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Sample records for addition primary cultures

  1. Additivity of Pyrethroid Actions on Sodium Influx in Cortical Neurons in Cerebrocortical Neurons in Primary Culture

    EPA Science Inventory

    BACKGROUND: Pyrethroid insecticides bind to voltage-gated sodium channels and modify their gating kinetics, thereby disrupting neuronal function. Although previous work has tested the additivity of pyrethroids in vivo, this has not been assessed directly at the primary molecular ...

  2. 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

  3. 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.

  4. 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

  5. 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.

  6. 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

  7. 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

  8. 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

  9. 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.

  10. 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

  11. 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…

  12. 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.…

  13. 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

  14. 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

  15. 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

  16. 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…

  17. 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

  18. 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

  19. 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…

  20. 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.

  1. 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'…

  2. 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.

  3. 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

  4. 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.

  5. 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.

  6. 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.

  7. 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

  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. 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

  14. 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.

  15. 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.

  16. 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

  17. 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

  18. 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.

  19. 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

  20. 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

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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

  8. 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

  9. 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

  10. 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

  11. Primary-tertiary diamine-catalyzed Michael addition of ketones to isatylidenemalononitrile derivatives.

    PubMed

    Kumar, Akshay; Chimni, Swapandeep Singh

    2014-01-01

    Simple primary-tertiary diamines easily derived from natural primary amino acids were used to catalyze the Michael addition of ketones with isatylidenemalononitrile derivatives. Diamine 1a in combination with D-CSA as an additive provided Michael adducts in high yield (up to 94%) and excellent enantioselectivity (up to 99%). The catalyst 1a was successfully used to catalyze the three-component version of the reaction by a domino Knoevenagel-Michael sequence. The Michael adduct 4a was transformed into spirooxindole 6 by a reduction with sodium borohydride in a highly enantioselective manner.

  12. 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

  13. 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

  14. 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

  15. 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.

  16. 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...

  17. 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.

  18. Strategies of Pre-Service Primary School Teachers for Solving Addition Problems with Negative Numbers

    ERIC Educational Resources Information Center

    Almeida, Rut; Bruno, Alicia

    2014-01-01

    This paper analyses the strategies used by pre-service primary school teachers for solving simple addition problems involving negative numbers. The findings reveal six different strategies that depend on the difficulty of the problem and, in particular, on the unknown quantity. We note that students use negative numbers in those problems they find…

  19. 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

  20. 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

  1. 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.

  2. 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.

  3. Glass Formation and Primary Solidification in Nd2Fe14B with TiC Additions

    NASA Astrophysics Data System (ADS)

    McCallum, R. W.; Willard, M. A.; Dennis, K. W.; Kramer, M. J.; Branagan, D. J.

    1996-03-01

    In bulk permanent magnets, the extrinsic properties relating to the microstructure determine the level of hard magnetic properties that is actually achieved. Alloy additions which affect the solidification behavior of the melt and then form precipitates which pin grain boundaries and therefor control grain size should be valuable in producing the uniform microstructure required for good magnetic properties. The addition of TiC to 2-14-1 results in a factor of three reduction of the quench rate required to produce amorphous material. In addition, the crystallization temperature of the glass is enhanced leading to enhanced nucleation and finer grain size during crystallization. Given the affect of TiC additions to the stochiometric melt, it is not surprising that the additions affect the range of primary solidification of the 2-14-1 phase. When TiC is added to the limit of liquid solubility, the limit 2-14-1 primary solidification is move farther from the stochiometric composition in the Nd rich region. Work at the Ames Laboratory, was supported by the U. S. Department of Energy under contract No. W-7405-ENG-82.

  4. 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

  5. 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.

  6. 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

  7. Additives

    NASA Technical Reports Server (NTRS)

    Smalheer, C. V.

    1973-01-01

    The chemistry of lubricant additives is discussed to show what the additives are chemically and what functions they perform in the lubrication of various kinds of equipment. Current theories regarding the mode of action of lubricant additives are presented. The additive groups discussed include the following: (1) detergents and dispersants, (2) corrosion inhibitors, (3) antioxidants, (4) viscosity index improvers, (5) pour point depressants, and (6) antifouling agents.

  8. 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

  9. 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...

  10. 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…

  11. 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.

  12. Additional requirements for leak-before-break application to primary coolant piping in Belgium

    SciTech Connect

    Roussel, G.

    1997-04-01

    Leak-Before-Break (LBB) technology has not been applied in the first design of the seven Pressurized Water Reactors the Belgian utility is currently operating. The design basis of these plants required to consider the dynamic effects associated with the ruptures to be postulated in the high energy piping. The application of the LBB technology to the existing plants has been recently approved by the Belgian Safety Authorities but with a limitation to the primary coolant loop. LBB analysis has been initiated for the Doel 3 and Tihange 2 plants to allow the withdrawal of some of the reactor coolant pump snubbers at both plants and not reinstall some of the restraints after steam generator replacement at Doel 3. LBB analysis was also found beneficial to demonstrate the acceptability of the primary components and piping to the new conditions resulting from power uprating and stretch-out operation. LBB analysis has been subsequently performed on the primary coolant loop of the Tihange I plant and is currently being performed for the Doel 4 plant. Application of the LBB to the primary coolant loop is based in Belgium on the U.S. Nuclear Regulatory Commission requirements. However the Belgian Safety Authorities required some additional analyses and put some restrictions on the benefits of the LBB analysis to maintain the global safety of the plant at a sufficient level. This paper develops the main steps of the safety evaluation performed by the Belgian Safety Authorities for accepting the application of the LBB technology to existing plants and summarizes the requirements asked for in addition to the U.S. Nuclear Regulatory Commission rules.

  13. 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.

  14. 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

  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. 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

  17. 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...

  18. 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.

  19. 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

  20. 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.

  1. 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…

  2. 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

  3. 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

  4. 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…

  5. 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…

  6. 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

  7. 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,…

  8. 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…

  9. 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…

  10. 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.

  11. 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.

  12. 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.

  13. 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

  14. 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

  15. 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.

  16. 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

  17. 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

  18. 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

  19. Emotional expression in music: contribution, linearity, and additivity of primary musical cues.

    PubMed

    Eerola, Tuomas; Friberg, Anders; Bresin, Roberto

    2013-01-01

    The aim of this study is to manipulate musical cues systematically to determine the aspects of music that contribute to emotional expression, and whether these cues operate in additive or interactive fashion, and whether the cue levels can be characterized as linear or non-linear. An optimized factorial design was used with six primary musical cues (mode, tempo, dynamics, articulation, timbre, and register) across four different music examples. Listeners rated 200 musical examples according to four perceived emotional characters (happy, sad, peaceful, and scary). The results exhibited robust effects for all cues and the ranked importance of these was established by multiple regression. The most important cue was mode followed by tempo, register, dynamics, articulation, and timbre, although the ranking varied across the emotions. The second main result suggested that most cue levels contributed to the emotions in a linear fashion, explaining 77-89% of variance in ratings. Quadratic encoding of cues did lead to minor but significant increases of the models (0-8%). Finally, the interactions between the cues were non-existent suggesting that the cues operate mostly in an additive fashion, corroborating recent findings on emotional expression in music (Juslin and Lindström, 2010). PMID:23908642

  20. 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.

  1. 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

  2. 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.

  3. 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

  4. 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.

  5. 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.

  6. 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

  7. 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.

  8. 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.

  9. 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

  10. 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

  11. 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

  12. 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.

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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.

  19. 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.

  20. 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

  1. 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.

  2. 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

  3. 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

  4. Effect of rate of addition of starter culture on textural characteristics of buffalo milk Feta type cheese during ripening.

    PubMed

    Kumar, Sanjeev; Kanawjia, S K; Kumar, Suryamani; Khatkar, Sunil

    2014-04-01

    The effect of rate of addition of starter culture on textural characteristics of buffalo milk Feta type cheese was investigated during ripening period up to two months. The textural characteristics of buffalo milk Feta type cheese in terms of hardness, cohesiveness, springiness, gumminess and chewiness were analyzed by using textural profile analyzer. The maximum hardness was found with cheese made using 1% culture, while the minimum was found with 2% culture. The cohesiveness and springiness decreased as the level of addition of starter culture increased. The chewiness of cheese also decreased, as the rate of addition of starter culture increased for cheese making. In addition to this, yield, moisture, fat, FDM, protein, salt and S/M of fresh buffalo milk Feta type cheese increased with the increase in rate of addition of starter culture; however, TS of experimental cheeses decreased. PMID:24741179

  5. Additively Manufactured Device for Dynamic Culture of Large Arrays of 3D Tissue Engineered Constructs.

    PubMed

    Costa, Pedro F; Hutmacher, Dietmar W; Theodoropoulos, Christina; Gomes, Manuela E; Reis, Rui L; Vaquette, Cédryck

    2015-04-22

    The ability to test large arrays of cell and biomaterial combinations in 3D environments is still rather limited in the context of tissue engineering and regenerative medicine. This limitation can be generally addressed by employing highly automated and reproducible methodologies. This study reports on the development of a highly versatile and upscalable method based on additive manufacturing for the fabrication of arrays of scaffolds, which are enclosed into individualized perfusion chambers. Devices containing eight scaffolds and their corresponding bioreactor chambers are simultaneously fabricated utilizing a dual extrusion additive manufacturing system. To demonstrate the versatility of the concept, the scaffolds, while enclosed into the device, are subsequently surface-coated with a biomimetic calcium phosphate layer by perfusion with simulated body fluid solution. 96 scaffolds are simultaneously seeded and cultured with human osteoblasts under highly controlled bidirectional perfusion dynamic conditions over 4 weeks. Both coated and noncoated resulting scaffolds show homogeneous cell distribution and high cell viability throughout the 4 weeks culture period and CaP-coated scaffolds result in a significantly increased cell number. The methodology developed in this work exemplifies the applicability of additive manufacturing as a tool for further automation of studies in the field of tissue engineering and regenerative medicine.

  6. 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

  7. 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.

  8. 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

  9. 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.

  10. 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,…

  11. 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…

  12. 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…

  13. 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…

  14. 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...

  15. 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…

  16. 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…

  17. 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

  18. 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

  19. 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

  20. 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.

  1. 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

  2. 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.

  3. Pharmacological profiling of native group II metabotropic glutamate receptors in primary cortical neuronal cultures using a FLIPR.

    PubMed

    Sanger, Helen; Hanna, Lydia; Colvin, Ellen M; Grubisha, Olivera; Ursu, Daniel; Heinz, Beverly A; Findlay, Jeremy D; Vivier, Richard G; Sher, Emanuele; Lodge, David; Monn, James A; Broad, Lisa M

    2013-03-01

    The group II metabotropic glutamate (mGlu) receptors comprised of the mGlu2 and mGlu3 receptor subtypes have gained recognition in recent years as potential targets for psychiatric disorders, including anxiety and schizophrenia. In addition to studies already indicating which subtype mediates the anxiolytic and anti-psychotic effects observed in disease models, studies to help further define the preferred properties of selective group II mGlu receptor ligands will be essential. Comparison of the in vitro properties of these ligands to their in vivo efficacy and tolerance profiles may help provide these additional insights. We have developed a relatively high-throughput native group II mGlu receptor functional assay to aid this characterisation. We have utilised dissociated primary cortical neuronal cultures, which after 7 days in vitro have formed functional synaptic connections and display periodic and spontaneous synchronised calcium (Ca(2+)) oscillations in response to intrinsic action potential bursts. We herein demonstrate that in addition to non-selective group II mGlu receptor agonists, (2R,4R)-APDC, LY379268 and DCG-IV, a selective mGlu2 agonist, LY541850, and mGlu2 positive allosteric modulators, BINA and CBiPES, inhibit the frequency of synchronised Ca(2+) oscillations in primary cultures of rat and mouse cortical neurons. Use of cultures from wild-type, mGlu2(-/-), mGlu3(-/-) and mGlu2/3(-/-) mice allowed us to further probe the contribution of mGlu2 and mGlu3, and revealed LY541850 to be a partial mGlu2 agonist and a full mGlu3 antagonist. Overnight pre-treatment of cultures with these ligands revealed a preferred desensitisation profile after treatment with a positive allosteric modulator. This article is part of a Special Issue entitled 'Metabotropic Glutamate Receptors'. PMID:22659090

  4. GISH analysis of disomic Brassica napus-Crambe abyssinica chromosome addition lines produced by microspore culture from monosomic addition lines.

    PubMed

    Wang, Youping; Sonntag, Karin; Rudloff, Eicke; Wehling, Peter; Snowdon, Rod J

    2006-02-01

    Two Brassica napus-Crambe abyssinica monosomic addition lines (2n=39, AACC plus a single chromosome from C. abyssinca) were obtained from the F(2) progeny of the asymmetric somatic hybrid. The alien chromosome from C. abyssinca in the addition line was clearly distinguished by genomic in situ hybridization (GISH). Twenty-seven microspore-derived plants from the addition lines were obtained. Fourteen seedlings were determined to be diploid plants (2n=38) arising from spontaneous chromosome doubling, while 13 seedlings were confirmed as haploid plants. Doubled haploid plants produced after treatment with colchicine and two disomic chromosome addition lines (2n=40, AACC plus a single pair of homologous chromosomes from C. abyssinca) could again be identified by GISH analysis. The lines are potentially useful for molecular genetic analysis of novel C. abyssinica genes or alleles contributing to traits relevant for oilseed rape (B. napus) breeding.

  5. 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.

  6. 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.

  7. 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

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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.

  13. 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.

  14. Effect of primary-zone equivalence ratio and hydrogen addition on exhaust emission in a hydrocarbon-fueled combustor

    NASA Technical Reports Server (NTRS)

    Norgren, C. T.; Ingebo, R. D.

    1974-01-01

    The effects of reducing the primary-zone equivalence ratio on the exhaust emission levels of oxides of nitrogen, carbon monoxide, and unburned hydrocarbons in experimental hydrocarbon-fueled combustor segments at simulated supersonic cruise and idle conditions were investigated. In addition, the effects of the injection of hydrogen fuel (up to 4 percent of the total weight of fuel) on the stability of the hydrocarbon flame and exhaust emissions were studied and compared with results obtained without hydrogen addition.

  15. Expanding access to primary care without additional budgets? A case study from Burkina Faso.

    PubMed

    Marschall, Paul; Flessa, Steffen

    2008-11-01

    The aim of this study is to demonstrate the impact of increased access to primary care on provider costs in the rural health district of Nouna, Burkina Faso. This study question is crucial for health care planning in this district, as other research work shows that the population has a higher need for health care services. From a public health perspective, an increase of utilisation of first-line health facilities would be necessary. However, the governmental budget that is needed to finance improved access was not known. The study is based on data of 2004 of a comprehensive provider cost information system. This database provides us with the actual costs of each primary health care facility (Centre de Santé et de Promotion Sociale, CSPS) in the health district. We determine the fixed and variable costs of each institution and calculate the average cost per service unit rendered in 2004. Based on the cost structure of each CSPS, we calculate the total costs if the demand for health care services increased. We conclude that the total provider costs of primary care (and therefore the governmental budget) would hardly rise if the coverage of the population were increased. This is mainly due to the fact that the highest variable costs are drugs, which are fully paid for by the customers (Bamako Initiative). The majority of other costs are fixed. Consequently, health care reforms that improve access to health care institutions must not fear dramatically increasing the costs of health care services. PMID:18197447

  16. 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.

  17. Nitrogen balancing and xylose addition enhances growth capacity and protein content in Chlorella minutissima cultures.

    PubMed

    Freitas, B C B; Esquível, M G; Matos, R G; Arraiano, C M; Morais, M G; Costa, J A V

    2016-10-01

    This study aimed to examine the metabolic changes in Chlorella minutissima cells grown under nitrogen-deficient conditions and with the addition of xylose. The cell density, maximum photochemical efficiency, and chlorophyll and lipid levels were measured. The expression of two photosynthetic proteins, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and the beta subunit (AtpB) of adenosine triphosphate synthase, were measured. Comparison of cells grown in medium with a 50% reduction in the nitrogen concentration versus the traditional medium solution revealed that the cells grown under nitrogen-deficient conditions exhibited an increased growth rate, higher maximum cell density (12.7×10(6)cellsmL(-1)), optimal PSII efficiency (0.69) and decreased lipid level (25.08%). This study has taken the first steps toward protein detection in Chlorella minutissima, and the results can be used to optimize the culturing of other microalgae. PMID:27359061

  18. Generalized Additive Mixed-Models for Pharmacology Using Integrated Discrete Multiple Organ Co-Culture

    PubMed Central

    Ingersoll, Thomas; Cole, Stephanie; Madren-Whalley, Janna; Booker, Lamont; Dorsey, Russell; Li, Albert; Salem, Harry

    2016-01-01

    Integrated Discrete Multiple Organ Co-culture (IDMOC) is emerging as an in-vitro alternative to in-vivo animal models for pharmacology studies. IDMOC allows dose-response relationships to be investigated at the tissue and organoid levels, yet, these relationships often exhibit responses that are far more complex than the binary responses often measured in whole animals. To accommodate departure from binary endpoints, IDMOC requires an expansion of analytic techniques beyond simple linear probit and logistic models familiar in toxicology. IDMOC dose-responses may be measured at continuous scales, exhibit significant non-linearity such as local maxima or minima, and may include non-independent measures. Generalized additive mixed-modeling (GAMM) provides an alternative description of dose-response that relaxes assumptions of independence and linearity. We compared GAMMs to traditional linear models for describing dose-response in IDMOC pharmacology studies. PMID:27110941

  19. 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

  20. Respiratory Syncytial Virus Uses CX3CR1 as a Receptor on Primary Human Airway Epithelial Cultures

    PubMed Central

    Johnson, Sara M.; McNally, Beth A.; Ioannidis, Ioannis; Flano, Emilio; Teng, Michael N.; Oomens, Antonius G.; Walsh, Edward E.; Peeples, Mark E.

    2015-01-01

    Respiratory syncytial virus (RSV) is the most frequent cause of lower respiratory disease in infants, but no vaccine or effective therapy is available. The initiation of RSV infection of immortalized cells is largely dependent on cell surface heparan sulfate (HS), a receptor for the RSV attachment (G) glycoprotein in immortalized cells. However, RSV infects the ciliated cells in primary well differentiated human airway epithelial (HAE) cultures via the apical surface, but HS is not detectable on this surface. Here we show that soluble HS inhibits infection of immortalized cells, but not HAE cultures, confirming that HS is not the receptor on HAE cultures. Conversely, a “non-neutralizing” monoclonal antibody against the G protein that does not block RSV infection of immortalized cells, does inhibit infection of HAE cultures. This antibody was previously shown to block the interaction between the G protein and the chemokine receptor CX3CR1 and we have mapped the binding site for this antibody to the CX3C motif and its surrounding region in the G protein. We show that CX3CR1 is present on the apical surface of ciliated cells in HAE cultures and especially on the cilia. RSV infection of HAE cultures is reduced by an antibody against CX3CR1 and by mutations in the G protein CX3C motif. Additionally, mice lacking CX3CR1 are less susceptible to RSV infection. These findings demonstrate that RSV uses CX3CR1 as a cellular receptor on HAE cultures and highlight the importance of using a physiologically relevant model to study virus entry and antibody neutralization. PMID:26658574

  1. Low cost production of 3D-printed devices and electrostimulation chambers for the culture of primary neurons

    PubMed Central

    Wardyn, Joanna D.; Sanderson, Chris; Swan, Laura E.; Stagi, Massimiliano

    2015-01-01

    The analysis of primary neurons is a basic requirement for many areas of neurobiology. However, the range of commercial systems available for culturing primary neurons is functionally limiting, and the expense of these devices is a barrier to both exploratory and large-scale studies. This is especially relevant as primary neurons often require unusual geometries and specialised coatings for optimum growth. Fortunately, the recent revolution in 3D printing offers the possibility to generate customised devices, which can support neuronal growth and constrain neurons in defined paths, thereby enabling many aspects of neuronal physiology to be studied with relative ease. In this article, we provide a detailed description of the system hardware and software required to produce affordable 3D-printed culture devices, which are also compatible with live-cell imaging. In addition, we also describe how to use these devices to grow and stimulate neurons within geometrically constrained compartments and provide examples to illustrate the practical utility and potential that these protocols offer for many aspects of experimental neurobiology. PMID:25962333

  2. Low cost production of 3D-printed devices and electrostimulation chambers for the culture of primary neurons.

    PubMed

    Wardyn, Joanna D; Sanderson, Chris; Swan, Laura E; Stagi, Massimiliano

    2015-08-15

    The analysis of primary neurons is a basic requirement for many areas of neurobiology. However, the range of commercial systems available for culturing primary neurons is functionally limiting, and the expense of these devices is a barrier to both exploratory and large-scale studies. This is especially relevant as primary neurons often require unusual geometries and specialised coatings for optimum growth. Fortunately, the recent revolution in 3D printing offers the possibility to generate customised devices, which can support neuronal growth and constrain neurons in defined paths, thereby enabling many aspects of neuronal physiology to be studied with relative ease. In this article, we provide a detailed description of the system hardware and software required to produce affordable 3D-printed culture devices, which are also compatible with live-cell imaging. In addition, we also describe how to use these devices to grow and stimulate neurons within geometrically constrained compartments and provide examples to illustrate the practical utility and potential that these protocols offer for many aspects of experimental neurobiology. PMID:25962333

  3. Low cost production of 3D-printed devices and electrostimulation chambers for the culture of primary neurons.

    PubMed

    Wardyn, Joanna D; Sanderson, Chris; Swan, Laura E; Stagi, Massimiliano

    2015-08-15

    The analysis of primary neurons is a basic requirement for many areas of neurobiology. However, the range of commercial systems available for culturing primary neurons is functionally limiting, and the expense of these devices is a barrier to both exploratory and large-scale studies. This is especially relevant as primary neurons often require unusual geometries and specialised coatings for optimum growth. Fortunately, the recent revolution in 3D printing offers the possibility to generate customised devices, which can support neuronal growth and constrain neurons in defined paths, thereby enabling many aspects of neuronal physiology to be studied with relative ease. In this article, we provide a detailed description of the system hardware and software required to produce affordable 3D-printed culture devices, which are also compatible with live-cell imaging. In addition, we also describe how to use these devices to grow and stimulate neurons within geometrically constrained compartments and provide examples to illustrate the practical utility and potential that these protocols offer for many aspects of experimental neurobiology.

  4. 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.

  5. Primary explosives: electrostatic discharge initiation, additive effect and its relation to thermal and explosive characteristics.

    PubMed

    Talawar, M B; Agrawal, A P; Anniyappan, M; Wani, D S; Bansode, M K; Gore, G M

    2006-09-21

    All explosives, under all conditions must be considered vulnerable to generation, accumulation and discharge of static charge. The low energy static hazards of the order as low as 2-3 mJ need to be guarded against in case of highly sensitive compounds namely primary explosives. The hazard is normally associated with manufacturing and filling operations due to discharge of static charge accumulated on a person supplying energy up to 20 mJ. To reduce the risk associated with static initiation hazard in the processing and handling of the explosives, the electrostatic sensitivity tests can provide an important input regarding electrostatic hazards. This paper presents electrostatic sensitivity data in terms of zero ignition probability data (E(SE0)) of some of the initiatory explosives such as nickel/cobalt hydrazinium nitrate, silver azide, lead azide and mercury salt of 5-nitro tetrazole. Similar data has also been presented for samples coated with polyvinyl pyrrolidone to study its effect on electrostatic sensitivity. The electrostatic spark sensitivity of some conventional and novel made to explain the increased spark sensitivity behavior on the basis of the possible primary explosives has been studied. The electrostatic spark sensitivity of primary explosives decreased in the order of AgN3 = NHN > PbN6 > MNT > CoHN > BNCP. A possible correlation of spark energy with approximation and assumption has been drawn with thermal, detonation and mechanical properties. The polyvinyl pyrrolidone coated samples followed the same order but interestingly with increased spark sensitivity. An attempt has been reasoning of dielectric nature of the materials or exothermic effects of decomposition products of PVP. The present work also reports the electrostatic spark sensitivity of cap compositions.

  6. 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

  7. [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.

  8. Cell contact as an independent factor modulating cardiac myocyte hypertrophy and survival in long-term primary culture

    NASA Technical Reports Server (NTRS)

    Clark, W. A.; Decker, M. L.; Behnke-Barclay, M.; Janes, D. M.; Decker, R. S.

    1998-01-01

    Cardiac myocytes maintained in cell culture develop hypertrophy both in response to mechanical loading as well as to receptor-mediated signaling mechanisms. However, it has been shown that the hypertrophic response to these stimuli may be modulated through effects of intercellular contact achieved by maintaining cells at different plating densities. In this study, we show that the myocyte plating density affects not only the hypertrophic response and features of the differentiated phenotype of isolated adult myocytes, but also plays a significant role influencing myocyte survival in vitro. The native rod-shaped phenotype of freshly isolated adult myocytes persists in an environment which minimizes myocyte attachment and spreading on the substratum. However, these conditions are not optimal for long-term maintenance of cultured adult cardiac myocytes. Conditions which promote myocyte attachment and spreading on the substratum, on the other hand, also promote the re-establishment of new intercellular contacts between myocytes. These contacts appear to play a significant role in the development of spontaneous activity, which enhances the redevelopment of highly differentiated contractile, junctional, and sarcoplasmic reticulum structures in the cultured adult cardiomyocyte. Although it has previously been shown that adult cardiac myocytes are typically quiescent in culture, the addition of beta-adrenergic agonists stimulates beating and myocyte hypertrophy, and thereby serves to increase the level of intercellular contact as well. However, in densely-plated cultures with intrinsically high levels of intercellular contact, spontaneous contractile activity develops without the addition of beta-adrenergic agonists. In this study, we compare the function, morphology, and natural history of adult feline cardiomyocytes which have been maintained in cultures with different levels of intercellular contact, with and without the addition of beta-adrenergic agonists

  9. 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…

  10. Cultural Dimensions of Feedback at an Australian University: A Study of International Students with English as an Additional Language

    ERIC Educational Resources Information Center

    Warner, Richard; Miller, Julia

    2015-01-01

    International students with English as an additional language face transitional challenges when entering a new academic culture. One such challenge involves optimising feedback to help foster their academic development, bearing in mind that feedback is not a culturally neutral entity (Nazif, A., Biswas, D., & Hilbig, R. (2004-2005). Towards an…

  11. 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.

  12. 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.…

  13. Role of residual additives in the cytotoxicity and cytokine release caused by polyvinyl chloride particles in pulmonary cell cultures.

    PubMed

    Xu, Haiyan; Dinsdale, David; Nemery, Benoit; Hoet, Peter H M

    2003-03-01

    Occupational exposure to polyvinyl chloride (PVC) dust has been linked to pulmonary disease. The aim of the present study was to investigate, in vitro, the role of additives in the cytotoxicity and the release of inflammatory mediators caused by PVC particles in different cells. We compared two types of emulsion PVC particles (E3 and E8) with their washed (hence, "additive-free") counterparts (W3 and W8). A positive control (crystalline SiO2, Min-U-Sil) and the pure additives, sodium lauryl sulfate (A3) and sodium alkylbenzenesulfonate (A8), were tested concurrently. Cytotoxicity (MTT assay) was assessed in primary cultures of rat alveolar macrophages, rat type II pneumocytes, and human alveolar macrophages (h-AM), and cultures of the A549 cell line (type II cell-derived) and the differentiated THP-1 cell line (macrophage-like). Hemolytic potential was assessed after a 2-h incubation with human erythrocytes. Cytokine release (IL-8, IL-6, and TNF-alpha) by A549 cells, THP-1 cells, and h-AM, was measured by ELISA after 4, 16, 24 and/or 48 h of exposure. Cytotoxicity and hemolytic activity of the washed particles were abolished or markedly decreased compared with their nonwashed forms. In A549 cells, E3 and E8 (2.5 mg/ml) caused a 3-fold increase in IL-8 release and a more than 10-fold increase in IL-6 release, whereas W3 and W8 did not elicit any significant response at similar concentrations. Compared with Min-U-Sil (0.1, 0.5, and 2.5 mg/ml), the response to E3 and E8 occurred later and was slightly lower (IL-8) or much more pronounced (IL-6). A3 and A8 exhibited similar responses to E3 and E8, at concentrations corresponding to those present in the particles. In conclusion, the in vitro cytotoxicity and inflammatory potential of some PVC particles appear to be mostly due to their residual additives.

  14. 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

  15. 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.

  16. 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.

  17. 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.

  18. 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

  19. 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.

  20. 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

  1. 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.

  2. 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

  3. 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

  4. 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.

  5. 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.

  6. 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.

  7. 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

  8. 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.

  9. 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

  10. 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.

  11. 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

  12. 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

  13. 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

  14. 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.

  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. Paleomagnetism of Silver Island, Keweenaw Peninsula, Michigan: Additional Support (?) for the Primary Curvature of the MCR

    NASA Astrophysics Data System (ADS)

    Diehl, J. F.; Durant, A. J.; Schepke, C.

    2009-05-01

    Silver Island lies between Eagle Harbor and Copper Harbor on the NW coastline of the Keweenaw Peninsula, Michigan. The island consists of a series of basaltic lava flows which dip to the north-northwest (356°) at an angle of approximately 38°. These flows constitute part of the Lake Shore Traps (LST), a series of interbedded lava flows within the Copper Harbor Conglomerate (CHC). The LST represent the youngest eruptive material associated with the 1.1 Ga Mid-Continent Rift (MCR). The most recent paleomagnetic study (now 15 years old) on the Lakeshore Traps (LST) defined three distinct directional clusters. Each cluster of directions corresponded to a different stratigraphic package of the LST within the CHC and all have extremely low between-site dispersion (s ˜ 4°) suggesting rather rapid emplacement of the LST packages. Consequently, each cluster has its own unique direction of magnetization. Since the lower two LST packages crop out along the coast line of the Peninsula with different structural trends, an opportunity was presented to test the conclusions of Hnat et al. (2006) that the curvature of the MCR was primary. To that end nine lava flows were sampled on Silver Island and their mean direction compared to the equivalent mean from lava flows sampled by Diehl and Haig (1994) from the tip of the Keweenaw Peninsula (upper lava flows of the Middle LST) which have an entirely different structural trend. Characteristic directions of magnetization of the Silver Island lava flows were isolated either using alternating field, thermal or a combination of both. The mean direction of magnetization for the nine sites is: D = 277.7°, I = 46.9°, α95 = 3.0°, k = 292.5. The mean direction recalculated from the Diehl and Haig study is: D = 277.8°, I = 40.6°, α95 = 2.9°, k = 315.7. Although the declinations of the two means are identical, interestingly the two means are statistically distinct at the 95% confidence level. Fold tests were inconclusive. Nevertheless

  19. Characterization and endocrine regulation of proliferation and differentiation of primary cultured preadipocytes from gilthead sea bream (Sparus aurata).

    PubMed

    Salmerón, C; Acerete, L; Gutiérrez, J; Navarro, I; Capilla, E

    2013-07-01

    A preadipocyte primary cell culture was established to gain knowledge about adipose tissue development in gilthead sea bream (Sparus aurata), one of the most extensively produced marine aquaculture species in the Mediterranean. The preadipocytes obtained from the stromal-vascular cell fraction of adipose tissue proliferated in culture, reaching confluence around day 8. At that time, the addition of an adipogenic medium promoted differentiation of the cells into mature adipocytes, which showed an enlarged cytoplasm filled with lipid droplets. First, cell proliferation and differentiation were analyzed under control and adipogenic conditions during culture development. Next, the effects of insulin, GH, and IGF-I on cell proliferation were evaluated at day 8. All peptides significantly stimulated proliferation of the cells after 48 h of incubation (P < 0.002 for GH and IGF-I and P < 0.05 for insulin), despite no differences were observed between the different doses tested. Subsequently, the effects of insulin and IGF-I maintaining differentiation when added to growth medium were studied at day 11, after 3 d of induction with adipogenic medium. The results showed that IGF-I is more potent than insulin enhancing differentiation (P < 0.01 for IGF-I compared with the control). In summary, a primary culture of gilthead sea bream preadipocytes has been characterized and the effects of several regulators of growth and development have been evaluated. This cellular system can be a good model to study the process of adipogenesis in fish, which may help improve the quality of the product in aquaculture.

  20. 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.

  1. 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.

  2. 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

  3. 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.

  4. 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

  5. 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.

  6. 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

  7. 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

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. Elicitation of silymarin in cell cultures of Silybum marianum: effect of subculture and repeated addition of methyl jasmonate.

    PubMed

    Sánchez-Sampedro, Maria Angeles; Fernández-Tárrago, Jorge; Corchete, Purificación

    2009-10-01

    Production of silymarin and the effect of the elicitor, methyl jasmonate (MeJA), was monitored in cell cultures of Silybum marianum over 4 years. Silymarin concentrations gradually declined after prolonged subculture, making the success of elicitor strategy limited in long-term cultures. The continuous presence of MeJA in cultures for an extended period was necessary for induction of silymarin accumulation. A repeated elicitor strategy was not a good option for improving silymarin productivity in batch cultures. Removal of medium from elicited cultures and addition of fresh medium avoided the toxic effects of elicitor accumulation, allowing the system to respond to a repeated MeJA treatment without loss of productivity.

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. 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

  19. 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

  20. 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

  1. 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.

  2. Expression of phospholipase A2 receptor in primary cultured podocytes derived from dog kidneys.

    PubMed

    Sugahara, Go; Kamiie, Junichi; Kobayashi, Ryosuke; Mineshige, Takayuki; Shirota, Kinji

    2016-06-01

    Phospholipase A2 receptor (PLA2R) expressed in human podocytes has been highlighted as a causative autoantigen of human idiopathic membranous nephropathy. However, its expression was found to be minimal or absent in murine and rat podocytes. In this study, immunofluorescence revealed the expression of PLA2R in the glomerular podocytes in the kidney tissue sections of dogs. We then attempted to culture canine podocytes and investigate the expression of PLA2R in these cells. Glomeruli were isolated from dog kidneys and cultured to obtain podocytes using nylon mesh-based isolation method as followed for isolating rat podocytes. The cultured cells expressed PLA2R mRNA and protein in addition to other podocyte markers (synaptopodin, podocin and nephrin). These results indicate that the canine podocytes express PLA2R.

  3. Expression of phospholipase A2 receptor in primary cultured podocytes derived from dog kidneys

    PubMed Central

    SUGAHARA, Go; KAMIIE, Junichi; KOBAYASHI, Ryosuke; MINESHIGE, Takayuki; SHIROTA, Kinji

    2016-01-01

    Phospholipase A2 receptor (PLA2R) expressed in human podocytes has been highlighted as a causative autoantigen of human idiopathic membranous nephropathy. However, its expression was found to be minimal or absent in murine and rat podocytes. In this study, immunofluorescence revealed the expression of PLA2R in the glomerular podocytes in the kidney tissue sections of dogs. We then attempted to culture canine podocytes and investigate the expression of PLA2R in these cells. Glomeruli were isolated from dog kidneys and cultured to obtain podocytes using nylon mesh-based isolation method as followed for isolating rat podocytes. The cultured cells expressed PLA2R mRNA and protein in addition to other podocyte markers (synaptopodin, podocin and nephrin). These results indicate that the canine podocytes express PLA2R. PMID:26854253

  4. 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

  5. EROD induction in microsomes and primary hepatocyte cultures prepared from individual double-crested cormorant embryo livers

    SciTech Connect

    Davis, J.A.; Fry, D.M.; Wilson, B.W.

    1994-12-31

    Double-crested cormorants (Phalacrocorax auritus) are being studied as a high trophic level indicator species for toxic contamination in San Francisco Bay and other locations on the California coast. In 1993, median ethoxyresorufin-o-deethylase (EROD) activities in embryo liver microsomes sampled from San Francisco Bay colonies were 4.2-fold and 2.3-fold higher than two coastal locations. In 1994 cormorant embryo livers from these same locations were again analyzed for liver microsomal EROD activity. In addition, basal and maximal EROD activity were measured in primary hepatocyte cultures from portions of the same livers used for microsomal EROD analysis to determine whether factors other than contaminant burdens in the egg contribute to the observed differences in activity. Differences in basal activity and induction capacity among the populations are discussed in the context of the hypothesis that polluted habitats select for increased metabolic capacity in affected populations.

  6. 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 ...

  7. 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…

  8. 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...

  9. 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.

  10. 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

  11. 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

  12. Induction of benzo(a)pyrene metabolism by 2,3,7,8-tetrachlorodibenzo-p-dioxin in primary cultures of adult rat hepatocytes: regulation by vitamin A

    SciTech Connect

    Steward, A.R.

    1982-01-01

    In order to develop a cellular model for studying mechanisms of enzyme induction and the effects of this induction on xenobiotic metabolism and cytotoxicity, the induction of benzo(a)pyrene (BaP) metabolism by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was investigated in primary hepatocyte cultures prepared from adult male rats and maintained in chemically-defined medium containing hormones. A derepression of induction was observed during the first 3 days in culture. Addition of 0.8 to 2.0 ..mu..g/ml of retinol acetate (RA) prevented about half of the derepression of induction occurring between 36 and 60 h in culture. Horse serum (10%) also blocked up to half of the observed derepression. Serum, however, also led to a 40% reduction in the partitioning of (/sup 3/H)TCDD from the medium into the hepatocytes. The derepresion of MFO induction in primary adult hepatocyte cultures may occur partly as a result of a deficiency of retinol. RA is hypothesized to slow the time course of induction by reducing the rate of protein turnover. RA may also partially block the shift in the dose-response curve for induction by TCDD by maintaining the normal metabolic regulation of the cytosolic receptor for TCDD. Addition of a physiological level of RA to the culture medium may therefore help to maintain the hepatocytes at a level of genetic expression more nearly representative of the intact liver.

  13. The potential of apolipoprotein E4 to act as a substrate for primary cultures of hippocampal neurons.

    PubMed

    Kim, Kwang-Min; Vicenty, Janice; Palmore, G Tayhas R

    2013-04-01

    The E4 isoform of apolipoprotein (apoE4) is known to be a major risk factor for Alzheimer's Disease (AD). Previous in vitro studies have shown apoE4 to have a negative effect on neuronal outgrowth when incubated with lipids. The effect of apoE4 itself on the development of neurons from the central nervous system (CNS), however, has not been well characterized. Consequently, apoE4 alone has not been pursued as a substrate for neuronal cultures. In this study, the effect of surface-bound apoE4 on developmental features of rat hippocampal neurons was examined. We show that apoE4 substrates elicit significantly enhanced values in all developmental features at day 2 of culture when compared to laminin (LN) substrates, which is the current substrate-of-choice for neuronal cultures. Interestingly, the adhesion of hippocampal neurons was found to be significantly lower on LN substrates than on glass substrates, but the axon lengths on both substrates were similar. In addition, this study demonstrates that the adhesion- and growth-enhancing effects of apoE4 substrates are not mediated by heparan sulfate proteoglycans (HSPGs), proteins that have been indicated to function as receptors or co-receptors for apoE4. In the absence of lipids, apoE4 appears to use an unknown pathway for up-regulating neuronal adhesion and neurite outgrowth. Our results indicate that apoE4 is better than LN as a substrate for primary cultures of CNS neurons and should be considered in the design of tissue engineered CNS.

  14. 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

  15. Revealing Additional Dimensions of Globalisation and Cultural Hegemony: A Response to Roland S. Persson's Call for Cultural Sensitivity in Gifted Studies

    ERIC Educational Resources Information Center

    Ambrose, Don

    2012-01-01

    In this commentary, the author finds the interdisciplinary approach of Roland S. Persson's (2012a) target article refreshing. Persson's (2012a) additional emphases on ethnocentricity, cultural bias and strong threads of influence from the global economy also are helpful. They shed light on some strong contextual influences that shape the…

  16. 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

  17. 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.

  18. 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

  19. 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

  20. A fundamental study of the oxidation behavior of SI primary reference fuels with propionaldehyde and DTBP as an additive

    NASA Astrophysics Data System (ADS)

    Johnson, Rodney

    In an effort to combine the benefits of SI and CI engines, Homogeneous Charge Compression Ignition (HCCI) engines are being developed. HCCI combustion is achieved by controlling the temperature, pressure, and composition of the fuel and air mixture so that autoignition occurs in proper phasing with the piston motion. This control system is fundamentally more challenging than using a spark plug or fuel injector to determine ignition timing as in SI and CI engines, respectively. As a result, this is a technical barrier that must be overcome to make HCCI engines applicable to a wide range of vehicles and viable for high volume production. One way to tailor the autoignition timing is to use small amounts of ignition enhancing additives. In this study, the effect of the addition of DTBP and propionaldehyde on the autoignition behavior of SI primary reference fuels was investigated. The present work was conducted in a new research facility built around a single cylinder Cooperative Fuels Research (CFR) octane rating engine but modified to run in HCCI mode. It focused on the effect of select oxygenated hydrocarbons on hydrocarbon fuel oxidation, specifically, the primary reference fuels n-heptane and iso-octane. This work was conducted under HCCI operating conditions. Previously, the operating parameters for this engine were validated for stable combustion under a wide range of operating parameters such as engine speeds, equivalence ratios, compression ratios and inlet manifold temperature. The stable operating range under these conditions was recorded and used for the present study. The major focus of this study was to examine the effect of the addition of DTBP or propionaldehyde on the oxidation behavior of SI primary reference fuels. Under every test condition the addition of the additives DTBP and propionaldehyde caused a change in fuel oxidation. DTBP always promoted fuel oxidation while propionaldehyde promoted oxidation for lower octane number fuels and delayed

  1. 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.

  2. 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

  3. 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

  4. 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.

  5. Alaskan Native Games--A Cross-Cultural Addition to the Physical Education Curriculum.

    ERIC Educational Resources Information Center

    Frey, Richard D.; Allen, Mike

    1989-01-01

    Importing traditional, yet unknown, physical activities from different cultures is an exciting way to add creativity and imagination to the physical education curriculum. Explanations, accompanied by teaching hints, are given of several traditional Alaskan native games which have been successfully used with K-Six students in the Anchorage School…

  6. 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

  7. 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

  8. Ischemia/Reperfusion Injury of Primary Porcine Cardiomyocytes in a Low-Shear Microfluidic Culture and Analysis Device

    PubMed Central

    Khanal, Grishma; Chung, Kiyong; Solis-Wever, Ximena; Johnson, Bradley; Pappas, Dimitri

    2011-01-01

    Ischemia/reperfusion (I/R) injury was induced in primary porcine cardiomyocytes in a low-shear microfluidic culture chip. The chip was capable of sustaining cardiomyocyte culture and inducing I/R injury by subjecting the cells to periods of hypoxia lasting 3-4 hours followed by normoxia. Mitochondrial membrane potential was assayed using MitoTracker Red to follow mitochondrial depolarization, the earliest stage of apoptosis. Cell adhesion and morphology were also determined simultaneously with fluorescence measurements. Changes in membrane potential were observed earlier than previously reported, with mitochondria becoming depolarized as early as 2 hours into the ischemia period. The cells with depolarized mitochondria were deemed apoptotic. Out of 38-61 cells per time frame, the fraction of apoptotic cells was found to be similar to control samples (3%) at two hours of ischemia, which increased up to 22% at the end of ischemia period as compared to 0% in the control samples. Morphological analysis of cells showed that 4 hours of ischemia followed by reperfusion produced blebbing cells within 2 hours of restoring oxygen to the chip. This approach is a versatile method for cardiomyocyte stress, and in future work additional analytical probes can be incorporated for a multi-analyte assays of cardiomyocyte apoptosis. PMID:21271001

  9. Immuno-electron microscopy of primary cell cultures from genetically modified animals in liquid by atmospheric scanning electron microscopy.

    PubMed

    Kinoshita, Takaaki; Mori, Yosio; Hirano, Kazumi; Sugimoto, Shinya; Okuda, Ken-ichi; Matsumoto, Shunsuke; Namiki, Takeshi; Ebihara, Tatsuhiko; Kawata, Masaaki; Nishiyama, Hidetoshi; Sato, Mari; Suga, Mitsuo; Higashiyama, Kenichi; Sonomoto, Kenji; Mizunoe, Yoshimitsu; Nishihara, Shoko; Sato, Chikara

    2014-04-01

    High-throughput immuno-electron microscopy is required to capture the protein-protein interactions realizing physiological functions. Atmospheric scanning electron microscopy (ASEM) allows in situ correlative light and electron microscopy of samples in liquid in an open atmospheric environment. Cells are cultured in a few milliliters of medium directly in the ASEM dish, which can be coated and transferred to an incubator as required. Here, cells were imaged by optical or fluorescence microscopy, and at high resolution by gold-labeled immuno-ASEM, sometimes with additional metal staining. Axonal partitioning of neurons was correlated with specific cytoskeletal structures, including microtubules, using primary-culture neurons from wild type Drosophila, and the involvement of ankyrin in the formation of the intra-axonal segmentation boundary was studied using neurons from an ankyrin-deficient mutant. Rubella virus replication producing anti-double-stranded RNA was captured at the host cell's plasma membrane. Fas receptosome formation was associated with clathrin internalization near the surface of primitive endoderm cells. Positively charged Nanogold clearly revealed the cell outlines of primitive endoderm cells, and the cell division of lactic acid bacteria. Based on these experiments, ASEM promises to allow the study of protein interactions in various complexes in a natural environment of aqueous liquid in the near future. PMID:24564988

  10. Reversal of opiate-induced apoptosis by human recombinant growth hormone in murine foetus primary hippocampal neuronal cell cultures.

    PubMed

    Svensson, Anne-Lie; Bucht, Nora; Hallberg, Mathias; Nyberg, Fred

    2008-05-20

    Previous studies have shown that chronic opiates may inhibit cell growth and trigger apoptosis leading to impaired cognitive capabilities in both humans and other mammals. In contrast, growth hormone (GH) has been demonstrated to stimulate cell growth and counteract apoptosis. GH has also been shown to improve learning and memory in both human and rodents. In this work, we demonstrate that GH may reverse opiate-induced apoptosis in cells derived from prenatal mouse hippocampus. Primary hippocampal cell cultures derived from 16-day-old fetal mouse neurons were treated with morphine for 7 days during growth in the absence or presence of recombinant human GH (rhGH). The release of lactate dehydrogenase (LDH) into the culture media and the level of cleaved caspase-3 were measured. Results indicate that morphine (15 microM) decreased the cell content in a concentration-dependent manner and increased LDH release and caspase-3 activity. Thus, fetal mouse neurons treated with morphine showed less viability compared with controls. Interestingly, the addition of rhGH (1 microM) counteracted the morphine-induced effect on the cell density. Furthermore, the hormone attenuated the effects on LHD release and caspase-3 activity elicited by morphine. These results suggest that the hormone is capable of preventing or even repairing morphine-induced damage to hippocampal cells.

  11. ZnO nanoparticle-induced oxidative stress triggers apoptosis by activating JNK signaling pathway in cultured primary astrocytes

    NASA Astrophysics Data System (ADS)

    Wang, Jieting; Deng, Xiaobei; Zhang, Fang; Chen, Deliang; Ding, Wenjun

    2014-03-01

    It has been documented in in vitro studies that zinc oxide nanoparticles (ZnO NPs) are capable of inducing oxidative stress, which plays a crucial role in ZnO NP-mediated apoptosis. However, the underlying molecular mechanism of apoptosis in neurocytes induced by ZnO NP exposure was not fully elucidated. In this study, we investigated the potential mechanisms of apoptosis provoked by ZnO NPs in cultured primary astrocytes by exploring the molecular signaling pathways triggered after ZnO NP exposure. ZnO NP exposure was found to reduce cell viability in MTT assays, increase lactate dehydrogenase (LDH) release, stimulate intracellular reactive oxygen species (ROS) generation, and elicit caspase-3 activation in a dose- and time-dependent manner. Apoptosis occurred after ZnO NP exposure as evidenced by nuclear condensation and poly(ADP-ribose) polymerase-1 (PARP) cleavage. A decrease in mitochondrial membrane potential (MMP) with a concomitant increase in the expression of Bax/Bcl-2 ratio suggested that the mitochondria also mediated the pathway involved in ZnO NP-induced apoptosis. In addition, exposure of the cultured cells to ZnO NPs led to phosphorylation of c-Jun N-terminal kinase (JNK), extracellular signal-related kinase (ERK), and p38 mitogen-activated protein kinase (p38 MAPK). Moreover, JNK inhibitor (SP600125) significantly reduced ZnO NP-induced cleaved PARP and cleaved caspase-3 expression, but not ERK inhibitor (U0126) or p38 MAPK inhibitor (SB203580), indicating that JNK signaling pathway is involved in ZnO NP-induced apoptosis in primary astrocytes.

  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. Viability of 'Candidatus Liberibacter asiaticus' prolonged by addition of citrus juice to culture medium.

    PubMed

    Parker, Jennifer K; Wisotsky, Sarah R; Johnson, Evan G; Hijaz, Faraj M; Killiny, Nabil; Hilf, Mark E; De La Fuente, Leonardo

    2014-01-01

    Huanglongbing, or citrus greening disease, is associated with infection by the phloem-limited bacterium 'Candidatus Liberibacter asiaticus'. Infection with 'Ca. L. asiaticus' is incurable; therefore, knowledge regarding 'Ca. L. asiaticus' biology and pathogenesis is essential to develop a treatment. However, 'Ca. L. asiaticus' cannot currently be successfully cultured, limiting its study. To gain insight into the conditions conducive for growth of 'Ca. L. asiaticus' in vitro, 'Ca. L. asiaticus' inoculum obtained from seed of fruit from infected pomelo trees (Citrus maxima 'Mato Buntan') was added to different media, and cell viability was monitored for up to 2 months using quantitative polymerase chain reaction in conjunction with ethidium monoazide. Media tested included one-third King's B (K), K with 50% juice from the infected fruit, K with 50% commercially available grapefruit juice, and 100% commercially available grapefruit juice. Results show that juice-containing media dramatically prolong viability compared with K in experiments reproduced during 2 years using different juice sources. Furthermore, biofilm formed at the air-liquid interface of juice cultures contained 'Ca. L. asiaticus' cells, though next-generation sequencing indicated that other bacterial genera were predominant. Chemical characterization of the media was conducted to discuss possible factors sustaining 'Ca. L. asiaticus' viability in vitro, which will contribute to future development of a culture medium for 'Ca. L. asiaticus'. PMID:23883155

  19. Viability of 'Candidatus Liberibacter asiaticus' prolonged by addition of citrus juice to culture medium.

    PubMed

    Parker, Jennifer K; Wisotsky, Sarah R; Johnson, Evan G; Hijaz, Faraj M; Killiny, Nabil; Hilf, Mark E; De La Fuente, Leonardo

    2014-01-01

    Huanglongbing, or citrus greening disease, is associated with infection by the phloem-limited bacterium 'Candidatus Liberibacter asiaticus'. Infection with 'Ca. L. asiaticus' is incurable; therefore, knowledge regarding 'Ca. L. asiaticus' biology and pathogenesis is essential to develop a treatment. However, 'Ca. L. asiaticus' cannot currently be successfully cultured, limiting its study. To gain insight into the conditions conducive for growth of 'Ca. L. asiaticus' in vitro, 'Ca. L. asiaticus' inoculum obtained from seed of fruit from infected pomelo trees (Citrus maxima 'Mato Buntan') was added to different media, and cell viability was monitored for up to 2 months using quantitative polymerase chain reaction in conjunction with ethidium monoazide. Media tested included one-third King's B (K), K with 50% juice from the infected fruit, K with 50% commercially available grapefruit juice, and 100% commercially available grapefruit juice. Results show that juice-containing media dramatically prolong viability compared with K in experiments reproduced during 2 years using different juice sources. Furthermore, biofilm formed at the air-liquid interface of juice cultures contained 'Ca. L. asiaticus' cells, though next-generation sequencing indicated that other bacterial genera were predominant. Chemical characterization of the media was conducted to discuss possible factors sustaining 'Ca. L. asiaticus' viability in vitro, which will contribute to future development of a culture medium for 'Ca. L. asiaticus'.

  20. Exogenous N addition enhances the responses of gross primary productivity to individual precipitation events in a temperate grassland.

    PubMed

    Guo, Qun; Hu, Zhong-Min; Li, Sheng-Gong; Yu, Gui-Rui; Sun, Xiao-Min; Li, Ling-Hao; Liang, Nai-Shen; Bai, Wen-Ming

    2016-06-06

    Predicted future shifts in the magnitude and frequency (larger but fewer) of precipitation events and enhanced nitrogen (N) deposition may interact to affect grassland productivity, but the effects of N enrichment on the productivity response to individual precipitation events remain unclear. In this study, we quantified the effects of N addition on the response patterns of gross primary productivity (GPP) to individual precipitation events of different sizes (Psize) in a temperate grassland in China. The results showed that N enrichment significantly increased the time-integrated amount of GPP in response to an individual precipitation event (GPPtotal), and the N-induced stimulation of GPP increased with increasing Psize. N enrichment rarely affected the duration of the GPP response, but it significantly stimulated the maximum absolute GPP response. Higher foliar N content might play an important role in the N-induced stimulation of GPP. GPPtotal in both the N-addition and control treatments increased linearly with Psize with similar Psize intercepts (approximately 5 mm, indicating a similar lower Psize threshold to stimulate the GPP response) but had a steeper slope under N addition. Our work indicates that the projected larger precipitation events will stimulate grassland productivity, and this stimulation might be amplified by increasing N deposition.

  1. Exogenous N addition enhances the responses of gross primary productivity to individual precipitation events in a temperate grassland

    NASA Astrophysics Data System (ADS)

    Guo, Qun; Hu, Zhong-Min; Li, Sheng-Gong; Yu, Gui-Rui; Sun, Xiao-Min; Li, Ling-Hao; Liang, Nai-Shen; Bai, Wen-Ming

    2016-06-01

    Predicted future shifts in the magnitude and frequency (larger but fewer) of precipitation events and enhanced nitrogen (N) deposition may interact to affect grassland productivity, but the effects of N enrichment on the productivity response to individual precipitation events remain unclear. In this study, we quantified the effects of N addition on the response patterns of gross primary productivity (GPP) to individual precipitation events of different sizes (Psize) in a temperate grassland in China. The results showed that N enrichment significantly increased the time-integrated amount of GPP in response to an individual precipitation event (GPPtotal), and the N-induced stimulation of GPP increased with increasing Psize. N enrichment rarely affected the duration of the GPP response, but it significantly stimulated the maximum absolute GPP response. Higher foliar N content might play an important role in the N-induced stimulation of GPP. GPPtotal in both the N-addition and control treatments increased linearly with Psize with similar Psize intercepts (approximately 5 mm, indicating a similar lower Psize threshold to stimulate the GPP response) but had a steeper slope under N addition. Our work indicates that the projected larger precipitation events will stimulate grassland productivity, and this stimulation might be amplified by increasing N deposition.

  2. Exogenous N addition enhances the responses of gross primary productivity to individual precipitation events in a temperate grassland

    PubMed Central

    Guo, Qun; Hu, Zhong-min; Li, Sheng-gong; Yu, Gui-rui; Sun, Xiao-min; Li, Ling-hao; Liang, Nai-shen; Bai, Wen-ming

    2016-01-01

    Predicted future shifts in the magnitude and frequency (larger but fewer) of precipitation events and enhanced nitrogen (N) deposition may interact to affect grassland productivity, but the effects of N enrichment on the productivity response to individual precipitation events remain unclear. In this study, we quantified the effects of N addition on the response patterns of gross primary productivity (GPP) to individual precipitation events of different sizes (Psize) in a temperate grassland in China. The results showed that N enrichment significantly increased the time-integrated amount of GPP in response to an individual precipitation event (GPPtotal), and the N-induced stimulation of GPP increased with increasing Psize. N enrichment rarely affected the duration of the GPP response, but it significantly stimulated the maximum absolute GPP response. Higher foliar N content might play an important role in the N-induced stimulation of GPP. GPPtotal in both the N-addition and control treatments increased linearly with Psize with similar Psize intercepts (approximately 5 mm, indicating a similar lower Psize threshold to stimulate the GPP response) but had a steeper slope under N addition. Our work indicates that the projected larger precipitation events will stimulate grassland productivity, and this stimulation might be amplified by increasing N deposition. PMID:27264386

  3. Exogenous N addition enhances the responses of gross primary productivity to individual precipitation events in a temperate grassland.

    PubMed

    Guo, Qun; Hu, Zhong-Min; Li, Sheng-Gong; Yu, Gui-Rui; Sun, Xiao-Min; Li, Ling-Hao; Liang, Nai-Shen; Bai, Wen-Ming

    2016-01-01

    Predicted future shifts in the magnitude and frequency (larger but fewer) of precipitation events and enhanced nitrogen (N) deposition may interact to affect grassland productivity, but the effects of N enrichment on the productivity response to individual precipitation events remain unclear. In this study, we quantified the effects of N addition on the response patterns of gross primary productivity (GPP) to individual precipitation events of different sizes (Psize) in a temperate grassland in China. The results showed that N enrichment significantly increased the time-integrated amount of GPP in response to an individual precipitation event (GPPtotal), and the N-induced stimulation of GPP increased with increasing Psize. N enrichment rarely affected the duration of the GPP response, but it significantly stimulated the maximum absolute GPP response. Higher foliar N content might play an important role in the N-induced stimulation of GPP. GPPtotal in both the N-addition and control treatments increased linearly with Psize with similar Psize intercepts (approximately 5 mm, indicating a similar lower Psize threshold to stimulate the GPP response) but had a steeper slope under N addition. Our work indicates that the projected larger precipitation events will stimulate grassland productivity, and this stimulation might be amplified by increasing N deposition. PMID:27264386

  4. 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

  5. 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.

  6. 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

  7. 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

  8. 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

  9. [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

  10. 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

  11. 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

  12. 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

  13. 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.

  14. 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.

  15. 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

  16. 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

  17. 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.

  18. What Do We Learn from Spheroid Culture Systems? Insights from Tumorspheres Derived from Primary Colon Cancer Tissue

    PubMed Central

    Qureshi-Baig, Komal; Ullmann, Pit; Rodriguez, Fabien; Frasquilho, Sónia; Nazarov, Petr V.; Haan, Serge; Letellier, Elisabeth

    2016-01-01

    Due to their self-renewal and tumorigenic properties, tumor-initiating cells (TICs) have been hypothesized to be important targets for colorectal cancer (CRC). However the study of TICs is hampered by the fact that the identification and culturing of TICs is still a subject of extensive debate. Floating three-dimensional spheroid cultures (SC) that grow in serum-free medium supplemented with growth factors are supposed to be enriched in TICs. We generated SC from fresh clinical tumor specimens and compared them to SC isolated from CRC cell-lines as well as to adherent differentiated counterparts. Patient-derived SC display self-renewal capacity and can induce serial transplantable tumors in immuno-deficient mice, which phenotypically resemble the tumor of origin. In addition, the original tumor tissue and established SC retain several similar CRC-relevant mutations. Primary SC express key stemness proteins such as SOX2, OCT4, NANOG and LGR5 and importantly show increased chemoresistance ability compared to their adherent differentiated counterparts and to cell line-derived SC. Strikingly, cells derived from spheroid or adherent differentiating culture conditions displayed similar self-renewal capacity and equally formed tumors in immune-deficient mice, suggesting that self-renewal and tumor-initiation capacity of TICs is not restricted to phenotypically immature spheroid cells, which we describe to be highly plastic and able to reacquire stem-cell traits even after long differentiation processes. Finally, we identified two genes among a sphere gene expression signature that predict disease relapse in CRC patients. Here we propose that SC derived from fresh patient tumor tissue present interesting phenotypic features that may have clinical relevance for chemoresistance and disease relapse and therefore represent a valuable tool to test for new CRC-therapies that overcome drug resistance. PMID:26745821

  19. What Do We Learn from Spheroid Culture Systems? Insights from Tumorspheres Derived from Primary Colon Cancer Tissue.

    PubMed

    Qureshi-Baig, Komal; Ullmann, Pit; Rodriguez, Fabien; Frasquilho, Sónia; Nazarov, Petr V; Haan, Serge; Letellier, Elisabeth

    2016-01-01

    Due to their self-renewal and tumorigenic properties, tumor-initiating cells (TICs) have been hypothesized to be important targets for colorectal cancer (CRC). However the study of TICs is hampered by the fact that the identification and culturing of TICs is still a subject of extensive debate. Floating three-dimensional spheroid cultures (SC) that grow in serum-free medium supplemented with growth factors are supposed to be enriched in TICs. We generated SC from fresh clinical tumor specimens and compared them to SC isolated from CRC cell-lines as well as to adherent differentiated counterparts. Patient-derived SC display self-renewal capacity and can induce serial transplantable tumors in immuno-deficient mice, which phenotypically resemble the tumor of origin. In addition, the original tumor tissue and established SC retain several similar CRC-relevant mutations. Primary SC express key stemness proteins such as SOX2, OCT4, NANOG and LGR5 and importantly show increased chemoresistance ability compared to their adherent differentiated counterparts and to cell line-derived SC. Strikingly, cells derived from spheroid or adherent differentiating culture conditions displayed similar self-renewal capacity and equally formed tumors in immune-deficient mice, suggesting that self-renewal and tumor-initiation capacity of TICs is not restricted to phenotypically immature spheroid cells, which we describe to be highly plastic and able to reacquire stem-cell traits even after long differentiation processes. Finally, we identified two genes among a sphere gene expression signature that predict disease relapse in CRC patients. Here we propose that SC derived from fresh patient tumor tissue present interesting phenotypic features that may have clinical relevance for chemoresistance and disease relapse and therefore represent a valuable tool to test for new CRC-therapies that overcome drug resistance. PMID:26745821

  20. Induction of rat jejunal epithelial cell expression of sucrase-isomaltase by glucocorticoids in primary cell culture and in vivo.

    PubMed

    Yeh, K Y; Yeh, M; Holt, P R

    1989-01-01

    The cell cycle phase that mediates the induction of intestinal sucrase-isomaltase (SI) expression by glucocorticoids was investigated by measuring migration rates of 3H-DNA-labeled and of SI-containing epithelial cells by autoradiography and indirect immunofluorescent staining after simultaneous administration of [3H]thymidine and cortisone to 12-d-old rat pups. By 24 and 48 h, lead 3H-DNA-labeled cells had migrated 7.8 and 12.4 cell positions higher on the villus than lead cells expressing SI. Cell migration rates from 12 to 24 h and 24 to 48 h were 0.68 and 0.97 cell position/h. Thus, commitment to SI expression occurred in cells 11.5-12.8 h after the S phase, which is calculated to be in the G1 phase. To determine whether committed cells need to replicate to express SI, cell differentiation was examined in primary cultures of crypt cells originating from corticosterone-treated rats. About two-thirds of cultured cells were retarded in the S phase after plating, as judged by no increase of DNA labeling indices, no change in epithelial cell number, and the absence of mitosis (less than 0.01%). The proportion of cells expressing SI increased from 0 to 6-8% between 12 and 24 h, and reached 48% 48 h after plating on collagen-coated dishes. SI expression did not occur in cells plated on glass or plastic surfaces. Pulse labeling with [35S]methionine confirmed that de novo synthesis of SI occurred in cell cultures. Thus, additional cell cycling of committed cells occurring in vivo is not obligatory for the expression of SI. PMID:2736328

  1. 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

  2. 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.

  3. 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

  4. 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

  5. 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

  6. Skeletal Muscle Satellite Cells: Background and Methods for Isolation and Analysis in a Primary Culture System

    PubMed Central

    Danoviz, Maria Elena; Yablonka-Reuveni, Zipora

    2012-01-01

    Summary Repair of adult skeletal muscle depends on satellite cells, myogenic stem cells located between the basal lamina and the plasmalemma of the myofiber. Standardized protocols for the isolation and culture of satellite cells are key tools for understanding cell autonomous and extrinsic factors that regulate their performance. Knowledge gained from such studies can contribute important insights to developing strategies for the improvement of muscle repair following trauma and in muscle wasting disorders. This chapter provides an introduction to satellite cell biology and further describes the basic protocol used in our laboratory to isolate and culture satellite cells from adult skeletal muscle. The cell culture conditions detailed herein support proliferation and differentiation of satellite cell progeny and the development of reserve cells, which are thought to reflect the in vivo self-renewal ability of satellite cells. Additionally, this chapter describes our standard immunostaining protocol that allows the characterization of satellite cell progeny by the temporal expression of characteristic transcription factors and structural proteins associated with different stages of myogenic progression. While emphasis is given here to the isolation and characterization of satellite cells from mouse hindlimb muscles, the protocols are suitable for other muscle types (such as diaphragm and extraocular muscles) and for muscles from other species, including chicken and rat. Altogether, the basic protocols described are straightforward and facilitate the study of diverse aspects of skeletal muscle stem cells. PMID:22130829

  7. Concentrated fed-batch cell culture increases manufacturing capacity without additional volumetric capacity.

    PubMed

    Yang, William C; Minkler, Daniel F; Kshirsagar, Rashmi; Ryll, Thomas; Huang, Yao-Ming

    2016-01-10

    Biomanufacturing factories of the future are transitioning from large, single-product facilities toward smaller, multi-product, flexible facilities. Flexible capacity allows companies to adapt to ever-changing pipeline and market demands. Concentrated fed-batch (CFB) cell culture enables flexible manufacturing capacity with limited volumetric capacity; it intensifies cell culture titers such that the output of a smaller facility can rival that of a larger facility. We tested this hypothesis at bench scale by developing a feeding strategy for CFB and applying it to two cell lines. CFB improved cell line A output by 105% and cell line B output by 70% compared to traditional fed-batch (TFB) processes. CFB did not greatly change cell line A product quality, but it improved cell line B charge heterogeneity, suggesting that CFB has both process and product quality benefits. We projected CFB output gains in the context of a 2000-L small-scale facility, but the output was lower than that of a 15,000-L large-scale TFB facility. CFB's high cell mass also complicated operations, eroded volumetric productivity, and showed our current processes require significant improvements in specific productivity in order to realize their full potential and savings in manufacturing. Thus, improving specific productivity can resolve CFB's cost, scale-up, and operability challenges. PMID:26521697

  8. Concentrated fed-batch cell culture increases manufacturing capacity without additional volumetric capacity.

    PubMed

    Yang, William C; Minkler, Daniel F; Kshirsagar, Rashmi; Ryll, Thomas; Huang, Yao-Ming

    2016-01-10

    Biomanufacturing factories of the future are transitioning from large, single-product facilities toward smaller, multi-product, flexible facilities. Flexible capacity allows companies to adapt to ever-changing pipeline and market demands. Concentrated fed-batch (CFB) cell culture enables flexible manufacturing capacity with limited volumetric capacity; it intensifies cell culture titers such that the output of a smaller facility can rival that of a larger facility. We tested this hypothesis at bench scale by developing a feeding strategy for CFB and applying it to two cell lines. CFB improved cell line A output by 105% and cell line B output by 70% compared to traditional fed-batch (TFB) processes. CFB did not greatly change cell line A product quality, but it improved cell line B charge heterogeneity, suggesting that CFB has both process and product quality benefits. We projected CFB output gains in the context of a 2000-L small-scale facility, but the output was lower than that of a 15,000-L large-scale TFB facility. CFB's high cell mass also complicated operations, eroded volumetric productivity, and showed our current processes require significant improvements in specific productivity in order to realize their full potential and savings in manufacturing. Thus, improving specific productivity can resolve CFB's cost, scale-up, and operability challenges.

  9. Computations on the primary photoreaction of Br2 with CO2: stepwise vs concerted addition of Br atoms.

    PubMed

    Xu, Kewei; Korter, Timothy M; Braiman, Mark S

    2015-04-01

    It was proposed previously that Br2-sensitized photolysis of liquid CO2 proceeds through a metastable primary photoproduct, CO2Br2. Possible mechanisms for such a photoreaction are explored here computationally. First, it is shown that the CO2Br radical is not stable in any geometry. This rules out a free-radical mechanism, for example, photochemical splitting of Br2 followed by stepwise addition of Br atoms to CO2-which in turn accounts for the lack of previously observed Br2+CO2 photochemistry in gas phases. A possible alternative mechanism in liquid phase is formation of a weakly bound CO2:Br2 complex, followed by concerted photoaddition of Br2. This hypothesis is suggested by the previously published spectroscopic detection of a binary CO2:Br2 complex in the supersonically cooled gas phase. We compute a global binding-energy minimum of -6.2 kJ mol(-1) for such complexes, in a linear geometry. Two additional local minima were computed for perpendicular (C2v) and nearly parallel asymmetric planar geometries, both with binding energies near -5.4 kJ mol(-1). In these two latter geometries, C-Br and O-Br bond distances are simultaneously in the range of 3.5-3.8 Å, that is, perhaps suitable for a concerted photoaddition under the temperature and pressure conditions where Br2 + CO2 photochemistry has been observed.

  10. Addition of in-vitro generated endothelial microparticles to von-Willebrand plasma improves primary and secondary hemostasis.

    PubMed

    Trummer, Arne; Werwitzke, Sonja; Wermes, Cornelia; Ganser, Arnold; Birschmann, Ingvild; Budde, Ulrich; Tiede, Andreas

    2014-03-01

    Increased endothelial microparticles (EMP) as markers for endothelial activation have been associated with worse outcomes in clinical prothrombotic situations. The procoagulant properties of EMP can be attributed to the expression of phospholipids, tissue factor and von-Willebrand factor on their surface. We therefore investigated whether addition of in-vitro generated EMP modifies hemostasis in plasma from patients with severe von-Willebrand disease (VWD). A large EMP pool was obtained from stimulated endothelial cell lines and EMP concentration was quantified by flow cytometry. The influence of EMP on primary and secondary hemostasis in VWD plasma was assessed using ristocetin-induced platelet aggregation (RIPA) and thrombin generation in a calibrated automated thrombogram (CAT), respectively. After addition of EMP, there was a significant increase in the maximal aggregation level in RIPA as well as a significant shortening of lag time and time-to-peak in CAT in comparison to control buffer. In summary, in vitro-generated EMP have the potential to improve hemostasis in severe VWD plasma and these results warrant further clinical reseach regarding their contribution to the clinical bleeding phenotype as well as their potential to improve replacement therapy.

  11. The effect of lactic acid bacterial starter culture and chemical additives on wilted rice straw silage.

    PubMed

    Wang, Yan-Su; Shi, Wei; Huang, Lin-Ting; Ding, Cheng-Long; Dai, Chuan-Chao

    2016-04-01

    Lactic acid bacteria (LAB) are suitable for rice straw silage fermentation, but have been studied rarely, and rice straw as raw material for ensiling is difficult because of its disadvantages, such as low nutrition for microbial activities and low abundances of natural populations of LAB. So we investigated the effect of application of LAB and chemical additives on the fermentation quality and microbial community of wilted rice straw silage. Treatment with chemical additives increased the concentrations of crude protein (CP), water soluble carbohydrate (WSC), acetic acid and lactic acid, reduced the concentrations of acid detergent fiber (ADF) and neutral detergent fiber (NDF), but did not effectively inhibit the growth of spoilage organisms. Inoculation with LABs did not improve the nutritional value of the silage because of poor growth of LABs in wilted rice straw. Inoculation with LAB and addition of chemical materials improved the quality of silage similar to the effects of addition of chemical materials alone. Growth of aerobic and facultatively anaerobic bacteria was inhibited by this mixed treatment and the LAB gradually dominated the microbial community. In summary, the fermentation quality of wilted rice straw silage had improved by addition of LAB and chemical materials. PMID:26429595

  12. The effect of lactic acid bacterial starter culture and chemical additives on wilted rice straw silage.

    PubMed

    Wang, Yan-Su; Shi, Wei; Huang, Lin-Ting; Ding, Cheng-Long; Dai, Chuan-Chao

    2016-04-01

    Lactic acid bacteria (LAB) are suitable for rice straw silage fermentation, but have been studied rarely, and rice straw as raw material for ensiling is difficult because of its disadvantages, such as low nutrition for microbial activities and low abundances of natural populations of LAB. So we investigated the effect of application of LAB and chemical additives on the fermentation quality and microbial community of wilted rice straw silage. Treatment with chemical additives increased the concentrations of crude protein (CP), water soluble carbohydrate (WSC), acetic acid and lactic acid, reduced the concentrations of acid detergent fiber (ADF) and neutral detergent fiber (NDF), but did not effectively inhibit the growth of spoilage organisms. Inoculation with LABs did not improve the nutritional value of the silage because of poor growth of LABs in wilted rice straw. Inoculation with LAB and addition of chemical materials improved the quality of silage similar to the effects of addition of chemical materials alone. Growth of aerobic and facultatively anaerobic bacteria was inhibited by this mixed treatment and the LAB gradually dominated the microbial community. In summary, the fermentation quality of wilted rice straw silage had improved by addition of LAB and chemical materials.

  13. 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

  14. 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

  15. 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.

  16. 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

  17. 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

  18. 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

  19. 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

  20. "I Think They Just Think it's Going to Go Away at Some Stage": Policy and Practice in Teaching English as an Additional Language in Irish Primary Schools

    ERIC Educational Resources Information Center

    Wallen, Matthew; Kelly-Holmes, Helen

    2006-01-01

    Due to growth in immigration to the Republic of Ireland, the number of language minority students enrolling in primary schools has increased substantially over the last 10 years. The Irish context is a particularly interesting one in that until recently Ireland was a country of net emigration with limited experience of cultural diversity. An…

  1. 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

  2. Use of cell lines and primary cultures to explore the capacity of rainbow trout to be a host for frog virus 3 (FV3).

    PubMed

    Pham, P H; Huang, Y J; Mosser, D D; Bols, N C

    2015-10-01

    The capacity of rainbow trout, Oncorhynchus mykiss, to be a host for frog virus 3 (FV3) was evaluated at the cellular level. Cell cultures from this species were tested for their ability to express FV3 major capsid protein (MCP) gene, to develop cytopathic effect (CPE), and to produce FV3. After FV3 addition, MCP transcripts were detected in six of six cell lines and in primary macrophage cultures. CPE developed in all cell culture systems, except primary lymphocytes. For the macrophage cell line, RTS11, and primary macrophages, cell death was by apoptosis because DNA laddering and Annexin staining were detected. By contrast, markers of apoptosis did not accompany CPE in three epithelial cell lines from the gill (RTgill-W1), intestine (RTgut-GC), and liver (RTL-W1) and in two fibroblast cell lines from gonads (RTG-2) and skin (RTHDF). Therefore, FV3 was able to enter and begin replicating in several cell types. Yet, FV3 was produced in only two cell lines, RTG-2 and RTL-W1, and only modestly. Overall, these results suggest that if tissue accessibility were possible, FV3 would have the capacity to induce injury, but the ability to replicate would be limited, likely making rainbow trout a poor host for FV3.

  3. 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

  4. 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

  5. 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.

  6. 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.

  7. 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

  8. 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

  9. 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.

  10. 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

  11. 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

  12. 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

  13. Raising Standards for Pupils Who Have English as an Additional Language (EAL) through Monitoring and Evaluation of Provision in Primary Schools

    ERIC Educational Resources Information Center

    Mistry, Malini; Sood, Krishan

    2012-01-01

    The aim of the research is to further knowledge and understand how monitoring and evaluation of pupils who have English as an additional language (EAL) is undertaken in primary schools. This is a comparative study across primary schools using qualitative approaches to help gain insight into current good practice and identify future needs in EAL.…

  14. 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.

  15. Characterization and comparative study of coal combustion residues from a primary and additional flue gas secondary desulfurization process

    SciTech Connect

    Gomes, S.; Francois, M.; Evrard, O.; Pellissier, C.

    1998-11-01

    An extensive characterization and comparative study was done on two flue gas desulfurization (FGD) residues derived from the same coal. LR residues (originated from Loire/Rhone in the south of Lyon, France) are obtained after a primary desulfurization process (SO{sub 2} is trapped by reaction with CaO at a temperature of about 1100 C), and LM residues (originating from La Maxe, near Metz in the east of France) are obtained after an additional secondary desulfurization process (SO{sub 2} is removed further by reaction with Ca(OH){sub 2} at a temperature of about 120 C). Various and complementary investigation methods were used to determine their chemical, physical, and mineralogical properties: x-ray fluorescence and diffraction, scanning electron microscopy, differential scanning calorimetry, thermogravimetry analysis, granulometric distribution, pycnometric density, BET specific surface area and pH, conductivity measurements, and chemical analysis of their insoluble fraction. The FGD residues contain basically two main components: a silico-aluminous fly ash part and calcic FGD phases. In the LR residues the two components can be considered as independent, whereas they are linked in the LM residues because chemical reactions have occurred, leading to the formation of silico-calcic gel CSH, hydrated aluminate AFm, and AFt phases.

  16. 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.

  17. 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…

  18. 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…

  19. 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.

  20. 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...

  1. 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

  2. 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.

  3. 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.

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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.

  9. 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

  10. 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

  11. 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.

  12. 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

  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. Cultural psychology.

    PubMed

    Heine, Steven J; Ruby, Matthew B

    2010-03-01

    Humans are a cultural species, constantly navigating a complex web of culturally bound practices, norms, and worldviews. This article provides a brief overview of the relatively young field of cultural psychology, which investigates the many ways psychology and culture interweave with one another. Highlighting the cultural nature of the human species, it draws upon research on cultural evolution, enculturation, and developmental processes. This review further summarizes a number of cultural differences in how people perceive the self, and the behavioral consequences that follow from these differences, in the domains of internal and external attribution styles, motivations for self-enhancement, approach/avoidance, primary and secondary control, as well as motivations for distinctiveness and conformity. Additionally, the review discusses research on the intersection of culture and emotion, as well as cultural differences in cognition, perception, and reasoning. Copyright © 2010 John Wiley & Sons, Ltd. For further resources related to this article, please visit the WIREs website.

  18. Inhibition of release of inflammatory mediators in primary and cultured cells by a Chinese herbal medicine formula for allergic rhinitis

    PubMed Central

    Lenon, George B; Xue, Charlie CL; Story, David F; Thien, Frank CK; McPhee, Sarah; Li, Chun G

    2007-01-01

    Background We demonstrated that a Chinese herbal formula, which we refer to as RCM-101, developed from a traditional Chinese medicine formula, reduced nasal and non-nasal symptoms of seasonal allergic rhinitis (SAR). The present study in primary and cultured cells was undertaken to investigate the effects of RCM-101 on the production/release of inflammatory mediators known to be involved in SAR. Methods Compound 48/80-induced histamine release was studied in rat peritoneal mast cells. Production of leukotriene B4 induced by the calcium ionophore A23187 was studied in porcine neutrophils using an HPLC assay and lipopolysaccharide-stimulated prostaglandin E2 production was studied in murine macrophage (Raw 264.7) cells by immune-enzyme assay. Expression of cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) was determined in Raw 264.7 cells, using western blotting techniques. Results RCM-101 (1–100 μg/mL) produced concentration-dependent inhibition of compound 48/80-induced histamine release from rat peritoneal mast cells and of lipopolysaccharide-stimulated prostaglandin E2 release from Raw 264.7 cells. Over the range 1 – 10 μg/mL, it inhibited A23187-induced leukotriene B4 production in porcine neutrophils. In addition, RCM-101 (100 μg/mL) inhibited the expression of COX-2 protein but did not affect that of COX-1. Conclusion The findings indicate that RCM-101 inhibits the release and/or synthesis of histamine, leukotriene B4 and prostaglandin E2 in cultured cells. These interactions of RCM-101 with multiple inflammatory mediators are likely to be related to its ability to reduce symptoms of allergic rhinitis. PMID:17302969

  19. Measurement and characterization of micronuclei in cultured primary lung cells of mice following inhalation exposure to benzene.

    PubMed

    Ranaldi, R; Bassani, B; Villani, P; Lombardi, C C; Tanzarella, C; Pacchierotti, F

    1998-09-01

    The genotoxic effects of benzene in lung cells of mice exposed to single acute doses by inhalation have been estimated by cytogenetic analysis of micronuclei in primary cultures of lung fibroblasts. Mice were nose-only exposed to 1000 p.p.m. for 30 or 60 min or to 3500 p.p.m. for 30 min and sacrificed 24 h after the end of exposure. Lung fibroblasts were cultured attached to coverslips for 72 h, the last 48 h in the presence of 0.75 microgram/ml cytochalasin B. Micronuclei were scored in binucleate cells. The mechanism(s) of micronucleus induction was characterized by immunofluorescent staining of kinetochore proteins (CREST staining), which allowed micronuclei due to chromosome loss (kinetochore-positive) to be distinguished from those produced by chromosome breakage (kinetochore-negative). Three- and 4-fold statistically significant increases in total micronucleus frequencies were observed in all benzene-exposed mice with respect to unexposed controls. The effect was neither concentration nor time dependent. This is compatible with a plateau dose-effect relationship for the effects on bone marrow, which is explained by saturation of metabolism. Both chromosome loss and chromosome breakage appear to contribute to micronucleus formation, suggesting that in addition to chromosome rearrangements, aneuploidy may be a relevant early genotoxic event associated with benzene carcinogenicity. Under the same treatment conditions no micronucleus induction could be shown in spleen lymphocytes, suggesting that with very short benzene exposures cells at the first contact site with local metabolizing capacity have a higher probability of genetic alterations potentially leading to neoplasia.

  20. Neuroprotective effects of lotus seedpod procyanidins on extremely low frequency electromagnetic field-induced neurotoxicity in primary cultured hippocampal neurons.

    PubMed

    Yin, Chunchun; Luo, Xiaoping; Duan, Yuqing; Duan, Wenyi; Zhang, Haihui; He, Yuanqing; Sun, Guibo; Sun, Xiaobo

    2016-08-01

    The present study investigated the protective effects of lotus seedpod procyanidins (LSPCs) on extremely low frequency electromagnetic field (ELF-EMF)-induced neurotoxicity in primary cultured rat hippocampal neurons and the underlying molecular mechanism. The results of MTT, morphological observation, superoxide dismutase (SOD) and malondialdehyde (MDA) assays showed that compared with control, incubating neurons under ELF-EMF exposure significantly decreased cell viability and increased the number of apoptotic cells, whereas LSPCs evidently protected the hippocampal neurons against ELF-EMF-induced cell damage. Moreover, a certain concentration of LSPCs inhibited the elevation of intracellular reactive oxygen species (ROS) and Ca(2+) level, as well as prevented the disruption of mitochondrial membrane potential induced by ELF-EMF exposure. In addition, supplementation with LSPCs could alleviate DNA damage, block cell cycle arrest at S phase, and inhibit apoptosis and necrosis of hippocampal neurons under ELF-EMF exposure. Further study demonstrated that LSPCs up-regulated the activations of Bcl-2, Bcl-xl proteins and suppressed the expressions of Bad, Bax proteins caused by ELF-EMF exposure. In conclusion, these findings revealed that LSPCs protected against ELF-EMF-induced neurotoxicity through inhibiting oxidative stress and mitochondrial apoptotic pathway. PMID:27470406

  1. Mitochondria-targeted antioxidant mitotempo protects mitochondrial function against amyloid beta toxicity in primary cultured mouse neurons.

    PubMed

    Hu, Hongtao; Li, Mo

    2016-09-01

    Mitochondrial defects including excess reactive oxygen species (ROS) production and compromised ATP generation are featured pathology in Alzheimer's disease (AD). Amyloid beta (Aβ)-mediated mitochondrial ROS overproduction disrupts intra-neuronal Redox balance, in turn exacerbating mitochondrial dysfunction leading to neuronal injury. Previous studies have found the beneficial effects of mitochondria-targeted antioxidants in preventing mitochondrial dysfunction and neuronal injury in AD animal and cell models, suggesting that mitochondrial ROS scavengers hold promise for the treatment of this neurological disorder. In this study, we have determined that mitotempo, a novel mitochondria-targeted antioxidant protects mitochondrial function from the toxicity of Aβ in primary cultured neurons. Our results showed that Aβ-promoted mitochondrial superoxide production and neuronal lipid oxidation were significantly suppressed by the application of mitotempo. Moreover, mitotempo also demonstrated protective effects on mitochondrial bioenergetics evidenced by preserved mitochondrial membrane potential, cytochrome c oxidase activity as well as ATP production. In addition, the Aβ-induced mitochondrial DNA (mtDNA) depletion and decreased expression levels of mtDNA replication-related DNA polymerase gamma (DNA pol γ) and Twinkle were substantially mitigated by mitotempo. Therefore, our study suggests that elimination of excess mitochondrial ROS rescues mitochondrial function in Aβ-insulted neruons; and mitotempo has the potential to be a promising therapeutic agent to protect mitochondrial and neuronal function in AD.

  2. Ginsenoside Rd attenuates Aβ25-35-induced oxidative stress and apoptosis in primary cultured hippocampal neurons.

    PubMed

    Liu, Juan-fang; Yan, Xiao-dong; Qi, Lin-song; Li, Ling; Hu, Geng-yao; Li, Peng; Zhao, Gang

    2015-09-01

    One of the most common pathological changes in Alzheimer's disease (AD) brain is the large number of amyloid β (Aβ) peptides accumulating in lesion areas. Ginsenosides are the most active components extracted from ginseng. Ginsenoside Rd (GRd) is a newly discovered saponin that has a stronger pharmacological activity than other ginsenosides, especially in neuroprotection. Here we examined the neuroprotective effects of GRd against neuronal insults induced by Aβ25-35 in primary cultured hippocampal neurons. A 10μM GRd treatment significantly prevented the loss of hippocampal neurons induced by Aβ25-35. In addition, GRd significantly ameliorated Aβ25-35-induced oxidative stress by decreasing the reactive oxygen species (ROS) production and malondialdehyde (MDA) level, and increasing the levels of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px); which is similar in treatments with 10μM of probucol (PB) and 100μM of edaravone (EDA). Moreover, our present study demonstrated that GRd significantly enhanced the expression of Bcl-2 mRNA, and decreased the expressions of Bax mRNA and Cyt c mRNA. GRd also downregulated the protein level of cleaved Caspase-3 compared to controls. These results highlighted the neuroprotective effects of GRd against Aβ25-35-induced oxidative stress and neuronal apoptosis, suggesting that this may be a promising therapeutics against AD. PMID:26111763

  3. Mitochondria-targeted antioxidant mitotempo protects mitochondrial function against amyloid beta toxicity in primary cultured mouse neurons.

    PubMed

    Hu, Hongtao; Li, Mo

    2016-09-01

    Mitochondrial defects including excess reactive oxygen species (ROS) production and compromised ATP generation are featured pathology in Alzheimer's disease (AD). Amyloid beta (Aβ)-mediated mitochondrial ROS overproduction disrupts intra-neuronal Redox balance, in turn exacerbating mitochondrial dysfunction leading to neuronal injury. Previous studies have found the beneficial effects of mitochondria-targeted antioxidants in preventing mitochondrial dysfunction and neuronal injury in AD animal and cell models, suggesting that mitochondrial ROS scavengers hold promise for the treatment of this neurological disorder. In this study, we have determined that mitotempo, a novel mitochondria-targeted antioxidant protects mitochondrial function from the toxicity of Aβ in primary cultured neurons. Our results showed that Aβ-promoted mitochondrial superoxide production and neuronal lipid oxidation were significantly suppressed by the application of mitotempo. Moreover, mitotempo also demonstrated protective effects on mitochondrial bioenergetics evidenced by preserved mitochondrial membrane potential, cytochrome c oxidase activity as well as ATP production. In addition, the Aβ-induced mitochondrial DNA (mtDNA) depletion and decreased expression levels of mtDNA replication-related DNA polymerase gamma (DNA pol γ) and Twinkle were substantially mitigated by mitotempo. Therefore, our study suggests that elimination of excess mitochondrial ROS rescues mitochondrial function in Aβ-insulted neruons; and mitotempo has the potential to be a promising therapeutic agent to protect mitochondrial and neuronal function in AD. PMID:27444386

  4. 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.

  5. 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

  6. 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

  7. 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

  8. 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…

  9. 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…

  10. 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

  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. In vitro Cultured Primary Roots Derived from Stem Segments of Cassava (Manihot esculenta) Can Behave Like Storage Organs

    PubMed Central

    Medina, Ricardo D.; Faloci, Mirta M.; Gonzalez, Ana M.; Mroginski, Luis A.

    2007-01-01

    Background and Aims Cassava (Manihot esculenta) has three adventitious root types: primary and secondary fibrous roots, and storage roots. Different adventitious root types can also regenerate from in vitro cultured segments. The aim of this study was to investigate aspects of in vitro production of storage roots. Methods Morphological and anatomical analyses were performed to identify and differentiate each root type. Twenty-nine clones were assayed to determine the effect of genotype on the capacity to form storage roots in vitro. The effects of cytokinins and auxins on the formation of storage roots in vitro were also examined. Key Results Primary roots formed in vitro and in vivo had similar tissue kinds; however, storage roots formed in vitro exhibited physiological specialization for storing starch. The only consistent diagnostic feature between secondary fibrous and storage roots was their functional differentiation. Anatomical analysis of the storage roots formed in vitro showed that radial expansion as a consequence of massive proliferation and enlargement of parenchymatous cells occurred in the middle cortex, but not from cambial activity as in roots formed in vivo. Cortical expansion could be related to dilatation growth favoured by hormone treatments. Starch deposition of storage roots formed in vitro was confined to cortical tissue and occurred earlier than in storage roots formed in vivo. Auxin and cytokinin supplementation were absolutely required for in vitro storage root regeneration; these roots were not able to develop secondary growth, but formed a tissue competent for starch storing. MS medium with 5 % sucrose plus 0·54 μm 1-naphthaleneacetic acid and 0·44 μm 6-benzylaminopurine was one of the most effective in stimulating the storage root formation. Genotypes differed significantly in their capacity to produce storage roots in vitro. Storage root formation was considerably affected by the segment's primary position and strongly

  16. 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

  17. 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

  18. Effects of anodizing parameters and heat treatment on nanotopographical features, bioactivity, and cell culture response of additively manufactured porous titanium.

    PubMed

    Amin Yavari, S; Chai, Y C; Böttger, A J; Wauthle, R; Schrooten, J; Weinans, H; Zadpoor, A A

    2015-06-01

    Anodizing could be used for bio-functionalization of the surfaces of titanium alloys. In this study, we use anodizing for creating nanotubes on the surface of porous titanium alloy bone substitutes manufactured using selective laser melting. Different sets of anodizing parameters (voltage: 10 or 20V anodizing time: 30min to 3h) are used for anodizing porous titanium structures that were later heat treated at 500°C. The nanotopographical features are examined using electron microscopy while the bioactivity of anodized surfaces is measured using immersion tests in the simulated body fluid (SBF). Moreover, the effects of anodizing and heat treatment on the performance of one representative anodized porous titanium structures are evaluated using in vitro cell culture assays using human periosteum-derived cells (hPDCs). It has been shown that while anodizing with different anodizing parameters results in very different nanotopographical features, i.e. nanotubes in the range of 20 to 55nm, anodized surfaces have limited apatite-forming ability regardless of the applied anodizing parameters. The results of in vitro cell culture show that both anodizing, and thus generation of regular nanotopographical feature, and heat treatment improve the cell culture response of porous titanium. In particular, cell proliferation measured using metabolic activity and DNA content was improved for anodized and heat treated as well as for anodized but not heat-treated specimens. Heat treatment additionally improved the cell attachment of porous titanium surfaces and upregulated expression of osteogenic markers. Anodized but not heat-treated specimens showed some limited signs of upregulated expression of osteogenic markers. In conclusion, while varying the anodizing parameters creates different nanotube structure, it does not improve apatite-forming ability of porous titanium. However, both anodizing and heat treatment at 500°C improve the cell culture response of porous titanium.

  19. Effects of anodizing parameters and heat treatment on nanotopographical features, bioactivity, and cell culture response of additively manufactured porous titanium.

    PubMed

    Amin Yavari, S; Chai, Y C; Böttger, A J; Wauthle, R; Schrooten, J; Weinans, H; Zadpoor, A A

    2015-06-01

    Anodizing could be used for bio-functionalization of the surfaces of titanium alloys. In this study, we use anodizing for creating nanotubes on the surface of porous titanium alloy bone substitutes manufactured using selective laser melting. Different sets of anodizing parameters (voltage: 10 or 20V anodizing time: 30min to 3h) are used for anodizing porous titanium structures that were later heat treated at 500°C. The nanotopographical features are examined using electron microscopy while the bioactivity of anodized surfaces is measured using immersion tests in the simulated body fluid (SBF). Moreover, the effects of anodizing and heat treatment on the performance of one representative anodized porous titanium structures are evaluated using in vitro cell culture assays using human periosteum-derived cells (hPDCs). It has been shown that while anodizing with different anodizing parameters results in very different nanotopographical features, i.e. nanotubes in the range of 20 to 55nm, anodized surfaces have limited apatite-forming ability regardless of the applied anodizing parameters. The results of in vitro cell culture show that both anodizing, and thus generation of regular nanotopographical feature, and heat treatment improve the cell culture response of porous titanium. In particular, cell proliferation measured using metabolic activity and DNA content was improved for anodized and heat treated as well as for anodized but not heat-treated specimens. Heat treatment additionally improved the cell attachment of porous titanium surfaces and upregulated expression of osteogenic markers. Anodized but not heat-treated specimens showed some limited signs of upregulated expression of osteogenic markers. In conclusion, while varying the anodizing parameters creates different nanotube structure, it does not improve apatite-forming ability of porous titanium. However, both anodizing and heat treatment at 500°C improve the cell culture response of porous titanium. PMID

  20. Bacterial biodegradation of melamine-contaminated aged soil: influence of different pre-culture media or addition of activation material.

    PubMed

    Hatakeyama, Takashi; Takagi, Kazuhiro

    2016-08-01

    This study aimed to investigate the biodegrading potential of Arthrobacter sp. MCO, Arthrobacter sp. CSP, and Nocardioides sp. ATD6 in melamine-contaminated upland soil (melamine: approx. 10.5 mg/kg dry weight) after 30 days of incubation. The soil sample used in this study had undergone annual treatment of lime nitrogen, which included melamine; it was aged for more than 10 years in field. When R2A broth was used as the pre-culture medium, Arthrobacter sp. MCO could degrade 55 % of melamine after 30 days of incubation, but the other strains could hardly degrade melamine (approximately 25 %). The addition of trimethylglycine (betaine) in soil as an activation material enhanced the degradation rate of melamine by each strain; more than 50 % of melamine was degraded by all strains after 30 days of incubation. In particular, strain MCO could degrade 72 % of melamine. When the strains were pre-cultured in R2A broth containing melamine, the degradation rate of melamine in soil increased remarkably. The highest (72 %) melamine degradation rate was noted when strain MCO was used with betaine addition.

  1. Bacterial biodegradation of melamine-contaminated aged soil: influence of different pre-culture media or addition of activation material.

    PubMed

    Hatakeyama, Takashi; Takagi, Kazuhiro

    2016-08-01

    This study aimed to investigate the biodegrading potential of Arthrobacter sp. MCO, Arthrobacter sp. CSP, and Nocardioides sp. ATD6 in melamine-contaminated upland soil (melamine: approx. 10.5 mg/kg dry weight) after 30 days of incubation. The soil sample used in this study had undergone annual treatment of lime nitrogen, which included melamine; it was aged for more than 10 years in field. When R2A broth was used as the pre-culture medium, Arthrobacter sp. MCO could degrade 55 % of melamine after 30 days of incubation, but the other strains could hardly degrade melamine (approximately 25 %). The addition of trimethylglycine (betaine) in soil as an activation material enhanced the degradation rate of melamine by each strain; more than 50 % of melamine was degraded by all strains after 30 days of incubation. In particular, strain MCO could degrade 72 % of melamine. When the strains were pre-cultured in R2A broth containing melamine, the degradation rate of melamine in soil increased remarkably. The highest (72 %) melamine degradation rate was noted when strain MCO was used with betaine addition. PMID:27080407

  2. 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.

  3. 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.

  4. Plasminogen activator inhibitor-1 promotes synaptogenesis and protects against aβ(1-42)-induced neurotoxicity in primary cultured hippocampal neurons.

    PubMed

    Cho, Harim; Joo, Yuyoung; Kim, Seonghan; Woo, Ran-Sook; Lee, Sang Hyung; Kim, Hye-Sun

    2013-01-01

    Plasminogen activator inhibitor-1 (PAI-1) is a soluble factor that is released from astrocytes, the most abundant type of glial cell in the brain. PAI-1 was initially identified as inhibiting two types of plasminogen activators, that is, tissue-type plasminogen and urokinase activators that are known to lead to the proteolytic degradation of the extracellular matrix. Recently, PAI-1 was reported to mediate the neuroprotective activity of transforming growth factor-β1 against N-methyl-D-aspartate receptor-mediated excitotoxicity and to be involved in angiogenesis following ischemic stroke, independently of the effects via the inhibition of tissue-type plasminogen and urokinase-type plasminogen activators. In this study, we examined whether PAI-1 influences synaptogenesis and neurotoxicity induced by amyloid beta peptide(1-42) (Aß(1-42)) in rat primary hippocampal neurons. Using immunostaining, treatment with PAI-1 for 24 h was found to significantly upregulate synaptophysin, postsynaptic density-95, and the polysialylated form of neural cell adhesion molecule, compared to treatment with vehicle alone. In addition, PAI-1 has neuroprotective effects against Aβ(1-42)-induced cytotoxicity in rat primary cultured hippocampal neurons. Taken together, our results suggest that PAI-1 has therapeutic potential in Alzheimer's disease by promoting synaptogenesis and by demonstrating neuroprotective effects against Aβ(1-42)-oligomer-induced neurotoxicity in rat primary cultured hippocampal neurons.

  5. 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

  6. 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.

  7. 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

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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

  13. Selection and optimization of transfection enhancer additives for increased virus-like particle production in HEK293 suspension cell cultures.

    PubMed

    Cervera, Laura; Fuenmayor, Javier; González-Domínguez, Irene; Gutiérrez-Granados, Sonia; Segura, Maria Mercedes; Gòdia, Francesc

    2015-12-01

    The manufacturing of biopharmaceuticals in mammalian cells typically relies on the use of stable producer cell lines. However, in recent years, transient gene expression has emerged as a suitable technology for rapid production of biopharmaceuticals. Transient gene expression is particularly well suited for early developmental phases, where several potential therapeutic targets need to be produced and tested in vivo. As a relatively new bioprocessing modality, a number of opportunities exist for improving cell culture productivity upon transient transfection. For instance, several compounds have shown positive effects on transient gene expression. These transfection enhancers either facilitate entry of PEI/DNA transfection complexes into the cell or nucleus or increase levels of gene expression. In this work, the potential of combining transfection enhancers to increase Gag-based virus-like particle production levels upon transfection of suspension-growing HEK 293 cells is evaluated. Using Plackett-Burman design of experiments, it is first tested the effect of eight transfection enhancers: trichostatin A, valproic acid, sodium butyrate, dimethyl sulfoxide (DMSO), lithium acetate, caffeine, hydroxyurea, and nocodazole. An optimal combination of compounds exhibiting the highest effect on gene expression levels was subsequently identified using a surface response experimental design. The optimal consisted on the addition of 20 mM lithium acetate, 3.36 mM valproic acid, and 5.04 mM caffeine which increased VLP production levels 3.8-fold, while maintaining cell culture viability at 94%. PMID:26278533

  14. 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

  15. 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

  16. 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.

  17. 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

  18. 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. 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

  19. The effect of human milk on DNA synthesis of neonatal rat hepatocytes in primary culture.

    PubMed

    Kohno, Y; Shiraki, K; Mura, T

    1991-03-01

    We studied the effect of human milk on DNA synthesis of neonatal hepatocytes to elucidate the physiologic role of human milk in growth of the liver. Neonatal hepatocytes were isolated from 5-d-old rats and cultured in serum-free medium. Human milk stimulated DNA synthesis of these hepatocytes in a concentration-dependent manner. The stimulatory activity of 7.5% (vol/vol) human milk plus 0.1 mumol/L insulin was five times that of control and was almost the same as that of 20 micrograms/L human epidermal growth factor (hEGF) plus insulin. The effect of human milk was additive with treatment with hEGF and insulin. The milk associated with prolonged jaundice of infants was significantly more active than the milk that was not associated with jaundice, although the concentration of hEGF was not different between the two types of milk. The mitogenic activity of milk was heat-labile, inactivated by DTT and stable after treatment with trypsin. Three peaks of the activity were detected in milk by gel filtration and the fraction containing proteins of molecular weight between 36,000 and 76,000 showed the highest activity. Anti-hEGF antibody did not inhibit this activity completely. These results suggested the presence of mitogens other than hEGF or a more active form of hEGF in human milk. The milk associated with breast-milk jaundice exerts a different influence on cell growth and may affect maturation of the liver function related to bilirubin metabolism. The mitogenic activity of milk might be important for growth and development of the liver in infants.

  20. Arsenic Compromises Conducting Airway Epithelial Barrier Properties in Primary Mouse and Immortalized Human Cell Cultures

    PubMed Central

    Sherwood, Cara L.; Liguori, Andrew E.; Olsen, Colin E.; Lantz, R. Clark; Burgess, Jefferey L.; Boitano, Scott

    2013-01-01

    Arsenic is a lung toxicant that can lead to respiratory illness through inhalation and ingestion, although the most common exposure is through contaminated drinking water. Lung effects reported from arsenic exposure include lung cancer and obstructive lung disease, as well as reductions in lung function and immune response. As part of their role in innate immune function, airway epithelial cells provide a barrier that protects underlying tissue from inhaled particulates, pathogens, and toxicants frequently found in inspired air. We evaluated the effects of a five-day exposure to environmentally relevant levels of arsenic {<4μM [~300 μg/L (ppb)] as NaAsO2} on airway epithelial barrier function and structure. In a primary mouse tracheal epithelial (MTE) cell model we found that both micromolar (3.9 μM) and submicromolar (0.8 μM) arsenic concentrations reduced transepithelial resistance, a measure of barrier function. Immunofluorescent staining of arsenic-treated MTE cells showed altered patterns of localization of the transmembrane tight junction proteins claudin (Cl) Cl-1, Cl-4, Cl-7 and occludin at cell-cell contacts when compared with untreated controls. To better quantify arsenic-induced changes in tight junction transmembrane proteins we conducted arsenic exposure experiments with an immortalized human bronchial epithelial cell line (16HBE14o-). We found that arsenic exposure significantly increased the protein expression of Cl-4 and occludin as well as the mRNA levels of Cl-4 and Cl-7 in these cells. Additionally, arsenic exposure resulted in altered phosphorylation of occludin. In summary, exposure to environmentally relevant levels of arsenic can alter both the function and structure of airway epithelial barrier constituents. These changes likely contribute to the observed arsenic-induced loss in basic innate immune defense and increased infection in the airway. PMID:24349408

  1. Adaptation and cross-cultural validation of the United States Primary Care Assessment Tool (expanded version) for use in South Africa

    PubMed Central

    Sayed, Abdul-Rauf; le Grange, Cynthia; Bhagwan, Susheela; Manga, Nayna

    2015-01-01

    Background Measuring primary care is important for health sector reform. The Primary Care Assessment Tool (PCAT) measures performance of elements essential for cost-effective care. Following minor adaptations prior to use in Cape Town in 2011, a few findings indicated a need to improve the content and cross-cultural validity for wider use in South Africa (SA). Aim This study aimed to validate the United States of America-developed PCAT before being used in a baseline measure of primary care performance prior to major reform. Setting Public sector primary care clinics, users, practitioners and managers in urban and rural districts in the Western Cape Province. Methods Face value evaluation of item phrasing and a combination of Delphi and Nominal Group Technique (NGT) methods with an expert panel and user focus group were used to obtain consensus on content relevant to SA. Original and new domains and items with > = 70% agreement were included in the South African version – ZA PCAT. Results All original PCAT domains achieved consensus on inclusion. One new domain, the primary healthcare (PHC) team, was added. Three of 95 original items achieved < 70% agreement, that is consensus to exclude as not relevant to SA; 19 new items were added. A few items needed minor rephrasing with local healthcare jargon. The demographic section was adapted to local socio-economic conditions. The adult PCAT was translated into isiXhosa and Afrikaans. Conclusion The PCAT is a valid measure of primary care performance in SA. The PHC team domain is an important addition, given its emphasis in PHC re-engineering. A combination of Delphi and NGT methods succeeded in obtaining consensus on a multi-domain, multi-item instrument in a resource- constrained environment. PMID:26245610

  2. In vitro primary sensitization and restimulation of hapten-specific T cells by fresh and cultured human epidermal Langerhans' cells.

    PubMed Central

    Moulon, C; Péguet-Navarro, J; Courtellemont, P; Redziniak, G; Schmitt, D

    1993-01-01

    We examined the capacity of human Langerhans's cells (LC) to sensitize autologous T cells to the trinitrophenyl hapten (TNP) in vitro. Two-day cultured Langerhans' cells, but not freshly prepared Langerhans' cells, can induce in vitro primary proliferative reactions to the TNP hapten. Using a CD45RA+ naive T-cell subset, similar results were found, therefore making the possibility of a previous in vivo T-cell contact with the hapten unlikely. The primary in vitro response was strongly inhibited by monoclonal antibodies to major histocompatibility complex (MHC) class I and II, CD4 antigens and ICAM-1 and LFA-3 adhesion molecules. Furthermore, we found that fresh LC can prime T cells to TNP, as revealed by a significant secondary T-cell proliferation after restimulation of the recovered T lymphocytes by fresh hapten-modified autologous LC. Nevertheless, the ability of these fresh LC to stimulate in vitro secondary hapten-specific T-cell proliferation was very limited in comparison with that of 2-day incubated Langerhans' cells. After secondary stimulation with TNP-cultured LC, sensitized T cells could be non-specifically expanded without losing hapten specificity. The TNP-specific T-cell lines were mostly of the CD4+ phenotype. The present findings extend previous studies in the mouse, showing that culture LC are potent antigen-presenting cells (APC) in primary hapten-dependent proliferation assays. Furthermore, this in vitro priming assay, using cultured human Langerhans' cells as APC, might be useful to analyse the early steps of T-cell sensitization and subsequently to develop in vitro predictive tests allowing detection of sensitizing compounds. PMID:7507088

  3. Primary cultured cells as sensitive in vitro model for assessment of toxicants--comparison to hepatocytes and gill epithelia.

    PubMed

    Zhou, Bingsheng; Liu, Chunsheng; Wang, Jingxian; Lam, Paul K S; Wu, Rudolf S S

    2006-11-16

    In an effort to develop cultured cell models for toxicity screening and environmental biomonitoring, we compared primary cultured gill epithelia and hepatocytes from freshwater tilapia (Oreochromis niloticus) to assess their sensitivity to AhR agonist toxicants. Epithelia were cultured on permeable supports (terephthalate membranes, "filters") and bathed on the apical with waterborne toxicants (pseudo in vivo asymmetrical culture conditions). Hepatocytes were cultured in multi-well plates and exposed to toxicants in culture medium. Cytochrome P4501A (measured as 7-Ethoxyresorufin-O-deethylase, EROD) was selected as a biomarker. For cultured gill epithelia, the integrity of the epithelia remained unchanged on exposure to model toxicants, such as 1,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), benzo(a)pyrene B[a]P, polychlorinated biphenyl (PCB) mixture (Aroclor 1254), and polybrominated diphenyl ether (PBDE) mixture (DE71). A good concentration-dependent response of EROD activity was clearly observed in both cultured gill epithelia and hepatocytes. The time-course response of EROD was measured as early as 3h, and was maximal after 6h of exposure to TCDD, B[a]P and Aroclor 1254. The estimated 6h EC50 for TCDD, B[a]P, and Aroclor 1254 was 1.2 x 10(-9), 5.7 x 10(-8) and 6.6 x 10(-6)M. For the cultured hepatocytes, time-course study showed that a significant induction of EROD took place at 18 h, and the maximal induction of EROD was observed at 24h after exposure. The estimated 24h EC50 for TCDD, B[a]P, and Aroclor 1254 was 1.4 x 10(-9), 8.1 x 10(-8) and 7.3 x 10(-6)M. There was no induction or inhibition of EROD in DE71 exposure to both gill epithelia and hepatocytes. The results show that cultured gill epithelia more rapidly induce EROD and are slightly more sensitive than cultured hepatocytes, and could be used as a rapid and sensitive tool for screening chemicals and monitoring environmental AhR agonist toxicants.

  4. Effects of transforming growth factor-β2 on myocilin expression and secretion in human primary cultured trabecular meshwork cells.

    PubMed

    Wu, Yuyu; Chen, Wanzhu; Guo, Maosheng; He, Qin; Hu, Yan

    2014-01-01

    High intraocular pressure (IOP) is a risk factor for primary open-angle glaucoma (POAG). The trabecular meshwork (TM), a reticular tissue in the outflow passage of the aqueous humor (AH), is a major contributor to intraocular outflow resistance. High levels of myocilin (MYOC), which is expressed in the TM, are associated with high IOP. Furthermore, transforming growth factor-β2 (TGF-β2) concentrations in human AH are significantly elevated in POAG patients. This study was designed to investigate the effects of TGF-β2 on MYOC expression and secretion in human primary cultured TM cells. Primary cultured human TM cells were treated with 0 (control group), 1, 10, and 100 ng/mL TGF-β2 for 12, 24, or 48 h. MYOC mRNA and protein expressions in TM cells and protein secretion in conditioned media were analyzed by semi-quantitative RT-PCR, Western blotting, and enzyme-linked immunosorbent assays (ELISA), respectively. TM cells treated with 1, 10, and, 100 ng/mL TGF-β2 for 48 h showed higher MYOC mRNA and protein expressions than those in the control group (0 ng/mL TGF-β2) (all P < 0.05). Treatment with TGF-β2 for 48 h also induced MYOC secretion in conditioned media in a dose-dependent manner (0 ng/mL: 7.107±1.163 pg/ml; 1 ng/mL: 7.879±1.894 pg/ml; 10 ng/mL: 8.063±1.181 pg/ml; 100 ng/mL: 8.902±0.699 pg/ml; all P < 0.05). In Conclusion, TGF-β2 induced MYOC expression and secretion in human primary cultured TM cells. Further investigations are required to confirm the involvement of these two factors in POAG pathogenesis.

  5. Vasopressin inhibits type-I collagen and albumin gene expression in primary cultures of adult rat hepatocytes

    SciTech Connect

    Chojkier, M.; Brenner, D.A.; Leffert, H.L.

    1989-06-05

    The mechanisms that regulate collagen gene expression in hepatic cells are poorly understood. Accelerated Ca2+ fluxes are associated with inhibiting collagen synthesis selectively in human fibroblasts. In suspension cultures of isolated hepatocytes, the Ca2+ agonist vasopressin increases cytosolic levels of free Ca2+. However, whether vasopressin's interactions with plasma membrane V1 receptors attenuate hepatic collagen production is unknown. We investigated this problem by studying vasopressin's effects on collagen synthesis and Ca2+ efflux in long-term primary cultures of differentiated and proliferation-competent adult rat hepatocytes. Twelve-day-old quiescent cultures were exposed to test substances and labeled with (5-3H)proline. Determinations of radioactivity in collagenase-sensitive and collagenase-resistant proteins were used to calculate the relative levels of collagen production. Synthetic (8-arg)vasopressin stimulated 45Ca2+ efflux within 1 min and inhibited hepatocyte collagen production within 3 h by 50%; overall rates of protein synthesis were not affected significantly. In cultures labeled with (35S)methionine, vasopressin also decreased the levels of newly synthesized and secreted albumin, but not fibrinogen, detected in specific immunoprecipitates analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Northern blot analyses using specific (32P)cDNA probes revealed 70% decreases in hybridizable levels of collagen alpha 1(I) mRNA in hepatocyte cultures treated with either vasopressin or Ca2+ ionophore A23187; hybridizable levels of albumin mRNA also fell approximately 50% following vasopressin treatment.

  6. Surgical extraction of human dorsal root ganglia from organ donors and preparation of primary sensory neuron cultures.

    PubMed

    Valtcheva, Manouela V; Copits, Bryan A; Davidson, Steve; Sheahan, Tayler D; Pullen, Melanie Y; McCall, Jordan G; Dikranian, Krikor; Gereau, Robert W

    2016-10-01

    Primary cultures of rodent sensory neurons are widely used to investigate the cellular and molecular mechanisms involved in pain, itch, nerve injury and regeneration. However, translation of these preclinical findings may be greatly improved by direct validation in human tissues. We have developed an approach to extract and culture human sensory neurons in collaboration with a local organ procurement organization (OPO). Here we describe the surgical procedure for extraction of human dorsal root ganglia (hDRG) and the necessary modifications to existing culture techniques to prepare viable adult human sensory neurons for functional studies. Dissociated sensory neurons can be maintained in culture for >10 d, and they are amenable to electrophysiological recording, calcium imaging and viral gene transfer. The entire process of extraction and culturing can be completed in <7 h, and it can be performed by trained graduate students. This approach can be applied at any institution with access to organ donors consenting to tissue donation for research, and is an invaluable resource for improving translational research. PMID:27606776

  7. Surgical extraction of human dorsal root ganglia from organ donors and preparation of primary sensory neuron cultures.

    PubMed

    Valtcheva, Manouela V; Copits, Bryan A; Davidson, Steve; Sheahan, Tayler D; Pullen, Melanie Y; McCall, Jordan G; Dikranian, Krikor; Gereau, Robert W

    2016-10-01

    Primary cultures of rodent sensory neurons are widely used to investigate the cellular and molecular mechanisms involved in pain, itch, nerve injury and regeneration. However, translation of these preclinical findings may be greatly improved by direct validation in human tissues. We have developed an approach to extract and culture human sensory neurons in collaboration with a local organ procurement organization (OPO). Here we describe the surgical procedure for extraction of human dorsal root ganglia (hDRG) and the necessary modifications to existing culture techniques to prepare viable adult human sensory neurons for functional studies. Dissociated sensory neurons can be maintained in culture for >10 d, and they are amenable to electrophysiological recording, calcium imaging and viral gene transfer. The entire process of extraction and culturing can be completed in <7 h, and it can be performed by trained graduate students. This approach can be applied at any institution with access to organ donors consenting to tissue donation for research, and is an invaluable resource for improving translational research.

  8. Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach.

    PubMed

    Vergara, M Natalia; Gutierrez, Christian; Canto-Soler, M Valeria

    2015-01-01

    The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying the biology of retinal neurons, particularly photoreceptors. The applications of this system go beyond basic research, as they can easily be adapted to high throughput technologies for drug development. However, genetic manipulation of retinal photoreceptors in these cultures has proven to be very challenging, posing an important limitation to the usefulness of the system. We have recently developed and validated an ex ovo plasmid electroporation technique that increases the rate of transfection of retinal cells in these cultures by five-fold compared to other currently available protocols(1). In this method embryonic chick eyes are enucleated at stage 27, the RPE is removed, and the retinal cup is placed in a plasmid-containing solution and electroporated using easily constructed custom-made electrodes. The retinas are then dissociated and cultured using standard procedures. This technique can be applied to overexpression studies as well as to the downregulation of gene expression, for example via the use of plasmid-driven RNAi technology, commonly achieving transgene expression in 25% of the photoreceptor population. The video format of the present publication will make this technology easily accessible to researchers in the field, enabling the study of gene function in primary retinal cultures. We have also included detailed explanations of the critical steps of this procedure for a successful outcome and reproducibility. PMID:26556302

  9. Regulation of troponin C synthesis in primary culture of chicken cardiac muscle cells.

    PubMed

    Malhotra, S B; Bag, J

    1987-01-01

    Cardiac myocyte cell culture from fourteen day old embryonic chicken heart was prepared. This cultured cell system was used to examine the regulation of troponin C (TnC) synthesis in cardiac muscle. To examine the regulation of TnC polypeptide synthesis, cardiac myocyte cells were pulse labelled with 35S-methionine at different days after plating. The synthesis of TnC was measured by determining the amount of radioactivity incorporated into the TnC polypeptide following separation by two dimensional gel electrophoresis. These measurements showed that TnC synthesis was maximum in 36 to 48 h old cultures and reached its lowest level in 4 day old cultures. This was in contrast to the synthesis of actin and tropomyosin. Synthesis of these polypeptides were lowest in 36 to 48 h old cultures and was maximum in 7 day old cultures. To examine whether the synthesis of TnC polypeptide paralleled the levels of TnC mRNA the sequences homologous to quail slow TnC cDNA clone were measured by hybridisation. The results showed that the decrease in the synthesis of troponin C polypeptide cannot be fully explained by the decrease in the steady state level of troponin C mRNA. The possibility of a role of translational control of troponin C mRNA in this process is discussed. PMID:2890096

  10. Cultured C2C12 cell lines as a model for assessment of bacterial attachment to bovine primary muscle cells.

    PubMed

    Zulfakar, Siti Shahara; White, Jason D; Ross, Tom; Tamplin, Mark L

    2013-06-01

    The mechanisms of bacterial attachment to meat tissues need to be understood to enhance meat safety interventions. However, little is known about attachment of foodborne pathogens to meat muscle cells. In this study, attachment of six Escherichia coli and two Salmonella strains to primary bovine muscle cells and a cultured muscle cell line, C2C12, was measured, including the effect of temperature. At 37°C, all but one strain (EC623) attached to C2C12 cells, whereas only five of eight strains (M23Sr, H10407, EC473, Sal1729a and Sal691) attached to primary cells. At 10 °C, two strains (H10407 and EC473) attached to C2C12 cells, compared to four strains (M23Sr, EC614, H10407 and Sal1729a) of primary cells. Comparing all strains at both temperatures, EC614 displayed the highest CFU per C2C12 cell (4.60±2.02CFU/muscle cell at 37 °C), whereas greater numbers of M23Sr attached per primary cell (51.88±39.43CFU/muscle cell at 37 °C). This study indicates that primary bovine muscle cells may provide a more relevant model system to study bacterial attachment to beef carcasses compared to cell lines such as C2C12.

  11. Neuroprotective role of thymoquinone against 1-methyl-4-phenylpyridinium-induced dopaminergic cell death in primary mesencephalic cell culture

    PubMed Central

    Radad, Khaled S.; Al-Shraim, Mubarak M.; Moustafa, Mahmoud F.; Rausch, Wolf-Dieter

    2015-01-01

    Objectives: To investigate potential mechanisms mediating the neuroprotective effect of thymoquinone (TQ) on dopaminergic neurons. Methods: This study was conducted in the Chemistry and Biochemistry Institute, University of Veterinary Medicine, Vienna, Austria between June and August 2013. Primary cultures were prepared from embryonic mouse mesencephala (OFI/SPF) at gestation day 14. Four sets of cultures were kept untreated, treated with TQ on the eighth day in vitro (DIV) for 4 days, treated with 1-methyl-4-phenylpyridinium (MPP+) on the tenth DIV for 48 hours and co-treated with thymoquinone and MPP+. On the twelfth DIV, cultures were subjected to immunohistochemistry against tyrosine hydroxylase and fluorescent staining using LysoTracker® Deep Red, 5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethyl benzimidazolylcarbocyanine (JC-1) and 4’,6-diamidino-2-phenylindole stains. Results: The MPP+ decreased the number of dopaminergic neurons by 40%, and increased the release of lactate dehydrogenase (LDH) into the culture medium. The TQ significantly rescued dopaminergic neurons and decreased the release of LDH at the concentrations of 0.1 and 1 µM. The TQ significantly shifted the red fluorescent intensity of the LysoTracker® Deep Red, increased the mitochondrial membrane potential as it increased the red:green florescent ratio of JC-1, and decreased MPP+-induced apoptotic cell death. Conclusion: The TQ protects dopaminergic neurons in primary mesencephalic culture by enhancing lysosomal degradation that clears damaged mitochondria and inhibits mitochondria-mediated apoptotic cell death. PMID:25630775

  12. [Influence of different gelatin concentration and lymphocyte isolation liquid on primary culture of umbilical cord blood derived adhesive cells].

    PubMed

    Zhang, Cheng; Chen, Xing-Hua; Zhang, Xi; Gao, Lei; Kong, Pei-Yan; Liu, Hong; Liang, Xue; Peng, Xian-Gui; Wang, Qing-Yu

    2008-12-01

    In order to study the influence of different gelatin concentrations, and lymphocyte isolation liquid on primary culture of umbilical cord blood-derived adhesive cells (hCBACs), the red blood cells of umbilical cord blood was separated by 3% and 6 % gelatin for detecting the effectiveness of sedimentation, then the adhesion rate at 48 hours, the day of initial expansion and the rate of culture success were detected for hCBACs cultured with CD34(+) cells after the mononuclear cells were separated by 6% gelatin followed by Ficoll and Percoll, and the morphological characteristics and growth status were observed by invert microscopy. Cytochemistry stain for nonspecific esterase stain (NSE), peroxidase (POX), periodic acid Schiff reaction (PAS) and alkali phosphatase (ALP) and immunocytochemistry labeling for CD31, CD45, CD68 and fibronectin (Fn) were detected. The results showed that 6 % gelatin was better than that 3% gelatin for red blood sedimentation. The Percoll was predominant over Ficoll in adhesion rate at 48 hours, the day of initial expansion, the time of initial formation of adhesive cell colony units, the time of maximal numbers of adhesive cell colony units, the the cell fusion time and ratio of culture success. 60% fibroblast-liked cells, 36% macrophage liked cells and 4% small-round cells were observed in cells isolated by both isolated methods. The cytochemistry stain for NSE, POX, PAS and ALP was similar in two groups, the difference was not statistically significant between these two groups. The immunocytochemistry labeling for CD31, CD45, CD68 and Fn was also similar in both groups and the difference was also not statistically significant between these two groups. It is concluded that the combination of 6% gelatin with Percoll is an ideal separation method for primary culture of hCBACs, which provides basic information for clinical application.

  13. Addition of Anti-Angiogenetic Therapy with Bevacizumab to Chemo- and Radiotherapy for Leptomeningeal Metastases in Primary Brain Tumors

    PubMed Central

    Burger, Michael C.; Zeiner, Pia S.; Jahnke, Kolja; Wagner, Marlies; Mittelbronn, Michel; Steinbach, Joachim P.

    2016-01-01

    Leptomeningeal dissemination of a primary brain tumor is a condition which is challenging to treat, as it often occurs in rather late disease stages in highly pretreated patients. Its prognosis is dismal and there is still no accepted standard of care. We report here a good clinical effect with a partial response in three out of nine patients and a stable disease with improvement on symptoms in two more patients following systemic anti-angiogenic treatment with bevacizumab (BEV) alone or in combination with chemo- and/or radiotherapy in a series of patients with leptomeningeal dissemination from primary brain tumors (diffuse astrocytoma WHO°II, anaplastic astrocytoma WHO°III, anaplastic oligodendroglioma WHO°III, primitive neuroectodermal tumor and glioblastoma, both WHO°IV). This translated into effective symptom control in five out of nine patients, but only moderate progression-free and overall survival times were reached. Partial responses as assessed by RANO criteria were observed in three patients (each one with anaplastic oligodendroglioma, primitive neuroectodermal tumor and glioblastoma). In these patients progression-free survival (PFS) intervals of 17, 10 and 20 weeks were achieved. In three patients (each one with diffuse astrocytoma, anaplastic astrocytoma and primitive neuroectodermal tumor) stable disease was observed with PFS of 13, 30 and 8 weeks. Another three patients (all with glioblastoma) were primary non-responders and deteriorated rapidly with PFS of 3 to 4 weeks. No severe adverse events were seen. These experiences suggest that the combination of BEV with more conventional therapy schemes with chemo- and/or radiotherapy may be a palliative treatment option for patients with leptomeningeal dissemination of brain tumors. PMID:27253224

  14. Adverse events analysis as an educational tool to improve patient safety culture in primary care: A randomized trial

    PubMed Central

    2011-01-01

    Background Patient safety is a leading item on the policy agenda of both major international health organizations and advanced countries generally. The quantitative description of the phenomena has given rise to intense concern with the issue in institutions and organizations, leading to a number of initiatives and research projects and the promotion of patient safety culture, with training becoming a priority both in Spain and internationally. To date, most studies have been conducted in a hospital setting, even though primary care is the type most commonly used by the public, in our experience. Our study aims to achieve the following: - Assess the registry of adverse events as an education tool to improve patient safety culture in the Family and Community Teaching Units of Galicia. - Find and analyze educational tools to improve patient safety culture in primary care. - Evaluate the applicability of the Hospital Survey on Patient Safety Culture by the Agency for Healthcare Research and Quality, Spanish version, in the context of primary health care. Design and methods Design Experimental unifactorial study of two groups, control and intervention. Study population Tutors and residents in Family and Community Medicine in last year of studies in Galicia, Spain. Sample From the population universe through voluntary participation. Twenty-seven tutor-resident units in each group required, randomly assigned. Intervention Residents and their respective tutor (tutor-resident pair) in teaching units on Family and Community Medicine from throughout Galicia will be invited to participate. Tutor-resident pair that agrees to participate will be sent the Hospital Survey on Patient Safety Culture. Then, tutor-resident pair will be assigned to each group-either intervention or control-through simple random sampling. The intervention group will receive specific training to record the adverse effects found in patients under their care, with subsequent feedback, after receiving

  15. Müller glia activation by VEGF-antagonizing drugs: An in vitro study on rat primary retinal cultures.

    PubMed

    Gaddini, Lucia; Varano, Monica; Matteucci, Andrea; Mallozzi, Cinzia; Villa, Marika; Pricci, Flavia; Malchiodi-Albedi, Fiorella

    2016-04-01

    The effects of the anti-Vascular Endothelial Growth Factor (VEGF) drugs ranibizumab and aflibercept were studied in Müller glia in primary mixed cultures from rat neonatal retina. Treatment with both agents induced activation of Müller glia, demonstrated by increased levels of Glial Fibrillary Acidic Protein. In addition, phosphorylated Extracellular-Regulated Kinase 1/2 (ERK 1/2) showed enhanced immunoreactivity in activated Müller glia. Treatment with aflibercept induced an increase in K(+) channel (Kir) 4.1 levels and both drugs upregulated Aquaporin 4 (AQP4) in activated Müller glia. The results show that VEGF-antagonizing drugs influence the homeostasis of Müller cells in primary retinal cultures, inducing an activated phenotype. Upregulation of Kir4.1 and AQP4 suggests that Müller glia activation following anti-VEGF drugs may not depict a detrimental gliotic reaction. Indeed, it could represent one of the mechanisms able to contribute to the therapeutic effects of these drugs, particularly in the presence of macular edema. PMID:26607807

  16. Swelling-activated chloride and potassium conductance in primary cultures of mouse proximal tubules. Implication of KCNE1 protein.

    PubMed

    Barrière, H; Rubera, I; Belfodil, R; Tauc, M; Tonnerieux, N; Poujeol, C; Barhanin, J; Poujeol, P

    2003-06-01

    Volume-sensitive chloride and potassium currents were studied, using the whole-cell clamp technique, in cultured wild-type mouse proximal convoluted tubule (PCT) epithelial cells and compared with those measured in PCT cells from null mutant kcne1 -/- mice. In wild-type PCT cells in primary culture, a Cl- conductance activated by cell swelling was identified. The initial current exhibited an outwardly rectifying current-voltage (I-V) relationship, whereas steady-state current showed decay at depolarized membrane potentials. The ion selectivity was I- > Br- > Cl- > > gluconate. This conductance was sensitive to 1 mM 4,4'-Diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), 0.1 mM 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and 1 mM diphenylamine-2-carboxylate (DPC). Osmotic stress also activated K+ currents. These currents are time-independent, activated at depolarized potentials, and inhibited by 0.5 mM quinidine, 5 mM barium, and 10 microM clofilium but are insensitive to 1 mM tetraethylammonium (TEA), 10 nM charybdotoxin (CTX), and 10 microM 293B. In contrast, the null mutation of kcne1 completely impaired volume-sensitive chloride and potassium currents in PCT. The transitory transfection of kcne1 restores both Cl- and K+ swelling-activated currents, confirming the implication of KCNE1 protein in the cell-volume regulation in PCT cells in primary cultures. PMID:12962276

  17. High CD49f expression is associated with osteosarcoma tumor progression: a study using patient-derived primary cell cultures

    PubMed Central

    Penfornis, Patrice; Cai, David Z; Harris, Michael R; Walker, Ryan; Licini, David; Fernandes, Joseph D A; Orr, Griffin; Koganti, Tejaswi; Hicks, Chindo; Induru, Spandana; Meyer, Mark S; Khokha, Rama; Barr, Jennifer; Pochampally, Radhika R

    2014-01-01

    Overall prognosis for osteosarcoma (OS) is poor despite aggressive treatment options. Limited access to primary tumors, technical challenges in processing OS tissues, and the lack of well-characterized primary cell cultures has hindered our ability to fully understand the properties of OS tumor initiation and progression. In this study, we have isolated and characterized cell cultures derived from four central high-grade human OS samples. Furthermore, we used the cell cultures to study the role of CD49f in OS progression. Recent studies have implicated CD49f in stemness and multipotency of both cancer stem cells and mesenchymal stem cells. Therefore, we investigated the role of CD49f in osteosarcomagenesis. First, single cell suspensions of tumor biopsies were subcultured and characterized for cell surface marker expression. Next, we characterized the growth and differentiation properties, sensitivity to chemotherapy drugs, and anchorage-independent growth. Xenograft assays showed that cell populations expressing CD49fhi/CD90lo cell phenotype produced an aggressive tumor. Multiple lines of evidence demonstrated that inhibiting CD49f decreased the tumor-forming ability. Furthermore, the CD49fhi/CD90lo cell population is generating more aggressive OS tumor growth and indicating this cell surface marker could be a potential candidate for the isolation of an aggressive cell type in OSs. PMID:24802970

  18. High CD49f expression is associated with osteosarcoma tumor progression: a study using patient-derived primary cell cultures.

    PubMed

    Penfornis, Patrice; Cai, David Z; Harris, Michael R; Walker, Ryan; Licini, David; Fernandes, Joseph D A; Orr, Griffin; Koganti, Tejaswi; Hicks, Chindo; Induru, Spandana; Meyer, Mark S; Khokha, Rama; Barr, Jennifer; Pochampally, Radhika R

    2014-08-01

    Overall prognosis for osteosarcoma (OS) is poor despite aggressive treatment options. Limited access to primary tumors, technical challenges in processing OS tissues, and the lack of well-characterized primary cell cultures has hindered our ability to fully understand the properties of OS tumor initiation and progression. In this study, we have isolated and characterized cell cultures derived from four central high-grade human OS samples. Furthermore, we used the cell cultures to study the role of CD49f in OS progression. Recent studies have implicated CD49f in stemness and multipotency of both cancer stem cells and mesenchymal stem cells. Therefore, we investigated the role of CD49f in osteosarcomagenesis. First, single cell suspensions of tumor biopsies were subcultured and characterized for cell surface marker expression. Next, we characterized the growth and differentiation properties, sensitivity to chemotherapy drugs, and anchorage-independent growth. Xenograft assays showed that cell populations expressing CD49f(hi) /CD90(lo) cell phenotype produced an aggressive tumor. Multiple lines of evidence demonstrated that inhibiting CD49f decreased the tumor-forming ability. Furthermore, the CD49f(hi) /CD90(lo) cell population is generating more aggressive OS tumor growth and indicating this cell surface marker could be a potential candidate for the isolation of an aggressive cell type in OSs. PMID:24802970

  19. Regulation of proximal tubular cell differentiation and proliferation in primary culture by matrix stiffness and ECM components.

    PubMed

    Chen, Wan-Chun; Lin, Hsi-Hui; Tang, Ming-Jer

    2014-09-15

    To explore whether matrix stiffness affects cell differentiation, proliferation, and transforming growth factor (TGF)-β1-induced epithelial-mesenchymal transition (EMT) in primary cultures of mouse proximal tubular epithelial cells (mPTECs), we used a soft matrix made from monomeric collagen type I-coated polyacrylamide gel or matrigel (MG). Both kinds of soft matrix benefited primary mPTECs to retain tubular-like morphology with differentiation and growth arrest and to evade TGF-β1-induced EMT. However, the potent effect of MG on mPTEC differentiation was suppressed by glutaraldehyde-induced cross-linking and subsequently stiffening MG or by an increasing ratio of collagen in the soft mixed gel. Culture media supplemented with MG also helped mPTECs to retain tubular-like morphology and a differentiated phenotype on stiff culture dishes as soft MG did. We further found that the protein level and activity of ERK were scaled with the matrix stiffness. U-0126, a MEK inhibitor, abolished the stiff matrix-induced dedifferentiation and proliferation. These data suggest that the ERK signaling pathway plays a vital role in matrix stiffness-regulated cell growth and differentiation. Taken together, both compliant property and specific MG signals from the matrix are required for the regulation of epithelial differentiation and proliferation. This study provides a basic understanding of how physical and chemical cues derived from the extracellular matrix regulate the physiological function of proximal tubules and the pathological development of renal fibrosis.

  20. Acrolein, a product of lipid peroxidation, inhibits glucose and glutamate uptake in primary neuronal cultures.

    PubMed

    Lovell, M A; Xie, C; Markesbery, W R

    2000-10-15

    Oxidative stress has been implicated in the pathogenesis of several neurodegenerative disorders including Alzheimer's disease (AD). Increased lipid peroxidation, decreased levels of polyunsaturated fatty acids, and increased levels of 4-hydroxynonenal (HNE), F(2)-isoprostanes, and F(4)-neuroprostanes are present in the brain in patients with AD. Acrolein, an alpha,beta-unsaturated aldehydic product of lipid peroxidation has been demonstrated to be approximately 100 times more reactive than HNE and is present in neurofibrillary tangles in the brain in AD. We recently demonstrated statistically significant elevated concentrations of extractable acrolein in the hippocampus/parahippocampal gyrus and amygdala in AD compared with age-matched control subjects. Concentrations of acrolein were two to five times those of HNE in the same samples. Treatment of hippocampal cultures with acrolein led to a time- and concentration-dependent decrease in cell survival as well as a concentration-dependent increase in intracellular calcium. In cortical neuron cultures, we now report that acrolein causes a concentration-dependent impairment of glutamate uptake and glucose transport in cortical neuron cultures. Treatment of cortical astrocyte cultures with acrolein led to the same pattern of impairment of glutamate uptake as observed in cortical neuron cultures. Collectively, these data demonstrate neurotoxicity mechanisms of arolein that might be important in the pathogenesis of neuron degeneration in AD.

  1. A standardized and reproducible protocol for serum-free monolayer culturing of primary paediatric brain tumours to be utilized for therapeutic assays.

    PubMed

    Sandén, Emma; Eberstål, Sofia; Visse, Edward; Siesjö, Peter; Darabi, Anna

    2015-01-01

    In vitro cultured brain tumour cells are indispensable tools for drug screening and therapeutic development. Serum-free culture conditions tentatively preserve the features of the original tumour, but commonly comprise neurosphere propagation, which is a technically challenging procedure. Here, we define a simple, non-expensive and reproducible serum-free cell culture protocol for establishment and propagation of primary paediatric brain tumour cultures as adherent monolayers. The success rates for establishment of primary cultures (including medulloblastomas, atypical rhabdoid tumour, ependymomas and astrocytomas) were 65% (11/17) and 78% (14/18) for sphere cultures and monolayers respectively. Monolayer culturing was particularly feasible for less aggressive tumour subsets, where neurosphere cultures could not be generated. We show by immunofluorescent labelling that monolayers display phenotypic similarities with corresponding sphere cultures and primary tumours, and secrete clinically relevant inflammatory factors, including PGE2, VEGF, IL-6, IL-8 and IL-15. Moreover, secretion of PGE2 was considerably reduced by treatment with the COX-2 inhibitor Valdecoxib, demonstrating the functional utility of our newly established monolayer for preclinical therapeutic assays. Our findings suggest that this culture method could increase the availability and comparability of clinically representative in vitro models of paediatric brain tumours, and encourages further molecular evaluation of serum-free monolayer cultures.

  2. A standardized and reproducible protocol for serum-free monolayer culturing of primary paediatric brain tumours to be utilized for therapeutic assays

    PubMed Central

    Sandén, Emma; Eberstål, Sofia; Visse, Edward; Siesjö, Peter; Darabi, Anna

    2015-01-01

    In vitro cultured brain tumour cells are indispensable tools for drug screening and therapeutic development. Serum-free culture conditions tentatively preserve the features of the original tumour, but commonly comprise neurosphere propagation, which is a technically challenging procedure. Here, we define a simple, non-expensive and reproducible serum-free cell culture protocol for establishment and propagation of primary paediatric brain tumour cultures as adherent monolayers. The success rates for establishment of primary cultures (including medulloblastomas, atypical rhabdoid tumour, ependymomas and astrocytomas) were 65% (11/17) and 78% (14/18) for sphere cultures and monolayers respectively. Monolayer culturing was particularly feasible for less aggressive tumour subsets, where neurosphere cultures could not be generated. We show by immunofluorescent labelling that monolayers display phenotypic similarities with corresponding sphere cultures and primary tumours, and secrete clinically relevant inflammatory factors, including PGE2, VEGF, IL-6, IL-8 and IL-15. Moreover, secretion of PGE2 was considerably reduced by treatment with the COX-2 inhibitor Valdecoxib, demonstrating the functional utility of our newly established monolayer for preclinical therapeutic assays. Our findings suggest that this culture method could increase the availability and comparability of clinically representative in vitro models of paediatric brain tumours, and encourages further molecular evaluation of serum-free monolayer cultures. PMID:26183281

  3. Microfluidic co-culture platform to quantify chemotaxis of primary stem cells.

    PubMed

    Tatárová, Z; Abbuehl, J P; Maerkl, S; Huelsken, J

    2016-05-21

    Functional analysis of primary tissue-specific stem cells is hampered by their rarity. Here we describe a greatly miniaturized microfluidic device for the multiplexed, quantitative analysis of the chemotactic properties of primary, bone marrow-derived mesenchymal stem cells (MSC). The device was integrated within a fully customized platform that both increased the viability of stem cells ex vivo and simplified manipulation during multidimensional acquisition. Since primary stem cells can be isolated only in limited number, we optimized the design for efficient cell trapping from low volume and low concentration cell suspensions. Using nanoliter volumes and automated microfluidic controls for pulsed medium supply, our platform is able to create stable gradients of chemoattractant secreted from mammalian producer cells within the device, as was visualized by a secreted NeonGreen fluorescent reporter. The design was functionally validated by a CXCL/CXCR ligand/receptor combination resulting in preferential migration of primary, non-passaged MSC. Stable gradient formation prolonged assay duration and resulted in enhanced response rates for slowly migrating stem cells. Time-lapse video microscopy facilitated determining a number of migratory properties based on single cell analysis. Jackknife-resampling revealed that our assay requires only 120 cells to obtain statistically significant results, enabling new approaches in the research on rare primary stem cells. Compartmentalization of the device not only facilitated such quantitative measurements but will also permit future, high-throughput functional screens. PMID:27137768

  4. Patient views on primary care services for epilepsy and areas where additional professional knowledge would be welcome.

    PubMed

    Chappell, B; Smithson, W H

    1998-12-01

    In the past decade there has been increasing interest in the part that general practice can play in the care of people with epilepsy. Primary care services for epilepsy vary from practice to practice. Some studies have suggested that people with epilepsy prefer secondary care services and are not keen for their epilepsy to be managed in general practice, but much of the data were collected in secondary care. This study collected data from various sources about present provision of services, patient satisfaction with services, views about service development, areas where GP knowledge may be improved and whether the site of data collection influenced the results. A questionnaire was piloted, then distributed and collected through branches of the British Epilepsy Association, general practice and secondary care clinics. Data collected were both quantitative and qualitative. One hundred and seventy-eight questionnaires were collected from three sources. The responders were a severe seizure group. Structured care in general practice was uncommon with 54% being seen only when needed. Dose and type of antiepileptic medication was rarely altered in general practice. Information about their condition was given to 44% of the responders by their GP. Sixty-one percent would prefer their epilepsy care to be 'shared' between primary and secondary services. The majority of patients were satisfied with GP services, felt they could easily discuss their epilepsy, but 58% felt they 'rarely' or 'never' received enough information about their condition in general practice. Satisfaction with GP care varied, dependent on where the data were collected. Patients would value more information and more time to discuss the effects of their epilepsy. In conclusion general practice care for epilepsy is still reactive. Patients value more information and more time to discuss implications. The data collection point affects the results; any conclusions about the organisation of epilepsy care should

  5. Additive effects of ulinastatin and docetaxel on growth of breast cancer xenograft in nude mice and expression of PGE2, IL-10, and IL-2 in primary breast cancer cells.

    PubMed

    Zhong, Biao; Shen, Hongyan; Sun, Xin; Wang, Hong; Zhang, Yonghua; Sun, Zhijun

    2012-05-01

    Ulinastatin is a broad-spectrum enzyme inhibitor extracted from urine. Previous data from our group suggested that ulinastatin could significantly inhibit proliferation of human breast MDA-MB-231 cells, growth of tumor xenograft in nude mice, and expression of interleukin (IL)-6 and IL-8. In the present study, we investigated whether there is an additive effect of ulinastatin and docetaxel on growth of breast cancer xenografts in nude mice and its possible mechanisms. Nude mice and primary human breast cancer cells were treated with phosphate buffered saline (PBS), ulinastatin, docetaxel, or ulinastatin plus docetaxel, respectively. Their effects on xenograft growth; expressions of cyclooxygenase-2 (COX2), prostaglandin E2 receptor 2 (EP2), IL-10, and IL-2; and secretion of prostaglandin E2 (PGE2) were examined using variety of methods, including semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR), Western blot, enzyme-linked immunosorbent (ELISA) assay, and immunohistochemistry SP method. The treatment with ulinastatin, docetaxel, or ulinastatin plus docetaxel could significantly (1) inhibit COX2 and IL-10 expression in primary tumor cells at both mRNA and protein levels, (2) reduce PGE2 secretion in culture supernatant (p<0.05), (3) inhibit COX2, EP2, and IL-10 protein levels in primary xenograft of nude mice, and (4) increase IL-2 expression (p<0.05) in primary xenografts of nude mice. In addition, ulinastatin and docetaxel had additive effects. We suggest that ulinastatin had similar effects of docetaxel and can enhance docetaxel's anticancer effects possibly by inhibiting COX2 expression, reducing PGE2 and EP2 expression and their binding, upregulating IL-2, and downregulating IL-10.

  6. Switchable stereocontrolled divergent synthesis induced by aza-Michael addition of deactivated primary amines under acid catalysis.

    PubMed

    Amara, Z; Drège, E; Troufflard, C; Retailleau, P; Tran Huu-Dau, M-E; Joseph, D

    2014-11-24

    Switchable tandem intramolecular aza-Michael/Michael and double aza-Michael reactions allow the oriented synthesis of highly functionalised cyclic skeletons. Conjugate addition of deactivated anilines triggers chemo- and stereo-divergent ring-closure reaction pathways with a striking selectivity depending on reaction conditions.

  7. Epoxypukalide Induces Proliferation and Protects against Cytokine-Mediated Apoptosis in Primary Cultures of Pancreatic β-Cells

    PubMed Central

    López-Acosta, José Francisco; Moreno-Amador, José Luis; Jiménez-Palomares, Margarita; Díaz-Marrero, Ana R.; Cueto, Mercedes; Perdomo, Germán; Cózar-Castellano, Irene

    2013-01-01

    There is an urgency to find new treatments for the devastating epidemic of diabetes. Pancreatic β-cells viability and function are impaired in the two most common forms of diabetes, type 1 and type 2. Regeneration of pancreatic β-cells has been proposed as a potential therapy for diabetes. In a preliminary study, we screened a collection of marine products for β-cell proliferation. One unique compound (epoxypukalide) showed capability to induce β-cell replication in the cell line INS1 832/13 and in primary rat cell cultures. Epoxypukalide was used to study β-cell proliferation by [3H]thymidine incorporation and BrdU incorporation followed by BrdU/insulin staining in primary cultures of rat islets. AKT and ERK1/2 signalling pathways were analyzed. Cell cycle activators, cyclin D2 and cyclin E, were detected by western-blot. Apoptosis was studied by TUNEL and cleaved caspase 3. β-cell function was measured by glucose-stimulated insulin secretion. Epoxypukalide induced 2.5-fold increase in β-cell proliferation; this effect was mediated by activation of ERK1/2 signalling pathway and upregulation of the cell cycle activators, cyclin D2 and cyclin E. Interestingly, epoxypukalide showed protection from basal (40% lower versus control) and cytokine-induced apoptosis (80% lower versus control). Finally, epoxypukalide did not impair β-cell function when measured by glucose-stimulated insulin secretion. In conclusion, epoxypukalide induces β-cell proliferation and protects against basal and cytokine-mediated β-cell death in primary cultures of rat islets. These findings may be translated into new treatments for diabetes. PMID:23300997

  8. Secretion of bioactive interleukin-6 and tumor necrosis factor-alpha proteins from primary cultured human fetal membrane chorion cells infected with influenza virus.

    PubMed

    Uchide, N; Suzuki, A; Ohyama, K; Bessho, T; Toyoda, H

    2006-01-01

    Influenza virus infection during pregnancy is implicated in one of the causes of premature delivery, abortion and stillbirth. Pro-inflammatory cytokines, such as interleukin (IL)-6 and tumor necrosis factor (TNF)-alpha produced by fetal membranes, are postulated to facilitate premature delivery. We investigated the secretion of IL-6 and TNF-alpha from primary cultured human fetal membrane chorion and amnion cells infected with influenza virus at protein and bioactivity levels in order to understand the pathology of premature delivery during influenza virus infection. Concentrations of IL-6 and TNF-alpha proteins were significantly increased in culture supernatants of chorion cells by influenza virus infection. Culture supernatants of the virus-infected chorion cells stimulated the proliferation of IL-6-sensitive 7-TD-1 cells and induced the cytolysis of TNF-alpha-sensitive L929 cells, both activities of which were inhibited by the addition of respective antibody, whereas no such phenomena were observed in amnion cells. The results demonstrated that only chorion cells secreted significant amounts of bioactive IL-6 and TNF-alpha proteins responding to influenza virus infection. The present study suggests a possibility that the secretion of bioactive IL-6 and TNF-alpha proteins from fetal membrane chorion cells is implicated in the pathogenesis of premature delivery during influenza virus infection. PMID:16122792

  9. Use of primary cell cultures to measure the late effects in the skins of rhesus monkeys irradiated with protons

    NASA Astrophysics Data System (ADS)

    Cox, A. B.; Wood, D. H.; Lett, J. T.

    Previous pilot investigations of the uses of primary cell cultures to study late damage in stem cells of the skin of the New Zealand white (NZW) rabbit and the rhesus monkey /1-3/, have been extended to individual monkeys exposed to 55 MeV protons. Protons of this energy have a larger range in tissue of (~2.6 cm) than the 32 MeV protons (~0.9 cm) to which the animals in our earlier studies had been exposed. Although the primary emphases in the current studies were improvement and simplification in the techniques and logistics of transportation of biopsies to a central analytical facility, comparison of the quantitative measurements obtained thus far for survival of stem cells in the skins from animals irradiated 21 years ago reveals that the effects of both proton energies are similar.

  10. The Effect of Mushroom Beta-Glucans from Solid Culture of Ganoderma lucidum on Inhibition of the Primary Tumor Metastasis

    PubMed Central

    Hung, Ming-Hsin; Chen, Sherwin; Wang, William; Lu, Chung-Lun

    2014-01-01

    This study evaluates the effect of mushroom beta-glucans (MBGS) derived from solid culture of Ganoderma lucidum on tumor inhibition by examining size of the primary tumor and rate of metastasis in Lewis lung carcinoma (LLC) bearing mice (C57BL/6), given oral administration of MBGS with radiation therapy. A previous result showed that MBGS enhances NK cell-mediated cytotoxicity in mice without LLC bearing in advance. Furthermore, applications of MBGS in conjunction with radiation therapy were effective in controlling tumor growth, and rate of metastasis, life threatening, and can potentially serve as a protective factor for wounds and hair loss that resulted from the overgrowth of primary tumor in LLC bearing mice. PMID:24799937

  11. Flavonoids Modulate the Proliferation of Neospora caninum in Glial Cell Primary Cultures

    PubMed Central

    Barbosa de Matos, Rosan; Braga-de-Souza, Suzana; Pena Seara Pitanga, Bruno; Amaral da Silva, Victor Diógenes; Viana de Jesus, Erica Etelvina; Morales Pinheiro, Alexandre; Dias Costa, Maria de Fátima; dos Santos El-Bacha, Ramon; de Oliveira Ribeiro, Cátia Suse

    2014-01-01

    Neospora caninum (Apicomplexa; Sarcocystidae) is a protozoan that causes abortion in cattle, horses, sheep, and dogs as well as neurological and dermatological diseases in dogs. In the central nervous system of dogs infected with N. caninum, cysts were detected that exhibited gliosis and meningitis. Flavonoids are polyphenolic compounds that exhibit antibacterial, antiparasitic, antifungal, and antiviral properties. In this study, we investigated the effects of flavonoids in a well-established in vitro model of N. caninum infection in glial cell cultures. Glial cells were treated individually with 10 different flavonoids, and a subset of cultures was also infected with the NC-1 strain of N. caninum. All of the flavonoids tested induced an increase in the metabolism of glial cells and many of them increased nitrite levels in cultures infected with NC-1 compared to controls and uninfected cultures. Among the flavonoids tested, 3',4'-dihydroxyflavone, 3',4',5,7-tetrahydroxyflavone (luteolin), and 3,3',4',5,6-pentahydroxyflavone (quercetin), also inhibited parasitophorous vacuole formation. Taken together, our findings show that flavonoids modulate glial cell responses, increase NO secretion, and interfere with N. caninum infection and proliferation. PMID:25548412

  12. The structural development of primary cultured hippocampal neurons on a graphene substrate.

    PubMed

    He, Zuhong; Zhang, Shasha; Song, Qin; Li, Wenyan; Liu, Dong; Li, Huawei; Tang, Mingliang; Chai, Renjie

    2016-10-01

    The potential of graphene-based nanomaterials as a neural interfacing material for neural repair and regeneration remains poorly understood. In the present study, the response to the graphene substrate by neurons was determined in a hippocampal culture model. The results revealed the growth and maturation of hippocampal cultures on graphene substrates were significantly improved compared to the commercial control. In details, graphene promoted growth cone growth and microtubule formation inside filopodia 24h after seeding as evidenced by a higher average number of filopodia emerging from growth cones, a longer average length of filopodia, and a larger growth cone area. Graphene also significantly boosted neurite sprouting and outgrowth. The dendritic length, the number of branch points, and the dendritic complex index were significantly improved on the graphene substrate during culture. Moreover, the spine density was enhanced and the maturation of dendritic spines from thin to stubby spines was significantly promoted on graphene at 21 days after seeding. Lastly, graphene significantly elevated the synapse density and synaptic activity in the hippocampal cultures. The present study highlights graphene's potential as a neural interfacing material for neural repair and regeneration and sheds light on the future biomedical applications of graphene-based nanomaterials. PMID:27395037

  13. A Conceptual Framework for the Cultural Integration of Cooperative Learning: A Thai Primary Mathematics Education Perspective

    ERIC Educational Resources Information Center

    Park, Ji Yong; Nuntrakune, Tippawan

    2013-01-01

    The Thailand education reform adopted cooperative learning to improve the quality of education. However, it has been reported that the introduction and maintenance of cooperative learning has been difficult and uncertain because of the cultural differences. The study proposed a conceptual framework developed based on making a connection between…

  14. Flow cytometric cell cycle analysis of somatic cells primary cultures established for bovine cloning.

    PubMed

    Katska, L; Bochenek, M; Kania, G; Ryñska, B; Smorag, Z

    2002-12-01

    An important factor governing developmental rates of somatic cloned embryos is the phase of the cell cycle of donor nuclei. The aim of this experiment was to investigate the distribution of cell cycle phases in bovine cumulus and fibroblast cells cultured using routine treatment, and under cell cycle-arresting treatments. The highest percentages of cumulus cells in the G0 + G1 stage were observed in uncultured, frozen/thawed cells originating from immature oocytes (79.8 +/- 2.2%), fresh and frozen/thawed cells from in vitro matured oocytes (84.1 +/- 6.2 and 77.8 +/- 5.7%, respectively), and in cycling cells (72.7 +/- 16.3 and 78.4 +/- 11.2%, respectively for cumulus cells from immature and in vitro matured oocytes). Serum starvation of cumulus cultures markedly decreased percentages of cells in G0 + G1, and prolonged starvation significantly increased (P < 0.05) percentages of cells in G2 + M phase. Culture of cumulus cells to confluency did not increase percentages of cells in G0 + G1. Contrary to findings in cumulus cells, significantly higher percentages of cells in G0 + G1 were apparent when fibroblast cells were cultured to confluency or serum starved, and significantly increased (P < 0.01) as the starvation period was prolonged. It is concluded that for particular cell types specific strategies should be used to attain improvements in the efficiency of cloning procedures.

  15. Modulation of protein synthesis and secretion by substratum in primary cultures of rat hepatocytes

    SciTech Connect

    Sudhakaran, P.R.; Stamatoglou, S.C.; Hughes, R.C.

    1986-12-01

    Hepatocytes isolated by perfusion of adult rat liver and cultured on substrata consisting of one or more of the major components of the liver biomatrix (fibronectin, laminin, type IV collagen) have been examined for the synthesis of defined proteins. Under these conditions, tyrosine amino transferase, a marker of hepatocyte function, is maintained at similar levels in response to dexamethasone over 5 days in culture on each substratum, and total cellular protein synthesis remains constant. By contrast, there is a rapid decrease in synthesis and secretion of albumin and a 3-7-fold increase in synthesis and section of ..cap alpha..-fetoprotein which are most marked on a laminin substratum, but least evident on type IV collagen, and an increased synthesis of fibronectin and type IV collagen. The newly synthesized matrix proteins are present in the cell layer as well as in cell secretions. The enhanced synthesis of fibronectin is less in cells seeded onto a fibronectin substratum than on laminin or type IV collagen substrata. These results indicate that hepatocytes cultured in serum-free medium on substrata composed of components of the liver biomatrix maintain certain functions of the differentiated state (tyrosine amino transferase), lose others (albumin secretion) and switch to increased synthesis of matrix components as well as fetal markers such as ..cap alpha..-fetoprotein. The magnitude of these effects depends on the substratum on which the hepatocytes are cultured.

  16. ACCUMULATION OF PBDE-47 IN PRIMARY CULTURES OF RAT NEOCORTICAL CELLS.

    EPA Science Inventory

    A number of recent studies have examined the neurotoxic actions of PBDEs using in vitro cell culture models. However, there is little data reporting the final concentration of PBDEs in cells after in vitro exposure to these compounds. To address this issue, the present study exam...

  17. Differential Recognition of Children's Cultural Practices in Middle Primary Literacy Classrooms

    ERIC Educational Resources Information Center

    Nixon, Helen; Comber, Barbara

    2006-01-01

    This paper argues that teachers' recognition of children's cultural practices is an important positive step in helping socio-economically disadvantaged children engage with school literacies. Based on 21 longitudinal case studies of children's literacy development over a 3-year period, the authors demonstrate that when children's knowledges and…

  18. Primary-Grade Students' Knowledge and Thinking about Communication 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. Little information exists about children's prior knowledge and thinking (including misconceptions) about these topics. This study was designed to provide such information with respect to the topic of…

  19. The Evaluation of the Culturally Different: Pre-School, Primary and Elementary Age Children.

    ERIC Educational Resources Information Center

    Jacobs, Stanley S.

    This report asserts that the evaluation of young children can be viewed as a classic case of evaluation of the culturally different. Emphasized is the fact that not only are the majority of tests developed with an adult's perspective concerning adequacy of directions, items, and formats, but also the evaluation of the products is carried out…

  20. Introduction: Primary-School Texts and Teaching Methods in the Wake of the Cultural Revolution

    ERIC Educational Resources Information Center

    Chinese Education, 1977

    1977-01-01

    Chinese elementary school primers of the post-cultural revolution period emphasize moral principles, military heroism, revolutionary ardor, and sacrifice of individual interest for the good of society. Considerable emphasis continues to be placed upon rote learning, tests, homework, and report cards, although apparently less than in pre-cultural…

  1. Review Symposium on "Culture and Pedagogy: International Comparisons in Primary Education," by Robin Alexander.

    ERIC Educational Resources Information Center

    Broadfoot, Patricia; Little, Angela W.

    2003-01-01

    Two reviews of an ambitious comparative study of elementary classroom practices and realities in the United States (Michigan), England, France, Russia, and India. There is no single definition of best practice in education, for both means and ends are culturally specific. Recent English educational policy emphasizing a generic definition and…

  2. A primary chicken tracheal cell culture system for the study of infection with avian respiratory viruses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A major route of infection of avian influenza virus (AIV) and Newcastle disease virus (NDV) in chickens is through cells of the airway epithelium. Here we describe the development and optimization of conditions for culture of tracheal epithelial cells from chicken embryos as well as their use in st...

  3. Data on acylglycerophosphate acyltransferase 4 (AGPAT4) during murine embryogenesis and in embryo-derived cultured primary neurons and glia.

    PubMed

    Bradley, Ryan M; Mardian, Emily B; Marvyn, Phillip M; Vasefi, Maryam S; Beazely, Michael A; Mielke, John G; Duncan, Robin E

    2016-03-01

    Whole mouse embryos at three developmental timepoints, embryonic (E) day E10.5, E14.5, and E18.5, were analyzed for Agpat4 mRNA expression. Primary cortical mouse cultures prepared from E18.5 mouse brains were used for immunohistochemistry. Our data show that Agpat4 is differentially expressed at three timepoints in murine embryogenesis and is immunodetectable in both neurons and glial cells derived from the developing mouse brain. This paper contains data related to research concurrently published in Bradley et al. (2015) [1].

  4. An intercellular regenerative calcium wave in porcine coronary artery endothelial cells in primary culture

    PubMed Central

    Domenighetti, Andrea Alberto; Bény, Jean-Louis; Chabaud, Fabienne; Frieden, Maud

    1998-01-01

    A regenerative calcium wave is an increase in cytosolic free calcium concentration ([Ca2+]i) which extends beyond the stimulated cells without decrement of amplitude, kinetics of [Ca2+]i increase and speed of propagation. The aim of the present study was to test the hypothesis that such a wave could be evoked by bradykinin stimulation and by scraping cultured endothelial cells from porcine coronary arteries. Calcium imaging was performed using the calcium-sensitive dye fura-2. A wound or a delivery of bradykinin to two to three cells on growing clusters of ≈300 cells caused an increase in [Ca2+]i which was propagated throughout the cluster in a regenerative manner over distances up to 400 μm. This wave spread through gap junctions since it was inhibited by the cell uncoupler palmitoleic acid. The same experiments performed in confluent cultures caused a rise in [Ca2+]i which failed to propagate in a regenerative way. The wave propagation probably failed because the confluent cells were less dye coupled than the growing cells. This was confirmed by immunohistology which detected a dramatic decrease in the number of connexin 40 gap junctions in the confluent cultures. The regenerative propagation of the wave was blocked by inhibitors of calcium-induced calcium release (CICR) and phospholipase C (PLC), and by suppression of extracellular calcium, but not by clamping the membrane potential with high-potassium solution. We conclude that regenerative intercellular calcium waves exist in cultured islets but not in confluent cultures of endothelial cells. An increase in [Ca2+]i is not sufficient to trigger a regenerative propagation. The PLC pathway, CICR and extracellular calcium are all necessary for a fully regenerated propagation. PMID:9782162

  5. Expression of a plasma membrane proteolipid during differentiation of neuronal and glial cells in primary culture.

    PubMed

    Shea, T B; Fischer, I; Sapirstein, V

    1986-09-01

    Plasma membrane proteolipid protein (PM-PLP) synthesis was examined in embryonic rat neurons and neonatal rat glial cells during differentiation in culture. Glial cultures were treated with 1 mM N6, O2, dibutyryl cyclic adenosine monophosphate (dbcAMP) following confluency to induce differentiation, which resulted in the elaboration of long cellular processes. However, no changes in the biosynthetic level of PM-PLP was observed during the differentiation of these cells. Neurons differentiated spontaneously in culture, forming cellular aggregates immediately following plating and elaborating a network of neurites over 7 days. The differentiation of neurons was accompanied by a seven-fold increase in PM-PLP synthesis with increases in biosynthetic increase in PM-PLP synthesis with increases in biosynthetic rate observed between days 1 and 3 and between days 3 and 7 in culture. Ultrastructural examination of neurons indicated that the Golgi apparatus was also developing during this period of time, with an increase in both the number of lamellae and generation of vesicles. The transport of PM-PLP to the plasma membrane was therefore examined in neurons at day 7 in culture by pulse labeling experiments with monensin and colchicine. Monensin (1 microM) was found to inhibit the appearance of radiolabeled PM-PLP in the plasma membrane by 63%, indicating that a functional Golgi apparatus is required for transport of PM-PLP to its target membrane. Colchicine (125 microM) also inhibited the appearance of newly synthesized PM-PLP in the plasma membrane by greater than 40%, suggesting that microtubules may also be required for PM-PLP transport to the plasma membrane. PMID:3016181

  6. Primary School Teachers' Views on a Holistic Approach to Facilitating the Acquisition of Musical Cultural Values

    ERIC Educational Resources Information Center

    Badjanova, Jelena

    2013-01-01

    Recent tendencies in education highlight the need to align the system of general education in Latvia with the tenets of sustainability. In keeping with this broad target orientation, this paper re-examines international and Latvian experiences and perspectives on the application of a holistic approach to the content of primary education. This…

  7. BOLISM OF ARSENITE IN CULTURED PRIMARY HEPATOCYTES FROM SIX MAMMALIAN SPECIES

    EPA Science Inventory

    Inorganic arsenic (iAs) is an environmental toxin and carcinogen. Biomethylation is the major pathway for the metabolism of iAs in many mammalian species, including the human. The liver is considered the primary site for iAs methylation and As (+3 oxidation state) methyltransfera...

  8. Diversity in Family Involvement in Children's Learning in English Primary Schools: Culture, Language and Identity

    ERIC Educational Resources Information Center

    Conteh, Jean; Kawashima, Yasuko

    2008-01-01

    Government policy in England has for many years encouraged parental involvement in their children's education. In response, most primary schools have developed a range of strategies designed to assist parents in supporting their children's learning at home, particularly in learning to read. However, it is a common assumption that parents from some…

  9. Health Imperatives in Primary Schools across Three Countries: Intersections of Class, Culture and Subjectivity

    ERIC Educational Resources Information Center

    Wright, Jan; Burrows, Lisette; Rich, Emma

    2012-01-01

    In this article, we want to focus on the impact of the new health imperatives on young children attending primary schools because the evidence from both our own and others work suggests that younger and younger children are talking in very negative and disturbing ways about themselves and their bodies. We see this in a context where in the name of…

  10. The Unreliability of MTT Assay in the Cytotoxic Test of Primary Cultured Glioblastoma Cells.

    PubMed

    Jo, Hwa Yeon; Kim, Yona; Park, Hyung Woo; Moon, Hyo Eun; Bae, Seongtae; Kim, JinWook; Kim, Dong Gyu; Paek, Sun Ha

    2015-09-01

    MTT assay is commonly used to assess the cellular cytotoxicity caused by anticancer drugs in glioblastomas. However, there have been some reports insisting that MTT assay exhibited non-specific intracellular reduction of tetrazolium which led to underestimated results of cytotoxicity. Here, we examine whether or not MTT assay can lead to incorrect information regarding alcohol-induced cytotoxicity on immortalized and primary glioblastoma cells. MTT assay was applied to assess the ethanol-induced cytotoxicity at various ethanol concentrations. The cellular cytotoxicity induced by different doses of ethanol was analyzed and compared through several cytotoxic assays. Ethanol-induced cytotoxicity observed through MTT assay on both cell types was shown to be ethanol dose-dependent below a 3% concentration. However, the cytotoxicity was shown to be markedly underestimated only in primary cells at a 5% concentration. RT-PCR and Western Blot showed increased expressions of pro-apoptotic proteins and decreased expressions of anti-apoptotic proteins in an ethanol dose-dependent manner in both cell types. Furthermore, we present a possible mechanism for the unreliable result of MTT assay. A high concentration of ethanol induces more severe membrane damage and increased intracellular concentration of NADH in primary cells which enhances the nonspecific reduction of tetrazolium salt. Together, our findings demonstrate that the cytotoxicity on primary cells could inaccurately be assessed when detected through MTT assay. Therefore, a careful interpretation is needed when one would analyze the cytotoxic results of MTT assay, and it is suggested that other assays must be accompanied to produce more reliable and accurate cytotoxic results on primary glioblastoma cells.

  11. Combined inhibition of HER1/EGFR and RAC1 results in a synergistic antiproliferative effect on established and primary cultured human glioblastoma cells.

    PubMed

    Karpel-Massler, Georg; Westhoff, M-Andrew; Zhou, Shaoxia; Nonnenmacher, Lisa; Dwucet, Annika; Kast, Richard E; Bachem, Max G; Wirtz, Christian R; Debatin, Klaus-Michael; Halatsch, Marc-Eric

    2013-09-01

    Glioblastoma is the most frequent brain tumor of glial origin in adults. With the best available standard-of-care, patients with this disease have a life expectancy of only approximately 15 months after diagnosis. Because the EGF receptor (HER1/EGFR) is one of the most commonly dysregulated oncogenes in glioblastoma, HER1/EGFR-targeted agents, such as erlotinib, were expected to provide a therapeutic benefit. However, their application in the clinical setting failed. Seeking an explanation for this finding, we previously identified several candidate genes for resistance of human glioblastoma cell lines toward erlotinib. On the basis of this panel of genes, we aimed at identifying drugs that synergistically enhance the antiproliferative effect of erlotinib on established and primary glioblastoma cell lines. We found that NSC23766, an inhibitor of RAC1, enhanced the antineoplastic effects of erlotinib in U87MG, T98MG, and A172MG glioblastoma cell lines for the most part in a synergistic or at least in an additive manner. In addition, the synergistic antiproliferative effect of erlotinib and NSC23766 was confirmed in primary cultured cells, indicating a common underlying cellular and molecular mechanism in glioblastoma. Therefore, agents that suppress RAC1 activation may be useful therapeutic partners for erlotinib in a combined targeted treatment of glioblastoma.

  12. Study of Silymarin and Vitamin E Protective Effects on Silver Nanoparticle Toxicity on Mice Liver Primary Cell Culture.

    PubMed

    Faedmaleki, Firouz; Shirazi, Farshad H; Ejtemaeimehr, Shahram; Anjarani, Soghra; Salarian, Amir-Ahmad; Ahmadi Ashtiani, Hamidreza; Rastegar, Hossein

    2016-02-01

    .7 ppm or µg/ml). Then the hepatoprotective effect of silymarin and vitamin E were experimented on silver nanoparticle toxicity on mice liver primary cell culture. The results showed that silymarin at 600 µg/ml and vitamin E at 2500 µmol/l have protective effects on silver nanoparticle toxicity on mice liver primary cell culture. Viability percentage of the primary liver cell of the mouse were exposed to silver nanoparticles at 121.7 ppm and co-treatment of silymarin, and vitamin E is more than viability percentage of the primary liver cell of the mouse were exposed to silver nanoparticles and silymarin or silver nanoparticles and vitamin E.

  13. Study of Silymarin and Vitamin E Protective Effects on Silver Nanoparticle Toxicity on Mice Liver Primary Cell Culture.

    PubMed

    Faedmaleki, Firouz; Shirazi, Farshad H; Ejtemaeimehr, Shahram; Anjarani, Soghra; Salarian, Amir-Ahmad; Ahmadi Ashtiani, Hamidreza; Rastegar, Hossein

    2016-02-01

    .7 ppm or µg/ml). Then the hepatoprotective effect of silymarin and vitamin E were experimented on silver nanoparticle toxicity on mice liver primary cell culture. The results showed that silymarin at 600 µg/ml and vitamin E at 2500 µmol/l have protective effects on silver nanoparticle toxicity on mice liver primary cell culture. Viability percentage of the primary liver cell of the mouse were exposed to silver nanoparticles at 121.7 ppm and co-treatment of silymarin, and vitamin E is more than viability percentage of the primary liver cell of the mouse were exposed to silver nanoparticles and silymarin or silver nanoparticles and vitamin E. PMID:26997594

  14. Space Takes Time: Concentration Dependent Output Codes from Primary Olfactory Networks Rapidly Provide Additional Information at Defined Discrimination Thresholds.

    PubMed

    Daly, Kevin C; Bradley, Samual; Chapman, Phillip D; Staudacher, Erich M; Tiede, Regina; Schachtner, Joachim

    2015-01-01

    As odor concentration increases, primary olfactory network representations expand in spatial distribution, temporal complexity and duration. However, the direct relationship between concentration dependent odor representations and the psychophysical thresholds of detection and discrimination is poorly understood. This relationship is absolutely critical as thresholds signify transition points whereby representations become meaningful to the organism. Here, we matched stimulus protocols for psychophysical assays and intracellular recordings of antennal lobe (AL) projection neurons (PNs) in the moth Manduca sexta to directly compare psychophysical thresholds and the output representations they elicit. We first behaviorally identified odor detection and discrimination thresholds across an odor dilution series for a panel of structurally similar odors. We then characterized spatiotemporal spiking patterns across a population of individually filled and identified AL PNs in response to those odors at concentrations below, at, and above identified thresholds. Using spatial and spatiotemporal based analyses we observed that each stimulus produced unique representations, even at sub-threshold concentrations. Mean response latency did not decrease and the percent glomerular activation did not increase with concentration until undiluted odor. Furthermore, correlations between spatial patterns for odor decreased, but only significantly with undiluted odor. Using time-integrated Euclidean distance (ED) measures, we determined that added spatiotemporal information was present at the discrimination but not detection threshold. This added information was evidenced by an increase in integrated distance between the sub-detection and discrimination threshold concentrations (of the same odor) that was not present in comparison of the sub-detection and detection threshold. After consideration of delays for information to reach the AL we find that it takes ~120-140 ms for the AL to

  15. Space Takes Time: Concentration Dependent Output Codes from Primary Olfactory Networks Rapidly Provide Additional Information at Defined Discrimination Thresholds

    PubMed Central

    Daly, Kevin C.; Bradley, Samual; Chapman, Phillip D.; Staudacher, Erich M.; Tiede, Regina; Schachtner, Joachim

    2016-01-01

    As odor concentration increases, primary olfactory network representations expand in spatial distribution, temporal complexity and duration. However, the direct relationship between concentration dependent odor representations and the psychophysical thresholds of detection and discrimination is poorly understood. This relationship is absolutely critical as thresholds signify transition points whereby representations become meaningful to the organism. Here, we matched stimulus protocols for psychophysical assays and intracellular recordings of antennal lobe (AL) projection neurons (PNs) in the moth Manduca sexta to directly compare psychophysical thresholds and the output representations they elicit. We first behaviorally identified odor detection and discrimination thresholds across an odor dilution series for a panel of structurally similar odors. We then characterized spatiotemporal spiking patterns across a population of individually filled and identified AL PNs in response to those odors at concentrations below, at, and above identified thresholds. Using spatial and spatiotemporal based analyses we observed that each stimulus produced unique representations, even at sub-threshold concentrations. Mean response latency did not decrease and the percent glomerular activation did not increase with concentration until undiluted odor. Furthermore, correlations between spatial patterns for odor decreased, but only significantly with undiluted odor. Using time-integrated Euclidean distance (ED) measures, we determined that added spatiotemporal information was present at the discrimination but not detection threshold. This added information was evidenced by an increase in integrated distance between the sub-detection and discrimination threshold concentrations (of the same odor) that was not present in comparison of the sub-detection and detection threshold. After consideration of delays for information to reach the AL we find that it takes ~120–140 ms for the AL to

  16. Differentiation between Glioblastoma Multiforme and Primary Cerebral Lymphoma: Additional Benefits of Quantitative Diffusion-Weighted MR Imaging

    PubMed Central

    Li, Chien Feng; Chen, Tai Yuan; Shu, Ginger; Kuo, Yu Ting; Lee, Yu Chang

    2016-01-01

    The differentiation between glioblastoma multiforme (GBM) and primary cerebral lymphoma (PCL) is important because the treatments are substantially different. The purpose of this article is to describe the MR imaging characteristics of GBM and PCL with emphasis on the quantitative ADC analysis in the tumor necrosis, the most strongly-enhanced tumor area, and the peritumoral edema. This retrospective cohort study collected 104 GBM (WHO grade IV) patients and 22 immune-competent PCL (diffuse large B cell lymphoma) patients. All these patients had pretreatment brain MR DWI and ADC imaging. Analysis of conventional MR imaging and quantitative ADC measurement including the tumor necrosis (ADCn), the most strongly-enhanced tumor area (ADCt), and the peritumoral edema (ADCe) were done. ROC analysis with optimal cut-off values and area-under-the ROC curve (AUC) was performed. For conventional MR imaging, there are statistical differences in tumor size, tumor location, tumor margin, and the presence of tumor necrosis between GBM and PCL. Quantitative ADC analysis shows that GBM tended to have significantly (P<0.05) higher ADC in the most strongly-enhanced area (ADCt) and lower ADC in the peritumoral edema (ADCe) as compared with PCL. Excellent AUC (0.94) with optimal sensitivity of 90% and specificity of 86% for differentiating between GBM and PCL was obtained by combination of ADC in the tumor necrosis (ADCn), the most strongly-enhanced tumor area (ADCt), and the peritumoral edema (ADCe). Besides, there are positive ADC gradients in the peritumoral edema in a subset of GBMs but not in the PCLs. Quantitative ADC analysis in these three areas can thus be implemented to improve diagnostic accuracy for these two brain tumor types. The histological correlation of the ADC difference deserves further investigation. PMID:27631626

  17. Differentiation between Glioblastoma Multiforme and Primary Cerebral Lymphoma: Additional Benefits of Quantitative Diffusion-Weighted MR Imaging.

    PubMed

    Ko, Ching Chung; Tai, Ming Hong; Li, Chien Feng; Chen, Tai Yuan; Chen, Jeon Hor; Shu, Ginger; Kuo, Yu Ting; Lee, Yu Chang

    2016-01-01

    The differentiation between glioblastoma multiforme (GBM) and primary cerebral lymphoma (PCL) is important because the treatments are substantially different. The purpose of this article is to describe the MR imaging characteristics of GBM and PCL with emphasis on the quantitative ADC analysis in the tumor necrosis, the most strongly-enhanced tumor area, and the peritumoral edema. This retrospective cohort study collected 104 GBM (WHO grade IV) patients and 22 immune-competent PCL (diffuse large B cell lymphoma) patients. All these patients had pretreatment brain MR DWI and ADC imaging. Analysis of conventional MR imaging and quantitative ADC measurement including the tumor necrosis (ADCn), the most strongly-enhanced tumor area (ADCt), and the peritumoral edema (ADCe) were done. ROC analysis with optimal cut-off values and area-under-the ROC curve (AUC) was performed. For conventional MR imaging, there are statistical differences in tumor size, tumor location, tumor margin, and the presence of tumor necrosis between GBM and PCL. Quantitative ADC analysis shows that GBM tended to have significantly (P<0.05) higher ADC in the most strongly-enhanced area (ADCt) and lower ADC in the peritumoral edema (ADCe) as compared with PCL. Excellent AUC (0.94) with optimal sensitivity of 90% and specificity of 86% for differentiating between GBM and PCL was obtained by combination of ADC in the tumor necrosis (ADCn), the most strongly-enhanced tumor area (ADCt), and the peritumoral edema (ADCe). Besides, there are positive ADC gradients in the peritumoral edema in a subset of GBMs but not in the PCLs. Quantitative ADC analysis in these three areas can thus be implemented to improve diagnostic accuracy for these two brain tumor types. The histological correlation of the ADC difference deserves further investigation. PMID:27631626

  18. Comparative effects of contraction and angiotensin II on growth of adult feline cardiocytes in primary culture

    NASA Technical Reports Server (NTRS)

    Wada, H.; Zile, M. R.; Ivester, C. T.; Cooper, G. 4th; McDermott, P. J.

    1996-01-01

    The purposes of this study were 1) to determine whether angiotensin II causes growth of adult feline cardiocytes in long-term culture, 2) to compare the growth effects of angiotensin II with those resulting from electrically stimulated contraction, and 3) to determine whether the anabolic effects of contraction are exerted via the angiotensin type 1 receptor. Adult feline cardiocytes were cultured on laminin-coated trays in a serum-free medium. Cardiocytes were either electrically stimulated to contract (1 Hz, 5-ms pulse duration, alternating polarity) or were nonstimulated and quiescent. Quiescent cells were studied as controls and after treatment with angiotensin II (10(-8) M), losartan (10(-6) M; an angiotensin type 1-receptor antagonist), or angiotensin II plus losartan. Contracting cells were studied in the presence and absence of angiotensin II or losartan. In quiescent cardiocytes, angiotensin II treatment on day 7 significantly increased protein synthesis rates by 22% and protein content per cell by 17%. The effects of angiotensin II were completely blocked by losartan. Electrically stimulated contraction on days 4 and 7 in culture significantly increased protein synthesis rate by 18 and 38% and protein content per cell by 19 and 46%, respectively. Angiotensin II treatment did not further increase protein synthesis rate or protein content in contracting cardiocytes. Furthermore, losartan did not block the anabolic effects of contraction on protein synthesis rates or protein content. In conclusion, angiotensin II can exert a modest anabolic effect on adult feline cardiocytes in culture. In contracting feline cardiocytes, angiotensin II has no effect on growth. Growth caused by electrically stimulated contraction occurs more rapidly and is greater in magnitude than that caused by angiotensin II. Growth of contracting adult feline cardiocytes is not dependent on activation of the angiotensin receptor.

  19. Airborne culturable fungi in naturally ventilated primary school environments in a subtropical climate

    NASA Astrophysics Data System (ADS)

    Salonen, Heidi; Duchaine, Caroline; Mazaheri, Mandana; Clifford, Sam; Morawska, Lidia

    2015-04-01

    There is currently a lack of reference values for indoor air fungal concentrations to allow for the interpretation of measurement results in subtropical school settings. Analysis of the results of this work established that, in the majority of properly maintained subtropical school buildings, without any major affecting events such as floods or visible mould or moisture contamination, indoor culturable fungi levels were driven by outdoor concentration. The results also allowed us to benchmark the "baseline range" concentrations for total culturable fungi, Penicillium spp., Cladosporium spp. and Aspergillus spp. in such school settings. The measured concentration of total culturable fungi and three individual fungal genera were estimated using Bayesian hierarchical modelling. Pooling of these estimates provided a predictive distribution for concentrations at an unobserved school. The results indicated that "baseline" indoor concentration levels for indoor total fungi, Penicillium spp., Cladosporium spp. and Aspergillus spp. in such school settings were generally ≤1450, ≤680, ≤480 and ≤90 cfu/m3, respectively, and elevated levels would indicate mould damage in building structures. The indoor/outdoor ratio for most classrooms had 95% credible intervals containing 1, indicating that fungi concentrations are generally the same indoors and outdoors at each school. Bayesian fixed effects regression modelling showed that increasing both temperature and humidity resulted in higher levels of fungi concentration.

  20. Quantitative Nuclease Protection Assays (qNPA) as Windows into Chemical-Induced Adaptive Response in Cultures of Primary Human Hepatocytes (Concentration and Time-Response)

    EPA Science Inventory

    Cultures of primary human hepatocytes have been shown to be dynamic in vitro model systems that retain liver-like functionality (e.g. metabolism, transport, induction). We have utilized these culture models to interrogate 309 ToxCast chemicals. The study design characterized both...

  1. Biological glass coating on ceramic materials: in vitro evaluation using primary osteoblast cultures from healthy and osteopenic rat bone.

    PubMed

    Torricelli, P; Verné, E; Brovarone, C V; Appendino, P; Rustichelli, F; Krajewski, A; Ravaglioli, A; Pierini, G; Fini, M; Giavaresi, G; Giardino, R

    2001-09-01

    ZrO2 and Al2O3 substrates were successfully coated by a double layer of a silica-based glass named RKKP, using a low-cost firing technique. RKKP is a glass well known for its bioactivity; therefore, a RKKP coating on Al2O3 or ZrO2, allows to combine the excellent mechanical properties of these strong ceramic substrates with its bioactivity. ZrO2 samples were easily coated using a double layer of RKKP by a simple enamelling technique. To accommodate the thermal expansion coefficient mismatch between Al2O3 and RK K P, this substrate was coated using a multilayered composite approach. All of the coatings were characterised from a morphological and compositional point of view, and an extensive biological evaluation was performed using fresh rat osteoblasts. Osteoblast primary cultures were derived from the trabecular bone of femoral condyles harvested from intact (NB) and osteopenic (OB) rats. After characterisation of their phenotype, osteoblasts were seeded on material samples of ZrO2 or Al2O3 coated with RKKP, and cultured for 7 days. Cell proliferation (MTT test) and cell differentiation (alkaline phosphatase activity) were evaluated at the end of the experiment, to assess osteoblast behaviour in the presence of biomaterials and determine if the results were related to the host bone quality. Results of both materials showed a good level of biocompatibility. In particular, MTT significant higher values were detected in NB cultures on ZrO2-RKKP samples; ALP activity significantly increased in NB cultures on Al2O3-RKKP and in OB cultures on both coated samples.

  2. A standardized laboratory and surgical method for in vitro culture isolation and expansion of primary human Tenon's fibroblasts.

    PubMed

    De Falco, Elena; Scafetta, Gaia; Napoletano, Chiara; Puca, Rosa; Vingolo, Enzo Maria; Ragona, Giuseppe; Iorio, Olga; Frati, Giacomo

    2013-06-01

    Good manufacturing practices guidelines require safer and standardized cell substrates especially for those cell therapy products to treat ocular diseases where fibroblasts are used as feeder layers. However, if these are unavailable for stem cells culturing, murine fibroblasts are regularly used, raising critical issues as accidentally transplanting xenogenous graft and adversely affecting stem cell clinical trials. Moreover, human fibroblasts play a significant role in testing novel ophthalmologic drugs. Accordingly, we developed a standardized laboratory and surgical approach to isolate normal and undamaged Tenon's fibroblasts to implement the setting up of banks for both stem cells-based ocular cell therapy and in vitro drug testing. A 2-3 cm(2) undamaged Tenon's biopsy was surgically obtained from 28 patients without mutually correlated ocular diseases. Nineteen dermal biopsies were used as control. Fibroblasts were isolated with or without collagenase, cultured in autologous, fetal bovine or AB serum, tested for viability by trypan blue, vimentin expression and standardized until passage 6. Successful Tenon's fibroblasts isolation was age dependent (P = 0.001) but not sex, pathology or surgery related. A significant rate of successful cultures were obtained when biopsies were not digested by collagenase (P = 0.013). Moreover, cultures in autologous or fetal bovine serum had comparable proliferative properties (P = 0.77; P = 0.82). Through our surgical and laboratory standardized procedure, we elucidated for the first time key points of this human primary culture system, the role of the autologous serum, comparing Tenon's and dermal fibroblasts. Our protocol may be clinically useful to reduce the risk above mentioned and may be potentially more effective for ophthalmological clinical purposes. PMID:22820760

  3. Conducting Museum Education Activities within the Context of Developing a Nature Culture in Primary School Students: MTA Natural History Museum Example

    ERIC Educational Resources Information Center

    Dilli, Rukiye

    2016-01-01

    The present study, aiming to develop nature culture in primary school students and to help them to become acquainted with their close environment, is a quasi-experimental study. Museum education activities were conducted with the study group which consisted of 128 fourth-grade primary school students. At the end of the study, the students gained…

  4. The Link Between Pre-School and Primary Education: Report on Visits to Pilot Projects in 8 Member States of the Council for Cultural Co-operation.

    ERIC Educational Resources Information Center

    Schumer-Strucksberg, M.

    This report presents a summary of visits to pilot projects which focused on the link between preschool and primary education in eight European countries of the Council for Cultural Cooperation. A brief overview of the problem of the relationships between preschool and primary education identifies priorities in the development of preschool…

  5. Phosphorylation in the C-terminal domain of Aquaporin-4 is required for Golgi transition in primary cultured astrocytes

    SciTech Connect

    Kadohira, Ikuko; Abe, Yoichiro Nuriya, Mutsuo; Sano, Kazumi; Tsuji, Shoji; Arimitsu, Takeshi; Yoshimura, Yasunori; Yasui, Masato

    2008-12-12

    Aquaporin-4 (AQP4) is expressed in the perivascular and subpial astrocytes end-feet in mammalian brain, and plays a critical component of an integrated water and potassium homeostasis. Here we examine whether AQP4 is phosphorylated in primary cultured mouse astrocytes. Astrocytes were metabolically labeled with [{sup 32}P]phosphoric acid, then AQP4 was immunoprecipitated with anti-AQP4 antibody. We observed that AQP4 was constitutively phosphorylated, which is reduced by treatment with protein kinase CK2 inhibitors. To elucidate the phosphorylation of AQP4 by CK2, myc-tagged wild-type or mutant AQP4 was transiently transfected in primary cultured astrocytes. Substitution of Ala residues for four putative CK2 phosphorylation sites in the C terminus abolished the phosphorylation of AQP4. Immunofluorescent microscopy revealed that the quadruple mutant was localized in the Golgi apparatus. These observations indicate that the C-terminal domain of AQP4 is constitutively phosphorylated at least in part by protein kinase CK2 and it is required for Golgi transition.

  6. Longitudinal Claudin Gene Expression Analyses in Canine Mammary Tissues and Thereof Derived Primary Cultures and Cell Lines

    PubMed Central

    Hammer, Susanne C.; Becker, Annegret; Rateitschak, Katja; Mohr, Annika; Lüder Ripoli, Florenza; Hennecke, Silvia; Junginger, Johannes; Hewicker-Trautwein, Marion; Brenig, Bertram; Ngezahayo, Anaclet; Nolte, Ingo; Murua Escobar, Hugo

    2016-01-01

    Human and canine mammary tumours show partial claudin expression deregulations. Further, claudins have been used for directed therapeutic approaches. However, the development of claudin targeting approaches requires stable claudin expressing cell lines. This study reports the establishment and characterisation of canine mammary tissue derived cell lines, analysing longitudinally the claudin-1, -3, -4 and -7 expressions in original tissue samples, primary cultures and developed cell lines. Primary cultures were derived from 17 canine mammary tissues: healthy, lobular hyperplasia, simple adenoma, complex adenoma, simple tubular carcinoma, complex carcinoma, carcinoma arising in a benign mixed tumour and benign mixed tissue. Cultivation was performed, if possible, until passage 30. Claudin mRNA and protein expressions were analysed by PCR, QuantiGene Plex Assay, immunocytochemistry and immunofluorescence. Further, cytokeratin expression was analysed immunocytochemically. Cultivation resulted in 11 established cell lines, eight showing epithelial character. In five of the early passages the claudin expressions decreased compared to the original tissues. In general, claudin expressions were diminished during cultivation. Three cell lines kept longitudinally claudin, as well as epithelial marker expressions, representing valuable tools for the development of claudin targeted anti-tumour therapies. PMID:27690019

  7. Stimulation of arachidonic acid metabolism in primary cultures of osteoblast-like cells by hormones and drugs

    SciTech Connect

    Feyen, J.H.; van der Wilt, G.; Moonen, P.; Di Bon, A.; Nijweide, P.J.

    1984-12-01

    The effects of parathyroid hormone (PTH), dihydroxycholecalciferol (1,25-(OH)2 D3), thrombin, epidermal growth factor (EGF) and 12-o-tetradecanoylphorbol-13-acetate (PMA) on the biosynthesis and release of arachidonic acid metabolites were studied in primary cultures of osteoblast-like cells isolated from 18-day-old chick embryo calvaria. Cells were labelled with (/sup 14/C)-arachidonic acid for 30 h. The radioactive eicosanoids were extracted from the cell culture media after a further 30 h stimulation period and analysed on a PRP-1 column by HPLC. The radioactive products were characterized by co-elution of (/sup 3/H) standard prostanoids. Osteoblasts showed a basal release of the prostanoids 6-keto-PGF1 alpha, TXB2, PGF2 alpha, PGE2, PGD2 and PGB2, the latter being the most abundant one. Indomethacin (10(-5) M) effectively inhibited the basal release, but not that of an as yet unidentified compound. The release of prostanoids was stimulated by PTH (2 U/ml), thrombin (0.4 NIH/ml), EGF (50 ng/ml) and PMA (25 ng/ml), the latter being by far the most potent one. 1,25-(OH)2D3 was found to slightly inhibit the prostanoid release. These results indicate: (1) primary cultures of osteoblasts synthesize several prostaglandins, thromboxane B2 and one unidentified product. (2) the action on bone of PTH and the various drugs tested may be, at least partly, mediated by an increased prostaglandin production by osteoblasts. Clearly this does not apply to 1,25-(OH)2D3.

  8. An in vitro triple cell co-culture model with primary cells mimicking the human alveolar epithelial barrier.

    PubMed

    Lehmann, Andrea D; Daum, Nicole; Bur, Michael; Lehr, Claus-Michael; Gehr, Peter; Rothen-Rutishauser, Barbara M

    2011-04-01

    A triple cell co-culture model was recently established by the authors, consisting of either A549 or 16HBE14o- epithelial cells, human blood monocyte-derived macrophages and dendritic cells, which offers the possibility to study the interaction of xenobiotics with those cells. The 16HBE14o- containing co-culture model mimics the airway epithelial barrier, whereas the A549 co-cultures mimic the alveolar type II-like epithelial barrier. The goal of the present work was to establish a new triple cell co-culture model composed of primary alveolar type I-like cells isolated from human lung biopsies (hAEpC) representing a more realistic alveolar epithelial barrier wall, since type I epithelial cells cover >93% of the alveolar surface. Monocultures of A549 and 16HBE14o- were morphologically and functionally compared with the hAEpC using laser scanning microscopy, as well as transmission electron microscopy, and by determining the epithelial integrity. The triple cell co-cultures were characterized using the same methods. It could be shown that the epithelial integrity of hAEpC (mean ± SD, 1180 ± 188 Ω cm(2)) was higher than in A549 (172 ± 59 Ω cm(2)) but similar to 16HBE14o- cells (1469 ± 156 Ω cm(2)). The triple cell co-culture model with hAEpC (1113 ± 30 Ω cm(2)) showed the highest integrity compared to the ones with A549 (93 ± 14 Ω cm(2)) and 16HBE14o- (558 ± 267 Ω cm(2)). The tight junction protein zonula occludens-1 in hAEpC and 16HBE14o- were more regularly expressed but not in A549. The epithelial alveolar model with hAEpC combined with two immune cells (i.e. macrophages and dendritic cells) will offer a novel and more realistic cell co-culture system to study possible cell interactions of inhaled xenobiotics and their toxic potential on the human alveolar type I epithelial wall.

  9. Skin punch biopsy explant culture for derivation of primary human fibroblasts.

    PubMed

    Vangipuram, Malini; Ting, Dennis; Kim, Sam; Diaz, Robert; Schüle, Birgitt

    2013-01-01

    Tissues and cell lines derived from an individual with disease are ideal sources to study disease-related cellular phenotypes. Patient-derived fibroblasts in this protocol have been successfully used in the derivation of induced pluripotent stem cells to model disease(1). Early passages of these fibroblasts can also be used for cell-based functional assays to study specific disease pathways, mechanisms(2) and subsequent drug screening approaches. The advantage of the presented protocol over enzymatic procedures are 1) the reproducibility of the technique from small amounts of tissue derived from older patients, e.g. patients affected with Parkinson's disease, 2) the technically simple approach over more challenging methodologies using enzymatic treatments, and 3) the time consideration: this protocol takes 15-20 min and can be performed immediately after biopsy arrival. Enzymatic treatments can take up to 4 hr and have the problems of overdigestion, reduction of cell viability and subsequent attachment of cells when not handled properly. This protocol describes the dissection and preparation of a 4-mm human skin biopsy for derivation of a fibroblast culture and has a very high success rate which is important when dealing with patient-derived tissue samples. In this culture, keratinocytes migrate out of the biopsy tissue within the first week after preparation. Fibroblasts appear 7-10 days after the first outgrowth of keratinocytes. DMEM high glucose media supplemented with 20% FBS favors the growth of fibroblasts over keratinocytes and fibroblasts will overgrow the keratinocytes. After 2 passages keratinocytes have been diluted out resulting in relatively homogenous fibroblast cultures which expresses the fibroblast marker SERPINH1 (HSP-47). Using this approach, 15-20 million fibroblasts can be derived in 4-8 weeks for cell banking. The skin dissection takes about 15-20 min, cells are then monitored once a day under the microscope, and media is changed every 2

  10. Prognostic value of trisomy 8 as a single anomaly and the influence of additional cytogenetic aberrations in primary myelodysplastic syndromes.

    PubMed

    Saumell, Sílvia; Florensa, Lourdes; Luño, Elisa; Sanzo, Carmen; Cañizo, Consuelo; Hernández, Jesus M; Cervera, José; Gallart, Miguel A; Carbonell, Félix; Collado, Rosa; Arenillas, Leonor; Pedro, Carme; Bargay, Joan; Nomdedeu, Benet; Xicoy, Blanca; Vallespí, Teresa; Raya, José M; Belloch, Luis; Sanz, Guillermo F; Solé, Francesc

    2012-11-01

    Trisomy 8 is the most common chromosomal gain in myelodysplastic syndromes (MDS), however, little is known about the features of MDS with isolated trisomy 8 and the influence of additional cytogenetic aberrations. We determined the characteristics and prognostic factors of 72 patients with trisomy 8 as a single anomaly and analysed also the impact of other aberrations added to trisomy 8 in another 62 patients. According to our study, MDS with isolated trisomy 8 was more frequent in men, with more than one cytopenia in most patients (62%) and having about 4% bone marrow blasts. The multivariate analysis demonstrated that platelet count and percentage bone marrow blasts had the strongest impact on overall survival (OS). The median OS for isolated trisomy 8, trisomy 8 plus one aberration (tr8 + 1), plus two (tr8 + 2) and plus three or more aberrations (tr8 + ≥3) was 34·3, 40, 23·4 and 5·8 months, respectively (P < 0·001). Trisomy 8 confers a poorer prognosis than a normal karyotype in MDS patients with ≥5% bone marrow blasts. This study supports the view that MDS with isolated trisomy 8 should be included in the intermediate cytogenetic risk group.

  11. Expression of two CYP1A genes in {beta}NF and TCDD-treated rainbow trout primary hepatocyte culture

    SciTech Connect

    Rabergh, C.M.; Lipsky, M.M.; Vroliijk, N.H.; Chen, T.T.

    1995-12-31

    In mammalian systems, it is well known that two CYP1A genes are expressed in response to environmental toxicants such as polycyclic aromatic hydrocarbons (PAHs) and TCDD (2,3,7,3-tetrachlorodibenzo-p-dioxin). The presence of two CYP1A genes in fish has been previously reported, though expression of these two genes has not been characterized. In this study, the authors examined the expression of these two genes in primary culture of rainbow trout hepatocytes treated with {beta}NF and TCDD. Hepatocytes were isolated by a modified two-step collagenase perfusion of the liver and cultured on polylysine coated dishes. The optimum time and concentration of induction was determined for both chemicals. The expression of the genes was also studied in long-term cultures up to 20 days. RNA was isolated by the method of Chomzynski and Sacchi and the RNase protection assay was used to detect the expression of the CYP1A genes by using antisense riboprobes specific for the MRNA of each gene. A differential concentration- and time-dependent expression of the two genes was observed in cells treated with {beta}NF and TCDD. Whether these two genes are paralogous, i.e., produced by gene duplication within the species, or whether one of them may in fact be an orthologue to a mammalian counterpart within the CYP1A family, remains to be determined.

  12. Effect of acute ethanol on beta-endorphin secretion from rat fetal hypothalamic neurons in primary cultures

    SciTech Connect

    Sarkar, D.K.; Minami, S. )

    1990-01-01

    To characterize the effect of ethanol on the hypothalamic {beta}-endorphin-containing neurons, rat fetal hypothalamic neurons were maintained in primary culture, and the secretion of {beta}-endorphin ({beta}-EP) was determined after ethanol challenges. Constant exposure to ethanol at doses of 6-50 mM produced a dose-dependent increase in basal secretion of {beta}-EP from these cultured cells. These doses of ethanol did not produce any significant effect on cell viability, DNA or protein content. The stimulated secretion of {beta}-EP following constant ethanol exposure is short-lasting. However, intermittent ethanol exposures maintained the ethanol stimulatory action on {beta}-EP secretion for a longer time. The magnitude of the {beta}-EP response to 50 mM ethanol is similar to that of the {beta}-EP response to 56 mM of potassium. Ethanol-stimulated {beta}-EP secretion required extracellular calcium and was blocked by a calcium channel blocker; a sodium channel blocker did not affect ethanol-stimulated secretion. These results suggest that the neuron culture system is a useful model for studying the cellular mechanisms involved in the ethanol-regulated hypothalamic opioid secretion.

  13. In utero electroporation followed by primary neuronal culture for studying gene function in subset of cortical neurons.

    PubMed

    Rice, Heather; Suth, Seiyam; Cavanaugh, William; Bai, Jilin; Young-Pearse, Tracy L

    2010-10-08

    In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect neuronal process outgrowth, shRNA or cDNA constructs can be introduced into primary neurons via chemical transfection or viral transduction. However, with primary cortical cells, a heterogeneous pool of cell types (glutamatergic neurons from different layers, inhibitory neurons, glial cells) are transfected using these methods. The use of in utero electroporation to introduce DNA constructs in the embryonic rodent cortex allows for certain subsets of cells to be targeted: while electroporation of early embryonic cortex targets deep layers of the cortex, electroporation at late embryonic timepoints targets more superficial layers. Further, differential placement of electrodes across the heads of individual embryos results in the targeting of dorsal-medial versus ventral-lateral regions of the cortex. Following electroporation, transfected cells can be dissected out, dissociated, and plated in vitro for quantitative analysis of neurite outgrowth. Here, we provide a step-by-step method to quantitatively measure neuronal process outgrowth in subsets of cortical cells. The basic protocol for in utero electroporation has been described in detail in two other JoVE articles from the Kriegstein lab. We will provide an overview of our protocol for in utero electroporation, focusing on the most important details, followed by a description of our protocol that applies in utero electroporation to the study of gene function in neuronal process outgrowth.

  14. Sensitivity to radiation of human normal, hyperthyroid, and neoplastic thyroid epithelial cells in primary culture

    SciTech Connect

    Miller, R.C.; Hiraoka, T.; Kopecky, K.J.; Nakamura, N.; Jones, M.P.; Ito, T.; Clifton, K.H.

    1987-07-01

    Samples of thyroid tissue removed surgically from 63 patients were cultured in vitro and exposed to X irradiation to investigate the radiosensitivities of various types of thyroid epithelial cells. A total of 76 samples were obtained, including neoplastic cells from patients with papillary carcinoma (PC) or follicular adenoma (FA), cells from hyperthyroidism (HY) patients, and normal cells from the surgical margins of PC and FA patients. Culturing of the cells was performed in a manner which has been shown to yield a predominance of epithelial cells. Results of colony formation assays indicated that cells from HY and FA patients were the least radiosensitive: when adjusted to the overall geometric mean plating efficiency of 5.5%, the average mean lethal dose Do was 97.6 cGy for HY cells and 96.7 and 94.3 cGy, respectively for neoplastic and normal cells from FA patients. Cells from PC patients were more radiosensitive, normal cells having an adjusted average Do of 85.0 cGy and PC cells a significantly lower average Do of 74.4 cGy. After allowing for this variation by cell type, in vitro radiosensitivity was not significantly related to age at surgery or sex. These results suggest that malignant thyroid cells may be especially radiosensitive.

  15. Primary cultures of cerebellar neurons: A unique model for studying benzodiazepine receptors

    SciTech Connect

    Allen, A.J.

    1988-01-01

    The binding of ({sup 3}H)flunitrazepam to intact cultures of cerebellar neurons was studied at 24{degree}C. Association of 1 nM ({sup 3}H)flunitrazepam was monophasic, k{sub +1} = 1.41 pmole{sup {minus}1} sec{sup {minus}1}. Dissociation was approximately monophasic, k{sub {minus}1} = 0.0145 sec{sup {minus}1}. The presence of 1 {mu}M diazepam in the diluting buffer significantly accelerated initial dissociation of ({sup 3}H)flunitrazepam. Saturation binding studies revealed a nonlinear Scatchard plot with a Hill coefficient (n{sub H}) of 0.81 and K{sub 0.5} = 28.7 nM. The data fit equally well a model with two independent binding sites, and a stoichiometric equation utilizing pairwise interactions between four sites. A newly developed HPLC method was used for rapid, sensitive, and quantitative detection of amino acid neutrotransmitters and adenosine released from cerebellar neurons in culture. In preliminary studies, this technique was coupled with a specially designed perfusion chamber to demonstrate that flunitrazepam enhances potassium-stimulated release of glutamate and GABA, and may inhibit basal release of taurine.

  16. Pigment Cell Differentiation in Sea Urchin Blastula-Derived Primary Cell Cultures

    PubMed Central

    Ageenko, Natalya V.; Kiselev, Konstantin V.; Dmitrenok, Pavel S.; Odintsova, Nelly A.

    2014-01-01

    The quinone pigments of sea urchins, specifically echinochrome and spinochromes, are known for their effective antioxidant, antibacterial, antifungal, and antitumor activities. We developed in vitro technology for inducing pigment differentiation in cell culture. The intensification of the pigment differentiation was accompanied by a simultaneous decrease in cell proliferation. The number of pigment cells was two-fold higher in the cells cultivated in the coelomic fluids of injured sea urchins than in those intact. The possible roles of the specific components of the coelomic fluids in the pigment differentiation process and the quantitative measurement of the production of naphthoquinone pigments during cultivation were examined by MALDI and electrospray ionization mass spectrometry. Echinochrome A and spinochrome E were produced by the cultivated cells of the sand dollar Scaphechinus mirabilis in all tested media, while only spinochromes were found in the cultivated cells of another sea urchin, Strongylocentrotus intermedius. The expression of genes associated with the induction of pigment differentiation was increased in cells cultivated in the presence of shikimic acid, a precursor of naphthoquinone pigments. Our results should contribute to the development of new techniques in marine biotechnology, including the generation of cell cultures producing complex bioactive compounds with therapeutic potential. PMID:24979272

  17. Effects of heavy ion to the primary culture of mouse brain cells

    NASA Technical Reports Server (NTRS)

    Nojima, Kumie; Nakadai, Taeko; Kohno, Yukio; Vazquez, Marcelo E.; Yasuda, Nakahiro; Nagaoka, Shunji

    2004-01-01

    To investigate effects of low dose heavy particle radiation to CNS system, we adopted mouse neonatal brain cells in culture being exposed to heavy ions by HIMAC at NIRS and NSRL at BNL. The applied dose varied from 0.05 Gy up to 2.0 Gy. The subsequent biological effects were evaluated by an induction of apoptosis and neuron survival focusing on the dependencies of the animal strains, SCID, B6, B6C3F1, C3H, used for brain cell culture, SCID was the most sensitive and C3H the least sensitive to particle radiation as evaluated by 10% apoptotic criterion. The LET dependency was compared with using SCID and B6 cells exposing to different ions (H, C, Ne, Si, Ar, and Fe). Although no detectable LET dependency was observed in the high LET (55-200 keV/micrometers) and low dose (<0.5 Gy) regions. The survivability profiles of the neurons were different in the mouse strains and ions. In this report, a result of memory and learning function to adult mice after whole-body and brain local irradiation at carbon ion and iron ion.

  18. Beyond Primary Prevention of Alcohol Use: A Culturally Specific Secondary Prevention Program for Mexican Heritage Adolescents

    PubMed Central

    Ayers, Stephanie; Gance-Cleveland, Bonnie; Mettler, Kathleen; Booth, Jaime

    2012-01-01

    Classroom-based primary prevention programs with adolescents are effective in inhibiting the onset of drug use, but these programs are not designed to directly address the unique needs of adolescents at higher risk of use or already using alcohol and other drugs. This article describes the initial efficacy evaluation of a companion psychosocial small group program which aims at addressing the needs of Mexican heritage students identified by their teachers as being at higher risk for substance use or already experimenting with alcohol and other drugs. The adolescent (7th grade) small group curricula, REAL Groups, is a secondary prevention program which supplements the primary classroom-based substance use prevention program, keepin’ it REAL. Following a mutual aid approach, a total of 109 7th grade students were referred by their teachers and participated in the REAL Groups. The remaining 252 7th grade students who did not participate served as the control group. To account for biased selection into REAL Groups, propensity score matching (PSM) was employed. The estimated average treatment effect for participants’ use of alcohol was calculated at the end of the 8th grade. Results indicate that alcohol use decreased among students who participated in the REAL Groups relative to matched students who did not participate. These findings suggest that REAL Groups may be an effective secondary prevention program for higher-risk Mexican heritage adolescents. PMID:22193861

  19. Claudins in a primary cultured puffer fish (Tetraodon nigroviridis) gill epithelium model alter in response to acute seawater exposure.

    PubMed

    Bui, Phuong; Kelly, Scott P

    2015-11-01

    Gill epithelium permeability and qualitative/quantitative aspects of gill claudin (cldn) tight junction (TJ) protein transcriptomics were examined with a primary cultured model gill epithelium developed using euryhaline puffer fish (Tetraodon nigroviridis) gills. The model was prepared using seawater-acclimated fish gills and was cultured on permeable cell culture filter supports. The model is composed of 1-2 confluent layers of gill pavement cells (PVCs), with the outer layer exhibiting prominent apical surface microridges and TJs between adjacent cells. During development of electrophysiological characteristics, the model exhibits a sigmoidal increase in transpithelial resistance (TER) and plateaus around 30 kΩcm(2). At this point paracellular movement of [(3)H]polyethylene glycol (PEG) 4000 was low at ~1.75 cm s(-1)×10(-7). When exposed to apical seawater (SW) epithelia exhibit a marked decrease in TER while PEG flux remained unchanged for at least 6 h. In association with this, transcript encoding cldn TJ proteins cldn3c, -23b, -27a, -27c, -32a and -33b increased during the first 6 h while cldn11a decreased. This suggests that these proteins are involved in maintaining barrier properties between gill PVCs of SW fishes. Gill cldn mRNA abundance also altered 6 and 12 h following abrupt SW exposure of puffer fish, but in a manner that differed qualitatively and quantitatively from the cultured model. This most likely reflects the cellular heterogeneity of whole tissue and/or the contribution of the endocrine system in intact fish. The current study provides insight into the physiological and transcriptomic response of euryhaline fish gill cells to a hyperosmotic environment.

  20. Claudins in a primary cultured puffer fish (Tetraodon nigroviridis) gill epithelium model alter in response to acute seawater exposure.

    PubMed

    Bui, Phuong; Kelly, Scott P

    2015-11-01

    Gill epithelium permeability and qualitative/quantitative aspects of gill claudin (cldn) tight junction (TJ) protein transcriptomics were examined with a primary cultured model gill epithelium developed using euryhaline puffer fish (Tetraodon nigroviridis) gills. The model was prepared using seawater-acclimated fish gills and was cultured on permeable cell culture filter supports. The model is composed of 1-2 confluent layers of gill pavement cells (PVCs), with the outer layer exhibiting prominent apical surface microridges and TJs between adjacent cells. During development of electrophysiological characteristics, the model exhibits a sigmoidal increase in transpithelial resistance (TER) and plateaus around 30 kΩcm(2). At this point paracellular movement of [(3)H]polyethylene glycol (PEG) 4000 was low at ~1.75 cm s(-1)×10(-7). When exposed to apical seawater (SW) epithelia exhibit a marked decrease in TER while PEG flux remained unchanged for at least 6 h. In association with this, transcript encoding cldn TJ proteins cldn3c, -23b, -27a, -27c, -32a and -33b increased during the first 6 h while cldn11a decreased. This suggests that these proteins are involved in maintaining barrier properties between gill PVCs of SW fishes. Gill cldn mRNA abundance also altered 6 and 12 h following abrupt SW exposure of puffer fish, but in a manner that differed qualitatively and quantitatively from the cultured model. This most likely reflects the cellular heterogeneity of whole tissue and/or the contribution of the endocrine system in intact fish. The current study provides insight into the physiological and transcriptomic response of euryhaline fish gill cells to a hyperosmotic environment. PMID:26239219

  1. Development of an in vitro quantal assay in primary cell cultures for a non-occluded baculo-like virus of penaeid shrimp.

    PubMed

    Tapay, L M; Lu, Y; Gose, R B; Nadala, E C; Brock, J A; Loh, P C

    1997-02-01

    An in vitro quantal assay (TCID50) for a non-occluded baculo-like virus isolate from naturally infected Penaeus japonicus obtained from China and experimentally infected P. stylirostris was developed using primary shrimp lymphoid cell cultures in Primaria 24-well tissue culture plates. The virus caused cytopathogenic effect (CPE) in the cell cultures as early as 2 day post-infection (p.i.). Initially, the cells rounded up and finally detached from the culture vessel as the infection progressed. At the present time, there is no established quantitative in vitro cell culture protocol for the assay of this baculo-like virus which has been reported by our laboratory to be highly pathogenic for P. stylirostris and P. vannamei, the two species of penaeid shrimp commercially cultured in Hawaii and the Western hemisphere. This quantal assay thus provides a simple and convenient method for the detection and assay of infectious virus in cultured penaeid shrimp.

  2. Political and cultural factors in achieving continuity with a primary health care provider at an Indian Health Service hospital.

    PubMed

    Dietrich, A J; Olson, A L

    1981-01-01

    A primary care system was established at Zuni-Ramah Indian Health Service Hospital and clinic in New Mexico. Continuity and coordination of care were added to a health care system that was already accountable, accessible, and comprehensive. The new system offered each patient a personal health care provider who worked as a member of a multidisciplinary team. In changing the health care system, special attention was given to its cultural and political setting, the village of Zuni. After thorough discussion with community and staff, community members' concerns about patients' privacy and free choice were better understood, and special efforts were made to safeguard them. Ongoing evaluation is essential to maintain continuity. Eight months after the primary care system was begun, 64 percent of patients who came for care had established a personal relationship with a health care provider. For 59 percent of the visits during the 1-month evaluation period, patients saw their regular provider and, for 82 percent, patients saw their provider or one of his or her team colleagues. These percentages include night and walk-in visits. The system required no extra funding or staff. The political process of planning and consultation helped anticipate and alleviate the community's concerns, but resistance from physician's assistants and some physicians was unexpected. A flexible approach has led to a gradual acceptance of this voluntary system. This experience with the people of Zuni village shows that a primary care system can be started in a rural Indian Health Service facility with minimal outside help. Apparent improvements in quality of care make the continuity of primary care worthy of further consideration in the IHS and similar health services systems.

  3. Two compartment model of diazepam biotransformation in an organotypical culture of primary human hepatocytes

    SciTech Connect

    Acikgoez, Ali; Karim, Najibulla; Giri, Shibashish; Schmidt-Heck, Wolfgang; Bader, Augustinus

    2009-01-15

    Drug biotransformation is one of the most important parameters of preclinical screening tests for the registration of new drug candidates. Conventional existing tests rely on nonhuman models which deliver an incomplete metabolic profile of drugs due to the lack of proper CYP450 expression as seen in human liver in vivo. In order to overcome this limitation, we used an organotypical model of human primary hepatocytes for the biotransformation of the drug diazepam with special reference to metabolites in both the cell matrix phase and supernatant and its interaction of three inducers (phenobarbital, dexamethasone, aroclor 1254) in different time responses (1, 2, 4, 8, 24 h). Phenobarbital showed the strongest inducing effect in generating desmethyldiazepam and induced up to a 150 fold increase in oxazepam-content which correlates with the increased availability of the precursor metabolites (temazepam and desmethyldiazepam). Aroclor 1254 and dexamethasone had the strongest inducing effect on temazepam and the second strongest on oxazepam. The strong and overlapping inductive role of phenobarbital strengthens the participation of CYP2B6 and CYP3A in diazepam N-demethylation and CYP3A in temazepam formation. Aroclor 1254 preferentially generated temazepam due to the interaction with CYP3A and potentially CYP2C19. In parallel we represented these data in the form of a mathematical model with two compartments explaining the dynamics of diazepam metabolism with the effect of these other inducers in human primary hepatocytes. The model consists of ten differential equations, with one for each concentration c{sub i,j} (i = diazepam, temazepam, desmethyldiazepam, oxazepam, other metabolites) and one for each compartment (j = cell matrix phase, supernatant), respectively. The parameters p{sub k} (k = 1, 2, 3, 4, 13) are rate constants describing the biotransformation of diazepam and its metabolites and the other parameters (k = 5, 6, 7, 8, 9, 10, 11, 12, 14, 15) explain the

  4. Inhibition of cytochrome P450s enhances (+)-usnic acid cytotoxicity in primary cultured rat hepatocytes.

    PubMed

    Shi, Qiang; Greenhaw, James; Salminen, William F

    2014-08-01

    (+)-Usnic acid (UA) is consumed as a dietary supplement to promote weight loss; however, dietary supplements containing UA have been associated with clinical cases of severe liver injury. UA has been shown to be hepatotoxic in rats and is extensively metabolized by hepatic cytochrome P450s (CYPs); therefore, we examined if UA metabolism results in the formation of cytotoxic metabolites or if metabolism is a detoxification process in primary rat hepatocytes. When CYP activity was suppressed by the non-isoenzyme-selective inhibitor SKF-525A (20 μM), or the CYP1A inhibitor alpha-naphthoflavone (10 μM), or the CYP3A inhibitor ketoconazole (25 μM), the cytotoxicity of UA at 3~6 μM after 3~20 h of exposure was significantly increased as measured by lactate dehydrogenase (LDH) leakage. At 2 h after UA exposure, an earlier time point prior to LDH release, these CYP inhibitors potentiated UA-induced inhibition of cellular respiration as determined by the Clark type oxygen electrode. Cellular adenosine triphosphate (ATP) depletion by UA was also exacerbated by these CYP inhibitors. The CYP2B/2C inhibitor, ticlopidine at 20 μM, showed no effects in parallel experiments. These data demonstrate that UA is bio-transformed to less toxic metabolites in rat primary hepatocytes, probably mainly by CYP1A and 3A, but not 2B/2C. Published 2013. This article is a U.S. Government work and is in the public domain in the USA.

  5. Characterizing primary human microglia: A comparative study with myeloid subsets and culture models.

    PubMed

    Melief, J; Sneeboer, M A M; Litjens, M; Ormel, P R; Palmen, S J M C; Huitinga, I; Kahn, R S; Hol, E M; de Witte, L D

    2016-11-01

    The biology of microglia has become subject to intense study, as they are widely recognized as crucial determinants of normal and pathologic brain functioning. While they are well studied in animal models, it is still strongly debated what specifies most accurately the phenotype and functioning of microglia in the human brain. In this study, we therefore isolated microglia from postmortem human brain tissue of corpus callosum (CC) and frontal cortex (CTX). The cells were phenotyped for a panel of typical microglia markers and genes involved in myeloid cell biology. Furthermore, their response to pro- and anti-inflammatory stimuli was assessed. The microglia were compared to key human myeloid cell subsets, including monocytes, monocyte-derived macrophages and monocyte-derived dendritic cells, and several commonly used microglial cell models. Protein and mRNA expression profiles partly differed between microglia isolated from CC and frontal cortex and were clearly distinct from other myeloid subsets. Microglia responded to both pro- (LPS or poly I:C) and anti-inflammatory (IL-4 or dexamethasone) stimuli. Interestingly, pro-inflammatory responses differed between microglia and monocyte-derived macrophages, as the former responded more strongly to poly I:C and the latter more strongly to LPS. Furthermore, we defined a large phenotypic discrepancy between primary human microglia and currently used microglial cell models and cell lines. In conclusion, we further delineated the unique and specific features that discriminate human microglia from other myeloid subsets, and we show that currently used cellular models only partly reflect the phenotype of primary human microglia. GLIA 2016;64:1857-1868.

  6. Preventing Cleavage of the Respiratory Syncytial Virus Attachment Protein in Vero Cells Rescues the Infectivity of Progeny Virus for Primary Human Airway Cultures

    PubMed Central

    Corry, Jacqueline; Johnson, Sara M.; Cornwell, Jessica

    2015-01-01

    ABSTRACT All live attenuated respiratory syncytial virus (RSV) vaccines that have advanced to clinical trials have been produced in Vero cells. The attachment (G) glycoprotein in virions produced in these cells is smaller than that produced in other immortalized cells due to cleavage. These virions are 5-fold less infectious for primary well-differentiated human airway epithelial (HAE) cell cultures. Because HAE cells are isolated directly from human airways, Vero cell-grown vaccine virus would very likely be similarly inefficient at initiating infection of the nasal epithelium following vaccination, and therefore, a larger inoculum would be required for effective vaccination. We hypothesized that Vero cell-derived virus containing an intact G protein would be more infectious for HAE cell cultures. Using protease inhibitors with increasing specificity, we identified cathepsin L to be the protease responsible for cleavage. Our evidence suggests that cleavage occurs in the late endosome or lysosome during endocytic recycling. Cathepsin L activity was 100-fold greater in Vero cells than in HeLa cells. In addition, cathepsin L was able to cleave the G protein in Vero cell-grown virions but not in HeLa cell-grown virions, suggesting a difference in G-protein posttranslational modification in the two cell lines. We identified by mutagenesis amino acids important for cleavage, and these amino acids included a likely cathepsin L cleavage site. Virus containing a modified, noncleavable G protein produced in Vero cells was 5-fold more infectious for HAE cells in culture, confirming our hypothesis and indicating the value of including such a mutation in future live attenuated RSV vaccines. IMPORTANCE Worldwide, RSV is the second leading infectious cause of infant death, but no vaccine is available. Experimental live attenuated RSV vaccines are grown in Vero cells, but during production the virion attachment (G) glycoprotein is cleaved. Virions containing a cleaved G protein

  7. Radiation Therapy in the Management of Head-and-Neck Cancer of Unknown Primary Origin: How Does the Addition of Concurrent Chemotherapy Affect the Therapeutic Ratio?

    SciTech Connect

    Chen, Allen M.; Farwell, D. Gregory; Lau, Derick H.; Li Baoqing; Luu, Quang; Donald, Paul J.

    2011-10-01

    Purpose: To determine how the addition of cisplatin-based concurrent chemotherapy to radiation therapy influences outcomes among a cohort of patients treated for head-and-neck cancer of unknown primary origin. Methods and Materials: The medical records of 60 consecutive patients treated by radiation therapy for squamous cell carcinoma of the head and neck presenting as cervical lymph node metastasis of occult primary origin were reviewed. Thirty-two patients (53%) were treated by concurrent chemoradiation, and 28 patients (47%) were treated by radiation therapy alone. Forty-five patients (75%) received radiation therapy after surgical resection, and 15 patients (25%) received primary radiation therapy. Thirty-five patients (58%) were treated by intensity-modulated radiotherapy. Results: The 2-year estimates of overall survival, local-regional control, and progression-free survival were 89%, 89%, and 79%, respectively, among patients treated by chemoradiation, compared to 90%, 92%, and 83%, respectively, among patients treated by radiation therapy alone (p > 0.05, for all). Exploratory analysis failed to identify any subset of patients who benefited from the addition of concurrent chemotherapy to radiation therapy. The use of concurrent chemotherapy was associated with a significantly increased incidence of Grade 3+ acute and late toxicity (p < 0.001, for both). Conclusions: Concurrent chemoradiation is associated with significant toxicity without a clear advantage to overall survival, local-regional control, and progression-free survival in the treatment of head-and-neck cancer of unknown primary origin. Although selection bias cannot be ignored, prospective data are needed to further address this question.

  8. Enhancement of anti-complementary and radical scavenging activities in the submerged culture of Cordyceps sinensis by addition of citrus peel.

    PubMed

    Choi, Jang Won; Ra, Kyung Soo; Kim, Seong Yeong; Yoon, Taek Joon; Yu, Kwang-Won; Shin, Kwang-Soon; Lee, Sam Pin; Suh, Hyung Joo

    2010-08-01

    To investigate the optimal conditions for the production of Cordyceps sinensis by the submerged culture method, glucosamine and exopolysaccharide (EPS) productivities were determined in culture broth containing different carbon sources, principally rice bran and citrus peel. An optimal medium composition (1.5% rice bran, 0.5% molasses, 3% CSL, 0.1% KH(2)PO(4), and 0.05% MgSO(4)) and the optimal condition (25 degrees C and 5-6 d culture time) for high EPS productivity with potent immune-stimulating activities were obtained. The addition of citrus peel to the culture of C. sinensis under the optimized conditions improved EPS productivity and glucosamine content. Furthermore, anti-complementary activity was higher (58.0-80.8%) using citrus peel as compared to no addition of citrus peel (48.2-68.7%). Antioxidant activity (AEAC value) of the citrus peel culture was high (284.3-384.6 mg/100g) compared to that of the culture without citrus peel (142.8-219.5mg/100g), indicating that the citrus peel helped enhance the anti-complementary and antioxidant activities of C. sinensis. PMID:20299211

  9. Assessing the prevalence of depression in Punjabi and English primary care attenders: the role of culture, physical illness and somatic symptoms.

    PubMed

    Bhui, Kamaldeep; Bhugra, Dinesh; Goldberg, David; Sauer, Justin; Tylee, Andre

    2004-09-01

    Previous studies exploring the prevalence of depression among South Asians reported inconsistent findings. Research artefacts due to sampling bias, measurements errors and a failure to include ethnographic methods may all explain this. We estimated the prevalence of depression, and variations of prevalence with culture, cultural adaptation, somatic symptoms and physical disability in a cross-sectional primary care survey of Punjabi and English attendees. We included a culture specific screening instrument, culturally adapted the instruments and offered bilingual interviews. We found that, compared with their English counterparts, depressive diagnoses were more common among Punjabis, Punjabi women, Punjabis with physical complaints and, contrary to expectation, even Punjabis with low scores for somatic symptoms.

  10. Simvastatin Inhibits Toll-like Receptor 8 (TLR8) Signaling in Primary Human Monocytes and Spontaneous Tumor Necrosis Factor Production from Rheumatoid Synovial Membrane Cultures

    PubMed Central

    Mullen, Lisa; Ferdjani, Jason; Sacre, Sandra

    2015-01-01

    Simvastatin has been shown to have antiinflammatory effects that are independent of its serum cholesterol lowering action, but the mechanisms by which these antiinflammatory effects are mediated have not been elucidated. To explore the mechanism involved, the effect of simvastatin on toll-like receptor (TLR) signaling in primary human monocytes was investigated. A short pretreatment with simvastatin dose-dependently inhibited the production of tumor necrosis factor (TNF)-α in response to TLR8 activation (but not TLR2, -4 or -5). Statins are known inhibitors of the cholesterol biosynthetic pathway, but, intriguingly, TLR8 inhibition could not be reversed by addition of mevalonate or geranylgeranyl pyrophosphate, downstream products of cholesterol biosynthesis. TLR8 signaling was examined in HEK 293 cells stably expressing TLR8, where simvastatin inhibited I kappa B kinase (IKK)α/β phosphorylation and subsequent nuclear factor (NF)-κB activation without affecting the pathway to activating protein-1 (AP-1). Because simvastatin has been reported to have antiinflammatory effects in RA patients and TLR8 signaling contributes to TNF production in human RA synovial tissue in culture, simvastatin was tested in these cultures. Simvastatin significantly inhibited the spontaneous release of TNF in this model, which was not reversed by mevalonate. Together, these results demonstrate a hitherto unrecognized mechanism of simvastatin inhibition of TLR8 signaling that may in part explain its beneficial antiinflammatory effects. PMID:26322850

  11. Cellular conservation of endangered midget buffalo (Lowland Anoa, Bubalus quarlesi) by establishment of primary cultured cell, and its immortalization with expression of cell cycle regulators.

    PubMed

    Fukuda, Tomokazu; Iino, Yuuka; Eitsuka, Takahiro; Onuma, Manabu; Katayama, Masafumi; Murata, Koichi; Inoue-Murayama, Miho; Hara, Kumiko; Isogai, Emiko; Kiyono, Tohru

    2016-10-01

    Lowland Anoa has become endangered due to hunting and human activity. Protection and breeding of endangered species in a controlled environment is the best way of conservation. However, it is not possible to adopt this approach for all endangered species because of the cost involved and the ever-increasing number of critically endangered species. In consideration of these limitations to the conventional conservation methods, we established a primary cell culture of endangered buffalo (Lowland Anoa, Bubalus quarlesi), for the preservation of this biological resource. In addition, we introduced human derived, mutant cyclin dependent kinase 4 (CDK4), Cyclin D, and telomerase reverse transcriptase (TERT) into the primary cells. The successful introduction of these three genes was confirmed by western blot with specific antibodies, and enzymatic activity. We also showed that the expression of mutant CDK4, Cyclin D, and TERT allows us to efficiently establish an immortalized cell line, with an intact chromosome pattern, from Lowland Anoa. To the best of our knowledge, this study is the first investigation that established an immortalized cell line of an endangered wild animal species.

  12. Cellular conservation of endangered midget buffalo (Lowland Anoa, Bubalus quarlesi) by establishment of primary cultured cell, and its immortalization with expression of cell cycle regulators.

    PubMed

    Fukuda, Tomokazu; Iino, Yuuka; Eitsuka, Takahiro; Onuma, Manabu; Katayama, Masafumi; Murata, Koichi; Inoue-Murayama, Miho; Hara, Kumiko; Isogai, Emiko; Kiyono, Tohru

    2016-10-01

    Lowland Anoa has become endangered due to hunting and human activity. Protection and breeding of endangered species in a controlled environment is the best way of conservation. However, it is not possible to adopt this approach for all endangered species because of the cost involved and the ever-increasing number of critically endangered species. In consideration of these limitations to the conventional conservation methods, we established a primary cell culture of endangered buffalo (Lowland Anoa, Bubalus quarlesi), for the preservation of this biological resource. In addition, we introduced human derived, mutant cyclin dependent kinase 4 (CDK4), Cyclin D, and telomerase reverse transcriptase (TERT) into the primary cells. The successful introduction of these three genes was confirmed by western blot with specific antibodies, and enzymatic activity. We also showed that the expression of mutant CDK4, Cyclin D, and TERT allows us to efficiently establish an immortalized cell line, with an intact chromosome pattern, from Lowland Anoa. To the best of our knowledge, this study is the first investigation that established an immortalized cell line of an endangered wild animal species. PMID:27449922

  13. An efficient and simple co-culture method for isolating primary human hepatic cells: Potential application for tumor microenvironment research.

    PubMed

    Dong, Wei; Lu, Aiguo; Zhao, Jingkun; Yin, Shuai; Ou, Baochi; Feng, Hao

    2016-10-01

    Co-cultivation of non-parenchymal cells (NPCs) and tumor cells from the same donor is important for metastatic cancer research. This study aimed to optimize a protocol for liver NPC isolation. Two novel 3D organotypic co‑culture models for hepatocyte, endothelial cell (EC) and Kupffer cell (KC) isolation were used. Long‑term cell co‑culture, density gradient centrifugation and magnetic‑activated cell sorting (MACS) were established. ECs were isolated from the co‑culture system; the purity of the ECs was 92±1.2%. The island‑like shape of hepatocytes was noted in the 3D co‑culture system, and spindle cells were found in the rest space. Immunofluorescence analysis showed a net structure; the connective tissue was positively stained with VE‑cadherin or CD68, which were ECs and KCs/macrophages. KCs were enriched in this system and separated by using selective adherence to plastic. Clec4f+ KCs consisted of 87±6.3% of these cells. Heterogeneous endothelium populations were detected, including sinusoid ECs, microvascular ECs and hepatic lymphatic vessel epithelial cells. In addition, hepatic progenitor cells were isolated and differentiated into hepatoblasts. Dendritic cells (DCs), invariant natural killer T (iNKT) cells were further separated by density gradient centrifugation and magnetic bead sorting. In the present study, high protein expression levels of desmin and GFAP were observed in the hepatic stellate cells (HSCs). Most of the HSCs were α‑SMA‑positive cells, which underlined the identity of activated HSCs. Intrahepatic human biliary epithelial cells (hBECs) were semi‑purified by centrifugation on a Percoll gradient and were further immunopurified. In conclusion, we provide an efficient long‑term culture method to obtain liver NPCs in sufficient number and purity. PMID:27498714

  14. Integrated and Culturally Relevant Care: A Model to Prepare Social Workers for Primary Care Behavioral Health Practice.

    PubMed

    Davis, Tamara S; Guada, Joe; Reno, Rebecca; Peck, Adriane; Evans, Shannon; Sigal, Laura Moskow; Swenson, Staci

    2015-01-01

    Policymakers and researchers emphasize needs for an integrated, effective, and efficient health care system to address well-documented disparities and inequities in care experienced by diverse populations. The Affordable Care Act, through its support of integrated health care, addresses social determinants of health with a goal of increasing access to care. Social work is poised to assume a central position in health care reform and integrated behavioral health, but must prepare practitioners to work alongside medical providers in health care settings. This article describes a social work field education model developed in partnership with community mental health and health care providers. The model, Integrated and Culturally Relevant Care, prepares social work students to provide behavioral health services in integrated primary care environments.

  15. Three diterpenes from Leonurus japonicus Houtt protect primary cultured rat cortical cells from glutamate-induced toxicity.

    PubMed

    Moon, Hyung-In

    2010-08-01

    To examine the cytoprotective effects of Leonurus japonicus Houtt, its protection against the glutamate-induced toxicity in primary cortical cultured neurons was tested. In order to clarify the cytoprotective mechanism(s) of this observed effect, isolation was performed to seek and identify active fractions and components. By such fractionation, three known diterpenes--prehispanolone (1), leojaponin (2) and iso-preleoheterin (3)--were isolated from the methanol extracts from the air-dried L. japonicus. Among these three compounds, leojaponin (2) exhibited significant cytoprotective activities against glutamate-induced toxicity, exhibiting cell viability of about 50%, at concentrations ranging from 0.1 microm to 10 microm. Therefore, the cytoprotective effect of L. japonicus might be due to the inhibition of glutamate-induced toxicity by the diterpenes it contains.

  16. Green Tea Polyphenols Attenuated Glutamate Excitotoxicity via Antioxidative and Antiapoptotic Pathway in the Primary Cultured Cortical Neurons.

    PubMed

    Cong, Lin; Cao, Chang; Cheng, Yong; Qin, Xiao-Yan

    2016-01-01

    Green tea polyphenols are a natural product which has antioxidative and antiapoptotic effects. It has been shown that glutamate excitotoxicity induced oxidative stress is linked to neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. In this study we explored the neuroprotective effect of green teen polyphenols against glutamate excitotoxicity in the primary cultured cortical neurons. We found that green tea polyphenols protected against glutamate induced neurotoxicity in the cortical neurons as measured by MTT and TUNEL assays. Green tea polyphenols were then showed to inhibit the glutamate induced ROS release and SOD activity reduction in the neurons. Furthermore, our results demonstrated that green tea polyphenols restored the dysfunction of mitochondrial pro- or antiapoptotic proteins Bax, Bcl-2, and caspase-3 caused by glutamate. Interestingly, the neuroprotective effect of green tea polyphenols was abrogated when the neurons were incubated with siBcl-2. Taken together, these results demonstrated that green tea polyphenols protected against glutamate excitotoxicity through antioxidative and antiapoptotic pathways.

  17. Regulation of UCP gene expression in brown adipocytes differentiated in primary culture. Effects of a new beta-adrenoceptor agonist.

    PubMed

    Champigny, O; Holloway, B R; Ricquier, D

    1992-07-01

    Primary cultures of precursor cells from mouse and rat brown adipose tissue (BAT) were used to study the effect of a new beta-agonist (ICI D7114) on the uncoupling protein (UCP) gene expression. ICI 215001 (the active metabolite of D7114) increased the expression of UCP and its mRNA in brown adipocytes differentiating in vitro in a dose-dependent manner. This stimulating effect was not inhibited by propranolol, a non-specific beta-antagonist, but was partially reduced by bupranolol, a beta 3-antagonist. No expression of UCP mRNA was ever induced by ICI 215001 in white adipocytes differentiated in vitro. It was concluded that the drug could affect the brown adipose cells through a beta 3-pathway. It could clearly modulate the expression of UCP in brown adipocytes differentiated in vitro, but was not able by itself to turn on the gene. PMID:1355051

  18. Achieving pH control in microalgal cultures through fed-batch addition of stoichiometrically-balanced growth media

    PubMed Central

    2013-01-01

    Background Lack of accounting for proton uptake and secretion has confounded interpretation of the stoichiometry of photosynthetic growth of algae. This is also problematic for achieving growth of microalgae to high cell concentrations which is necessary to improve productivity and the economic feasibility of commercial-scale chemical production systems. Since microalgae are capable of consuming both nitrate and ammonium, this represents an opportunity to balance culture pH based on a nitrogen feeding strategy that does not utilize gas-phase CO2 buffering. Stoichiometry suggests that approximately 36 weight%N-NH4+ (balance nitrogen as NO3-) would minimize the proton imbalance and permit high-density photoautotrophic growth as it does in higher plant tissue culture. However, algal media almost exclusively utilize nitrate, and ammonium is often viewed as ‘toxic’ to algae. Results The microalgae Chlorella vulgaris and Chlamydomonas reinhardtii exclusively u