL cells respond appropriately to hypoxia, hypertonicity, and stimulation by desmopressin, and they can be transfected. The establishment of this primary culture system will allow the investigation of TAL cells obtained from genetically modified mouse models, providing a critical tool for understanding the role of that segment in health and disease. PMID:23887378

  4. Understanding the culture of primary health care: implications for clinical practice.

    PubMed

    Camillo, Pat

    2004-01-01

    A qualitative, ethnographic study was undertaken to determine whether older women experienced barriers to health care related to gender and power relations within biomedical culture. A feminist perspective was utilized, incorporating concepts from critical medical anthropology. Data collection methods included individual interviews, focus groups and participant observation. The participants were active in guiding the research and validating the findings. Barriers related to gender and age were observed during primary health care visits, although they were not always directly apparent to the women. There is evidence to suggest that older women's ability to access primary health care depends on the degree of cultural connectedness they encounter within their particular health care facility. Using the findings of this study, a theoretical model is proposed to understand the culture of primary health care within a critical and cultural context. PMID:15587545

  5. Investigating the establishment of primary cell culture from different abalone (Haliotis midae) tissues.

    PubMed

    van der Merwe, Mathilde; Auzoux-Bordenave, Stéphanie; Niesler, Carola; Roodt-Wilding, Rouvay

    2010-07-01

    The abalone, Haliotis midae, is the most valuable commodity in South African aquaculture. The increasing demand for marine shellfish has stimulated research on the biology and physiology of target species in order to improve knowledge on growth, nutritional requirements and pathogen identification. The slow growth rate and long generation time of abalone restrict efficient design of in vivo experiments. Therefore, in vitro systems present an attractive alternative for short term experimentation. The use of marine invertebrate cell cultures as a standardised and controlled system to study growth, endocrinology and disease contributes to the understanding of the biology of economically important molluscs. This paper investigates the suitability of two different H. midae tissues, larval and haemocyte, for establishing primary cell cultures. Cell cultures are assessed in terms of culture initiation, cell yield, longevity and susceptibility to contamination. Haliotis midae haemocytes are shown to be a more feasible tissue for primary cell culture as it could be maintained without contamination more readily than larval cell cultures. The usefulness of short term primary haemocyte cultures is demonstrated here with a growth factor trial. Haemocyte cultures can furthermore be used to relate phenotypic changes at the cellular level to changes in gene expression at the molecular level.

  6. Investigating the establishment of primary cell culture from different abalone (Haliotis midae) tissues

    PubMed Central

    Auzoux-Bordenave, Stéphanie; Niesler, Carola; Roodt-Wilding, Rouvay

    2010-01-01

    The abalone, Haliotis midae, is the most valuable commodity in South African aquaculture. The increasing demand for marine shellfish has stimulated research on the biology and physiology of target species in order to improve knowledge on growth, nutritional requirements and pathogen identification. The slow growth rate and long generation time of abalone restrict efficient design of in vivo experiments. Therefore, in vitro systems present an attractive alternative for short term experimentation. The use of marine invertebrate cell cultures as a standardised and controlled system to study growth, endocrinology and disease contributes to the understanding of the biology of economically important molluscs. This paper investigates the suitability of two different H. midae tissues, larval and haemocyte, for establishing primary cell cultures. Cell cultures are assessed in terms of culture initiation, cell yield, longevity and susceptibility to contamination. Haliotis midae haemocytes are shown to be a more feasible tissue for primary cell culture as it could be maintained without contamination more readily than larval cell cultures. The usefulness of short term primary haemocyte cultures is demonstrated here with a growth factor trial. Haemocyte cultures can furthermore be used to relate phenotypic changes at the cellular level to changes in gene expression at the molecular level. PMID:20680682

  7. Changes of Brain Connectivity in the Primary Motor Cortex After Subcortical Stroke: A Multimodal Magnetic Resonance Imaging Study.

    PubMed

    Li, Yongxin; Wang, Defeng; Zhang, Heye; Wang, Ya; Wu, Ping; Zhang, Hongwu; Yang, Yang; Huang, Wenhua

    2016-02-01

    The authors investigated the changes in connectivity networks of the bilateral primary motor cortex (M1) of subcortical stroke patients using a multimodal neuroimaging approach with antiplatelet therapy. Nineteen patients were scanned at 2 time points: before and 1 month after the treatment. The authors assessed the resting-state functional connectivity (FC) and probabilistic fiber tracking of left and right M1 of every patient, and then compared these results to the 15 healthy controls. The authors also evaluated the correlations between the neuroimaging results and clinical scores.Compared with the controls, the patients showed a significant decrease of FC in the contralateral motor cortex before treatment, and the disrupted FC was restored after treatment. The fiber tracking results in the controls indicated that the body of the corpus callosum should be the main pathway connecting the M1 and contralateral hemispheres. All patients exhibited reduced probability of structural connectivity within this pathway before treatment and which was restored after treatment. Significant correlations were also found in these patients between the connectivity results and clinical scores, which might imply that the connectivity of M1 can be used to evaluate the motor skills in stroke patients.These findings can help elucidate the neural mechanisms responsible for the brain connectivity recovery after stroke.

  8. Measurement tools and process indicators of patient safety culture in primary care. A mixed methods study by the LINNEAUS collaboration on patient safety in primary care

    PubMed Central

    Parker, Dianne; Wensing, Michel; Esmail, Aneez; Valderas, Jose M

    2015-01-01

    ABSTRACT Background: There is little guidance available to healthcare practitioners about what tools they might use to assess the patient safety culture. Objective: To identify useful tools for assessing patient safety culture in primary care organizations in Europe; to identify those aspects of performance that should be assessed when investigating the relationship between safety culture and performance in primary care. Methods: Two consensus-based studies were carried out, in which subject matter experts and primary healthcare professionals from several EU states rated (a) the applicability to their healthcare system of several existing safety culture assessment tools and (b) the appropriateness and usefulness of a range of potential indicators of a positive patient safety culture to primary care settings. The safety culture tools were field-tested in four countries to ascertain any challenges and issues arising when used in primary care. Results: The two existing tools that received the most favourable ratings were the Manchester patient safety framework (MaPsAF primary care version) and the Agency for healthcare research and quality survey (medical office version). Several potential safety culture process indicators were identified. The one that emerged as offering the best combination of appropriateness and usefulness related to the collection of data on adverse patient events. Conclusion: Two tools, one quantitative and one qualitative, were identified as applicable and useful in assessing patient safety culture in primary care settings in Europe. Safety culture indicators in primary care should focus on the processes rather than the outcomes of care. PMID:26339832

  9. Efficient establishment of primary fibroblast cultures from the hawksbill sea turtle (Eretmochelys imbricata).

    PubMed

    Fukuda, Tomokazu; Kurita, Jun; Saito, Tomomi; Yuasa, Kei; Kurita, Masanobu; Donai, Kenichiro; Nitto, Hiroshi; Soichi, Makoto; Nishimori, Katsuhiko; Uchida, Takafumi; Isogai, Emiko; Onuma, Manabu; Sone, Hideko; Oseko, Norihisa; Inoue-Murayama, Miho

    2012-12-01

    The hawksbill sea turtle (Eretmochelys imbricata) is a critically endangered species at a risk of extinction. Preservation of the genomic and cellular information of endangered animals is important for future genetic and biological studies. Here, we report the efficient establishment of primary fibroblast cultures from skin tissue of the hawksbill sea turtle. We succeeded in establishing 19 primary cultures from 20 hawksbill sea turtle individuals (a success rate of 95%). These cells exhibited a fibroblast-like morphology and grew optimally at a temperature of 26°C, but experienced a loss of viability when cultured at 37°C. Chromosomal analysis using the primary cells derived here revealed that hawksbill sea turtles have a 2n = 56 karyotype. Furthermore, we showed that our primary cell cultures are free of several fish-related viruses, and this finding is important for preservation purposes. To our knowledge, this report is the first to describe primary cell cultures established from normal tissues of the hawksbill sea turtle. The results will contribute to the preservation of biodiversity, especially for the sea turtles that are critically endangered owing to human activities.

  10. [THE DISTRIBUTION OF CORTICO-THALAMIC PROJECTIONS OF DIFFERENT OF DIFFERENT SOMATOTOPIC REPRESENTATIONS OF PRIMARY MOTOR AND SENSORY CORTEX].

    PubMed

    Ipekchyan, N M; Badalyan, S A

    2016-01-01

    The peculiarities of localization and distribution of cortico-thalamic efferents of different somatotopical representations of primary motor (MI) and sensory (SI) cortex were studied in cat brain. MI efferent fibers (4y, 6ab areas) preferentially projected to ventral posterolateral and medial (VPL, VPM), ventrolateral (VL), and reticular (R) nuclei, localized in rostral part of the thalamus (T), as opposed to SI (areas 1, 2, 3a, 3b), which projected preferentially to caudal part of T, VPL, VPM and R nuclei. Latero-medial organization of cortico-thalamic connections was demonstrated, with predominant localization of cortical representation of hindlimbs in the lateral part of VPL, of forelimbs--in the medial part of VPL, of face and head--also in VM and VPM. Quantitative analysis of the distribution of corticothalamic efferents of different somatotopical representations of MI has demonstrated the most extensive, massive connections with T nuclei (VPL, VL, R) of the motor representation of forelimb, followed by the representation of hindlimb, trunk and, finally, the minimal projection of the representation of face and head. As opposed to motor representation of the forelimb and also of the face and head, with uniform distribution of fibers in VPL, VL and R, the number of efferents of motor representation of hindlimb, passing in VL, was almost 2.5 time lower than in VPL and R, whereas the representation of trunk had the predominant projection to VL. Dominant cortico-thalamic connection suggests greater involvement of T nuclei studied in the realization of functional specialization of certain somatotopical representations of MI. PMID:27487657

  11. Low-frequency rTMS inhibitory effects in the primary motor cortex: Insights from TMS-evoked potentials.

    PubMed

    Casula, Elias P; Tarantino, Vincenza; Basso, Demis; Arcara, Giorgio; Marino, Giuliana; Toffolo, Gianna Maria; Rothwell, John C; Bisiacchi, Patrizia S

    2014-09-01

    The neuromodulatory effects of repetitive transcranial magnetic stimulation (rTMS) have been mostly investigated by peripheral motor-evoked potentials (MEPs). New TMS-compatible EEG systems allow a direct investigation of the stimulation effects through the analysis of TMS-evoked potentials (TEPs). We investigated the effects of 1-Hz rTMS over the primary motor cortex (M1) of 15 healthy volunteers on TEP evoked by single pulse TMS over the same area. A second experiment in which rTMS was delivered over the primary visual cortex (V1) of 15 healthy volunteers was conducted to examine the spatial specificity of the effects. Single-pulse TMS evoked four main components: P30, N45, P60 and N100. M1-rTMS resulted in a significant decrease of MEP amplitude and in a significant increase of P60 and N100 amplitude. There was no effect after V1-rTMS. 1-Hz rTMS appears to increase the amount of inhibition following a TMS pulse, as demonstrated by the higher N100 and P60, which are thought to originate from GABAb-mediated inhibitory post-synaptic potentials. Our results confirm the reliability of the TMS-evoked N100 as a marker of cortical inhibition and provide insight into the neuromodulatory effects of 1-Hz rTMS. The present finding could be of relevance for therapeutic and diagnostic purposes.

  12. Basolateral Cl channels in primary airway epithelial cultures.

    PubMed

    Fischer, Horst; Illek, Beate; Finkbeiner, Walter E; Widdicombe, Jonathan H

    2007-06-01

    Salt and water absorption and secretion across the airway epithelium are important for maintaining the thin film of liquid lining the surface of the airway epithelium. Movement of Cl across the apical membrane involves the CFTR Cl channel; however, conductive pathways for Cl movement across the basolateral membrane have been little studied. Here, we determined the regulation and single-channel properties of the Cl conductance (G(Cl)) in airway surface epithelia using epithelial cultures from human or bovine trachea and freshly isolated ciliated cells from the human nasal epithelium. In Ussing chamber studies, a swelling-activated basolateral G(Cl) was found, which was further stimulated by forskolin and blocked by N-phenylanthranilic acid (DPC) = sucrose > flufenamic acid = niflumic acid = glibenclamide > CdCl(2) = 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) = DIDS = ZnCl(2) > tamoxifen > 4,4'-dinitro-2,2'-stilbene-disulfonate disodium salt (DNDS). In whole cell patch-clamp experiments, three types of G(Cl) were identified: 1) a voltage-activated, DIDS- (but not Cd-) blockable and osmosensitive G(Cl); 2) an inwardly rectifying, hyperpolarization-activated and Cd-sensitive G(Cl); and 3) a forskolin-activated, linear G(Cl), which was insensitive to Cd and DIDS. In cell-attached patch-clamp recordings, the basolateral pole of isolated ciliated cells expressed three types of Cl channels: 1) an outwardly rectifying, swelling-activated Cl channel; 2) a strongly inwardly rectifying Cl channel; and 3) a forskolin-activated, low-conductance channel. We propose that, depending on the driving force for Cl across the apical membrane, basolateral Cl channels confine Cl(-) secretion or support transcellular Cl(-) absorption.

  13. 9 CFR 3.15 - Primary conveyances (motor vehicle, rail, air, and marine).

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ..., Care, Treatment, and Transportation of Dogs and Cats 1 Transportation Standards § 3.15 Primary... transport dogs and cats must be designed, constructed, and maintained in a manner that at all times protects... being transported in it. (c) Each primary enclosure containing dogs or cats must be positioned in...

  14. 9 CFR 3.15 - Primary conveyances (motor vehicle, rail, air, and marine).

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ..., Care, Treatment, and Transportation of Dogs and Cats 1 Transportation Standards § 3.15 Primary... transport dogs and cats must be designed, constructed, and maintained in a manner that at all times protects... being transported in it. (c) Each primary enclosure containing dogs or cats must be positioned in...

  15. 9 CFR 3.15 - Primary conveyances (motor vehicle, rail, air, and marine).

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ..., Care, Treatment, and Transportation of Dogs and Cats 1 Transportation Standards § 3.15 Primary... transport dogs and cats must be designed, constructed, and maintained in a manner that at all times protects... being transported in it. (c) Each primary enclosure containing dogs or cats must be positioned in...

  16. 9 CFR 3.15 - Primary conveyances (motor vehicle, rail, air, and marine).

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ..., Care, Treatment, and Transportation of Dogs and Cats 1 Transportation Standards § 3.15 Primary... transport dogs and cats must be designed, constructed, and maintained in a manner that at all times protects... being transported in it. (c) Each primary enclosure containing dogs or cats must be positioned in...

  17. Lab on a chip-based hepatic sinusoidal system simulator for optimal primary hepatocyte culture.

    PubMed

    Choi, Yoon Young; Kim, Jaehyung; Lee, Sang-Hoon; Kim, Dong-Sik

    2016-08-01

    Primary hepatocyte cultures have been used in studies on liver disease, physiology, and pharmacology. While they are an important tool for in vitro liver studies, maintaining liver-specific characteristics of hepatocytes in vitro is difficult, as these cells rapidly lose their unique characteristics and functions. Portal flow is an important condition to preserve primary hepatocyte functions and liver regeneration in vivo. We have developed a microfluidic chip that does not require bulky peripheral devices or an external power source to investigate the relationship between hepatocyte functional maintenance and flow rates. In our culture system, two types of microfluidic devices were used as scaffolds: a monolayer- and a concave chamber-based device. Under flow conditions, our chips improved albumin and urea secretion rates after 13 days compared to that of the static chips. Reverse transcription polymerase chain reaction demonstrated that hepatocyte-specific gene expression was significantly higher at 13 days under flow conditions than when using static chips. For both two-dimensional and three-dimensional culture on the chips, flow resulted in the best performance of the hepatocyte culture in vitro. We demonstrated that flow improves the viability and efficiency of long-term culture of primary hepatocytes and plays a key role in hepatocyte function. These results suggest that this flow system has the potential for long-term hepatocyte cultures as well as a technique for three-dimensional culture. PMID:27334878

  18. Conditions for initiating Lake Victoria haplochromine (Oreochromis esculentus) primary cell cultures from caudal fin biopsies.

    PubMed

    Filice, Melissa; Lee, C; Mastromonaco, Gabriela F

    2014-10-01

    The global decline of freshwater fishes has created a need to cryopreserve biological materials from endangered species in an effort to conserve the biodiversity within this taxon. Since maternal gametes and embryos from fish are difficult to cryopreserve, somatic cells obtained from caudal fins have become an increasingly popular resource as they contain both maternal and paternal DNA ensuring valuable traits are not lost from the population. Somatic cells stored in cryobanks can be used to supplement endangered populations with genetically valuable offspring with the use of assisted reproductive technologies. However, initiating primary cell cultures from caudal fin biopsies of endangered species can be challenging as standardized protocols have not yet been developed. The objective of this study was to identify culture conditions, including antibiotic supplementation, biopsy size, and culture temperature, suitable for establishing primary cell cultures of ngege (Oreochromis esculentus), a critically endangered African cichlid. Six-millimeter caudal fin biopsies provided sufficient material to develop a primary cell culture when incubated at 25°C using standard fish cell culture medium containing 1× Primocin. Further investigation and application of these culture conditions for other endangered freshwater fishes is necessary. PMID:24985486

  19. Spinal cord organotypic slice cultures for the study of regenerating motor axon interactions with 3D scaffolds.

    PubMed

    Gerardo-Nava, Jose; Hodde, Dorothee; Katona, Istvan; Bozkurt, Ahmet; Grehl, Torsten; Steinbusch, Harry W M; Weis, Joachim; Brook, Gary A

    2014-05-01

    Numerous in-vitro techniques exist for investigating the influence of 3D substrate topography on sensory axon growth. However, simple and cost-effective methods for studying post-natal motor axon interactions with such substrates are lacking. Here, spinal cord organotypic slice cultures (OSC) from post-natal day 7-9 rat pups were presented with spinal nerve roots, or blocks of fibrin hydrogel or 3D microporous collagen scaffolds to investigate motor axon-substrate interactions. By 7-14 days, axons from motor neuronal pools extended into the explanted nerve roots, growing along Schwann cell processes and demonstrating a full range of axon-Schwann cell interactions, from simple ensheathment to concentric wrapping by Schwann cell processes and the formation of compact myelin within a basal lamina sheath. Extensive motor axon regeneration and all stages of axon-Schwann interactions were also supported within the longitudinally orientated microporous framework of the 3D collagen scaffold. In stark contrast, the simple fibrin hydrogel only supported axon growth and cell migration over its surface. The relative ease of demonstrating such motor axon regeneration through the microporous 3D framework by immunofluorescence, two-photon microscopy and transmission electron microscopy strongly supports the adoption of this technique for assaying the influence of substrate topography and functionalization in regenerative bioengineering.

  20. Hydroxylation, conjugation and sulfation of bile acids in primary monolayer cultures of rat hepatocytes

    SciTech Connect

    Princen, H.M.; Meijer, P.

    1988-08-15

    Hydroxylation of lithocholic, chenodeoxycholic, deoxycholic and cholic acids was studied in monolayers of rat hepatocytes cultured for 76 h. The majority of added lithocholic and chenodeoxycholic acids was metabolized to beta-muricholic acid (56-76%). A small part of these bile acids (9%), however, and a considerable amount of deoxycholic and cholic acids (21%) were converted into metabolites more polar than cholic acid in the first culture period. Formation of these compounds decreased during the last day of culture. Bile acids synthesized after addition of (4-/sup 14/C)-cholesterol were almost entirely (97%) sulfated and/or conjugated, predominantly with taurine (54-66%), during culture. Sulfated bile acids were mainly composed of free bile acids. The ability of hepatocytes to sulfurylate bile acids declined with culture age. Thus, rat hepatocytes in primary monolayer culture are capable to sulfurylate bile acids and to hydroxylate trihydroxylated bile acids, suggesting formation of polyhydroxylated metabolites.

  1. Organization Complexity and Primary Care Providers' Perceptions of Quality Improvement Culture Within the Veterans Health Administration.

    PubMed

    Korom-Djakovic, Danijela; Canamucio, Anne; Lempa, Michele; Yano, Elizabeth M; Long, Judith A

    2016-01-01

    This study examined how aspects of quality improvement (QI) culture changed during the introduction of the Veterans Health Administration (VHA) patient-centered medical home initiative and how they were influenced by existing organizational factors, including VHA facility complexity and practice location. A voluntary survey, measuring primary care providers' (PCPs') perspectives on QI culture at their primary care clinics, was administered in 2010 and 2012. Participants were 320 PCPs from hospital- and community-based primary care practices in Pennsylvania, West Virginia, Delaware, New Jersey, New York, and Ohio. PCPs in community-based outpatient clinics reported an improvement in established processes for QI, and communication and cooperation from 2010 to 2012. However, their peers in hospital-based clinics did not report any significant improvements in QI culture. In both years, compared with high-complexity facilities, medium- and low-complexity facilities had better scores on the scales assessing established processes for QI, and communication and cooperation.

  2. Cellular Microenvironment Dictates Androgen Production by Murine Fetal Leydig Cells in Primary Culture1

    PubMed Central

    Carney, Colleen M.; Muszynski, Jessica L.; Strotman, Lindsay N.; Lewis, Samantha R.; O'Connell, Rachel L.; Beebe, David J.; Theberge, Ashleigh B.; Jorgensen, Joan S.

    2014-01-01

    ABSTRACT Despite the fact that fetal Leydig cells are recognized as the primary source of androgens in male embryos, the mechanisms by which steroidogenesis occurs within the developing testis remain unclear. A genetic approach was used to visualize and isolate fetal Leydig cells from remaining cells within developing mouse testes. Cyp11a1-Cre mice were bred to mT/mG dual reporter mice to target membrane-tagged enhanced green fluorescent protein (GFP) within steroidogenic cells, whereas other cells expressed membrane-tagged tandem-dimer tomato red. Fetal Leydig cell identity was validated using double-labeled immunohistochemistry against GFP and the steroidogenic enzyme 3beta-HSD, and cells were successfully isolated as indicated by qPCR results from sorted cell populations. Because fetal Leydig cells must collaborate with neighboring cells to synthesize testosterone, we hypothesized that the fetal Leydig cell microenvironment defined their capacity for androgen production. Microfluidic culture devices were used to measure androstenedione and testosterone production of fetal Leydig cells that were cultured in cell-cell contact within a mixed population, were isolated but remained in medium contact via compartmentalized co-culture with other testicular cells, or were isolated and cultured alone. Results showed that fetal Leydig cells maintained their identity and steroidogenic activity for 3–5 days in primary culture. Microenvironment dictated proficiency of testosterone production. As expected, fetal Leydig cells produced androstenedione but not testosterone when cultured in isolation. More testosterone accumulated in medium from mixed cultures than from compartmentalized co-cultures initially; however, co-cultures maintained testosterone synthesis for a longer time. These data suggest that a combination of cell-cell contact and soluble factors constitute the ideal microenvironment for fetal Leydig cell activity in primary culture. PMID:25143354

  3. Cellular microenvironment dictates androgen production by murine fetal Leydig cells in primary culture.

    PubMed

    Carney, Colleen M; Muszynski, Jessica L; Strotman, Lindsay N; Lewis, Samantha R; O'Connell, Rachel L; Beebe, David J; Theberge, Ashleigh B; Jorgensen, Joan S

    2014-10-01

    Despite the fact that fetal Leydig cells are recognized as the primary source of androgens in male embryos, the mechanisms by which steroidogenesis occurs within the developing testis remain unclear. A genetic approach was used to visualize and isolate fetal Leydig cells from remaining cells within developing mouse testes. Cyp11a1-Cre mice were bred to mT/mG dual reporter mice to target membrane-tagged enhanced green fluorescent protein (GFP) within steroidogenic cells, whereas other cells expressed membrane-tagged tandem-dimer tomato red. Fetal Leydig cell identity was validated using double-labeled immunohistochemistry against GFP and the steroidogenic enzyme 3beta-HSD, and cells were successfully isolated as indicated by qPCR results from sorted cell populations. Because fetal Leydig cells must collaborate with neighboring cells to synthesize testosterone, we hypothesized that the fetal Leydig cell microenvironment defined their capacity for androgen production. Microfluidic culture devices were used to measure androstenedione and testosterone production of fetal Leydig cells that were cultured in cell-cell contact within a mixed population, were isolated but remained in medium contact via compartmentalized co-culture with other testicular cells, or were isolated and cultured alone. Results showed that fetal Leydig cells maintained their identity and steroidogenic activity for 3-5 days in primary culture. Microenvironment dictated proficiency of testosterone production. As expected, fetal Leydig cells produced androstenedione but not testosterone when cultured in isolation. More testosterone accumulated in medium from mixed cultures than from compartmentalized co-cultures initially; however, co-cultures maintained testosterone synthesis for a longer time. These data suggest that a combination of cell-cell contact and soluble factors constitute the ideal microenvironment for fetal Leydig cell activity in primary culture. PMID:25143354

  4. Cocaine Causes Apoptotic Death in Rat Mesencephalon and Striatum Primary Cultures.

    PubMed

    Lepsch, Lucilia B; Planeta, Cleopatra S; Scavone, Critoforo

    2015-01-01

    To study cocaine's toxic effects in vitro, we have used primary mesencephalic and striatal cultures from rat embryonic brain. Treatment with cocaine causes a dramatic increase in DNA fragmentation in both primary cultures. The toxicity induced by cocaine was paralleled with a concomitant decrease in the microtubule associated protein 2 (MAP2) and/or neuronal nucleus protein (NeuN) staining. We also observed in both cultures that the cell death caused by cocaine was induced by an apoptotic mechanism, confirmed by TUNEL assay. Therefore, the present paper shows that cocaine causes apoptotic cell death and inhibition of the neurite prolongation in striatal and mesencephalic cell culture. These data suggest that if similar neuronal damage could be produced in the developing human brain, it could account for the qualitative or quantitative defects in neuronal pathways that cause a major handicap in brain function following prenatal exposure to cocaine. PMID:26295051

  5. Cocaine Causes Apoptotic Death in Rat Mesencephalon and Striatum Primary Cultures

    PubMed Central

    Lepsch, Lucilia B.; Planeta, Cleopatra S.; Scavone, Critoforo

    2015-01-01

    To study cocaine's toxic effects in vitro, we have used primary mesencephalic and striatal cultures from rat embryonic brain. Treatment with cocaine causes a dramatic increase in DNA fragmentation in both primary cultures. The toxicity induced by cocaine was paralleled with a concomitant decrease in the microtubule associated protein 2 (MAP2) and/or neuronal nucleus protein (NeuN) staining. We also observed in both cultures that the cell death caused by cocaine was induced by an apoptotic mechanism, confirmed by TUNEL assay. Therefore, the present paper shows that cocaine causes apoptotic cell death and inhibition of the neurite prolongation in striatal and mesencephalic cell culture. These data suggest that if similar neuronal damage could be produced in the developing human brain, it could account for the qualitative or quantitative defects in neuronal pathways that cause a major handicap in brain function following prenatal exposure to cocaine. PMID:26295051

  6. 9 CFR 3.114 - Primary conveyances (motor vehicle, rail, air and marine).

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... safety and comfort of the marine mammals contained within at all times. All primary conveyances used must... species involved and to provide for the safety and comfort of the marine mammal, or other...

  7. Health system factors affecting communication with pediatricians: gendered work culture in primary care.

    PubMed

    Lynch, Sean

    2011-01-01

    This qualitative study examined the roles that practice setting, education level, and gender may play in social workers' communication satisfaction with pediatricians. Taking an ethnographic approach, the researcher interviewed social workers and pediatricians who worked together to provide mental health services in primary care. The results suggested that gender at the health system level may be an issue and that gendered work culture in primary care was a factor in communication. In particular, reimbursement, an aspect of the gendered work culture, was a substantial communication barrier, and the implications for Medicaid billing are discussed. PMID:22085327

  8. Establishment of primary cell culture from the temperate symbiotic cnidarian, Anemonia viridis.

    PubMed

    Barnay-Verdier, Stéphanie; Dall'osso, Diane; Joli, Nathalie; Olivré, Juliette; Priouzeau, Fabrice; Zamoum, Thamilla; Merle, Pierre-Laurent; Furla, Paola

    2013-10-01

    The temperate symbiotic sea anemone Anemonia viridis, a member of the Cnidaria phylum, is a relevant experimental model to investigate the molecular and cellular events involved in the preservation or in the rupture of the symbiosis between the animal cells and their symbiotic microalgae, commonly named zooxanthellae. In order to increase research tools for this model, we developed a primary culture from A. viridis animal cells. By adapting enzymatic dissociation protocols, we isolated animal host cells from a whole tentacle in regeneration state. Each plating resulted in a heterogeneous primary culture consisted of free zooxanthellae and many regular, small rounded and adherent cells (of 3-5 μm diameter). Molecular analyses conducted on primary cultures, maintained for 2 weeks, confirmed a specific signature of A. viridis cells. Further serial dilutions and micromanipulation allowed us to obtain homogenous primary cultures of the small rounded cells, corresponding to A. viridis "epithelial-like cells". The maintenance and the propagation over a 4 weeks period of primary cells provide, for in vitro cnidarian studies, a preliminary step for further investigations on cnidarian cellular pathways notably in regard to symbiosis interactions. PMID:23595421

  9. GABA and Glutamate in Children with Primary Complex Motor Stereotypies: A 1H MRS Study at 7T

    PubMed Central

    Harris, A. D.; Singer, H. S.; Horska, A.; Kline, T.; Ryan, M.; Edden, R. A. E.; Mahone, E. Mark

    2015-01-01

    Background and Purpose Complex motor stereotypies (CMS) are rhythmic, repetitive, fixed, purposeful but purposeless movements that stop with distraction. They can occur in otherwise normal healthy children (primary stereotypies), as well in those with autism spectrum disorders (secondary stereotypies). The underlying neurobiological basis for these movements is unknown, but thought to involve cortical-striatal-thalamo-cortical pathways. In order to further clarify potential neurochemical alterations, GABA, glutamate (Glu), glutamine (Gln), N-acetyl aspartate (NAA) and choline (Cho) levels were measured in four frontostriatal regions, using 1H MRS at 7T. Materials and Methods A total of 18 children with primary CMS and 24 typically developing controls, ages 5-10 years completed MRS at 7T. Single voxel STEAM acquisitions from the anterior cingulate cortex (ACC), premotor cortex (PMC), dorsolateral prefrontal cortex (DLPFC) and striatum were obtained and metabolites were quantified with respect to creatine using LCModel. Results The 7T scan was well tolerated by all participants. Compared to controls, children with CMS had lower levels of GABA ACC (GABA/Cr, p=0.049; GABA/Glu: p=0.051) and striatum (GABA/Cr: p= 0.028; GABA/Glu: p=0.0037), but not the DLPFC or PMC. Glu, Gln, NAA, and Cho levels did not differ between groups in any of the aforementioned regions. Within the CMS group, reduced GABA/Cr in the ACC was significantly associated with greater severity of motor stereotypies (r=-0.59, p= 0.021). Conclusions These results suggest possible GABAergic dysfunction within corticostriatal pathways in children with primary CMS. PMID:26542237

  10. Evaluation of an in vitro muscle contraction model in mouse primary cultured myotubes.

    PubMed

    Manabe, Yasuko; Ogino, Shinya; Ito, Miyuki; Furuichi, Yasuro; Takagi, Mayumi; Yamada, Mio; Goto-Inoue, Naoko; Ono, Yusuke; Fujii, Nobuharu L

    2016-03-15

    To construct an in vitro contraction model with the primary cultured myotubes, we isolated satellite cells from the mouse extensor digitorum longus. Differentiated myotubes possessed a greater number of sarcomere assemblies and higher expression levels of myosin heavy chain, cytochrome c oxidase IV, and myoglobin than in C2C12 myotubes. In agreement with these results regarding the sarcomere assemblies and protein expressions, the primary myotubes showed higher contractile activity stimulated by the electric pulses than that in the C2C12 myotubes. These data suggest that mouse primary myotubes will be a valuable research tool as an in vitro muscle contraction model. PMID:26548957

  11. Epidermal growth factor receptor numbers in male and female mouse primary hepatocyte cultures.

    PubMed

    Benveniste, R; Danoff, T M; Ilekis, J; Craig, H R

    1988-10-01

    Epidermal growth factor receptors (EGF-R) were measured in adult male and female mouse primary hepatocyte cultures. On culture day 1, female hepatocytes had significantly fewer EGF-R than male hepatocytes (1.3 x 10(4) versus 6.2 x 10(5) per cell). Over the next three days, morphological changes consistent with progressive heptocyte dedifferentiation were observed. During this period, EGF-R numbers progressively increased in female cultures and decreased in male cultures, and by day 4 the sexual difference in EGF-R numbers was obliterated. These results indicate that a relationship exists between the degree of differentiation in hepatocyte cultures and the expression of EGF-R on the cell surface.

  12. Final report for measurement of primary particulate matter emissions from light-duty motor vehicles

    SciTech Connect

    Norbeck, J. M.; Durbin, T. D.; Truex, T. J.

    1998-12-31

    This report describes the results of a particulate emissions study conducted at the University of California, Riverside, College of Engineering-Center for Environmental Research and Technology (CE-CERT) from September of 1996 to August of 1997. The goal of this program was to expand the database of particulate emissions measurements from motor vehicles to include larger numbers of representative in-use vehicles. This work was co-sponsored by the Coordinating Research Council (CRC), the South Coast Air Quality Management District (SCAQMD), and the National Renewable Energy Laboratory (NREL) and was part of a larger study of particulate emissions being conducted in several states under sponsorship by CRC. For this work, FTP particulate mass emission rates were determined for gasoline and diesel vehicles, along with the fractions of particulates below 2.5 and 10 microns aerodynamic diameter. A total of 129 gasoline-fueled vehicles and 19 diesel-fueled vehicles were tested as part of the program.

  13. Alterations in primary motor cortex neurotransmission and gene expression in hemi-parkinsonian rats with drug-induced dyskinesia.

    PubMed

    Lindenbach, D; Conti, M M; Ostock, C Y; Dupre, K B; Bishop, C

    2015-12-01

    Treatment of Parkinson's disease (PD) with dopamine replacement relieves symptoms of poverty of movement, but often causes drug-induced dyskinesias. Accumulating clinical and pre-clinical evidence suggests that the primary motor cortex (M1) is involved in the pathophysiology of PD and that modulating cortical activity may be a therapeutic target in PD and dyskinesia. However, surprisingly little is known about how M1 neurotransmitter tone or gene expression is altered in PD, dyskinesia or associated animal models. The present study utilized the rat unilateral 6-hydroxydopamine (6-OHDA) model of PD/dyskinesia to characterize structural and functional changes taking place in M1 monoamine innervation and gene expression. 6-OHDA caused dopamine pathology in M1, although the lesion was less severe than in the striatum. Rats with 6-OHDA lesions showed a PD motor impairment and developed dyskinesia when given L-DOPA or the D1 receptor agonist, SKF81297. M1 expression of two immediate-early genes (c-Fos and ARC) was strongly enhanced by either L-DOPA or SKF81297. At the same time, expression of genes specifically involved in glutamate and GABA signaling were either modestly affected or unchanged by lesion and/or treatment. We conclude that M1 neurotransmission and signal transduction in the rat 6-OHDA model of PD/dyskinesia mirror features of human PD, supporting the utility of the model to study M1 dysfunction in PD and the elucidation of novel pathophysiological mechanisms and therapeutic targets. PMID:26363150

  14. Excitability changes in the left primary motor cortex innervating the hand muscles induced during speech about hand or leg movements.

    PubMed

    Onmyoji, Yusuke; Kubota, Shinji; Hirano, Masato; Tanaka, Megumi; Morishita, Takuya; Uehara, Kazumasa; Funase, Kozo

    2015-05-01

    In the present study, we used transcranial magnetic stimulation (TMS) to investigate the changes in the excitability of the left primary motor cortex (M1) innervating the hand muscles and in short-interval intracortical inhibition (SICI) during speech describing hand or leg movements. In experiment 1, we investigated the effects of the contents of speech on the amplitude of the motor evoked potentials (MEPs) induced during reading aloud and silent reading. In experiment 2, we repeated experiment 1 with an additional condition, the non-vocal oral movement (No-Voc OM) condition, and investigated the change in SICI induced in each condition using the paired TMS paradigm. The MEP observed in the reading aloud and No-Voc OM conditions exhibited significantly greater amplitudes than those seen in the silent reading conditions, irrespective of the content of the sentences spoken by the subjects or the timing of the TMS. There were no significant differences in SICI between the experimental conditions. Our findings suggest that the increased excitability of the left M1 hand area detected during speech was mainly caused by speech-related oral movements and the activation of language processing-related brain functions. The increased left M1 excitability was probably also mediated by neural mechanisms other than reduced SICI; i.e., disinhibition.

  15. Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry.

    PubMed

    Ali, M Yakut; Anand, Sandeep V; Tangella, Krishnarao; Ramkumar, Davendra; Saif, Taher A

    2015-01-01

    Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion receptors and associated signal transduction membrane proteins, the cytoskeletal architecture, and molecular motors. Mechanosensitivity of different cancer cells in vitro are investigated primarily with immortalized cell lines or murine derived primary cells, not with primary human cancer cells. Hence, little is known about the mechanosensitivity of primary human colon cancer cells in vitro. Here, an optimized protocol is developed that describes the isolation of primary human colon cells from healthy and cancerous surgical human tissue samples. Isolated colon cells are then successfully cultured on soft (2 kPa stiffness) and stiff (10 kPa stiffness) polyacrylamide hydrogels and rigid polystyrene (~3.6 GPa stiffness) substrates functionalized by an extracellular matrix (fibronectin in this case). Fluorescent microbeads are embedded in soft gels near the cell culture surface, and traction assay is performed to assess cellular contractile stresses using free open access software. In addition, immunofluorescence microscopy on different stiffness substrates provides useful information about primary cell morphology, cytoskeleton organization and vinculin containing focal adhesions as a function of substrate rigidity. PMID:26065530

  16. 9 CFR 3.114 - Primary conveyances (motor vehicle, rail, air and marine).

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ..., rail, air and marine). 3.114 Section 3.114 Animals and Animal Products ANIMAL AND PLANT HEALTH..., Care, Treatment, and Transportation of Marine Mammals Transportation Standards § 3.114 Primary... safeguards (such as, but not limited to, cooling the animal with cold water, adding ice to...

  17. 9 CFR 3.37 - Primary conveyances (motor vehicle, rail, air, and marine).

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ..., Care, Treatment, and Transportation of Guinea Pigs and Hamsters Transportation Standards § 3.37 Primary... transporting live guinea pigs and hamsters shall be designed and constructed to protect the health, and ensure the safety and comfort of the live guinea pigs and hamsters at all times. (b) The animal cargo...

  18. 9 CFR 3.37 - Primary conveyances (motor vehicle, rail, air, and marine).

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ..., Care, Treatment, and Transportation of Guinea Pigs and Hamsters Transportation Standards § 3.37 Primary... transporting live guinea pigs and hamsters shall be designed and constructed to protect the health, and ensure the safety and comfort of the live guinea pigs and hamsters at all times. (b) The animal cargo...

  19. 9 CFR 3.37 - Primary conveyances (motor vehicle, rail, air, and marine).

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ..., Care, Treatment, and Transportation of Guinea Pigs and Hamsters Transportation Standards § 3.37 Primary... transporting live guinea pigs and hamsters shall be designed and constructed to protect the health, and ensure the safety and comfort of the live guinea pigs and hamsters at all times. (b) The animal cargo...

  20. 9 CFR 3.62 - Primary conveyances (motor vehicle, rail, air, and marine).

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ..., Care, Treatment and Transportation of Rabbits Transportation Standards § 3.62 Primary conveyances... transporting live rabbits shall be designed and constructed to protect the health, and ensure the safety and comfort of the rabbits contained therein at all times. (b) The animal cargo space shall be constructed...

  1. 9 CFR 3.62 - Primary conveyances (motor vehicle, rail, air, and marine).

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ..., Care, Treatment and Transportation of Rabbits Transportation Standards § 3.62 Primary conveyances... transporting live rabbits shall be designed and constructed to protect the health, and ensure the safety and comfort of the rabbits contained therein at all times. (b) The animal cargo space shall be constructed...

  2. 9 CFR 3.62 - Primary conveyances (motor vehicle, rail, air, and marine).

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ..., Care, Treatment and Transportation of Rabbits Transportation Standards § 3.62 Primary conveyances... transporting live rabbits shall be designed and constructed to protect the health, and ensure the safety and comfort of the rabbits contained therein at all times. (b) The animal cargo space shall be constructed...

  3. Development of Quality Assurance System in Culture and Nation Character Education in Primary Education in Indonesia

    ERIC Educational Resources Information Center

    Susilana, Rudi; Asra

    2013-01-01

    The purpose of national education is to develop skills and build dignified national character and civilization in educating nation life (Act No. 20, 2003). The paper describes a system of quality assurance in culture and character education in primary education. This study employs the six sigma model which consists of the formula DMAIC (Define,…

  4. Promoting Cultural Understandings through Song across the Tasman: Pre-Service Primary Teacher Education

    ERIC Educational Resources Information Center

    Joseph, Dawn; Trinick, Robyn

    2016-01-01

    As tertiary music educators across the Tasman we argue that music, particularly song, is an effective medium for teaching and learning about non-western music when preparing generalist primary Pre-Service Teachers (PSTs). Using "voice" as a portable and accessible vehicle to transmit cultural understandings, we draw on the Zimbabwean…

  5. Resource and Production, A Primary Unit in Cultural Geography. Pupil Text and Workbook and Teacher Manual.

    ERIC Educational Resources Information Center

    Imperatore, William

    This is an instructional unit in cultural geography for the primary grades. The major objective of the unit, which is comprised of a Pupil Text/Workbook and Teacher Manual, is to develop the geographic concepts labeled resource and production. Teaching strategies used include the Pestalozzian method of asking leading questions to draw the students…

  6. Primary-Grade Students' Knowledge and Thinking about Shelter as a Cultural Universal.

    ERIC Educational Resources Information Center

    Brophy, Jere; Alleman, Janet

    The traditional K-3 social studies curriculum has focused on food, clothing, shelter, communication, transportation, and other cultural universals. A study was designed to provide information with respect to the topic of shelter, and in the process, to assess claims that primary grade students do not need instruction in the topic because they…

  7. CHANGES IN GENE EXPRESSION DURING DIFFERENTIATION OF CULTURED HUMAN PRIMARY BRONCHIAL EPITHELIAL CELLS

    EPA Science Inventory

    Primary airway epithelial cell cultures are a useful tool for the in vitro study of normal bronchial cell differentiation and function, airway disease mechanisms, and pathogens and toxin response. Growth of these cells at an air-liquid interface for several days results in the f...

  8. Culturally Relevant Literature: What Matters Most to Primary-Age Urban Learners

    ERIC Educational Resources Information Center

    Cartledge, Gwendolyn; Keesey, Susan; Bennett, Jessica G.; Ramnath, Rajiv; Council, Morris R., III.

    2016-01-01

    The ratings and rationales primary-age urban learners gave culturally relevant reading passages was the focus of this descriptive study. First- and second-grade students each read 30 researcher-developed passages reflecting the students' immediate and historical backgrounds. The students rated the passages and gave a reason for their ratings. A…

  9. Knowing in Primary Physical Education in the UK: Negotiating Movement Culture

    ERIC Educational Resources Information Center

    Ward, Gavin; Quennerstedt, Mikael

    2015-01-01

    This paper aims to understand how pupils and teachers actions-in-context constitute being-a-pupil and being-a-teacher within a primary school physical education (PE) movement culture. Dewey and Bentley's theory of transaction, which views organism-in-environment-as-a-whole, enables the researcher to explore how actions-in-ongoing activities…

  10. Glutamate receptor subunit expression in primary neuronal and secondary glial cultures.

    PubMed

    Janssens, N; Lesage, A S

    2001-06-01

    We report on the expression of ionotropic glutamate receptor subunits in primary neuronal cultures from rat cortex, hippocampus and cerebellum and of metabotropic glutamate (mGlu) receptor subtypes in these neuronal cultures as well as in cortical astroglial cultures. We found that the NMDA receptor (NR) subunits NR1, NR2A and NR2B were expressed in all three cultures. Each of the three cultures showed also expression of the four AMPA receptor subunits. Although RT-PCR detected mRNA of all kainate (KA) subunits in the three cultures, western blot showed only expression of Glu6 and KA2 receptor subunits. The expression analysis of mGlu receptors indicated the presence of all mGlu receptor subtype mRNAs in the three neuronal cultures, except for mGlu2 receptor mRNA, which was not detected in the cortical and cerebellar culture. mGlu1a/alpha, -2/3 and -5 receptor proteins were present in all three cultures, whereas mGlu4a and mGlu8a receptor proteins were not detected. Astroglial cultures were grown in either serum-containing or chemically defined medium. Only mGlu5 receptor protein was found in astroglial cultures grown in serum-containing medium. When astrocytes were cultured in chemically defined medium, mGlu3, -5 and -8 receptor mRNAs were detected, but at the protein level, still only mGlu5 receptor was found. PMID:11413230

  11. A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies

    PubMed Central

    Parikh, Mukti R.; Belch, Andrew R.; Pilarski, Linda M; Kirshner, Julia

    2014-01-01

    Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods rely on the ability of cells either to attach to a solid substratum of a tissue culture dish or to grow in suspension in liquid medium. Multiple immortal cell lines have been created and grown using such approaches, however, these methods frequently fail when primary cells need to be grown ex vivo. Such failure has been attributed to the absence of the appropriate extracellular matrix components of the tissue microenvironment from the standard systems where tissue culture plastic is used as a surface for cell growth. Extracellular matrix is an integral component of the tissue microenvironment and its presence is crucial for the maintenance of physiological functions such as cell polarization, survival, and proliferation. Here we present a 3-dimensional tissue culture method where primary bone marrow cells are grown in extracellular matrix formulated to recapitulate the microenvironment of the human bone (rBM system). Embedded in the extracellular matrix, cells are supplied with nutrients through the medium supplemented with human plasma, thus providing a comprehensive system where cell survival and proliferation can be sustained for up to 30 days while maintaining the cellular composition of the primary tissue. Using the rBM system we have successfully grown primary bone marrow cells from normal donors and patients with amyloidosis, and various hematological malignancies. The rBM system allows for direct, in-matrix real time visualization of the cell behavior and evaluation of preclinical efficacy of novel therapeutics. Moreover, cells can be isolated from the rBM and subsequently used for in vivo transplantation, cell sorting, flow cytometry, and nucleic acid and protein analysis. Taken together, the rBM method provides a reliable system for the growth of primary bone marrow cells under physiological conditions

  12. Prolactin mediates neuroprotection against excitotoxicity in primary cell cultures of hippocampal neurons via its receptor.

    PubMed

    Vergara-Castañeda, E; Grattan, D R; Pasantes-Morales, H; Pérez-Domínguez, M; Cabrera-Reyes, E A; Morales, T; Cerbón, M

    2016-04-01

    Recently it has been reported that prolactin (PRL) exerts a neuroprotective effect against excitotoxicity in hippocampus in the rat in vivo models. However, the exact mechanism by which PRL mediates this effect is not completely understood. The aim of our study was to assess whether prolactin exerts neuroprotection against excitotoxicity in an in vitro model using primary cell cultures of hippocampal neurons, and to determine whether this effect is mediated via the prolactin receptor (PRLR). Primary cell cultures of rat hippocampal neurons were used in all experiments, gene expression was evaluated by RT-qPCR, and protein expression was assessed by Western blot analysis and immunocytochemistry. Cell viability was assessed by using the MTT method. The results demonstrated that PRL treatment of neurons from primary cultures did not modify cell viability, but that it exerted a neuroprotective effect, with cells treated with PRL showing a significant increase of viability after glutamate (Glu)--induced excitotoxicity as compared with neurons treated with Glu alone. Cultured neurons expressed mRNA for both PRL and its receptor (PRLR), and both PRL and PRLR expression levels changed after the excitotoxic insult. Interestingly, the PRLR protein was detected as two main isoforms of 100 and 40 kDa as compared with that expressed in hypothalamic cells, which was present only as a 30 kDa variant. On the other hand, PRL was not detected in neuron cultures, either by western blot or by immunohistochemistry. Neuroprotection induced by PRL was significantly blocked by specific oligonucleotides against PRLR, thus suggesting that the PRL role is mediated by its receptor expressed in these neurons. The overall results indicated that PRL induces neuroprotection in neurons from primary cell cultures. PMID:26874070

  13. Prolactin mediates neuroprotection against excitotoxicity in primary cell cultures of hippocampal neurons via its receptor.

    PubMed

    Vergara-Castañeda, E; Grattan, D R; Pasantes-Morales, H; Pérez-Domínguez, M; Cabrera-Reyes, E A; Morales, T; Cerbón, M

    2016-04-01

    Recently it has been reported that prolactin (PRL) exerts a neuroprotective effect against excitotoxicity in hippocampus in the rat in vivo models. However, the exact mechanism by which PRL mediates this effect is not completely understood. The aim of our study was to assess whether prolactin exerts neuroprotection against excitotoxicity in an in vitro model using primary cell cultures of hippocampal neurons, and to determine whether this effect is mediated via the prolactin receptor (PRLR). Primary cell cultures of rat hippocampal neurons were used in all experiments, gene expression was evaluated by RT-qPCR, and protein expression was assessed by Western blot analysis and immunocytochemistry. Cell viability was assessed by using the MTT method. The results demonstrated that PRL treatment of neurons from primary cultures did not modify cell viability, but that it exerted a neuroprotective effect, with cells treated with PRL showing a significant increase of viability after glutamate (Glu)--induced excitotoxicity as compared with neurons treated with Glu alone. Cultured neurons expressed mRNA for both PRL and its receptor (PRLR), and both PRL and PRLR expression levels changed after the excitotoxic insult. Interestingly, the PRLR protein was detected as two main isoforms of 100 and 40 kDa as compared with that expressed in hypothalamic cells, which was present only as a 30 kDa variant. On the other hand, PRL was not detected in neuron cultures, either by western blot or by immunohistochemistry. Neuroprotection induced by PRL was significantly blocked by specific oligonucleotides against PRLR, thus suggesting that the PRL role is mediated by its receptor expressed in these neurons. The overall results indicated that PRL induces neuroprotection in neurons from primary cell cultures.

  14. Primary cell cultures from sea urchin ovaries: a new experimental tool.

    PubMed

    Mercurio, Silvia; Di Benedetto, Cristiano; Sugni, Michela; Candia Carnevali, M Daniela

    2014-02-01

    In the present work, primary cell cultures from ovaries of the edible sea urchin Paracentrotus lividus were developed in order to provide a simple and versatile experimental tool for researches in echinoderm reproductive biology. Ovary cell phenotypes were identified and characterized by different microscopic techniques. Although cell cultures could be produced from ovaries at all stages of maturation, the cells appeared healthier and viable, displaying a higher survival rate, when ovaries at early stages of gametogenesis were used. In terms of culture medium, ovarian cells were successfully cultured in modified Leibovitz-15 medium, whereas poor results were obtained in minimum essential medium Eagle and medium 199. Different substrates were tested, but ovarian cells completely adhered only on poly-L-lysine. To improve in vitro conditions and stimulate cell proliferation, different serum-supplements were tested. Fetal calf serum and an originally developed pluteus extract were detrimental to cell survival, apparently accelerating processes of cell death. In contrast, cells cultured with sea urchin egg extract appeared larger and healthier, displaying an increased longevity that allowed maintaining them for up to 1 month. Overall, our study provides new experimental bases and procedures for producing successfully long-term primary cell cultures from sea urchin ovaries offering a good potential to study echinoid oogenesis in a controlled system and to investigate different aspects of echinoderm endocrinology and reproductive biology.

  15. Trichostatin A, a critical factor in maintaining the functional differentiation of primary cultured rat hepatocytes

    SciTech Connect

    Henkens, Tom . E-mail: Tom.Henkens@vub.ac.be; Papeleu, Peggy; Elaut, Greetje; Vinken, Mathieu; Rogiers, Vera; Vanhaecke, Tamara

    2007-01-01

    Histone deacetylase inhibitors (HDI) have been shown to increase differentiation-related gene expression in several tumor-derived cell lines by hyperacetylating core histones. Effects of HDI on primary cultured cells, however, have hardly been investigated. In the present study, the ability of trichostatin A (TSA), a prototype hydroxamate HDI, to counteract the loss of liver-specific functions in primary rat hepatocyte cultures has been investigated. Upon exposure to TSA, it was found that the cell viability of the cultured hepatocytes and their albumin secretion as a function of culture time were increased. TSA-treated hepatocytes also better maintained cytochrome P450 (CYP)-mediated phase I biotransformation capacity, whereas the activity of phase II glutathione S-transferases (GST) was not affected. Western blot and qRT-PCR analysis of CYP1A1, CYP2B1 and CYP3A11 protein and mRNA levels, respectively, further revealed that TSA acts at the transcriptional level. In addition, protein expression levels of the liver-enriched transcription factors (LETFs) hepatic nuclear factor 4 alpha (HNF4{alpha}) and CCAAT/enhancer binding protein alpha (C/EBP{alpha}) were accordingly increased by TSA throughout culture time. In conclusion, these findings indicate that TSA plays a major role in the preservation of the differentiated hepatic phenotype in culture. It is suggested that the effects of TSA on CYP gene expression are mediated via controlling the expression of LETFs.

  16. Reusable Solid Rocket Motor - Accomplishments, Lessons, and a Culture of Success

    NASA Technical Reports Server (NTRS)

    Moore, Dennis R.; Phelps, Willie J.

    2011-01-01

    The Reusable Solid Rocket Motor represents the largest solid rocket motor ever flown and the only human rated solid motor. Each Reusable Solid Rocket Motor (RSRM) provides approximately 3-million lb of thrust to lift the integrated Space Shuttle vehicle from the launch pad. The motors burn out approximately 2 minutes later, separate from the vehicle and are recovered and refurbished. The size of the motor and the need for high reliability were challenges. Thrust shaping, via shaping of the propellant grain, was needed to limit structural loads during ascent. The motor design evolved through several block upgrades to increase performance and to increase safety and reliability. A major redesign occurred after STS-51L with the Redesigned Solid Rocket Motor. Significant improvements in the joint sealing systems were added. Design improvements continued throughout the Program via block changes with a number of innovations including development of low temperature o-ring materials and incorporation of a unique carbon fiber rope thermal barrier material. Recovery of the motors and post flight inspection improved understanding of hardware performance, and led to key design improvements. Because of the multidecade program duration material obsolescence was addressed, and requalification of materials and vendors was sometimes needed. Thermal protection systems and ablatives were used to protect the motor cases and nozzle structures. Significant understanding of design and manufacturing features of the ablatives was developed during the program resulting in optimization of design features and processing parameters. The project advanced technology in eliminating ozone-depleting materials in manufacturing processes and the development of an asbestos-free case insulation. Manufacturing processes for the large motor components were unique and safety in the manufacturing environment was a special concern. Transportation and handling approaches were also needed for the large

  17. Multi-channel NIRS of the primary motor cortex to discriminate hand from foot activity

    NASA Astrophysics Data System (ADS)

    Koenraadt, K. L. M.; Duysens, J.; Smeenk, M.; Keijsers, N. L. W.

    2012-08-01

    The poor spatial resolution of near-infrared spectroscopy (NIRS) makes it difficult to distinguish two closely located cortical areas from each other. Here, a combination of multi-channel NIRS and a centre of gravity (CoG) approach (widely accepted in the field of transcranial magnetic stimulation; TMS) was used to discriminate between closely located cortical areas activated during hand and foot movements. Similarly, the possibility of separating the more anteriorly represented discrete movements from rhythmic movements was studied. Thirteen healthy right-handed subjects performed rhythmic or discrete (‘task’) hand or foot (‘extremity’) tapping. Hemodynamic responses were measured using an 8-channel NIRS setup. For oxyhemoglobin (OHb) and deoxyhemoglobin (HHb), a CoG was determined for each condition using the mean hemodynamic responses and the coordinates of the channels. Significant hemodynamic responses were found for hand and foot movements. Based on the HHb responses, the NIRS-CoG of hand movements was located 0.6 cm more laterally compared to the NIRS-CoG of foot movements. For OHb responses no difference in NIRS-CoG was found for ‘extremity’ nor for ‘task’. This is the first NIRS study showing hemodynamic responses for isolated foot movements. Furthermore, HHb responses have the potential to be used in multi-channel NIRS experiments requiring differential activation of motor cortex areas linked to either hand or foot movements.

  18. Primary care units in Emilia-Romagna, Italy: an assessment of organizational culture.

    PubMed

    Pracilio, Valerie P; Keith, Scott W; McAna, John; Rossi, Giuseppina; Brianti, Ettore; Fabi, Massimo; Maio, Vittorio

    2014-01-01

    This study investigates the organizational culture and associated characteristics of the newly established primary care units (PCUs)-collaborative teams of general practitioners (GPs) who provide patients with integrated health care services-in the Emilia-Romagna Region (RER), Italy. A survey instrument covering 6 cultural dimensions was administered to all 301 GPs in 21 PCUs in the Local Health Authority (LHA) of Parma, RER; the response rate was 79.1%. Management style, organizational trust, and collegiality proved to be more important aspects of PCU organizational culture than information sharing, quality, and cohesiveness. Cultural dimension scores were positively associated with certain characteristics of the PCUs including larger PCU size and greater proportion of older GPs. The presence of female GPs in the PCUs had a negative impact on collegiality, organizational trust, and quality. Feedback collected through this assessment will be useful to the RER and LHAs for evaluating and guiding improvements in the PCUs.

  19. Extracellular matrix components influence DNA synthesis of rat hepatocytes in primary culture

    SciTech Connect

    Sawada, N.; Tomomura, A.; Sattler, C.A.; Sattler, G.L.; Kleinman, H.K.; Pitot, H.C.

    1986-12-01

    The effects of several extracellular matrix components (EMCs) - fibronectin (Fn), laminin (Ln), type I (C-I) and type IV (C-IV) collagen - on DNA synthesis in rat hepatocytes in primary culture were examined by both quantitative scintillation spectrometry and autoradiography of (/sup 3/H)thymidine incorporation. Hepatocytes cultured on Fn showed the most active DNA synthesis initiated by epidermal growth factor (EGF) with decreasing levels of (/sup 3/H)thymidine uptake exhibited in the cell cultured on C-IV, C-I, and Ln, respectively. The decreasing level of DNA synthesis in hepatocytes cultured on Fn, C-IV, C-I, and Ln respectively was not influenced by cell density. The number of EGF receptors of hepatocytes was also not influenced by EMCs. These data suggest that EMCs modify hepatocyte DNA synthesis by means of post-EGF-receptor mechanisms which are regulated by both growth factors and cell density.

  20. Differential induction of Pax genes by NGF and BDNF in cerebellar primary cultures

    PubMed Central

    1994-01-01

    The Pax genes encode sequence-specific DNA binding transcription factors that are expressed in embryonic development of the nervous system. Primary neuronal cell cultures derived from the cerebellar cortex of embryonic day 14, newborn and 7-d old mice, were used to investigate the cell-type specific expression patterns of three members of the murine paired box containing gene family (Pax gene family), in vitro. Cell types which express Pax-2, Pax-3, and Pax-6 RNA in primary cultures correspond to those found in regions of the cerebellum which show RNA signals in sections of the developing mouse brain. To find mechanisms regulating Pax gene expression during cerebellar development, the differential regulation of Pax-2, Pax-3, and Pax-6 by NGF and BDNF, two structurally related neurotrophins, was studied in such primary cultures. Pax-2 and Pax-6 RNA increased slightly by 1 h and remained elevated throughout a 24-h treatment with BDNF and NGF. Pax-3 RNA was not detected in newborn cultures, but underwent a rapid (1 h) and transient (2 h) induction upon treatment with either BDNF or NGF. No response was seen with EGF or FGF. Cycloheximide treatment amplified Pax-3 induction and prolonged the signal. Thus, Pax-3 induction resembles that of the immediate-early gene c-fos, which transduces growth factor signals during the development of particular neuronal/glial cell types. The changes in Pax expression were inductive rather than trophic. PMID:8163557

  1. Preclinical Assessment of the Anticancer Drug Response of Plexiform Neurofibroma Tissue Using Primary Cultures

    PubMed Central

    Mautner, Victor-F.; Friedrich, Reinhard E.; Kluwe, Lan

    2015-01-01

    Background and Purpose Individualized drug testing for tumors using a strategy analogous to antibiotic tests for infectious diseases would be highly desirable for personalized and individualized cancer care. Methods Primary cultures containing tumor and nontumor stromal cells were utilized in a novel strategy to test drug responses with respect to both efficacy and specificity. The strategy tested in this pilot study was implemented using four primary cultures derived from plexiform neurofibromas. Responses to two cytotoxic drugs (nilotinib and imatinib) were measured by following dose-dependent changes in the proportions of tumor and nontumor cells, determined by staining them with cell-type-specific antibodies. The viability of the cultured cells and the cytotoxic effect of the drugs were also measured using proliferation and cytotoxicity assays. Results The total number of cells decreased after the drug treatment, in accordance with the observed reduction in proliferation and increased cytotoxic effect upon incubation with the two anticancer drugs. The proportions of Schwann cells and fibroblasts changed dose-dependently, although the patterns of change varied between the tumor samples (from different sources) and between the two drugs. The highly variable in vitro drug responses probably reflect the large variations in the responses of tumors to therapies between individual patients in vivo. Conclusions These preliminary results suggest that the concept of assessing in vitro drug responses using primary cultures is feasible, but demands the extensive further development of an application for preclinical drug selection and drug discovery. PMID:25851896

  2. A Simplified Method for Ultra-Low Density, Long-Term Primary Hippocampal Neuron Culture.

    PubMed

    Lu, Zhongming; Piechowicz, Mariel; Qiu, Shenfeng

    2016-01-01

    Culturing primary hippocampal neurons in vitro facilitates mechanistic interrogation of many aspects of neuronal development. Dissociated embryonic hippocampal neurons can often grow successfully on glass coverslips at high density under serum-free conditions, but low density cultures typically require a supply of trophic factors by co-culturing them with a glia feeder layer, preparation of which can be time-consuming and laborious. In addition, the presence of glia may confound interpretation of results and preclude studies on neuron-specific mechanisms. Here, a simplified method is presented for ultra-low density (~2,000 neurons/cm2), long-term (>3 months) primary hippocampal neuron culture that is under serum free conditions and without glia cell support. Low density neurons are grown on poly-D-lysine coated coverslips, and flipped on high density neurons grown in a 24-well plate. Instead of using paraffin dots to create a space between the two neuronal layers, the experimenters can simply etch the plastic bottom of the well, on which the high density neurons reside, to create a microspace conducive to low density neuron growth. The co-culture can be easily maintained for >3 months without significant loss of low density neurons, thus facilitating the morphological and physiological study of these neurons. To illustrate this successful culture condition, data are provided to show profuse synapse formation in low density cells after prolonged culture. This co-culture system also facilitates the survival of sparse individual neurons grown in islands of poly-D-lysine substrates and thus the formation of autaptic connections. PMID:27022758

  3. Reusable Solid Rocket Motor - Accomplishment, Lessons, and a Culture of Success

    NASA Technical Reports Server (NTRS)

    Moore, D. R.; Phelps, W. J.

    2011-01-01

    The Reusable Solid Rocket Motor (RSRM) represents the largest solid rocket motor (SRM) ever flown and the only human-rated solid motor. High reliability of the RSRM has been the result of challenges addressed and lessons learned. Advancements have resulted by applying attention to process control, testing, and postflight through timely and thorough communication in dealing with all issues. A structured and disciplined approach was taken to identify and disposition all concerns. Careful consideration and application of alternate opinions was embraced. Focus was placed on process control, ground test programs, and postflight assessment. Process control is mandatory for an SRM, because an acceptance test of the delivered product is not feasible. The RSRM maintained both full-scale and subscale test articles, which enabled continuous improvement of design and evaluation of process control and material behavior. Additionally RSRM reliability was achieved through attention to detail in post flight assessment to observe any shift in performance. The postflight analysis and inspections provided invaluable reliability data as it enables observation of actual flight performance, most of which would not be available if the motors were not recovered. RSRM reusability offered unique opportunities to learn about the hardware. NASA is moving forward with the Space Launch System that incorporates propulsion systems that takes advantage of the heritage Shuttle and Ares solid motor programs. These unique challenges, features of the RSRM, materials and manufacturing issues, and design improvements will be discussed in the paper.

  4. Bicuculline induces synapse formation on primary cultured accessory olfactory bulb neurons.

    PubMed

    Kato-Negishi, Midori; Muramoto, Kazuyo; Kawahara, Masahiro; Hosoda, Ritsuko; Kuroda, Yoichiro; Ichikawa, Masumi

    2003-09-01

    To investigate the roles of the GABAergic inhibitory system of accessory olfactory bulb (AOB) in pheromonal memory formation, we have developed a primary culture system of AOB neurons, which had numerous excitatory and inhibitory synapses. Using this culture system of AOB neurons, we examined the correlation in rats between neuronal excitation and synaptic morphology by bicuculline-induced disinhibition of cultured AOB neurons. The exposure to bicuculline induced long-lasting oscillatory changes in the intracellular calcium level ([Ca2+]in) of cultured non-GABAergic multipolar neurons, which were identified as mitral/tufted cells (MT cells). These MT cells exhibited the appearance of dendritic filopodia structures after a 10-min treatment with bicuculline. By labelling presynaptic terminals with FM4-64, the appearance of new presynaptic terminals was clearly observed on newly formed filopodia after 120 min treatment with bicuculline. These results suggest that bicuculline-induced [Ca2+]in oscillation of MT cells induces the growth of filopodia and subsequently the formation of new presynaptic terminals. Furthermore, tetrodotoxin or the deprivation of extracellular calcium blocked bicuculline-induced synapse formation. The present results indicate that the long-lasting [Ca2+]in oscillation caused by bicuculline-induced disinhibition of cultured MT cells is significantly implicated in the mechanism underlying synapse formation on cultured AOB neurons. Our established culture system of AOB neurons will aid in clarifying the mechanism of synapse formation between AOB neurons and the molecular mechanism of pheromonal memory formation. PMID:14511315

  5. Disparate effects of serum on basal and evoked NFAT activity in primary astrocyte cultures.

    PubMed

    Furman, Jennifer L; Artiushin, Irina A; Norris, Christopher M

    2010-01-29

    In astrocytes, the Ca(2+)-dependent protein phosphatase calcineurin (CN) strongly regulates neuro-immune/inflammatory cascades through activation of the transcription factor, nuclear factor of activated T cells (NFAT). While primary cell cultures provide a useful model system for investigating astrocytic CN/NFAT signaling, variable results may arise both within and across labs because of differences in culture conditions. Here, we determined the extent to which serum and cell confluency affect basal and evoked astrocytic NFAT activity in primary cortical astrocyte cultures. Cells were grown to either approximately 50% or >90% confluency, pre-loaded with an NFAT-luciferase reporter construct, and maintained for 16 h in medium with or without 10% fetal bovine serum (FBS). NFAT-dependent luciferase expression was then measured 5h after treatment with vehicle alone to assess basal NFAT activity, or with Ca(2+) mobilizers and IL-1 beta to assess evoked activity. The results revealed significantly higher levels of basal NFAT activity in FBS-containing medium, regardless of cell confluency. Conversely, evoked NFAT activation was significantly lower in serum-containing medium, with an even greater inhibition observed in confluent cultures. Application of 10% FBS to serum-free astrocyte cultures quickly evoked a roughly seven-fold increase in NFAT activity that was significantly reduced by co-delivery of neutralizing agents for IL-1 beta, TNFalpha, and/or IFN gamma, suggesting that serum occludes evoked NFAT activation through a cytokine-based mechanism. Together, the results demonstrate that the presence of serum and cell confluency have a major impact on CN/NFAT signaling in primary astrocyte cultures and therefore must be taken into consideration when using this model system.

  6. Isolation, primary culture and morphological characterization of oenocytes from Aedes aegypti pupae.

    PubMed

    Martins, G F; Guedes, B A M; Silva, L M; Serrão, J E; Fortes-Dias, C L; Ramalho-Ortigão, J M; Pimenta, P F P

    2011-04-01

    Oenocytes are ectodermic cells that participate in a number of critical physiological roles such as detoxification and lipid storage and metabolism in insects. In light of the lack of information on oenocytes from Aedes aegypti and the potential role of these cells in the biology of this major yellow fever and dengue vector, we developed a protocol to purify and maintain Ae. aegypti pupa oenocytes in primary culture. Ae. aegypti oenocytes were cultured as clustered and as isolated ovoid cells with a smooth surface. Our results demonstrate that these cells remain viable in cell culture for at least two months. We also investigated their morphology in vivo and in vitro using light, confocal, scanning and transmission electron microscopes. This work is the first successful attempt in isolating and maintaining Ae. aegypti oenocytes in culture, and a significant step towards understanding the role of this cell type in this important disease vector. The purification and the development of primary cultures of insect oenocytes will allow future studies of their metabolism in producing and secreting compounds.

  7. Lithium treatment elongates primary cilia in the mouse brain and in cultured cells

    SciTech Connect

    Miyoshi, Ko; Kasahara, Kyosuke; Miyazaki, Ikuko; Asanuma, Masato

    2009-10-30

    The molecular mechanisms underlying the therapeutic effects of lithium, a first-line antimanic mood stabilizer, have not yet been fully elucidated. Treatment of the algae Chlamydomonas reinhardtii with lithium has been shown to induce elongation of their flagella, which are analogous structures to vertebrate cilia. In the mouse brain, adenylyl cyclase 3 (AC3) and certain neuropeptide receptors colocalize to the primary cilium of neuronal cells, suggesting a chemosensory function for the primary cilium in the nervous system. Here we show that lithium treatment elongates primary cilia in the mouse brain and in cultured cells. Brain sections from mice chronically fed with Li{sub 2}CO{sub 3} were subjected to immunofluorescence study. Primary cilia carrying both AC3 and the receptor for melanin-concentrating hormone (MCH) were elongated in the dorsal striatum and nucleus accumbens of lithium-fed mice, as compared to those of control animals. Moreover, lithium-treated NIH3T3 cells and cultured striatal neurons exhibited elongation of the primary cilia. The present results provide initial evidence that a psychotropic agent can affect ciliary length in the central nervous system, and furthermore suggest that lithium exerts its therapeutic effects via the upregulation of cilia-mediated MCH sensing. These findings thus contribute novel insights into the pathophysiology of bipolar mood disorder and other psychiatric diseases.

  8. Axitinib affects cell viability and migration of a primary foetal lung adenocarcinoma culture.

    PubMed

    Menna, Cecilia; De Falco, Elena; Pacini, Luca; Scafetta, Gaia; Ruggieri, Paola; Puca, Rosa; Petrozza, Vincenzo; Ciccone, Anna Maria; Rendina, Erino Angelo; Calogero, Antonella; Ibrahim, Mohsen

    2014-01-01

    Fetal lung adenocarcinoma (FLAC) is a rare variant of lung adenocarcinoma. Studies regarding FLAC have been based only on histopathological observations, thus representative in vitro models of FLAC cultures are unavailable. We have established and characterized a human primary FLAC cell culture, exploring its biology, chemosensitivity, and migration. FLAC cells and specimen showed significant upregulation of VEGF165 and HIF-1α mRNA levels. This observation was confirmed by in vitro chemosensitivity and migration assay, showing that only Axitinib was comparable to Cisplatin treatment. We provide a suitable in vitro model to further investigate the nature of this rare type of cancer. PMID:24380379

  9. Isolated Primary Blast Inhibits Long-Term Potentiation in Organotypic Hippocampal Slice Cultures.

    PubMed

    Vogel, Edward W; Effgen, Gwen B; Patel, Tapan P; Meaney, David F; Bass, Cameron R Dale; Morrison, Barclay

    2016-04-01

    Over the last 13 years, traumatic brain injury (TBI) has affected over 230,000 U.S. service members through the conflicts in Iraq and Afghanistan, mostly as a result of exposure to blast events. Blast-induced TBI (bTBI) is multi-phasic, with the penetrating and inertia-driven phases having been extensively studied. The effects of primary blast injury, caused by the shockwave interacting with the brain, remain unclear. Earlier in vivo studies in mice and rats have reported mixed results for primary blast effects on behavior and memory. Using a previously developed shock tube and in vitro sample receiver, we investigated the effect of isolated primary blast on the electrophysiological function of rat organotypic hippocampal slice cultures (OHSC). We found that pure primary blast exposure inhibited long-term potentiation (LTP), the electrophysiological correlate of memory, with a threshold between 9 and 39 kPa·ms impulse. This deficit occurred well below a previously identified threshold for cell death (184 kPa·ms), supporting our previously published finding that primary blast can cause changes in brain function in the absence of cell death. Other functional measures such as spontaneous activity, network synchronization, stimulus-response curves, and paired-pulse ratios (PPRs) were less affected by primary blast exposure, as compared with LTP. This is the first study to identify a tissue-level tolerance threshold for electrophysiological changes in neuronal function to isolated primary blast.

  10. Primary motor cortex changes after amputation correlate with phantom limb pain and the ability to move the phantom limb.

    PubMed

    Raffin, Estelle; Richard, Nathalie; Giraux, Pascal; Reilly, Karen T

    2016-04-15

    A substantial body of evidence documents massive reorganization of primary sensory and motor cortices following hand amputation, the extent of which is correlated with phantom limb pain. Many therapies for phantom limb pain are based upon the idea that plastic changes after amputation are maladaptive and attempt to normalize representations of cortical areas adjacent to the hand area. Recent data suggest, however, that higher levels of phantom pain are associated with stronger local activity and more structural integrity in the missing hand area rather than with reorganization of neighbouring body parts. While these models appear to be mutually exclusive they could co-exist, and one reason for the apparent discrepancy between them might be that no single study has examined the organisation of lip, elbow, and hand movements in the same participants. In this study we thoroughly examined the 3D anatomy of the central sulcus and BOLD responses during movements of the hand, elbow, and lips using MRI techniques in 11 upper-limb amputees and 17 healthy control subjects. We observed different reorganizational patterns for all three body parts as the former hand area showed few signs of reorganization, but the lip and elbow representations reorganized and shifted towards the hand area. We also found that poorer voluntary control and higher levels of pain in the phantom limb were powerful drivers of the lip and elbow topological changes. In addition to providing further support for the maladaptative plasticity model, we demonstrate for the first time that motor capacities of the phantom limb correlate with post-amputation reorganization, and that this reorganization is not limited to the face and hand representations but also includes the proximal upper-limb. PMID:26854561

  11. Primary motor cortex changes after amputation correlate with phantom limb pain and the ability to move the phantom limb.

    PubMed

    Raffin, Estelle; Richard, Nathalie; Giraux, Pascal; Reilly, Karen T

    2016-04-15

    A substantial body of evidence documents massive reorganization of primary sensory and motor cortices following hand amputation, the extent of which is correlated with phantom limb pain. Many therapies for phantom limb pain are based upon the idea that plastic changes after amputation are maladaptive and attempt to normalize representations of cortical areas adjacent to the hand area. Recent data suggest, however, that higher levels of phantom pain are associated with stronger local activity and more structural integrity in the missing hand area rather than with reorganization of neighbouring body parts. While these models appear to be mutually exclusive they could co-exist, and one reason for the apparent discrepancy between them might be that no single study has examined the organisation of lip, elbow, and hand movements in the same participants. In this study we thoroughly examined the 3D anatomy of the central sulcus and BOLD responses during movements of the hand, elbow, and lips using MRI techniques in 11 upper-limb amputees and 17 healthy control subjects. We observed different reorganizational patterns for all three body parts as the former hand area showed few signs of reorganization, but the lip and elbow representations reorganized and shifted towards the hand area. We also found that poorer voluntary control and higher levels of pain in the phantom limb were powerful drivers of the lip and elbow topological changes. In addition to providing further support for the maladaptative plasticity model, we demonstrate for the first time that motor capacities of the phantom limb correlate with post-amputation reorganization, and that this reorganization is not limited to the face and hand representations but also includes the proximal upper-limb.

  12. Peripheral facial nerve lesions induce changes in the firing properties of primary motor cortex layer 5 pyramidal cells.

    PubMed

    Múnera, A; Cuestas, D M; Troncoso, J

    2012-10-25

    Facial nerve lesions elicit long-lasting changes in vibrissal primary motor cortex (M1) muscular representation in rodents. Reorganization of cortical representation has been attributed to potentiation of preexisting horizontal connections coming from neighboring muscle representation. However, changes in layer 5 pyramidal neuron activity induced by facial nerve lesion have not yet been explored. To do so, the effect of irreversible facial nerve injury on electrophysiological properties of layer 5 pyramidal neurons was characterized. Twenty-four adult male Wistar rats were randomly subjected to two experimental treatments: either surgical transection of mandibular and buccal branches of the facial nerve (n=18) or sham surgery (n=6). Unitary and population activity of vibrissal M1 layer 5 pyramidal neurons recorded in vivo under general anesthesia was compared between sham-operated and facial nerve-injured animals. Injured animals were allowed either one (n=6), three (n=6), or five (n=6) weeks recovery before recording in order to characterize the evolution of changes in electrophysiological activity. As compared to control, facial nerve-injured animals displayed the following sustained and significant changes in spontaneous activity: increased basal firing frequency, decreased spike-associated local field oscillation amplitude, and decreased spontaneous theta burst firing frequency. Significant changes in evoked-activity with whisker pad stimulation included: increased short latency population spike amplitude, decreased long latency population oscillations amplitude and frequency, and decreased peak frequency during evoked single-unit burst firing. Taken together, such changes demonstrate that peripheral facial nerve lesions induce robust and sustained changes of layer 5 pyramidal neurons in vibrissal motor cortex. PMID:22877641

  13. Responses of single corticospinal neurons to intracortical stimulation of primary motor and premotor cortex in the anesthetized macaque monkey.

    PubMed

    Maier, Marc A; Kirkwood, Peter A; Brochier, Thomas; Lemon, Roger N

    2013-06-01

    The responses of individual primate corticospinal neurons to localized electrical stimulation of primary motor (M1) and of ventral premotor cortex (area F5) are poorly documented. To rectify this and to study interactions between responses from these areas, we recorded corticospinal axons, identified by pyramidal tract stimulation, in the cervical spinal cord of three chloralose-anesthetized macaque monkeys. Single stimuli (≤400 μA) were delivered to the hand area of M1 or F5 through intracortical microwire arrays. Only 14/112 (13%) axons showed responses to M1 stimuli that indicated direct intracortical activation of corticospinal neurons (D-responses); no D-responses were seen from F5. In contrast, 62 axons (55%) exhibited consistent later responses to M1 stimulation, corresponding to indirect activation (I-responses), showing that single-pulse intracortical stimulation of motor areas can result in trans-synaptic activation of a high proportion of the corticospinal output. A combined latency histogram of all axon responses was nonperiodic, clearly different from the periodic surface-recorded corticospinal volleys. This was readily explained by correcting for conduction velocities of individual axons. D-responding axons, taken as originating in neurons close to the M1 stimulating electrodes, showed more I-responses from M1 than those without a D-response, and 8/10 of these axons also responded to F5 stimulation. Altogether, 33% of tested axons responded to F5 stimulation, most of which also showed I-responses from M1. These excitatory effects are in keeping with facilitation of hand muscles evoked from F5 being relayed via M1. This was further demonstrated by facilitation of test responses from M1 by conditioning F5 stimuli. PMID:23536718

  14. Effects of Trichostatin A on drug uptake transporters in primary rat hepatocyte cultures.

    PubMed

    Ramboer, Eva; Rogiers, Vera; Vanhaecke, Tamara; Vinken, Mathieu

    2015-01-01

    The present study was set up to investigate the effects of Trichostatin A (TSA), a prototypical epigenetic modifier, on the expression and activity of hepatic drug uptake transporters in primary cultured rat hepatocytes. To this end, the expression of the sinusoidal transporters sodium-dependent taurocholate cotransporting polypeptide (Ntcp) and organic anion transporting polypeptide 4 (Oatp4) was monitored by real-time quantitative reverse transcriptase polymerase chain reaction analysis and immunoblotting. The activity of the uptake transporters was analyzed using radiolabeled substrates and chemical inhibitors. Downregulation of the expression and activity of Oatp4 and Ntcp was observed as a function of the cultivation time and could not be counteracted by TSA. In conclusion, the epigenetic modifier TSA does not seem to exert a positive effect on the expression and activity of the investigated uptake transporters in primary rat hepatocyte cultures. PMID:26648816

  15. [Primary health care reform and implications for the organizational culture of Health Center Groups in Portugal].

    PubMed

    Leone, Claudia; Dussault, Gilles; Lapão, Luís Velez

    2014-01-01

    The health sector's increasing complexity poses major challenges for administrators. There is considerable consensus on workforce quality as a key determinant of success for any health reform. This study aimed to explore the changes introduced by an action-training intervention in the organizational culture of the 73 executive directors of Health Center Groups (ACES) in Portugal during the primary health care reform. The study covers two periods, before and after the one-year ACES training, during which the data were collected and analyzed. The Competing Values Framework allowed observing that after the ACES action-training intervention, the perceptions of the executive directors regarding their organizational culture were more aligned with the practices and values defended by the primary health care reform. The study highlights the need to continue monitoring results over different time periods to elaborate further conclusions.

  16. Primary fibroblasts cultures reveal TDP-43 abnormalities in amyotrophic lateral sclerosis patients with and without SOD1 mutations.

    PubMed

    Sabatelli, Mario; Zollino, Marcella; Conte, Amelia; Del Grande, Alessandra; Marangi, Giuseppe; Lucchini, Matteo; Mirabella, Massimiliano; Romano, Angela; Piacentini, Roberto; Bisogni, Giulia; Lattante, Serena; Luigetti, Marco; Rossini, Paolo Maria; Moncada, Alice

    2015-05-01

    TAR DNA-binding protein 43 (TDP-43) is a major component of the pathologic inclusions observed in the motor neurons of amyotrophic lateral sclerosis (ALS) patients. We examined TDP-43 expression in primary fibroblasts cultures from 22 ALS patients, including cases with SOD1 (n = 4), TARDBP (n = 4), FUS (n = 2), and C9ORF72 (n = 3) mutations and 9 patients without genetic defect. By using a phosphorylation-independent antibody, 15 patients showed notable alterations of TDP-43 level in the nuclear or cytoplasmic compartments. In particular, a marked accumulation of TDP-43 was observed in the cytoplasm of all cases with C9ORF72 and TARDBP mutations, 1 patient with FUS mutation and 3 patients without genetic defect. Patients with SOD1 mutations revealed a significant reduction of TDP-43 in the nuclei without cytoplasmic mislocalization. These changes were associated with the presence of truncated and phosphorylated TDP-43 species. Our results show that fibroblasts recapitulate some of hallmark TDP-43 abnormalities observed in neuronal cells. The reduction of full-length TDP-43 level in mutant SOD1 cells indicates that at least some SOD1 mutations alter TDP-43 metabolism.

  17. Inhalation of primary motor vehicle emissions: Effects of urbanpopulation and land area

    SciTech Connect

    Marshall, Julian D.; McKone, Thomas E.; Nazaroff, William W.

    2004-06-14

    Urban population density can influence transportation demand, as expressed through average daily vehicle-kilometers traveled per capita (VKT). In turn, changes in transportation demand influence total passenger vehicle emissions. Population density can also influence the fraction of total emissions that are inhaled by the exposed urban population. Equations are presented that describe these relationships for an idealized representation of an urban area. Using analytic solutions to these equations, we investigate the effect of three changes in urban population and urban land area (infill, sprawl, and constant-density growth) on per capita inhalation intake of primary pollutants from passenger vehicles. The magnitude of these effects depends on density-emissions elasticity ({var_epsilon}{sub e}), a normalized derivative relating change in population density to change in vehicle emissions. For example, if urban population increases, per capita intake is less with infill development than with constant-density growth if {var_epsilon}{sub e} is less than -0.5, while for {var_epsilon}{sub e} greater than -0.5 the reverse is true.

  18. Habitus and Flow in Primary School Musical Practice: Relations between Family Musical Cultural Capital, Optimal Experience and Music Participation

    ERIC Educational Resources Information Center

    Valenzuela, Rafael; Codina, Nuria

    2014-01-01

    Based on Bourdieu's idea that cultural capital is strongly related to family context, we describe the relations between family musical cultural capital and optimal experience during compulsory primary school musical practice. We analyse whether children from families with higher levels of musical cultural capital, and specifically with regard…

  19. Human Immunodeficiency Virus Type 1 Coat Protein Neurotoxicity Mediated by Nitric Oxide in Primary Cortical Cultures

    NASA Astrophysics Data System (ADS)

    Dawson, Valina L.; Dawson, Ted M.; Uhl, George R.; Snyder, Solomon H.

    1993-04-01

    The human immunodeficiency virus type 1 coat protein, gp120, kills neurons in primary cortical cultures at low picomolar concentrations. The toxicity requires external glutamate and calcium and is blocked by glutamate receptor antagonists. Nitric oxide (NO) contributes to gp120 toxicity, since nitroarginine, an inhibitor of NO synthase, prevents toxicity as does deletion of arginine from the incubation medium and hemoglobin, which binds NO. Superoxide dismutase also attenuates toxicity, implying a role for superoxide anions.

  20. The Cultural Aspect of Foreign Languages Teaching at Primary School in Turkey

    ERIC Educational Resources Information Center

    Sadik, Turkoglu; Omer, Kocer

    2011-01-01

    Learning a foreign language is a new concept at primary school in Turkey. Even if the Minister of National Education has been thinking of it for a long time, it has only been compulsory since 2006. Pedagogues are still doing research on the way to teach a new language to children. One of the ideas is to base this new learning on cultural contents.…

  1. Characterization of a primary brown adipocyte culture system derived from human fetal interscapular fat

    PubMed Central

    Seiler, Sarah E; Xu, Dan; Ho, Jia-Pei; Lo, Kinyui Alice; Buehrer, Benjamin M; Ludlow, Y John W; Kovalik, Jean-Paul; Sun, Lei

    2015-01-01

    Brown fat has gained widespread attention as a potential therapeutic target to treat obesity and associated metabolic disorders. Indeed, the anti-obesity potential of multiple targets to stimulate both brown adipocyte differentiation and recruitment have been verified in rodent models. However, their therapeutic potential in humans is unknown due to the lack of a human primary brown adipocyte cell culture system. Likewise, the lack of a well-characterized human model has limited the discovery of novel targets for the activation of human brown fat. To address this current need, we aimed to identify and describe the first primary brown adipocyte cell culture system from human fetal interscapular brown adipose tissue. Pre-adipocytes isolated from non-viable human fetal interscapular tissue were expanded and cryopreserved. Cells were then thawed and plated alongside adult human subcutaneous and omental pre-adipocytes for subsequent differentiation and phenotypic characterization. Interscapular pre-adipocytes in cell culture differentiated into mature adipocytes that were morphologically indistinguishable from the adult white depots. Throughout differentiation, cultured human fetal interscapular adipocytes demonstrated increased expression of classical brown fat markers compared to subcutaneous and omental cells. Further, functional analysis revealed an elevation in fatty acid oxidation as well as maximal and uncoupled oxygen consumption in interscapular brown adipocytes compared to white control cells. These data collectively identify the brown phenotype of these cells. Thus, our primary cell culture system derived from non-viable human fetal interscapular brown adipose tissue provides a valuable tool for the study of human brown adipocyte biology and for the development of anti-obesity therapeutics. PMID:26451287

  2. Corticospinal Inputs to Primate Motoneurons Innervating the Forelimb from Two Divisions of Primary Motor Cortex and Area 3a

    PubMed Central

    Witham, Claire L.; Fisher, Karen M.; Edgley, Steve A.

    2016-01-01

    Previous anatomical work in primates has suggested that only corticospinal axons originating in caudal primary motor cortex (“new M1”) and area 3a make monosynaptic cortico-motoneuronal connections with limb motoneurons. By contrast, the more rostral “old M1” is proposed to control motoneurons disynaptically via spinal interneurons. In six macaque monkeys, we examined the effects from focal stimulation within old and new M1 and area 3a on 135 antidromically identified motoneurons projecting to the upper limb. EPSPs with segmental latency shorter than 1.2 ms were classified as definitively monosynaptic; these were seen only after stimulation within new M1 or at the new M1/3a border (incidence 6.6% and 1.3%, respectively; total n = 27). However, most responses had longer latencies. Using measures of the response facilitation after a second stimulus compared with the first, and the reduction in response latency after a third stimulus compared with the first, we classified these late responses as likely mediated by either long-latency monosynaptic (n = 108) or non-monosynaptic linkages (n = 108). Both old and new M1 generated putative long-latency monosynaptic and non-monosynaptic effects; the majority of responses from area 3a were non-monosynaptic. Both types of responses from new M1 had significantly greater amplitude than those from old M1. We suggest that slowly conducting corticospinal fibers from old M1 generate weak late monosynaptic effects in motoneurons. These may represent a stage in control of primate motoneurons by the cortex intermediate between disynaptic output via an interposed interneuron seen in nonprimates and the fast direct monosynaptic connections present in new M1. SIGNIFICANCE STATEMENT The corticospinal tract in Old World primates makes monosynaptic connections to motoneurons; previous anatomical work suggests that these connections come only from corticospinal tract (CST) neurons in the subdivision of primary motor cortex within the

  3. Diabetes increases susceptibility of primary cultures of rat proximal tubular cells to chemically induced injury

    SciTech Connect

    Zhong Qing; Terlecky, Stanley R.; Lash, Lawrence H.

    2009-11-15

    Diabetic nephropathy is characterized by increased oxidative stress and mitochondrial dysfunction. In the present study, we prepared primary cultures of proximal tubular (PT) cells from diabetic rats 30 days after an ip injection of streptozotocin and compared their susceptibility to oxidants (tert-butyl hydroperoxide, methyl vinyl ketone) and a mitochondrial toxicant (antimycin A) with that of PT cells isolated from age-matched control rats, to test the hypothesis that PT cells from diabetic rats exhibit more cellular and mitochondrial injury than those from control rats when exposed to these toxicants. PT cells from diabetic rats exhibited higher basal levels of reactive oxygen species (ROS) and higher mitochondrial membrane potential, demonstrating that the PT cells maintain the diabetic phenotype in primary culture. Incubation with either the oxidants or mitochondrial toxicant resulted in greater necrotic and apoptotic cell death, greater evidence of morphological damage, greater increases in ROS, and greater decreases in mitochondrial membrane potential in PT cells from diabetic rats than in those from control rats. Pretreatment with either the antioxidant N-acetyl-L-cysteine or a catalase mimetic provided equivalent protection of PT cells from both diabetic and control rats. Despite the greater susceptibility to oxidative and mitochondrial injury, both cytoplasmic and mitochondrial glutathione concentrations were markedly higher in PT cells from diabetic rats, suggesting an upregulation of antioxidant processes in diabetic kidney. These results support the hypothesis that primary cultures of PT cells from diabetic rats are a valid model in which to study renal cellular function in the diabetic state.

  4. Polystyrene-coated micropallets for culture and separation of primary muscle cells

    PubMed Central

    Detwiler, David A.; Dobes, Nicholas C.; Sims, Christopher E.; Kornegay, Joseph N.; Allbritton, Nancy L.

    2011-01-01

    Despite identification of a large number of adult stem cell types, current primary cell isolation and identification techniques yield heterogeneous samples, making detailed biological studies challenging. To identify subsets of isolated cells, technologies capable of simultaneous cell culture and cloning are necessary. Micropallet arrays, a new cloning platform for adherent cell types, hold great potential. However, the microstructures composing these arrays are fabricated from an epoxy photoresist 1002F, a growth surface unsuitable for many cell types. Optimization of the microstructures’ surface properties was conducted for the culture of satellite cells, primary muscle cells for which improved cell isolation techniques are desired. A variety of surface materials were screened for satellite cell adhesion and proliferation and compared to their optimal substrate, gelatin-coated Petri dishes. A 1-μm thick, polystyrene copolymer was applied to the microstructures by contact-printing. A negatively charged copolymer of 5% acrylic acid in 95% styrene was found to be equivalent to the control Petri dishes for cell adhesion and proliferation. Cells cultured on control dishes and optimal copolymer-coated surfaces maintained an undifferentiated state and showed similar mRNA expression for two genes indicative of cell differentiation during a standard differentiation protocol. Experiments using additional contact-printed layers of extracellular matrix proteins collagen and gelatin showed no further improvements. This micropallet coating strategy is readily adaptable to optimize the array surface for other types of primary cells. PMID:22159513

  5. A hybrid substratum for primary hepatocyte culture that enhances hepatic functionality with low serum dependency

    PubMed Central

    Meng, Qingyuan; Tao, Chunsheng; Qiu, Zhiye; Akaike, Toshihiro; Cui, Fuzhai; Wang, Xiumei

    2015-01-01

    Cell culture systems have proven to be crucial for the in vitro maintenance of primary hepatocytes and the preservation of hepatic functional expression at a high level. A poly-(N-p-vinylbenzyl-4-O-β-D-galactopyranosyl-D-gluconamide) matrix can recognize cells and promote liver function in a spheroid structure because of a specific galactose–asialoglycoprotein receptor interaction. Meanwhile, a fusion protein, E-cadherin-Fc, when incubated with various cells, has shown an enhancing effect on cellular viability and metabolism. Therefore, a hybrid substratum was developed for biomedical applications by using both of these materials to combine their advantages for primary hepatocyte cultures. The isolated cells showed a monolayer aggregate morphology on the coimmobilized surface and displayed higher functional expression than cells on traditional matrices. Furthermore, the hybrid system, in which the highest levels of cell adhesion and hepatocellular metabolism were achieved with the addition of 1% fetal bovine serum, showed a lower serum dependency than the collagen/gelatin-coated surface. Accordingly, this substrate may attenuate the negative effects of serum and further contribute to establishing a defined culture system for primary hepatocytes. PMID:25848252

  6. Proteomic and metabolomic responses to connexin43 silencing in primary hepatocyte cultures.

    PubMed

    Vinken, Mathieu; Maes, Michaël; Cavill, Rachel; Valkenborg, Dirk; Ellis, James K; Decrock, Elke; Leybaert, Luc; Staes, An; Gevaert, Kris; Oliveira, André G; Menezes, Gustavo B; Cogliati, Bruno; Dagli, Maria Lúcia Zaidan; Ebbels, Timothy M D; Witters, Erwin; Keun, Hector C; Vanhaecke, Tamara; Rogiers, Vera

    2013-05-01

    Freshly established cultures of primary hepatocytes progressively adopt a foetal-like phenotype and display increased production of connexin43. The latter is a multifaceted cellular entity with variable subcellular locations, including the mitochondrial compartment. Cx43 forms hemichannels and gap junctions that are involved in a plethora of physiological and pathological processes, such as apoptosis. The present study was conducted with the goal of shedding more light onto the role of connexin43 in primary hepatocyte cultures. Connexin43 expression was suppressed by means of RNA interference technology, and the overall outcome of this treatment on the hepatocellular proteome and metabolome was investigated using tandem mass tag-based differential protein profiling and (1)H NMR spectroscopy, respectively. Global protein profiling revealed a number of targets of the connexin43 knock-down procedure, including mitochondrial proteins (heat shock protein 60, glucose-regulated protein 75, thiosulphate sulphurtransferase and adenosine triphosphate synthase) and detoxifying enzymes (glutathione S-transferase μ 2 and cytochrome P450 2C70). At the metabolomic level, connexin43 silencing caused no overt changes, though there was some evidence for a subtle increase in intracellular glycine quantities. Collectively, these data could further substantiate the established existence of a mitochondrial connexin pool and could be reconciled with the previously reported involvement of connexin43 signalling in spontaneously occurring apoptosis in primary hepatocyte cultures.

  7. Rotenone induces cell death in primary dopaminergic culture by increasing ROS production and inhibiting mitochondrial respiration.

    PubMed

    Radad, Khaled; Rausch, Wolf-Dieter; Gille, Gabriele

    2006-09-01

    Although the definite etiology of Parkinson's disease is still unclear, increasing evidence has suggested an important role for environmental factors such as exposure to pesticides in increasing the risk of developing Parkinson's disease. In the present study, primary cultures prepared from embryonic mouse mesencephala were applied to investigate the toxic effects and underlying mechanisms of rotenone-induced neuronal cell death relevant to Parkinson's disease. Results revealed that rotenone destroyed dopaminergic neurons in a dose- and time-dependent manner. Consistent with the cytotoxic effect of rotenone as evidenced by dopaminergic cell loss, it significantly increased the release of lactate dehydrogenase into the culture medium, the number of necrotic cells in the culture and the number of nuclei showing apoptotic features. Rotenone exerted toxicity by decreasing the mitochondrial membrane potential, increasing reactive oxygen species production and shifting respiration to a more anaerobic state.

  8. Primary Retinal Cultures as a Tool for Modeling Diabetic Retinopathy: An Overview

    PubMed Central

    Varano, Monica; Mallozzi, Cinzia; Gaddini, Lucia; Formisano, Giuseppe; Pricci, Flavia

    2015-01-01

    Experimental models of diabetic retinopathy (DR) have had a crucial role in the comprehension of the pathophysiology of the disease and the identification of new therapeutic strategies. Most of these studies have been conducted in vivo, in animal models. However, a significant contribution has also been provided by studies on retinal cultures, especially regarding the effects of the potentially toxic components of the diabetic milieu on retinal cell homeostasis, the characterization of the mechanisms on the basis of retinal damage, and the identification of potentially protective molecules. In this review, we highlight the contribution given by primary retinal cultures to the study of DR, focusing on early neuroglial impairment. We also speculate on possible themes into which studies based on retinal cell cultures could provide deeper insight. PMID:25688355

  9. Infection of primary cultures of murine splenic and thymic cells with coxsackievirus B4.

    PubMed

    Jaïdane, Hela; Gharbi, Jawhar; Lobert, Pierre-Emmanuel; Caloone, Delphine; Lucas, Bernadette; Sané, Famara; Idziorek, Thierry; Romond, Marie-Bénédicte; Aouni, Mahjoub; Hober, Didier

    2008-01-01

    Infection of primary cultures of total splenic and thymic cells from BALB/c and C3H/HeN mice with CVB4 E2 and JVB strains has been investigated. The presence of positive-strand viral RNA within cells was determined by semi-nested RT-PCR, and viral replication was attested by detection of intracellular negative-strand viral RNA and by release of infectious particles in culture supernatants. Viral replication occurred with both CVB4 strains to an extent dependent on the genetic background of the host. No interferon-alpha production was detected in the supernatants of CVB4-infected cultures using biological titration. Together these results suggest that infection of splenic and thymic cells can play a role in virus dissemination, and therefore in the pathophysiology of CVB4 infections.

  10. Effects of Carbon Ions on Primary Cultures of Mouse Brain Cells

    NASA Astrophysics Data System (ADS)

    Nojima, K.; Ando, K.; Fujiwara, H.; Ando, S.

    Primary mixed cultures of astrocytes and microglia were obtained from neonatal mice, and were irradiated with high-LET carbon ions. Immunohistochemical staining showed astrocytes survived more prominently than microglia. Tagged with specific antibodies, astrocytes and microglia surviving after irradiation were counted by flow cytometry. Decreases in the number of microglia and astrocytes were detected at a dose as small as 2 Gy when Day 5 cultures were irradiated with 13 keV/μm carbon ions. When the cultures were irradiated on Day 10, the dose-dependent decrease of microglia was more prominent for 13 keV/μun carbon ions than 70 keV/μm carbon ions. Astrocytes showed a marginal decrease at Day 10 and Day 14. We concluded that microglia are more sensitive than astrocytes to carbon ions and X-rays, and that the radiosensitivity of microglia depends on both differentiation/proliferation status and radiation quality

  11. Culture-based identification of pigmented Porphyromonas and Prevotella species in primary endodontic infections

    PubMed Central

    Rajaram, Anuradha; Kotrashetti, Vijayalakshmi S.; Somannavar, Pradeep D.; Ingalagi, Preeti; Bhat, Kishore

    2016-01-01

    Background. The most common species isolated from primary endodontic infections are black-pigmented bacteria. These species are implicated in apical abscess formation due to their proteolytic activity and are fastidious in nature. Therefore, the present study was carried out to evaluate the presence and identification of various pigmented Porphyromonas and Prevotella species in the infected root canal through culture-based techniques. Methods. Thirty-one patients with primary endodontic infections were selected. Using sterile paper points, samples were collected from the root canals after access opening and prior to obturation, which were cultured using blood and kanamycin blood agar. Subsequently, biochemical test was used to identify the species and the results were analyzed using percentage comparison analysis, McNemar and chi-squared tests, Wilcoxon match pair test and paired t-test. Results. Out of 31 samples 26 were positive for black-pigmented organisms; the predominantly isolated species were Prevotella followed by Porphyromonas. In Porphyromonas only P. gingivalis was isolated. One of the interesting features was isolation of P. gingivalis through culture, which is otherwise very difficult to isolate through culture. Conclusion. The presence of Prevotella and Porphyromonas species suggests that a significant role is played by these organisms in the pathogenesis of endodontic infections.

  12. Culture-based identification of pigmented Porphyromonas and Prevotella species in primary endodontic infections

    PubMed Central

    Rajaram, Anuradha; Kotrashetti, Vijayalakshmi S.; Somannavar, Pradeep D.; Ingalagi, Preeti; Bhat, Kishore

    2016-01-01

    Background. The most common species isolated from primary endodontic infections are black-pigmented bacteria. These species are implicated in apical abscess formation due to their proteolytic activity and are fastidious in nature. Therefore, the present study was carried out to evaluate the presence and identification of various pigmented Porphyromonas and Prevotella species in the infected root canal through culture-based techniques. Methods. Thirty-one patients with primary endodontic infections were selected. Using sterile paper points, samples were collected from the root canals after access opening and prior to obturation, which were cultured using blood and kanamycin blood agar. Subsequently, biochemical test was used to identify the species and the results were analyzed using percentage comparison analysis, McNemar and chi-squared tests, Wilcoxon match pair test and paired t-test. Results. Out of 31 samples 26 were positive for black-pigmented organisms; the predominantly isolated species were Prevotella followed by Porphyromonas. In Porphyromonas only P. gingivalis was isolated. One of the interesting features was isolation of P. gingivalis through culture, which is otherwise very difficult to isolate through culture. Conclusion. The presence of Prevotella and Porphyromonas species suggests that a significant role is played by these organisms in the pathogenesis of endodontic infections. PMID:27651878

  13. Mechanism of soluble beta-amyloid 25-35 neurotoxicity in primary cultured rat cortical neurons.

    PubMed

    Wang, Yong; Liu, Lili; Hu, Weimin; Li, Guanglai

    2016-04-01

    This study aimed to determine the effects of different concentrations of soluble beta-amyloid 25-35 (Aβ25-35) on cell viability, calcium overload, and PI3K-p85 expression in cultured cortical rat neurons. Primary cultured cerebral cortical neurons of newborn rats were divided randomly into six groups. Five groups were treated with soluble Aβ25-35 at concentrations of 10nmol/L, 100nmol/L, 1μmol/L, 10μmol/L, or 30μmol/L. Cell Counting Kit-8 staining was used to measure cell viability, laser-scanning confocal imaging was used to detect changes in intracellular free calcium concentration, and western blot assay was used to measure neuronal PI3K-p85 expression. Soluble Aβ25-35 was found to reduce cell viability and induce calcium overload in primary cultured rat cerebral cortical neurons, in a concentration-dependent manner. At certain concentrations, soluble Aβ25-35 also increased neuronal PI3K-p85 expression. These findings reveal that soluble Aβ25-35 reduces the viability of cultured cerebral cortical rat neurons. The neurotoxicity mechanism may involve calcium overload and disruption of insulin signal transduction pathways. PMID:26940239

  14. Photodynamic therapy (PDT) of endometrium primary cultures serving as an in-vitro model for endometriosis

    NASA Astrophysics Data System (ADS)

    Herter, Wiebke; Viereck, Volker; Keckstein, J.; Steiner, Rudolf W.; Rueck, Angelika C.

    1994-05-01

    As a new treatment model for endometriosis, photodynamic therapy (PDT) was applied to endometrium cultures. Endometriosis is a benign disease. Therefore primary cultures were used instead of cell lines. Endometrium is composed of epithelial and stromal cells which can also be found in primary culture. While stromal cells take a polygonal shape in culture, epithelial cells form cell colonies. PSIII (Photasan III), which is similar to hematorporphyrin derivate (HpD), meso-tetra (4-sulfonatophenyl) porphyrin (TPPS4), which posses a high fluorescence quantum yield and may be useful in fluorescence diagnosis of subtle endometriotic spots, and methylene blue (MB), a vital dye with phototoxic properties, were used as photosensitizers. Different sensitizer concentrations and incubation times were applied. The highest phototoxicity was observed for PSIII; TPPS4 and MB were less phototoxic. We compared our results with the sensitivity of cell lines described in the literature. The necessary irradiation to destroy stromal cells was relatively high but still in the same dimension as for cell lines. However they were even more sensitive than epithelial cells. This was true for all sensitizers used.

  15. Photodynamic treatment (PDT) of endometrium primary cultures serving as an in-vitro-model for endometriosis

    NASA Astrophysics Data System (ADS)

    Werter, Wiebke; Viereck, Volker; Keckstein, J.; Steiner, Rudolf W.; Rueck, Angelika C.

    1994-05-01

    As a new treatment model for endometriosis, photodynamic therapy (PDT) was applied to endometrium cultures. Endometriosis is a benign disease. Therefore primary cultures were used instead of cell lines. Endometrium is composed of epithelial and stromal cells which can also be found in primary culture. While stromal cells take a polygonal shape in culture, epithelial cells form cell colonies. PSIII (Photasan III), which is similar to hematorporphyrin derivate (HpD), meso-tetra (4-sulfonatophenyl) porphyrin (TPPS4), which posses a high fluorescence quantum yield and may be useful in fluorescence diagnosis of subtle endometriotic spots, and methylene blue (MB), a vital dye with phototoxic properties, were used as photosensitizers. Different sensitizer concentrations and incubation times were applied. The highest phototoxicity was observed for PSIII; TPPS4 and MB were less phototoxic. We compared our results with the sensitivity of cell lines described in the literature. The necessary irradiation to destroy stromal cells was relatively high but still in the same dimension as for cell lines. However they were even more sensitive than epithelial cells. This was true for all sensitizers used.

  16. Mechanism of soluble beta-amyloid 25-35 neurotoxicity in primary cultured rat cortical neurons.

    PubMed

    Wang, Yong; Liu, Lili; Hu, Weimin; Li, Guanglai

    2016-04-01

    This study aimed to determine the effects of different concentrations of soluble beta-amyloid 25-35 (Aβ25-35) on cell viability, calcium overload, and PI3K-p85 expression in cultured cortical rat neurons. Primary cultured cerebral cortical neurons of newborn rats were divided randomly into six groups. Five groups were treated with soluble Aβ25-35 at concentrations of 10nmol/L, 100nmol/L, 1μmol/L, 10μmol/L, or 30μmol/L. Cell Counting Kit-8 staining was used to measure cell viability, laser-scanning confocal imaging was used to detect changes in intracellular free calcium concentration, and western blot assay was used to measure neuronal PI3K-p85 expression. Soluble Aβ25-35 was found to reduce cell viability and induce calcium overload in primary cultured rat cerebral cortical neurons, in a concentration-dependent manner. At certain concentrations, soluble Aβ25-35 also increased neuronal PI3K-p85 expression. These findings reveal that soluble Aβ25-35 reduces the viability of cultured cerebral cortical rat neurons. The neurotoxicity mechanism may involve calcium overload and disruption of insulin signal transduction pathways.

  17. Culture-based identification of pigmented Porphyromonas and Prevotella species in primary endodontic infections.

    PubMed

    Rajaram, Anuradha; Kotrashetti, Vijayalakshmi S; Somannavar, Pradeep D; Ingalagi, Preeti; Bhat, Kishore

    2016-01-01

    Background. The most common species isolated from primary endodontic infections are black-pigmented bacteria. These species are implicated in apical abscess formation due to their proteolytic activity and are fastidious in nature. Therefore, the present study was carried out to evaluate the presence and identification of various pigmented Porphyromonas and Prevotella species in the infected root canal through culture-based techniques. Methods. Thirty-one patients with primary endodontic infections were selected. Using sterile paper points, samples were collected from the root canals after access opening and prior to obturation, which were cultured using blood and kanamycin blood agar. Subsequently, biochemical test was used to identify the species and the results were analyzed using percentage comparison analysis, McNemar and chi-squared tests, Wilcoxon match pair test and paired t-test. Results. Out of 31 samples 26 were positive for black-pigmented organisms; the predominantly isolated species were Prevotella followed by Porphyromonas. In Porphyromonas only P. gingivalis was isolated. One of the interesting features was isolation of P. gingivalis through culture, which is otherwise very difficult to isolate through culture. Conclusion . The presence of Prevotella and Porphyromonas species suggests that a significant role is played by these organisms in the pathogenesis of endodontic infections. PMID:27651878

  18. Senescence Process in Primary Wilms' Tumor Cell Culture Induced by p53 Independent p21 Expression

    PubMed Central

    Theerakitthanakul, Korkiat; Saetang, Jirakrit; Kruatong, Jirasak; Graidist, Potchanapond; Raungrut, Pritsana; Kayasut, Kanita; Sangkhathat, Surasak

    2016-01-01

    Wilms tumor (WT) is an embryonal tumor occurring in developing kidney tissue. WT cells showing invasive cancer characteristics, also retain renal stem cell behaviours. In-vitro culture of WT is hampered by limited replicative potential. This study aimed to establish a longterm culture of WT cells to enable the study of molecular events to attempt to explain its cellular senescence. Methods: Primary cell cultures from fresh WT tumor specimen were established. Of 5 cultures tried, only 1 could be propagated for more than 7 passages. One culture, identified as PSU-SK-1, could be maintained > 35 passages and was then subjected to molecular characterization and evaluation for cancer characteristics. The cells consistently harbored concomitant mutations of CTNNB1 (Ser45Pro) and WT1 (Arg413Stop) thorough the cultivation. On Transwell invasion assays, the cells exhibited migration and invasion at 55% and 27% capability of the lung cancer cells, A549. On gelatin zymography, PSU-SK-1 showed high expression of the matrix metaloproteinase. The cells exhibited continuous proliferation with 24-hour doubling time until passages 28-30 when the growth slowed, showing increased cell size, retention of cells in G1/S proportion and positive β-galactosidase staining. As with those evidence of senescence in advanced cell passages, expression of p21 and cyclin D1 increased when the expression of β-catenin and its downstream protein, TCF, declined. There was also loss-of-expression of p53 in this cell line. In conclusion, cellular senescence was responsible for limited proliferation in the primary culture of WT, which was also associated with increased expression of p21 and was independent of p53 expression. Decreased activation of the Wnt signalling might explain the induction of p21 expression. PMID:27698927

  19. Senescence Process in Primary Wilms' Tumor Cell Culture Induced by p53 Independent p21 Expression

    PubMed Central

    Theerakitthanakul, Korkiat; Khrueathong, Jeerasak; Kruatong, Jirasak; Graidist, Potchanapond; Raungrut, Pritsana; Kayasut, Kanita; Sangkhathat, Surasak

    2016-01-01

    Wilms tumor (WT) is an embryonal tumor occurring in developing kidney tissue. WT cells showing invasive cancer characteristics, also retain renal stem cell behaviours. In-vitro culture of WT is hampered by limited replicative potential. This study aimed to establish a longterm culture of WT cells to enable the study of molecular events to attempt to explain its cellular senescence. Methods: Primary cell cultures from fresh WT tumor specimen were established. Of 5 cultures tried, only 1 could be propagated for more than 7 passages. One culture, identified as PSU-SK-1, could be maintained > 35 passages and was then subjected to molecular characterization and evaluation for cancer characteristics. The cells consistently harbored concomitant mutations of CTNNB1 (Ser45Pro) and WT1 (Arg413Stop) thorough the cultivation. On Transwell invasion assays, the cells exhibited migration and invasion at 55% and 27% capability of the lung cancer cells, A549. On gelatin zymography, PSU-SK-1 showed high expression of the matrix metaloproteinase. The cells exhibited continuous proliferation with 24-hour doubling time until passages 28-30 when the growth slowed, showing increased cell size, retention of cells in G1/S proportion and positive β-galactosidase staining. As with those evidence of senescence in advanced cell passages, expression of p21 and cyclin D1 increased when the expression of β-catenin and its downstream protein, TCF, declined. There was also loss-of-expression of p53 in this cell line. In conclusion, cellular senescence was responsible for limited proliferation in the primary culture of WT, which was also associated with increased expression of p21 and was independent of p53 expression. Decreased activation of the Wnt signalling might explain the induction of p21 expression.

  20. IL-8 secretion in primary cultures of prostate cells is associated with prostate cancer aggressiveness

    PubMed Central

    Neveu, Bertrand; Moreel, Xavier; Deschênes-Rompré, Marie-Pier; Bergeron, Alain; LaRue, Hélène; Ayari, Cherifa; Fradet, Yves; Fradet, Vincent

    2014-01-01

    Background Chronic inflammation is believed to be a major factor in prostate cancer initiation and promotion and has been studied using prostate cancer cells and immortalized cell lines. However, little is known about the contribution of normal cells to the prostatic microenvironment and inflammation. We aim to study the contribution of normal prostate epithelial cells to prostate inflammation and to link the inflammatory status of normal cells to prostate cancer aggressiveness. Materials and methods Short-term primary cell cultures of normal epithelial prostate cells were derived from prostate biopsies from 25 men undergoing radical prostatectomy, cystoprostatectomy, or organ donation. Cells were treated with polyinosinic:polycytidylic acid, a mimic of double-stranded viral RNA and a potent inducer of the inflammatory response. Secretion of interleukin (IL)-8 in the cell culture medium by untreated and treated cells was measured and we determined the association between IL-8 levels in these primary cell cultures and prostate cancer characteristics. The Fligner–Policello test was used to compare the groups. Results Baseline and induced IL-8 secretion were highly variable between cultured cells from different patients. This variation was not related to drug use, past medical history, age, or preoperative prostate-specific antigen value. Nonetheless, an elevated secretion of IL-8 from normal cultured epithelial cells was associated with prostate cancer aggressiveness (P=0.0005). Conclusion The baseline secretion of IL-8 from normal prostate epithelial cells in culture is strongly correlated with cancer aggressiveness and may drive prostate cancer carcinogenesis. A better characterization of individual prostate microenvironment may provide a basis for personalized treatment and for monitoring the effects of strategies aimed at preventing aggressive prostate cancer. PMID:24892030

  1. Chronic lithium treatment increased intracellular S100ß levels in rat primary neuronal culture.

    PubMed

    Emamghoreishi, Masoumeh; Keshavarz, Mojtaba; Nekooeian, Ali Akbar

    2015-01-01

    S100ß a neurotrophic factor mainly released by astrocytes, has been implicated in the pathogenesis of bipolar disorder. Thus, lithium may exert its neuroprotective effects to some extent through S100ß. Furthermore, the possible effects of lithium on astrocytes as well as on interactions between neurons and astrocytes as a part of its mechanisms of actions are unknown. This study was undertaken to determine the effect of lithium on S100β in neurons, astrocytes and a mixture of neurons and astrocytes. Rat primary astrocyte, neuronal and mixed neuro-astroglia cultures were prepared from cortices of 18-day's embryos. Cell cultures were exposed to lithium (1mM) or vehicle for 1day (acute) or 7 days (chronic). RT-PCR and ELISA determined S100β mRNA and intra- and extracellular protein levels. Chronic lithium treatment significantly increased intracellular S100β in neuronal and neuro-astroglia cultures in comparison to control cultures (P<0.05). Acute and chronic lithium treatments exerted no significant effects on intracellular S100β protein levels in astrocytes, and extracellular S100β protein levels in three studied cultures as compared to control cultures. Acute and chronic lithium treatments did not significantly alter S100β mRNA levels in three studied cultures, compared to control cultures. Chronic lithium treatment increased intracellular S100ß protein levels in a cell-type specific manner which may favor its neuroprotective action. The findings of this study suggest that lithium may exert its neuroprotective action, at least partly, by increasing neuronal S100ß level, with no effect on astrocytes or interaction between neurons and astrocytes.

  2. Decoding 3-D Reach and Grasp Kinematics from High-Frequency Local Field Potentials in Primate Primary Motor Cortex

    PubMed Central

    Zhuang, Jun; Vargas-Irwin, Carlos; Donoghue, John P.

    2011-01-01

    Intracortical microelectrode array recordings generate a variety of neural signals with potential application as control signals in neural interface systems. Previous studies have focused on single and multiunit activity, as well as low frequency local field potentials (LFPs), but have not explored higher frequency (>200 Hz) LFPs. In addition, the potential to decode three dimensional (3-D) reach and grasp kinematics based on LFPs has not been demonstrated. Here, we use mutual information and decoding analyses to probe the information content about 3-D reaching and grasping of 7 different LFP frequency bands in the range of 0.3 Hz – 400 Hz. LFPs were recorded via 96-microelectrode arrays in primary motor cortex (M1) of two monkeys performing free reaching to grasp moving objects. Mutual information analyses revealed that higher frequency bands (e.g. 100 – 200 Hz and 200 – 400 Hz) carried the most information about the examined kinematics. Furthermore, Kalman filter decoding revealed that broadband high frequency LFPs, likely reflecting multiunit activity, provided the best decoding performance as well as substantial accuracy in reconstructing reach kinematics, grasp aperture and aperture velocity. These results indicate that LFPs, especially high frequency bands, could be useful signals for neural interfaces controlling 3-D reach and grasp kinematics. PMID:20403782

  3. Different Patterns of Cortical Inputs to Subregions of the Primary Motor Cortex Hand Representation in Cebus apella

    PubMed Central

    Dea, Melvin; Hamadjida, Adjia; Elgbeili, Guillaume; Quessy, Stephan; Dancause, Numa

    2016-01-01

    The primary motor cortex (M1) plays an essential role in the control of hand movements in primates and is part of a complex cortical sensorimotor network involving multiple premotor and parietal areas. In a previous study in squirrel monkeys, we found that the ventral premotor cortex (PMv) projected mainly to 3 regions within the M1 forearm representation [rostro-medial (RM), rostro-lateral (RL), and caudo-lateral (CL)] with very few caudo-medial (CM) projections. These results suggest that projections from premotor areas to M1 are not uniform, but rather segregated into subregions. The goal of the present work was to study how inputs from diverse areas of the ipsilateral cortical network are organized within the M1 hand representation. In Cebus apella, different retrograde neuroanatomical tracers were injected in 4 subregions of the hand area of M1 (RM, RL, CM, and CL). We found a different pattern of input to each subregion of M1. RM receives inputs predominantly from dorsal premotor cortex, RL from PMv, CM from area 5, and CL from area 2. These results support that the M1 hand representation is composed of several subregions, each part of a unique cortical network. PMID:26966266

  4. Different Patterns of Cortical Inputs to Subregions of the Primary Motor Cortex Hand Representation in Cebus apella.

    PubMed

    Dea, Melvin; Hamadjida, Adjia; Elgbeili, Guillaume; Quessy, Stephan; Dancause, Numa

    2016-04-01

    The primary motor cortex (M1) plays an essential role in the control of hand movements in primates and is part of a complex cortical sensorimotor network involving multiple premotor and parietal areas. In a previous study in squirrel monkeys, we found that the ventral premotor cortex (PMv) projected mainly to 3 regions within the M1 forearm representation [rostro-medial (RM), rostro-lateral (RL), and caudo-lateral (CL)] with very few caudo-medial (CM) projections. These results suggest that projections from premotor areas to M1 are not uniform, but rather segregated into subregions. The goal of the present work was to study how inputs from diverse areas of the ipsilateral cortical network are organized within the M1 hand representation. In Cebus apella, different retrograde neuroanatomical tracers were injected in 4 subregions of the hand area of M1 (RM, RL, CM, and CL). We found a different pattern of input to each subregion of M1. RM receives inputs predominantly from dorsal premotor cortex, RL from PMv, CM from area 5, and CL from area 2. These results support that the M1 hand representation is composed of several subregions, each part of a unique cortical network.

  5. The implementation of a social constructivist approach in primary science education in Confucian heritage culture: the case of Vietnam

    NASA Astrophysics Data System (ADS)

    Hằng, Ngô Vũ Thu; Meijer, Marijn Roland; Bulte, Astrid M. W.; Pilot, Albert

    2015-09-01

    Social constructivism has been increasingly studied and implemented in science school education. Nevertheless, there is a lack of holistic studies on the implementation of social constructivist approach in primary science education in Confucian heritage culture. This study aims to determine to what extent a social constructivist approach is implemented in primary science education in Confucian heritage culture and to give explanations for the implementation from a cultural perspective. Findings reveal that in Confucian heritage culture a social constructivist approach has so far not implemented well in primary science education. The implementation has been considerably influenced by Confucian heritage culture, which has characteristics divergent from and aligning with those of social constructivism. This study indicates a need for design-based research on social constructivism-based science curriculum for Confucian heritage culture.

  6. The development of a method for the preparation of rat intestinal epithelial cell primary cultures.

    PubMed

    Evans, G S; Flint, N; Somers, A S; Eyden, B; Potten, C S

    1992-01-01

    We describe a reproducible method for growing small intestinal epithelium (derived from the suckling rat intestine) in short-term (primary) cultures. Optimal culture conditions were determined by quantitative assays of proliferation (i.e. changes in cellularity and DNA synthesis). Isolation of the epithelia and, significantly, preservation of its three-dimensional integrity was achieved using a collagenase/dispase digestion technique. Purification of the epithelium was also facilitated by the use of a simple differential sedimentation method. The results presented below support the idea that proliferation of normal gut epithelium ex vivo is initially dependent upon the maintenance of the structural integrity of this tissue and upon factors produced by heterologous mesenchymal cells. Proliferation in vitro was also critically dependent upon the quality of the medium and constituents used. Cultures reached confluence within 10-14 days and consisted of epithelial colonies together with varying amounts of smooth-muscle-like cells. Cultures have been maintained for periods up to one month, but the longer-term potential for growth by sub-culturing has not been examined. Strategies for reducing the proliferation of these non-epithelial cells are also described.

  7. Characterization of a primary bile ductular cell culture from the livers of rats during extrahepatic cholestasis.

    PubMed Central

    Sirica, A. E.; Sattler, C. A.; Cihla, H. P.

    1985-01-01

    The establishment of novel bile ductular cell cultures was accomplished with the use of explants of a hyperplastic bile ductular tissue preparation obtained from rat livers at 10 to 15 weeks after bile duct ligation or a bile ductular cell fraction isolated from this tissue preparation by a procedure involving Percoll density gradient centrifugation. Observations made on these primary explant and monolayer bile ductular cell cultures were limited to the first 3 days of culture where the morphologic features of the bile ductular epithelium remained fairly well preserved, while fibroblast contamination was found to be very low. These cultured cells also retained over this period a high specific activity for the bile ductular cell marker enzyme gamma-glutamyl transpeptidase, as well as possessed measurable but decreasing specific activities for leucine aminopeptidase and alkaline phosphatase. Karyotypic analysis of the cultured monolayer cells further showed them to be diploid. In addition, preliminary transplantation studies demonstrated the presence of well-differentiated bile ductular-like structures following inoculation of the freshly isolated bile ductular cell fraction into the interscapular fat pads of recipient rats. Images Figure 2 Figure 1 Figure 3 Figure 4 Figure 5 Figure 6 PMID:2861743

  8. Characterization of the liver-macrophages isolated from a mixed primary culture of neonatal swine hepatocytes.

    PubMed

    Kitani, Hiroshi; Yoshioka, Miyako; Takenouchi, Takato; Sato, Mitsuru; Yamanaka, Noriko

    2014-01-01

    We recently developed a novel procedure to obtain liver-macrophages in sufficient number and purity using a mixed primary culture of rat and bovine hepatocytes. In this study, we aim to apply this method to the neonatal swine liver. Swine parenchymal hepatocytes were isolated by a two-step collagenase perfusion method and cultured in T75 culture flasks. Similar to the rat and bovine cells, the swine hepatocytes retained an epithelial cell morphology for only a few days and progressively changed into fibroblastic cells. After 5-13 days of culture, macrophage-like cells actively proliferated on the mixed fibroblastic cell sheet. Gentle shaking of the culture flask followed by the transfer and brief incubation of the culture supernatant resulted in a quick and selective adhesion of macrophage-like cells to a plastic dish surface. After rinsing dishes with saline, the attached macrophage-like cells were collected at a yield of 10(6) cells per T75 culture flask at 2-3 day intervals for more than 3 weeks. The isolated cells displayed a typical macrophage morphology and were strongly positive for macrophage markers, such as CD172a, Iba-1 and KT022, but negative for cytokeratin, desmin and α-smooth muscle actin, indicating a highly purified macrophage population. The isolated cells exhibited phagocytosis of polystyrene microbeads and a release of inflammatory cytokines upon lipopolysaccharide stimulation. This shaking and attachment method is applicable to the swine liver and provides a sufficient number of macrophages without any need of complex laboratory equipments. PMID:24707456

  9. Development of a quantal assay in primary shrimp cell culture for yellow head baculovirus (YBV) of penaeid shrimp.

    PubMed

    Lu, Y; Tapay, L M; Loh, P C; Brock, J A; Gose, R

    1995-03-01

    A 50% tissue culture infectious dose assay (TCID50) using primary culture of shrimp lymphoid organ (Oka) cells was developed for the quantitative titration of yellow-head baculovirus (YBV), a newly isolated virus of penaeid shrimp. The assay protocol includes the use of Primaria-grade 96-well tissue culture plates to grow the primary lymphoid organ cells of penaeid shrimp. A 15% gill suspension from YBV-infected shrimp was determined to have an infectious virus titer of 5 x 10(5.75) TCID50/ml. This report represents the first convenient assay protocol using cell culture derived from penaeid shrimp to titer a shrimp virus.

  10. Dual signal transduction pathways activated by TSH receptors in rat primary tanycyte cultures.

    PubMed

    Bolborea, Matei; Helfer, Gisela; Ebling, Francis J P; Barrett, Perry

    2015-06-01

    Tanycytes play multiple roles in hypothalamic functions, including sensing peripheral nutrients and metabolic hormones, regulating neurosecretion and mediating seasonal cycles of reproduction and metabolic physiology. This last function reflects the expression of TSH receptors in tanycytes, which detect photoperiod-regulated changes in TSH secretion from the neighbouring pars tuberalis. The present overall aim was to determine the signal transduction pathway by which TSH signals in tanycytes. Expression of the TSH receptor in tanycytes of 10-day-old Sprague Dawley rats was observed by in situ hybridisation. Primary ependymal cell cultures prepared from 10-day-old rats were found by immunohistochemistry to express vimentin but not GFAP and by PCR to express mRNA for Dio2, Gpr50, Darpp-32 and Tsh receptors that are characteristic of tanycytes. Treatment of primary tanycyte/ependymal cultures with TSH (100  IU/l) increased cAMP as assessed by ELISA and induced a cAMP-independent increase in the phosphorylation of ERK1/2 as assessed by western blot analysis. Furthermore, TSH (100  IU/l) stimulated a 2.17-fold increase in Dio2 mRNA expression. We conclude that TSH signal transduction in cultured tanycytes signals via Gαs to increase cAMP and via an alternative G protein to increase phosphorylation of ERK1/2. PMID:25878058

  11. Establishment of primary cultures for mouse ameloblasts as a model of their lifetime

    SciTech Connect

    Suzawa, Tetsuo . E-mail: suzawa@dent.showa-u.ac.jp; Itoh, Nao; Takahashi, Naoyuki; Katagiri, Takenobu; Morimura, Naoko; Kobayashi, Yasuna; Yamamoto, Toshinori; Kamijo, Ryutaro

    2006-07-07

    To understand how the properties of ameloblasts are spatiotemporally regulated during amelogenesis, two primary cultures of ameloblasts in different stages of differentiation were established from mouse enamel epithelium. Mouse primary ameloblasts (MPAs) prepared from immature enamel epithelium (MPA-I) could proliferate, whereas those from mature enamel epithelium (MPA-M) could not. MPA-M but not MPA-I caused apoptosis during culture. The mRNA expression of amelogenin, a marker of immature ameloblasts, was down-regulated, and that of enamel matrix serine proteiase-1, a marker of mature ameloblasts, was induced in MPA-I during culture. Using green fluorescence protein as a reporter, a visualized reporter system was established to analyze the promoter activity of the amelogenin gene. The region between -1102 bp and -261 bp was required for the reporter expression in MPA-I. These results suggest that MPAs are valuable in vitro models for investigation of ameloblast biology, and that the visualized system is useful for promoter analysis in MPAs.

  12. Electron microscopy of primary cell cultures in solution and correlative optical microscopy using ASEM.

    PubMed

    Hirano, Kazumi; Kinoshita, Takaaki; Uemura, Takeshi; Motohashi, Hozumi; Watanabe, Yohei; Ebihara, Tatsuhiko; Nishiyama, Hidetoshi; Sato, Mari; Suga, Mitsuo; Maruyama, Yuusuke; Tsuji, Noriko M; Yamamoto, Masayuki; Nishihara, Shoko; Sato, Chikara

    2014-08-01

    Correlative light-electron microscopy of cells in a natural environment of aqueous liquid facilitates high-throughput observation of protein complex formation. ASEM allows the inverted SEM to observe the wet sample from below, while an optical microscope observes it from above quasi-simultaneously. The disposable ASEM dish with a silicon nitride (SiN) film window can be coated variously to realize the primary-culture of substrate-sensitive cells in a few milliliters of culture medium in a stable incubator environment. Neuron differentiation, neural networking, proplatelet-formation and phagocytosis were captured by optical or fluorescence microscopy, and imaged at high resolution by gold-labeled immuno-ASEM with/without metal staining. Fas expression on the cell surface was visualized, correlated to the spatial distribution of F-actin. Axonal partitioning was studied using primary-culture neurons, and presynaptic induction by GluRδ2-N-terminus-linked fluorescent magnetic beads was correlated to the presynaptic-marker Bassoon. Further, megakaryocytes secreting proplatelets were captured, and P-selectins with adherence activity were localized to some of the granules present by immuno-ASEM. The phagocytosis of lactic acid bacteria by dendritic cells was also imaged. Based on these studies, ASEM correlative microscopy promises to allow the study of various mesoscopic-scale dynamics in the near future. PMID:24216127

  13. Low-shear modelled microgravity environment maintains morphology and differentiated functionality of primary porcine hepatocyte cultures.

    PubMed

    Nelson, Leonard J; Walker, Simon W; Hayes, Peter C; Plevris, John N

    2010-01-01

    Hepatocytes cultured in conventional static culture rapidly lose polarity and differentiated function. This could be explained by gravity-induced sedimentation, which prevents formation of complete three-dimensional (3D) cell-cell/cell-matrix interactions and disrupts integrin-mediated signals (including the most abundant hepatic integrin alpha(5)beta(1)), important for cellular polarity and differentiation. Cell culture in a low fluid shear modelled microgravity (about 10(-2) g) environment promotes spatial colocation/self-aggregation of dissociated cells and induction of 3D differentiated liver morphology. Previously, we demonstrated the utility of a NASA rotary bioreactor in maintaining key metabolic functions and 3D aggregate formation of high-density primary porcine hepatocyte cultures over 21 days. Using serum-free chemically defined medium, without confounding interactions of exogenous bioscaffolding or bioenhancing surface materials, we investigated features of hepatic cellular polarity and differentiated functionality, including expression of hepatic integrin alpha(5), as markers of functional morphology. We report here that in the absence of exogenous biomatrix scaffolding, hepatocytes cultured in serum-free chemically defined medium in a microgravity environment rapidly (<24 h) form macroscopic (2-5 mm), compacted 3D hepatospheroid structures consisting of a shell of glycogen-positive viable cells circumscribing a core of eosinophilic cells. The spheroid shell layers exhibited ultrastructural, morphological and functional features of differentiated, polarized hepatic tissue including strong expression of the integrin alpha(5) subunit, functional bile canaliculi, albumin synthesis, and fine ultrastructure reminiscent of in vivo hepatic tissue. The low fluid shear microgravity environment may promote tissue-like self-organization of dissociated cells, and offer advantages over spheroids cultured in conventional formats to delineate optimal conditions for

  14. Functional expression of voltage-gated sodium channels in primary cultures of human cervical cancer.

    PubMed

    Diaz, Daniel; Delgadillo, Dulce Maria; Hernández-Gallegos, Elizabeth; Ramírez-Domínguez, Martha Eugenia; Hinojosa, Luz María; Ortiz, Cindy Sharon; Berumen, Jaime; Camacho, Javier; Gomora, Juan Carlos

    2007-02-01

    Cervical cancer (CaC) is the third most frequent cause of death from cancer among women in the world and the first in females of developing countries. Several ion channels are upregulated in cancer, actually potassium channels have been suggested as tumor markers and therapeutic targets for CaC. Voltage-gated sodium channels (VGSC) activity is involved in proliferation, motility, and invasion of prostate and breast cancer cells; however, the participation of this type of channels in CaC has not been explored. In the present study, we identified both at the molecular and electrophysiological level VGSC in primary cultures from human cervical carcinoma biopsies. With the whole cell patch clamp technique, we isolated and identified a voltage-gated Na(+) current as the main component of the inward current in all investigated cells. Sodium current was characterized by its kinetics, voltage dependence, sensitivity to tetrodotoxin (TTX) block and dependence to [Na(+)](o). By analyzing the expression of mRNAs encoding TTX-sensitive Na(+) channel alpha subunits with standard RT-PCR and specific primers, we detected Na(v)1.2, Na(v)1.4, Na(v)1.6, and Na(v)1.7 transcripts in total RNA obtained from primary cultures and biopsies of CaC. Restriction enzyme analysis of PCR products was consistent with the molecular nature of the corresponding genes. Notably, only transcripts for Na(v)1.4 sodium channels were detected in biopsies from normal cervix. The results show for the first time the functional expression of VGSC in primary cultures from human CaC, and suggest that these channels might be considered as potential molecular markers for this type of cancer.

  15. Gypenosides protects dopaminergic neurons in primary culture against MPP(+)-induced oxidative injury.

    PubMed

    Wang, Peng; Niu, Le; Guo, Xiao-Dong; Gao, Li; Li, Wei-Xin; Jia, Dong; Wang, Xue-Lian; Ma, Lian-Ting; Gao, Guo-Dong

    2010-10-30

    Oxidative injury has been implicated in the etiology of Parkinson's disease (PD). Gypenosides (GPs), the saponins extract derived from the Gynostemma pentaphyllum, has various bioactivities. In this study, GPs was investigated for its neuroprotective effects on the 1-methyl-4-phenylpyridinium ion (MPP(+))-induced oxidative injury of dopaminergic neurons in primary nigral culture. It was found that GPs pretreatment, cotreatment or posttreatment significantly and dose-dependently attenuated MPP(+)-induced oxidative damage, reduction of dopamine uptake, loss of tyrosine hydrolase (TH)-immunopositive neurons and degeneration of TH-immunopositive neurites. However, the preventive effect of GPs was more potential than its therapeutical effect. Most importantly, the neuroprotective effect of GPs may be attributed to GPs-induced strengthened antioxidation as manifested by significantly increased glutathione content and enhanced activity of glutathione peroxidase, catalyze and superoxide dismutase in nigral culture. The neuroprotective effects of GPs are specific for dopaminergic neurons and it may have therapeutic potential in the treatment of PD.

  16. Utilization of supplemental methionine sources by primary cultures of chick hepatocytes

    SciTech Connect

    Dibner, J.J.

    1983-10-01

    Utilization of 2-hydroxy-4-(methylthio) butanoic acid (HMB) as a substrate for protein synthesis was studied by using primary cultures of chick liver cells. Cultures were prepared by enzymatic dissociation of livers from week old Hubbard broiler chicks and were maintained for 4 days under nonproliferative conditions. Hepatocyte differentiation was verified by using dexamethasone induction of tyrosine aminotransferase activity. Conversion of (14C)HMB to L-methionine was shown by chromatographic analysis of hepatocyte protein hydrolysate and incorporation into protein was proven by cycloheximide inhibition of synthesis. When incorporation of HMB was compared to that of DL-methionine (DLM) equimolar quantities of the two sources were found in liver cell protein. These results support, at a cellular level, the conclusion that HMB and DLM are biochemically equivalent sources of methionine for protein synthesis.

  17. Genetic basis of triticale breeding (x triticale). IV. Embryo culture for synthesizing primary hexaploid triticales

    SciTech Connect

    Gordei, I.A.; Khodortsova, L.F.

    1986-07-01

    Results are reported on enhancing the efficiency of embryo culture for synthesizing primary hexaploid triticales (AABBRR, 2n = 42). The antioxidant tomatoside has a positive effect on the reduction of progamous incompatibility of wheat with rye and increases the output of wheat-rye amphihaploids. It has been established that irradiation of embryos, cultured on nutrient medium, with helium-neon laser, increases significantly (P < 0.01) the regeneration frequency of the wheat-rye hybrid embryos. The highest frequency (40%) of amphidiploids was obtained by treating the plants with 0.15% colchicine through roots during the tillering phase. Hexaploid triticales from 11 cross combinations between tetraploid wheats (AABB, 2n = 28) and diploid rye (RR, 2n = 14) formed the initial material for breeding.

  18. Influence of several extracellular matrix components in primary cultures of bovine mammary epithelial cells.

    PubMed

    Delabarre, S; Claudon, C; Laurent, F

    1997-02-01

    Mammary epithelial cells, obtained from lactating cows, were cultured onto inserts coated with several components of extracellular matrix. The influence of these components upon the maintenance of differentiation has been determinated. Every day, alpha S1-casein secretion was measured by radioimmunoassay (RIA) in apical and basal compartments. Reorganization of functional tight junctions was evaluated by measurement of transepithelial electrical resistance (TER). On EHS matrix, cells underwent alveolar structures and never established TER. alpha S1-casein secretion strongly fluctuated with the day of culture. When plated onto fibronectin, cells reorganized a typical pavement and established TER. Nevertheless, TER and casein secretion highly fluctuated. On laminin-coated inserts, a few cells bound to the substratum, dedifferentiated, and proliferated to confluency within 9 days. TER progressively increased to a stable level after 15 days. Casein was not recovered after 6 days. Cells on type I collagen-coated inserts reorganized an epithelial pavement within 2 days and quickly established a stable TER. They secreted apically high levels of casein during 2 weeks. As cells maintained their biochemical differentiation, the culture on type I collagen-coated inserts seems an efficient model for primary culture of bovine mammary epithelial cells and allows studies of polarized alpha S1-casein secretion.

  19. Effects of growth factors and trefoil peptides on migration and replication in primary oxyntic cultures.

    PubMed

    Kato, K; Chen, M C; Nguyen, M; Lehmann, F S; Podolsky, D K; Soll, A H

    1999-05-01

    Restitution, the lateral migration of cells over an intact basement membrane, maintains mucosal integrity. We studied the regulation of migration and proliferation of enzyme-dispersed canine oxyntic mucosa cells in primary culture. Confluent monolayers were wounded and cultured in serum-free medium, and cells migrating into the wound were counted. [3H]thymidine incorporation into DNA was studied using subconfluent cultures. Considerable migration occurred in untreated monolayers; however, epidermal growth factor (EGF), transforming growth factor (TGF)-alpha, basic fibroblast growth factor (bFGF), insulin-like growth factor I (IGF-I), two trefoil peptides, and interleukin (IL)-1beta further enhanced migration. The specific EGF receptor (EGFR) monoclonal antibody, MAb-528, inhibited both basal and TGF-alpha- or IL-1beta-stimulated migration, but not the response to trefoil peptide, bFGF, or IGF-I. Exogenous TGF-beta inhibited cell proliferation but did not alter migration. Immunoneutralization with anti-TGF-beta blocked the response to exogenous TGF-beta and produced a small enhancement of basal thymidine incorporation but did not attenuate basal or TGF-alpha-stimulated migration. In conclusion, endogenous EGFR ligands regulate proliferation and migration. TGF-beta inhibits mitogenesis; it did not upregulate migration in these cultures. Although bFGF, IGF-I, and IL-1beta enhance gastric epithelial migration, only IL-1beta acted in a TGF-alpha-dependent fashion.

  20. Additional survey on genotoxicity of natural anthraquinones in the hepatocyte primary culture/DNA repair assay.

    PubMed

    Mori, H; Yoshimi, N; Iwata, H; Tanaka, T; Kawai, K; Sankawa, U

    1988-08-01

    Genotoxicity of fungal anthraquinones of islandicin, iridoskyrin and (-) rubroskyrin, and a colorant of insect origin, cochineal and its component, carminic acid, an anthraquinone, was examined in the hepatocyte primary culture/DNA repair test. The results were compared with that of versicolorin A, an anthraquinone with bisfuran ring, which had been proved to be genotoxic on this assay. All of these anthraquinones, differently from versicolorin A did not show clear response of DNA repair. The results suggest that these agents are not genotoxic carcinogens. PMID:3193483

  1. Primary monolayer culture of adult mouse hepatocytes -- a model for the study of hepatotropic viruses.

    PubMed

    Arnheiter, H

    1980-01-01

    Primary monolayer cultures of adult mouse hepatocytes isolated by collagenase perfusion of the liver in situ were exposed to 2 hepatotropic viruses, an avian influenza A virus adapted to grow in mouse liver in vivo and a herpes simplex type I virus. Influenza virus infection led to lysis ofindividual hepatocytes and total monolayer destruction within 18 to 120 hours after infection according to the virus dose used. Virus replication was evidenced by assaying hepatocyte supernates for hemagglutinin and infectivity, by immunofluorescent staining and by electron microscopy. Herpes virus infection resulted in polykaryocyte formation followed by nuclear pycnosis and cell lysis. Virus replication was assayed by titration of supernate infectivity.

  2. Antidepressants regulate glucocorticoid receptor messenger RNA concentrations in primary neuronal cultures.

    PubMed

    Pepin, M C; Beaulieu, S; Barden, N

    1989-07-01

    Increased cortisol secretion, caused by hyperactivity of the brain-pituitary-adrenal axis, and non-suppression of cortisol secretion following dexamethasone administration are two characteristics frequently associated with major depression or the depressed phase of bipolar illness. Antidepressants, irrespective of their selective inhibitory actions on the re-uptake of serotonin or of norepinephrine, modify glucocorticoid receptor messenger RNA concentrations in primary cultures of rat hypothalamic or amygdaloid neurons in a biphasic manner, with predominant stimulatory effects. This suggests a mechanism whereby antidepressants, by restoring the sensitivity of the limbic-hypothalamic system to glucocorticoid feedback inhibition, reverse the hyperactivity of the brain-pituitary-adrenal axis.

  3. Continual electric field stimulation preserves contractile function of adult ventricular myocytes in primary culture.

    PubMed

    Berger, H J; Prasad, S K; Davidoff, A J; Pimental, D; Ellingsen, O; Marsh, J D; Smith, T W; Kelly, R A

    1994-01-01

    To model with greater fidelity the electromechanical function of freshly isolated heart muscle cells in primary culture, we describe a technique for the continual electrical stimulation of adult myocytes at physiological frequencies for several days. A reusable plastic cover was constructed to fit standard, disposable 175-cm2 tissue culture flasks and to hold parallel graphite electrodes along the long axis of each flask, which treated a uniform electric field that resulted in a capture efficiency of ventricular myocytes of 75-80%. Computer-controlled amplifiers were designed to be capable of driving a number of flasks concurrently, each containing up to 4 x 10(6) myocytes, over a range of stimulation frequencies (from 0.1 to 7.0 Hz) with reversal of electrode polarity after each stimulus to prevent the development of pH gradients around each electrode. Unlike quiescent, unstimulated myocytes, the amplitude of contraction, and velocities of shortening and relaxation did not change in myocytes paced at 3-5 Hz for up to 72 h. The maintenance of normal contractile function in paced myocytes required mechanical contraction per se, since paced myocytes that remained quiescent due to the inclusion of 2.5 microM verapamil in the culture medium for 48 h also exhibited a decline in contractility when paced after verapamil removal. Similarly, pacing increased peak calcium current compared with quiescent cells that had not been paced. Thus myocyte contraction at physiological frequencies induced by continual uniform electric field stimulation in short-term primary culture in defining medium maintains some biophysical parameters of myocyte phenotype that are similar to those observed in freshly isolated adult ventricular myocytes.

  4. Neuroprotective Effect of Carnosine on Primary Culture of Rat Cerebellar Cells under Oxidative Stress.

    PubMed

    Lopachev, A V; Lopacheva, O M; Abaimov, D A; Koroleva, O V; Vladychenskaya, E A; Erukhimovich, A A; Fedorova, T N

    2016-05-01

    Dipeptide carnosine (β-alanyl-L-histidine) is a natural antioxidant, but its protective effect under oxidative stress induced by neurotoxins is studied insufficiently. In this work, we show the neuroprotective effect of carnosine in primary cultures of rat cerebellar cells under oxidative stress induced by 1 mM 2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH), which directly generates free radicals both in the medium and in the cells, and 20 nM rotenone, which increases the amount of intracellular reactive oxygen species (ROS). In both models, adding 2 mM carnosine to the incubation medium decreased cell death calculated using fluorescence microscopy and enhanced cell viability estimated by the MTT assay. The antioxidant effect of carnosine inside cultured cells was demonstrated using the fluorescence probe dichlorofluorescein. Carnosine reduced by half the increase in the number of ROS in neurons induced by 20 nM rotenone. Using iron-induced chemiluminescence, we showed that preincubation of primary neuronal cultures with 2 mM carnosine prevents the decrease in endogenous antioxidant potential of cells induced by 1 mM AAPH and 20 nM rotenone. Using liquid chromatography-mass spectrometry, we showed that a 10-min incubation of neuronal cultures with 2 mM carnosine leads to a 14.5-fold increase in carnosine content in cell lysates. Thus, carnosine is able to penetrate neurons and exerts an antioxidant effect. Western blot analysis revealed the presence of the peptide transporter PEPT2 in rat cerebellar cells, which suggests the possibility of carnosine transport into the cells. At the same time, Western blot analysis showed no carnosine-induced changes in the level of apoptosis regulating proteins of the Bcl-2 family and in the phosphorylation of MAP kinases, which suggests that carnosine could have minimal or no side effects on proliferation and apoptosis control systems in normal cells. PMID:27297901

  5. Primary culture and plasmid electroporation of the murine organ of Corti.

    PubMed

    Parker, Mark; Brugeaud, Aurore; Edge, Albert S B

    2010-02-04

    In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells. An increasingly essential tool in auditory research is the isolation and in vitro culture of the organ explant. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants. This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary

  6. Primary culture and plasmid electroporation of the murine organ of Corti.

    PubMed

    Parker, Mark; Brugeaud, Aurore; Edge, Albert S B

    2010-01-01

    In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells. An increasingly essential tool in auditory research is the isolation and in vitro culture of the organ explant. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants. This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary

  7. The Relationship between Central Visual Field Damage and Motor Vehicle Collisions in Primary Open-Angle Glaucoma Patients

    PubMed Central

    Yuki, Kenya; Asaoka, Ryo; Tsubota, Kazuo

    2014-01-01

    Purpose To investigate the relationship between visual field (VF) damage and history of motor vehicle collisions (MVCs) in subjects with primary open-angle glaucoma (POAG). Methods MVC history and driving habits were recorded using patient questionnaires in 247 POAG patients. Patients' driving attitudes (carefulness) were estimated using Rasch analysis. The relationship between MVC outcomes and 52 total deviation (TD) values of integrated binocular VF (IVF), better and worse visual acuities (VAs), age and gender was analyzed using principal component analysis and logistic regression. Results 51 patients had the history of MVCs. Significant difference was observed between patients with and without history of MVCs only for: better VA, a single TD value in the superior-right VF, and the typical distance driven in a week (unpaired t-test, p = 0.002, 0.015 and 0.006, respectively). There was not a significant relationship between MVCs and mean deviation (MD) of IVF (p = 0.41, logistic regression). None of the principal components were significantly correlated with MVC outcome (p>0.05, polynomial logistic regression analysis). There was a significant relationship between IVF MD and Rasch derived Person parameter (R2 = 0.023, p = 0.0095). There was also a significant positive relationship between MVCs and the distance driven in a week (p = 0.005, logistic regression). Conclusions In this study of POAG patients, MVCs were not related to central binocular VF damage. These results suggest the relationship between visual function and driving is not straightforward, and careful consideration should be given when predicting patients' driving ability using their VF. PMID:25545660

  8. Effects of methylmercury on primary cultured rat hepatocytes: Cell injury and inhibition of growth factor stimulated DNA synthesis

    SciTech Connect

    Tanno, Keiichi; Fukazawa, Toshiyuki; Tajima, Shizuko; Fujiki, Motoo )

    1992-08-01

    Many more studies deal with the toxicity of methylmercury on nervous tissue than on its toxicity to the liver. Methylmercury accumulates in the liver in higher concentrations than brain and the liver has the primary function of detoxifying methylmercury. According to recent studies, hepatocyte mitochondrial membranes are destroyed by methylmercury and DNA synthesis is inhibited by methylmercury during hepatocyte regeneration. Methylmercury alters the membrane ion permeability of isolate skate hepatocytes, and inhibits the metal-sensitive alcohol dehydrogenase and glutathione reductase of primary cultured rat hepatocytes. However, little is known about the effect of methylmercury on hepatocyte proliferation in primary cultured rat hepatocytes. We therefore used the primary cultured rat hepatocytes to investigate the effects of methylmercury on cell injury and growth factor stimulate DNA synthesis. The primary effect of methylmercury is to inhibit hepatocyte proliferation rather than to cause direct cell injury. 16 refs., 4 figs.

  9. Xenobiotic-Metabolizing Enzyme and Transporter Gene Expression in Primary Cultures of Human Hepatocytes Modulated by Toxcast Chemicals

    EPA Science Inventory

    Primary human hepatocyte cultures are useful in vitro model systems of human liver because when cultured under appropriate conditions the hepatocytes retain liver-like functionality such as metabolism, transport, and cell signaling. This model system was used to characterize the ...

  10. Chinese Cultural Education in Post-Colonial Hong Kong: Primary School Chinese Language Teachers' Belief and Practice

    ERIC Educational Resources Information Center

    Kwan, Ming Kai Marko

    2010-01-01

    Before 1997, no formal curriculum on Chinese cultural education for primary schools was developed in Hong Kong although the education authority had started to introduce some items of Chinese cultural learning into the Chinese language syllabus when the Target Oriented Curriculum was implemented in 1996. However, such items were incorporated into…

  11. Evaluation of cytokine gene expression after avian influenza virus infection in avian cell lines and primary cell cultures

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The innate immune responses elicited by avian influenza virus (AIV) infection has been studied by measuring cytokine gene expression by relative real time PCR (rRT-PCR) in vitro, using both cell lines and primary cell cultures. Continuous cell lines offer advantages over the use of primary cell cult...

  12. GABA uptake in astrocytes in primary cultures: coupling with two sodium ions.

    PubMed

    Larsson, O M; Hertz, L; Schousboe, A

    1980-01-01

    The influence of sodium ions on GABA uptake into astrocytes in primary cultures has been investigated performing kinetic analysis of GABA uptake at different sodium concentrations in the range 16 to 151 mM. These investigations reveal that sodium affects both the Km and the Vmax of the saturable component of the astroglial GABA uptake. Uptake rates as a function of the sodium concentration at high GABA concentrations (greater than or equal to 50 microM) were clearly sigmoid whereas at lower GABA concentrations this sigmoid shape was not obvious. Accordingly, Hill plots of the sodium dependency at high GABA concentrations exhibited straight lines with slopes of 2.0 to 2.5, suggesting that the coupling ratio between sodium and GABA is at least two. Corresponding Hill plots at lower GABA concentrations exhibited slopes of 1.6 to 1.8. Moreover, plots of 1/v versus 1/Na2 gave better fits to straight lines than plots of 1/v versus 1/Na which were curvilinear upward. Again, this curvilinearity was more pronounced at high GABA concentrations that at low GABA concentrations. From these results it is concluded that GABA uptake into astrocytes in primary cultures requires the binding of at least two sodium ions per GABA molecule transported.

  13. Using Photobleaching to Measure Spindle Microtubule Dynamics in Primary Cultures of Dividing Drosophila Meiotic Spermatocytes

    PubMed Central

    2015-01-01

    In dividing animal cells, a microtubule (MT)-based bipolar spindle governs chromosome movement. Current models propose that the spindle facilitates and/or generates translocating forces by regionally depolymerizing the kinetochore fibers (k-fibers) that bind each chromosome. It is unclear how conserved these sites and the resultant chromosome-moving mechanisms are between different dividing cell types because of the technical challenges of quantitatively studying MTs in many specimens. In particular, our knowledge of MT kinetics during the sperm-producing male meiotic divisions remains in its infancy. In this study, I use an easy-to-implement photobleaching-based assay for measuring spindle MT dynamics in primary cultures of meiotic spermatocytes isolated from the fruit fly Drosophila melanogaster. By use of standard scanning confocal microscopy features, fiducial marks were photobleached on fluorescent protein (FP)-tagged MTs. These were followed by time-lapse imaging during different division stages, and their displacement rates were calculated using public domain software. I find that k-fibers continually shorten at their poles during metaphase and anaphase A through the process of MT flux. Anaphase chromosome movement is complemented by Pac-Man, the shortening of the k-fiber at its chromosomal interface. Thus, Drosophila spermatocytes share the sites of spindle dynamism and mechanisms of chromosome movement with mitotic cells. The data reveal the applicability of the photobleaching assay for measuring MT dynamics in primary cultures. This approach can be readily applied to other systems. PMID:25802491

  14. Replication of human immunodeficiency virus type 1 in primary dendritic cell cultures.

    PubMed Central

    Langhoff, E; Terwilliger, E F; Bos, H J; Kalland, K H; Poznansky, M C; Bacon, O M; Haseltine, W A

    1991-01-01

    The ability of the human immunodeficiency virus type 1 (HIV-1) to replicate in primary blood dendritic cells was investigated. Dendritic cells compose less than 1% of the circulating leukocytes and are nondividing cells. Highly purified preparations of dendritic cells were obtained using recent advances in cell fractionation. The results of these experiments show that dendritic cells, in contrast to monocytes and T cells, support the active replication of all strains of HIV-1 tested, including T-cell tropic and monocyte/macrophage tropic isolates. The dendritic cell cultures supported much more virus production than did cultures of primary unseparated T cells, CD4+ T cells, and adherent as well as nonadherent monocytes. Replication of HIV-1 in dendritic cells produces no noticeable cytopathic effect nor does it decrease total cell number. The ability of the nonreplicating dendritic cells to support high levels of replication of HIV-1 suggests that this antigen-presenting cell population, which is also capable of supporting clonal T-cell growth, may play a central role in HIV pathogenesis, serving as a source of continued infection of CD4+ T cells and as a reservoir of virus infection. Images PMID:1910172

  15. Protective effect of butylated hydroxylanisole against hydrogen peroxide-induced apoptosis in primary cultured mouse hepatocytes

    PubMed Central

    Hwang, Geun Hye; Jeon, Yu Jin; Han, Ho Jae; Park, Soo Hyun; Baek, Kyoung Min; Chang, Woochul; Kim, Joong Sun; Kim, Lark Kyun; Lee, You-Mie; Lee, Sangkyu; Bae, Jong-Sup; Jee, Jun-Goo

    2015-01-01

    Butylated hydroxyanisole (BHA) is a synthetic phenolic compound consisting of a mixture of two isomeric organic compounds: 2-tert-butyl-4-hydroxyanisole and 3-tert-butyl-4-hydroxyanisole. We examined the effect of BHA against hydrogen peroxide (H2O2)-induced apoptosis in primary cultured mouse hepatocytes. Cell viability was significantly decreased by H2O2 in a dose-dependent manner. Additionally, H2O2 treatment increased Bax, decreased Bcl-2, and promoted PARP-1 cleavage in a dose-dependent manner. Pretreatment with BHA before exposure to H2O2 significantly attenuated the H2O2-induced decrease of cell viability. H2O2 exposure resulted in an increase of intracellular reactive oxygen species (ROS) generation that was significantly inhibited by pretreatment with BHA or N-acetyl-cysteine (NAC, an ROS scavenger). H2O2-induced decrease of cell viability was also attenuated by pretreatment with BHA and NAC. Furthermore, H2O2-induced increase of Bax, decrease of Bcl-2, and PARP-1 cleavage was also inhibited by BHA. Taken together, results of this investigation demonstrated that BHA protects primary cultured mouse hepatocytes against H2O2-induced apoptosis by inhibiting ROS generation. PMID:25798044

  16. Protective effect of memantine against Doxorubicin toxicity in primary neuronal cell cultures: influence a development stage.

    PubMed

    Jantas, D; Lason, W

    2009-01-01

    One of the serious unwanted effects of the anthracycline anticancer drug doxorubicin (Dox, adriamycin) is its neurotoxicity, which can be evoked by the activation of extracellular (FAS/CD95/Apo-1) pathway of apoptosis in cells. Since memantine, a clinically used N-methyl-D: -aspartic acid (NMDA) receptor antagonist, shows antiapoptotic action in several models of neuronal cell damage, in this study we evaluated the effect of memantine on the cell death induced by Dox in primary neuronal cell cultures. First, we investigated the effect of different concentrations of Dox (0.1-5 microM) on mouse neocortical, hippocampal, striatal, and cerebellar neurons on 7- and 12-day in vitro (DIV). The 7 DIV neuronal cell cultures were more prone to Dox-induced cell death than 12 DIV cultures. The cerebellar neurons were the most resistant to Dox-induced apoptosis in comparison to neuronal cell cultures derived from the forebrain. Memantine (0.1-2 microM) attenuated the Dox-evoked lactate dehydrogenase release in 7 DIV neuronal cell cultures with no significant effect on 12 DIV cultures. The ameliorating effect of memantine on Dox-mediated cell death was also confirmed by an increase in cell viability measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction assay. There was no effect of memantine on Dox-induced caspase-8 and -3 activity and Dox-evoked decrease in mitochondrial potential, although attenuation in the number of cells with apoptotic DNA fragmentation was observed. We also showed that the antiapoptotic effect of memantine in our model was NMDA receptor-independent, since two other antagonists of this receptor, MK-801 and AP-5, did not attenuate Dox-induced cell death. Furthermore, memantine did not influence the Dox-evoked increase in cytoplasmic Ca2+ level. The obtained data suggest developmental regulation of both, the Dox-mediated neurotoxicity and efficacy of memantine in alleviating the Dox-induced cell damage in neuronal cell cultures

  17. Primary lateral sclerosis: upper-motor-predominant amyotrophic lateral sclerosis with frontotemporal lobar degeneration--immunohistochemical and biochemical analyses of TDP-43.

    PubMed

    Kosaka, Takayuki; Fu, Yong-Juan; Shiga, Atsushi; Ishidaira, Haruka; Tan, Chun-Feng; Tani, Takashi; Koike, Ryoko; Onodera, Osamu; Nishizawa, Masatoyo; Kakita, Akiyoshi; Takahashi, Hitoshi

    2012-08-01

    Primary lateral sclerosis (PLS) is clinically defined as a disorder selectively affecting the upper motor neuron (UMN) system. However, recently it has also been considered that PLS is heterogeneous in its clinical presentation. To elucidate the association of PLS, or disorders mimicking PLS, with 43-kDa TAR DNA-binding protein (TDP-43) abnormality, we examined two adult patients with motor neuron disease, which clinically was limited almost entirely to the UMN system, and was followed by progressive frontotemporal atrophy. In the present study, the distribution and severity, and biochemical profile of phosphorylated TDP-43 (pTDP-43) in the brains and spinal cords were examined immunohistochemically and biochemically. Pathologically, in both cases, frontotemporal lobar degeneration with ubiquitin inclusions (FTLD-U) was evident, with the most severe degeneration in the motor cortex. An important feature in both cases was the presence of Bunina bodies and/or ubiquitin inclusions, albeit very rarely, in the well preserved lower motor neurons. The amygdala and neostriatum were also affected. pTDP-43 immunohistochemistry revealed the presence of many positively stained neuronal cytoplamic inclusions (NCIs) and dystrophic neurites/neuropil threads in the affected frontotemporal cortex and subcortical gray matter. By contrast, such pTDP-43 lesions, including NCIs, were observed in only a few lower motor neurons. pTDP-43 immunoblotting revealed that fragments of ∼25-kDa were present in the cortices, but not in the spinal cord in both cases. Genetically, neither of the patients had any mutation in the TDP-43 gene. In conclusion, we consider that although PLS may be a clinically significant disease entity, at autopsy, the majority of such clinical cases would present as upper-motor-predominant amyotrophic lateral sclerosis with FTLD-TDP. PMID:22098653

  18. An imbalance in progenitor cell populations reflects tumour progression in breast cancer primary culture models

    PubMed Central

    2011-01-01

    Background Many factors influence breast cancer progression, including the ability of progenitor cells to sustain or increase net tumour cell numbers. Our aim was to define whether alterations in putative progenitor populations could predict clinicopathological factors of prognostic importance for cancer progression. Methods Primary cultures were established from human breast tumour and adjacent non-tumour tissue. Putative progenitor cell populations were isolated based on co-expression or concomitant absence of the epithelial and myoepithelial markers EPCAM and CALLA respectively. Results Significant reductions in cellular senescence were observed in tumour versus non-tumour cultures, accompanied by a stepwise increase in proliferation:senescence ratios. A novel correlation between tumour aggressiveness and an imbalance of putative progenitor subpopulations was also observed. Specifically, an increased double-negative (DN) to double-positive (DP) ratio distinguished aggressive tumours of high grade, estrogen receptor-negativity or HER2-positivity. The DN:DP ratio was also higher in malignant MDA-MB-231 cells relative to non-tumourogenic MCF-10A cells. Ultrastructural analysis of the DN subpopulation in an invasive tumour culture revealed enrichment in lipofuscin bodies, markers of ageing or senescent cells. Conclusions Our results suggest that an imbalance in tumour progenitor subpopulations imbalances the functional relationship between proliferation and senescence, creating a microenvironment favouring tumour progression. PMID:21521500

  19. Establishment, characterization, and toxicological application of loggerhead sea turtle (Caretta caretta) primary skin fibroblast cell cultures.

    PubMed

    Webb, Sarah J; Zychowski, Gregory V; Bauman, Sandy W; Higgins, Benjamin M; Raudsepp, Terje; Gollahon, Lauren S; Wooten, Kimberly J; Cole, Jennifer M; Godard-Codding, Céline

    2014-12-16

    Pollution is a well-known threat to sea turtles but its impact is poorly understood. In vitro toxicity testing presents a promising avenue to assess and monitor the effects of environmental pollutants in these animals within the legal constraints of their endangered status. Reptilian cell cultures are rare and, in sea turtles, largely derived from animals affected by tumors. Here we describe the full characterization of primary skin fibroblast cell cultures derived from biopsies of multiple healthy loggerhead sea turtles (Caretta caretta), and the subsequent optimization of traditional in vitro toxicity assays to reptilian cells. Characterization included validating fibroblast cells by morphology and immunocytochemistry, and optimizing culture conditions by use of growth curve assays with a fractional factorial experimental design. Two cell viability assays, MTT and lactate dehydrogenase (LDH), and an assay measuring cytochrome P4501A (CYP1A) expression by quantitative PCR were optimized in the characterized cells. MTT and LDH assays confirmed cytotoxicity of perfluorooctanoic acid at 500 μM following 72 and 96 h exposures while CYP1A5 induction was detected after 72 h exposure to 0.1-10 μM benzo[a]pyrene. This research demonstrates the validity of in vitro toxicity testing in sea turtles and highlights the need to optimize mammalian assays to reptilian cells.

  20. Establishment, characterization, and toxicological application of loggerhead sea turtle (Caretta caretta) primary skin fibroblast cell cultures.

    PubMed

    Webb, Sarah J; Zychowski, Gregory V; Bauman, Sandy W; Higgins, Benjamin M; Raudsepp, Terje; Gollahon, Lauren S; Wooten, Kimberly J; Cole, Jennifer M; Godard-Codding, Céline

    2014-12-16

    Pollution is a well-known threat to sea turtles but its impact is poorly understood. In vitro toxicity testing presents a promising avenue to assess and monitor the effects of environmental pollutants in these animals within the legal constraints of their endangered status. Reptilian cell cultures are rare and, in sea turtles, largely derived from animals affected by tumors. Here we describe the full characterization of primary skin fibroblast cell cultures derived from biopsies of multiple healthy loggerhead sea turtles (Caretta caretta), and the subsequent optimization of traditional in vitro toxicity assays to reptilian cells. Characterization included validating fibroblast cells by morphology and immunocytochemistry, and optimizing culture conditions by use of growth curve assays with a fractional factorial experimental design. Two cell viability assays, MTT and lactate dehydrogenase (LDH), and an assay measuring cytochrome P4501A (CYP1A) expression by quantitative PCR were optimized in the characterized cells. MTT and LDH assays confirmed cytotoxicity of perfluorooctanoic acid at 500 μM following 72 and 96 h exposures while CYP1A5 induction was detected after 72 h exposure to 0.1-10 μM benzo[a]pyrene. This research demonstrates the validity of in vitro toxicity testing in sea turtles and highlights the need to optimize mammalian assays to reptilian cells. PMID:25384208

  1. Analysis of cell identity, morphology, apoptosis and mitotic activity in a primary neural cell culture system in Drosophila

    PubMed Central

    2012-01-01

    In Drosophila, most neurogenetic research is carried out in vivo. Mammalian research demonstrates that primary cell culture techniques provide a powerful model to address cell autonomous and non-autonomous processes outside their endogenous environment. We developed a cell culture system in Drosophila using wildtype and genetically manipulated primary neural tissue for long-term observations. We assessed the molecular identity of distinct neural cell types by immunolabeling and genetically expressed fluorescent cell markers. We monitored mitotic activity of cell cultures derived from wildtype and tumorous larval brains. Our system provides a powerful approach to unveil developmental processes in the nervous system and to complement studies in vivo. PMID:22554060

  2. Role of porphyrin sequestration in the biogenesis of iron-laden astrocytic inclusions in primary culture.

    PubMed

    Schipper, H M; Small, L; Wang, X; Brawer, J R

    2002-01-01

    Astrocytes in subcortical regions of the mammalian brain progressively accumulate iron-rich, autofluorecent cytoplasmic inclusions as a function of aging. Cysteamine (CSH) accelerates the appearance of this senescent glial phenotype in situ and in primary rat astroglial cultures. Porphyrins have been implicated as the source of orange-red autofluorescence in these glial inclusions. Yet, CSH has been shown to suppress porphyrin-heme biosynthesis in cultured astroglia. To determine whether porphyrin biosynthesis or sequestration participates in the biogenesis of these glial inclusions, the porphyrin precursor, (3)H-delta-aminolevulinic acid ((3)H-ALA) was administered to CSH-exposed and control rat astroglial cultures followed by light and electron microscopic autoradiography. Control cultures exhibited faint orange-red autofluorescence, intense (3)H-ALA labeling, numerous normal mitochondria and few cytoplasmic inclusions. In these cells, (3)H-ALA labeling largely occurred over normal mitochondria. The CSH-treated astroglia exhibited diminished (3)H-ALA labeling and contained numerous orange-red autofluorescent inclusions. The latter manifested internal compartments delimited by double membranes characteristic of damaged mitochondria. The complement of normal mitochondria in the CSH-exposed cells was markedly reduced. In the CSH-treated cells, (3)H-ALA labeling predominated over the large multi-compartmental inclusions. CSH attenuates de novo porphyrin-heme biosynthesis in astroglia but may induce punctate orange-red autofluorescence in the cytoplasm of these cells by promoting large numbers of damaged, porphyrin-containing mitochondria to form tight aggregates within the nascent gliosomes.

  3. Apical vacuole formation by gastric parietal cells in primary culture: effect of low extracellular Ca2+

    PubMed Central

    Nakada, Stephanie L.; Machen, Terry E.; Forte, John G.

    2012-01-01

    In primary culture, the gastric parietal cell's deeply invaginated apical membrane, seen in microscopy by phalloidin binding to F-actin (concentrated in microvilli and a subapical web), is engulfed into the cell, separated from the basolateral membrane (which then becomes the complete plasma membrane), and converted, from a lacy interconnected system of canaliculi, into several separate vacuoles. In this study, vacuolar morphology was achieved by 71% of parietal cells 8 h after typical collagenase digestion of rabbit gastric mucosa, but the tight-junctional protein zonula occludens-1 (ZO-1) was completely delocalized after ∼2 h, when cells were ready for culturing. Use of low-Ca2+ medium (4 mM EGTA) to release cells quickly from gastric glands yielded parietal cells in which ZO-1 was seen in a small spot or ring, a localization quickly lost if these cells were then cultured in normal Ca2+ but remaining up to 20 h if they were cultured in low Ca2+. The cells in low Ca2+ mostly retained, at 20 h, an intermediate morphology of many bulbous canalicular expansions (“prevacuoles”), seemingly with narrow interconnections. Histamine stimulation of 20-h cells with intermediate morphology caused colocalization of proton-pumping H-K-ATPase with canaliculi and prevacuoles but little swelling of those structures, consistent with a remaining apical pore through which secreted acid could escape. Apparent canalicular interconnections, lack of stimulated swelling, and lingering ZO-1 staining indicate inhibition of membrane fission processes that separate apical from basolateral membrane and vacuoles from each other, suggesting an important role for extracellular Ca2+ in these, and possibly other, endocytotic processes. PMID:23099641

  4. Mechanisms of chloroform and carbon tetrachloride toxicity in primary cultured mouse hepatocytes

    SciTech Connect

    Ruch, R.J.; Klaunig, J.E.; Schultz, N.E.; Askari, A.B.; Lacher, D.A.; Pereira, M.A.; Goldblatt, P.J.

    1986-11-01

    Mechanisms of chloroform (CHCl/sub 3/) and carbon tetrachloride (CCl/sub 4/) toxicity to primary cultured male B6C3F1 mouse hepatocytes were investigated. The cytotoxicity of both CHCl/sub 3/ and CCl/sub 4/ was dose- and duration-dependent. Maximal hepatocyte toxicity, as determined by lactate dehydrogenase leakage into the culture medium, occurred with the highest concentrations of CHCl/sub 3/ (5 mM) and CCl/sub 4/ (2.5 mM) used and with the longest duration of treatment (20 hr). CCl/sub 4/ was approximately 16 times more toxic than CHCl/sub 3/ to the hepatocytes. The toxicity of these compounds was decreased by adding the mixed function oxidase system (MFOS) inhibitor, SKF-525A (25..mu..M) to the cultures. The addition of diethyl maleate (0.25 mM), which depletes intracellular glutathione (GSH)-potentiated CHCl/sub 3/ and CCl/sub 4/ toxicity. The toxicity of CHCl/sub 3/ and CCl/sub 4/ could also be decreased by adding the antioxidants N,N'-diphenyl-p-phenylenediamine (DPPD) (25..mu..M), ..cap alpha..-tocopherol acetate (Vitamin E) (0.1 mM), or superoxide dismutase (SOD) (100 U/mL) to the cultures. These results suggest that: in mouse hepatocytes, both CHCl/sub 3/ and CCl/sub 4/ are metabolized to toxic components by the MFOS; GSH plays a role in detoxifying those metabolites; free radicals are produced during the metabolism of CHCl/sub 3/ and CCl/sub 4/; and free radicals may be important mediators of the toxicity of these two halomethanes.

  5. Evaluation of primary and secondary production using wastewater as a culture medium.

    PubMed

    Nandini, S; Ramírez-García, Pedro; Sarma, S S S

    2010-10-01

    The ability of rotifers and cladocerans to convert primary to secondary production in wastewaters was tested. Scenedesmus acutus was cultured on Bold's (defined) medium, wastewater from the tertiary phase of water treatment and a mixture of both. The algal growth rates (µ) ranged from 0.4 to 0.7 day⁻¹, being highest in defined medium. The demographic characteristics of Brachionus rubens and Moina macrocopa were tested using algae at a density of 1.0 x 10⁶ cells mL⁻¹. Into each test jar, we introduced 20 neonates (< 12-h-old) of either B. rubens or M. macrocopa. Daily (for M. macrocopa) or twice a day (for B. rubens), dead adults and the neonates were enumerated and removed. Average life-span and generation time of B. rubens were not significantly influenced by the algal treatment type. Gross and net reproductive rates were significantly influenced by the medium on which the algae was cultured; in the case of B. rubens, they ranged from 20-36 and 10-22 offspring female⁻¹; the corresponding values for M. macrocopa were higher (38-110 and 13-31 offspring female⁻¹, respectively). The rate of population increase was higher for Brachionus (0.41-0.65 day⁻¹)) compared to Moina (0.28-0.57 day⁻¹). Brachionus had significantly higher growth rates on algae cultured on Bold medium than on treated wastewater while Moina grew significantly better on Scenedesmus cultured on Bold medium or a mixture of treated wastewater and Bold medium than on treated wastewater alone. PMID:19748945

  6. Extracellular matrix-dependent differentiation of rabbit tracheal epithelial cells in primary culture.

    PubMed

    Baeza-Squiban, A; Boisvieux-Ulrich, E; Guilianelli, C; Houcine, O; Geraud, G; Guennou, C; Marano, F

    1994-01-01

    The differentiation of tracheal epithelial cells in primary culture was investigated according to the nature of the extracellular matrix used. Cultures obtained by the explant technique were realized on a type I collagen substratum either as a thin, dried coating or as a thick, hydrated gel supplemented with culture medium and serum. These two types of substratum induced distinct cell morphology and cytokeratin expression in the explant derived cells. Where cells are less proliferating (from Day 7 to 10 of culture), differentiation was evaluated by morphologic ultrastructural observations, immunocytochemical detection of cytokeratins, and determination of cytokeratin pattern by biochemical analysis. The epithelium obtained on gel was multilayered, with small, round basal cells under large, flattened upper cells. The determination of the keratin pattern expressed by cells grown on gel revealed an expression of keratin 13, already considered as a specific marker of squamous metaplasia, that diminished with retinoic acid treatment. Present results demonstrated by confocal microscopy that K13-positive cells were large upper cells with a dense keratin network, whereas lower cells were positively stained with a specific monoclonal antibody to basal cells (KB37). Moreover, keratin neosynthesis analysis pointed out a higher expression of K6, a marker of hyperproliferation, on gel than on coating. All these data suggest a differentiation of rabbit tracheal epithelial cells grown on gel toward squamous metaplasia. By contrast, the epithelium observed on coating is nearly a monolayer of very large and spread out cells. No K13-positive cells were observed, but an increase in the synthesis of simple epithelium marker (K18) was detected. These two substrata, similar in composition and different in structure, induce separate differentiation and appear as good tools to explore the mechanisms of differentiation of epithelial tracheal cells.

  7. Effect of Transcranial Direct Current Stimulation over the Primary Motor Cortex on Cerebral Blood Flow: A Time Course Study Using Near-infrared Spectroscopy.

    PubMed

    Takai, Haruna; Tsubaki, Atsuhiro; Sugawara, Kazuhiro; Miyaguchi, Shota; Oyanagi, Keiichi; Matsumoto, Takuya; Onishi, Hideaki; Yamamoto, Noriaki

    2016-01-01

    Transcranial direct current stimulation (tDCS) is a noninvasive brain stimulation technique that is applied during stroke rehabilitation. The purpose of this study was to examine diachronic intracranial hemodynamic changes using near-infrared spectroscopy (NIRS) during tDCS applied to the primary motor cortex (M1). Seven healthy volunteers were tested during real stimulation (anodal and cathodal) and during sham stimulation. Stimulation lasted 20 min and NIRS data were collected for about 23 min including the baseline. NIRS probe holders were positioned over the entire contralateral sensory motor area. Compared to the sham condition, both anodal and cathodal stimulation resulted in significantly lower oxyhemoglobin (O2Hb) concentrations in the contralateral premotor cortex (PMC), supplementary motor area (SMA), and M1 (p<0.01). Particularly in the SMA, the O2Hb concentration during anodal stimulation was significantly lower than that during the sham condition (p<0.01), while the O2Hb concentration during cathodal stimulation was lower than that during anodal stimulation (p<0.01). In addition, in the primary sensory cortex, the O2Hb concentration during anodal stimulation was significantly higher than the concentrations during both cathodal stimulation and the sham condition (p<0.05). The factor of time did not demonstrate significant differences. These results suggest that both anodal and cathodal tDCS cause widespread changes in cerebral blood flow, not only in the area immediately under the electrode, but also in other areas of the cortex. PMID:26782230

  8. Layer 5 Pyramidal Neurons' Dendritic Remodeling and Increased Microglial Density in Primary Motor Cortex in a Murine Model of Facial Paralysis.

    PubMed

    Urrego, Diana; Troncoso, Julieta; Múnera, Alejandro

    2015-01-01

    This work was aimed at characterizing structural changes in primary motor cortex layer 5 pyramidal neurons and their relationship with microglial density induced by facial nerve lesion using a murine facial paralysis model. Adult transgenic mice, expressing green fluorescent protein in microglia and yellow fluorescent protein in projecting neurons, were submitted to either unilateral section of the facial nerve or sham surgery. Injured animals were sacrificed either 1 or 3 weeks after surgery. Two-photon excitation microscopy was then used for evaluating both layer 5 pyramidal neurons and microglia in vibrissal primary motor cortex (vM1). It was found that facial nerve lesion induced long-lasting changes in the dendritic morphology of vM1 layer 5 pyramidal neurons and in their surrounding microglia. Dendritic arborization of the pyramidal cells underwent overall shrinkage. Apical dendrites suffered transient shortening while basal dendrites displayed sustained shortening. Moreover, dendrites suffered transient spine pruning. Significantly higher microglial cell density was found surrounding vM1 layer 5 pyramidal neurons after facial nerve lesion with morphological bias towards the activated phenotype. These results suggest that facial nerve lesions elicit active dendrite remodeling due to pyramidal neuron and microglia interaction, which could be the pathophysiological underpinning of some neuropathic motor sequelae in humans.

  9. Understanding metal homeostasis in primary cultured neurons. Studies using single neuron subcellular and quantitative metallomics.

    PubMed

    Colvin, Robert A; Lai, Barry; Holmes, William R; Lee, Daewoo

    2015-07-01

    The purpose of this study was to demonstrate how single cell quantitative and subcellular metallomics inform us about both the spatial distribution and cellular mechanisms of metal buffering and homeostasis in primary cultured neurons from embryonic rat brain, which are often used as models of human disease involving metal dyshomeostasis. The present studies utilized synchrotron radiation X-ray fluorescence (SRXRF) and focused primarily on zinc and iron, two abundant metals in neurons that have been implicated in the pathogenesis of neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. Total single cell contents for calcium, iron, zinc, copper, manganese, and nickel were determined. Resting steady state zinc showed a diffuse distribution in both soma and processes, best defined by the mass profile of the neuron with an enrichment in the nucleus compared with the cytoplasm. Zinc buffering and homeostasis was studied using two modes of cellular zinc loading - transporter and ionophore (pyrithione) mediated. Single neuron zinc contents were shown to statistically significantly increase by either loading method - ionophore: 160 million to 7 billion; transporter 160 million to 280 million atoms per neuronal soma. The newly acquired and buffered zinc still showed a diffuse distribution. Soma and processes have about equal abilities to take up zinc via transporter mediated pathways. Copper levels are distributed diffusely as well, but are relatively higher in the processes relative to zinc levels. Prior studies have observed iron puncta in certain cell types, but others have not. In the present study, iron puncta were characterized in several primary neuronal types. The results show that iron puncta could be found in all neuronal types studied and can account for up to 50% of the total steady state content of iron in neuronal soma. Although other metals can be present in iron puncta, they are predominantly iron containing and do not appear to be

  10. Understanding metal homeostasis in primary cultured neurons. Studies using single neuron subcellular and quantitative metallomics.

    PubMed

    Colvin, Robert A; Lai, Barry; Holmes, William R; Lee, Daewoo

    2015-07-01

    The purpose of this study was to demonstrate how single cell quantitative and subcellular metallomics inform us about both the spatial distribution and cellular mechanisms of metal buffering and homeostasis in primary cultured neurons from embryonic rat brain, which are often used as models of human disease involving metal dyshomeostasis. The present studies utilized synchrotron radiation X-ray fluorescence (SRXRF) and focused primarily on zinc and iron, two abundant metals in neurons that have been implicated in the pathogenesis of neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. Total single cell contents for calcium, iron, zinc, copper, manganese, and nickel were determined. Resting steady state zinc showed a diffuse distribution in both soma and processes, best defined by the mass profile of the neuron with an enrichment in the nucleus compared with the cytoplasm. Zinc buffering and homeostasis was studied using two modes of cellular zinc loading - transporter and ionophore (pyrithione) mediated. Single neuron zinc contents were shown to statistically significantly increase by either loading method - ionophore: 160 million to 7 billion; transporter 160 million to 280 million atoms per neuronal soma. The newly acquired and buffered zinc still showed a diffuse distribution. Soma and processes have about equal abilities to take up zinc via transporter mediated pathways. Copper levels are distributed diffusely as well, but are relatively higher in the processes relative to zinc levels. Prior studies have observed iron puncta in certain cell types, but others have not. In the present study, iron puncta were characterized in several primary neuronal types. The results show that iron puncta could be found in all neuronal types studied and can account for up to 50% of the total steady state content of iron in neuronal soma. Although other metals can be present in iron puncta, they are predominantly iron containing and do not appear to be

  11. Improved protocols for protein and RNA isolation from three-dimensional collagen sandwich cultures of primary hepatocytes.

    PubMed

    Heidebrecht, F; Schulz, I; Keller, M; Behrens, S-E; Bader, A

    2009-10-01

    The sandwich culture is the most widely used long-term culture system for functional primary hepatocytes. Despite its advantages, the currently available protocols for protein and RNA extraction are either time-consuming or contain steps that may skewer the results. This paper describes improved protocols for RNA and protein extraction from sandwich cultures that are easy to perform, require short working time, and use no additional enzymatic reactions that could change the expression profile of the cells. The quality of the RNA is excellent, allowing also applications requiring high purity such as microarrays. In general, the protocols are suited for any cells in 3D collagen culture. PMID:19539596

  12. Sex and strain differences in the hepatocyte primary culture/DNA repair test

    SciTech Connect

    McQueen, C.A.; Way, B.M. )

    1991-01-01

    The hepatocyte primary culture (HPC)/DNA repair test was developed using hepatocytes isolated from male F-344 rats. A number of genetic polymorphisms have been shown to occur in inbred strains of rats, which may lead to variation in biotransformation of xenobiotics resulting in differences in susceptibility to genotoxins. The effect of the strain utilized as a source of hepatocytes was investigated with female Lewis, F-344, and DA rats. Variation was observed when hepatocytes from the three strains were exposed to aflatoxin B{sub 1} (AFB{sub 1}). No clearcut strain differences were seen when cells were exposed to diethylnitrosamine (DEN) or 2-acetylaminofluorene. These results demonstrate that both the strain and the sex of the animal used as a source of hepatocytes can affect the HPC/DNA repair test.

  13. Metabolic effects of ethanol on primary cell cultures of rat skeletal muscle.

    PubMed

    Garriga, Judit; Fernández-Solá, Joaquim; Adanero, Ester; Urbano-Márquez, Alvaro; Cussó, Roser

    2005-01-01

    Individuals who have consumed alcohol chronically accumulate glycogen in their skeletal muscles. Changes in the energy balance caused by alcohol consumption might lead to alcoholic myopathy. Experimental models used in the past, such as with skeletal muscle biopsy samples of alcohol-dependent individuals or in animal models, do not distinguish between direct effects and indirect effects (i.e., alterations to the nervous or endocrine system) of alcohol. In the current study, we evaluated the direct effect of ethanol on skeletal muscle glycogen concentrations and related glycolytic pathways. We measured the changes in metabolite concentrations and enzyme activities of carbohydrate metabolism in primary cell cultures of rat skeletal muscle exposed to ethanol for two periods. The concentrations of glycolytic metabolites and the activities of several enzymes that regulate glucose and glycogen metabolism were measured. After a short exposure to ethanol (6 h), glucose metabolism slowed. After 48 h of exposure, glycogen accumulation was observed.

  14. Antiadipogenic properties of retinol in primary cultured differentiating human adipocyte precursor cells.

    PubMed

    Garcia, E; Lacasa, D; Agli, B; Giudicelli, Y; Castelli, D

    2000-04-01

    The aim of this study was to investigate the effect of retinol on the human adipose conversion process using primary cultured human adipocyte precursor cells. When these cells were seeded in a medium containing retinol (concentrations ranging from 3.5 nM to 3.5 muM), cell proliferation was slightly inhibited by high concentrations of retinol, as demonstrated by cell counting and [(3)H]-thymidine incorporation. Moreover, the differentiation capacities of these cells were markedly and dose-dependently inhibited by retinol, as shown by the reduced expression of the lipogenic enzyme glycerol-3-phosphate dehydrogenase and by microscopic morphological analysis. These results strongly suggest that retinol, by inhibiting the ability of human preadipocytes to convert into mature adipocytes, could be of potential interest in the prevention of human adipose tissue development in general and of cellulitis in particular. PMID:18503465

  15. Developmental changes of neuron-specific enolase and neurofilament proteins in primary neural culture.

    PubMed

    Schilling, K; Scherbaum, C; Pilgrim, C

    1988-01-01

    The expression of neuron-specific enolase (NSE) and neurofilament (NF) proteins in primary dissociated cell cultures derived from 14-day-old fetal rat diencephalon was studied by immunocytochemistry and quantitative western-blot techniques. Both neuronal marker proteins, NSE and NF, can be detected as early as day 2 in vitro. They show pronounced quantitative increases during the time period studied (12 days), the relative change being highest during the first few days in vitro (DIV). The molar ratio of the medium weight NF to the heavy NF polypeptide is 9.1 after 2 DIV and 2.6 after 12 DIV. Phosphorylation of the heavy NF polypeptide increases steadily during cultivation. Comparison of these results to in vivo data reported in the literature suggests that, qualitatively, neuronal development in vitro follows the pattern observed in vivo, but at an accelerated pace.

  16. Primary hepatocyte cultures as in vitro tools for toxicity testing: quo vadis?

    PubMed

    Vinken, Mathieu; Vanhaecke, Tamara; Rogiers, Vera

    2012-04-01

    Cultures of primary hepatocytes are versatile tools that can serve many in vitro toxicity testing purposes. However, they cope with dedifferentiation, a process that is already initiated during the hepatocyte isolation procedure and that is manifested as the progressive loss of functionality upon subsequent cultivation. A number of strategies to prevent dedifferentiation have been introduced over the last decades, all which aim at re-establishing the in vivo hepatocyte micro-environment in vitro, but that are of merely limited success. Recent mechanistic insight into the mechanisms that underlie hepatocyte dedifferentiation has opened new avenues for the development of novel approaches that target the actual causes of this deteriorative process and thus for the generation of a long-term hepatic in vitro tool. Such experimental system is urgently needed, especially in the light of the stringent European legislative modifications that are currently encountered by the pharmaceutical, chemical and, particularly, the cosmetic industry.

  17. Generation, culture and flow-cytometric characterization of primary mouse macrophages.

    PubMed

    Schleicher, Ulrike; Bogdan, Christian

    2009-01-01

    Macrophages are not only host cells for many pathogens, but also fulfill several key functions in the innate and adaptive immune response, including the release of pro- and anti-inflammatory cytokines, the generation of organic and inorganic autacoids, the phagocytosis and killing of intracellular microorganisms or tumor cells, and the degradation and presentation of antigens. Several of these functions are shared by other immune cells, including dendritic cells, granulocytes, NK cells, and/or T lymphocytes. Thus, the analysis of macrophage functions in vitro using primary mouse cell populations requires standardized methods for the generation and culture of macrophages that guarantee high cell purity as well as the absence of stimulatory microbial contaminants. This chapter presents methodology to achieve these aims.

  18. Neurotoxic potential and cellular uptake of T-2 toxin in human astrocytes in primary culture.

    PubMed

    Weidner, Maria; Lenczyk, Marlies; Schwerdt, Gerald; Gekle, Michael; Humpf, Hans-Ulrich

    2013-03-18

    The trichothecene mycotoxin T-2 toxin, which is produced by fungi of the Fusarium species, is a worldwide occurring contaminant of cereal based food and feed. The cytotoxic properties of T-2 toxin are already well described with apoptosis being a major mechanism of action in various cell lines as well as in primary cells of different origin. However, only few data on neurotoxic properties of T-2 toxin are reported so far, but in vivo studies showed different effects of T-2 toxin on behavior as well as on levels of brain amines in animals. To further investigate the cytotoxic properties of T-2 toxin on cells derived from brain tissue, normal human astrocytes in primary culture (NHA) were used in this study. Besides studies of cytotoxicity, apoptosis (caspase-3-activation, Annexin V) and necrosis (LDH-release), the cellular uptake and metabolism of T-2 toxin in NHA was analyzed and compared to the uptake in an established human cell line (HT-29). The results show that human astrocytes were highly sensitive to the cytotoxic properties of T-2 toxin, and apoptosis, induced at low concentrations, was identified for the first time as the mechanism of toxic action in NHA. Furthermore, a strong accumulation of T-2 toxin in NHA and HT-29 cells was detected, and T-2 toxin was subjected to metabolism leading to HT-2 toxin, a commonly found metabolite after T-2 toxin incubation in both cell types. This formation seems to occur within the cells since incubations of T-2 toxin with cell depleted culture medium did not lead to any degradation of the parent toxin. The results of this study emphasize the neurotoxic potential of T-2 toxin in human astrocytes at low concentrations after short incubation times. PMID:23363530

  19. Designing a primary science curriculum in a globalizing world: How do social constructivism and Vietnamese culture meet?

    NASA Astrophysics Data System (ADS)

    Hằng, Ngô Vũ Thu; Meijer, Marijn Roland; Bulte, Astrid M. W.; Pilot, Albert

    2016-03-01

    The implementation of social constructivist approaches to learning science in primary education in Vietnamese culture as an example of Confucian heritage culture remains challenging and problematic. This theoretical paper focuses on the initial phase of a design-based research approach; that is, the description of the design of a formal, written curriculum for primary science education in which features of social constructivist approaches to learning are synthesized with essential aspects of Vietnamese culture. The written design comprises learning aims, a framework that is the synthesis of learning functions, learning settings and educational expectations for learning phases, and exemplary curriculum units. Learning aims are formulated to comprehensively develop scientific knowledge, skills, and attitudes toward science for primary students. Derived from these learning aims, the designed framework consists of four learning phases respectively labeled as Engagement, Experience, Exchange, and Follow-up. The designed framework refers to knowledge of the "nature of science" education and characteristics of Vietnamese culture as an example of Confucian heritage culture. The curriculum design aims to serve as an educational product that addresses previously analyzed problems of primary science education in the Vietnamese culture in a globalizing world.

  20. Sensitivity of primary fibroblasts in culture to atmospheric oxygen does not correlate with species lifespan

    PubMed Central

    Patrick, Alison; Seluanov, Michael; Hwang, Chaewon; Tam, Jonathan; Khan, Tanya; Morgenstern, Ari; Wiener, Lauren; Vazquez, Juan M.; Zafar, Hiba; Wen, Robert; Muratkalyeva, Malika; Doerig, Katherine; Zagorulya, Maria; Cole, Lauren; Catalano, Sophia; Lobo Ladd, Aliny AB; Coppi, A. Augusto; Coşkun, Yüksel; Tian, Xiao; Ablaeva, Julia; Nevo, Eviatar; Gladyshev, Vadim N.; Zhang, Zhengdong D.; Vijg, Jan; Seluanov, Andrei; Gorbunova, Vera

    2016-01-01

    Differences in the way human and mouse fibroblasts experience senescence in culture had long puzzled researchers. While senescence of human cells is mediated by telomere shortening, Parrinello et al. demonstrated that senescence of mouse cells is caused by extreme oxygen sensitivity. It was hypothesized that the striking difference in oxygen sensitivity between mouse and human cells explains their different rates of aging. To test if this hypothesis is broadly applicable, we cultured cells from 16 rodent species with diverse lifespans in 3% and 21% oxygen and compared their growth rates. Unexpectedly, fibroblasts derived from laboratory mouse strains were the only cells demonstrating extreme sensitivity to oxygen. Cells from hamster, muskrat, woodchuck, capybara, blind mole rat, paca, squirrel, beaver, naked mole rat and wild-caught mice were mildly sensitive to oxygen, while cells from rat, gerbil, deer mouse, chipmunk, guinea pig and chinchilla showed no difference in the growth rate between 3% and 21% oxygen. We conclude that, although the growth of primary fibroblasts is generally improved by maintaining cells in 3% oxygen, the extreme oxygen sensitivity is a peculiarity of laboratory mouse strains, possibly related to their very long telomeres, and fibroblast oxygen sensitivity does not directly correlate with species' lifespan. PMID:27163160

  1. Cholesteryl ester transfer protein gene expression during differentiation of human preadipocytes to adipocytes in primary culture.

    PubMed

    Gauthier, B; Robb, M; McPherson, R

    1999-02-01

    The expression pattern of the CETP gene in relationship to that of LPL, adipsin, PPARgamma, C/EBPalpha, ADD1/SREBPI and actin was examined by RT-PCR during differentiation of human fibroblastic preadipocytes to adipocytes in primary culture. Preadipocytes were isolated from subcutaneous fat obtained from healthy female subjects undergoing mammary reduction procedures, and induced to differentiate in culture. Morphologically, adipogenesis was confirmed by the accumulation of lipid droplets in cells. We show that the gene encoding CETP is expressed in preadipocytes and is present throughout differentiation as compared to LPL and adipsin which were detected in the majority of samples by day 2 or 3 of adipogenesis. The transcription factors, PPARgamma, ADD1/SREBP1 and C/EBPalpha were expressed by day 2, concomitant with the appearance of LPL and adipsin but subsequent to the appearance of CETP. CETP mRNA was not detectable in human skin fibroblasts. These studies demonstrate that CETP. expression is induced at an early stage of commitment to the adipocyte lineage and may be activated by transcription factor(s), which are not members of the PPAR, ADD1/SREBP1 or C/EBP families. PMID:10030381

  2. Tightly bound nuclear progesterone receptor is not phosphorylated in primary chick oviduct cultures.

    PubMed Central

    Garcia, T; Jung-Testas, I; Baulieu, E E

    1986-01-01

    Oviduct cells from estradiol-treated chicks were grown in primary culture. After 3-5 days of culture in medium containing estradiol, 90% of the cellular progesterone binding sites were detected in the cytosol. After exposure to [3H]progesterone at 37 degrees C, 80% of the progesterone binding sites were found in nuclear fractions. Progesterone receptor phosphorylation was assessed after incubating the cells with [32P]orthophosphate. Receptor components were immunoprecipitated with a specific polyclonal antibody (IgG-G3) and analyzed by NaDodSO4/PAGE and autoradiography. In the cytosol, constant amounts of 32P-labeled 110-kDa subunit (the B subunit, one of the progesterone-binding components of the receptor) and of the non-steroid-binding heat shock protein hsp90 were found, whether cells had been exposed to progesterone or not. No 32P-labeled 79-kDa subunit (the A subunit, another progesterone-binding subunit) was detected. Various procedures were used to solubilize nuclear progesterone receptor (0.5 M KCl, micrococcal nuclease, NaDodSO4), and in no case was 32P-labeled B subunit detected in the extracts. However, nonradioactive B subunit was detected by immunoblot in a nuclear KCl extract of progesterone-treated cells. These results suggest that the fraction of the B subunit that becomes strongly attached to nuclear structures is not phosphorylated upon exposure of cells to progesterone. Images PMID:3463987

  3. [Propagation of the HTV in primary human embryonic kidney and lung cell culture].

    PubMed

    Liu, B; Dai, J; Wang, X; Wang, X; Shen, G

    1994-08-01

    2 strains of Hantaan virus (HTV, 76-118, Hubei-114) have been propagated successfully in cultured primary human embryonic kidney (HEK) and lung (HEL) cells. Cytopathic effect (CPE) was observed in the two kind of cells on day 5 to 7 postinoculation which showed the cell became round and clustered, then detached. The replicating peak of the Hubei-114 in two kinds of cell cultures appeared on the 11th day and another strain on the 14th or 17th day after infection. The ultrastructure changes were observed with EM and IEM, which stained by ICGT before embedding. It was discovered that the mitochondia atrophied and decreased, and inclusion bodies in the cytoplasma of HEK and KEL cells. A large amount of gold granulae were found in the inclusion bodies and the virions were seen occasionally. Contamination with other agents have been ruled out. Our data suggest that the replicating characters of HTV in these cell systems might be possible for the pathogenicity of HFRS for human. PMID:7801638

  4. Sensitivity of primary fibroblasts in culture to atmospheric oxygen does not correlate with species lifespan.

    PubMed

    Patrick, Alison; Seluanov, Michael; Hwang, Chaewon; Tam, Jonathan; Khan, Tanya; Morgenstern, Ari; Wiener, Lauren; Vazquez, Juan M; Zafar, Hiba; Wen, Robert; Muratkalyeva, Malika; Doerig, Katherine; Zagorulya, Maria; Cole, Lauren; Catalano, Sophia; Lobo Ladd, Aliny Ab; Coppi, A Augusto; Coşkun, Yüksel; Tian, Xiao; Ablaeva, Julia; Nevo, Eviatar; Gladyshev, Vadim N; Zhang, Zhengdong D; Vijg, Jan; Seluanov, Andrei; Gorbunova, Vera

    2016-05-01

    Differences in the way human and mouse fibroblasts experience senescence in culture had long puzzled researchers. While senescence of human cells is mediated by telomere shortening, Parrinello et al. demonstrated that senescence of mouse cells is caused by extreme oxygen sensitivity. It was hypothesized that the striking difference in oxygen sensitivity between mouse and human cells explains their different rates of aging. To test if this hypothesis is broadly applicable, we cultured cells from 16 rodent species with diverse lifespans in 3% and 21% oxygen and compared their growth rates. Unexpectedly, fibroblasts derived from laboratory mouse strains were the only cells demonstrating extreme sensitivity to oxygen. Cells from hamster, muskrat, woodchuck, capybara, blind mole rat, paca, squirrel, beaver, naked mole rat and wild-caught mice were mildly sensitive to oxygen, while cells from rat, gerbil, deer mouse, chipmunk, guinea pig and chinchilla showed no difference in the growth rate between 3% and 21% oxygen. We conclude that, although the growth of primary fibroblasts is generally improved by maintaining cells in 3% oxygen, the extreme oxygen sensitivity is a peculiarity of laboratory mouse strains, possibly related to their very long telomeres, and fibroblast oxygen sensitivity does not directly correlate with species' lifespan. PMID:27163160

  5. Primary neuronal-astrocytic co-culture platform for neurotoxicity assessment of di-(2-ethylhexyl) phthalate.

    PubMed

    Wu, Yang; Li, Ke; Zuo, Haoxiao; Yuan, Ye; Sun, Yi; Yang, Xu

    2014-05-01

    Plastics such as polyvinyl chlorides (PVC) are widely used in many indoor constructed environments; however, their unbound chemicals, such as di-(2-ethylhexyl) phthalates (DEHP), can leach into the surrounding environment. This study focused on DEHP's effect on the central nervous system by determining the precise DEHP content in mice brain tissue after exposure to the chemical, to evaluate the specific exposure range. Primary neuronal-astrocyte co-culture systems were used as in vitro models for chemical hazard identification of DEHP. Oxidative stress was hypothesized as a probable mechanism involved, and therefore the total reactive oxygen species (ROS) concentration was determined as a biomarker of oxidative stress. In addition, NeuriteTracer, a neurite tracing plugin with ImageJ, was used to develop an assay for neurotoxicity to provide quantitative measurements of neurological parameters, such as neuronal number, neuron count and neurite length, all of which could indicate neurotoxic effects. The results showed that with 1 nmol/L DEHP exposure, there was a significant increase in ROS concentrations, indicating that the neuronal-astrocyte cultures were injured due to exposure to DEHP. In response, astrocyte proliferation (gliosis) was initiated, serving as a mechanism to maintain a homeostatic environment for neurons and protect neurons from toxic chemicals. There is a need to assess the cumulative effects of DEHP in animals to evaluate the possible uptake and effects on the human neuronal system from exposure to DEHP in the indoor environment.

  6. Substance P receptors in primary cultures of cortical astrocytes from the mouse.

    PubMed Central

    Torrens, Y; Beaujouan, J C; Saffroy, M; Daguet de Montety, M C; Bergström, L; Glowinski, J

    1986-01-01

    Binding sites for substance P were labeled on intact cortical glial cells from newborn mice in primary culture using 125I-labeled Bolton-Hunter-labeled substance P. Maximal specific binding (95% of total binding) was reached after 2-3 weeks in culture. The binding was saturable, reversible, and temperature dependent. Scatchard and Hill analysis revealed a single population of noninteracting high-affinity binding sites (Kd, 0.33 nM; Bmax, 14.4 fmol per dish). Competition studies made with tachykinins and substance P analogues indicated that the characteristics of the 125I-labeled Bolton-Hunter labeled substance P binding sites on glial cells were identical to those on rat brain synaptosomes. 125I-labeled Bolton-Hunter labeled substance P binding sites were visualized by autoradiography, and differences in the intensity of labeling were seen among astrocytes. Substance P was found to stimulate phosphatidylinositol turnover; the EC50 value (0.36 nM) was identical to the IC50 value (0.38 nM) determined in binding studies. 125I-labeled Bolton-Hunter labeled substance P binding sites were also found on astrocytes derived from other brain structures and from the spinal cord of mice. Images PMID:2431412

  7. Biosynthesis and polarized distribution of neutral endopeptidase in primary cultures of kidney proximal tubule cells.

    PubMed Central

    Jalal, F; Dehbi, M; Berteloot, A; Crine, P

    1994-01-01

    When cultured in defined medium, kidney proximal convoluted tubule (PCT) cells form a homogeneous population and retain a number of differentiated functions. To characterize this cell system further as a functional model of epithelial polarity, we investigated the biogenic pathway of neutral endopeptidase (NEP), one of the most abundant microvillar membrane proteins in intestinal and kidney cells. We showed that, in contrast with some tumoral cell lines, RNA extracted from PCT cells shows the presence of a single mRNA species encoding NEP. Pulse-chase studies followed by selective immunoprecipitation of NEP molecules present either at the cell surface or in intracellular cell compartments showed that newly synthesized NEP molecules reached the cell surface as early as 30 min after the beginning of the chase with maximum cell surface expression at 60 min. When grown on semipermeable supports, PCT cells were found to target NEP exclusively to the apical plasma membrane. Similar results have been described using MDCK cells to study targeting of recombinant NEP. Thus primary cultures of PCT cells represent a new model with which to investigate the biogenic pathway of endogenous proteins in native epithelial cells. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:7945190

  8. Information services for primary care: the organizational culture of general practice and the information needs of partnerships and primary care groups.

    PubMed

    Bryant, S L

    1999-09-01

    In a primary-care led National Health Service it is imperative for librarians not only to develop user-centred services for health professionals based in the community but also to facilitate information management within Primary Care Groups. In this article recent research in the field is discussed, and challenges intrinsic to delivering information services to primary care are identified. Drawing on the experience of one Practice Librarian in the Aylesbury area, the importance of organizational culture is considered, along with its implications for making successful approaches to partnerships. Five factors that motivated these practices to contract the services of an independent librarian are identified. The information needs of Primary Care Groups are discussed and the essential characteristics of future service provision are noted.

  9. Effects of repetitive low-pressure explosive blast on primary neurons and mixed cultures.

    PubMed

    Zander, Nicole E; Piehler, Thuvan; Banton, Rohan; Benjamin, Richard

    2016-09-01

    Repetitive mild traumatic brain injury represents a considerable health concern, particularly for athletes and military personnel. For blast-induced brain injury, threshold shock-impulse levels required to induce such injuries and cumulative effects with single and/or multiple exposures are not well characterized. Currently, there is no established in vitro experimental model with blast pressure waves generated by live explosives. This study presents results of primary neurons and mixed cultures subjected to our unique in vitro indoor experimental platform that uses real military explosive charges to probe the effects of primary explosive blast at the cellular level. The effects of the blast on membrane permeability, generation of reactive oxygen species (ROS), uptake of sodium ions, intracellular calcium, and release of glutamate were probed 2 and 24 hr postblast. Significant changes in membrane permeability and sodium uptake among the sham, single-blast-injured, and triple-blast-injured samples were observed. A significant increase in ROS and glutamate release was observed for the triple-blast-injured samples compared with the sham. Changes in intracellular calcium were not significant. These results suggest that blast exposure disrupts the integrity of the plasma membrane, leading to the upset of ion homeostasis, formation of ROS, and glutamate release. Published 2016. †This article is a U.S. Government work and is in the public domain in the USA.

  10. Effects of repetitive low-pressure explosive blast on primary neurons and mixed cultures.

    PubMed

    Zander, Nicole E; Piehler, Thuvan; Banton, Rohan; Benjamin, Richard

    2016-09-01

    Repetitive mild traumatic brain injury represents a considerable health concern, particularly for athletes and military personnel. For blast-induced brain injury, threshold shock-impulse levels required to induce such injuries and cumulative effects with single and/or multiple exposures are not well characterized. Currently, there is no established in vitro experimental model with blast pressure waves generated by live explosives. This study presents results of primary neurons and mixed cultures subjected to our unique in vitro indoor experimental platform that uses real military explosive charges to probe the effects of primary explosive blast at the cellular level. The effects of the blast on membrane permeability, generation of reactive oxygen species (ROS), uptake of sodium ions, intracellular calcium, and release of glutamate were probed 2 and 24 hr postblast. Significant changes in membrane permeability and sodium uptake among the sham, single-blast-injured, and triple-blast-injured samples were observed. A significant increase in ROS and glutamate release was observed for the triple-blast-injured samples compared with the sham. Changes in intracellular calcium were not significant. These results suggest that blast exposure disrupts the integrity of the plasma membrane, leading to the upset of ion homeostasis, formation of ROS, and glutamate release. Published 2016. †This article is a U.S. Government work and is in the public domain in the USA. PMID:27317559

  11. Cadmium-induced damage to primary cultures of rat Leydig cells.

    PubMed

    Yang, Jian-Ming; Arnush, Marc; Chen, Qiong-Yu; Wu, Xiang-Dong; Pang, Bing; Jiang, Xue-Zhi

    2003-01-01

    The mechanism of testicular toxicity of cadmium is poorly understood. Previous studies focusing on cadmium-related changes in testicular histopathology have implicated testicular blood vessel damage as the main cause of cadmium toxicity. To further explore the toxic effects of cadmium on testis, we isolated and cultured rat Leydig cells, exposed to 10, 20, and 40 microM of cadmium chloride (base doses). After 24 h of exposure, cells and supernatants were harvested to examine cytotoxicity and genotoxicity of cadmium. The results show that both cell viability and concentration of testosterone excretion in primary Leydig cells are significantly lower in cadmium-exposed groups compared to the controls. Changes in testosterone excretion with human chorionic gonadotropin (hCG) stimulation is especially profound. The contents of malondialdehyde (MDA) and the activity of glutathione peroxidase (GSH-Px) in exposed groups are significantly higher than those in the control group, but the activity of superoxide dismutase (SOD) is lower. The number of cells with DNA single strand breaks and the levels of cellular DNA damage in all three exposure groups are significantly higher than in controls. These results indicate that cadmium is directly toxic to primary Leydig cells, and that the decreased percentage of normal cells and the increased level of DNA damage in cadmium-exposed Leydig cells may be responsible for decreased testosterone secretion. PMID:14555193

  12. Analysis of changes in the expression pattern of claudins using salivary acinar cells in primary culture.

    PubMed

    Fujita-Yoshigaki, Junko

    2011-01-01

    Primary saliva is produced from blood plasma in the acini of salivary glands and is modified by ion adsorption and secretion as the saliva passes through the ducts. In rodents, acinar cells of salivary glands express claudin-3 but not claudin-4, whereas duct cells express both claudins-3 and -4. The distinct claudin expression patterns may reflect differences in the permeability of tight junctions between acinar and duct cells. To analyze the role of claudins in salivary glands, we established a system for the primary culture of parotid acinar cells, where the expression patterns of claudins are remarkably changed. Real-time RT-PCR and immunoblot analyses reveal that the expression levels of claudins-4 and -6 increased, whereas claudins-3 and -10 decreased. We found that the signal to induce those changes is triggered during cell isolation and is mediated by Src and p38 MAP kinase. Here, we introduce the methods used to determine the signal pathway that induces the change in claudin expression.

  13. Evidence for reduced Cl- and increased Na+ permeability in cystic fibrosis human primary cell cultures.

    PubMed

    Boucher, R C; Cotton, C U; Gatzy, J T; Knowles, M R; Yankaskas, J R

    1988-11-01

    1. Employing a primary cell culture system and intracellular microelectrodes, we quantitated and compared the Na+ and Cl- pathways in apical membranes of normal and cystic fibrosis (CF) human airway epithelia. 2. Like the transepithelial difference (PD) in situ, the PD of CF epithelia in culture (-27 +/- 4 mV, mean +/- S.E.M.; n = 28) exceeded the PD of normal epithelia (-10 +/- 1 mV; n = 22). The raised PD principally reflected an increase in the rate of active transport (equivalent short circuit, Ieq) for CF epithelia (61 +/- 9 microA cm-2) as compared with normal epithelia (23 +/- 3 microA cm-2). No significant differences in transepithelial resistance were detected. 3. As indicated by ion replacement studies (gluconate for Cl-), the apical membrane of normal cells exhibits an apical membrane Cl- conductance (GCl) that can be activated by isoprenaline. CF cells do not exhibit an apical membrane GCl, nor can a GCl be activated by isoprenaline. 4. CF cells exhibited a larger amiloride-sensitive Ieq and amiloride-sensitive apical membrane conductance (GNa) than normal cells. Further, the amiloride-sensitive Ieq was increased by isoprenaline in CF but not normal airway epithelia. 5. Equivalent circuit analysis yielded evidence for a more positive electromotive force (EMF) across the apical membrane and a more negative EMF across the basolateral membrane of CF cells as compared with normal cells. Baseline resistances of the apical (Ra) and basolateral (Rb) membranes did not differ for normal and CF cells. 6. Estimates of the resistance of the paracellular path to ion flow (Rs) by equivalent circuit analysis or ion substitution detected no differences in Rs between CF and normal cells. 7. We conclude that abnormalities in both cellular Cl- permeability (reduced) and Na+ permeability (increased) are characteristic of the cultured CF respiratory epithelial cell. These data suggest that a defect in the regulation of apical membrane permeabilities is a central feature of

  14. Slow conduction in mixed cultured strands of primary ventricular cells and stem cell-derived cardiomyocytes.

    PubMed

    Kucera, Jan P; Prudat, Yann; Marcu, Irene C; Azzarito, Michela; Ullrich, Nina D

    2015-01-01

    Modern concepts for the treatment of myocardial diseases focus on novel cell therapeutic strategies involving stem cell-derived cardiomyocytes (SCMs). However, functional integration of SCMs requires similar electrophysiological properties as primary cardiomyocytes (PCMs) and the ability to establish intercellular connections with host myocytes in order to contribute to the electrical and mechanical activity of the heart. The aim of this project was to investigate the properties of cardiac conduction in a co-culture approach using SCMs and PCMs in cultured cell strands. Murine embryonic SCMs were pooled with fetal ventricular cells and seeded in predefined proportions on microelectrode arrays to form patterned strands of mixed cells. Conduction velocity (CV) was measured during steady state pacing. SCM excitability was estimated from action potentials measured in single cells using the patch clamp technique. Experiments were complemented with computer simulations of conduction using a detailed model of cellular architecture in mixed cell strands. CV was significantly lower in strands composed purely of SCMs (5.5 ± 1.5 cm/s, n = 11) as compared to PCMs (34.9 ± 2.9 cm/s, n = 21) at similar refractoriness (100% SCMs: 122 ± 25 ms, n = 9; 100% PCMs: 139 ± 67 ms, n = 14). In mixed strands combining both cell types, CV was higher than in pure SCMs strands, but always lower than in 100% PCM strands. Computer simulations demonstrated that both intercellular coupling and electrical excitability limit CV. These data provide evidence that in cultures of murine ventricular cardiomyocytes, SCMs cannot restore CV to control levels resulting in slow conduction, which may lead to reentry circuits and arrhythmias.

  15. Primary cilia expression in bone marrow in response to mechanical stimulation in explant bioreactor culture.

    PubMed

    Coughlin, T R; Schiavi, J; Alyssa Varsanik, M; Voisin, M; Birmingham, E; Haugh, M G; McNamara, L M; Niebur, G L

    2016-01-01

    Bone marrow contains a multitude of mechanically sensitive cells that may participate in mechanotransduction. Primary cilia are sensory organelles expressed on mesenchymal stem cells (MSCs), osteoblasts, osteocytes, and other cell types that sense fluid flow in monolayer culture. In marrow, cilia could similarly facilitate the sensation of relative motion between adjacent cells or interstitial fluid. The goal of this study was to determine the response of cilia to mechanical stimulation of the marrow. Bioreactors were used to supply trabecular bone explants with low magnitude mechanical stimulation (LMMS) of 0.3 ×g at 30 Hz for 1 h/d, 5 d/week, inducing shear stresses in the marrow. Four groups were studied: unstimulated (UNSTIM), stimulated (LMMS), and with and without chloral hydrate (UNSTIM+CH and LMMS+CH, respectively), which was used to disrupt cilia. After 19 days of culture, immunohistochemistry for acetylated α-tubulin revealed that more cells expressed cilia in culture compared to in vivo controls. Stimulation decreased the number of cells expressing cilia in untreated explants, but not in CH-treated explants. MSCs represented a greater fraction of marrow cells in the untreated explants than CH-treated explants. MSCs harvested from the stimulated groups were more proliferative than in the unstimulated explants, but this effect was absent from CH treated explants. In contrast to the marrow, neither LMMS nor CH treatment affected bone formation as measured by mineralising surface. Computational models indicated that LMMS does not induce bone strain, and the reported effects were thus attributed to shear stress in the marrow. From a clinical perspective, genetic or pharmaceutical alterations of cilia expression may affect marrow health and function. PMID:27434268

  16. Slow conduction in mixed cultured strands of primary ventricular cells and stem cell-derived cardiomyocytes

    PubMed Central

    Kucera, Jan P.; Prudat, Yann; Marcu, Irene C.; Azzarito, Michela; Ullrich, Nina D.

    2015-01-01

    Modern concepts for the treatment of myocardial diseases focus on novel cell therapeutic strategies involving stem cell-derived cardiomyocytes (SCMs). However, functional integration of SCMs requires similar electrophysiological properties as primary cardiomyocytes (PCMs) and the ability to establish intercellular connections with host myocytes in order to contribute to the electrical and mechanical activity of the heart. The aim of this project was to investigate the properties of cardiac conduction in a co-culture approach using SCMs and PCMs in cultured cell strands. Murine embryonic SCMs were pooled with fetal ventricular cells and seeded in predefined proportions on microelectrode arrays to form patterned strands of mixed cells. Conduction velocity (CV) was measured during steady state pacing. SCM excitability was estimated from action potentials measured in single cells using the patch clamp technique. Experiments were complemented with computer simulations of conduction using a detailed model of cellular architecture in mixed cell strands. CV was significantly lower in strands composed purely of SCMs (5.5 ± 1.5 cm/s, n = 11) as compared to PCMs (34.9 ± 2.9 cm/s, n = 21) at similar refractoriness (100% SCMs: 122 ± 25 ms, n = 9; 100% PCMs: 139 ± 67 ms, n = 14). In mixed strands combining both cell types, CV was higher than in pure SCMs strands, but always lower than in 100% PCM strands. Computer simulations demonstrated that both intercellular coupling and electrical excitability limit CV. These data provide evidence that in cultures of murine ventricular cardiomyocytes, SCMs cannot restore CV to control levels resulting in slow conduction, which may lead to reentry circuits and arrhythmias. PMID:26442264

  17. Preparation of primary myogenic precursor cell/myoblast cultures from basal vertebrate lineages.

    PubMed

    Froehlich, Jacob Michael; Seiliez, Iban; Gabillard, Jean-Charles; Biga, Peggy R

    2014-01-01

    Due to the inherent difficulty and time involved with studying the myogenic program in vivo, primary culture systems derived from the resident adult stem cells of skeletal muscle, the myogenic precursor cells (MPCs), have proven indispensible to our understanding of mammalian skeletal muscle development and growth. Particularly among the basal taxa of Vertebrata, however, data are limited describing the molecular mechanisms controlling the self-renewal, proliferation, and differentiation of MPCs. Of particular interest are potential mechanisms that underlie the ability of basal vertebrates to undergo considerable postlarval skeletal myofiber hyperplasia (i.e. teleost fish) and full regeneration following appendage loss (i.e. urodele amphibians). Additionally, the use of cultured myoblasts could aid in the understanding of regeneration and the recapitulation of the myogenic program and the differences between them. To this end, we describe in detail a robust and efficient protocol (and variations therein) for isolating and maintaining MPCs and their progeny, myoblasts and immature myotubes, in cell culture as a platform for understanding the evolution of the myogenic program, beginning with the more basal vertebrates. Capitalizing on the model organism status of the zebrafish (Danio rerio), we report on the application of this protocol to small fishes of the cyprinid clade Danioninae. In tandem, this protocol can be utilized to realize a broader comparative approach by isolating MPCs from the Mexican axolotl (Ambystoma mexicanum) and even laboratory rodents. This protocol is now widely used in studying myogenesis in several fish species, including rainbow trout, salmon, and sea bream(1-4). PMID:24835774

  18. Mutant human FUS Is ubiquitously mislocalized and generates persistent stress granules in primary cultured transgenic zebrafish cells.

    PubMed

    Acosta, Jamie Rae; Goldsbury, Claire; Winnick, Claire; Badrock, Andrew P; Fraser, Stuart T; Laird, Angela S; Hall, Thomas E; Don, Emily K; Fifita, Jennifer A; Blair, Ian P; Nicholson, Garth A; Cole, Nicholas J

    2014-01-01

    FUS mutations can occur in familial amyotrophic lateral sclerosis (fALS), a neurodegenerative disease with cytoplasmic FUS inclusion bodies in motor neurons. To investigate FUS pathology, we generated transgenic zebrafish expressing GFP-tagged wild-type or fALS (R521C) human FUS. Cell cultures were made from these zebrafish and the subcellular localization of human FUS and the generation of stress granule (SG) inclusions examined in different cell types, including differentiated motor neurons. We demonstrate that mutant FUS is mislocalized from the nucleus to the cytosol to a similar extent in motor neurons and all other cell types. Both wild-type and R521C FUS localized to SGs in zebrafish cells, demonstrating an intrinsic ability of human FUS to accumulate in SGs irrespective of the presence of disease-associated mutations or specific cell type. However, elevation in relative cytosolic to nuclear FUS by the R521C mutation led to a significant increase in SG assembly and persistence within a sub population of vulnerable cells, although these cells were not selectively motor neurons.

  19. Development and Validation of a Test Instrument for the Assessment of Basic Motor Competencies in Primary School

    ERIC Educational Resources Information Center

    Herrmann, Christian; Gerlach, Erin; Seelig, Harald

    2015-01-01

    A central aim of Physical Education (PE) is the promotion of basic motor competencies ("Motorische Basiskompetenzen" [MOBAK]). These are the necessary prerequisites for developing a physically active lifestyle. Valid test instruments are needed for the evaluation of the effect of PE. For this purpose, we developed a test instrument for…

  20. The Contribution of Primary Motor Cortex Is Essential for Probabilistic Implicit Sequence Learning: Evidence from Theta Burst Magnetic Stimulation

    ERIC Educational Resources Information Center

    Wilkinson, Leonora; Teo, James T.; Obeso, Ignacio; Rothwell, John C.; Jahanshahi, Marjan

    2010-01-01

    Theta burst transcranial magnetic stimulation (TBS) is considered to produce plastic changes in human motor cortex. Here, we examined the inhibitory and excitatory effects of TBS on implicit sequence learning using a probabilistic serial reaction time paradigm. We investigated the involvement of several cortical regions associated with implicit…

  1. Protective effects of isoatriplicolide tiglate from Paulownia coreana against glutamate-induced neurotoxicity in primary cultured rat cortical cells.

    PubMed

    Chung, Ill-Min; Kim, Eun-Hye; Jeon, Hyun-Seok; Moon, Hyung-In

    2010-06-01

    To examine the neuroprotective effects of Paulownia coreana, we tested its protection against the glutamate-induced neurotoxicity to primary cultured cortical neurons. An aqueous extract of the plants exhibited significant protection against glutamate-induced toxicity in primary cultured rat cortical cells. In order to clarify the neuroprotective mechanism(s) of this observed effect, isolation was performed to seek and identify active fractions and components. By such fractionation, one bioactive sesquiterpene lactone, isoatriplicolide tiglate, was isolated, which exhibited significant neuroprotective activities against glutamate-induced toxicity, exhibiting cell viability of about 50%, at concentrations ranging from 0.1 microM to 10 microM. PMID:20614807

  2. Feasibility of Primary Tumor Culture Models and Preclinical Prediction Assays for Head and Neck Cancer: A Narrative Review

    PubMed Central

    Dohmen, Amy J. C.; Swartz, Justin E.; Van Den Brekel, Michiel W. M.; Willems, Stefan M.; Spijker, René; Neefjes, Jacques; Zuur, Charlotte L.

    2015-01-01

    Primary human tumor culture models allow for individualized drug sensitivity testing and are therefore a promising technique to achieve personalized treatment for cancer patients. This would especially be of interest for patients with advanced stage head and neck cancer. They are extensively treated with surgery, usually in combination with high-dose cisplatin chemoradiation. However, adding cisplatin to radiotherapy is associated with an increase in severe acute toxicity, while conferring only a minor overall survival benefit. Hence, there is a strong need for a preclinical model to identify patients that will respond to the intended treatment regimen and to test novel drugs. One of such models is the technique of culturing primary human tumor tissue. This review discusses the feasibility and success rate of existing primary head and neck tumor culturing techniques and their corresponding chemo- and radiosensitivity assays. A comprehensive literature search was performed and success factors for culturing in vitro are debated, together with the actual value of these models as preclinical prediction assay for individual patients. With this review, we aim to fill a gap in the understanding of primary culture models from head and neck tumors, with potential importance for other tumor types as well. PMID:26343729

  3. Integrated economic and experimental framework for screening of primary recovery technologies for high cell density CHO cultures.

    PubMed

    Popova, Daria; Stonier, Adam; Pain, David; Titchener-Hooker, Nigel J; Farid, Suzanne S

    2016-07-01

    Increases in mammalian cell culture titres and densities have placed significant demands on primary recovery operation performance. This article presents a methodology which aims to screen rapidly and evaluate primary recovery technologies for their scope for technically feasible and cost-effective operation in the context of high cell density mammalian cell cultures. It was applied to assess the performance of current (centrifugation and depth filtration options) and alternative (tangential flow filtration (TFF)) primary recovery strategies. Cell culture test materials (CCTM) were generated to simulate the most demanding cell culture conditions selected as a screening challenge for the technologies. The performance of these technology options was assessed using lab scale and ultra scale-down (USD) mimics requiring 25-110mL volumes for centrifugation and depth filtration and TFF screening experiments respectively. A centrifugation and depth filtration combination as well as both of the alternative technologies met the performance selection criteria. A detailed process economics evaluation was carried out at three scales of manufacturing (2,000L, 10,000L, 20,000L), where alternative primary recovery options were shown to potentially provide a more cost-effective primary recovery process in the future. This assessment process and the study results can aid technology selection to identify the most effective option for a specific scenario.

  4. Integrated economic and experimental framework for screening of primary recovery technologies for high cell density CHO cultures

    PubMed Central

    Popova, Daria; Stonier, Adam; Pain, David; Titchener‐Hooker, Nigel J.

    2016-01-01

    Abstract Increases in mammalian cell culture titres and densities have placed significant demands on primary recovery operation performance. This article presents a methodology which aims to screen rapidly and evaluate primary recovery technologies for their scope for technically feasible and cost‐effective operation in the context of high cell density mammalian cell cultures. It was applied to assess the performance of current (centrifugation and depth filtration options) and alternative (tangential flow filtration (TFF)) primary recovery strategies. Cell culture test materials (CCTM) were generated to simulate the most demanding cell culture conditions selected as a screening challenge for the technologies. The performance of these technology options was assessed using lab scale and ultra scale‐down (USD) mimics requiring 25–110mL volumes for centrifugation and depth filtration and TFF screening experiments respectively. A centrifugation and depth filtration combination as well as both of the alternative technologies met the performance selection criteria. A detailed process economics evaluation was carried out at three scales of manufacturing (2,000L, 10,000L, 20,000L), where alternative primary recovery options were shown to potentially provide a more cost‐effective primary recovery process in the future. This assessment process and the study results can aid technology selection to identify the most effective option for a specific scenario. PMID:27067803

  5. Integrated economic and experimental framework for screening of primary recovery technologies for high cell density CHO cultures.

    PubMed

    Popova, Daria; Stonier, Adam; Pain, David; Titchener-Hooker, Nigel J; Farid, Suzanne S

    2016-07-01

    Increases in mammalian cell culture titres and densities have placed significant demands on primary recovery operation performance. This article presents a methodology which aims to screen rapidly and evaluate primary recovery technologies for their scope for technically feasible and cost-effective operation in the context of high cell density mammalian cell cultures. It was applied to assess the performance of current (centrifugation and depth filtration options) and alternative (tangential flow filtration (TFF)) primary recovery strategies. Cell culture test materials (CCTM) were generated to simulate the most demanding cell culture conditions selected as a screening challenge for the technologies. The performance of these technology options was assessed using lab scale and ultra scale-down (USD) mimics requiring 25-110mL volumes for centrifugation and depth filtration and TFF screening experiments respectively. A centrifugation and depth filtration combination as well as both of the alternative technologies met the performance selection criteria. A detailed process economics evaluation was carried out at three scales of manufacturing (2,000L, 10,000L, 20,000L), where alternative primary recovery options were shown to potentially provide a more cost-effective primary recovery process in the future. This assessment process and the study results can aid technology selection to identify the most effective option for a specific scenario. PMID:27067803

  6. Hemispheric Asymmetry of Frequency-Dependent Suppression in the Ipsilateral Primary Motor Cortex During Finger Movement: A Functional Magnetic Resonance Imaging Study

    PubMed Central

    Hayashi, Masamichi J.; Saito, Daisuke N.; Aramaki, Yu; Asai, Tatsuya; Fujibayashi, Yasuhisa

    2008-01-01

    Electrophysiological studies have suggested that the activity of the primary motor cortex (M1) during ipsilateral hand movement reflects both the ipsilateral innervation and the transcallosal inhibitory control from its counterpart in the opposite hemisphere, and that their asymmetry might cause hand dominancy. To examine the asymmetry of the involvement of the ipsilateral motor cortex during a unimanual motor task under frequency stress, we conducted block-design functional magnetic resonance imaging with 22 normal right-handed subjects. The task involved visually cued unimanual opponent finger movement at various rates. The contralateral M1 showed symmetric frequency-dependent activation. The ipsilateral M1 showed task-related deactivation at low frequencies without laterality. As the frequency of the left-hand movement increased, the left M1 showed a gradual decrease in the deactivation. This data suggests a frequency-dependent increased involvement of the left M1 in ipsilateral hand control. By contrast, the right M1 showed more prominent deactivation as the frequency of the right-hand movement increased. This suggests that there is an increased transcallosal inhibition from the left M1 to the right M1, which overwhelms the right M1 activation during ipsilateral hand movement. These results demonstrate the dominance of the left M1 in both ipsilateral innervation and transcallosal inhibition in right-handed individuals. PMID:18413350

  7. Regulation of sulfotransferase gene expression by glucocorticoid hormones and xenobiotics in primary rat hepatocyte culture.

    PubMed

    Runge-Morris, M

    1998-02-20

    In the rat liver, hydroxysteroid sulfotransferase-a (HST-a) and aryl sulfotransferase IV (ASTIV) represent two major rat hepatic sulfotransferases that are important to xenobiotic metabolism. Prototypic CYP1A1 and CYP2B/3A inducers regulate rat hepatic sulfotransferase gene expression although not necessarily in a coordinate direction. It has been previously reported that in vivo treatment with CYP1A1 inducer 3-methylcholanthrene (3-MC) suppresses rat hepatic HST-a mRNA expression in a dose-dependent manner. Similarly, HST-a and ASTIV mRNA levels become suppressed or induced, respectively, following in vivo treatment with phenobarbital (PB)-like CYP2B/3A inducers or prototypic CYP3A inducers such as glucocorticoid hormones. In the whole animal, sulfotransferase gene expression is modulated by members of the hypothalamic/pituitary-adrenal gonadal hormone axis. However, studies in primary rat hepatocyte culture suggest that prototypic P450 inducers regulate HST-a and ASTIV gene expression directly at the level of the hepatocyte. Glucocorticoid-mediated sulfotransferase expression was compared with the regulation of tyrosine amino transferase (TAT), a gene that is transcriptionally regulated by ligand bound glucocorticoid receptor. It was found that lower doses of dexamethasone (DEX, 10(-7) M) produced concomitant increases in ASTIV and TAT mRNA expression, whereas HST-a mRNA expression continued to rise as the DEX dose was increased through 10(-5) M. When hepatocytes were co-incubated with DEX and antiglucocorticoid/antiprogestin RU-486, DEX-stimulated HST-a mRNA expression was not significantly inhibited by RU-486, but ASTIV and TAT mRNA expression were inhibited to a similar extent. The results suggested that ASTIV, like TAT, is likely regulated by a classical glucocorticoid receptor mediated mechanism, whereas HST-a is probably regulated by glucocorticoids via an alternative mechanism. In contrast to the positive effects of glucocorticoid hormones, HST-a and ASTIV

  8. Primary Human Uterine Leiomyoma Cell Culture Quality Control: Some Properties of Myometrial Cells Cultured under Serum Deprivation Conditions in the Presence of Ovarian Steroids

    PubMed Central

    Sumikawa, Joana Tomomi; Batista, Fabrício Pereira; Paredes-Gamero, Edgar J.; Girão, Manoel J. B. C.; Oliva, Maria Luiza V.

    2016-01-01

    Cell culture is considered the standard media used in research to emulate the in vivo cell environment. Crucial in vivo experiments cannot be conducted in humans and depend on in vitro methodologies such as cell culture systems. However, some procedures involving the quality control of cells in culture have been gradually neglected by failing to acknowledge that primary cells and cell lines change over time in culture. Thus, we report methods based on our experience for monitoring primary cell culture of human myometrial cells derived from uterine leiomyoma. We standardized the best procedure of tissue dissociation required for the study of multiple genetic marker systems that include species-specific antigens, expression of myofibroblast or myoblast markers, growth curve, serum deprivation, starvation by cell cycle synchronization, culture on collagen coated plates, and 17 β-estradiol (E2) and progesterone (P4) effects. The results showed that primary myometrial cells from patients with uterine leiomyoma displayed myoblast phenotypes before and after in vitro cultivation, and leiomyoma cells differentiated into mature myocyte cells under the appropriate differentiation-inducing conditions (serum deprivation). These cells grew well on collagen coated plates and responded to E2 and P4, which may drive myometrial and leiomyoma cells to proliferate and adhere into a focal adhesion complex involvement in a paracrine manner. The establishment of these techniques as routine procedures will improve the understanding of the myometrial physiology and pathogenesis of myometrium-derived diseases such as leiomyoma. Mimicking the in vivo environment of fibrotic conditions can prevent false results and enhance results that are based on cell culture integrity. PMID:27391384

  9. Promotion of growth and differentiation of rat ductular oval cells in primary culture.

    PubMed

    Germain, L; Noël, M; Gourdeau, H; Marceau, N

    1988-01-15

    Oval cells emerging in rat liver at the early period of 3-methyl-4-dimethylaminoazobenzene treatment constitute a mixed epithelial cell compartment with respect to alpha-fetoprotein (AFP) and cytokeratin differential expression, and include a subpopulation which exhibits a phenotype intermediate between ductular cells and hepatocytes (Germain et al., Cancer Res., 45:673-681, 1985). In the present study we have examined the developmental potential of ductular oval cells in primary culture and after in vivo transfer. The use of monoclonal and polyclonal antibodies directed against cytokeratins of Mr 39,000 (CK39), 52,000 (CK52), and 55,000 (CK55) and vimentin, and also monoclonal antibodies against exposed surface components of oval cells (BDS7) and normal hepatocytes (HES6) allowed us to establish the ductular phenotype of the oval cells. A highly enriched preparation of oval cells was obtained by perfusion/digestion of the liver with collagenase, treatment of the cell suspension with trypsin and DNase, selective removal of hepatocytes by panning using the anti-HES6 antibody, and cell separation by isopyknic centrifugation in a Percoll gradient. The procedure yielded about 8 x 10(7) cells, of which 95% expressed CK39, CK52, and BDS7, 84% gamma-glutamyl transpeptidase, and 5% albumin and AFP. The primary response of cultured oval cells to various combinations of growth and differentiation promoting factors was evaluated with respect to their capacity to initiate DNA synthesis as measured by [3H]thymidine labeling from day 1 to 3, and/or to produce albumin and AFP and express tyrosine aminotransferase. Culture in the presence of either serum or clot blood extract resulted in a low proliferative activity with less than 5% of the nuclei being labeled. Over a 5-day period, fusion of a large portion of the oval cells led to multinucleated cells. When the cells were cultured in the presence of an elaborate combination of supplements [minimum essential medium containing 1 m

  10. Intracellular degradation of chemically functionalized carbon nanotubes using a long-term primary microglial culture model

    NASA Astrophysics Data System (ADS)

    Bussy, Cyrill; Hadad, Caroline; Prato, Maurizio; Bianco, Alberto; Kostarelos, Kostas

    2015-12-01

    Chemically functionalized carbon nanotubes (f-CNTs) have been used in proof-of-concept studies to alleviate debilitating neurological conditions. Previous in vivo observations in brain tissue have suggested that microglia - acting as resident macrophages of the brain - play a critical role in the internalization of f-CNTs and their partial in situ biodegradation following a stereotactic administration in the cortex. At the same time, several reports have indicated that immune cells such as neutrophils, eosinophils and even macrophages could participate in the processing of carbon nanomaterials via oxidation processes leading to degradation, with surface properties acting as modulators of CNT biodegradability. In this study we questioned whether degradability of f-CNTs within microglia could be modulated depending on the type of surface functionalization used. We investigated the kinetics of degradation of multi-walled carbon nanotubes (MWNTs) functionalized via different chemical strategies that were internalized within isolated primary microglia over three months. A cellular model of rat primary microglia that can be maintained in cell culture for a long period of time was first developed. The Raman structural signature of the internalized f-CNTs was then studied directly in cells over a period of up to three months, following a single exposure to a non-cytotoxic concentration of three different f-CNTs (carboxylated, aminated and both carboxylated and aminated). Structural modifications suggesting partial but continuous degradation were observed for all nanotubes irrespective of their surface functionalization. Carboxylation was shown to promote more pronounced structural changes inside microglia over the first two weeks of the study.Chemically functionalized carbon nanotubes (f-CNTs) have been used in proof-of-concept studies to alleviate debilitating neurological conditions. Previous in vivo observations in brain tissue have suggested that microglia - acting as

  11. Development of a Selective Culture Medium for Primary Isolation of the Main Brucella Species▿

    PubMed Central

    De Miguel, M. J.; Marín, C. M.; Muñoz, P. M.; Dieste, L.; Grilló, M. J.; Blasco, J. M.

    2011-01-01

    Bacteriological diagnosis of brucellosis is performed by culturing animal samples directly on both Farrell medium (FM) and modified Thayer-Martin medium (mTM). However, despite inhibiting most contaminating microorganisms, FM also inhibits the growth of Brucella ovis and some B. melitensis and B. abortus strains. In contrast, mTM is adequate for growth of all Brucella species but only partially inhibitory for contaminants. Moreover, the performance of both culture media for isolating B. suis has never been established properly. We first determined the performance of both media for B. suis isolation, proving that FM significantly inhibits B. suis growth. We also determined the susceptibility of B. suis to the antibiotics contained in both selective media, proving that nalidixic acid and bacitracin are highly inhibitory, thus explaining the reduced performance of FM for B. suis isolation. Based on these results, a new selective medium (CITA) containing vancomycin, colistin, nystatin, nitrofurantoin, and amphotericin B was tested for isolation of the main Brucella species, including B. suis. CITA's performance was evaluated using reference contaminant strains but also field samples taken from brucella-infected animals or animals suspected of infection. CITA inhibited most contaminant microorganisms but allowed the growth of all Brucella species, to levels similar to those for both the control medium without antibiotics and mTM. Moreover, CITA medium was more sensitive than both mTM and FM for isolating all Brucella species from field samples. Altogether, these results demonstrate the adequate performance of CITA medium for the primary isolation of the main Brucella species, including B. suis. PMID:21270216

  12. Biological effects of inorganic arsenic on primary cultures of rat astrocytes.

    PubMed

    Catanzaro, Irene; Schiera, Gabriella; Sciandrello, Giulia; Barbata, Giusi; Caradonna, Fabio; Proia, Patrizia; Di Liegro, Italia

    2010-10-01

    It is well established that inorganic arsenic induces neurotoxic effects and neurological defects in humans and laboratory animals. The cellular and molecular mechanisms of its actions, however, remain elusive. Herein we report the effects of arsenite (NaAsO2) on primary cultures of rat astrocytes. Cells underwent induction of heat shock protein 70 only at the highest doses of inorganic arsenic (30 and 60 microM), suggesting a high threshold to respond to stress. We also investigated arsenic genotoxicity with the comet assay. Interestingly, although cells treated with 10 microM arsenite for 24 h maintained >70% viability, with respect to untreated cells, high DNA damage was already observed. Since arsenic is not known to be a direct-acting genotoxic agent, we investigated the possibility that its effects are due, in astrocytes as well, to ROS formation, as already described for other cell types. However, FACS analysis after CM-H2DCFDA staining did not evidence any significant increase of ROS production while, on the contrary, at the highest arsenite concentrations used, ROS production decreased. Concordantly, we found that, if most cells in the culture are still alive (i.e. up to 10 microM arsenite), they show a treatment-dependent increase in the concentration of SOD1. On the other hand, SOD2 concentration did not change. Finally, we found that astrocytes also synthesize PIPPin, an RNA-binding protein, the concentration of which was recently reported to change in response to stress induced by cadmium. Here we also report that, in cells exposed to high doses of arsenite, an anti-PIPPin antibody-positive faster migrating protein appears. PMID:20818482

  13. The neurotoxicity of hallucinogenic amphetamines in primary cultures of hippocampal neurons.

    PubMed

    Capela, João Paulo; da Costa Araújo, Silvana; Costa, Vera Marisa; Ruscher, Karsten; Fernandes, Eduarda; Bastos, Maria de Lourdes; Dirnagl, Ulrich; Meisel, Andreas; Carvalho, Félix

    2013-01-01

    3,4-Methylenedioxymethamphetamine (MDMA or "Ecstasy") and 2,5-dimethoxy-4-iodoamphetamine hydrochloride (DOI) are hallucinogenic amphetamines with addictive properties. The hippocampus is involved in learning and memory and seems particularly vulnerable to amphetamine's neurotoxicity. We evaluated the neurotoxicity of DOI and MDMA in primary neuronal cultures of hippocampus obtained from Wistar rat embryos (E-17 to E-19). Mature neurons after 10 days in culture were exposed for 24 or 48 h either to MDMA (100-800 μM) or DOI (10-100 μM). Both the lactate dehydrogenase (LDH) release and the tetrazolium-based (MTT) assays revealed a concentration- and time-dependent neuronal death and mitochondrial dysfunction after exposure to both drugs. Both drugs promoted a significant increase in caspase-8 and caspase-3 activities. At concentrations that produced similar levels of neuronal death, DOI promoted a higher increase in the activity of both caspases than MDMA. In the mitochondrial fraction of neurons exposed 24h to DOI or MDMA, we found a significant increase in the 67 kDa band of apoptosis inducing factor (AIF) by Western blot. Moreover, 24h exposure to DOI promoted an increase in cytochrome c in the cytoplasmatic fraction of neurons. Pre-treatment with an antibody raised against the 5-HT(2A)-receptor (an irreversible antagonist) greatly attenuated neuronal death promoted by 48 h exposure to DOI or MDMA. In conclusion, hallucinogenic amphetamines promoted programmed neuronal death involving both the mitochondria machinery and the extrinsic cell death key regulators. Death was dependent, at least in part, on the stimulation of the 5-HT(2A)-receptors.

  14. Ouabain-induced changes in MAP kinase phosphorylation in primary culture of rat cerebellar cells.

    PubMed

    Lopachev, Alexander V; Lopacheva, Olga M; Osipova, Ekaterina A; Vladychenskaya, Elizaveta A; Smolyaninova, Larisa V; Fedorova, Tatiana N; Koroleva, Olga V; Akkuratov, Evgeny E

    2016-07-01

    Cardiotonic steroid (CTS) ouabain is a well-established inhibitor of Na,K-ATPase capable of inducing signalling processes including changes in the activity of the mitogen activated protein kinases (MAPK) in various cell types. With increasing evidence of endogenous CTS in the blood and cerebrospinal fluid, it is of particular interest to study ouabain-induced signalling in neurons, especially the activation of MAPK, because they are the key kinases activated in response to extracellular signals and regulating cell survival, proliferation and apoptosis. In this study we investigated the effect of ouabain on the level of phosphorylation of three MAPK (ERK1/2, JNK and p38) and on cell survival in the primary culture of rat cerebellar cells. Using Western blotting we described the time course and concentration dependence of phosphorylation for ERK1/2, JNK and p38 in response to ouabain. We discovered that ouabain at a concentration of 1 μM does not cause cell death in cultured neurons while it changes the phosphorylation level of the three MAPK: ERK1/2 is phosphorylated transiently, p38 shows sustained phosphorylation, and JNK is dephosphorylated after a long-term incubation. We showed that ERK1/2 phosphorylation increase does not depend on ouabain-induced calcium increase and p38 activation. Changes in p38 phosphorylation, which is independent from ERK1/2 activation, are calcium dependent. Changes in JNK phosphorylation are calcium dependent and also depend on ERK1/2 and p38 activation. Ten-micromolar ouabain leads to cell death, and we conclude that different effects of 1-μM and 10-μM ouabain depend on different ERK1/2 and p38 phosphorylation profiles. Copyright © 2016 John Wiley & Sons, Ltd.

  15. Live-cell imaging of autophagy induction and autophagosome-lysosome fusion in primary cultured neurons

    PubMed Central

    Bains, Mona; Heidenreich, Kim A.

    2009-01-01

    The discovery that impaired autophagy is linked to a wide variety of prominent diseases including cancer and neurodegeneration has lead to an explosion of research in this area. Methodologies that allow investigators to observe and quantify the autophagic process will clearly advance our knowledge of how this process contributes to the pathophysiology of many clinical disorders. The recent identification of essential autophagy genes in higher eukaryotes has made it possible to analyze autophagy in mammalian cells that express autophagy proteins tagged with fluorescent markers. This chapter describes such methods using primary cultured neurons that undergo up-regulation of autophagy when trophic factors are removed from their medium. The prolonged up-regulated autophagy, in turn, contributes to the death of these neurons, thus providing a model to examine the relationship between enhanced autophagy and cell death. Neurons are isolated from the cerebellum of postnatal day 7 rat pups and cultured in the presence of trophic factors and depolarizing concentrations of potassium. Once established, the neurons are transfected with an adeno-viral vector expressing MAP1-LC3 with red fluorescent protein (RFP). MAP1-LC3 is the mammalian homologue of the yeast autophagosomal marker Atg8 and when tagged to GFP or RFP, it is the most widely used marker for autophagosomes. Once expression is stable, autophagy is induced by removing trophic factors. At various time points after inducing autophagy, the neurons are stained with LysoSensor Green (a pH-dependent lysosome marker) and Hoechst (a DNA marker) and subjected to live-cell imaging. In some cases, time-lapse imaging is used to examine the step-wise process of autophagy in live neurons. PMID:19216905

  16. Phase Dependency of the Human Primary Motor Cortex and Cholinergic Inhibition Cancelation During Beta tACS

    PubMed Central

    Guerra, Andrea; Pogosyan, Alek; Nowak, Magdalena; Tan, Huiling; Ferreri, Florinda; Di Lazzaro, Vincenzo; Brown, Peter

    2016-01-01

    The human motor cortex has a tendency to resonant activity at about 20 Hz so stimulation should more readily entrain neuronal populations at this frequency. We investigated whether and how different interneuronal circuits contribute to such resonance by using transcranial magnetic stimulation (TMS) during transcranial alternating current stimulation (tACS) at motor (20 Hz) and a nonmotor resonance frequency (7 Hz). We tested different TMS interneuronal protocols and triggered TMS pulses at different tACS phases. The effect of cholinergic short-latency afferent inhibition (SAI) was abolished by 20 Hz tACS, linking cortical beta activity to sensorimotor integration. However, this effect occurred regardless of the tACS phase. In contrast, 20 Hz tACS selectively modulated MEP size according to the phase of tACS during single pulse, GABAAergic short-interval intracortical inhibition (SICI) and glutamatergic intracortical facilitation (ICF). For SICI this phase effect was more marked during 20 Hz stimulation. Phase modulation of SICI also depended on whether or not spontaneous beta activity occurred at ~20 Hz, supporting an interaction effect between tACS and underlying circuit resonances. The present study provides in vivo evidence linking cortical beta activity to sensorimotor integration, and for beta oscillations in motor cortex being promoted by resonance in GABAAergic interneuronal circuits. PMID:27522077

  17. Acute effects of alcohol on stimulus-induced gamma oscillations in human primary visual and motor cortices.

    PubMed

    Campbell, Anne E; Sumner, Petroc; Singh, Krish D; Muthukumaraswamy, Suresh D

    2014-08-01

    Alcohol is a rich drug affecting both the γ-amino butyric acid (GABA) and glutamatergic neurotransmitter systems. Recent findings from both modeling and pharmacological manipulation have indicated a link between GABAergic activity and oscillations measured in the gamma frequency range (30-80 Hz), but there are no previous reports of alcohol's modulation of gamma-band activity measured by magnetoencephalography (MEG) or electroencephalography (EEG). In this single-blind, placebo-controlled crossover study, 16 participants completed two study days, on one day of which they consumed a dose of 0.8 g/kg alcohol, and on the other day a placebo. MEG recordings of brain activity were taken before and after beverage consumption, using visual grating and finger abduction paradigms known to induce gamma-band activity in the visual and motor cortices respectively. Time-frequency analyses of beamformer source reconstructions in the visual cortex showed that alcohol increased peak gamma amplitude and decreased peak frequency. For the motor task, alcohol increased gamma amplitude in the motor cortex. These data support the notion that gamma oscillations are dependent, in part, on the balance between excitation and inhibition. Disruption of this balance by alcohol, by increasing GABAergic inhibition at GABAA receptors and decreasing glutamatergic excitation at N-methyl-D-aspartic acid receptors, alters both the amplitude and frequency of gamma oscillations. The findings provide further insight into the neuropharmacological action of alcohol.

  18. Cytotoxicity Study on Luminescent Nanocrystals Containing Phospholipid Micelles in Primary Cultures of Rat Astrocytes.

    PubMed

    Latronico, Tiziana; Depalo, Nicoletta; Valente, Gianpiero; Fanizza, Elisabetta; Laquintana, Valentino; Denora, Nunzio; Fasano, Anna; Striccoli, Marinella; Colella, Matilde; Agostiano, Angela; Curri, M Lucia; Liuzzi, Grazia Maria

    2016-01-01

    Luminescent colloidal nanocrystals (NCs) are emerging as a new tool in neuroscience field, representing superior optical probes for cellular imaging and medical diagnosis of neurological disorders with respect to organic fluorophores. However, only a limited number of studies have, so far, explored NC applications in primary neurons, glia and related cells. Indeed astrocytes, as resident cells in the central nervous system (CNS), play an important pathogenic role in several neurodegenerative and neuroinflammatory diseases, therefore enhanced imaging tools for their thorough investigation are strongly amenable. Here, a comprehensive and systematic study on the in vitro toxicological effect of core-shell type luminescent CdSe@ZnS NCs incorporated in polyethylene glycol (PEG) terminated phospholipid micelles on primary cultures of rat astrocytes was carried out. Cytotoxicity response of empty micelles based on PEG modified phospholipids was compared to that of their NC containing counterpart, in order to investigate the effect on cell viability of both inorganic NCs and micelles protecting NC surface. Furthermore, since the surface charge and chemistry influence cell interaction and toxicity, effect of two different functional groups terminating PEG-modified phospholipid micelles, namely amine and carboxyl group, respectively, was evaluated against bare micelles, showing that carboxyl group was less toxic. The ability of PEG-lipid micelles to be internalized into the cells was qualitatively and quantitatively assessed by fluorescence microscopy and photoluminescence (PL) assay. The results of the experiments clearly demonstrate that, once incorporated into the micelles, a low, not toxic, concentration of NCs is sufficient to be distinctly detected within cells. The overall study provides essential indications to define the optimal experimental conditions to effectively and profitably use the proposed luminescent colloidal NCs as optical probe for future in vivo

  19. Intracellular degradation of chemically functionalized carbon nanotubes using a long-term primary microglial culture model.

    PubMed

    Bussy, Cyrill; Hadad, Caroline; Prato, Maurizio; Bianco, Alberto; Kostarelos, Kostas

    2016-01-01

    Chemically functionalized carbon nanotubes (f-CNTs) have been used in proof-of-concept studies to alleviate debilitating neurological conditions. Previous in vivo observations in brain tissue have suggested that microglia - acting as resident macrophages of the brain - play a critical role in the internalization of f-CNTs and their partial in situ biodegradation following a stereotactic administration in the cortex. At the same time, several reports have indicated that immune cells such as neutrophils, eosinophils and even macrophages could participate in the processing of carbon nanomaterials via oxidation processes leading to degradation, with surface properties acting as modulators of CNT biodegradability. In this study we questioned whether degradability of f-CNTs within microglia could be modulated depending on the type of surface functionalization used. We investigated the kinetics of degradation of multi-walled carbon nanotubes (MWNTs) functionalized via different chemical strategies that were internalized within isolated primary microglia over three months. A cellular model of rat primary microglia that can be maintained in cell culture for a long period of time was first developed. The Raman structural signature of the internalized f-CNTs was then studied directly in cells over a period of up to three months, following a single exposure to a non-cytotoxic concentration of three different f-CNTs (carboxylated, aminated and both carboxylated and aminated). Structural modifications suggesting partial but continuous degradation were observed for all nanotubes irrespective of their surface functionalization. Carboxylation was shown to promote more pronounced structural changes inside microglia over the first two weeks of the study.

  20. Cytotoxicity Study on Luminescent Nanocrystals Containing Phospholipid Micelles in Primary Cultures of Rat Astrocytes

    PubMed Central

    Valente, Gianpiero; Fanizza, Elisabetta; Laquintana, Valentino; Denora, Nunzio; Fasano, Anna; Striccoli, Marinella; Colella, Matilde; Agostiano, Angela; Curri, M. Lucia; Liuzzi, Grazia Maria

    2016-01-01

    Luminescent colloidal nanocrystals (NCs) are emerging as a new tool in neuroscience field, representing superior optical probes for cellular imaging and medical diagnosis of neurological disorders with respect to organic fluorophores. However, only a limited number of studies have, so far, explored NC applications in primary neurons, glia and related cells. Indeed astrocytes, as resident cells in the central nervous system (CNS), play an important pathogenic role in several neurodegenerative and neuroinflammatory diseases, therefore enhanced imaging tools for their thorough investigation are strongly amenable. Here, a comprehensive and systematic study on the in vitro toxicological effect of core-shell type luminescent CdSe@ZnS NCs incorporated in polyethylene glycol (PEG) terminated phospholipid micelles on primary cultures of rat astrocytes was carried out. Cytotoxicity response of empty micelles based on PEG modified phospholipids was compared to that of their NC containing counterpart, in order to investigate the effect on cell viability of both inorganic NCs and micelles protecting NC surface. Furthermore, since the surface charge and chemistry influence cell interaction and toxicity, effect of two different functional groups terminating PEG-modified phospholipid micelles, namely amine and carboxyl group, respectively, was evaluated against bare micelles, showing that carboxyl group was less toxic. The ability of PEG-lipid micelles to be internalized into the cells was qualitatively and quantitatively assessed by fluorescence microscopy and photoluminescence (PL) assay. The results of the experiments clearly demonstrate that, once incorporated into the micelles, a low, not toxic, concentration of NCs is sufficient to be distinctly detected within cells. The overall study provides essential indications to define the optimal experimental conditions to effectively and profitably use the proposed luminescent colloidal NCs as optical probe for future in vivo

  1. Copper Nanoparticles and Copper Sulphate Induced Cytotoxicity in Hepatocyte Primary Cultures of Epinephelus coioides

    PubMed Central

    Wang, Tao; Chen, Xiaoyan; Long, Xiaohua; Liu, Zhaopu; Yan, Shaohua

    2016-01-01

    Copper nanoparticles (Cu-NPs) were widely used in various industrial and commercial applications. The aim of this study was to analyze the cytotoxicity of Cu-NPs on primary hepatocytes of E.coioides compared with copper sulphate (CuSO4). Cultured cells were exposed to 0 or 2.4 mg Cu L-1 as CuSO4or Cu-NPs for 24-h. Results showed either form of Cu caused a dramatic loss in cell viability, more so in the CuSO4 than Cu-NPs treatment. Compared to control, either CuSO4 or Cu-NPs significantly increased reactive oxygen species(ROS) and malondialdehyde(MDA) concentration in hepatocytes by overwhelming total superoxide dismutase (T-SOD) activity, catalase(CAT) activity and glutathione(GSH) concentration. In addition, the antioxidative-related genes [SOD (Cu/Zn), SOD (Mn), CAT, GPx4] were also down-regulated. The apoptosis and necrosis percentage was significantly higher after the CuSO4 or Cu-NPs treatment than the control. The apoptosis was induced by the increased cytochrome c concentration in the cytosol and elevated caspase-3, caspase-8 and caspase-9 activities. Additionally, the apoptosis-related genes (p53, p38β and TNF-α) and protein (p53 protein) were up-regulated after the CuSO4 or Cu-NPs treatment, with CuSO4 exposure having a greater effect than Cu-NPs. In conclusion, Cu-NPs had similar types of toxic effects as CuSO4 on primary hepatocytes of E.coioides, but toxicity of CuSO4 was more severe than that of Cu-NPs. PMID:26890000

  2. Copper Nanoparticles and Copper Sulphate Induced Cytotoxicity in Hepatocyte Primary Cultures of Epinephelus coioides.

    PubMed

    Wang, Tao; Chen, Xiaoyan; Long, Xiaohua; Liu, Zhaopu; Yan, Shaohua

    2016-01-01

    Copper nanoparticles (Cu-NPs) were widely used in various industrial and commercial applications. The aim of this study was to analyze the cytotoxicity of Cu-NPs on primary hepatocytes of E.coioides compared with copper sulphate (CuSO4). Cultured cells were exposed to 0 or 2.4 mg Cu L-1 as CuSO4or Cu-NPs for 24-h. Results showed either form of Cu caused a dramatic loss in cell viability, more so in the CuSO4 than Cu-NPs treatment. Compared to control, either CuSO4 or Cu-NPs significantly increased reactive oxygen species(ROS) and malondialdehyde(MDA) concentration in hepatocytes by overwhelming total superoxide dismutase (T-SOD) activity, catalase(CAT) activity and glutathione(GSH) concentration. In addition, the antioxidative-related genes [SOD (Cu/Zn), SOD (Mn), CAT, GPx4] were also down-regulated. The apoptosis and necrosis percentage was significantly higher after the CuSO4 or Cu-NPs treatment than the control. The apoptosis was induced by the increased cytochrome c concentration in the cytosol and elevated caspase-3, caspase-8 and caspase-9 activities. Additionally, the apoptosis-related genes (p53, p38β and TNF-α) and protein (p53 protein) were up-regulated after the CuSO4 or Cu-NPs treatment, with CuSO4 exposure having a greater effect than Cu-NPs. In conclusion, Cu-NPs had similar types of toxic effects as CuSO4 on primary hepatocytes of E.coioides, but toxicity of CuSO4 was more severe than that of Cu-NPs.

  3. Intracellular degradation of chemically functionalized carbon nanotubes using a long-term primary microglial culture model.

    PubMed

    Bussy, Cyrill; Hadad, Caroline; Prato, Maurizio; Bianco, Alberto; Kostarelos, Kostas

    2016-01-01

    Chemically functionalized carbon nanotubes (f-CNTs) have been used in proof-of-concept studies to alleviate debilitating neurological conditions. Previous in vivo observations in brain tissue have suggested that microglia - acting as resident macrophages of the brain - play a critical role in the internalization of f-CNTs and their partial in situ biodegradation following a stereotactic administration in the cortex. At the same time, several reports have indicated that immune cells such as neutrophils, eosinophils and even macrophages could participate in the processing of carbon nanomaterials via oxidation processes leading to degradation, with surface properties acting as modulators of CNT biodegradability. In this study we questioned whether degradability of f-CNTs within microglia could be modulated depending on the type of surface functionalization used. We investigated the kinetics of degradation of multi-walled carbon nanotubes (MWNTs) functionalized via different chemical strategies that were internalized within isolated primary microglia over three months. A cellular model of rat primary microglia that can be maintained in cell culture for a long period of time was first developed. The Raman structural signature of the internalized f-CNTs was then studied directly in cells over a period of up to three months, following a single exposure to a non-cytotoxic concentration of three different f-CNTs (carboxylated, aminated and both carboxylated and aminated). Structural modifications suggesting partial but continuous degradation were observed for all nanotubes irrespective of their surface functionalization. Carboxylation was shown to promote more pronounced structural changes inside microglia over the first two weeks of the study. PMID:26647092

  4. Cytotoxicity Study on Luminescent Nanocrystals Containing Phospholipid Micelles in Primary Cultures of Rat Astrocytes.

    PubMed

    Latronico, Tiziana; Depalo, Nicoletta; Valente, Gianpiero; Fanizza, Elisabetta; Laquintana, Valentino; Denora, Nunzio; Fasano, Anna; Striccoli, Marinella; Colella, Matilde; Agostiano, Angela; Curri, M Lucia; Liuzzi, Grazia Maria

    2016-01-01

    Luminescent colloidal nanocrystals (NCs) are emerging as a new tool in neuroscience field, representing superior optical probes for cellular imaging and medical diagnosis of neurological disorders with respect to organic fluorophores. However, only a limited number of studies have, so far, explored NC applications in primary neurons, glia and related cells. Indeed astrocytes, as resident cells in the central nervous system (CNS), play an important pathogenic role in several neurodegenerative and neuroinflammatory diseases, therefore enhanced imaging tools for their thorough investigation are strongly amenable. Here, a comprehensive and systematic study on the in vitro toxicological effect of core-shell type luminescent CdSe@ZnS NCs incorporated in polyethylene glycol (PEG) terminated phospholipid micelles on primary cultures of rat astrocytes was carried out. Cytotoxicity response of empty micelles based on PEG modified phospholipids was compared to that of their NC containing counterpart, in order to investigate the effect on cell viability of both inorganic NCs and micelles protecting NC surface. Furthermore, since the surface charge and chemistry influence cell interaction and toxicity, effect of two different functional groups terminating PEG-modified phospholipid micelles, namely amine and carboxyl group, respectively, was evaluated against bare micelles, showing that carboxyl group was less toxic. The ability of PEG-lipid micelles to be internalized into the cells was qualitatively and quantitatively assessed by fluorescence microscopy and photoluminescence (PL) assay. The results of the experiments clearly demonstrate that, once incorporated into the micelles, a low, not toxic, concentration of NCs is sufficient to be distinctly detected within cells. The overall study provides essential indications to define the optimal experimental conditions to effectively and profitably use the proposed luminescent colloidal NCs as optical probe for future in vivo

  5. A validation study of the use of near-infrared spectroscopy imaging in primary and secondary motor areas of the human brain.

    PubMed

    Drenckhahn, Christoph; Koch, Stefan P; Dümmler, Johannes; Kohl-Bareis, Matthias; Steinbrink, Jens; Dreier, Jens P

    2015-08-01

    The electroencephalographically measured Bereitschafts (readiness)-potential in the supplementary motor area (SMA) serves as a signature of the preparation of motor activity. Using a multichannel, noninvasive near-infrared spectroscopy (NIRS) imager, we studied the vascular correlate of the readiness potential. Sixteen healthy subjects performed a self-paced or externally triggered motor task in a single or repetitive pattern, while NIRS simultaneously recorded the task-related responses of deoxygenated hemoglobin (HbR) in the primary motor area (M1) and the SMA. Right-hand movements in the repetitive sequence trial elicited a significantly greater HbR response in both the SMA and the left M1 compared to left-hand movements. During the single sequence condition, the HbR response in the SMA, but not in the M1, was significantly greater for self-paced than for externally cued movements. Nonetheless, an unequivocal temporal delay was not found between the SMA and M1. Near-infrared spectroscopy is a promising, noninvasive bedside tool for the neuromonitoring of epileptic seizures or cortical spreading depolarizations (CSDs) in patients with epilepsy, stroke, or brain trauma because these pathological events are associated with typical spatial and temporal changes in HbR. Propagation is a characteristic feature of these events which importantly supports their identification and characterization in invasive recordings. Unfortunately, the present noninvasive study failed to show a temporal delay during self-paced movements between the SMA and M1 as a vascular correlate of the readiness potential. Although this result does not exclude, in principle, the possibility that scalp-NIRS can detect a temporal delay between different regions during epileptic seizures or CSDs, it strongly suggests that further technological development of NIRS should focus on both improved spatial and temporal resolution. This article is part of a Special Issue entitled Status Epilepticus.

  6. A validation study of the use of near-infrared spectroscopy imaging in primary and secondary motor areas of the human brain.

    PubMed

    Drenckhahn, Christoph; Koch, Stefan P; Dümmler, Johannes; Kohl-Bareis, Matthias; Steinbrink, Jens; Dreier, Jens P

    2015-08-01

    The electroencephalographically measured Bereitschafts (readiness)-potential in the supplementary motor area (SMA) serves as a signature of the preparation of motor activity. Using a multichannel, noninvasive near-infrared spectroscopy (NIRS) imager, we studied the vascular correlate of the readiness potential. Sixteen healthy subjects performed a self-paced or externally triggered motor task in a single or repetitive pattern, while NIRS simultaneously recorded the task-related responses of deoxygenated hemoglobin (HbR) in the primary motor area (M1) and the SMA. Right-hand movements in the repetitive sequence trial elicited a significantly greater HbR response in both the SMA and the left M1 compared to left-hand movements. During the single sequence condition, the HbR response in the SMA, but not in the M1, was significantly greater for self-paced than for externally cued movements. Nonetheless, an unequivocal temporal delay was not found between the SMA and M1. Near-infrared spectroscopy is a promising, noninvasive bedside tool for the neuromonitoring of epileptic seizures or cortical spreading depolarizations (CSDs) in patients with epilepsy, stroke, or brain trauma because these pathological events are associated with typical spatial and temporal changes in HbR. Propagation is a characteristic feature of these events which importantly supports their identification and characterization in invasive recordings. Unfortunately, the present noninvasive study failed to show a temporal delay during self-paced movements between the SMA and M1 as a vascular correlate of the readiness potential. Although this result does not exclude, in principle, the possibility that scalp-NIRS can detect a temporal delay between different regions during epileptic seizures or CSDs, it strongly suggests that further technological development of NIRS should focus on both improved spatial and temporal resolution. This article is part of a Special Issue entitled Status Epilepticus. PMID

  7. Efficient and sustained gene expression in primary T lymphocytes and primary and cultured tumor cells mediated by adeno-associated virus plasmid DNA complexed to cationic liposomes.

    PubMed

    Philip, R; Brunette, E; Kilinski, L; Murugesh, D; McNally, M A; Ucar, K; Rosenblatt, J; Okarma, T B; Lebkowski, J S

    1994-04-01

    We have used cationic liposomes to facilitate adeno-associated virus (AAV) plasmid transfections of primary and cultured cell types. AAV plasmid DNA complexed with liposomes showed levels of expression several fold higher than those of complexes with standard plasmids. In addition, long-term expression (> 30 days) of the gene, unlike the transient expression demonstrated by typical liposome-mediated transfection with standard plasmids, was observed. Southern analysis of chromosomal DNA further substantiated the hypothesis that the long-term expression was due to the presence of the transgene in the AAV plasmid-transfected group and not in the standard plasmid-transfected group. AAV plasmid-liposome complexes induced levels of transgene expression comparable to those obtained by recombinant AAV transduction. Primary breast, ovarian, and lung tumor cells were transfectable with the AAV plasmid DNA-liposome complexes. Transfected primary and cultured tumor cells were able to express transgene product even after lethal irradiation. High-level gene expression was also observed in freshly isolated CD3+, CD4+, and CD8+ T cells from normal human peripheral blood. Transfection efficiency ranged from 10 to 50% as assessed by intracellular interleukin-2 levels in interleukin-2-transfected cells. The ability to express transgenes in primary tumor and lymphoid cells may be applied toward tumor vaccine studies and protocols which may eventually permit highly specific modulation of the cellular immune response in cancer and AIDS.

  8. Efficient and sustained gene expression in primary T lymphocytes and primary and cultured tumor cells mediated by adeno-associated virus plasmid DNA complexed to cationic liposomes.

    PubMed Central

    Philip, R; Brunette, E; Kilinski, L; Murugesh, D; McNally, M A; Ucar, K; Rosenblatt, J; Okarma, T B; Lebkowski, J S

    1994-01-01

    We have used cationic liposomes to facilitate adeno-associated virus (AAV) plasmid transfections of primary and cultured cell types. AAV plasmid DNA complexed with liposomes showed levels of expression several fold higher than those of complexes with standard plasmids. In addition, long-term expression (> 30 days) of the gene, unlike the transient expression demonstrated by typical liposome-mediated transfection with standard plasmids, was observed. Southern analysis of chromosomal DNA further substantiated the hypothesis that the long-term expression was due to the presence of the transgene in the AAV plasmid-transfected group and not in the standard plasmid-transfected group. AAV plasmid-liposome complexes induced levels of transgene expression comparable to those obtained by recombinant AAV transduction. Primary breast, ovarian, and lung tumor cells were transfectable with the AAV plasmid DNA-liposome complexes. Transfected primary and cultured tumor cells were able to express transgene product even after lethal irradiation. High-level gene expression was also observed in freshly isolated CD3+, CD4+, and CD8+ T cells from normal human peripheral blood. Transfection efficiency ranged from 10 to 50% as assessed by intracellular interleukin-2 levels in interleukin-2-transfected cells. The ability to express transgenes in primary tumor and lymphoid cells may be applied toward tumor vaccine studies and protocols which may eventually permit highly specific modulation of the cellular immune response in cancer and AIDS. Images PMID:8139545

  9. Lack of systematic effects of the 5-hydroxytryptamine 3 receptor antagonist ICS 205-930 on gastric emptying and antral motor activity in patients with primary anorexia nervosa.

    PubMed Central

    Stacher, G; Bergmann, H; Granser-Vacariu, G V; Wiesnagrotzki, S; Wenzelabatzi, T A; Gaupmann, G; Kugi, A; Steinringer, H; Schneider, C; Höbart, J

    1991-01-01

    1. The 5-hydroxytryptamine 3 receptor antagonist, ICS 205-930, has been reported to have potent effects on gastric smooth muscle and to enhance gastric emptying in animals, but findings in man have been inconsistent. 2. This study investigated the effects of ICS 205-930 on gastric emptying of an isotopically labelled semisolid 1168 kJ meal and on antral contractility in patients with primary anorexia nervosa, a condition frequently associated with impaired gastric motor function. 3. Thirteen female patients (age 18-39 years, median 22 years; percentage of ideal body weight 52-90%, median 66%) participated each in two studies, in which 0.15-0.18 mg kg-1 ICS 205-930 or placebo were infused i.v. in crossover, double-blind fashion. Gastric emptying and antral contractility were recorded scintigraphically for 50 min. 4. ICS 205-930 did not affect gastric emptying: the mean percentage of meal remaining in the stomach after 50 min (69.6% +/- 3.2 s.e. mean) was nearly identical to that after placebo (70.7 +/- 3.3%). 5. Amplitude, frequency and propagation velocity of antral contractions differed only little after ICS 205-930 and placebo, respectively. 6. The results show that ICS 205-930 has no effect on the impaired gastric motor activity in primary anorexia nervosa and thus provide further evidence that the compound does not have prominent prokinetic effects in man. PMID:1768560

  10. No effects of 20 Hz-rTMS of the primary motor cortex in vegetative state: A randomised, sham-controlled study.

    PubMed

    Cincotta, Massimo; Giovannelli, Fabio; Chiaramonti, Roberta; Bianco, Giovanni; Godone, Marco; Battista, Donato; Cardinali, Consuelo; Borgheresi, Alessandra; Sighinolfi, Antonella; D'Avanzo, Anna Maria; Breschi, Marco; Dine, Ylli; Lino, Mario; Zaccara, Gaetano; Viggiano, Maria Pia; Rossi, Simone

    2015-10-01

    We assessed the effects of a non-invasive neuromodulatory intervention with repetitive transcranial magnetic stimulation (rTMS) of the motor cortex in patients with vegetative state (VS) by a randomised, sham-controlled study with a cross-over design. Eleven patients classified as being in VS (9 post-anoxic, 2 post-traumatic, time elapsed from the injury 9-85 months) were included in the study. Real or sham 20 Hz rTMS were applied to the left primary motor cortex (M1) for 5 consecutive days. Primary outcome measures were changes in the JFK Coma Recovery Scale-Revised (CRS-R) scale total score and Clinical Global Impression Improvement (CGI-I) scale. Additional measures were EEG changes and impression of the patients' relatives using the CGI-I scale. Evaluations were blindly performed at baseline, after the first day of treatment, immediately after the end of the 5-days treatment, 1 week and 1 month later. Slight changes observed in the CRS-R and CGI-I scores did not significantly differ between real or sham stimulation conditions. EEG was not significantly changed on average, although spots of brain reactivity were occasionally found underneath the stimulation point. Findings did not provide evidence of therapeutic effect of 20 Hz rTMS of the M1 in chronic VS, at least with conventional coils and current safety parameters. Therefore, they might be useful to better allocate human and financial resources in future trials.

  11. A primary neuron culture system for the study of herpes simplex virus latency and reactivation.

    PubMed

    Kobayashi, Mariko; Kim, Ju-Youn; Camarena, Vladimir; Roehm, Pamela C; Chao, Moses V; Wilson, Angus C; Mohr, Ian

    2012-04-02

    Herpes simplex virus type-1 (HSV-1) establishes a life-long latent infection in peripheral neurons. This latent reservoir is the source of recurrent reactivation events that ensure transmission and contribute to clinical disease. Current antivirals do not impact the latent reservoir and there are no vaccines. While the molecular details of lytic replication are well-characterized, mechanisms controlling latency in neurons remain elusive. Our present understanding of latency is derived from in vivo studies using small animal models, which have been indispensable for defining viral gene requirements and the role of immune responses. However, it is impossible to distinguish specific effects on the virus-neuron relationship from more general consequences of infection mediated by immune or non-neuronal support cells in live animals. In addition, animal experimentation is costly, time-consuming, and limited in terms of available options for manipulating host processes. To overcome these limitations, a neuron-only system is desperately needed that reproduces the in vivo characteristics of latency and reactivation but offers the benefits of tissue culture in terms of homogeneity and accessibility. Here we present an in vitro model utilizing cultured primary sympathetic neurons from rat superior cervical ganglia (SCG) (Figure 1) to study HSV-1 latency and reactivation that fits most if not all of the desired criteria. After eliminating non-neuronal cells, near-homogeneous TrkA(+) neuron cultures are infected with HSV-1 in the presence of acyclovir (ACV) to suppress lytic replication. Following ACV removal, non-productive HSV-1 infections that faithfully exhibit accepted hallmarks of latency are efficiently established. Notably, lytic mRNAs, proteins, and infectious virus become undetectable, even in the absence of selection, but latency-associated transcript (LAT) expression persists in neuronal nuclei. Viral genomes are maintained at an average copy number of 25 per neuron

  12. Functional differentiation and alveolar morphogenesis of primary mammary cultures on reconstituted basement membrane

    SciTech Connect

    BARCELLOS-HOFF, M. H; AGGELER, J.; RAM, T. G; BISSELL, M. J

    1989-02-01

    An essential feature of mammary gland differentiation during pregnancy is the formation of alveoli composed of polarized epithelial cells, which, under the influence of lactogenic hormones, secrete vectorially and sequester milk proteins. Previous culture studies have described either organization of cells polarized towards lumina containing little or no demonstrable tissue-specific protein, or establishment of functional secretory cells exhibiting little or no glandular architecture. In this paper, we report that tissue-specific vectorial secretion coincides with the formation of functional alveoli-like structures by primary mammary epithelial cells cultured on a reconstituted basement membrane matrix (derived from Engelbreth-Holm-Swarm murine tumour). Morphogenesis of these unique three-dimensional structures was initiated by cell-directed remodelling of the exogenous matrix leading to reorganization of cells into matrixensheathed aggregates by 24 h after plating. The aggregates subsequently cavitated, so that by day 6 the cells were organized into hollow spheres in which apical cell surfaces faced lumina sealed by tight junctions and basal surfaces were surrounded by a distinct basal lamina. The profiles of proteins secreted into the apical (luminal) and basal (medium) compartments indicated that these alveoli-like structures were capable of an appreciable amount of vectorial secretion. Immunoprecipitation with a broad spectrum milk antiserum showed that more than 80% of caseins were secreted into the lumina, whereas iron-binding proteins (both lactoferrin and transferrin) were present in comparable amounts in each compartment. Thus, these mammary cells established protein targeting pathways directing milk-specific proteins to the luminal compartment. A time course monitoring secretory activity demonstrated that establishment of tissue-specific vectorial secretion and increased total and milk protein secretion coincided with functional alveolar

  13. Comparisons of mouse mesenchymal stem cells in primary adherent culture of compact bone fragments and whole bone marrow.

    PubMed

    Cai, Yiting; Liu, Tianshu; Fang, Fang; Xiong, Chengliang; Shen, Shiliang

    2015-01-01

    The purification of mouse bone marrow mesenchymal stem cells (BMSCs) by using the standard method of whole bone marrow adherence to plastic still remains ineffective. An increasing number of studies have indicated compact bone as an alternative source of BMSCs. We isolated BMSCs from cultured compact bone fragments and investigated the proliferative capacity, surface immunophenotypes, and osteogenic and adipogenic differentiations of the cells after the first trypsinization. The fragment culture was based on the fact that BMSCs were assembled in compact bones. Thus, the procedure included flushing bone marrow out of bone cavity and culturing the fragments without any collagenase digestion. The cell yield from cultured fragments was slightly less than that from cultured bone marrow using the same bone quantity. However, the trypsinized cells from cultured fragments exhibited significantly higher proliferation and were accompanied with more CD90 and CD44 expressions and less CD45 expression. The osteogenic and adipogenic differentiation capacity of cells from cultured fragments were better than those of cells from bone marrow. The directly adherent culture of compact bone is suitable for mouse BMSC isolation, and more BMSCs with potentially improved proliferation capacity can be obtained in the primary culture.

  14. Effect of low molecular weight epidermal material upon DNA synthesis in primary cultures of newborn rat keratinocytes

    SciTech Connect

    Abler, A.S.

    1985-01-01

    The objective of this study was to isolate inhibitors of replicative DNA synthesis from newborn rat epidermis. The strategy for this study was to assay epidermal extracts for inhibitors of DNA synthesis in primary cultures of newborn rat keratinocytes. DNA synthesis was measured as the incorporation of /sup 4/H-TdR into acid precipitable material. The low molecular weight fraction, LMWF (less than 10Kd), of an aqueous epidermal extract was found to contain activity that inhibits replicative DNA synthesis in primary cultures. The inhibitory activity of the LMWD was detected in a novel assay utilizing primary cultures that were synchronized at the G1/S boundary with the DNA polymerase alpha inhibitor, aphidicolin. LMWF caused a dose dependent inhibition of replicative DNA synthesis as measured by the incorporation of /sup 3/H-TdR into acid precipitable material. The magnitude of the inhibitory effect for a given dose of LMWF was dependent upon the duration of exposure to that dose. The results presented in this investigation suggest that newborn rat epidermis contains a small polypeptide factor that inhibits replicative DNA synthesis in primary culture of newborn rat keratinocytes.

  15. The Implementation of a Social Constructivist Approach in Primary Science Education in Confucian Heritage Culture: The Case of Vietnam

    ERIC Educational Resources Information Center

    H?ng, Ngô Vu Thu; Meijer, Marijn Roland; Bulte, Astrid M. W.; Pilot, Albert

    2015-01-01

    Social constructivism has been increasingly studied and implemented in science school education. Nevertheless, there is a lack of holistic studies on the implementation of social constructivist approach in primary science education in Confucian heritage culture. This study aims to determine to what extent a social constructivist approach is…

  16. The Possible Cultural Consequences for Children as They Learn to Read in English at Primary Three in Singapore

    ERIC Educational Resources Information Center

    Jones, Sally Ann

    2010-01-01

    This article presents the findings of an observation study of reading lessons in English at Primary Three in Singapore. The aims of the study were first to establish whether a common pedagogy to teach reading in English exists at this transition year of children's schooling, and second what the cultural effects of this pedagogy might be for…

  17. Transformation of primary cultures of shrimp (Penaeus stylirostris) lymphoid (Oka) organ with Simian virus-40 (T) antigen.

    PubMed

    Tapay, L M; Lu, Y; Brock, J A; Nadala, E C; Loh, P C

    1995-05-01

    Primary cultures of lymphoid (Oka) organ from Penaeus stylirostris were transformed with naked or Lipofectin-mediated pSV-3 neo, a shuttle vector containing the tumor (T) antigen gene from Simian virus-40. The transformed cells, OKTr-1 and OKTr-23, exhibited the following characteristics: rounded morphology forming grapelike aggregates, loosely adhesive, increased growth rate in Medium-199, resistance to G-418 (a neomycin analog marker in the shuttle vector), cloning efficiencies of 68.7% and 36.7% in soft agarose, respectively, and stability in liquid nitrogen storage. Immunofluorescence staining (IFA) of the transformed cells using a monoclonal antibody against SV-40 tumor antigen showed positive results. In contrast, primary cell cultures exhibited fibroblast-like morphology and formed a tight, adhesive monolayer on the surface of the culture vessel. They were sensitive to G-418, and showed negative results with IFA. To date, OKTr-1 and OKTr-23 have undergone 44 and 18 passages, respectively. Primary cultures of the lymphoid organ have not been successfully passaged beyond the primary stage.

  18. Development and characterization of a primary culture of chicken embryonic tracheal epithelial cells and their use in avian studies

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A major route of infection of avian influenza is through cells of the airway epithelium. To study the molecular mechanism of infection and early host responses we created a primary chicken tracheal cell culture. Epithelial cells were isolated from the trachea of 18 day old chicken embryos and cult...

  19. Role of TASK2 potassium channels regarding volume regulation in primary cultures of mouse proximal tubules.

    PubMed

    Barriere, Herve; Belfodil, Radia; Rubera, Isabelle; Tauc, Michel; Lesage, Florian; Poujeol, Chantal; Guy, Nicolas; Barhanin, Jacques; Poujeol, Philippe

    2003-08-01

    Several papers reported the role of TASK2 channels in cell volume regulation and regulatory volume decrease (RVD). To check the possibility that the TASK2 channel modulates the RVD process in kidney, we performed primary cultures of proximal convoluted tubules (PCT) and distal convoluted tubules (DCT) from wild-type and TASK2 knockout (KO) mice. In KO mice, the TASK2 coding sequence was in part replaced by the lac-Z gene. This allows for the precise localization of TASK2 in kidney sections using beta-galactosidase staining. TASK2 was only localized in PCT cells. K+ currents were analyzed by the whole-cell clamp technique with 125 mM K-gluconate in the pipette and 140 mM Na-gluconate in the bath. In PCT cells from wild-type mice, hypotonicity induced swelling-activated K+ currents insensitive to 1 mM tetraethylammonium, 10 nM charybdotoxin, and 10 microM 293B, but blocked by 500 microM quinidine and 10 microM clofilium. These currents were increased in alkaline pH and decreased in acidic pH. In PCT cells from TASK2 KO, swelling-activated K+ currents were completely impaired. In conclusion, the TASK2 channel is expressed in kidney proximal cells and could be the swelling-activated K+ channel responsible for the cell volume regulation process during osmolyte absorptions in the proximal tubules. PMID:12860925

  20. Toxicity evaluation of new agricultural fungicides in primary cultured cortical neurons.

    PubMed

    Regueiro, Jorge; Olguín, Nair; Simal-Gándara, Jesús; Suñol, Cristina

    2015-07-01

    Fungicides are crucial for food protection as well as for the production of crops of suitable quality and quantity to provide a viable economic return. Like other pesticides, fungicides are widely sprayed on agricultural land, especially in wine-growing areas, from where they can move-off after application. Furthermore, residues of these agrochemicals can remain on crops after harvest and even after some food processing operations, being a major exposure pathway. Although a relatively low toxicity has been claimed for this kind of compounds, information about their neurotoxicity is still scarce. In the present study, nine fungicides recently approved for agricultural uses in the EU - ametoctradin, boscalid, cyazofamid, dimethomorph, fenhexamid, kresoxim-methyl, mepanipyrim, metrafenone and pyraclostrobin - have been evaluated for their toxicity in primary cultured mouse cortical neurons. Exposure to 0.1-100µM for 7 days in vitro resulted in a dose-dependent toxicity in the MTT cell viability assay. Strobilurin fungicides kresoxim-methyl (KR) and pyraclostrobin (PY) were the most neurotoxic compounds (lethal concentration 50 were in the low micromolar and nanomolar levels, respectively) causing a rapid raise in intracellular calcium [Ca(2+)]i and strong depolarization of mitochondrial membrane potential. KR- and PY-induced cell death was reversed by the calcium channels blockers MK-801 and verapamil, suggesting that calcium entry through NMDA receptors and voltage-operated calcium channels are involved in KR- and PY-induced neurotoxicity. These results highlight the need for further evaluation of their neurotoxic effects in vivo.

  1. Phosphatidylcholine resynthesis from components of internalized phospholipids in rat granular pneumocytes in primary culture

    SciTech Connect

    Chander, A.; Reicherter, J.; Fisher, A.B.

    1986-05-01

    Uptake, degradation and reutilization of surfactant phospholipids was investigated by incubating granular pneumocytes in primary culture with 0.2 mM liposomal phosphatidylcholine containing (/sup 3/H-methyl)choline labeled dipalmitoyl PC. Trypsin-resistant cell associated liposome radioactivity in PC declined steadily with time of incubation to 50% of total radioactivity by 140 min. In the water soluble fraction, most of the radioactivity was present in glycerophosphorylcholine which increased steadily to 13% of total cell associated radioactivity. While the proportion of radioactivity in choline remained unchanged, it increased with time in CDP-choline and phosphorylcholine suggesting reutilization of choline for PC resynthesis. In lamellar bodies isolated from these cells, less than 10% of PC label was present in unsaturated PC. In the microsomal fraction the label in unsaturated PC at 21 min was 56% of total PC which increased to 71% by 140 min of incubation with liposomes (slope = 0.19%/min; r = 0.67) suggesting metabolic reutilization of dipalmitoyl PC in this compartment. These observations indicate that granular pneumocytes degrade internalized PC and resynthesize PC de novo from degradation products.

  2. PPAR-γ Impairment Alters Peroxisome Functionality in Primary Astrocyte Cell Cultures

    PubMed Central

    Di Cesare Mannelli, Lorenzo; Zanardelli, Matteo; Micheli, Laura; Ghelardini, Carla

    2014-01-01

    Peroxisomes provide glial cells with protective functions against the harmful effects of H2O2 on neurons and peroxisome impairment results in nervous lesions. Agonists of the γ-subtype of the Peroxisome-Proliferator-Activated-Receptors (PPAR) have been proposed as neuroprotective agents in neurodegenerative disorders. Nevertheless, the role of PPAR-γ alterations in pathophysiological mechanisms and the relevance of peroxisome functions in the PPAR-γ effects are not yet clear. In a primary cell culture of rat astrocytes, the irreversible PPAR-γ antagonist GW9662 concentration-dependently decreased the activity of catalase, the most important antioxidant defense enzyme in peroxisomes. Catalase functionality recovered in a few days and the PPAR-γ agonist rosiglitazone promoted reversal of enzymatic damage. The reversible antagonist G3335 reduced both the activity and expression of catalase in a rosiglitazone-prevented manner. G3335 reduced also the glutathione reductase expression, indicating that enzyme involved in glutathione regeneration was compromised. Neither the PPAR-α target gene Acyl-Coenzyme-A-oxidase-1 nor the mitochondrial detoxifying enzyme NADH:ubiquinone-oxidoreductase (NDFUS3) was altered by PPAR-γ inhibition. In conclusion, PPAR-γ inhibition induced impairment of catalase in astrocytes. A general decrease of the antioxidant defenses of the cell suggests that a PPAR-γ hypofunction could participate in neurodegenerative mechanisms through peroxisomal damage. This series of experiments could be a useful model for studying compounds able to restore peroxisome functionality. PMID:24729976

  3. Effect of Carnosine in Experimental Arthritis and on Primary Culture Chondrocytes.

    PubMed

    Ponist, S; Drafi, F; Kuncirova, V; Mihalova, D; Rackova, L; Danisovic, L; Ondrejickova, O; Tumova, I; Trunova, O; Fedorova, T; Bauerova, K

    2016-01-01

    Carnosine's (CARN) anti-inflammatory potential in autoimmune diseases has been but scarcely investigated as yet. The aim of this study was to evaluate the therapeutic potential of CARN in rat adjuvant arthritis, in the model of carrageenan induced hind paw edema (CARA), and also in primary culture of chondrocytes under H2O2 injury. The experiments were done on healthy animals, arthritic animals, and arthritic animals with oral administration of CARN in a daily dose of 150 mg/kg b.w. during 28 days as well as animals with CARA treated by a single administration of CARN in the same dose. CARN beneficially affected hind paw volume and changes in body weight on day 14 and reduced hind paw swelling in CARA. Markers of oxidative stress in plasma and brain (malondialdehyde, 4-hydroxynonenal, protein carbonyls, and lag time of lipid peroxidation) and also activity of gamma-glutamyltransferase were significantly corrected by CARN. CARN also reduced IL-1alpha in plasma. Suppression of intracellular oxidant levels was also observed in chondrocytes pretreated with CARN. Our results obtained on two animal models showed that CARN has systemic anti-inflammatory activity and protected rat brain and chondrocytes from oxidative stress. This finding suggests that CARN might be beneficial for treatment of arthritic diseases. PMID:26885252

  4. A novel method for primary neuronal culture and characterization under different high temperature.

    PubMed

    Zhang, Tao; Hu, Huaiqiang; Tao, Zhen; Niu, Bing; Jiao, Shusheng; Zhang, Jun; Li, Yiyang; Cao, Bingzhen

    2016-09-01

    Heatstroke is a big threat to human health; however, the characteristic of pathological changes of neurons during heatstroke development remains unclear. Here, using an in vitro model of primary cultured neurons from newborn Wistar rats, we investigated the effects of the different combinations of high temperature (37, 39, 41, 43, 45, and 47°C) and exposure time (45 min and 1 h) on the neurons. We found that, under the treatment of 45 min-heat, the neurons could resist high temperature up to 45°C, and under the treatment of 1 h-heat, the mortality of neurons increased as the temperature rises. After heating for 1 h, only a small minority of the neurons died under 41 and 43°C, which primarily occurred in the form of apoptosis. Up to 45°C for 1 h, most neurons occurred to necrosis. Meaningfully, some necrotic neurons expressed specific fried egg-like morphology. Our findings suggest that different high temperatures and exposure times were two key factors influencing the death of neurons. Under the high temperature (below 43°C for 1 h) similar to heatstroke, it just led a small percentage of neurons to apoptosis, and anti-apoptosis controls for preventing and treating heatstroke are promising. PMID:27130681

  5. Neurogenic and neurotrophic effects of BDNF peptides in mouse hippocampal primary neuronal cell cultures.

    PubMed

    Cardenas-Aguayo, Maria del Carmen; Kazim, Syed Faraz; Grundke-Iqbal, Inge; Iqbal, Khalid

    2013-01-01

    The level of brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, is down regulated in Alzheimer's disease (AD), Parkinson's disease (PD), depression, stress, and anxiety; conversely the level of this neurotrophin is increased in autism spectrum disorders. Thus, modulating the level of BDNF can be a potential therapeutic approach for nervous system pathologies. In the present study, we designed five different tetra peptides (peptides B-1 to B-5) corresponding to different active regions of BDNF. These tetra peptides were found to be non-toxic, and they induced the expression of neuronal markers in mouse embryonic day 18 (E18) primary hippocampal neuronal cultures. Additionally, peptide B-5 induced the expression of BDNF and its receptor, TrkB, suggesting a positive feedback mechanism. The BDNF peptides induced only a moderate activation (phosphorylation at Tyr 706) of the TrkB receptor, which could be blocked by the Trk's inhibitor, K252a. Peptide B-3, when combined with BDNF, potentiated the survival effect of this neurotrophin on H(2)O(2)-treated E18 hippocampal cells. Peptides B-3 and B-5 were found to work as partial agonists and as partial antagonists competing with BDNF to activate the TrkB receptor in a dose-dependent manner. Taken together, these results suggest that the described BDNF tetra peptides are neurotrophic, can modulate BDNF signaling in a partial agonist/antagonist way, and offer a novel therapeutic approach to neural pathologies where BDNF levels are dysregulated. PMID:23320097

  6. Autophagy Plays a Cytoprotective Role During Cadmium-Induced Oxidative Damage in Primary Neuronal Cultures.

    PubMed

    Wang, Tao; Wang, Qiwen; Song, Ruilong; Zhang, Yajing; Zhang, Kangbao; Yuan, Yan; Bian, Jianchun; Liu, Xuezhong; Gu, Jianhong; Liu, Zongping

    2015-12-01

    Cadmium (Cd) induces significant oxidative damage in cells. Recently, it was reported that autophagy could be induced by Cd in neurons. However, little is known about the role of reactive oxygen species (ROS) during Cd-induced autophagy. In our study, we examined the cross-talk between ROS and autophagy by using N-acetyl cysteine (NAC, an antioxidant) and chloroquine (CQ, a pharmacological inhibitor of autophagy) in a primary rat neuronal cell cultures. We observed accumulation of acidic vesicular organelles and the increased expression of endogenous protein light chain 3 (LC3) in Cd-treated neurons, revealing that Cd induced a high level of autophagy. Moreover, increased levels of ROS were observed in neurons treated with Cd, showing that ROS accumulation was closely associated with neuron's exposure to Cd. Furthermore, we found that autophagy was inhibited by using CQ and/or NAC with further aggravation of mitochondrial damage, lactate dehydrogenase (LDH) leakage and hypoploid apoptotic cell number in Cd-treated neurons. These results proved that autophagy has a cytoprotective role during Cd-induced toxicity in neurons, and it can prevent the oxidative damage. These findings may enable the development of novel therapeutic strategies for neurological diseases.

  7. Ambroxol inhibits rhinovirus infection in primary cultures of human tracheal epithelial cells.

    PubMed

    Yamaya, Mutsuo; Nishimura, Hidekazu; Nadine, Lusamba Kalonji; Ota, Chiharu; Kubo, Hiroshi; Nagatomi, Ryoichi

    2014-04-01

    The mucolytic drug ambroxol hydrochloride reduces the production of pro-inflammatory cytokines and the frequency of exacerbation in patients with chronic obstructive pulmonary disease (COPD). However, the inhibitory effects of ambroxol on rhinovirus infection, the major cause of COPD exacerbations, have not been studied. We examined the effects of ambroxol on type 14 rhinovirus (RV14) infection, a major RV group, in primary cultures of human tracheal epithelial cells. RV14 infection increased virus titers and cytokine content in the supernatants and RV14 RNA in the cells. Ambroxol (100 nM) reduced RV14 titers and cytokine concentrations of interleukin (IL)-1β, IL-6 and IL-8 in the supernatants and RV14 RNA in the cells after RV14 infection, in addition to reducing susceptibility to RV14 infection. Ambroxol also reduced the expression of intercellular adhesion molecule-1 (ICAM-1), the receptor for RV14, and the number of acidic endosomes from which RV14 RNA enters the cytoplasm. In addition, ambroxol reduced the activation of the transcription factor nuclear factor kappa B (NF-κB) in the nucleus. These results suggest that ambroxol inhibits RV14 infection partly by reducing ICAM-1 and acidic endosomes via the inhibition of NF-κB activation. Ambroxol may modulate airway inflammation by reducing the production of cytokines in rhinovirus infection.

  8. Hepatocyte growth factor enhances the barrier function in primary cultures of rat brain microvascular endothelial cells.

    PubMed

    Yamada, Narumi; Nakagawa, Shinsuke; Horai, Shoji; Tanaka, Kunihiko; Deli, Maria A; Yatsuhashi, Hiroshi; Niwa, Masami

    2014-03-01

    The effects of hepatocyte growth factor (HGF) on barrier functions were investigated by a blood-brain barrier (BBB) in vitro model comprising a primary culture of rat brain capillary endothelial cells (RBEC). In order to examine the response of the peripheral endothelial cells to HGF, human umbilical vascular endothelial cells (HUVEC) and human dermal microvascular endothelial cells (HMVEC) were also treated with HGF. HGF decreased the permeability of RBEC to sodium fluorescein and Evans blue albumin, and dose-dependently increased transendothelial electrical resistance (TEER) in RBEC. HGF altered the immunochemical staining pattern of F-actin bands and made ZO-1 staining more distinct on the linear cell borders in RBEC. In contrast, HGF increased sodium fluorescein and Evans blue albumin permeability in HMVEC and HUVEC, and decreased TEER in HMVEC. In HMVEC, HGF reduced cortical actin bands and increased stress fiber density, and increased the zipper-like appearance of ZO-1 staining. Western blot analysis showed that HGF significantly increased the amount of ZO-1 and VE-cadherin. HGF seems to act on the BBB to strengthen BBB integrity. These findings indicated that cytoskeletal rearrangement and cell-cell adhesion, such as through VE-cadherin and ZO-1, are candidate mechanisms for the influence of HGF on the BBB. The possibility that HGF has therapeutic significance in protecting the BBB from damage needs to be considered. PMID:24370951

  9. Ambroxol inhibits rhinovirus infection in primary cultures of human tracheal epithelial cells.

    PubMed

    Yamaya, Mutsuo; Nishimura, Hidekazu; Nadine, Lusamba Kalonji; Ota, Chiharu; Kubo, Hiroshi; Nagatomi, Ryoichi

    2014-04-01

    The mucolytic drug ambroxol hydrochloride reduces the production of pro-inflammatory cytokines and the frequency of exacerbation in patients with chronic obstructive pulmonary disease (COPD). However, the inhibitory effects of ambroxol on rhinovirus infection, the major cause of COPD exacerbations, have not been studied. We examined the effects of ambroxol on type 14 rhinovirus (RV14) infection, a major RV group, in primary cultures of human tracheal epithelial cells. RV14 infection increased virus titers and cytokine content in the supernatants and RV14 RNA in the cells. Ambroxol (100 nM) reduced RV14 titers and cytokine concentrations of interleukin (IL)-1β, IL-6 and IL-8 in the supernatants and RV14 RNA in the cells after RV14 infection, in addition to reducing susceptibility to RV14 infection. Ambroxol also reduced the expression of intercellular adhesion molecule-1 (ICAM-1), the receptor for RV14, and the number of acidic endosomes from which RV14 RNA enters the cytoplasm. In addition, ambroxol reduced the activation of the transcription factor nuclear factor kappa B (NF-κB) in the nucleus. These results suggest that ambroxol inhibits RV14 infection partly by reducing ICAM-1 and acidic endosomes via the inhibition of NF-κB activation. Ambroxol may modulate airway inflammation by reducing the production of cytokines in rhinovirus infection. PMID:23856970

  10. Cultural responses to pain in UK children of primary school age: a mixed-methods study.

    PubMed

    Azize, Pary M; Endacott, Ruth; Cattani, Allegra; Humphreys, Ann

    2014-06-01

    Pain-measurement tools are often criticized for not addressing the influence of culture and ethnicity on pain. This study examined how children who speak English as a primary or additional language discuss pain. Two methods were used in six focus group interviews with 34 children aged 4-7 years: (i) use of drawings from the Pediatric Pain Inventory to capture the language used by children to describe pain; and (ii) observation of the children's placing of pain drawings on red/amber/green paper to denote perceived severity of pain. The findings demonstrated that children with English as an additional language used less elaborate language when talking about pain, but tended to talk about the pictures prior to deciding where they should be placed. For these children, there was a positive significant relationship between language, age, and length of stay in the UK. The children's placement of pain drawings varied according to language background, sex, and age. The findings emphasize the need for sufficient time to assess pain adequately in children who do not speak English as a first language.

  11. Biosynthesis and secretion of alpha 1 acute-phase globulin in primary cultures of rat hepatocytes.

    PubMed

    Bauer, J; Kurdowska, A; Tran-Thi, T A; Budek, W; Koj, A; Decker, K; Heinrich, P C

    1985-01-15

    Experimental inflammation in rats led to a sevenfold increase in serum levels of alpha 1 acute-phase globulin. This increase is correlated with elevated levels of translatable mRNA for alpha 1 acute-phase globulin in the liver. Biosynthesis and secretion of alpha 1 acute-phase globulin were studied in rat hepatocyte primary cultures. An intracellular form of alpha 1 acute-phase globulin with an apparent relative molecular mass of 63 500 and a secreted form of 68 000 were found. The intracellular form of alpha 1 acute-phase globulin could be deglycosylated by endoglucosaminidase H treatment indicating that its oligosaccharide chains were of the high-mannose type. The secreted form of alpha 1 acute-phase globulin was not sensitive to endoglucosaminidase H, but was susceptible to the action of sialidase reflecting carbohydrate side-chains of the complex type. Pulse-chase experiments revealed a precursor-product relationship for the high-mannose and the complex type alpha 1 acute-phase globulin. In the hepatocyte medium newly synthesized alpha 1 acute-phase globulin was detected 30 min after the pulse. Unglycosylated alpha 1 acute-phase globulin was found in the cells as well as in the medium when the transfer of oligosaccharide chains onto the polypeptide chains was blocked by tunicamycin. Tunicamycin led to a marked delay in alpha 1 acute-phase globulin secretion. PMID:2578391

  12. Effects of iron on rainbow trout gill cells in primary culture.

    PubMed

    Leguen, Isabelle; Peron, Sandrine; Prunet, Patrick

    2011-10-01

    This study investigated the effects of iron in the form of iron sulphate (FeSO(4)·7H(2)O), over the range 0.01-1 mM on rainbow trout primary gill cells cultured on semi-permeable membranes. The endpoints measured were cell proliferation, mucous cell numbers, area of mucus in mucous cells, ultrastructural analysis and transepithelial resistance. Regardless of the concentration, FeSO(4) did not modify the apical surface of pavement cells (microridge) and mucous cells. However, at 1 mM, this metal reduced cell numbers, by inhibiting cell proliferation and causing cell death, and induced a decrease in transepithelial resistance. It is interesting to note that cell numbers were also reduced in the presence of 0.5 mM iron salt, although this reduction did not modify transepithelial resistance. FeSO(4) reduced mucous cell number but did not change mucus area in mucous cells suggesting that this metal could induce a discharge of mucous cells, but mucus secretion would be total and not partial. In conclusion, our in vitro model has allowed to study some toxic effect but also resistance of gill epithelium in presence of iron.

  13. Ectopic lignification in primary cellulose-deficient cell walls of maize cell suspension cultures.

    PubMed

    Mélida, Hugo; Largo-Gosens, Asier; Novo-Uzal, Esther; Santiago, Rogelio; Pomar, Federico; García, Pedro; García-Angulo, Penélope; Acebes, José Luis; Álvarez, Jesús; Encina, Antonio

    2015-04-01

    Maize (Zea mays L.) suspension-cultured cells with up to 70% less cellulose were obtained by stepwise habituation to dichlobenil (DCB), a cellulose biosynthesis inhibitor. Cellulose deficiency was accompanied by marked changes in cell wall matrix polysaccharides and phenolics as revealed by Fourier transform infrared (FTIR) spectroscopy. Cell wall compositional analysis indicated that the cellulose-deficient cell walls showed an enhancement of highly branched and cross-linked arabinoxylans, as well as an increased content in ferulic acid, diferulates and p-coumaric acid, and the presence of a polymer that stained positive for phloroglucinol. In accordance with this, cellulose-deficient cell walls showed a fivefold increase in Klason-type lignin. Thioacidolysis/GC-MS analysis of cellulose-deficient cell walls indicated the presence of a lignin-like polymer with a Syringyl/Guaiacyl ratio of 1.45, which differed from the sensu stricto stress-related lignin that arose in response to short-term DCB-treatments. Gene expression analysis of these cells indicated an overexpression of genes specific for the biosynthesis of monolignol units of lignin. A study of stress signaling pathways revealed an overexpression of some of the jasmonate signaling pathway genes, which might trigger ectopic lignification in response to cell wall integrity disruptions. In summary, the structural plasticity of primary cell walls is proven, since a lignification process is possible in response to cellulose impoverishment. PMID:25735403

  14. Effect of Carnosine in Experimental Arthritis and on Primary Culture Chondrocytes

    PubMed Central

    Ponist, S.; Drafi, F.; Kuncirova, V.; Mihalova, D.; Rackova, L.; Danisovic, L.; Ondrejickova, O.; Tumova, I.; Trunova, O.; Fedorova, T.; Bauerova, K.

    2016-01-01

    Carnosine's (CARN) anti-inflammatory potential in autoimmune diseases has been but scarcely investigated as yet. The aim of this study was to evaluate the therapeutic potential of CARN in rat adjuvant arthritis, in the model of carrageenan induced hind paw edema (CARA), and also in primary culture of chondrocytes under H2O2 injury. The experiments were done on healthy animals, arthritic animals, and arthritic animals with oral administration of CARN in a daily dose of 150 mg/kg b.w. during 28 days as well as animals with CARA treated by a single administration of CARN in the same dose. CARN beneficially affected hind paw volume and changes in body weight on day 14 and reduced hind paw swelling in CARA. Markers of oxidative stress in plasma and brain (malondialdehyde, 4-hydroxynonenal, protein carbonyls, and lag time of lipid peroxidation) and also activity of gamma-glutamyltransferase were significantly corrected by CARN. CARN also reduced IL-1alpha in plasma. Suppression of intracellular oxidant levels was also observed in chondrocytes pretreated with CARN. Our results obtained on two animal models showed that CARN has systemic anti-inflammatory activity and protected rat brain and chondrocytes from oxidative stress. This finding suggests that CARN might be beneficial for treatment of arthritic diseases. PMID:26885252

  15. Differential in vitro effects of chemotherapeutic agents on primary cultures of human ovarian carcinoma.

    PubMed

    Kornblith, P; Ochs, R L; Wells, A; Gabrin, M J; Piwowar, J; Chattopadhyay, A; George, L D; Burholt, D

    2004-01-01

    The treatment of ovarian cancer principally relies on the use of platinum and taxane chemotherapeutic agents. Short-term clinical results have been encouraging, but long-term responses remain limited. In this report, an in vitro assay system that utilizes cells grown from human tumor explants has been used to quantitatively evaluate responses to relevant concentrations of alternative chemotherapeutic agents. The results suggest that there are significant differences in the responses of explant-derived cultured cells to the different agents tested. In an evaluation of 276 primary ovarian cancer specimens, five nonstandard drugs were tested in 51 cases. Of these 51 cases, cyclophosphamide had the highest rate of response at 67%, followed by doxorubicin at 61%, gemcitabine at 49%, etoposide at 48%, and topotecan at 14%. Venn diagrams, representing the in vitro responses to the platins and taxanes, as well as the responses to the nonstandard drugs, illustrate that there clearly are distinct differences among patients in a given population. These data underscore the potential importance of evaluating each patient's response to a number of different drugs to optimize the therapeutic decision-making process. PMID:15304154

  16. Ectopic lignification in primary cellulose-deficient cell walls of maize cell suspension cultures.

    PubMed

    Mélida, Hugo; Largo-Gosens, Asier; Novo-Uzal, Esther; Santiago, Rogelio; Pomar, Federico; García, Pedro; García-Angulo, Penélope; Acebes, José Luis; Álvarez, Jesús; Encina, Antonio

    2015-04-01

    Maize (Zea mays L.) suspension-cultured cells with up to 70% less cellulose were obtained by stepwise habituation to dichlobenil (DCB), a cellulose biosynthesis inhibitor. Cellulose deficiency was accompanied by marked changes in cell wall matrix polysaccharides and phenolics as revealed by Fourier transform infrared (FTIR) spectroscopy. Cell wall compositional analysis indicated that the cellulose-deficient cell walls showed an enhancement of highly branched and cross-linked arabinoxylans, as well as an increased content in ferulic acid, diferulates and p-coumaric acid, and the presence of a polymer that stained positive for phloroglucinol. In accordance with this, cellulose-deficient cell walls showed a fivefold increase in Klason-type lignin. Thioacidolysis/GC-MS analysis of cellulose-deficient cell walls indicated the presence of a lignin-like polymer with a Syringyl/Guaiacyl ratio of 1.45, which differed from the sensu stricto stress-related lignin that arose in response to short-term DCB-treatments. Gene expression analysis of these cells indicated an overexpression of genes specific for the biosynthesis of monolignol units of lignin. A study of stress signaling pathways revealed an overexpression of some of the jasmonate signaling pathway genes, which might trigger ectopic lignification in response to cell wall integrity disruptions. In summary, the structural plasticity of primary cell walls is proven, since a lignification process is possible in response to cellulose impoverishment.

  17. Astrocytes Enhance Streptococcus suis-Glial Cell Interaction in Primary Astrocyte-Microglial Cell Co-Cultures.

    PubMed

    Seele, Jana; Nau, Roland; Prajeeth, Chittappen K; Stangel, Martin; Valentin-Weigand, Peter; Seitz, Maren

    2016-06-13

    Streptococcus (S.) suis infections are the most common cause of meningitis in pigs. Moreover, S. suis is a zoonotic pathogen, which can lead to meningitis in humans, mainly in adults. We assume that glial cells may play a crucial role in host-pathogen interactions during S. suis infection of the central nervous system. Glial cells are considered to possess important functions during inflammation and injury of the brain in bacterial meningitis. In the present study, we established primary astrocyte-microglial cell co-cultures to investigate interactions of S. suis with glial cells. For this purpose, microglial cells and astrocytes were isolated from new-born mouse brains and characterized by flow cytometry, followed by the establishment of astrocyte and microglial cell mono-cultures as well as astrocyte-microglial cell co-cultures. In addition, we prepared microglial cell mono-cultures co-incubated with uninfected astrocyte mono-culture supernatants and astrocyte mono-cultures co-incubated with uninfected microglial cell mono-culture supernatants. After infection of the different cell cultures with S. suis, bacteria-cell association was mainly observed with microglial cells and most prominently with a non-encapsulated mutant of S. suis. A time-dependent induction of NO release was found only in the co-cultures and after co-incubation of microglial cells with uninfected supernatants of astrocyte mono-cultures mainly after infection with the capsular mutant. Only moderate cytotoxic effects were found in co-cultured glial cells after infection with S. suis. Taken together, astrocytes and astrocyte supernatants increased interaction of microglial cells with S. suis. Astrocyte-microglial cell co-cultures are suitable to study S. suis infections and bacteria-cell association as well as NO release by microglial cells was enhanced in the presence of astrocytes.

  18. Astrocytes Enhance Streptococcus suis-Glial Cell Interaction in Primary Astrocyte-Microglial Cell Co-Cultures

    PubMed Central

    Seele, Jana; Nau, Roland; Prajeeth, Chittappen K.; Stangel, Martin; Valentin-Weigand, Peter; Seitz, Maren

    2016-01-01

    Streptococcus (S.) suis infections are the most common cause of meningitis in pigs. Moreover, S. suis is a zoonotic pathogen, which can lead to meningitis in humans, mainly in adults. We assume that glial cells may play a crucial role in host-pathogen interactions during S. suis infection of the central nervous system. Glial cells are considered to possess important functions during inflammation and injury of the brain in bacterial meningitis. In the present study, we established primary astrocyte-microglial cell co-cultures to investigate interactions of S. suis with glial cells. For this purpose, microglial cells and astrocytes were isolated from new-born mouse brains and characterized by flow cytometry, followed by the establishment of astrocyte and microglial cell mono-cultures as well as astrocyte-microglial cell co-cultures. In addition, we prepared microglial cell mono-cultures co-incubated with uninfected astrocyte mono-culture supernatants and astrocyte mono-cultures co-incubated with uninfected microglial cell mono-culture supernatants. After infection of the different cell cultures with S. suis, bacteria-cell association was mainly observed with microglial cells and most prominently with a non-encapsulated mutant of S. suis. A time-dependent induction of NO release was found only in the co-cultures and after co-incubation of microglial cells with uninfected supernatants of astrocyte mono-cultures mainly after infection with the capsular mutant. Only moderate cytotoxic effects were found in co-cultured glial cells after infection with S. suis. Taken together, astrocytes and astrocyte supernatants increased interaction of microglial cells with S. suis. Astrocyte-microglial cell co-cultures are suitable to study S. suis infections and bacteria-cell association as well as NO release by microglial cells was enhanced in the presence of astrocytes. PMID:27304968

  19. Human adipose-derived mesenchymal stem cells improve motor functions and are neuroprotective in the 6-hydroxydopamine-rat model for Parkinson's disease when cultured in monolayer cultures but suppress hippocampal neurogenesis and hippocampal memory function when cultured in spheroids.

    PubMed

    Berg, Jürgen; Roch, Manfred; Altschüler, Jennifer; Winter, Christine; Schwerk, Anne; Kurtz, Andreas; Steiner, Barbara

    2015-02-01

    Adult human adipose-derived mesenchymal stem cells (MSC) have been reported to induce neuroprotective effects in models for Parkinson's disease (PD). However, these effects strongly depend on the most optimal application of the transplant. In the present study we compared monolayer-cultured (aMSC) and spheroid (sMSC) MSC following transplantation into the substantia nigra (SN) of 6-OHDA lesioned rats regarding effects on the local microenvironment, degeneration of dopaminergic neurons, neurogenesis in the hippocampal DG as well as motor and memory function in the 6-OHDA-rat model for PD. aMSC transplantation significantly increased tyrosine hydroxylase (TH) and brain-derived neurotrophic factor (BDNF) levels in the SN, increased the levels of the glial fibrillary acidic protein (GFAP) and improved motor functions compared to untreated and sMSC treated animals. In contrast, sMSC grafting induced an increased local microgliosis, decreased TH levels in the SN and reduced numbers of newly generated cells in the dentate gyrus (DG) without yet affecting hippocampal learning and memory function. We conclude that the neuroprotective potential of adipose-derived MSC in the rat model of PD crucially depends on the applied cellular phenotype.

  20. Expression of genes coding for antioxidant enzymes and heat shock proteins is altered in primary cultures of rat hepatocytes.

    PubMed

    Van Remmen, H; Williams, M D; Heydari, A R; Takahashi, R; Chung, H Y; Yu, B P; Richardson, A

    1996-02-01

    The expression of genes for heat shock proteins in the HSP70 family and genes for antioxidant enzymes was studied in rat hepatocytes cultured in either L-15 or Williams E media on a collagen matrix for up to 48 hours. The mRNA transcripts for the heat shock proteins hsp70, hsc70, and grp78 were induced dramatically when hepatocytes were cultured in L-15, and to a lesser extent when cultured in Williams E. The increase in hsp70 and hsc70 mRNA levels in the cultured hepatocytes was correlated with an increase in the nuclear transcription of these two genes and the binding activity of the heat shock transcription factor to the heat shock element. Culturing rat hepatocytes in either L-15 or Williams E resulted in a decrease in the levels of the mRNA transcripts for catalase and glutathione peroxidase and the activities of these two enzymes. However, the expression of Cu/Zn-superoxide dismutase, i.e., the level of the mRNA transcript or the enzymatic activity, did not change appreciably when hepatocytes were cultured for up to 48 hours. The decline in catalase and glutathione peroxidase expression in the cultured hepatocytes was correlated with a decrease in the GSH/GSSG ratio and an increase in lipid peroxidation. These data show that the expression of several genes involved in cellular protection change when hepatocytes are placed in primary cultures. Therefore, one must be careful in extrapolating from primary cultures to the liver in vivo, especially when studying processes that might be affected by heat shock proteins or antioxidant enzymes.

  1. Long-term human primary hepatocyte cultures in a microfluidic liver biochip show maintenance of mRNA levels and higher drug metabolism compared with Petri cultures.

    PubMed

    Jellali, Rachid; Bricks, Thibault; Jacques, Sébastien; Fleury, Marie-José; Paullier, Patrick; Merlier, Franck; Leclerc, Eric

    2016-07-01

    Human primary hepatocytes were cultivated in a microfluidic bioreactor and in Petri dishes for 13 days. mRNA kinetics in biochips showed an increase in the levels of CYP2B6, CYP2C19, CYP2C8, CYP3A4, CYP1A2, CYP2D6, HNF4a, SULT1A1, UGT1A1 mRNA related genes when compared with post extraction levels. In addition, comparison with Petri dishes showed higher levels of CYP2B6, CYP2C19, CYP2C8, CYP3A4, CYP1A2, CYP2D6 related genes at the end of culture. Functional assays illustrated a higher urea and albumin production over the period of culture in biochips. Bioreactor drug metabolism (midazolam and phenacetin) was not superior to the Petri dish after 2 days of culture. The CYP3A4 midazolam metabolism was maintained in biochips after 13 days of culture, whereas it was almost undetectable in Petri dishes. This led to a 5000-fold higher value of the metabolic ratio in the biochips. CYP1A2 phenacetin metabolism was found to be higher in biochips after 5, 9 and 13 days of culture. Thus, a 100-fold higher metabolic ratio of APAP in biochips was measured after 13 days of perfusion. These results demonstrated functional primary human hepatocyte culture in the bioreactor in a long-term culture. Copyright © 2016 John Wiley & Sons, Ltd.

  2. Growth and differentiation of primary and passaged equine bronchial epithelial cells under conventional and air-liquid-interface culture conditions

    PubMed Central

    2011-01-01

    Background Horses develop recurrent airway obstruction (RAO) that resembles human bronchial asthma. Differentiated primary equine bronchial epithelial cells (EBEC) in culture that closely mimic the airway cells in vivo would be useful to investigate the contribution of bronchial epithelium in inflammation of airway diseases. However, because isolation and characterization of EBEC cultures has been limited, we modified and optimized techniques of generating and culturing EBECs from healthy horses to mimic in vivo conditions. Results Large numbers of EBEC were obtained by trypsin digestion and successfully grown for up to 2 passages with or without serum. However, serum or ultroser G proved to be essential for EBEC differentiation on membrane inserts at ALI. A pseudo-stratified muco-ciliary epithelium with basal cells was observed at differentiation. Further, transepithelial resistance (TEER) was more consistent and higher in P1 cultures compared to P0 cultures while ciliation was delayed in P1 cultures. Conclusions This study provides an efficient method for obtaining a high-yield of EBECs and for generating highly differentiated cultures. These EBEC cultures can be used to study the formation of tight junction or to identify epithelial-derived inflammatory factors that contribute to lung diseases such as asthma. PMID:21649893

  3. Endothelins are involved in regulating the proliferation and differentiation of mouse epidermal melanocytes in serum-free primary culture.

    PubMed

    Hirobe, T

    2001-11-01

    Mouse epidermal melanoblasts preferentially proliferated from disaggregated epidermal cell suspensions derived from newborn mouse skin in serum-free melanoblast-defined medium (MDM). After 14 d, almost all keratinocytes that existed predominantly in the early stage of primary culture died, and pure cultures of melanoblasts were obtained. Epidermal melanoblasts dramatically increased in number in MDMDF consisting of MDM supplemented with dibutyryl adenosine 3':5'-cyclic monophosphate (DBcAMP) and basic fibroblast growth factor (bFGF). Epidermal melanocytes increased in number in MDMD consisting of MDM supplemented with DBcAMP. On the other hand, epidermal melanocytes were induced to differentiate in MDMM consisting of MDM supplemented with alpha-melanocyte-stimulating hormone (MSH). Pure cultured primary melanoblasts or melanocytes in MDMDF or MDMD were further cultured with MDMDF or MDMD supplemented with endothelin (ET)-1, -2, or -3 from 14 d. A dramatic increase in the number of melanoblasts or melanocytes was observed after 7 d; however, no increase in the number of melanoblasts or melanocytes was observed in the absence of ET-1, -2, or -3. The increase in the number of melanoblasts or melanocytes was comparable with that of melanoblasts or melanocytes cocultured with secondary keratinocytes in MDMDF or MDMD. Also, pure cultured primary melanoblasts in MDM were further cultured with MDMM supplemented with ET-1, -2, or -3 from 14 d. A dramatic increase in the percentage of melanocytes in the melanoblast-melanocyte population was observed after 7 d; however, no increase in the percentage of melanocytes was observed in the absence of ET-1, -2, or -3. The increase was comparable with that of melanocytes cocultured with secondary keratinocytes in MDMM. Moreover, anti-ET-1, -2, and -3 antibodies inhibited both the proliferation of melanoblasts or melanocytes in MDMDF or MDMD and the differentiation of melanocytes in MDMM in primary culture. These results suggest that

  4. Toxicity of green tea extracts and their constituents in rat hepatocytes in primary culture.

    PubMed

    Schmidt, M; Schmitz, H-J; Baumgart, A; Guédon, D; Netsch, M I; Kreuter, M-H; Schmidlin, C B; Schrenk, D

    2005-02-01

    Recent reports on sporadic cases of liver disorders (acute hepatitis, icterus, hepatocellular necrosis) after ingestion of dietary supplements based on hydro-alcoholic extracts from green tea leaves led to restrictions of the marketing of such products in certain countries of the EU. Since green tea is considered to exert a number of beneficial health effects, and, therefore, green tea products are widely used as dietary supplements, we were interested in the possible mechanism of hepatotoxicity of green tea extracts and in the components involved in such effects. Seven hours after seeding on collagen, rat hepatocytes in primary culture were treated with various hydro-alcoholic green tea extracts (two different native 80% ethanolic dry extracts and an 80% ethanolic dry extract cleared from lipophilic compounds). Cells were washed, and reduction of resazurin, used as a viability parameter monitoring intact mitochondrial function, was determined. It was found that all seven green tea extracts examined enhanced resazurin reduction significantly at a concentration range of 100-500 microg/ml medium, while a significant decrease was observed at 1-3mg/ml medium. Decreased levels were concomitant with abundant necrosis as observed by microscopic inspection of the cultures and with increased leakage of lactate dehydrogenase activity from the cells. In a separate series of experiments, the green tea constituents (-)-epicatechin, (-)-epigallocatechin-3-gallate, caffeine and theanine were tested at concentrations reflecting their levels in a typical green tea extract. Synthetic (+)-epigallocatechin (200 microM) was used for comparison. Cytotoxicity was found with (-)-epigallocatechin-3-gallate only. The concomitant addition of 0.25 mM ascorbate/0.05 mM alpha-tocopherol had no influence on cytotoxicity. In conclusion, our results suggest that high concentrations of green tea extract can exert acute toxicity in rat liver cells. (-)-Epigallocatechin-3-gallate seems to be a key

  5. N-deacetyl ketoconazole-induced hepatotoxicity in a primary culture system of rat hepatocytes.

    PubMed

    Rodriguez, R J; Acosta, D

    1997-02-28

    Ketoconazole (KT) is an azole antifungal agent that has been associated with hepatotoxicity. The mechanism of its hepatotoxicity has not yet been resolved. It has been suggested that a reactive metabolite may be the cause of toxicity because the hepatic injury does not appear to be mediated through an immunoallergic mechanism. Several metabolites of KT have been reported in the literature of which the deacetylated metabolite, N-deacetyl ketoconazole (DAK), is the major metabolite which undergoes further metabolism by the flavin-containing monooxygenases (FMO) to form a potentially toxic dialdehyde. The objective of this study was to evaluate DAK's cytotoxicity and the role of FMO in a primary culture system of rat hepatocytes. Cytotoxicity was evaluated by measuring the leakage of the cytosolic enzyme, lactate dehydrogenase (LDH), into the medium and by assessing mitochondrial reduction of 3-(4,5-dimethythiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT). The cultures were exposed to various concentrations of DAK (20-160 microM) for 0.5-4 h. There was a significant increase (P < 0.05) in LDH leakage and an immediate decrease in MTT reduction (P < 0.05) as early as 0.5 h. The MTT reduction assay appeared to be more sensitive than the LDH assay in that lower concentrations were needed to observe a 50% reduction of MTT (107, 90, 75, 58 microM DAK at 0.5, 1.0, 2.0 and 4.0 h, respectively). The concentrations to observe 50% LDH leakage from the hepatocytes were 155, 133, 100, 70 microM DAK at 0.5, 1.0, 2.0 and 4.0 h, respectively. Moreover, co-treatment with methimazole, a competitive substrate for FMO, produced a significant decrease (P < 0.05) in % LDH leakage as early as 0.5 h, when compared to cells treated solely with DAK. Also, the toxicity was significantly (P < 0.05) enhanced as early as 0.5 h by n-octylamine, a known positive effector for FMO. These results demonstrate that DAK is a more potent cytotoxicant than its parent compound, KT, as reported previously

  6. Toxicity of green tea extracts and their constituents in rat hepatocytes in primary culture.

    PubMed

    Schmidt, M; Schmitz, H-J; Baumgart, A; Guédon, D; Netsch, M I; Kreuter, M-H; Schmidlin, C B; Schrenk, D

    2005-02-01

    Recent reports on sporadic cases of liver disorders (acute hepatitis, icterus, hepatocellular necrosis) after ingestion of dietary supplements based on hydro-alcoholic extracts from green tea leaves led to restrictions of the marketing of such products in certain countries of the EU. Since green tea is considered to exert a number of beneficial health effects, and, therefore, green tea products are widely used as dietary supplements, we were interested in the possible mechanism of hepatotoxicity of green tea extracts and in the components involved in such effects. Seven hours after seeding on collagen, rat hepatocytes in primary culture were treated with various hydro-alcoholic green tea extracts (two different native 80% ethanolic dry extracts and an 80% ethanolic dry extract cleared from lipophilic compounds). Cells were washed, and reduction of resazurin, used as a viability parameter monitoring intact mitochondrial function, was determined. It was found that all seven green tea extracts examined enhanced resazurin reduction significantly at a concentration range of 100-500 microg/ml medium, while a significant decrease was observed at 1-3mg/ml medium. Decreased levels were concomitant with abundant necrosis as observed by microscopic inspection of the cultures and with increased leakage of lactate dehydrogenase activity from the cells. In a separate series of experiments, the green tea constituents (-)-epicatechin, (-)-epigallocatechin-3-gallate, caffeine and theanine were tested at concentrations reflecting their levels in a typical green tea extract. Synthetic (+)-epigallocatechin (200 microM) was used for comparison. Cytotoxicity was found with (-)-epigallocatechin-3-gallate only. The concomitant addition of 0.25 mM ascorbate/0.05 mM alpha-tocopherol had no influence on cytotoxicity. In conclusion, our results suggest that high concentrations of green tea extract can exert acute toxicity in rat liver cells. (-)-Epigallocatechin-3-gallate seems to be a key

  7. African American Identity and a Theory for Primary Cultural Instructional Design

    ERIC Educational Resources Information Center

    Thomas, Michael K.; Columbus, Marco A.

    2010-01-01

    This article is on the strange confluence of culture, identity, learning, and systemic design. We argue that the work of instructional design is, essentially, work on culture and identity. A person's culture and identity fully and inextricably situate their thought, action, and interaction. For this reason, this inherent situatedness of culture…

  8. Organizational Culture in a Successful Primary School: An Ethnographic Case Study

    ERIC Educational Resources Information Center

    Negis-Isik, Ayse; Gursel, Musa

    2013-01-01

    Even though they are perceived similar from outside, all schools have distinct characteristics and a culture that differ them from other schools. School culture, is one of the important factors that play role in school efficiency and success. The purpose of this study was to examine the culture of a successful school profoundly. This study was a…

  9. Structural specificity of steroids in stimulating DNA synthesis and protooncogene expression in primary rat hepatocyte cultures.

    PubMed

    Lee, C H; Edwards, A M

    2002-05-01

    Among the chemical compounds of varied structure which possess liver tumour-promoting are steroids, such as estrogens, pregnenolone derivatives and anabolic steroids. Although the mechanism(s) of tumour promotion in liver by these xenobiotics is not well understood, it is clear that growth stimulation is one important element in their action. As a basis for better defining whether steroids stimulate growth by a common mechanism or fall into sub-groups with differing actions, the effects of 46 steroids on DNA synthesis and the expression of protooncogenes c-fos and c-myc were examined in primary cultures of normal rat hepatocytes. Tentative groupings of steroids have been identified based on apparent structural requirements for stimulation of DNA synthesis, and effects of auxiliary factors in modulating this growth stimulus. For a "progestin" group, insulin appeared to be permissive for stimulation of DNA synthesis, and presence of an ester or hydroxyl group at 17alpha-position in combination with a non-polar group at C(6) appeared to be required for stimulation. For the pregnenes, dexamethasone was stimulatory. Structural requirements include a non-polar substitution at 16alpha-position and presence of a 6alpha-methyl group. Androgens were weak or ineffective stimulators of DNA synthesis. Anabolic steroids were weak to strong stimulators and alteration to A ring structure in combination with non-polar substitution at 17alpha-position appeared to be required for the activity. With the exception of the anabolic steroid, dianabol, there do not appear to be strong correlation between ability to stimulate DNA synthesis and ability to induce protooncogene expression among the steroids. This study provides a starting point for future more detailed examination of growth-stimulatory mechanism(s) of action of steroids in the liver. PMID:12127039

  10. Stimulation of albumin gene transcription by insulin in primary cultures of rat hepatocytes

    SciTech Connect

    Lloyd, C.E.; Kalinyak, J.E.; Hutson, S.M.; Jefferson, L.S.

    1987-02-01

    The first goal of the work reported here was to prepare single-stranded DNA sequences for use in studies on the regulation of albumin gene expression. A double-stranded rat albumin cDNA clone was subcloned into the bacteriophage vector M13mp7. Single-stranded recombinant clones were screened for albumin sequences containing either the mRNA strand or the complementary strand. Two clones were selected that contained the 1200 nucleotide long 3' end of the albumin sequence. DNA from the clone containing the mRNA strand was used as a template for DNA polymerase I to prepare a radiolabeled, single-stranded cDNA to albumin mRNA. This radiolabeled cDNA probe was used to quantitate the relative abundance of albumin mRNA in samples of total cellular RNA. DNA from the clone containing the complementary strand was used to measure relative rates of albumin gene transcription in isolated nuclei. The second goal was to use the single-stranded DNA probes to investigate the mechanism of the insulin-mediated stimulation of albumin synthesis in primary cultures of rat hepatocytes. Addition of insulin to hepatocytes maintained in a chemically defined, serum-free medium for 40 h in the absence of any hormones resulted in a specific 1.5- to 2.5-fold stimulation of albumin gene transcription that was maximal at 3 h and was maintained above control values for at least 24 h. The rate of albumin gene transcription in nuclei isolated from livers of diabetic rats was reduced to 50% of the value recorded in control nuclei. Taken together, these findings demonstrate that insulin regulates synthesis of albumin at the level of gene transcription.

  11. Pregnane X Receptor Modulates the Inflammatory Response in Primary Cultures of Hepatocytes

    PubMed Central

    Sun, Mengxi; Cui, Wenqi; Woody, Sarah K.

    2015-01-01

    Bacterial sepsis is characterized by a rapid increase in the expression of inflammatory mediators to initiate the acute phase response in liver. Inflammatory mediator release is counterbalanced by the coordinated expression of anti-inflammatory molecules such as interleukin 1 receptor antagonist (IL1-Ra) through time. This study determined whether activation of pregnane X receptor (PXR, NR1I2) alters the lipopolysaccharide (LPS)-inducible gene expression program in primary cultures of hepatocytes (PCHs). Preactivation of PXR for 24 hours in PCHs isolated from wild-type mice suppressed the subsequent LPS-inducible expression of the key inflammatory mediators interleukin 1β (IL-1β), interleukin 6 (IL-6), and tumor necrosis factor α (TNFα) but not in PCHs isolated from Pxr-null (PXR-knockout [KO]) mice. Basal expression of key inflammatory cytokines was elevated in PCHs from PXR-KO mice. Stimulation of PCHs from PXR-KO mice with LPS alone produced enhanced levels of IL-1β when compared with wild-type mice. Experiments performed using PCHs from both humanized-PXR transgenic mice as well as human donors indicate that prolonged activation of PXR produces an increased secretion of IL1-Ra from cells through time. Our data reveal a working model that describes a pivotal role for PXR in both inhibiting as well as in resolving the inflammatory response in hepatocytes. Understanding the molecular details of how PXR is converted from a positive regulator of drug-metabolizing enzymes into a transcriptional suppressor of inflammation in liver will provide new pharmacologic strategies for modulating inflammatory-related diseases in the liver and intestine. PMID:25527709

  12. Environmental monitoring of urban streams using a primary fish gill cell culture system (FIGCS).

    PubMed

    Schnell, Sabine; Bawa-Allah, Kafilat; Otitoloju, Adebayo; Hogstrand, Christer; Miller, Thomas H; Barron, Leon P; Bury, Nic R

    2015-10-01

    The primary fish gill cell culture system (FIGCS) is an in vitro technique which has the potential to replace animals in whole effluent toxicity tests. In the current study FIGCS were transported into the field and exposed to filtered (0.2μm) river water for 24h from 4 sites, on 2 different sampling dates. Sites 1 and 2 are situated in an urban catchment (River Wandle, London, UK) with site 1 downstream of a sewage treatment work; site 3 is located in a suburban park (River Cray, Kent, UK), and site 4 is more rural (River Darent, Kent, UK). The change in transepithelial electrical resistance (TER), the expression of the metal responsive genes metallothionein A (mta) and B (mtb), cytochrome P450 1A1 (cyp1a1) and 3A27 (cyp3a27), involved in phase 1 metabolism, were assessed following exposure to sample water for 24h. TER was comparable between FIGCS exposed to 0.2μm filtered river water and those exposed to synthetic moderately soft water for 24h. During the first sampling time, there was an increase in mta, cyp1a1 and cyp3a27 gene expression in epithelium exposed to water from sites 1 and 2, and during the second sampling period an increase in cyp3a27 gene expression at sites 1 and 4. Urban river water is a complex mixture of contaminants (e.g., metals, pesticides, pharmaceuticals and polyaromatic hydrocarbons) and the increase in the expression of genes encoding mta, cyp1a1 and cyp3a27 in FIGCS is indicative of the presence of biologically active pollutants.

  13. Highly purified hexachlorobenzene induces cytochrome P4501A in primary cultures of chicken embryo hepatocytes

    SciTech Connect

    Mundy, Lukas J.; Jones, Stephanie P.; Crump, Doug; Herve, Jessica C.; Konstantinov, Alex; Utley, Fiona; Potter, David; Kennedy, Sean W.

    2010-11-01

    Some uncertainty exists regarding the purity of hexachlorobenzene (HCB) used in past toxicity studies. It has been suggested that reported toxic and biochemical effects initially attributed to HCB exposure may have actually been elicited by contamination of HCB by polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs). Herein, primary cultures of chicken embryo hepatocytes (CEH) were used to compare the potencies of two lots of reagent-grade hexachlorobenzene (HCB-old [HCB-O] and HCB-new [HCB-N]), highly purified HCB (HCB-P) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) as inducers of ethoxyresorufin O-deethylase (EROD) activity, cytochrome P4501A4 (CYP1A4) messenger ribonucleic acid (mRNA) and CYP1A5 mRNA. The study also compared the EROD- and CYP1A4/5 mRNA-inducing potencies of HCB to the potencies of two mono-ortho substituted polychlorinated biphenyls (PCBs), 2,3,3',4,4'-pentachlorobiphenyl (PCB 105) and 2,3'4,4',5-pentachlorobiphenyl (PCB 118). HCB-O, HCB-N and HCB-P all induced EROD activity and up-regulated CYP1A4 and CYP1A5 mRNAs. Induction was not caused by contamination of HCB with PCDDs or PCDFs. Based upon a comparison of the EC{sub 50} and EC{sub threshold} values for EROD and CYP1A4/5 mRNA concentration-response curves, the potency of HCB relative to the potency of TCDD was 0.0001, and was similar to that of PCB 105 and PCB 118. The maximal EROD activity and CYP1A4/5 mRNA expression differed greatly between HCB and TCDD, and may contribute to an overestimation of the ReP value calculated for highly purified HCB.

  14. The biosynthesis of acute-phase proteins in primary cultures of rat hepatocytes.

    PubMed

    Andus, T; Gross, V; Tran-Thi, T A; Schreiber, G; Nagashima, M; Heinrich, P C

    1983-07-01

    The biosynthesis and secretion of alpha 2-macroglobulin, transferrin, alpha 1-acid glycoprotein and alpha 1-proteinase inhibitor were studied in rat hepatocyte primary cultures. After labeling with [35S]methionine, two forms, which can be separated electrophoretically differing by molecular weight, were found for each of the four glycoproteins. The following molecular weights were estimated for the intracellular precursors and the secreted forms: alpha 2-macroglobulin, 176 000 and 182 000; transferrin, 84 000 and 86 000; alpha 1-acid glycoprotein, 39 000 and 43 000-60 000; alpha 1-proteinase inhibitor, 49 000 and 54 000. Carbohydrate moieties could be removed from intracellular forms by treatment with endoglucosaminidase H indicating that their oligosaccharide chains were of the high-mannose type. The extracellular forms were sensitive to sialidase. They incorporated [3H]galactose and [3H]fucose showing that their oligosaccharide chains were of the complex type. Pulse-chase experiments revealed a precursor-product relationship for the high-mannose and the complex type glycoproteins. In the hepatocyte medium newly synthesized albumin was detected after 30 min and newly synthesized glycoproteins after 60 min. Unglycosylated alpha 2-macroglobulin (162 000), transferrin (79 000), alpha 1-acid glycoprotein (23 000), and alpha 1-proteinase inhibitor (41 000) were found in the cells as well as in the medium, when the transfer of oligosaccharide chains onto the polypeptide chains was blocked by tunicamycin. Tunicamycin led to a marked reduction of the secretion of alpha 2-macroglobulin, alpha 1-acid glycoprotein and alpha 1-proteinase inhibitor, whereas the secretion of transferrin was less affected. PMID:6602705

  15. Human Primary Trophoblast Cell Culture Model to Study the Protective Effects of Melatonin Against Hypoxia/reoxygenation-induced Disruption.

    PubMed

    Sagrillo-Fagundes, Lucas; Clabault, Hélène; Laurent, Laetitia; Hudon-Thibeault, Andrée-Anne; Salustiano, Eugênia Maria Assunção; Fortier, Marlène; Bienvenue-Pariseault, Josianne; Wong Yen, Philippe; Sanderson, J Thomas; Vaillancourt, Cathy

    2016-01-01

    This protocol describes how villous cytotrophoblast cells are isolated from placentas at term by successive enzymatic digestions, followed by density centrifugation, media gradient isolation and immunomagnetic purification. As observed in vivo, mononucleated villous cytotrophoblast cells in primary culture differentiate into multinucleated syncytiotrophoblast cells after 72 hr. Compared to normoxia (8% O2), villous cytotrophoblast cells that undergo hypoxia/reoxygenation (0.5% / 8% O2) undergo increased oxidative stress and intrinsic apoptosis, similar to that observed in vivo in pregnancy complications such as preeclampsia, preterm birth, and intrauterine growth restriction. In this context, primary villous trophoblasts cultured under hypoxia/reoxygenation conditions represent a unique experimental system to better understand the mechanisms and signalling pathways that are altered in human placenta and facilitate the search for effective drugs that protect against certain pregnancy disorders. Human villous trophoblasts produce melatonin and express its synthesizing enzymes and receptors. Melatonin has been suggested as a treatment for preeclampsia and intrauterine growth restriction because of its protective antioxidant effects. In the primary villous cytotrophoblast cell model described in this paper, melatonin has no effect on trophoblast cells in normoxic state but restores the redox balance of syncytiotrophoblast cells disrupted by hypoxia/reoxygenation. Thus, human villous trophoblast cells in primary culture are an excellent approach to study the mechanisms behind the protective effects of melatonin on placental function during hypoxia/reoxygenation. PMID:27500522

  16. Integration of Herbal Medicine in Primary Care in Israel: A Jewish-Arab Cross-Cultural Perspective

    PubMed Central

    Ben-Arye, Eran; Lev, Efraim; Keshet, Yael; Schiff, Elad

    2011-01-01

    Herbal medicine is a prominent complementary and alternative medicine (CAM) modality in Israel based on the country's natural diversity and impressive cultural mosaic. In this study, we compared cross-cultural perspectives of patients attending primary care clinics in northern Israel on herbal medicine specifically and CAM generally, and the possibility of integrating them within primary care. Research assistants administered a questionnaire to consecutive patients attending seven primary care clinics. About 2184 of 3713 respondents (59%) defined themselves as Muslims, Christians or Druze (henceforth Arabs) and 1529 (41%) as Jews. Arab respondents reported more use of herbs during the previous year (35 versus 27.8% P = .004) and of more consultations with herbal practitioners (P < .0001). Druze reported the highest rate of herbal consultations (67.9%) and Ashkenazi Jews the lowest rate (45.2%). About 27.5% of respondents supported adding a herbal practitioner to their clinic's medical team if CAM were to be integrated within primary care. Both Arabs and Jews report considerable usage of herbal medicine, with Arabs using it significantly more. Cross-cultural perspectives are warranted in the study of herbal medicine use in the Arab and Jewish societies. PMID:19864354

  17. Postnatal developmental profile of neurons and glia in motor nuclei of the brainstem and spinal cord, and its comparison with organotypic slice cultures.

    PubMed

    Cifra, Alessandra; Mazzone, Graciela L; Nani, Francesca; Nistri, Andrea; Mladinic, Miranda

    2012-08-01

    In vitro preparations of the neonatal rat spinal cord or brainstem are useful to investigate the organization of motor networks and their dysfunction in neurological disease models. Long-term spinal cord organotypic cultures can extend our understanding of such pathophysiological processes over longer times. It is, however, surprising that detailed descriptions of the type (and number) of neurons and glia in such preparations are currently unavailable to evaluate cell-selectivity of experimental damage. The focus of the present immunohistochemical study is the novel characterization of the cell population in the lumbar locomotor region of the rat spinal cord and in the brainstem motor nucleus hypoglossus at 0-4 postnatal days, and its comparison with spinal organotypic cultures at 2-22 days in vitro. In the nucleus hypoglossus, neurons were 40% of all cells and 80% of these were motoneurons. Astrocytes (35% of total cells) were the main glial cells, while microglia was <10%. In the spinal gray matter, the highest neuronal density was in the dorsal horn (>80%) and the lowest in the ventral horn (≤57%) with inverse astroglia numbers and few microglia. The number of neurons (including motoneurons) and astrocytes was stable after birth. Like in the spinal cord, motoneurons in organotypic spinal culture were <10% of ventral horn cells, with neurons <40%, and the rest made up by glia. The present report indicates a comparable degree of neuronal and glial maturation in brainstem and spinal motor nuclei, and that this condition is also observed in 3-week-old organotypic cultures.

  18. Spared Primary Motor Cortex and The Presence of MEP in Cerebral Palsy Dictate the Responsiveness to tDCS during Gait Training.

    PubMed

    Grecco, Luanda A Collange; Oliveira, Claudia Santos; Galli, Manuela; Cosmo, Camila; Duarte, Natália de Almeida Carvalho; Zanon, Nelci; Edwards, Dylan J; Fregni, Felipe

    2016-01-01

    The current priority of investigations involving transcranial direct current stimulation (tDCS) and neurorehabilitation is to identify biomarkers associated with the positive results of the interventions such that respondent and non-respondent patients can be identified in the early phases of treatment. The aims were to determine whether: (1) present motor evoked potential (MEP); and (2) injuries involving the primary motor cortex, are associated with tDCS-enhancement in functional outcome following gait training in children with cerebral palsy (CP). We reviewed the data from our parallel, randomized, sham-controlled, double-blind studies. Fifty-six children with spastic CP received gait training (either treadmill training or virtual reality training) and tDCS (active or sham). Univariate and multivariate logistic regression analyses were employed to identify clinical, neurophysiologic and neuroanatomic predictors associated with the responsiveness to treatment with tDCS. MEP presence during the initial evaluation and the subcortical injury were associated with positive effects in the functional results. The logistic regression revealed that present MEP was a significant predictor for the six-minute walk test (6MWT; p = 0.003) and gait speed (p = 0.028), whereas the subcortical injury was a significant predictor of gait kinematics (p = 0.013) and gross motor function (p = 0.021). In this preliminary study involving children with CP, two important prediction factors of good responses to anodal tDCS combined with gait training were identified. Apparently, MEP (integrity of the corticospinal tract) and subcortical location of the brain injury exerted different influences on aspects related to gait, such as velocity and kinematics. PMID:27486393

  19. Relationships among low-frequency local field potentials, spiking activity, and three-dimensional reach and grasp kinematics in primary motor and ventral premotor cortices.

    PubMed

    Bansal, Arjun K; Vargas-Irwin, Carlos E; Truccolo, Wilson; Donoghue, John P

    2011-04-01

    A prominent feature of motor cortex field potentials during movement is a distinctive low-frequency local field potential (lf-LFP) (<4 Hz), referred to as the movement event-related potential (mEP). The lf-LFP appears to be a global signal related to regional synaptic input, but its relationship to nearby output signaled by single unit spiking activity (SUA) or to movement remains to be established. Previous studies comparing information in primary motor cortex (MI) lf-LFPs and SUA in the context of planar reaching tasks concluded that lf-LFPs have more information than spikes about movement. However, the relative performance of these signals was based on a small number of simultaneously recorded channels and units, or for data averaged across sessions, which could miss information of larger-scale spiking populations. Here, we simultaneously recorded LFPs and SUA from two 96-microelectrode arrays implanted in two major motor cortical areas, MI and ventral premotor (PMv), while monkeys freely reached for and grasped objects swinging in front of them. We compared arm end point and grip aperture kinematics' decoding accuracy for lf-LFP and SUA ensembles. The results show that lf-LFPs provide enough information to reconstruct kinematics in both areas with little difference in decoding performance between MI and PMv. Individual lf-LFP channels often provided more accurate decoding of single kinematic variables than any one single unit. However, the decoding performance of the best single unit among the large population usually exceeded that of the best single lf-LFP channel. Furthermore, ensembles of SUA outperformed the pool of lf-LFP channels, in disagreement with the previously reported superiority of lf-LFP decoding. Decoding results suggest that information in lf-LFPs recorded from intracortical arrays may allow the reconstruction of reach and grasp for real-time neuroprosthetic applications, thus potentially supplementing the ability to decode these same features

  20. Spared Primary Motor Cortex and The Presence of MEP in Cerebral Palsy Dictate the Responsiveness to tDCS during Gait Training.

    PubMed

    Grecco, Luanda A Collange; Oliveira, Claudia Santos; Galli, Manuela; Cosmo, Camila; Duarte, Natália de Almeida Carvalho; Zanon, Nelci; Edwards, Dylan J; Fregni, Felipe

    2016-01-01

    The current priority of investigations involving transcranial direct current stimulation (tDCS) and neurorehabilitation is to identify biomarkers associated with the positive results of the interventions such that respondent and non-respondent patients can be identified in the early phases of treatment. The aims were to determine whether: (1) present motor evoked potential (MEP); and (2) injuries involving the primary motor cortex, are associated with tDCS-enhancement in functional outcome following gait training in children with cerebral palsy (CP). We reviewed the data from our parallel, randomized, sham-controlled, double-blind studies. Fifty-six children with spastic CP received gait training (either treadmill training or virtual reality training) and tDCS (active or sham). Univariate and multivariate logistic regression analyses were employed to identify clinical, neurophysiologic and neuroanatomic predictors associated with the responsiveness to treatment with tDCS. MEP presence during the initial evaluation and the subcortical injury were associated with positive effects in the functional results. The logistic regression revealed that present MEP was a significant predictor for the six-minute walk test (6MWT; p = 0.003) and gait speed (p = 0.028), whereas the subcortical injury was a significant predictor of gait kinematics (p = 0.013) and gross motor function (p = 0.021). In this preliminary study involving children with CP, two important prediction factors of good responses to anodal tDCS combined with gait training were identified. Apparently, MEP (integrity of the corticospinal tract) and subcortical location of the brain injury exerted different influences on aspects related to gait, such as velocity and kinematics.

  1. Spared Primary Motor Cortex and The Presence of MEP in Cerebral Palsy Dictate the Responsiveness to tDCS during Gait Training

    PubMed Central

    Grecco, Luanda A. Collange; Oliveira, Claudia Santos; Galli, Manuela; Cosmo, Camila; Duarte, Natália de Almeida Carvalho; Zanon, Nelci; Edwards, Dylan J.; Fregni, Felipe

    2016-01-01

    The current priority of investigations involving transcranial direct current stimulation (tDCS) and neurorehabilitation is to identify biomarkers associated with the positive results of the interventions such that respondent and non-respondent patients can be identified in the early phases of treatment. The aims were to determine whether: (1) present motor evoked potential (MEP); and (2) injuries involving the primary motor cortex, are associated with tDCS-enhancement in functional outcome following gait training in children with cerebral palsy (CP). We reviewed the data from our parallel, randomized, sham-controlled, double-blind studies. Fifty-six children with spastic CP received gait training (either treadmill training or virtual reality training) and tDCS (active or sham). Univariate and multivariate logistic regression analyses were employed to identify clinical, neurophysiologic and neuroanatomic predictors associated with the responsiveness to treatment with tDCS. MEP presence during the initial evaluation and the subcortical injury were associated with positive effects in the functional results. The logistic regression revealed that present MEP was a significant predictor for the six-minute walk test (6MWT; p = 0.003) and gait speed (p = 0.028), whereas the subcortical injury was a significant predictor of gait kinematics (p = 0.013) and gross motor function (p = 0.021). In this preliminary study involving children with CP, two important prediction factors of good responses to anodal tDCS combined with gait training were identified. Apparently, MEP (integrity of the corticospinal tract) and subcortical location of the brain injury exerted different influences on aspects related to gait, such as velocity and kinematics. PMID:27486393

  2. A Guide to Childhood Motor Stereotypies, Tic Disorders and the Tourette Spectrum for the Primary Care Practitioner

    PubMed Central

    Mills, Sarah; Hedderly, Tammy

    2014-01-01

    Movement disorders presenting in childhood are often complex and a heterogenous group of difficulties which can be a minefield for the primary care doctor. The recent activities of the European Society for the Study of Tourette Syndrome (ESSTS) have included publication of European clinical guidelines for Tourette syndrome and other Tic disorders aimed at guiding paediatricians and psychiatrists in managing these children. This paper aims to summarise the key points for primary care teams and impart important facts and general information on related childhood movement disorders in early development. PMID:24757265

  3. A soluble biocompatible guanidine-containing polyamidoamine as promoter of primary brain cell adhesion and in vitro cell culturing

    NASA Astrophysics Data System (ADS)

    Tonna, Noemi; Bianco, Fabio; Matteoli, Michela; Cagnoli, Cinzia; Antonucci, Flavia; Manfredi, Amedea; Mauro, Nicolò; Ranucci, Elisabetta; Ferruti, Paolo

    2014-08-01

    This paper reports on a novel application of an amphoteric water-soluble polyamidoamine named AGMA1 bearing 4-butylguanidine pendants. AGMA1 is an amphoteric, prevailingly cationic polyelectrolyte with isoelectric point of about 10. At pH 7.4 it is zwitterionic with an average of 0.55 excess positive charges per unit, notwithstanding it is highly biocompatible. In this work, it was found that AGMA1 surface-adsorbed on cell culturing coverslips exhibits excellent properties as adhesion and proliferation promoter of primary brain cells such as microglia, as well as of hippocampal neurons and astrocytes. Microglia cells cultured on AGMA1-coated coverslips substrate displayed the typical resting, ramified morphology of those cultured on poly-L-lysine and poly-L-ornithine, employed as reference substrates. Mixed cultures of primary astrocytes and neuronal cells grown on AGMA1- and poly-L-lysine coated coverslips were morphologically undistinguishable. On both substrates, neurons differentiated axon and dendrites and eventually established perfectly functional synaptic contacts. Quantitative immunocytochemical staining revealed no difference between AGMA1 and poly-L-lysine. Electrophysiological experiments allowed recording neuron spontaneous activity on AGMA1. In addition, cell cultures on both AGMA1 and PLL displayed comparable excitatory and inhibitory neurotransmission, demonstrating that the synaptic contacts formed were fully functional.

  4. Primary culture of glial cells from mouse sympathetic cervical ganglion: a valuable tool for studying glial cell biology.

    PubMed

    de Almeida-Leite, Camila Megale; Arantes, Rosa Maria Esteves

    2010-12-15

    Central nervous system glial cells as astrocytes and microglia have been investigated in vitro and many intracellular pathways have been clarified upon various stimuli. Peripheral glial cells, however, are not as deeply investigated in vitro despite its importance role in inflammatory and neurodegenerative diseases. Based on our previous experience of culturing neuronal cells, our objective was to standardize and morphologically characterize a primary culture of mouse superior cervical ganglion glial cells in order to obtain a useful tool to study peripheral glial cell biology. Superior cervical ganglia from neonatal C57BL6 mice were enzymatically and mechanically dissociated and cells were plated on diluted Matrigel coated wells in a final concentration of 10,000cells/well. Five to 8 days post plating, glial cell cultures were fixed for morphological and immunocytochemical characterization. Glial cells showed a flat and irregular shape, two or three long cytoplasm processes, and round, oval or long shaped nuclei, with regular outline. Cell proliferation and mitosis were detected both qualitative and quantitatively. Glial cells were able to maintain their phenotype in our culture model including immunoreactivity against glial cell marker GFAP. This is the first description of immunocytochemical characterization of mouse sympathetic cervical ganglion glial cells in primary culture. This work discusses the uses and limitations of our model as a tool to study many aspects of peripheral glial cell biology.

  5. Characterization of primary organic aerosol emissions from meat cooking, trash burning, and motor vehicles with high-resolution aerosol mass spectrometry and comparison with ambient and chamber observations.

    PubMed

    Mohr, Claudia; Huffman, Alex; Cubison, Michael J; Aiken, Allison C; Docherty, Kenneth S; Kimmel, Joel R; Ulbrich, Ingrid M; Hannigan, Michael; Jimenez, Jose L

    2009-04-01

    Organic aerosol (OA) emissions from motor vehicles, meat-cooking and trash burning are analyzed here using a high-resolution aerosol mass spectrometer (AMS). High resolution data show that aerosols emitted by combustion engines and plastic burning are dominated by hydrocarbon-like organic compounds. Meat cooking and especially paper burning emissions contain significant fractions of oxygenated organic compounds; however, their unit-resolution mass spectral signatures are very similar to those from ambient hydrocarbon-like OA, and very different from the mass spectra of ambient secondary or oxygenated OA (OOA). Thus, primary OA from these sources is unlikelyto be a significant direct source of ambient OOA. There are significant differences in high-resolution tracer m/zs that may be useful for differentiating some of these sources. Unlike in most ambient spectra, all of these sources have low total m/z 44 and this signal is not dominated by the CO2+ ion. All sources have high m/z 57, which is low during high OOA ambient periods. Spectra from paper burning are similar to some types of biomass burning OA, with elevated m/z 60. Meat cooking aerosols also have slightly elevated m/z 60, whereas motor vehicle emissions have very low signal at this m/z.

  6. Non-invasive modulation of somatosensory evoked potentials by the application of static magnetic fields over the primary and supplementary motor cortices

    PubMed Central

    Kirimoto, Hikari; Asao, Akihiko; Tamaki, Hiroyuki; Onishi, Hideaki

    2016-01-01

    This study was performed to investigate the possibility of non-invasive modulation of SEPs by the application of transcranial static magnetic field stimulation (tSMS) over the primary motor cortex (M1) and supplementary motor cortex (SMA), and to measure the strength of the NdFeB magnetic field by using a gaussmeter. An NdFeB magnet or a non-magnetic stainless steel cylinder (for sham stimulation) was settled on the scalp over M1 and SMA of 14 subjects for periods of 15 min. SEPs following right median nerve stimulation were recorded before and immediately after, 5 min after, and 10 min after tSMS from sites C3′ and F3. Amplitudes of the N33 component of SEPs at C3′ significantly decreased immediately after tSMS over M1 by up to 20%. However, tSMS over the SMA did not affect the amplitude of any of the SEP components. At a distance of 2–3 cm (rough depth of the cortex), magnetic field strength was in the range of 110–190 mT. Our results that tSMS over M1 can reduce the amplitude of SEPs are consistent with those of low-frequency repeated TMS and cathodal tDCS studies. Therefore, tSMS could be a useful tool for modulating cortical somatosensory processing. PMID:27698365

  7. Reduction of lipofuscin by centrophenoxine and chlorpromazine in the neurons of rat cerebral hemisphere in primary culture.

    PubMed

    Ohtani, R; Kawashima, S

    1983-01-01

    Cells from neonatal rat cerebral hemispheres were dispersed by trypsin and cultured for 32 days. Histochemical, fluorescence, and electron microscopic analyses demonstrated that lipofuscin pigments increased in neuronal and non-neuronal cells in primary culture according to the lapse of time. When centrophenoxine (10(-4) or 5 X 10(-4) M) or chlorpromazine (10(-6) or 10(-5) M) was added to the medium, the accumulation of lipofuscin pigments in neurons was significantly reduced. However, the effects of these agents were not detected in non-neuronal cells.

  8. How to change organisational culture: Action research in a South African public sector primary care facility

    PubMed Central

    De Sa, Angela; Christodoulou, Maria

    2016-01-01

    Background Organisational culture is a key factor in both patient and staff experience of the healthcare services. Patient satisfaction, staff engagement and performance are related to this experience. The department of health in the Western Cape espouses a values-based culture characterised by caring, competence, accountability, integrity, responsiveness and respect. However, transformation of the existing culture is required to achieve this vision. Aim To explore how to transform the organisational culture in line with the desired values. Setting Retreat Community Health Centre, Cape Town, South Africa. Methods Participatory action research with the leadership engaged with action and reflection over a period of 18 months. Change in the organisational culture was measured at baseline and after 18 months by means of a cultural values assessment (CVA) survey. The three key leaders at the health centre also completed a 360-degree leadership values assessment (LVA) and had 6 months of coaching. Results Cultural entropy was reduced from 33 to 13% indicating significant transformation of organisational culture. The key driver of this transformation was change in the leadership style and functioning. Retreat health centre shifted from a culture that emphasised hierarchy, authority, command and control to one that established a greater sense of cohesion, shared vision, open communication, appreciation, respect, fairness and accountability. Conclusion Transformation of organisational culture was possible through a participatory process that focused on the leadership style, communication and building relationships by means of CVA and feedback, 360-degree LVA, feedback and coaching and action learning in a co-operative inquiry group. PMID:27608671

  9. No effects of 20 Hz-rTMS of the primary motor cortex in vegetative state: A randomised, sham-controlled study.

    PubMed

    Cincotta, Massimo; Giovannelli, Fabio; Chiaramonti, Roberta; Bianco, Giovanni; Godone, Marco; Battista, Donato; Cardinali, Consuelo; Borgheresi, Alessandra; Sighinolfi, Antonella; D'Avanzo, Anna Maria; Breschi, Marco; Dine, Ylli; Lino, Mario; Zaccara, Gaetano; Viggiano, Maria Pia; Rossi, Simone

    2015-10-01

    We assessed the effects of a non-invasive neuromodulatory intervention with repetitive transcranial magnetic stimulation (rTMS) of the motor cortex in patients with vegetative state (VS) by a randomised, sham-controlled study with a cross-over design. Eleven patients classified as being in VS (9 post-anoxic, 2 post-traumatic, time elapsed from the injury 9-85 months) were included in the study. Real or sham 20 Hz rTMS were applied to the left primary motor cortex (M1) for 5 consecutive days. Primary outcome measures were changes in the JFK Coma Recovery Scale-Revised (CRS-R) scale total score and Clinical Global Impression Improvement (CGI-I) scale. Additional measures were EEG changes and impression of the patients' relatives using the CGI-I scale. Evaluations were blindly performed at baseline, after the first day of treatment, immediately after the end of the 5-days treatment, 1 week and 1 month later. Slight changes observed in the CRS-R and CGI-I scores did not significantly differ between real or sham stimulation conditions. EEG was not significantly changed on average, although spots of brain reactivity were occasionally found underneath the stimulation point. Findings did not provide evidence of therapeutic effect of 20 Hz rTMS of the M1 in chronic VS, at least with conventional coils and current safety parameters. Therefore, they might be useful to better allocate human and financial resources in future trials. PMID:26301875

  10. Fermentation and growth response of a primary poultry isolate of Salmonella typhimurium grown under strict anaerobic conditions in continuous culture and amino acid-limited batch culture.

    PubMed

    Maciorowski, K G; Nisbet, D J; Ha, S D; Corrier, D E; DeLoach, J R; Ricke, S C

    1997-01-01

    Salmonella typhimurium is a significant hazard to consumer health that is carried asymptomatically in poultry gastrointestinal tracts. Nurmi cultures may prevent Salmonella colonization in young chicks, but the mechanism of competitive exclusion is unclear. Modeling Salmonella's metabolism in pure culture may allow for greater definition in choosing strains for Nurmi cultures. The growth rates and affinity constants of S. typhimurium growing in amino acid-limited conditions were determined in batch culture and compared to primary poultry isolates of cecal strains. Serine and NH4Cl were the best N sources for growth of all organisms tested in this study. The fermentation response of S. typhimurium was also monitored in continuous culture at a slow dilution rate of 0.021 h-1. S. typhimurium was found to adapt to VL media, with trends in protein disappearance, Yglucose, and Yprotein. This may show that amino acid or protein concentrations may be an integral component of the initial establishment of S. typhimurium in the cecum.

  11. Fermentation and growth response of a primary poultry isolate of Salmonella typhimurium grown under strict anaerobic conditions in continuous culture and amino acid-limited batch culture.

    PubMed

    Maciorowski, K G; Nisbet, D J; Ha, S D; Corrier, D E; DeLoach, J R; Ricke, S C

    1997-01-01

    Salmonella typhimurium is a significant hazard to consumer health that is carried asymptomatically in poultry gastrointestinal tracts. Nurmi cultures may prevent Salmonella colonization in young chicks, but the mechanism of competitive exclusion is unclear. Modeling Salmonella's metabolism in pure culture may allow for greater definition in choosing strains for Nurmi cultures. The growth rates and affinity constants of S. typhimurium growing in amino acid-limited conditions were determined in batch culture and compared to primary poultry isolates of cecal strains. Serine and NH4Cl were the best N sources for growth of all organisms tested in this study. The fermentation response of S. typhimurium was also monitored in continuous culture at a slow dilution rate of 0.021 h-1. S. typhimurium was found to adapt to VL media, with trends in protein disappearance, Yglucose, and Yprotein. This may show that amino acid or protein concentrations may be an integral component of the initial establishment of S. typhimurium in the cecum. PMID:9192013

  12. Effect of aging on GHRF-induced growth hormone release from anterior pituitary cells in primary culture

    SciTech Connect

    Spik, K.W.; Boyd, R.L.; Sonntag, W.E.

    1991-03-01

    Five criteria were developed to validate the primary cell culture model for comparison of GRF-induced release of growth hormone in pituitary tissue from aging animals. Pituitaries from young (5-mo), middle-aged (14-mo), and old (24-mo) male Fischer 344 rats were dispersed using either trypsin/trypsin inhibitor or dispase and compared with respect to the number of pituitary cells recovered, cell viability, 3H-leucine incorporation into total protein, time course for recovery of optimal response to GRF, and the dose-relationship for GRF-induced release of growth hormone 2, 4, and 6 days after dispersal. Results indicated that direct comparison of cellular responses between tissues from young, middle-aged, and old rats in primary cell culture is confounded by variations in time for recovery of optimal responses, the effects of the enzymes used for dispersal, and the methods used to express the data.

  13. Kit ligand promotes the transition from primordial to primary follicles after in vitro culture of ovine ovarian tissue.

    PubMed

    Cavalcante, A Y P; Gouveia, B B; Barberino, R S; Lins, T L B G; Santos, L P; Gonçalves, R J S; Celestino, J J H; Matos, M H T

    2016-08-01

    This study evaluated the effects of kit ligand (KL) on the morphology and development of ovine preantral follicles (fresh control) and after 7 days of in vitro culture in α-Minimal Essential Medium (α-MEM; control medium) or the presence of KL (1, 10, 50, 100 or 200 ng/ml). There was an increase in the percentage of primary follicles at the concentration of 100 ng/ml KL, compared with the fresh control, control medium (α-MEM) and the other KL concentrations. Follicle diameter was significantly higher than the control medium only at concentrations of 50 and 100 ng/ml KL. In conclusion, 100 ng/ml KL promoted the transition from primordial to primary follicles (follicular activation) after in vitro culture of ovine ovarian tissue.

  14. The primary culture of mirror carp snout and caudal fin tissues and the isolation of Koi herpesvirus.

    PubMed

    Zhou, Jingxiang; Wang, Hao; Zhu, Xia; Li, Xingwei; Lv, Wenliang; Zhang, Dongming

    2013-10-01

    The explosive Koi herpesvirus (KHV) epidemic has caused the deaths of a large number of carp and carp variants and has produced serious economic losses. The mirror carp (Cyprinus carpio var. specularis) exhibits strong environmental adaptability and its primary cells can be used to isolate KHV. This study utilized the tissue explant method to systematically investigate primary cell culture conditions for mirror carp snout and caudal fin tissues. We demonstrated that cells from these two tissue types had strong adaptability, and when cultured in Medium 199 (M199) containing 20% serum at 26 to 30°C, the cells from the snout and caudal fin tissues exhibited the fastest egress and proliferation. Inoculation of these two cell types with KHV-infected fish kidney tissues produced typical cytopathic effects; additionally, identification by electron microscopy, and PCR indicated that KHV could be isolated from both cell types. PMID:23893087

  15. Water permeability of primary mouse keratinocyte cultures grown at the air-liquid interface

    SciTech Connect

    Cumpstone, M.B.; Kennedy, A.H.; Harmon, C.S.; Potts, R.O.

    1989-04-01

    In order to study the development of the epidermal permeability barrier in vitro, tritiated water (HTO) flux was measured across murine keratinocytes cultured at the air-liquid interface. Using a micro-diffusion technique, it was shown that air-liquid cultures form areas where the water diffusion is comparable to that of intact neonatal mouse skin. When water permeability is measured over a large area of the culture surface, however, significantly higher flux is obtained. These results show that under the culture conditions used, areas of water barrier comparable to intact neonatal mouse skin coexist with regions of less complete barrier formation.

  16. The functional alterations associated with motor imagery training: a comparison between motor execution and motor imagery of sequential finger tapping

    NASA Astrophysics Data System (ADS)

    Zhang, Hang; Yao, Li; Long, Zhiying

    2011-03-01

    Motor imagery training, as an effective strategy, has been more and more applied to mental disorders rehabilitation and motor skill learning. Studies on the neural mechanism underlying motor imagery have suggested that such effectiveness may be related to the functional congruence between motor execution and motor imagery. However, as compared to the studies on motor imagery, the studies on motor imagery training are much fewer. The functional alterations associated with motor imagery training and the effectiveness of motor imagery training on motor performance improvement still needs further investigation. Using fMRI, we employed a sequential finger tapping paradigm to explore the functional alterations associated with motor imagery training in both motor execution and motor imagery task. We hypothesized through 14 consecutive days motor imagery training, the motor performance could be improved and the functional congruence between motor execution and motor imagery would be sustained form pre-training phase to post-training phase. Our results confirmed the effectiveness of motor imagery training in improving motor performance and demonstrated in both pre and post-training phases, motor imagery and motor execution consistently sustained the congruence in functional neuroanatomy, including SMA (supplementary motor cortex), PMA (premotor area); M1( primary motor cortex) and cerebellum. Moreover, for both execution and imagery tasks, a similar functional alteration was observed in fusiform through motor imagery training. These findings provided an insight into the effectiveness of motor imagery training and suggested its potential therapeutic value in motor rehabilitation.

  17. The susceptibility of primary cultured rhesus macaque kidney epithelial cells to rhesus cytomegalovirus strains.

    PubMed

    Yue, Yujuan; Kaur, Amitinder; Lilja, Anders; Diamond, Don J; Walter, Mark R; Barry, Peter A

    2016-06-01

    Kidney epithelial cells are common targets for human and rhesus cytomegalovirus (HCMV and RhCMV) in vivo, and represent an important reservoir for long-term CMV shedding in urine. To better understand the role of kidney epithelial cells in primate CMV natural history, primary cultures of rhesus macaque kidney epithelial cells (MKE) were established and tested for infectivity by five RhCMV strains, including two wild-type strains (UCD52 and UCD59) and three strains containing different coding contents in UL/b'. The latter strains included 180.92 [containing an intact RhUL128-RhUL130-R hUL131 (RhUL128L) locus but deleted for the UL/b' RhUL148-rh167-loci], 68-1 (RhUL128L-defective and fibroblast-tropic) and BRh68-1.2 (the RhUL128L-repaired version of 68-1). As demonstrated by RhCMV cytopathic effect, plaque formation, growth kinetics and early virus entry, we showed that MKE were differentially susceptible to RhCMV infection, related to UL/b' coding contents of the different strains. UCD52 and UCD59 replicated vigorously in MKE, 68-1 replicated poorly, and 180.92 grew with intermediate kinetics. Reconstitution of RhUL128L in 68-1 (BRh68-1.2) restored its replication efficiency in MKE as compared to UCD52 and UCD59, consistent with the essential role of UL128L for HCMV epithelial tropism. Further analysis revealed that the UL/b' UL148-rh167-loci deletion in 180.92 impaired RhUL132 (rh160) expression. Given that 180.92 retains an intact RhUL128L, but genetically or functionally lacks genes from RhUL132 (rh160) to rh167 in UL/b', its attenuated infection efficiency indicated that, along with RhUL128L, an additional protein(s) encoded within the UL/b' RhUL132 (rh160)-rh167 region (potentially, RhUL132 and/or RhUL148) is indispensable for efficient replication in MKE. PMID:26974598

  18. Cryopreservation and in vitro culture of primary cell types from lung tissue of a stranded pygmy sperm whale (Kogia breviceps).

    PubMed

    Annalaura Mancia; Spyropoulos, Demetri D; McFee, Wayne E; Newton, Danforth A; Baatz, John E

    2012-01-01

    Current models for in vitro studies of tissue function and physiology, including responses to hypoxia or environmental toxins, are limited and rely heavily on standard 2-dimensional (2-D) cultures with immortalized murine or human cell lines. To develop a new more powerful model system, we have pursued methods to establish and expand cultures of primary lung cell types and reconstituted tissues from marine mammals. What little is known about the physiology of the deep-sea diving pygmy sperm whale (PSW), Kogia breviceps, comes primarily from stranding events that occur along the coast of the southeastern United States. Thus, development of a method for preserving live tissues and retrieving live cells from deceased stranded individuals was initiated. This report documents successful cryopreservation of PSW lung tissue. We established in vitro cultures of primary lung cell types from tissue fragments that had been cryopreserved several months earlier at the stranding event. Dissociation of cryopreserved lung tissues readily provides a variety of primary cell types that, to varying degrees, can be expanded and further studied/manipulated in cell culture. In addition, PSW-specific molecular markers have been developed that permitted the monitoring of fibroblast, alveolar type II, and vascular endothelial cell types. Reconstitution of 3-D cultures of lung tissues with these cell types is now underway. This novel system may facilitate the development of rare or disease-specific lung tissue models (e.g., to test causes of PSW stranding events and lead to improved treatments for pulmonary hypertension or reperfusion injury in humans). Also, the establishment of a "living" tissue bank biorepository for rare/endangered species could serve multiple purposes as surrogates for freshly isolated samples.

  19. Differential stimulation of neurotrophin release by the biocompatible nano-material (carbon nanotube) in primary cultured neurons.

    PubMed

    Kim, Yun Gi; Kim, Jong Wan; Pyeon, Hee Jang; Hyun, Jung Keun; Hwang, Ji-Young; Choi, Seong-Jun; Lee, Ja-Yeon; Deák, Ferenc; Kim, Hae-Won; Lee, Young Il

    2014-01-01

    In order to develop novel, effective therapies for central nervous system regeneration, it is essential to better understand the role of neurotrophic factors and to design, accordingly, better artificial scaffolds to support both neurite outgrowth and synapse formation. Both nerve growth factor and brain-derived neurotrophic factor are major factors in neural survival, development, synaptogenesis, and synaptic connectivity of primary cultured neurons. As a prime candidate coating material for such neural cultures, carbon nanotubes offer unique structural, mechanical, and electrical properties. In this study, carbon nanotubes coated glass-coverslips were used as the matrix of a primary neural culture system used to investigate the effects of carbon nanotubes on neurite outgrowth and nerve growth factor/brain-derived neurotrophic factor release and expression. For these purposes, we performed comparative analyses of primary cultured neurons on carbon nanotubes coated, non-coated, and Matrigel-coated coverslips. The morphological findings showed definite carbon nanotubes effects on the neurite outgrowths and synaptogenic figures in both cortical and hippocampal neurons when compared with the non-coated negative control. Although the carbon nanotubes did not change neurotrophin expression levels, it stimulated brain-derived neurotrophic factor release into the media from both types of neurons. Accordingly, we suggest a different mechanism of action between carbon nanotubes and Matrigel in relation to the specific neurotrophic factors. Since carbon nanotubes supply long-term extracellular molecular cues for the survival and neurite outgrowths of cultured neurons, the results from this study will contribute to an understanding of carbon nanotubes biological effects and provide new insight into their role in the secretion of neurotrophic factors.

  20. Development of a primary mouse intestinal epithelial cell monolayer culture system to evaluate factors that modulate IgA transcytosis

    PubMed Central

    Moon, Clara; VanDussen, Kelli L.; Miyoshi, Hiroyuki; Stappenbeck, Thaddeus S.

    2013-01-01

    There is significant interest in the use of primary intestinal epithelial cells in monolayer culture to model intestinal biology. However, it has proven to be challenging to create functional, differentiated monolayers using current culture methods, likely due to the difficulty in expanding these cells. Here, we adapted our recently developed method for the culture of intestinal epithelial spheroids to establish primary epithelial cell monolayers from the colon of multiple genetic mouse strains. These monolayers contained differentiated epithelial cells that displayed robust transepithelial electrical resistance. We then functionally tested them by examining IgA transcytosis across Transwells. IgA transcytosis required induction of polymeric immunoglobulin receptor (pIgR) expression, which could be stimulated by a combination of LPS and inhibition of γ-secretase. In agreement with previous studies using immortalized cell lines, we found that TNFα, IL-1β, IL-17 and heat-killed microbes also stimulated pIgR expression and IgA transcytosis. We used wild-type and knockout cells to establish that amongst these cytokines, IL-17 was the most potent inducer of pIgR expression/IgA transcytosis. IFNγ however did not induce pIgR expression, and instead led to cell death. This new method will allow the use of primary cells for studies of intestinal physiology. PMID:24220295

  1. Activation of antioxidant response element in mouse primary cortical cultures with sesquiterpene lactones isolated from Tanacetum parthenium

    PubMed Central

    Fischedick, Justin T; Standiford, Miranda; Johnson, Delinda A.; De Vos, Ric C.H.; Todorović, Slađana; Banjanac, Tijana; Verpoorte, Rob; Johnson, Jeffrey A.

    2012-01-01

    Tanacetum parthenium (Asteraceae) produces biologically active sesquiterpene lactones (SL). Nuclear factor E2-related factor 2 (Nrf2) is a transcription factor known to activate a series of genes termed the antioxidant response element (ARE). Activation of the Nrf2/ARE may be useful for the treatment of neurodegenerative disease. In this study we isolated 11 sesquiterpene lactones from T. parthenium with centrifugal partition chromatography and semi-preparative HPLC. Compounds were screened in-vitro for their ability to activate the ARE on primary mouse cortical cultures as well as for their toxicity towards the cultures. All sesquiterpene lactones containing the α-methylene-γ-lactone moiety were able to activate the ARE although a number of compounds displayed significant cellular toxicity towards the cultures. The structure activity relationship of the sesquiterpene lactones indicate that the guaianolides isolated were more active and less toxic then the germacranolides. PMID:22923197

  2. Establishment of a primary hepatocyte culture from the small Indian mongoose (Herpestes auropunctatus) and distribution of mercury in liver tissue.

    PubMed

    Horai, Sawako; Yanagi, Kumiko; Kaname, Tadashi; Yamamoto, Masatatsu; Watanabe, Izumi; Ogura, Go; Abe, Shintaro; Tanabe, Shinsuke; Furukawa, Tatsuhiko

    2014-11-01

    The present study established a primary hepatocyte culture for the small Indian mongoose (Herpestes auropunctatus). To determine the suitable medium for growing the primary hepatic cells of this species, we compared the condition of cells cultured in three media that are frequently used for mammalian cell culture: Dulbecco's Modified Eagle's Medium, RPMI-1640, and William's E. Of these, William's E medium was best suited for culturing the hepatic cells of this species. Using periodic acid-Schiff staining and ultrastructural observations, we demonstrated the cells collected from mongoose livers were hepatocytes. To evaluate the distribution of mercury (Hg) in the liver tissue, we carried out autometallography staining. Most of the Hg compounds were found in the central region of hepatic lobules. Smooth endoplasmic reticulum, which plays a role inxenobiotic metabolism, lipid/cholesterol metabolism, and the digestion and detoxification of lipophilic substances is grown in this area. This suggested that Hg colocalized with smooth endoplasmic reticulum. The results of the present study could be useful to identify the detoxification systems of wildlife with high Hg content in the body, and to evaluate the susceptibility of wildlife to Hg toxicity. PMID:25142347

  3. Perceptions of culturally competent diabetes management in a primary care practice.

    PubMed

    Kirk, Julienne K; Hildebrandt, Carol; Davis, Stephen; Crandall, Sonia J; Siciliano, Alissa B; Marion, Gail S

    2014-01-01

    To evaluate whether clinicians consider the impact of culture on diabetes management, a survey was mailed to 300 randomly selected patients > or = 50 years with type 2 diabetes and 153 surveys were returned. Data were correlated with A1C values. African Americans (AA) and non-Hispanic whites (NHW), (91.9%, 97.0%) respectively, reported clinicians discussed benefits of controlling blood sugar but did not discuss effects of cultural issues on glucose control (< or = 50%). AAs perceived clinicians were more accommodating of their cultural preferences than did NHWs (49.2% versus 30.6%) (P < .05). Females (51.9%) (P < .01) reported that clinicians acknowledged the importance of their cultural beliefs with a slightly higher percentage for African American females (54.8%) versus non-Hispanic White females (48.6%). Understanding the patient's and clinician's views of cultural beliefs as they relate to diabetes self-management can provide perspectives to guide care.

  4. Maintenance of Hepatic Functions in Primary Human Hepatocytes Cultured on Xeno-Free and Chemical Defined Human Recombinant Laminins.

    PubMed

    Watanabe, Masaaki; Zemack, Helen; Johansson, Helene; Hagbard, Louise; Jorns, Carl; Li, Meng; Ellis, Ewa

    2016-01-01

    Refined methods for maintaining specific functions of isolated hepatocytes under xeno-free and chemical defined conditions is of great importance for the development of hepatocyte research and regenerative therapy. Laminins, a large family of heterotrimeric basement membrane adhesion proteins, are highly cell and tissue type specific components of the extracellular matrix and strongly influence the behavior and function of associated cells and/or tissues. However, detailed biological functions of many laminin isoforms are still to be evaluated. In this study, we determined the distribution of laminin isoforms in human liver tissue and isolated primary human hepatocytes by western blot analysis, and investigated the efficacy of different human recombinant laminin isoforms on hepatic functions during culture. Protein expressions of laminin-chain α2, α3, α4, β1, β3, γ1, and γ2 were detected in both isolated human hepatocytes and liver tissue. No α1 and α5 expression could be detected in liver tissue or hepatocytes. Hepatocytes were isolated from five different individual livers, and cultured on human recombinant laminin isoforms -111, -211, -221, -332, -411, -421, -511, and -521 (Biolamina AB), matrigel (extracted from Engelbreth-Holm-Swarm sarcoma), or collagen type IV (Collagen). Hepatocytes cultured on laminin showed characteristic hexagonal shape in a flat cell monolayer. Viability, double stranded DNA concentration, and Ki67 expression for hepatocytes cultured for six days on laminin were comparable to those cultured on EHS and Collagen. Hepatocytes cultured on laminin also displayed production of human albumin, alpha-1-antitrypsin, bile acids, and gene expression of liver-enriched factors, such as hepatocyte nuclear factor 4 alpha, glucose-6-phosphate, cytochrome P450 3A4, and multidrug resistance-associated protein 2. We conclude that all forms of human recombinant laminin tested maintain cell viability and liver-specific functions of primary human

  5. Maintenance of Hepatic Functions in Primary Human Hepatocytes Cultured on Xeno-Free and Chemical Defined Human Recombinant Laminins

    PubMed Central

    Watanabe, Masaaki; Zemack, Helen; Johansson, Helene; Hagbard, Louise; Jorns, Carl; Li, Meng; Ellis, Ewa

    2016-01-01

    Refined methods for maintaining specific functions of isolated hepatocytes under xeno-free and chemical defined conditions is of great importance for the development of hepatocyte research and regenerative therapy. Laminins, a large family of heterotrimeric basement membrane adhesion proteins, are highly cell and tissue type specific components of the extracellular matrix and strongly influence the behavior and function of associated cells and/or tissues. Ho