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Sample records for primary cultured normal

  1. Cell-type Selective Phototoxicity Achieved with Chlorophyll-a Derived Photosensitizers in a Co-culture System of Primary Human Tumor and Normal Lung Cells

    PubMed Central

    Tracy, Erin C.; Bowman, Mary J.; Pandey, Ravindra K.; Henderson, Barbara W.; Baumann, Heinz

    2011-01-01

    The ATP-dependent transporter ABCG2 exports certain photosensitizers (PS) from cells, implying that the enhanced expression of ABCG2 by cancer cells may confer resistance to photodynamic therapy (PDT) mediated by those PS. In 35 patient-derived primary cultures of lung epithelial and stromal cells, PS with different subcellular localization and affinity for ABCG2 displayed cell-type specific retention both independent and dependent on ABCG2. In the majority of cases, the ABCG2 substrate 2-[1-hexyloxyethyl]-2-devinyl pyropheophorbide-a (HPPH) was lost from fibroblastic cells more rapidly than from their epithelial counterparts, even in the absence of detectable ABCG2 expression, facilitating selective eradication by PDT of epithelial over fibroblastic cells in tumor/stroma co-cultures. Pairwise comparison of normal and transformed epithelial cells also identified tumor cells with elevated or reduced retention of HPPH, depending on ABCG2. Enhanced ABCG2 expression led to the selective PDT survival of tumor cells in tumor/stroma co-cultures. This survival pattern was reversible through HPPH derivatives that are not ABCG2 substrates or the ABCG2 inhibitor imatinib mesylate. PS retention, not differences in subcellular distribution or cell signaling responses, was determining cell type selective death by PDT. These data suggest that up-front knowledge of tumor characteristics, specifically ABCG2 status, could be helpful in individualized PDT treatment design. PMID:21883244

  2. Cytotoxicity of HBD3 for dendritic cells, normal human epidermal keratinocytes, hTERT keratinocytes, and primary oral gingival epithelial keratinocytes in cell culture conditions.

    PubMed

    Leelakanok, Nattawut; Fischer, Carol L; Bates, Amber M; Guthmiller, Janet M; Johnson, Georgia K; Salem, Aliasger K; Brogden, Kim A; Brogden, Nicole K

    2015-12-01

    Human β-defensin 3 (HBD3) is a prominent host defense peptide. In our recent work, we observed that HBD3 modulates pro-inflammatory agonist-induced chemokine and cytokine responses in human myeloid dendritic cells (DCs), often at 20.0 μM concentrations. Since HBD3 can be cytotoxic in some circumstances, it is necessary to assess its cytotoxicity for DCs, normal human epidermal keratinocytes (NHEKs), human telomerase reverse transcriptase (hTERT) keratinocytes, and primary oral gingival epithelial (GE) keratinocytes in different cell culture conditions. Cells, in serum free media with resazurin and in complete media with 10% fetal bovine serum and resazurin, were incubated with 5, 10, 20, and 40 μM HBD3. Cytotoxicity was determined by measuring metabolic conversion of resazurin to resorufin. The lethal dose 50 (LD50, mean μM±Std Err) values were determined from the median fluorescent intensities of test concentrations compared to live and killed cell controls. The LD50 value range of HBD3 was 18.2-35.9 μM in serum-free media for DCs, NHEKs, hTERT keratinocytes, and GE keratinocytes, and >40.0 μM in complete media. Thus, HBD3 was cytotoxic at higher concentrations, which must be considered in future studies of HBD3-modulated chemokine and cytokine responses in vitro. PMID:26367466

  3. Cultured normal mammalian tissue and process

    NASA Technical Reports Server (NTRS)

    Goodwin, Thomas J. (Inventor); Prewett, Tacey L. (Inventor); Wolf, David A. (Inventor); Spaulding, Glenn F. (Inventor)

    1993-01-01

    Normal mammalian tissue and the culturing process has been developed for the three groups of organ, structural and blood tissue. The cells are grown in vitro under microgravity culture conditions and form three dimensional cell aggregates with normal cell function. The microgravity culture conditions may be microgravity or simulated microgravity created in a horizontal rotating wall culture vessel.

  4. Effects of insulin, dexamethasone and cytokines on {alpha}{sub 1}-acid glycoprotein gene expression in primary cultures of normal rat hepatocytes

    SciTech Connect

    Barraud, B.; Balavoine, S.; Feldmann, G.; Lardeux, B.

    1996-04-01

    While the effects of insulin, dexamethasone and cytokines on {alpha}{sub 1}-acid glycoprotein gene expression have been investigated in various hepatoma cell lines, the individual and combined effects of these components on the expression of this gene have been rarely studied in cultured normal rat hepatocytes. In this cell model, we have shown that mRNA levels of {alpha}{sub 1}-acid glycoprotein were not decreased at least during the first 24 h of culture under basal conditions. During these short-term cultures, the expression of {alpha}{sub 1}-acid glycoprotein in normal hepatocytes showed a high degree of responsiveness to dexamethasone alone (20-fold increase) and to dexamethasone associated with various cytokines (interleukin-1{beta}, interleukin-6 and tumor necrosis factor {alpha}) with a 40 to 100-fold increase depending on the cytokine. Insulin alone did not modify {alpha}{sub 1}-acid glycoprotein mRNA; however, this hormone exerted a positive effect (about 50% increase) in the presence of dexamethasone or dexamethasone with cytokines. These results indicate that the regulation of {alpha}{sub 1}-acid glycoprotein in cultured normal rat hepatocytes presents major differences when compared to reported observations in rat hepatoma cell lines. 49 refs., 2 figs., 2 tabs.

  5. 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.

  6. Kinetics and regulation of human keratinocyte stem cell growth in short-term primary ex vivo culture. Cooperative growth factors from psoriatic lesional T lymphocytes stimulate proliferation among psoriatic uninvolved, but not normal, stem keratinocytes.

    PubMed Central

    Bata-Csorgo, Z; Hammerberg, C; Voorhees, J J; Cooper, K D

    1995-01-01

    Flow cytometric analysis of primary ex vivo keratinocyte cultures demonstrated that stem cells, (beta 1 integrin+, keratin 1/keratin 10 [K1/K10-], proliferating cell nuclear antigen [PCNA-] [Bata-Csorgo, Zs., C. Hammerberg, J. J. Voorhees, and K. D. Cooper. 1993. J. Exp. Med. 178:1271-1281]) establish such cultures. This methodology also enabled the quantitation of synchronized recruitment of these cells from G0 into G1 of the cell cycle (PCNA expression), which preceded bright beta 1 integrin expression. (beta 1 integrinbright expression has been shown to be a characteristic feature of keratinocyte stem cells in culture (Jones, P. H., and F. M. Watt. 1993. Cell. 73:713-724). Using the above assay, we determined whether lesional T lymphocytes in psoriasis could be directly responsible for the induction of the stem cell hyperproliferation that is characteristic of this disease. Indeed, CD4+ T lymphocytes, cloned from lesional psoriatic skin and stimulated by immobilized anti-CD3 plus fibronectin, promoted psoriatic uninvolved keratinocyte stem cell proliferation via soluble factors. This induction appeared to be through accelerated recruitment of stem cells from their quiescent state (G0) into cell cycle. By contrast, normal keratinocyte stem cells exhibited no such growth stimulation. Supernatants exhibiting growth induction all contained high levels of GM-CSF and gamma-IFN, low IL-3 and TNF-alpha, and variable IL-4. Only anti-gamma-IFN antibody was able to neutralize growth stimulatory activity of the supernatants on psoriatic uninvolved keratinocyte stem cells. However, because recombinant gamma-IFN alone inhibited growth in this assay, these data suggest that, in psoriasis, gamma-IFN acts cooperatively with other growth factors in the immune induction of cell cycle progression by the normally quiescent stem cell keratinocytes. Images PMID:7529261

  7. 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.

  8. 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

  9. 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

  10. 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…

  11. 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

  12. 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

  13. 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.…

  14. 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…

  15. Human alveolar epithelial type II cells in primary culture.

    PubMed

    Mao, Pu; Wu, Songling; Li, Jianchun; Fu, Wei; He, Weiqun; Liu, Xiaoqing; Slutsky, Arthur S; Zhang, Haibo; Li, Yimin

    2015-02-01

    Alveolar epithelial type II (AEII) cells are a key structure and defender in the lung but also are the targets in many lung diseases, including acute respiratory distress syndrome, ventilator-induced lung injury, and pulmonary fibrosis. We sought to establish an optimized method for high yielding and long maintenance of characteristics of primary human AEII cells to facilitate the investigation of the mechanisms of lung diseases at the cellular and molecular levels. Adult human peripheral normal lung tissues of oncologic patients undergoing lung resection were collected. The AEII cells were isolated and identified by the expression of pro-surfactant protein (SP)C, epithelial sodium channel (αENaC) and cytokeratin (CK)-8, the lamellar bodies specific for AEII cells, and confirmed by the histology using electron microscopy. The phenotype of AEII cells was characterized by the expression of surfactant proteins (SP-A, SP-B, SP-C, SP-D), CK-8, KL-6, αENaC, and aquaporin (AQP)-3, which was maintained over 20 days. The biological activity of the primary human AEII cells producing SP-C, cytokines, and intercellular adhesion molecule-1 was vigorous in response to stimulation with tumor necrosis factor-α. We have modified previous methods and optimized a method for isolation of high purity and long maintenance of the human AEII cell phenotype in primary culture. This method provides an important tool for studies aiming at elucidating the molecular mechanisms of lung diseases exclusively in AEII cells. PMID:25677546

  16. Human alveolar epithelial type II cells in primary culture

    PubMed Central

    Mao, Pu; Wu, Songling; Li, Jianchun; Fu, Wei; He, Weiqun; Liu, Xiaoqing; Slutsky, Arthur S; Zhang, Haibo; Li, Yimin

    2015-01-01

    Alveolar epithelial type II (AEII) cells are a key structure and defender in the lung but also are the targets in many lung diseases, including acute respiratory distress syndrome, ventilator-induced lung injury, and pulmonary fibrosis. We sought to establish an optimized method for high yielding and long maintenance of characteristics of primary human AEII cells to facilitate the investigation of the mechanisms of lung diseases at the cellular and molecular levels. Adult human peripheral normal lung tissues of oncologic patients undergoing lung resection were collected. The AEII cells were isolated and identified by the expression of pro-surfactant protein (SP)C, epithelial sodium channel (αENaC) and cytokeratin (CK)-8, the lamellar bodies specific for AEII cells, and confirmed by the histology using electron microscopy. The phenotype of AEII cells was characterized by the expression of surfactant proteins (SP-A, SP-B, SP-C, SP-D), CK-8, KL-6, αENaC, and aquaporin (AQP)-3, which was maintained over 20 days. The biological activity of the primary human AEII cells producing SP-C, cytokines, and intercellular adhesion molecule-1 was vigorous in response to stimulation with tumor necrosis factor-α. We have modified previous methods and optimized a method for isolation of high purity and long maintenance of the human AEII cell phenotype in primary culture. This method provides an important tool for studies aiming at elucidating the molecular mechanisms of lung diseases exclusively in AEII cells. PMID:25677546

  17. 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…

  18. 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...

  19. Mapping the cellular and molecular heterogeneity of normal and malignant breast tissues and cultured cell lines

    PubMed Central

    2010-01-01

    Introduction Normal and neoplastic breast tissues are comprised of heterogeneous populations of epithelial cells exhibiting various degrees of maturation and differentiation. While cultured cell lines have been derived from both normal and malignant tissues, it remains unclear to what extent they retain similar levels of differentiation and heterogeneity as that found within breast tissues. Methods We used 12 reduction mammoplasty tissues, 15 primary breast cancer tissues, and 20 human breast epithelial cell lines (16 cancer lines, 4 normal lines) to perform flow cytometry for CD44, CD24, epithelial cell adhesion molecule (EpCAM), and CD49f expression, as well as immunohistochemistry, and in vivo tumor xenograft formation studies to extensively analyze the molecular and cellular characteristics of breast epithelial cell lineages. Results Human breast tissues contain four distinguishable epithelial differentiation states (two luminal phenotypes and two basal phenotypes) that differ on the basis of CD24, EpCAM and CD49f expression. Primary human breast cancer tissues also contain these four cellular states, but in altered proportions compared to normal tissues. In contrast, cultured cancer cell lines are enriched for rare basal and mesenchymal epithelial phenotypes, which are normally present in small numbers within human tissues. Similarly, cultured normal human mammary epithelial cell lines are enriched for rare basal and mesenchymal phenotypes that represent a minor fraction of cells within reduction mammoplasty tissues. Furthermore, although normal human mammary epithelial cell lines exhibit features of bi-potent progenitor cells they are unable to differentiate into mature luminal breast epithelial cells under standard culture conditions. Conclusions As a group breast cancer cell lines represent the heterogeneity of human breast tumors, but individually they exhibit increased lineage-restricted profiles that fall short of truly representing the intratumoral

  20. 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

  1. 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

  2. Provider and clinic cultural competence in a primary care setting.

    PubMed

    Paez, Kathryn A; Allen, Jerilyn K; Carson, Kathryn A; Cooper, Lisa A

    2008-03-01

    A multilevel approach that enhances the cultural competence of clinicians and healthcare systems is suggested as one solution to reducing racial/ethnic disparities in healthcare. The primary objective of this cross-sectional study was to determine if there is a relationship between the cultural competence of primary care providers and the clinics where they work. Forty-nine providers from 23 clinics in Baltimore, Maryland and Wilmington, Delaware, USA completed an on-line survey which included items assessing provider and clinic cultural competence. Using simple linear regression, it was found that providers with attitudes reflecting greater cultural motivation to learn were more likely to work in clinics with a higher percent of nonwhite staff, and those offering cultural diversity training and culturally adapted patient education materials. More culturally appropriate provider behavior was associated with a higher percent of nonwhite staff in the clinic, and culturally adapted patient education materials. Enhancing provider and clinic cultural competence may be synergistic strategies for reducing healthcare disparities. PMID:18164114

  3. Provider and Clinic Cultural Competence in a Primary Care Setting

    PubMed Central

    Paez, Kathryn A; Allen, Jerilyn K; Carson, Kathryn A; Cooper, Lisa A

    2008-01-01

    A multilevel approach that enhances the cultural competence of clinicians and healthcare systems is suggested as one solution to reducing racial/ethnic disparities in healthcare. The primary objective of this cross-sectional study was to determine if there is a relationship between the cultural competence of primary care providers and the clinics where they work. Forty-nine providers from 23 clinics in Baltimore, Maryland and Wilmington, Delaware, USA. completed an on-line survey which included items assessing provider and clinic cultural competence. Using simple linear regression, it was found that providers with attitudes reflecting greater cultural motivation to learn were more likely to work in clinics with a higher percent of nonwhite staff, and those offering cultural diversity training and culturally adapted patient education materials. More culturally appropriate provider behavior was associated with a higher percent of nonwhite staff in the clinic, and culturally adapted patient education materials. Enhancing provider and clinic cultural competence may be synergistic strategies for reducing healthcare disparities. PMID:18164114

  4. CCDC65 Mutation Causes Primary Ciliary Dyskinesia with Normal Ultrastructure and Hyperkinetic Cilia

    PubMed Central

    Horani, Amjad; Brody, Steven L.; Ferkol, Thomas W.; Shoseyov, David; Wasserman, Mollie G.; Ta-shma, Asaf; Wilson, Kate S.; Bayly, Philip V.; Amirav, Israel; Cohen-Cymberknoh, Malena; Dutcher, Susan K.; Elpeleg, Orly; Kerem, Eitan

    2013-01-01

    Background Primary ciliary dyskinesia (PCD) is a genetic disorder characterized by impaired ciliary function, leading to chronic sinopulmonary disease. The genetic causes of PCD are still evolving, while the diagnosis is often dependent on finding a ciliary ultrastructural abnormality and immotile cilia. Here we report a novel gene associated with PCD but without ciliary ultrastructural abnormalities evident by transmission electron microscopy, but with dyskinetic cilia beating. Methods Genetic linkage analysis was performed in a family with a PCD subject. Gene expression was studied in Chlamydomonas reinhardtii and human airway epithelial cells, using RNA assays and immunostaining. The phenotypic effects of candidate gene mutations were determined in primary culture human tracheobronchial epithelial cells transduced with gene targeted shRNA sequences. Video-microscopy was used to evaluate cilia motion. Results A single novel mutation in CCDC65, which created a termination codon at position 293, was identified in a subject with typical clinical features of PCD. CCDC65, an orthologue of the Chlamydomonas nexin-dynein regulatory complex protein DRC2, was localized to the cilia of normal nasal epithelial cells but was absent in those from the proband. CCDC65 expression was up-regulated during ciliogenesis in cultured airway epithelial cells, as was DRC2 in C. reinhardtii following deflagellation. Nasal epithelial cells from the affected individual and CCDC65-specific shRNA transduced normal airway epithelial cells had stiff and dyskinetic cilia beating patterns compared to control cells. Moreover, Gas8, a nexin-dynein regulatory complex component previously identified to associate with CCDC65, was absent in airway cells from the PCD subject and CCDC65-silenced cells. Conclusion Mutation in CCDC65, a nexin-dynein regulatory complex member, resulted in a frameshift mutation and PCD. The affected individual had altered cilia beating patterns, and no detectable

  5. 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

  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. Primary cancer cell culture: mammary-optimized vs conditional reprogramming.

    PubMed

    Alamri, Ahmad M; Kang, Keunsoo; Groeneveld, Svenja; Wang, Weisheng; Zhong, Xiaogang; Kallakury, Bhaskar; Hennighausen, Lothar; Liu, Xuefeng; Furth, Priscilla A

    2016-07-01

    The impact of different culture conditions on biology of primary cancer cells is not always addressed. Here, conditional reprogramming (CRC) was compared with mammary-optimized EpiCult-B (EpiC) for primary mammary epithelial cell isolation and propagation, allograft generation, and genome-wide transcriptional consequences using cancer and non-cancer mammary tissue from mice with different dosages of Brca1 and p53 Selective comparison to DMEM was included. Primary cultures were established with all three media, but CRC was most efficient for initial isolation (P<0.05). Allograft development was faster using cells grown in EpiC compared with CRC (P<0.05). Transcriptome comparison of paired CRC and EpiC cultures revealed 1700 differentially expressed genes by passage 20. CRC promoted Trp53 gene family upregulation and increased expression of epithelial differentiation genes, whereas EpiC elevated expression of epithelial-mesenchymal transition genes. Differences did not persist in allografts where both methods yielded allografts with relatively similar transcriptomes. Restricting passage (<7) reduced numbers of differentially expressed genes below 50. In conclusion, CRC was most efficient for initial cell isolation but EpiC was quicker for allograft generation. The extensive culture-specific gene expression patterns that emerged with longer passage could be limited by reducing passage number when both culture transcriptomes were equally similar to that of the primary tissue. Defining impact of culture condition and passage on the transcriptome of primary cells could assist experimental design and interpretation. For example, differences that appear with passage and culture condition are potentially exploitable for comparative studies targeting specific biological networks in different transcriptional environments. PMID:27267121

  8. Primary and Reversible Pisa Syndrome in Juvenile Normal Pressure Hydrocephalus

    PubMed Central

    Leon-Sarmiento, Fidias E.; Pradilla, Gustavo; del Rosario Zambrano, Maria

    2012-01-01

    Objective To report a case of Pisa syndrome in a patient with idiopathic normal pressure hydrocephalus, who had never been exposed to psychotropic medications. Methods A 26 years-old, Colombian, male patient, was referred because he had cognitive abnormalities, gait disturbances and urinary incontinence. This patient also displayed pleurothotonos. Neurofunctional evaluation of sensory and motor integration at peripheral and central nervous system levels were done. Results Pisa syndrome disappeared after spinal tap drainage with further gait, balance and behavioral improvement. A brainstem-thalamocortical deregulation of the central sensory and motor programming, due to the chaotic enlargement of brain ventricles was thought to be the pathophysiological mechanism underlying this case. Conclusion NPH must not be longer considered as an exclusive geriatric disorder. Further, uncommon movement disorders may appear with this disorder, which should be carefully approached to avoid iatrogenic and deleterious pharmacological interventions. PMID:23794788

  9. Rapid Selection of Mesenchymal Stem and Progenitor Cells in Primary Prostate Stromal Cultures

    PubMed Central

    Brennen, W. Nathaniel; Kisteman, L. Nelleke; Isaacs, John T.

    2016-01-01

    BACKGROUND Carcinoma-associated fibroblasts (CAFs) are a dominant component of the tumor microenvironment with pro-tumorigenic properties. Despite this knowledge, their physiologic origins remain poorly understood. Mesenchymal stem cells (MSCs) can be recruited from the bone marrow to areas of tissue damage and inflammation, including prostate cancer. MSCs can generate and have many overlapping properties with CAFs in preclinical models. METHODS Multiparameter flow cytometry and multipotent differentiation assays used to define MSCs in primary prostate stromal cultures derived from young (>25 yrs) organ donors and prostate cancer patients compared with bone marrow-derived stromal cultures. Population doubling times, population doublings, cell size, and differentiation potential determined under multiple culture conditions, including normoxia, hypoxia, and a variety of media. TGF-β measured by ELISA. RESULTS MSCs and stromal progenitors are not only present in normal and malignant prostate tissue, but are quickly selected for in primary stromal cultures derived from these tissues; becoming the dominant population within just a few passages. Growth potential inversely associated with TGF-β concentrations. All conditions generated populations with an average cell diameter >15 μm. All cultures tested had the ability to undergo osteogenic and chondrogenic differentiation, but unlike bone marrow-derived MSCs, primary stromal cultures derived from normal prostate tissue lack adipogenic differentiation potential. In contrast, a subset of stromal cultures derived from prostate cancer patients retain the ability to differentiate into adipocytes; a property that is significantly suppressed under hypoxic conditions in both bone marrow- and prostate-derived MSCs. CONCLUSIONS Primary prostate stromal cultures are highly enriched in cells with an MSC or stromal progenitor phenotype. The use of primary cultures such as these to study CAFs raises interesting implications when

  10. LIVER REGENERATION STUDIES WITH RAT HEPATOCYTES IN PRIMARY CULTURE

    EPA Science Inventory

    Adult rat parenchymal hepatocytes in primary culture can be induced to enter into DNA synthesis and mitosis. The optimal conditions for hepatocyte replication are low plating density (less than 10,000 cells/sq cm) and 50% serum from two-thirds partially hepatectomized rats (48 hr...

  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. Neurotrophic effect of isoquinoline derivatives in primary cortical culture.

    PubMed

    Ueda, Y; Nakanishi, H; Yoshida, K

    1999-01-01

    Recent studies indicate that the N-methyl-D-aspartate (NMDA) antagonist, (+)-1-methyl-1-phenyl-1,2,3,4-tetrahydroisoquinoline hydrochloride (FR 115427), enhanced neuronal survival in primary culture of cortical neurons from mouse embryos. In the present study isoquinoline derivatives were examined for the neurotrophic activity in primary culture of cortical neurons and were also examined for anti-NMDA activity. In spite of varying level of anti-NMDA activity, isoquinoline derivatives enhanced neuronal survival at the concentration of 10 microM. To elucidate of the mechanisms of neurotrophic activity in primary cortical culture, nicardipine and flunarizine, known calcium channel blockers, were also tested. Neither nicardipine nor flunarizine showed neurotrophic activity up to the doses causing toxicity in cultured neurons. NBQX, an AMPA receptor antagonist, was also tested for neurotrophic activity. However no enhancement of neuronal survival was observed. These data suggest that one of the mechanisms to promote neuronal survival may depend on the structure of isoquinoline ring. Moreover neurotrophic activity observed in our culture systems might not relate on anti-NMDA activity, blockade of voltage dependent L-type calcium channels and antagonization of AMPA receptor. PMID:10530799

  13. A rapid procedure for flow cytometric DNA analysis in cultures of normal and transformed epidermal cells.

    PubMed

    Tennenbaum, T; Giloh, H; Fusenig, N E; Kapitulnik, J

    1988-06-01

    A simple, rapid, and highly reproducible procedure for flow cytometric DNA analysis has been adapted for studying cell cycle kinetics in epidermal cell cultures. The preparation of cell nuclei and their staining with the fluorescent dye propidium iodide were performed directly on the culture dish, without prior suspension and fixation of the cells. Singly dispersed nuclei were produced by mild trypsinization of cells in the presence of the nonionic detergent Nonidet P-40 and spermine. The culture dishes could be kept frozen for prolonged periods of time before trypsinization and staining, without affecting either the recovery of nuclei or the cell cycle distribution profiles. This remarkable stability of cell nuclei greatly simplified the analysis of multiple samples in cell cycle kinetic studies. This method was used to analyze the cell cycle distribution in cultures of normal and transformed mouse epidermal cells, human colon carcinoma cells, primary bovine aortic endothelial cells, and fibroblastic and myogenic cell lines. This procedure should be very useful in studying growth kinetics, differentiation, and transformation of epidermal as well as other adherent cell types. PMID:2453587

  14. Epithelial cell cultures from normal and cancerous human tissues.

    PubMed

    Owens, R B; Smith, H S; Nelson-Rees, W A; Springer, E L

    1976-04-01

    Thirty epithelial cell strains were isolated from human carcinomas and normal epithelial tissues by collagenase digestion and selective removal of fibroblasts with trypsin-Versene. Most strains were obtained from metastatic carcinomas or epithelia of the urinary and intestinal tracts. The success rate for growth of both neoplastic and normal tissues (excluding skin) was 38%. Six of these strains showed gross morphologic and chromosome changes typical of malignant cells. Nine resembled normal epithelium. The other 15 exhibited some degree of morphologic change from normal. PMID:176412

  15. 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

  16. 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

  17. Alpha particle induced DNA damage and repair in normal cultured thyrocytes of different proliferation status.

    PubMed

    Lyckesvärd, Madeleine Nordén; Delle, Ulla; Kahu, Helena; Lindegren, Sture; Jensen, Holger; Bäck, Tom; Swanpalmer, John; Elmroth, Kecke

    2014-07-01

    Childhood exposure to ionizing radiation increases the risk of developing thyroid cancer later in life and this is suggested to be due to higher proliferation of the young thyroid. The interest of using high-LET alpha particles from Astatine-211 ((211)At), concentrated in the thyroid by the same mechanism as (131)I [1], in cancer treatment has increased during recent years because of its high efficiency in inducing biological damage and beneficial dose distribution when compared to low-LET radiation. Most knowledge of the DNA damage response in thyroid is from studies using low-LET irradiation and much less is known of high-LET irradiation. In this paper we investigated the DNA damage response and biological consequences to photons from Cobolt-60 ((60)Co) and alpha particles from (211)At in normal primary thyrocytes of different cell cycle status. For both radiation qualities the intensity levels of γH2AX decreased during the first 24h in both cycling and stationary cultures and complete repair was seen in all cultures but cycling cells exposed to (211)At. Compared to stationary cells alpha particles were more harmful for cycling cultures, an effect also seen at the pChk2 levels. Increasing ratios of micronuclei per cell nuclei were seen up to 1Gy (211)At. We found that primary thyrocytes were much more sensitive to alpha particle exposure compared with low-LET photons. Calculations of the relative biological effectiveness yielded higher RBE for cycling cells compared with stationary cultures at a modest level of damage, clearly demonstrating that cell cycle status influences the relative effectiveness of alpha particles. PMID:24769180

  18. 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

  19. 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

  20. The American abortion debate: culture war or normal discourse?

    PubMed

    Dillon, M

    1995-01-01

    This paper investigates whether James Hunter's culture war thesis is an apt characterization of the American abortion debate. The author focuses on three arguments central to Hunter's analysis: 1) that the abortion debate involves two paradigmatically opposed world views; 2) that debate about abortion, since it involves moral discourse, is structurally different than other political debates; and 3) that the new alignments in abortion politics are culturally significant. Examining existing research in each of these three domains, the author finds that the debate over abortion is more complex than suggested by Hunter. World views of pro-life and pro-choice activists, for example, share a commitment to some overlapping values; the argumentative structure of abortion discourse has a pattern rather similar to that of political debate more generally, and new alignments on abortion, such as that between the Catholic Church and the Southern Baptist Convention, do not displace historically embedded differences in symbolic resources and cultural orientation. As suggested by the author, it may be more helpful, therefore, to think of the abortion debate as an ongoing public conversation about America's cultural tradition and how it should be variously expressed in contemporary laws and practices. PMID:12320388

  1. 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

  2. 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

  3. Tetracycline-induced steatosis in primary canine hepatocyte cultures.

    PubMed

    Amacher, D E; Martin, B A

    1997-12-01

    Primary hepatocyte cultures prepared from male beagle dog liver were used to determine susceptibility of the canine liver to tetracycline-induced steatosis. The effects of the drug on mitochondrial lipid metabolism and intracellular triglyceride accumulation were monitored at the same time that steatosis was detected by light microscopy and quantitated using lipid-specific stains. Exposure of primary canine hepatocyte cultures to tetracycline for 24-48 h resulted in concentration-dependent, significant increases in the Oil Red O-stained lipid inclusions. Microscopic examination of the total stained areas suggested that increases over control levels were due primarily to the increase in the size of the lipid inclusions rather than in the number. Biochemical analyses for triglyceride content and histological staining with Nile red, another neutral lipid-specific dye, confirmed a specific increase in intracellular triglyceride following a 24-h exposure to noncytotoxic levels of tetracycline beta-oxidation studies based on the oxidation of [14C]palmitic acid or [14C]palmitoyl carnitine demonstrated a concentration-dependent inhibition of mitochondrial but not peroxisomal beta-oxidation in hepatocytes after a 24-h exposure to tetracycline. In vitro incubation of tetracycline with mitochondria isolated from dog liver showed similar concentration-dependent inhibition. This study clearly indicates that the canine hepatocyte is susceptible to tetracycline-induced steatosis. Triglyceride accumulation was concomitant with the inhibition of mitochondrial lipid metabolism, indicating that this is a primary mechanism leading to steatosis in dog hepatocytes following tetracycline exposure. PMID:9441722

  4. Epidermal growth factor receptor expression in primary cultured human colorectal carcinoma cells.

    PubMed Central

    Tong, W. M.; Ellinger, A.; Sheinin, Y.; Cross, H. S.

    1998-01-01

    In situ hybridization on human colon tissue demonstrates that epidermal growth factor receptor (EGFR) mRNA expression is strongly increased during tumour progression. To obtain test systems to evaluate the relevance of growth factor action during carcinogenesis, primary cultures from human colorectal carcinomas were established. EGFR distribution was determined in 2 of the 27 primary cultures and was compared with that in well-defined subclones derived from the Caco-2 cell line, which has the unique property to differentiate spontaneously in vitro in a manner similar to normal enterocytes. The primary carcinoma-derived cells had up to three-fold higher total EGFR levels than the Caco-2 subclones and a basal mitotic rate at least fourfold higher. The EGFR affinity constant is 0.26 nmol l(-1), which is similar to that reported in Caco-2 cells. The proliferation rate of Caco-2 cells is mainly induced by EGF from the basolateral cell surface where the majority of receptors are located, whereas primary cultures are strongly stimulated from the apical side also. This corresponds to a three- to fivefold higher level of EGFR at the apical cell surface. This redistribution of EGFR to apical plasma membranes in advanced colon carcinoma cells suggests that autocrine growth factors in the colon lumen may play a significant role during tumour progression. Images Figure 1 Figure 2 PMID:9667648

  5. Nucleoside transport in primary cultured rabbit tracheal epithelial cells.

    PubMed

    Mathias, Neil R; Wu, Sharon K; Kim, Kwang-Jin; Lee, Vincent H L

    2005-01-01

    The present study aimed at elucidating the mechanisms of nucleoside transport in primary cultured rabbit tracheal epithelial cells (RTEC) grown on a permeable filter support. Uptake of (3)H-uridine, the model nucleoside substrate, from the apical fluid of primary cultured RTEC was examined with respect to its dependence on Na(+), substrate concentration, temperature and its sensitivity to inhibitors, other nucleosides and antiviral nucleoside analogs. Apical (3)H-uridine uptake in primary cultured RTEC was strongly dependent on an inward Na(+) gradient and temperature. Ten micromolar nitro-benzyl-mercapto-purine-ribose (NBMPR) (an inhibitor of es-type nucleoside transport in the nanomolar range) did not further inhibit this process. (3)H-uridine uptake from apical fluid was inhibited by basolateral ouabain (10 microM) and apical phloridzin (100 microM), indicating that uptake may involve a secondary active transport process. Uridine uptake was saturable with a K(m) of 3.4 +/- 1.8 microM and the V(max) of 24.3 +/- 5.2 pmoles/mg protein/30 s. Inhibition studies indicated that nucleoside analogs that have a substitution on the nucleobase competed with uridine uptake from apical fluid, but those with modifications on the ribose sugar including acyclic analogs were ineffective. The pattern of inhibition of apical (3)H-uridine, (3)H-inosine and (3)H-thymidine uptake into RTEC cells by physiological nucleosides was consistent with multiple systems: A pyrimidine-selective transport system (CNT1); a broad nucleoside substrate transport system that excludes inosine (CNT4) and an equilibrative NBMPR-insensitive nucleoside transport system (ei type). These results indicate that the presence of apically located nucleoside transporters in the epithelial cells lining the upper respiratory tract can lead to a high accumulation of nucleosides in the trachea. At least one Na(+)-dependent, secondary, active transport process may mediate the apical absorption of nucleosides or

  6. 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. PMID:26001598

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

    PubMed

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

    2011-10-01

    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 μm 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 cells and their interactions with MVECL in response to pharmacological interventions and in the processes of disease, repair and regeneration. PMID:21624488

  8. 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

  9. Antisense oligodeoxynucleotide to the cystic fibrosis gene inhibits anion transport in normal cultured sweat duct cells

    SciTech Connect

    Sorscher, E.J.; Kirk, K.L.; Weaver, M.L.; Jilling, T.; Blalock, J.E.; LeBoeuf, R.D. )

    1991-09-01

    The authors have tested the hypothesis that the cystic fibrosis (CF) gene product, called the CF transmembrane conductance regulator (CFTR), mediates anion transport in normal human sweat duct cells. Sweat duct cells in primary culture were treated with oligodeoxynucleotides that were antisense to the CFTR gene transcript in order to block the expression of the wild-type CFTR. Anion transport in CFTR transcript antisense-treated cells was then assessed with a halide-specific dye, 6-methoxy-N-(3-sulfopropryl)quinolinium, and fluorescent digital imaging microscopy to monitor halide influx and efflux from single sweat duct cells. Antisense oligodeoxynucleotide treatment for 24 hr virtually abolished Cl{sup {minus}} transport in sweat duct cells compared with untreated cells or control cells treated with sense oligodeoxynucleotides. Br{sup {minus}} uptake into sweat duct cells was also blocked after a 24-hr CFTR transcript antisense treatments, but not after treatments for only 4 hr. Lower concentrations of antisense oligodeoxynucleotides were less effective at inhibiting Cl{sup {minus}} transport. These results indicate that oligodeoxynucleotides that are antisense to CFTR transcript inhibit sweat duct Cl{sup {minus}} permeability in both a time-dependent and dose-dependent manner. This approach provides evidence that inhibition of the expression of the wild-type CFTR gene in a normal, untransfected epithelial cell results in an inhibition of Cl{sup {minus}} permeability.

  10. Antisense oligodeoxynucleotide to the cystic fibrosis gene inhibits anion transport in normal cultured sweat duct cells.

    PubMed Central

    Sorscher, E J; Kirk, K L; Weaver, M L; Jilling, T; Blalock, J E; LeBoeuf, R D

    1991-01-01

    We have tested the hypothesis that the cystic fibrosis (CF) gene product, called the CF transmembrane conductance regulator (CFTR), mediates anion transport in normal human sweat duct cells. Sweat duct cells in primary culture were treated with oligodeoxynucleotides that were antisense to the CFTR gene transcript in order to block the expression of the wild-type CFTR. Anion transport in CFTR transcript antisense-treated cells was then assessed with a halide-specific dye, 6-methoxy-N-(3-sulfopropyl)quinolinium, and fluorescent digital imaging microscopy to monitor halide influx and efflux from single sweat duct cells. Antisense oligodeoxynucleotide treatment (3.9 or 1.3 microM) for 24 hr virtually abolished Cl- transport in sweat duct cells compared with untreated cells or control cells treated with sense oligodeoxynucleotides. Br- uptake into sweat duct cells was also blocked after a 24-hr CFTR transcript antisense treatment, but not after treatment for only 4 hr. Lower concentrations of antisense oligodeoxynucleotides were less effective at inhibiting Cl- transport. These results indicate that oligodeoxynucleotides that are antisense to CFTR transcript inhibit sweat duct Cl- permeability in both a time-dependent and dose-dependent manner. This approach provides evidence that inhibition of the expression of the wild-type CFTR gene in a normal, untransfected epithelial cell results in an inhibition of Cl- permeability. Images PMID:1715578

  11. 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. PMID:26690781

  12. 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.

  13. Normality, Deviance, Identity, Cultural Tracking and School Achievement: The Case of Ethiopian Jews in Israel

    ERIC Educational Resources Information Center

    Berhanu, Girma

    2005-01-01

    This article reveals the intrinsic connection between the constructs normality, identity, meaning, cultural tracking, and school achievement. In particular, it illuminates the indirect connection between cultural tracking and a reduction in the meaningful engagement of school tasks. As documented elsewhere, learning proceeds in a meaningful…

  14. The use of primary sentence stress by normal, aphasic, and autistic children.

    PubMed

    Baltaxe, C A; Guthrie, D

    1987-06-01

    Primary sentence stress is an important aspect of the English prosodic system. Its adequate use is a prerequisite in the development of normal intonation patterns. This study examined the use of primary sentence stress in autistic children with mean length of utterance (MLU) scores between 1.9 and 4.1 morphemes. Normal and aphasic subjects at similar MLU levels served as contrast groups. The primary sentence task required that the subjects verbally respond to a request for information and provide a description of a play situation. Toy manipulation was used to elicit the desired responses. Listener judgment served as the basis for analyzing results. Although all subjects were able to perform the task, differences were seen in the number of correct responses and in the pattern of stress misassignment. These results are at variance with a prediction of stress placement on grammatical grounds. An explanation is offered, based on pragmatic considerations and cognitive developmental trends in young children. PMID:2440848

  15. 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

  16. Preliminary study of spectral features of normal and malignant cell cultures

    NASA Astrophysics Data System (ADS)

    Atif, M.; Farooq, W. A.; Siddiqui, Maqsood A.; Al-Khedhairy, Abdulaziz A.

    2016-04-01

    In this study the fluorescence emission spectra of normal and malignant cell cultures were recorded at an excitation wavelength of 290 nm, corresponding to the higher fluorescence intensity at 350 nm (due to tryptophan) of three malignant cells and normal cells. Similarly, Stokes shift spectra were recorded for normal and malignant cell cultures with a shift, Δλ, of 70 nm. The Stokes shift shows the existence of discriminating features between normal and carcinoma cell lines due to the higher concentration of phenylalanine and tryptophan in carcinoma cell lines which are completely absent in normal cell lines. Hence, both the emission spectra and the Stokes shift spectra showed considerably different spectral features between the normal and malignant cells. The preliminary studies indicate the potential application of fluorescence spectroscopy for cancer detection using the spectral features of biofluorophores.

  17. Cellular and molecular characterization of a novel primary osteoblast culture from the vertebrate model organism Xenopus tropicalis.

    PubMed

    Bertin, Ariana; Hanna, Patricia; Otarola, Gaston; Fritz, Alan; Henriquez, Juan Pablo; Marcellini, Sylvain

    2015-04-01

    Osteogenesis is the fundamental process by which bones are formed, maintained and regenerated. The osteoblasts deposit the bone mineralized matrix by secreting large amounts of extracellular proteins and by allowing the biochemical conditions for the nucleation of hydroxyapatite crystals. Normal bone formation requires a tight control of osteoblastic activity, and therefore, osteoblasts represent a major focus of interest in biomedical research. Several crucial features of osteogenesis can be readily recapitulated using murine, avian and fish primary and immortalized osteoblastic cultures. Here, we describe a novel and straightforward in vitro culture of primary osteoblasts from the amphibian Xenopus tropicalis, a major vertebrate model organism. X. tropicalis osteoblasts can readily be extracted from the frontoparietal bone of pre-metamorphosing tadpole skulls by series of gentle protease treatments. Such primary cultures efficiently proliferate and can conveniently be grown at room temperature, in the absence of CO2, on a variety of substrates. X. tropicalis primary osteoblasts express well-characterized genes known to be active during osteogenesis of teleost fish, chick, mouse and human. Upon differentiation, such cultures mineralize and activate DMP1, an osteocyte-specific gene. Importantly, X. tropicalis primary osteoblasts can be efficiently transfected and respond to the forced activation of the bone morphogenetic protein pathway by increasing their nuclear levels of phospho-Smad. Therefore, this novel primary culture is amenable to experimental manipulations and represents a valuable tool for improving our understanding of the complex network of molecular interactions that govern vertebrate bone formation. PMID:25371327

  18. Testing gene therapy vectors in human primary nasal epithelial cultures

    PubMed Central

    Cao, Huibi; Ouyang, Hong; Ip, Wan; Du, Kai; Duan, Wenming; Avolio, Julie; Wu, Jing; Duan, Cathleen; Yeger, Herman; Bear, Christine E; Gonska, Tanja; Hu, Jim; Moraes, Theo J

    2015-01-01

    Cystic fibrosis (CF) results from mutations in the CF transmembrane conductance regulator (CFTR) gene, which codes for a chloride/bicarbonate channel in the apical epithelial membranes. CFTR dysfunction results in a multisystem disease including the development of life limiting lung disease. The possibility of a cure for CF by replacing defective CFTR has led to different approaches for CF gene therapy; all of which ultimately have to be tested in preclinical model systems. Primary human nasal epithelial cultures (HNECs) derived from nasal turbinate brushing were used to test the efficiency of a helper-dependent adenoviral (HD-Ad) vector expressing CFTR. HD-Ad-CFTR transduction resulted in functional expression of CFTR at the apical membrane in nasal epithelial cells obtained from CF patients. These results suggest that HNECs can be used for preclinical testing of gene therapy vectors in CF. PMID:26730394

  19. Cryopreservation of primary cell cultures of marine invertebrates.

    PubMed

    Odintsova, N; Kiselev, K; Sanina, N; Kostetsky, E

    2001-01-01

    Primary cell cultures obtained from somatic and larval tissues of bivalve molluscs and from embryos of sea urchins were frozen to -196 degrees C by two-step freezing using 10% dimethyl sulfoxide (DMSO) or/and trehalose (3-30 mg/ml) as cryoprotectants. We estimated both cell viability and the RNA synthetic activity after freeze-thaw. Total lipid extracts from the tissues of echinoderms examined as possible cryoprotective agents demonstrated a weak cryoprotective capacity. Mussel lipid extract was found to possess a considerable cryoprotective activity. Cryoprotective capacity of tested lipids correlated with their thermotropic behaviour. DMSO + trehalose combination was shown to be a favourable cryoprotectant and sea urchin blastula cells the most freezing-tolerant cells. PMID:11788872

  20. Testing gene therapy vectors in human primary nasal epithelial cultures.

    PubMed

    Cao, Huibi; Ouyang, Hong; Ip, Wan; Du, Kai; Duan, Wenming; Avolio, Julie; Wu, Jing; Duan, Cathleen; Yeger, Herman; Bear, Christine E; Gonska, Tanja; Hu, Jim; Moraes, Theo J

    2015-01-01

    Cystic fibrosis (CF) results from mutations in the CF transmembrane conductance regulator (CFTR) gene, which codes for a chloride/bicarbonate channel in the apical epithelial membranes. CFTR dysfunction results in a multisystem disease including the development of life limiting lung disease. The possibility of a cure for CF by replacing defective CFTR has led to different approaches for CF gene therapy; all of which ultimately have to be tested in preclinical model systems. Primary human nasal epithelial cultures (HNECs) derived from nasal turbinate brushing were used to test the efficiency of a helper-dependent adenoviral (HD-Ad) vector expressing CFTR. HD-Ad-CFTR transduction resulted in functional expression of CFTR at the apical membrane in nasal epithelial cells obtained from CF patients. These results suggest that HNECs can be used for preclinical testing of gene therapy vectors in CF. PMID:26730394

  1. Preparation of Extracellular Matrices Produced by Cultured and Primary Fibroblasts.

    PubMed

    Franco-Barraza, Janusz; Beacham, Dorothy A; Amatangelo, Michael D; Cukierman, Edna

    2016-01-01

    Fibroblasts secrete and organize extracellular matrix (ECM), which provides structural support for their adhesion, migration, and tissue organization, besides regulating cellular functions such as growth and survival. Cell-to-matrix interactions are vital for vertebrate development. Disorders in these processes have been associated with fibrosis, developmental malformations, cancer, and other diseases. This unit describes a method for preparing a three-dimensional matrix derived from fibroblastic cells; the matrix is three-dimensional, cell and debris free, and attached to a two-dimensional culture surface. Cell adhesion and spreading are normal on these matrices. This matrix can also be compressed into a two-dimensional matrix and solubilized to study the matrix biochemically. © 2016 by John Wiley & Sons, Inc. PMID:27245425

  2. Characterization of a unique technique for culturing primary adult human epithelial progenitor/“stem cells”

    PubMed Central

    2012-01-01

    Background Primary keratinocytes derived from epidermis, oral mucosa, and urothelium are used in construction of cell based wound healing devices and in regenerative medicine. This study presents in vitro technology that rapidly expands keratinocytes in culture by growing monolayers under large volumes of serum-free, essential fatty acid free, low calcium medium that is replaced every 24 hrs. Methods Primary cell cultures were produced from epidermal skin, oral mucosa and ureter by trypsinization of tissue. Cells were grown using Epilife medium with growth factors under high medium volumes. Once densely confluent, the keratinocyte monolayer produced cells in suspension in the overlying medium that can be harvested every 24 hrs. over a 7–10 day period. The cell suspension (approximately 8 X 105 cells/ml) is poured into a new flask to form another confluent monolayer over 2–4 days. This new culture, in turn produced additional cell suspensions that when serially passed expand the cell strain over 2–3 months, without the use of enzymes to split the cultures. The cell suspension, called epithelial Pop Up Keratinocytes (ePUKs) were analyzed for culture expansion, cell size and glucose utilization, attachment to carrier beads, micro-spheroid formation, induction of keratinocyte differentiation, and characterized by immunohistochemistry. Results The ePUKs expanded greatly in culture, attached to carrier beads, did not form micro-spheroids, used approximately 50% of medium glucose over 24 hrs., contained a greater portion of smaller diameter cells (8–10 microns), reverted to classical appearing cultures when returned to routine feeding schedules (48 hrs. and 15 ml/T-75 flask) and can be differentiated by either adding 1.2 mM medium calcium, or essential fatty acids. The ePUK cells are identified as cycling (Ki67 expressing) basal cells (p63, K14 expressing). Conclusions Using this primary culture technique, large quantities of epithelial cells can

  3. Biopsies of the normal-appearing urothelium in primary bladder cancer

    PubMed Central

    Librenjak, Davor; Novakovic, Zana Saratlija; Situm, Marijan; Milostic, Kazimir; Duvnjak, Mario

    2010-01-01

    Aim: The aim of the study was to determine the incidence of "positive" findings in biopsies of the normal-appearing urothelium near primary cancer and their influence on therapeutic decisions. Materials and Methods: Between January 2001 and October 2008, in 230 patients with primary bladder cancer during initial resection of tumor, we also performed random biopsy of surrounding normal-appearing urothelium. We analyzed retrospectively the number and type of positive biopsy findings and their impact on further treatment. Results: There were 40% of patients (92/230) whose normal-appearing urothelium biopsy revealed pathological findings such as tumor tissue, Tis, and dysplasia. In 24.4% of patients, the stage of the primary tumor was Ta (32/131), in 50% it was T1 stage (30/61), and in 79% T2 stage (30/38). When we assessed the grade of malignancy, we found 18% of biopsies with G1 tumors (16/88), 33% with G2 tumors (19/59), and 69% with G3 tumors (57/83). Tumor tissue that was found in the normal-appearing urothelium in biopsy specimens in 13% of patients was in stage Ta (17/131), in 16% it was T1 stage (10/61), and in 39% of patients, the tumor was in T2 stage (15/38). Pathological findings of random biopsies were crucial in changing therapeutical decisions in 4.6% (9/192) of patients. Conclusion: Biopsy of the normal-appearing urothelial tissue is easy to perform and may help in identifying patients with high risk of disease progression and recurrence. Based on our results and results from the literature we recommend this simple tool as part of the routine management during transurethral resection of primary bladder cancer. PMID:20882158

  4. ATP stimulates calcium influx in primary astrocyte cultures

    SciTech Connect

    Neary, J.T.; van Breemen, C.; Forster, E.; Norenberg, L.O.; Norenberg, M.D.

    1988-12-30

    The effect of ATP and other purines on /sup 45/Ca uptake was studied in primary cultures of rat astrocytes. Treatment of the cells with ATP for 1 to 30 min brought about an increase in cellular /sup 45/Ca. Stimulation of calcium influx by ATP was investigated using a 90 sec exposure to /sup 45/Ca and over a concentration range of 0.1 nM to 3 mM; a biphasic dose-response curve was obtained with EC50 values of 0.3 nM and 9 uM, indicating the presence of low and high affinity purinergic binding sites. Similar levels of /sup 45/Ca influx at 90 sec were observed with ATP, ADP and adenosine (all at 100 uM). Prior treatment of the cultures with LaCl3 blocked the purine-induced /sup 45/Ca influx. These findings indicate that one pathway for calcium entry in astrocytes involves purinergic receptor-operated, calcium channels.

  5. 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.

  6. Hypoxanthine-Guanine Phosphoribosyltransferase Deficiency: Activity in Normal, Mutant, and Heterozygote-Cultured Human Skin Fibroblasts

    PubMed Central

    Fujimoto, Wilfred Y.; Seegmiller, J. Edwin

    1970-01-01

    Cultured skin fibroblasts from patients deficient for the enzyme hypoxanthine-guanine phosphoribosyltransferase (PRT) activity show very low but nevertheless significant levels of apparent PRT enzyme despite absence of detectable activity (<0.004% of normal) in erythrocytes of the same patients. In fibroblasts this mutant enzyme is more heat labile than the normal enzyme. These findings indicate that PRT deficiency in this disorder is not due to a deletion mutation of the PRT locus. Individual cultured skin fibroblasts from heterozygote females for PRT deficiency show normal, intermediate, or very low levels of PRT activity. The mosaicism demonstrated in the heterozygotes for this X-linked disorder accounts for the cells with normal and very low activities of PRT. Intermediate activity can best be explained by the phenomenon of metabolic cooperation presumably from the transfer of either PRT enzyme or messenger RNA, from normal to mutant cells. Images PMID:5267139

  7. 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

  8. Stimulation and suppression of aldosterone in plasma of normal man and in primary aldosteronism

    PubMed Central

    Horton, R.

    1969-01-01

    The effect of stimulating and suppressive influences on plasma aldosterone in normal man and in patients with primary aldosteronism were studied using a sensitive double-isotope derivative assay for aldosterone. In normal sitting subjects, values were 9.2±0.9 (SE) mμg/100 ml and in subjects supine for 1 hr plasma aldosterone was 5.2±0.4 (SE) mμg/100 ml. Adrenocorticotropic hormone (ACTH), 0.5 U/hr, produced a rise of 46.8±22 (SE) mμg which was similar to the 1-hr effect of an infusion of a synthetic ACTH (β1-24, Cortrosyn). Angiotensin II in pressor amounts also increased plasma aldosterone 21.5±2.9 (SE) without change in plasma cortisol, whereas a subpressor dose ([unk]) had minimal effect. Fludrocortisone, 1.2 mg/day for 3 days, suppressed plasma aldosterone levels to 1.8±0.7 (SE) mμg/100 ml in five normal sitting subjects (P < 0.01); however, dexamethasone, 2 mg/day for 1-2 days, did not lower aldosterone concentration in plasma. In six patients with primary aldosteronism, plasma aldosterone on a normal sodium diet was 39.1±4.4 (SE) which differed significantly from normal sitting or supine subjects (P < 0.001). In contrast to the normal subjects, neither a pressor infusion of angiotensin II for 1 hr, nor fludrocortisone, 1.2 mg/day for 3 days, impressively altered plasma aldosterone levels. This approach appears to be useful for the study of the acute physiology and control mechanisms of aldosterone production in normal and hypertensive man. PMID:4307457

  9. Stages of Cell Cannibalism--Entosis--in Normal Human Keratinocyte Culture.

    PubMed

    Garanina, A S; Khashba, L A; Onishchenko, G E

    2015-11-01

    Entosis is a type of cell cannibalism during which one cell penetrates into another cell and usually dies inside it. Researchers mainly pay attention to initial and final stages of entosis. Besides, tumor cells in suspension are the primary object of studies. In the present study, we investigated morphological changes of both cells-participants of entosis during this process. The substrate-dependent culture of human normal keratinocytes HaCaT was chosen for the work. A combination of light microscopy and scanning electron microscopy was used to prove that one cell was completely surrounded by the plasma membrane of another cell. We investigated such "cell-in-cell" structures and described the structural and functional changes of both cells during entosis. The outer cell nucleus localization and shape were changed. Gradual degradation of the inner cell nucleus and of the junctions between the inner and the outer cells was revealed. Moreover, repeated redistribution of the outer cell membrane organelles (Golgi apparatus, lysosomes, mitochondria, and autophagosomes), rearrangement of its cytoskeleton, and change in the lysosomal, autophagosomal, and mitochondrial state in both entotic cells were observed during entosis. On the basis of these data, we divided entosis into five stages that make it possible to systematize description of this type of cell death. PMID:26615438

  10. Lithium chloride induces mesenchymal‑to‑epithelial reverting transition in primary colon cancer cell cultures.

    PubMed

    Costabile, Valeria; Duraturo, Francesca; Delrio, Paolo; Rega, Daniela; Pace, Ugo; Liccardo, Raffaella; Rossi, Giovanni Battista; Genesio, Rita; Nitsch, Lucio; Izzo, Paola; De Rosa, Marina

    2015-05-01

    Epithelial‑to‑mesenchymal transition (EMT) confers stem cell‑like phenotype and more motile properties to carcinoma cells. During EMT, the expression of E‑cadherin decreases, resulting in loss of cell‑cell adhesion and increased migration. Expression of Twist1 and other pleiotropic transcription factors, such as Snail, is known to activate EMT. We established primary colon cancer cell cultures from samples of operated patients and validated cultures by cytogenetic and molecular biology approaches. Western blot assay, quantitative real‑time PCR and immunofluorescence were performed to investigate the expression of E‑cadherin, vimentin, β‑catenin, cytokeratin‑20 and ‑18, Twist1, Snail, CD44, cyclooxygenase‑2 (COX2), Sox2, Oct4 and Nanog. Moreover, cell differentiation was induced by incubation with LiCl‑containing medium for 10 days. We observed that these primary colorectal cancer (CRC) cells lost expression of the E‑cadherin epithelial marker, which was instead expressed in cancer and normal colon mucosa of the same patient, while overexpressed vimentin (mesenchymal marker), Twist1, Snail (EMT markers) and COX2. Cytokeratin‑18 was expressed both in tissues and cell cultures. Expression of stem cell markers, such as CD44, Oct4 and Nanog, were also observed. Following differentiation with the glycogen synthase kinase 3β (GSK3β) inhibitor LiCl, the cells began to express E‑cadherin and, at once, Twist1 and Snail expression was strongly downregulated, suggesting a MET‑reverting process. In conclusion, we established primary colon mesenchymal cancer cell cultures expressing mesenchymal and epithelial biomarkers together with high level of EMT transcription factors. We propose that they could represent a good model for studying EMT and its reverting mechanism, the mesenchymal‑to‑epithelial transition (MET). Our observation indicates that LiCl, a GSK3β inhibitor, induces MET in vitro, suggesting that LiCl and GSK3β could represent

  11. Ultrasonic differentiation of normal versus malignant breast epithelial cells in monolayer cultures.

    PubMed

    Doyle, Timothy E; Goodrich, Jeffrey B; Ambrose, Brady J; Patel, Hemang; Kwon, Soonjo; Pearson, Lee H

    2010-11-01

    Normal and malignant mammary epithelial cells were studied using laboratory measurements, wavelet analysis, and numerical simulations of monolayer cell cultures to determine whether microscopic breast cancer can be detected in vitro with high-frequency ultrasound. Pulse-echo waveforms were acquired by immersing a broadband, unfocused 50-MHz transducer in the growth media of cell culture well plates and collecting the first reflection from the well bottoms. The simulations included a multilayer pulse-reflection model and a model of two-dimensional arrays of spherical cells and nuclei. The results show that normal and malignant cells produce time-domain signals and spectral features that are significantly different. PMID:21110531

  12. 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

  13. 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...

  14. Human Airway Primary Epithelial Cells Show Distinct Architectures on Membrane Supports Under Different Culture Conditions.

    PubMed

    Min, Kyoung Ah; Rosania, Gus R; Shin, Meong Cheol

    2016-06-01

    To facilitate drug development for lung delivery, it is highly demanding to establish appropriate airway epithelial cell models as transport barriers to evaluate pharmacokinetic profiles of drug molecules. Besides the cancer-derived cell lines, as the primary cell model, normal human bronchial epithelial (NHBE) cells have been used for drug screenings because of physiological relevance to in vivo. Therefore, to accurately interpret drug transport data in NHBE measured by different laboratories, it is important to know biophysical characteristics of NHBE grown on membranes in different culture conditions. In this study, NHBE was grown on the polyester membrane in a different medium and its transport barrier properties as well as cell architectures were fully characterized by functional assays and confocal imaging throughout the days of cultures. Moreover, NHBE cells on inserts in a different medium were subject to either of air-interfaced culture (AIC) or liquid-covered culture (LCC) condition. Cells in the AIC condition were cultivated on the membrane with medium in the basolateral side only, whereas cells with medium in apical and basolateral sides under the LCC condition. Quantitative microscopic imaging with biophysical examination revealed distinct multilayered architectures of differentiated NHBE cells, suggesting NHBE as functional cell barriers for the lung-targeting drug transport. PMID:26818810

  15. Chemosensitivity testing of primary cultures of Merkel cell cancer.

    PubMed

    Kearsley, J H; Hurst, T; Khoo, S K

    1993-10-01

    Twenty-seven tumor specimens from patients with Merkel cell carcinoma (MCC) were tested for chemosensitivity against a battery of nine cytotoxic drugs in a short-term antimetabolic assay measuring inhibition of thymidine incorporation. Dose-response curves were constructed by plotting drug concentration in micrograms/ml versus % control [3H]thymidine incorporation. Specimens were considered 'sensitive' to a drug if, at the approximate peak plasma concentration (PPC), the inhibition of [3H]thymidine was greater than 50% when compared with untreated control primary cultures. The assay revealed a 'sensitive' tumor in 19 of 20 specimens and 16 of 17 patients had a tumor that was 'sensitive' to at least one drug tested in the assay system. The highest sensitivity in order of frequency was found with doxorubicin, epirubicin, cyclophosphamide, etoposide and cisplatin. At least 40% of the tumors were 'sensitive' to these five drugs. Cyclophosphamide was chosen as the most active drug (at PPC) in 10 of 19 assays (53%), etoposide in seven of 17 (41%), doxorubicin in four of 19 (21%), chlorambucil in one of 12 (8%) and cisplatin in one of 18 (5%) of assays. Though our results are preliminary, we have identified for the first time a range of cytotoxic drugs which appear effective against MCC in vitro. Our main task now is to determine whether our in vitro predictive assay will correlate with clinical benefit to the patient. PMID:8292815

  16. Characterization of cultured cholangiocytes isolated from livers of patients with primary sclerosing cholangitis.

    PubMed

    Tabibian, James H; Trussoni, Christy E; O'Hara, Steven P; Splinter, Patrick L; Heimbach, Julie K; LaRusso, Nicholas F

    2014-10-01

    Primary sclerosing cholangitis (PSC) is a chronic, idiopathic cholangiopathy. The role of cholangiocytes (biliary epithelial cells) in PSC pathogenesis is unknown and remains an active area of research. Here, through cellular, molecular and next-generation sequencing (NGS) methods, we characterize and identify phenotypic and signaling features of isolated PSC patient-derived cholangiocytes. We isolated cholangiocytes from stage 4 PSC patient liver explants by dissection, differential filtration and immune-magnetic bead separation. We maintained cholangiocytes in culture and assessed for: (i) cholangiocyte, cell adhesion and inflammatory markers; (ii) proliferation rate; (iii) transepithelial electrical resistance (TEER); (iv) cellular senescence; and (v) transcriptomic profiles by NGS. We used two well-established normal human cholangiocyte cell lines (H69 and NHC) as controls. Isolated PSC cells expressed cholangiocyte (eg, cytokeratin 7 and 19) and epithelial cell adhesion markers (EPCAM, ICAM) and were negative for hepatocyte and myofibroblast markers (albumin, α-actin). Proliferation rate was lower for PSC compared with normal cholangiocytes (4 vs 2 days, respectively, P<0.01). Maximum TEER was also lower in PSC compared with normal cholangiocytes (100 vs 145 Ωcm(2), P<0.05). Interleukin-6 (IL-6) and IL-8 (protein and mRNA) were both increased compared with NHCs and H69s (all P<0.01). The proportion of cholangiocytes staining positive for senescence-associated β-galactosidase was higher in PSC cholangiocytes compared with NHCs (48% vs 5%, P<0.01). Finally, NGS confirmed cholangiocyte marker expression in isolated PSC cholangiocytes and extended our findings regarding pro-inflammatory and senescence-associated signaling. In conclusion, we have demonstrated that high-purity cholangiocytes can be isolated from human PSC liver and grown in primary culture. Isolated PSC cholangiocytes exhibit a phenotype that may reflect their in vivo contribution to disease and

  17. Characterization of cultured cholangiocytes isolated from livers of patients with primary sclerosing cholangitis

    PubMed Central

    Tabibian, James H.; Trussoni, Christy E.; O’Hara, Steven P.; Splinter, Patrick L.; Heimbach, Julie K.; LaRusso, Nicholas F.

    2014-01-01

    Primary sclerosing cholangitis (PSC) is a chronic, idiopathic cholangiopathy. The role of biliary epithelial cells, i.e. cholangiocytes, in PSC pathogenesis is unknown and remains an active area of research. Here, through cellular, molecular, and next-generation sequencing (NGS) methods, we characterize and identify phenotypic and signaling features of isolated PSC patient-derived cholangiocytes. We isolated cholangiocytes from stage 4 PSC patient liver explants by dissection, differential filtration, and immune-magnetic bead separation. We maintained cholangiocytes in culture and assessed for: i) cholangiocyte, cell adhesion, and inflammatory markers; ii) proliferation rate; iii) transepithelial electrical resistance (TEER); iv) cellular senescence; and v) transcriptomic profiles by NGS. We used two well-established normal human cholangiocyte cell lines (H69 and NHC) as controls. Isolated PSC cells expressed cholangiocyte (e.g. cytokeratin 7 and 19) and epithelial cell adhesion markers (EPCAM, ICAM) and were negative for hepatocyte and myofibroblast markers (albumin, α-actin). Proliferation rate was lower for PSC compared to normal cholangiocytes (4 vs. 2 days, respectively, p<0.01). Maximum TEER was also lower in PSC compared to normal cholangiocytes (100 vs. 145 Ωcm2, p<0.05). IL-6 and IL-8 (protein and mRNA) were both increased compared to NHCs and H69s (all p<0.01). The proportion of cholangiocytes staining positive for senescence-associated β-galactosidase was higher in PSC cholangiocytes compared to NHCs (48% vs. 5%, p<0.01). Lastly, NGS confirmed cholangiocyte marker expression in isolated PSC cholangiocytes and extended our findings regarding pro-inflammatory and senescence-associated signaling. In conclusion, we have demonstrated that high-purity cholangiocytes can be isolated from human PSC liver and grown in primary culture. Isolated PSC cholangiocytes exhibit a phenotype that may reflect their in vivo contribution to disease and serve as a vital

  18. Phosphorylation of intracellular proteins related to the multihormonal regulation of prolactin: comparison of normal anterior pituitary cells in culture with the tumor-derived GH cell lines

    SciTech Connect

    Beretta, L.; Boutterin, M.C.; Sobel, A.

    1988-01-01

    We have previously identified a group of cytoplasmic phosphoproteins (proteins 1-11) whose phosphorylation could be related, on a pharmacological basis, to the multihormonal regulation of PRL synthesis and release in the anterior pituitary tumor-derived GH cell lines. Phosphoproteins with identical migration properties on two-dimensional electrophoresis gels were also detectable in normal rat anterior pituitary cells in culture. We designed appropriate culture and (/sup 32/P) phosphate-labeling conditions allowing to analyze the regulation of the phosphorylation of these proteins in normal pituitary cells. TRH, 12-O-tetradecanoylphorbol-13-acetate, and vasoactive intestinal peptide induced the same qualitative changes in phosphorylation of proteins 1-11 in normal as in GH cells. Quantitative differences observed are most likely due to the heterogeneity of primary pituitary cultures. Phosphorylation changes affecting proteins 14-16, not previously detected in GH cells, were also observed with normal anterior pituitary cells. GH cell lines have lost the sensitivity of pituitary lactotrophs for dopamine, an important physiological inhibitor of PRL synthesis and release. In normal anterior pituitary cells in culture, dopamine inhibited also the TRH-stimulated phosphorylation of proteins 1-10, thus strengthening the correlation between phosphorylation of these proteins and multihormonal regulation of pituitary cell functions. Our results indicate: 1) that the same phosphoproteins as in GH cells are related to the multihormonal regulation of nontumoral, normal anterior pituitary cells in culture; 2) that dopamine acts by interfering with the phosphorylation of these proteins.

  19. Regulation of heme metabolism in normal and sideroblastic bone marrow cells in culture

    SciTech Connect

    Ibraham, N.G.; Lutton, J.D.; Hoffman, R.; Levere, R.D.

    1985-05-01

    Heme metabolism was examined in developing in vitro erythroid colonies (CFUE) and in bone marrow samples taken directly from four normal donors and four patients with sideroblastic anemia. Maximum activities of delta-aminolevulinic acid synthase (ALAS), ALA dehydratase (ALAD), and /sup 14/C-ALA incorporation into heme were achieved in normal marrow CFUE after 8 days of culture, whereas heme oxygenase progressively decreased to low levels of activity during the same period. Assays on nucleated bone marrow cells taken directly from patients revealed that ALAS activity was considerably reduced in idiopathic sideroblastic anemia (IASA) and X-linked sideroblastic anemia (X-SA) bone marrow specimens, whereas the activity increased more than twofold (normal levels) when cells were assayed from 8-day CFUE. In all cases, ALAD activity appeared to be within normal levels. Measurement of heme synthesis revealed that normal levels of /sup 14/C-ALA incorporation into heme were achieved in IASA cells but were reduced in X-SA cells. In marked contrast to levels in normal cells, heme oxygenase was found to be significantly elevated (two- to fourfold) in bone marrow cells taken directly from patients with IASA and X-SA. Results from this study demonstrate that IASA and X-SA bone marrow cells have disturbances in ALAS and heme metabolism, and that erythropoiesis (CFUE) can be restored to normal levels when cells are cultured in methylcellulose.

  20. Effect of iprodione, a dicarboximide fungicide, on primary cultured rainbow trout (Oncorhynchus mykiss) hepatocytes.

    PubMed

    Radice, S; Ferraris, M; Marabini, L; Grande, S; Chiesara, E

    2001-09-01

    As is known from literature, iprodione, a dicarboximide fungicide, has a highly specific action, with a capacity to cause oxidative damage through production of free oxygen radicals (ROS), but it does not appear to be species selective. Since this substance is able to diffuse in water, evaluation of its capacity to induce oxidative damage in an aquatic organism such as the rainbow trout (Oncorhynchus mykiss) was considered of particular interest. A study was, therefore, undertaken to investigate the effect of iprodione on free radicals (ROS) and malondialdehyde (MDA) production, reduced glutathione (GSH) content and catalase activity (CAT), in primary cultured trout hepatocytes, following treatment with 0.2, 0.3 and 0.4 mM concentrations for a 24-h period. The iprodione 0.3 and 0.4 mM concentrations increased both ROS and MDA production and decreased GSH content and CAT activity. These results suggest that iprodione is able to produce oxidative damage in primary cultured fish hepatocytes, thus confirming that its action is specific, but not species selective. It is also well known that ROS production in fungi is due to interaction with the flavin enzyme NADPH cytochrome c reductase to the extent that the normal electron flow from NADPH to cytochrome c is blocked. In contrast, we observed that, in primary cultured trout hepatocytes, iprodione appears to have no effect on NADPH cytochrome c reductase activity. It is, therefore, possible to presume that the mechanism of oxidative damage in trout hepatocytes differs from that observed in fungi. Moreover, our experiments also demonstrate that iprodione is able to induce "in vitro" CYP1A1, leading to the conclusion that the production of ROS is due to this phenomenon. PMID:11451425

  1. Measurement of Primary Ejecta From Normal Incident Hypervelocity Impact on Lunar Regolith Simulant

    NASA Technical Reports Server (NTRS)

    Edwards, David L.; Cooke, William; Moser, Danielle; Swift, Wesley

    2007-01-01

    The National Aeronautics and Space Administration (NASA) continues to make progress toward long-term lunar habitation. Critical to the design of a lunar habitat is an understanding of the lunar surface environment. A subject for further definition is the lunar primary ejecta environment. The document NASA SP-8013 was developed for the Apollo program and is the latest definition of the primary ejecta environment. There is concern that NASA SP-8013 may over-estimate the lunar primary ejecta environment. NASA's Meteoroid Environment Office (MEO) has initiated several tasks to improve the accuracy of our understanding of the lunar surface primary ejecta environment. This paper reports the results of experiments on projectile impact into pumice targets, simulating lunar regolith. The Ames Vertical Gun Range (AVGR) was used to accelerate spherical Pyrex projectiles of 0.29g to velocities ranging between 2.5 km/s and 5.18 km/s. Impact on the pumice target occurred at normal incidence. The ejected particles were detected by thin aluminum foil targets placed around the pumice target in a 0.5 Torr vacuum. A simplistic technique to characterize the ejected particles was formulated. Improvements to this technique will be discussed for implementation in future tests.

  2. 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

  3. Turnover of inositol pentakisphosphates, inositol hexakisphosphate and diphosphoinositol polyphosphates in primary cultured hepatocytes.

    PubMed Central

    Glennon, M C; Shears, S B

    1993-01-01

    We have used a non-transformed cell model, the primary cultured hepatocyte, to explore the turnover of inositol hexakisphosphate, multiple isomers of inositol pentakisphosphate and two novel diphosphoinositol polyphosphates. All of these compounds gradually accumulated radioactivity throughout a 70 h period of labelling with [3H]inositol. However, a rapid metabolic rate was revealed upon inhibition of diphosphoinositol polyphosphate biphosphatase(s) with 1 mM fluoride for 40 min: this treatment elevated levels of [3H]diphosphoinositol polyphosphates up to 10-fold, indicating that their cellular pools were normally turning over at least 10 times every 40 min. This was accompanied by a turnover of about 10% of the pool of inositol hexakisphosphate. Control experiments established that 200 nM vasopressin brought about a typical activation of phospholipase C in hepatocytes after 62 h of primary culture. This agonist treatment did not affect steady-state levels of [3H]inositol pentakisphosphates, [3H]inositol hexakisphosphate or [3H]diphosphoinositol polyphosphates. However, prolonged treatment of hepatocytes with 2 microM thapsigargin reduced steady-state levels of [3H]diphosphoinositol polyphosphates by 50-70%. This effect of thapsigargin was also observed in the presence of fluoride, indicating that thapsigargin inhibited the rate of synthesis of diphosphoinositol polyphosphates. PMID:8343137

  4. Low calcium culture condition induces mesenchymal cell-like phenotype in normal human epidermal keratinocytes

    SciTech Connect

    Takagi, Ryo; Yamato, Masayuki; Murakami, Daisuke; Sugiyama, Hiroaki; Okano, Teruo

    2011-08-26

    Highlights: {yields} Normal human epidermal keratinocytes serially cultured under low calcium concentration were cytokeratin and vimentin double positive cells. {yields} The human keratinocytes expressed some epithelial stem/progenitor cell makers, mesenchymal cell markers, and markers of epithelial-mesenchymal transition. {yields} Mesenchymal cell-like phenotype in the keratinocytes was suppressed under high-calcium condition. -- Abstract: Epithelial-mesenchymal transition (EMT) is an important cellular phenomenon in organ developments, cancer invasions, and wound healing, and many types of transformed cell lines are used for investigating for molecular mechanisms of EMT. However, there are few reports for EMT in normal human epithelial cells, which are non-transformed or non-immortalized cells, in vitro. Therefore, normal human epidermal keratinocytes (NHEK) serially cultured in low-calcium concentration medium (LCM) were used for investigating relations between differentiation and proliferation and mesenchymal-like phenotype in the present study, since long-term cultivation of NHEK is achieved in LCM. Interestingly, NHEK serially cultured in LCM consisted essentially of cytokeratin-vimentin double positive cells (98%), although the NHEK exhibited differentiation under high-calcium culture condition with 3T3 feeder layer. The vimentin expression was suppressed under high-calcium condition. These results may indicate the importance of mesenchymal-like phenotype for serially cultivation of NHEK in vitro.

  5. Cellular differentiation hierarchies in normal and culture-adapted human embryonic stem cells.

    PubMed

    Enver, Tariq; Soneji, Shamit; Joshi, Chirag; Brown, John; Iborra, Francisco; Orntoft, Torben; Thykjaer, Thomas; Maltby, Edna; Smith, Kath; Abu Dawud, Raed; Jones, Mark; Matin, Maryam; Gokhale, Paul; Draper, Jonathan; Andrews, Peter W

    2005-11-01

    Human embryonic stem cell (HESC) lines vary in their characteristics and behaviour not only because they are derived from genetically outbred populations, but also because they may undergo progressive adaptation upon long-term culture in vitro. Such adaptation may reflect selection of variants with altered propensity for survival and retention of an undifferentiated phenotype. Elucidating the mechanisms involved will be important for understanding normal self-renewal and commitment to differentiation and for validating the safety of HESC-based therapy. We have investigated this process of adaptation at the cellular and molecular levels through a comparison of early passage (normal) and late passage (adapted) sublines of a single HESC line, H7. To account for spontaneous differentiation that occurs in HESC cultures, we sorted cells for SSEA3, which marks undifferentiated HESC. We show that the gene expression programmes of the adapted cells partially reflected their aberrant karyotype, but also resulted from a failure in X-inactivation, emphasizing the importance in adaptation of karyotypically silent epigenetic changes. On the basis of growth potential, ability to re-initiate ES cultures and global transcription profiles, we propose a cellular differentiation hierarchy for maintenance cultures of HESC: normal SSEA3+ cells represent pluripotent stem cells. Normal SSEA3- cells have exited this compartment, but retain multilineage differentiation potential. However, adapted SSEA3+ and SSEA3- cells co-segregate within the stem cell territory, implying that adaptation reflects an alteration in the balance between self-renewal and differentiation. As this balance is also an essential feature of cancer, the mechanisms of culture adaptation may mirror those of oncogenesis and tumour progression. PMID:16159889

  6. 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

  7. Human colonic crypts in culture: segregation of immunochemical markers in normal versus adenoma-derived

    PubMed Central

    Dame, Michael K; Jiang, Yan; Appelman, Henry D; Copley, Kelly D; McClintock, Shannon D; Aslam, Muhammad Nadeem; Attili, Durga; Elmunzer, B Joseph; Brenner, Dean E; Varani, James; Turgeon, D Kim

    2014-01-01

    In order to advance a culture model of human colonic neoplasia, we developed methods for the isolation and in vitro maintenance of intact colonic crypts from normal human colon tissue and adenomas. Crypts were maintained in three-dimensional Matrigel culture with a simple, serum-free, low Ca2+ (0.15 mM) medium. Intact colonic crypts from normal human mucosa were viably maintained for 3–5 days with preservation of the in situ crypt-like architecture, presenting a distinct base and apex. Abnormal structures from adenoma tissue could be maintained through multiple passages (up to months), with expanding buds/tubules. Immunohistochemical markers for intestinal stem cells (Lgr5), growth (Ki67), differentiation (E-cadherin, cytokeratin 20 (CK20) and mucin 2 (MUC2)) and epithelial turnover (Bax, cleaved Caspase-3), paralleled the changes in function. The epithelial cells in normal crypts followed the physiological sequence of progression from proliferation to differentiation to dissolution in a spatially and temporally appropriate manner. Lgr5 expression was seen in a few basal cells of freshly isolated crypts, but was not detected after 1–3 days in culture. After 24 h in culture, crypts from normal colonic tissue continued to show strong Ki67 and MUC2 expression at the crypt base, with a gradual decrease over time such that by days 3–4 Ki67 was not expressed. The differentiation marker CK20 increased over the same period, eventually becoming intense throughout the whole crypt. In adenoma-derived structures, expression of markers for all stages of progression persisted for the entire time in culture. Lgr5 showed expression in a few select cells after months in culture. Ki67 and MUC2 were largely associated with the proliferative budding regions while CK20 was localized to the parent structure. This ex vivo culture model of normal and adenomatous crypts provides a readily accessible tool to help understand the growth and differentiation process in human colonic

  8. 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

  9. Primary culture of axolotl spinal cord ependymal cells.

    PubMed

    Chernoff, E A; Munck, C M; Mendelsohn, L G; Egar, M W

    1990-01-01

    In order to examine the role of ependymal cells in the spinal cord regeneration of urodele amphibians, procedures were established to identify and culture these cells. Cell isolation and culture conditions were determined for ependymal cells from larval and adult axolotls (Ambystoma mexicanum). Dissociated cells prepared from intact spinal cords were cultured on fibronectin- or laminin-coated dishes. Dissociated cells attached more rapidly to fibronectin, but attached and spread on both fibronectin and laminin. Essentially pure populations of ependymal cells were obtained by removing 2 week old ependymal outgrowth from lesion sites of adult spinal cords. These ependymal outgrowths attached and grew only on fibronectin-coated dishes. Growth and trophic factors were tested to formulate a medium that would support ependymal cell proliferation. The necessary peptide hormones were PDGF, EGF, and insulin. TGF-beta(1) affected the organization of cell outgrowth. Initially, longterm culture required the presence of high levels of axolotl serum. Addition of purified bovine hemaglobin in the culture medium reduced the serum requirement. Outgrowth from expiants was subcultured by transferring groups of cells. Intrinsic markers were used to identify ependymal cells in culture. The ependymal cells have characteristic ring-shaped nucleoli in both intact axolotl spinal cords and in culture. Indirect immunofluorescence examination of intermediate filaments showed that ependymal cells were glial fibrillary acidic protein (GFAP) negative and vimentin positive in culture. Identification of dividing cells was made using (3)H-thymidine incorporation and autoradiography, and by the presence of mitotic figures in the cultured cells. PMID:18620322

  10. 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

  11. Effects of nutritional and hormonal factors on the metabolism of retinol-binding protein by primary cultures of rat hepatocytes

    SciTech Connect

    Dixon, J.L.; Goodman, D.S.

    1987-01-01

    Studies were conducted to explore hormonal and nutritional factors that might be involved in the regulation of retinol-binding protein (RBP) synthesis and secretion by the liver. The studies employed primary cultures of hepatocytes from normal rats. When cells were cultured in Dulbecco's modified Eagle's medium alone, a high rate of RBP secretion was observed initially, which declined and became quite low by 24 hr. Supplementing the medium with amino acids maintained RBP and albumin secretion at moderate (but less than initial) rates for at least 3 days. Further addition of dexamethasone maintained the production and secretion rates of RBP, transthyretin, and albumin close to the initial rates for up to 3-5 days in culture as measured by radioimmunoassay. Hormonally treated hepatocytes produced and secreted RBP, transthyretin, and albumin at both absolute and relative rates similar to physiological values, as estimated from rates reported by others from studies in vivo and with perfused livers. Glucagon addition partially maintained the secretion rates of these 3 proteins, but less effectively than did dexamethasone. A number of other hormones, added singly or in combination, did not affect RBP production or secretion. Addition of retinol to the cultured normal hepatocytes was without effect upon RBP secretion. These studies show that supplementing the culture medium of hepatocytes with amino acids and dexamethasone maintains RBP production and secretion for several days. In normal hepatocytes, with ample supply of retinol available within the cell, addition of exogenous retinol does not appear to influence RBP metabolism or secretion by the cells.

  12. 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…

  13. 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...

  14. Primary culture of human Schwann and schwannoma cells: Improved and simplified protocol

    PubMed Central

    Dilwali, Sonam; Patel, Pratik B.; Roberts, Daniel S.; Basinsky, Gina M.; Harris, Gordon J.; Emerick, Kevin; Stankovic, Konstantina M.

    2014-01-01

    Primary culture of human Schwann cells (SCs) and vestibular schwannoma (VS) cells are invaluable tools to investigate SC physiology and VS pathobiology, and to devise effective pharmacotherapies against VS, which are sorely needed. However, existing culture protocols, in aiming to create robust, pure cultures, employ methods that can lead to loss of biological characteristics of the original cells, potentially resulting in misleading biological findings. We have developed a minimally manipulative method to culture primary human SC and VS cells, without the use of selective mitogens, toxins, or time-consuming and potentially transformative laboratory techniques. Schwann cell purity was quantified longitudinally using S100 staining in SC cultures derived from the great auricular nerve and VS cultures followed for 7 and 12 weeks, respectively. SC cultures retained approximately ≥85% purity for 2 weeks. VS cultures retained approximately ≥80% purity for the majority of the span of 12 weeks, with maximal purity of 87% at 2 weeks. The VS cultures showed high level of biological similarity (68% on average) to their respective parent tumors, as assessed using a protein array featuring 41 growth factors and receptors. Apoptosis rate in vitro negatively correlated with tumor volume. Our results, obtained using a faster, simplified culturing method than previously utilized, indicate that highly pure, primary human SC and VS cultures can be established with minimal manipulation, reaching maximal purity at 2 weeks of culture. The VS cultures recapitulate the parent tumors' biology to a great degree, making them relevant models to investigate VS pathobiology. PMID:24910344

  15. Influence of free residual chlorine on cultured human epidermal keratinocytes from normal skin and hypertrophic scars.

    PubMed

    Matsumoto, Y; Mori, H; Hayakawa, A; Ohashi, M

    1995-07-01

    In Japan, public health regulations state that the water in rinsing pools used before swimming should contain 50-100 mg/l of chlorine. We examined the influence of chlorination at high concentrations in rinsing pools on the skin using cultured human epidermal keratinocytes from normal skin and hypertrophic scars. Chlorination of cell culture for 15 min with 200 mg/l of free residual chlorine proved cytotoxic to both types of keratinocytes as did 100 mg/l of free residual chlorine for 1 or 3 consecutive days. Keratinocytes from hypertrophic scars, when cultivated in 100 mg/l of free residual chlorine, were more vulnerable to chlorine than those from normal skin. Cell characteristics of cultured keratinocytes from hypertrophic scars may be somewhat different from those of normal skin. The phenomena observed in this experimental model of the skin suggest that people exposed to chlorine in rinsing pools at concentrations in excess of 200 mg/l for about 15 min before swimming are at risk of developing cutaneous disorders, especially at sites of injury, e.g. scars. PMID:7577833

  16. Parents' Perceptions of Primary Health Care Physiotherapy With Preterm Infants: Normalization, Clarity, and Trust.

    PubMed

    Håkstad, Ragnhild B; Obstfelder, Aud; Øberg, Gunn Kristin

    2016-08-01

    Having a preterm infant is a life-altering event for parents. The use of interventions intended to support the parents is recommended. In this study, we investigated how parents' perceptions of physiotherapy in primary health care influenced their adaptation to caring for a preterm child. We conducted 17 interviews involving parents of seven infants, at infants' corrected age (CA) 3, 6, and 12 months. The analysis was a systematic text condensation, connecting to theory of participatory sense-making. The parents described a progression toward a new normalcy in the setting of persistent uncertainty. Physiotherapists can ameliorate this uncertainty and support the parents' progression toward normalization, by providing knowledge and acknowledging both the child as subject and the parent-child relationship. Via embodied interaction and the exploration of their child's capacity, the parents learn about their children's individuality and gain the confidence necessary to support and care for their children in everyday life. PMID:26449495

  17. Human colon tissue in organ culture: preservation of normal and neoplastic characteristics

    PubMed Central

    Bhagavathula, Narasimharao; Mankey, Cohra; DaSilva, Marissa; Paruchuri, Tejaswi; Aslam, Muhammad Nadeem; Varani, James

    2009-01-01

    Normal and neoplastic human colon tissue obtained at surgery was used to establish conditions for organ culture. Optimal conditions included an atmosphere of 5% CO2 and 95% O2; tissue partially submerged with mucosa at the gas interface; and serum-free medium with 1.5 mM Ca2+ and a number of growth supplements. Histological, histochemical, and immunohistochemical features that distinguish normal and neoplastic tissue were preserved over a 2-d period. With normal tissue, this included the presence of elongated crypts with small, densely packed cells at the crypt base and mucin-containing goblet cells in the upper portion. Ki67 staining, for proliferating cells, was confined to the lower third of the crypt, while expression of extracellular calcium-sensing receptor was seen in the upper third and surface epithelium. E-cadherin and β-catenin were expressed throughout the epithelium and confined to the cell surface. In tumor tissue, the same disorganized, abnormal glandular structures seen at time zero were present after 2 d. The majority of cells in these structures were mucin-poor, but occasional goblet cells were seen and mucin staining was present. Ki67 staining was seen throughout the abnormal epithelium and calcium-sensing receptor expression was weak and variable. E-cadherin was seen at the cell surface (similar to normal tissue), but in some places, there was diffuse cytoplasmic staining. Finally, intense cytoplasmic and nuclear β-catenin staining was observed in cultured neoplastic tissue. PMID:19915935

  18. Pericellular oxygen concentration of cultured primary human trophoblasts

    PubMed Central

    Chen, Baosheng; Longtine, Mark S.; Nelson, D. Michael

    2012-01-01

    Introduction Oxygen is pivotal in placental development and function. In vitro culture of human trophoblasts provides a useful model to study this phenomenon, but a hotly debated issue is whether or not the oxygen tension of the culture conditions mimics in vivo conditions. We tested the hypothesis that ambient oxygen tensions in culture reflect the pericellular oxygen levels. Methods We used a microelectrode oxygen sensor to measure the concentration of dissolved oxygen in the culture medium equilibrated with 21%, 8% or <0.5% oxygen. Results The concentration of oxygen in medium without cells resembled that in the ambient atmosphere. The oxygen concentration present in medium bathing trophoblasts was remarkably dependent on the depth within the medium where sampling occurred, and the oxygen concentration within the overlying atmosphere was not reflected in medium immediately adjacent to the cells. Indeed, the pericellular oxygen concentration was in a range that most would consider severe hypoxia, at ≤ 0.6% oxygen or about 4.6 mm Hg, when the overlying atmosphere was 21% oxygen. Conclusions We conclude that culture conditions of 21% oxygen are unable to replicate the pO2 of 40–60 mm Hg commonly attributed to the maternal blood in the intervillous space in the second and third trimesters of pregnancy. We further surmise that oxygen atmospheres in culture conditions between 0.5% and 21% provide different oxygen fluxes in the immediate pericellular environment yet can still yield insights into the responses of human trophoblast to different oxygen conditions. PMID:23211472

  19. Inhibition of Primary ADP-Induced Platelet Aggregation in Normal Subjects after Administration of Nitrofurantoin (Furadantin)

    PubMed Central

    Rossi, Ennio C.; Levin, Nathan W.

    1973-01-01

    The evidence indicating that platelets may play a role in the occurrence of certain thromboembolic phenomena has stimulated a search for inhibitors of platelet function. This report presents data to indicate that nitrofurantoin is a potent inhibitor of primary ADP-induced platelet aggregation. The addition of 10 μM nitrofurantoin to citrated platelet-rich plasma obtained from 12 normal subjects produced a 29±6% (2 SD) inhibition of the velocity of platelet aggregation induced by 2 μM ADP. The inhibitory effect of nitrofurantoin demonstrated competitive kinetics in respect to ADP. The intravenous (180 mg) or oral (200 mg) administration of nitrofurantoin produced a serum nitrofurantoin concentration ranging from 2.7 to 23 μM in 28 normal subjects. Platelet-rich plasma obtained from these subjects demonstrated inhibition of the velocity of ADP-induced platelet aggregation that correlated with the log of the serum nitrofurantoin concentration (P < 0.001). Collagen-induced platelet aggregation was also inhibited in a dose-related manner, and the bleeding time was significantly prolonged in the two subjects with the highest serum nitrofurantoin concentration. These studies indicate that nitrofurantoin in vivo inhibits platelet function to a degree that is proportional to the serum nitrofurantoin concentration. PMID:4729043

  20. Production of Normal Mammalian Organ Culture Using a Medium Containing Mem-Alpha, Leibovitz L 15, Glucose Galactose Fructose

    NASA Technical Reports Server (NTRS)

    Goodwin, Thomas J. (Inventor); Wolf, David A. (Inventor); Spaulding, Glenn F. (Inventor); Prewett, Tacey L. (Inventor)

    1999-01-01

    Normal mammalian tissue and the culturing process has been developed for the three groups of organ, structural and blood tissue. The cells are grown in vitro under micro- gravity culture conditions and form three dimensional cells aggregates with normal cell function. The microgravity culture conditions may be microgravity or simulated microgravity created in a horizontal rotating wall culture vessel. The medium used for culturing the cells, especially a mixture of epithelial and mesenchymal cells contains a mixture of Mem-alpha and Leibovits L15 supplemented with glucose, galactose and fructose.

  1. 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

  2. Differentiation of normal and cultured preneoplastic tracheal epithellal cells in rats: importance of epithelial mesenchymal interactions

    SciTech Connect

    Terzaghi, M.; Klein-Szanto, A.J.P.

    1980-11-01

    Changes in the dependence on mesenchymal tissues for survival and differentiation in inbred F344 female rats were investigated in tracheal epithelial cells exposed to 7,12-dimethylbenz(a)anthracene (DMBA) and 12-O-tetradecanoylphorbol 13-acetate (TPA). Fresh suspensions of normal tracheal epithelium or cultured preneoplastic cells were inoculated into isolated organ segments (trachea, esophagus, bladder, or small intestine) or into Dacron containers that were then implanted subdermally into isogenic recipients. At various times after cell inoculation and implantation, tissues were removed for histologic evaluation. Normal cells inoculated into frozen-thawed trachea, esophagus, bladder, and intestine yielded a regular mucociliary epithelium. Normal cell inocula did not, however, survive in trachea previously heated (100/sup 0/C), fixed in ethanol, or digested with collagenese; nor did normal cells survive in Dacron containers unless tracheal fibroblasts plus epithelial cells were inoculated together. DMBA- and TPA-exposed cell populations with increased growth capacity in vitro survived and differentiated on all of the above substrates. For survival and differentiation in vivo, preneoplastic cells appeared to have less stringent substrate requirements than did normal cells. Application of the described techniques to the study of changes occurring early in the development of neoplastic disease is discussed.

  3. 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...

  4. 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

  5. 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…

  6. [Primary cultures of human umbilical chord vein endothelial cells: a biological model for studying enterococcal infection mechanisms].

    PubMed

    Chiriboga, Carlos Andrés; Fontanilla, Marta Raquel

    2004-12-01

    Although enterococcus bacteria are normal human intestinal flora, they rank as the third most common pathogen involved in hospital acquired infections. Generally, these bacteria are considered extracellular pathogens; however, an increasing number of reports indicate invasiveness to epithelial cell lines and macrophages. Despite their importance as nosocomial infection agents in patients suffering bacteremias and endocarditis, their interaction with endothelial cells has not been fully described. Herein, the nosocomial Enterococcus faecalis isolate Ef2890 from a hospitalized patient was exposed to cultured human venous endothelial cells from the umbilical chord. When the primary cell cultures were inoculated with Ef2890 and treated with bactericidal antibiotics to kill extracellular and adhered bacteria, intracellular bacteria were recovered and plated 4 h post-infection. These observations indicate that cell cultures provide a valuable biological model to study interactions between endothelium and enterococci. PMID:15678808

  7. 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

  8. 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

  9. Hepatobiliary disposition in primary cultures of dog and monkey hepatocytes.

    PubMed

    Rose, Kelly A; Kostrubsky, Vsevolod; Sahi, Jasminder

    2006-01-01

    Hepatobiliary transporters are a major route for elimination of xenobiotics and endogenous products. In vitro hepatobiliary models have been reported for human and rat, but not for the other preclinical species used in safety evaluation. We have established methodologies for culturing dog and monkey hepatocytes with optimal bile canalicular formation and function, using a sandwich culture comprising rigid collagen substratum and gelled collagen overlay. Hepatic uptake utilizing sinusoidal transporters and biliary excretion through canalicular transporters were assessed using the bile salt taurocholate, salicylate (negative control), and the Bsep inhibitors cyclosporin A (CsA) and glyburide. There was significant taurocholate and salicylate canalicular efflux in dog and monkey hepatocytes, although the amount of salicylate transported was one thousandth that of taurocholate. Species differences were observed, as glyburide significantly inhibited taurocholate uptake in monkey (64% at 10 microM) but not dog hepatocytes, and inhibited taurocholate efflux in dog (100% at 10 microM) but not monkey hepatocytes. CsA did not inhibit bile salt uptake and significantly inhibited canalicular efflux in dog (at 0.1 microM) and monkey (at 1 and 10 microM) hepatocyte cultures. These results suggest that glyburide is a bile salt uptake inhibitor in monkey but not in dog hepatocytes and that CsA inhibits bile salt canalicular efflux but not basolateral uptake in these species. We have established dog and monkey hepatocytes in sandwich culture with intact bile canalicular formation and function. The differences observed in taurocholate transport between dog and monkey hepatocytes may be indicative of in vivo species differences. PMID:16749858

  10. Learning and Teaching about Cultural Universals in Primary-Grade Social Studies.

    ERIC Educational Resources Information Center

    Brophy, Jere; Alleman, Janet

    2002-01-01

    Argues that topical units on cultural universals are well suited for introducing primary grade students to social studies, although the units need to be more powerful than those in leading textbooks. Notes a study supporting the feasibility of cultural universals units in first and second grade classrooms. Suggests guidelines for lesson plans,…

  11. EFFECT OF NONGENOTOXIC ENVIRONMENTAL CONTAMINATION ON CHOLESTEROL AND DNA SYNTHESIS IN CULTURED PRIMARY RAT HEPATOCYTES

    EPA Science Inventory

    The effect of certain reputedly non genotoxic agents on cholesterol and DNA synthesis was investigated in cultured rat primary hepatocytes and liver slices. epatocytes in culture were incubated for 48, 60, and 72 hrs with one of the following chemicals; namely, chloroform (CHCl3)...

  12. 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…

  13. Stimulation of Mucosal Mast Cell Growth in Normal and Nude Rat Bone Marrow Cultures

    NASA Astrophysics Data System (ADS)

    Haig, David M.; McMenamin, Christine; Gunneberg, Christian; Woodbury, Richard; Jarrett, Ellen E. E.

    1983-07-01

    Mast cells with the morphological and biochemical properties of mucosal mast cells (MMC) appear and proliferate to form the predominant cell type in rat bone marrow cultures stimulated with factors from antigen- or mitogen-activated lymphocytes. Conditioned media causing a selective proliferation of MMC were derived from mesenteric lymph node cells of Nippostrongylus brasiliensis-infected rats restimulated in vitro with specific antigen or from normal or infected rat mesenteric lymph node cells stimulated with concanavalin A. MMC growth factor is not produced by T-cell-depleted mesenteric lymph node cells or by the mesenteric lymph node cells of athymic rats. By contrast, MMC precursors are present in the bone marrow of athymic rats and are normally receptive to the growth factor produced by the lymphocytes of thymus-intact rats. The thymus dependence of MMC hyperplasia is thus based on the requirement of a thymus-independent precursor for a T-cell-derived growth promoter.

  14. The numerical stroop effect in primary school children: a comparison of low, normal, and high achievers.

    PubMed

    Heine, Angela; Tamm, Sascha; De Smedt, Bert; Schneider, Michael; Thaler, Verena; Torbeyns, Joke; Stern, Elsbeth; Verschaffel, Lieven; Jacobs, Arthur

    2010-01-01

    Sixty-six primary school children were selected, of which 21 scored low on a standardized math achievement test, 23 were normal, and 22 high achievers. In a numerical Stroop experiment, children were asked to make numerical and physical size comparisons on digit pairs. The effects of congruity and numerical distance were determined. All children exhibited congruity and distance effects in the numerical comparison. In the physical comparison, children of all performance groups showed Stroop effects when the numerical distance between the digits was large but failed to show them when the distance was small. Numerical distance effects depended on the congruity condition, with a typical effect of distance in the congruent, and a reversed distance effect in the incongruent condition. Our results are hard to reconcile with theories that suggest that deficits in the automaticity of numerical processing can be related to differential math achievement levels. Immaturity in the precision of mappings between numbers and their numerical magnitudes might be better suited to explain the Stroop effects in children. However, as the results for the high achievers demonstrate, in addition to numerical processing capacity per se, domain-general functions might play a crucial role in Stroop performance, too. PMID:20437281

  15. Primary cultures of rabbit renal proximal tubule cells: I. Growth and biochemical characteristics.

    PubMed

    Aleo, M D; Taub, M L; Nickerson, P A; Kostyniak, P J

    1989-09-01

    Before the usefulness of a new in vitro model can be ascertained, the model must be properly defined and characterized. This study presents the growth rate and biochemical characteristics of rabbit renal proximal tubule cells in primary culture over a 2-wk culture period. When grown in a hormonally defined, antibiotic-free medium these cells form confluent monolayer cultures within 7 d after plating. Multicellular dome formation, an indicator of transepithelial solute transport, was expressed after confluent cultures were formed. The activity of the cytosolic enzyme, lactate dehydrogenase, and the lysosomal enzyme, N-acetyl-glucosaminidase, increased 14- and 2-fold during the first 8 d of culture, respectively. In contrast, the activity of a brush border enzyme, alkaline phosphatase, decreased 85% within the first 8 d of culture. Release of these enzyme markers into the culture medium, which are routinely used to measure cytotoxicity, stabilized after 8 d in culture. The ratio of cellular protein to DNA changed according to the state of cellular growth. Values rose from 0.035 mg protein/micrograms DNA in preconfluent cultures to 0.059 mg protein/micrograms DNA in confluent cultures. These results document the characteristics of a primary proximal tubule cell culture system for future studies in in vitro toxicology. PMID:2793776

  16. 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,…

  17. 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…

  18. 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…

  19. 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'…

  20. Culture and Classroom Reform: The Case of the District Primary Education Project, India.

    ERIC Educational Resources Information Center

    Clarke, Prema

    2003-01-01

    The impact of the international donor-supported District Primary Education Programme on the thinking and actions of 234 primary school teachers in South India was influenced by four cultural constructs: "openness to regulation,""sense of duty," and views on social hierarchy and knowledge acquisition. Teachers were receptive to new methods…

  1. 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. PMID:17977040

  2. 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

  3. 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

  4. Oscillating activity of a calcium-activated K+ channel in normal and cancerous mammary cells in culture.

    PubMed

    Enomoto, K; Furuya, K; Maeno, T; Edwards, C; Oka, T

    1991-01-01

    Calcium-activated potassium channels were the channels most frequently observed in primary cultured normal mammary cell and in the established mammary tumor cell, MMT060562. In both cells, single-channel and whole-cell clamp recordings sometimes showed slow oscillations of the Ca2(+)-gated K+ current. The characteristics of the Ca2(+)-activated K+ channels in normal and cancerous mammary cells were quite similar. The slope conductances changed from 8 to 70 pS depending on the mode of recording and the ionic composition in the patch electrode. The open probability of this channel increased between 0.1 to 1 microM of the intracellular Ca2+, but it was independent of the membrane potential. Charybdotoxin reduced the activity of the Ca2(+)-activated K+ channel and the oscillation of the membrane current, but apamin had no apparent effect. The application of tetraethylammonium (TEA) from outside and BaCl2 from inside of the cell diminished the activity of the channel. The properties of this channel were different from those of both the large conductance (BK or MAXI K) and small conductance (SK) type Ca2(+)-activated K+ channels. PMID:1710671

  5. Functional gait outcomes for idiopathic normal pressure hydrocephalus after primary endoscopic third ventriculostomy.

    PubMed

    Sankey, Eric W; Jusué-Torres, Ignacio; Elder, Benjamin D; Goodwin, C Rory; Batra, Sachin; Hoffberger, Jamie; Lu, Jennifer; Blitz, Ari M; Rigamonti, Daniele

    2015-08-01

    We evaluated if patients with idiopathic normal pressure hydrocephalus (iNPH) showed functional improvement after primary endoscopic third ventriculostomy (ETV). The efficacy of ETV for iNPH remains controversial. We retrospectively reviewed 10 consecutive patients treated between 2009 and 2011 with ETV for iNPH. Seven patients with a median age of 73 years (range: 60-80) who underwent a primary ETV for iNPH were included for analysis. Median follow-up was 39 months (range: 26-46). Post-ETV stoma and aqueductal and cisternal flows were confirmed via high resolution, gradient echo and phase contrast MRI. Post-ETV timed up and go (TUG) and Tinetti performance oriented mobility assessment scores were compared to pre- and post-lumbar puncture (LP) values. A second LP was performed if ETV failed to sustain the observed improvement after initial LP. Patients who demonstrated ETV failure were subsequently shunted. Compared to pre-LP TUG and Tinetti values of 14.00 seconds (range: 12.00-23.00) and 22 (range: 16-24), post-LP scores improved to 11.00 seconds (range: 8.64-15.00; p=0.06) and 25 (range: 24-28; p=0.02), respectively. ETV failed to sustain this improvement with slight worsening between pre-LP and post-ETV TUG and Tinetti scores. Improvement from pre-LP assessment was regained after shunting and at last follow-up with TUG and Tinetti scores of 12.97 seconds (range: 9.00-18.00; p=0.250) and 25 (range: 18-27; p=0.07), and 11.87 seconds (range: 8.27-18.50; p=0.152) and 23 (range: 18-26; p=0.382), respectively. Despite stoma patency, ETV failed to sustain functional improvement seen after LP, however, improvement was regained after subsequent shunting suggesting that shunt placement remains the preferred treatment for iNPH. PMID:25979256

  6. Metabolism of ochratoxin A by primary cultures of rat hepatocytes.

    PubMed Central

    Hansen, C E; Dueland, S; Drevon, C A; Størmer, F C

    1982-01-01

    Association of ochratoxin A with cultured rat hepatocytes occurs at 4 degrees C, and the saturation level in the medium is 0.3 mM ochratoxin A, with maximal binding after 60 min. At 37 degrees C the level of cell-associated ochratoxin A increased up to 6 h and remained at 2 nmol of toxin per mg of cell protein for 30 h. With increasing concentrations of ochratoxin A, increasing amounts of the toxin accumulated in the cells; saturation occurred at a concentration of 0.3 mM. Ochratoxin A was metabolized by hepatocytes at 37 degrees. (4R)-4-Hydroxyochratoxin A appeared in the medium at a maximal level (about 30 nmol/mg of cell protein) at an ochratoxin A concentration of 0.25 mM after 48 h of incubation. Small amounts of (4S)-4-hydroxyochratoxin A were detected only after incubation for 22 h or longer. PMID:7103484

  7. Comparison of Prelaminar Thickness between Primary Open Angle Glaucoma and Normal Tension Glaucoma Patients

    PubMed Central

    Jung, Youn Hea; Park, Hae-Young L.; Jung, Kyoung In; Park, Chan Kee

    2015-01-01

    Main Objective The thinning of prelaminar tissue and prelamina cupping is known to occur by ischemia, as we see in anterior ischemic optic neuropathy. Since normal tension glaucoma (NTG) is thought to be more related to vascular factor than in primary open-angle glaucoma (POAG), we hypothesized that prelamina thinning may occur prominently in NTG patients. This study investigated the difference in prelaminar tissue thickness between patients with POAG and NTG and verified the factors related to prelaminar thinning. Methods Complete ophthalmic examination including standard automatic perimetry was performed in all patients. The prelaminar tissue thickness was measured in all patients by performing enhanced depth imaging with a Heidelberg Spectralis Optical Coherence Tomography. The retinal nerve fiber layer and optic nerve head parameters were obtained using the Heidelberg Retina Tomography II and Cirrus Optical Coherence Tomography. Various ocular factors and their relationships with prelaminar thickness were analyzed. Results The mean prelaminar tissue thickness was significantly thinner in patients with POAG than in those with NTG. The difference in the prelaminar thickness between patients with POAG and those with NTG was greater in the early field defect group than in the moderate and severe field groups. In multivariate analysis, the mean prelaminar thickness was related to the intraocular pressure, mean deviation, cup-disc ratio, and cup volume. Conclusions The prelaminar tissue was thinner in patients with POAG than in patients with NTG, and intraocular pressure had a strong influence on the prelaminar thickness in both POAG and NTG. This may indicate that mechanical compression is the main pathogenic factor in both POAG and NTG. PMID:25793734

  8. Measurement of cation movement in primary cultures using fluorescent dyes.

    PubMed

    Reynolds, I J

    2001-05-01

    Ca(2+), Na(+), K(+), and Mg(2+) have a central role in neuronal excitability. The concentration of these cations in the cytoplasm of neurons (generically termed [ion(+)]i) provides a marker of the excitation state of the neurons, and may also illuminate the activity of specific signaling mechanisms that involve Ca(2+)- or Mg(2+)-activated enzymes. The measurement of [ion(+)]i in cultured neurons is achieved with the use of an ion-sensitive fluorescent dye in combination with equipment designed to quantitatively measure fluorescence. Specificity is obtained by choosing dyes with the appropriate selectivity for the ion of interest. Measurements of steady state ion concentrations can be made, as well as measurements of the net difference between ion movement into the cytoplasm (in response to a stimulus) and the physiological buffering of that ion. The procedures in this unit for loading and recording from dyes are broadly similar for each ion when ratiometric dyes are used as described, and can readily be modified for use with single-wavelength dyes. Support protocols are provided for calibration of individual dyes, which can be more problematic. PMID:18428522

  9. Metastasis suppressor 1 regulates neurite outgrowth in primary neuron cultures.

    PubMed

    Yu, Juan; Lin, Shuyun; Wang, Mei; Liang, Lijun; Zou, Zijiao; Zhou, Xinfeng; Wang, Meichi; Chen, Ping; Wang, Ying

    2016-10-01

    Metastasis suppressor 1 (MTSS1) or missing in metastasis (MIM) is an actin- and membrane-binding protein with tumor suppressor functions. MTSS1 is important for cell morphology, motility, metastasis. The role of MTSS1 in cell morphology has been widely investigated in non-neuronal tissues; however the role of MTSS1 in neurite outgrowth remains unclear. Here we investigated the effect of MTSS1 on neurite outgrowth in primary cerebellar granule and hippocampal neurons of mouse. We found that overexpression of MTSS1 in cerebellar granule neurons significantly enhanced dendrite elaboration but inhibited axon elongation. This phenotype was significantly reduced by deletion of the Wiskott-Aldrich homology 2 (WH2) motif and point mutation in the insulin receptor substrate p53 (IRSp53) and MIM/MTSS1 homology (IMD) domain. Furthermore, inhibition of Rac1 activity or blocking of phosphatidyl inositol phosphates (PIPs) signaling decreased the effect of MTSS1 markedly. In accordance with the over-expression data, knockdown of MTSS1 in cerebellar granule neurons could increase the axon length but decrease the dendrite length and the number of dendrites. In addition, MTSS1 knock down in embryonic hippocampal neurons suppressed neurite branching and reduced dendrite length. Our findings have demonstrated that MTSS1 modulates neuronal morphology, possibly through a Rac1-PIPs signaling pathway. PMID:27401056

  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. 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

  12. Patient safety culture in primary care: developing a theoretical framework for practical use

    PubMed Central

    Kirk, Susan; Parker, Dianne; Claridge, Tanya; Esmail, Aneez; Marshall, Martin

    2007-01-01

    Objective Great importance has been attached to a culture of safe practice in healthcare organisations, but it has proved difficult to engage frontline staff with this complex concept. The present study aimed to develop and test a framework for making the concept of safety culture meaningful and accessible to managers and frontline staff, and facilitating discussion of ways to improve team/organisational safety culture. Setting Eight primary care trusts and a sample of their associated general practices in north west England. Methods In phase 1 a comprehensive review of the literature and a postal survey of experts helped identify the key dimensions of safety culture in primary care. Semistructured interviews with 30 clinicians and managers explored the application of these dimensions to an established theory of organisational maturity. In phase 2 the face validity and utility of the framework was assessed in 33 interviews and 14 focus groups. Results Nine dimensions were identified through which safety culture is expressed in primary care organisations. Organisational descriptions were developed for how these dimensions might be characterised at five levels of organisational maturity. The resulting framework conceptualises patient safety culture as multidimensional and dynamic, and seems to have a high level of face validity and utility within primary care. It aids clinicians' and managers' understanding of the concept of safety culture and promotes discussion within teams about their safety culture maturity. Conclusions The framework moves the agenda on from rhetoric about the importance of safety culture to a way of understanding why and how the shared values of staff working within a healthcare organisation may be operationalised to create a safe environment for patient care. PMID:17693682

  13. Reasoning Abilities in Primary School: A Pilot Study on Poor Achievers vs. Normal Achievers in Computer Game Tasks

    ERIC Educational Resources Information Center

    Dagnino, Francesca Maria; Ballauri, Margherita; Benigno, Vincenza; Caponetto, Ilaria; Pesenti, Elia

    2013-01-01

    This paper presents the results of preliminary research on the assessment of reasoning abilities in primary school poor achievers vs. normal achievers using computer game tasks. Subjects were evaluated by means of cognitive assessment on logical abilities and academic skills. The aim of this study is to better understand the relationship between…

  14. Response to hypogravity of normal in vitro cultured follicular cells from thyroid

    NASA Astrophysics Data System (ADS)

    Meli, Antonella; Perrella, Giuseppina; Curcio, Francesco; Saverio, F.; Impiombato, Ambesi

    Aim of this investigation is the study of molecular modifications occurring in differentiated mammalian cells exposed to gravitational changes. The test system chosen is a well characterized clone of differentiated, normal thyroid follicular cells (FRTL5) in long-term culture. As a follow-up to our recent experiment performed during the MASER-7 sounding rocket mission, flown for European Space Agency by Swedish Space Corporation in May 1996, we evaluated FRTL5 cells responses to Thyroid Stimulating Hormone dependent cAMP production under acute hypogravity conditions obtained in a fast rotating clinostat. Following this approach, we evaluated the FRTL5 cells response to TSH under microgravity conditions in order to optimize experimental tools and strategies in preparation to, and in between real flight missions.

  15. 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

    2016-03-01

    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. PMID:25414376

  16. 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

  17. 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

  18. 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

  19. 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

  20. Primary culture of venom glands from the Brazilian armed spider, Phoneutria nigriventer (Araneae, Ctenidae).

    PubMed

    Silva, Luciana Maria; Lages, Carolina Pereira; Venuto, Thiago; Lima, Rebeca Mascarenhas; Diniz, Marcelo Vasconcellos; Valentim, Cláudia L Lage; Baba, Elio Hideo; Pimenta, Paulo Filemon P; Fortes-Dias, Consuelo L

    2008-03-01

    Phoneutria spider venoms are a rich source of bioactive components. The limited amounts of crude material available, however, can be considered as a major hindrance for a faster development in the field. In the present study, we attempted to establish primary cultures of venom glands of Phoneutria nigriventer as an alternative, in vitro source of venom. Three different developmental stages were tried as starting materials: whole embryo (inside the cocoon), nymph (early after cocoon hatching) and young adult (1 year after cocoon hatching). The embryonic cells remained in suspension in the primary cultures, with no signs of adhesion or differentiation, for about 6 months. Nevertheless, this culture was useful for the first chromosome C-banding of Phoneutria. An average of 29+/-1 acrocentric chromosomes were found. Striated muscle cells were the only kind of cells in the culture of venom glands from Phoneutria nymphs. The most promising results were achieved with 1-year-old specimens. Besides muscle, adherent epithelial cells were also obtained in culture. Although these cells remained in culture for a short time (up to 48 h) immunochemical analysis of the culture supernatant evidenced the presence of Phoneutria venom components. This can be considered as a first step toward the functional cultures of venom glands of Phoneutria spiders. PMID:18068746

  1. Primary culture of purified Leydig cells isolated from adult rat testes.

    PubMed

    Browning, J Y; Heindel, J J; Grotjan, H E

    1983-02-01

    Methods for isolating highly purified Leydig cells permit the study of acute responses and biochemical properties of Leydig cells independent of other testicular cell types. The present study describes the development of a primary culture system for purified Leydig cells from adult rats in which the cells retain their ability to secrete testosterone for at least 72 h in culture. When Leydig cells were cultured in tissue culture medium 199--0.1% BSA (M199-BSA), basal testosterone secretion declined by 72 h, whereas hCGB-stimulated testosterone secretion was reduced by 48 h. Changing the culture medium twice daily or adding 0.5% fetal calf serum (fcs) enhanced basal and gonadotropin-stimulated testosterone secretion at 72 h in culture, although responsiveness to hCG was reduced to 57% of that in freshly isolated cells. Incubation of Leydig cells in the defined culture medium Dulbecco's Modified Eagles-Ham's F-12 (1:1, vol/vol) supplemented with 15 mM Hepes buffer, transferrin, insulin, and epidermal growth factor (DHG:F12 + Hepes + TIE) in either the presence or absence of 0.5% fcs yielded functional Leydig cells for longer intervals in culture. Furthermore, testosterone secretion was greater in DHG:F12 + Hepes + TIE than in M199-BSA at all time intervals tested. In DHG:F12 + Hepes + TIE, basal and gonadotropin-stimulated testosterone production by Leydig cells were maintained for 72 h in culture. Degenerative changes in morphology were apparent in some cells at 72 h, but not at earlier times in culture. This primary culture system for isolated Leydig cells provides a valuable tool to examine the temporally regulated events in Leydig cell function. PMID:6848362

  2. Differential susceptibility of primary cultured human skin cells to hypericin PDT in an in vitro model.

    PubMed

    Popovic, A; Wiggins, T; Davids, L M

    2015-08-01

    Skin cancer is the most common cancer worldwide, and its incidence rate in South Africa is increasing. Photodynamic therapy (PDT) has been shown to be an effective treatment modality, through topical administration, for treatment of non-melanoma skin cancers. Our group investigates hypericin-induced PDT (HYP-PDT) for the treatment of both non-melanoma and melanoma skin cancers. However, a prerequisite for effective cancer treatments is efficient and selective targeting of the tumoral cells with minimal collateral damage to the surrounding normal cells, as it is well established that cancer therapies have bystander effects on normal cells in the body, often causing undesirable side effects. The aim of this study was to investigate the cellular and molecular effects of HYP-PDT on normal primary human keratinocytes (Kc), melanocytes (Mc) and fibroblasts (Fb) in an in vitro tissue culture model which represented both the epidermal and dermal cellular compartments of human skin. Cell viability analysis revealed a differential cytotoxic response to a range of HYP-PDT doses in all the human skin cell types, showing that Fb (LD50=1.75μM) were the most susceptible to HYP-PDT, followed by Mc (LD50=3.5μM) and Kc (LD50>4μM HYP-PDT) These results correlated with the morphological analysis which displayed distinct morphological changes in Fb and Mc, 24h post treatment with non-lethal (1μM) and lethal (3μM) doses of HYP-PDT, but the highest HYP-PDT doses had no effect on Kc morphology. Fluorescent microscopy displayed cytoplasmic localization of HYP in all the 3 skin cell types and additionally, HYP was excluded from the nuclei in all the cell types. Intracellular ROS levels measured in Fb at 3μM HYP-PDT, displayed a significant 3.8 fold (p<0.05) increase in ROS, but no significant difference in ROS levels occurred in Mc or Kc. Furthermore, 64% (p<0.005) early apoptotic Fb and 20% (p<0.05) early apoptotic Mc were evident; using fluorescence activated cell sorting (FACS), 24

  3. Naïve adult stem cells isolation from primary human fibroblast cultures.

    PubMed

    Wenzel, Vera; Roedl, Daniela; Ring, Johannes; Djabali, Karima

    2013-01-01

    Over the last decade, several adult stem cell populations have been identified in human skin (1-4). The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory (5, 6). These early studies described a multipotent precursor cell population from adult mammalian dermis (5). These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons (5). Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers (6). Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies. Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies (7). The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable (7). These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures (7). We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are

  4. Spectrum of Cytogenomic Abnormalities Revealed by Array Comparative Genomic Hybridization on Products of Conception Culture Failure and Normal Karyotype Samples.

    PubMed

    Zhou, Qinghua; Wu, Shen-Yin; Amato, Katherine; DiAdamo, Autumn; Li, Peining

    2016-03-20

    Approximately 30% of pregnancies after implantation end up in spontaneous abortions, and 50% of them are caused by chromosomal abnormalities. However, the spectrum of genomic copy number variants (CNVs) in products of conception (POC) and the underlying gene-dosage-sensitive mechanisms causing spontaneous abortions remain largely unknown. In this study, array comparative genomic hybridization (aCGH) analysis was performed as a salvage procedure for 128 POC culture failure (POC-CF) samples and as a supplemental procedure for 106 POC normal karyotype (POC-NK) samples. Chromosomal abnormalities were detected in 10% of POC-CF and pathogenic CNVs were detected in 3.9% of POC-CF and 5.7% of POC-NK samples. Compiled results from this study and relevant case series through a literature review demonstrated an abnormality detection rate (ADR) of 35% for chromosomal abnormalities in POC-CF samples, 3.7% for pathogenic CNVs in POC-CF samples, and 4.6% for pathogenic CNVs in POC-NK samples. Ingenuity Pathway Analysis (IPA) was performed on the genes from pathogenic CNVs found in POC samples. The denoted primary gene networks suggested that apoptosis and cell proliferation pathways are involved in miscarriage. In summary, a similar spectrum of cytogenomic abnormalities was observed in POC culture success and POC-CF samples. A threshold effect correlating the number of dosage-sensitive genes in a chromosome with the observed frequency of autosomal trisomy is proposed. A rationalized approach using firstly fluorescence in situ hybridization (FISH) testing with probes of chromosomes X/Y/18, 13/21, and 15/16/22 for common aneuploidies and polyploidies and secondly aCGH for other cytogenomic abnormalities is recommended for POC-CF samples. PMID:27020032

  5. Control of thyrotropin glycosylation in normal rat pituitary cells in culture: effect of thyrotropin-releasing hormone

    SciTech Connect

    Ponsin, G.; Mornex, R.

    1983-08-01

    Regulation of glycosylation of TSH was studied in primary cultures of normal rat pituitary cells. (3H)Glucosamine or (3H)proline incorporation into immunoprecipitable TSH and trichloroacetic acid-precipitable proteins was measured after incubation periods ranging from 4-72 h. TSH release was assessed by RIA of TSH in the medium. TRH (30 nM) specifically increased the glycosylation of TSH despite the fact that it did not stimulate (3H)proline incorporation into the hormone even after 72 h of continuous labeling. The TRH-stimulated (3H)glucosamine-labeled TSH was completely recovered in the incubation medium. Effective concentrations of TRH were in the same range as those necessary for stimulation of TSH release (10(-10) - 10(-6) M). Somatostatin (50 nM) and T3 (10 microM) antagonized TRH effects on both TSH release and glycosylation. Stages of TSH glycosylation were discriminated by the addition to the culture medium of tunicamycin (10 micrograms/ml) or monensin (25 microM), which are known to inhibit core and terminal glycosylation of proteins, respectively. Medium (3H)glucosamine-labeled TSH was fully glycosylated, whereas a large part of the intracellular hormone was only core glycosylated. This suggests that terminal glycosylation of TSH could be related to hormone secretion. TRH stimulated essentially only terminal glycosylation of TSH. No alteration of core glycosylation of the hormone was observed after TRH treatment. The stimulating effect of TRH on terminal glycosylation of TSH is probably related to its ability to stimulate hormone release.

  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. 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…

  8. 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,…

  9. 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…

  10. The Perceptions of Primary School Teachers on Principal Cultural Leadership Behaviors

    ERIC Educational Resources Information Center

    Karakose, Turgut

    2008-01-01

    The purpose of this study was to investigate the perceptions of primary school teachers on principal cultural leadership behaviors, and examine the opinions of the participating teachers according to their various occupational characteristics. The study is descriptive in nature and evaluates the teachers' perceptions by using the Cultural…

  11. Cultures of Engagement in Challenging Circumstances: Four Lebanese Primary Schools in Urban Beirut

    ERIC Educational Resources Information Center

    Nabhani, Mona; Busher, Hugh; Bahous, Rima

    2012-01-01

    This study of four private primary schools in Beirut, Lebanon, investigated why the children in the schools appeared to out-perform their peers in other schools. The study investigated the cultures that teachers and principals constructed in schools with children and their parents, wondering whether they would exhibit characteristics said to…

  12. 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…

  13. 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…

  14. Inhibition of DNA synthesis by chemical carcinogens in cultures of initiated and normal proliferating rat hepatocytes

    SciTech Connect

    Novicki, D.L.; Rosenberg, M.R.; Michalopoulos, G.

    1985-01-01

    Rat hepatocytes in primary culture can be stimulated to replicate under the influence of rat serum and sparse plating conditions. Higher replication rates are induced by serum from two-thirds partially hepatectomized rats. The effects of carcinogens and noncarcinogens on the ability of hepatocytes to synthesize DNA were examined by measuring the incorporation of (3H)thymidine by liquid scintillation counting and autoradiography. Hepatocyte DNA synthesis was not decreased by ethanol or dimethyl sulfoxide at concentrations less than 0.5%. No effect was observed when 0.1 mM ketamine, Nembutal, hypoxanthine, sucrose, ascorbic acid, or benzo(e)pyrene was added to cultures of replicating hepatocytes. Estrogen, testosterone, tryptophan, and vitamin E inhibited DNA synthesis by approximately 50% at 0.1 mM, a concentration at which toxicity was noticeable. Several carcinogens requiring metabolic activation as well as the direct-acting carcinogen N-methyl-N'-nitro-N-nitrosoguanidine interfered with DNA synthesis. Aflatoxin B1 inhibited DNA synthesis by 50% (ID50) at concentrations between 1 X 10(-8) and 1 X 10(-7) M. The ID50 for 2-acetylaminofluorene was between 1 X 10(-7) and 1 X 10(-6) M. Benzo(a)pyrene and 3'-methyl-4-dimethylaminoazobenzene inhibited DNA synthesis 50% between 1 X 10(-5) and 1 X 10(-4) M. Diethylnitrosamine and dimethylnitrosamine (ID50 between 1 X 10(-4) and 5 X 10(-4) M) and 1- and 2-naphthylamine (ID50 between 1 X 10(-5) and 5 X 10(-4) M) caused inhibition of DNA synthesis at concentrations which overlapped with concentrations that caused measurable toxicity.

  15. Ultrastructural analysis of primary human urethral epithelial cell cultures infected with Neisseria gonorrhoeae.

    PubMed

    Harvey, H A; Ketterer, M R; Preston, A; Lubaroff, D; Williams, R; Apicella, M A

    1997-06-01

    In men with gonococcal urethritis, the urethral epithelial cell is a site of infection. To study the pathogenesis of gonorrhea in this cell type, we have developed a method to culture primary human urethral epithelial cells obtained at the time of urologic surgery. Fluorescent analysis demonstrated that 100% of the cells stained for keratin. Microscopic analyses indicated that these epithelial cells arrayed in a pattern similar to that seen in urethral epithelium. Using immunoelectron and confocal microscopy, we compared the infection process seen in primary cells with events occurring during natural infection of the same cell type in men with gonococcal urethritis. Immunoelectron microscopy studies of cells infected with Neisseria gonorrhoeae 1291 Opa+ P+ showed adherence of organisms to the epithelial cell membrane, pedestal formation with evidence of intimate association between the gonococcal and the epithelial cell membranes, and intracellular gonococci present in vacuoles. Confocal studies of primary urethral epithelial cells showed actin polymerization upon infection. Polyclonal antibodies to the asialoglycoprotein receptor (ASGP-R) demonstrated the presence of this receptor on infected cells in the primary urethral cell culture. In situ hybridization using a fluorescent-labeled probe specific to the ASGP-R mRNA demonstrated this message in uninfected and infected cells. These features were identical to those seen in urethral epithelial cells in exudates from males with gonorrhea. Infection of primary urethral cells in culture mimics events seen in natural infection and will allow detailed molecular analysis of gonococcal pathogenesis in a human epithelial cell which is commonly infected. PMID:9169783

  16. Tamoxifen Induces Expression of Immune Response-Related Genes in Cultured Normal Human Mammary Epithelial Cells

    PubMed Central

    Schild-Hay, Laura J.; Leil, Tarek A.; Divi, Rao L.; Olivero, Ofelia, A.; Weston, Ainsley; Poirier, Miriam C.

    2008-01-01

    Use of tamoxifen (TAM) is associated with a 50% reduction in breast cancer incidence and an increase in endometrial cancer incidence. Here, we documented TAM-induced gene expression changes in cultured normal human mammary epithelial cells (NHMEC strains numbered 5, 16 and 40), established from tissue taken at reduction mammoplasty from 3 individuals. Cells exposed to 0, 10 or 50 μM TAM for 48 hours were evaluated for (E)-α-(deoxyguanosin-N2-yl)-tamoxifen (dG-N2-TAM) adduct formation by TAM-DNA (DNA modified with dG-N2-TAM) chemiluminescence immunoassay (CIA), gene expression changes using NCI DNA-oligonucleotide microarray, and real time (RT)-PCR. At 48 hr, cells exposed to 10 μM and 50 μM TAM were 85.6% and 48.4% viable, respectively, and there were no measurable dG-N2-TAM adducts. For microarray, cells were exposed to 10 μM TAM and genes with expression changes of ≥ 3-fold were as follows: thirteen genes up-regulated and one down-related for strain 16; seventeen genes up-regulated for strain 5; and eleven genes up-regulated for strain 40. Interferon-inducible genes (IFITM1, IFIT1, IFNA1, MXI and GIP3), and a potassium ion channel (KCNJ1) were up-regulated in all 3 strains. No significant expression changes were found for genes related to estrogen or xenobiotic metabolism. RT-PCR revealed up-regulation of interferon α (IFNA1) and confirmed the TAM-induced up-regulation of the genes identified by microarray, with the exception of GIP3 and MX1, which were not up-regulated in strain 40. Induction of interferon-related genes in the three NHMEC strains suggests that, in addition to hormonal effects, TAM exposure may enhance immune response in normal breast tissue. PMID:19155303

  17. 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

  18. 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.

  19. 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. PMID:24002666

  20. 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

  1. Localization of sarcomeric proteins during myofibril assembly in cultured mouse primary skeletal myotubes.

    PubMed

    White, Jennifer; Barro, Marietta V; Makarenkova, Helen P; Sanger, Joseph W; Sanger, Jean M

    2014-09-01

    It is important to understand how muscle forms normally in order to understand muscle diseases that result in abnormal muscle formation. Although the structure of myofibrils is well understood, the process through which the myofibril components form organized contractile units is not clear. Based on the staining of muscle proteins in avian embryonic cardiomyocytes, we previously proposed that myofibrils formation occurred in steps that began with premyofibrils followed by nascent myofibrils and ending with mature myofibrils. The purpose of this study was to determine whether the premyofibril model of myofibrillogenesis developed from studies developed from studies in avian cardiomyocytes was supported by our current studies of myofibril assembly in mouse skeletal muscle. Emphasis was on establishing how the key sarcomeric proteins, F-actin, nonmuscle myosin II, muscle myosin II, and α-actinin were organized in the three stages of myofibril assembly. The results also test previous reports that nonmuscle myosins II A and B are components of the Z-bands of mature myofibrils, data that are inconsistent with the premyofibril model. We have also determined that in mouse muscle cells, telethonin is a late assembling protein that is present only in the Z-bands of mature myofibrils. This result of using specific telethonin antibodies supports the approach of using YFP-tagged proteins to determine where and when these YFP-sarcomeric fusion proteins are localized. The data presented in this study on cultures of primary mouse skeletal myocytes are consistent with the premyofibril model of myofibrillogenesis previously proposed for both avian cardiac and skeletal muscle cells. PMID:25125171

  2. Measuring safety culture in Dutch primary care: psychometric characteristics of the SCOPE-PC questionnaire

    PubMed Central

    2013-01-01

    Background Patient safety has been a priority in primary healthcare in the last years. The prevailing culture is seen as an important condition for patient safety in practice and several tools to measure patient safety culture have therefore been developed. Although Dutch primary care consists of different professions, such as general practice, dental care, dietetics, physiotherapy and midwifery, a safety culture questionnaire was only available for general practices. The purpose of this study was to modify and validate this existing questionnaire to a generic questionnaire for all professions in Dutch primary care. Methods A validated Dutch questionnaire for general practices was modified to make it usable for all Dutch primary care professions. Subsequently, this questionnaire was administered to a random sample of 2400 practices from eleven primary care professions. The instrument’s factor structure, reliability and validity were examined using confirmatory and explorative factor analyses. Results 921 questionnaires were returned. Of these, 615 were eligible for factor analysis. The resulting SCOPE-PC questionnaire consisted of seven dimensions: ‘open communication and learning from errors’, ‘handover and teamwork’, ‘adequate procedures and working conditions’, ‘patient safety management’, ‘support and fellowship’, ‘intention to report events’ and ‘organisational learning’ with a total of 41 items. All dimensions had good reliability with Cronbach’s alphas ranging from 0.70 – 0.90, and the questionnaire had a good construct validity. Conclusions The SCOPE-PC questionnaire has sound psychometric characteristics for use by the different professions in Dutch primary care to gain insight in their safety culture. PMID:24044750

  3. Molecular pathway activation features linked with transition from normal skin to primary and metastatic melanomas in human.

    PubMed

    Shepelin, Denis; Korzinkin, Mikhail; Vanyushina, Anna; Aliper, Alexander; Borisov, Nicolas; Vasilov, Raif; Zhukov, Nikolay; Sokov, Dmitry; Prassolov, Vladimir; Gaifullin, Nurshat; Zhavoronkov, Alex; Bhullar, Bhupinder; Buzdin, Anton

    2016-01-01

    Melanoma is the most aggressive and dangerous type of skin cancer, but its molecular mechanisms remain largely unclear. For transcriptomic data of 478 primary and metastatic melanoma, nevi and normal skin samples, we performed high-throughput analysis of intracellular molecular networks including 592 signaling and metabolic pathways. We showed that at the molecular pathway level, the formation of nevi largely resembles transition from normal skin to primary melanoma. Using a combination of bioinformatic machine learning algorithms, we identified 44 characteristic signaling and metabolic pathways connected with the formation of nevi, development of primary melanoma, and its metastases. We created a model describing formation and progression of melanoma at the level of molecular pathway activation. We discovered six novel associations between activation of metabolic molecular pathways and progression of melanoma: for allopregnanolone biosynthesis, L-carnitine biosynthesis, zymosterol biosynthesis (inhibited in melanoma), fructose 2, 6-bisphosphate synthesis and dephosphorylation, resolvin D biosynthesis (activated in melanoma), D-myo-inositol hexakisphosphate biosynthesis (activated in primary, inhibited in metastatic melanoma). Finally, we discovered fourteen tightly coordinated functional clusters of molecular pathways. This study helps to decode molecular mechanisms underlying the development of melanoma. PMID:26624979

  4. Molecular pathway activation features linked with transition from normal skin to primary and metastatic melanomas in human

    PubMed Central

    Shepelin, Denis; Korzinkin, Mikhail; Vanyushina, Anna; Aliper, Alexander; Borisov, Nicolas; Vasilov, Raif; Zhukov, Nikolay; Sokov, Dmitry; Prassolov, Vladimir; Gaifullin, Nurshat; Zhavoronkov, Alex; Bhullar, Bhupinder; Buzdin, Anton

    2016-01-01

    Melanoma is the most aggressive and dangerous type of skin cancer, but its molecular mechanisms remain largely unclear. For transcriptomic data of 478 primary and metastatic melanoma, nevi and normal skin samples, we performed high-throughput analysis of intracellular molecular networks including 592 signaling and metabolic pathways. We showed that at the molecular pathway level, the formation of nevi largely resembles transition from normal skin to primary melanoma. Using a combination of bioinformatic machine learning algorithms, we identified 44 characteristic signaling and metabolic pathways connected with the formation of nevi, development of primary melanoma, and its metastases. We created a model describing formation and progression of melanoma at the level of molecular pathway activation. We discovered six novel associations between activation of metabolic molecular pathways and progression of melanoma: for allopregnanolone biosynthesis, L-carnitine biosynthesis, zymosterol biosynthesis (inhibited in melanoma), fructose 2, 6-bisphosphate synthesis and dephosphorylation, resolvin D biosynthesis (activated in melanoma), D-myo-inositol hexakisphosphate biosynthesis (activated in primary, inhibited in metastatic melanoma). Finally, we discovered fourteen tightly coordinated functional clusters of molecular pathways. This study helps to decode molecular mechanisms underlying the development of melanoma. PMID:26624979

  5. Effect of urokinase on the proliferation of primary cultures of human prostatic cells

    SciTech Connect

    Kirchheimer, J.C.; Wojta, J.; Hienert, G.; Christ, G.; Heger, M.E.; Pflueger, H.B.; Binder, B.R.

    1987-11-01

    The effects of exogenously added urokinase type plasminogen activator, tissue type plasminogen activator, plasmin and thrombin on the proliferation of primary cultures of cells derived from prostatic hyperplasia or prostatic carcinomas were investigated by measuring the incorporation of /sup 3/H-thymidine into the cultures. Addition of urokinase type plasminogen activator (1.35 x 10(-9) M) or thrombin (10(-7) M) to the culture medium caused a two-fold increase of /sup 3/H-thymidine incorporation, regardless of the origin of the prostatic cells. Tissue type plasminogen activator did not alter the rate of /sup 3/H-thymidine incorporation, whereas plasmin caused a 25% decrease of /sup 3/H-thymidine incorporation in all cultures.

  6. 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. PMID:24006025

  7. Optic Disc Perfusion in Primary Open Angle and Normal Tension Glaucoma Eyes Using Optical Coherence Tomography-Based Microangiography

    PubMed Central

    Wen, Joanne C.; Zhang, Qinqin; Xin, Chen; Gupta, Divakar; Mudumbai, Raghu C.; Johnstone, Murray A.; Wang, Ruikang K.; Chen, Philip P.

    2016-01-01

    Purpose To investigate optic disc perfusion differences in normal, primary open-angle glaucoma (POAG), and normal tension glaucoma (NTG) eyes using optical microangiography (OMAG) based optical coherence tomography (OCT) angiography technique. Design Cross-sectional, observational study. Subjects Twenty-eight normal, 30 POAG, and 31 NTG subjects. Methods One eye from each subject was scanned with a 68 kHz Cirrus HD-OCT 5,000-based OMAG prototype system centered at the optic nerve head (ONH) (Carl Zeiss Meditec Inc, Dublin, CA). Microvascular images were generated from the OMAG dataset by detecting the differences in OCT signal between consecutive B-scans. The pre-laminar layer (preLC) was isolated by a semi-automatic segmentation program. Main Outcome Measures Optic disc perfusion, quantified as flux, vessel area density, and normalized flux (flux normalized by the vessel area) within the ONH. Results Glaucomatous eyes had significantly lower optic disc perfusion in preLC in all three perfusion metrics (p<0.0001) compared to normal eyes. The visual field (VF) mean deviation (MD) and pattern standard deviation (PSD) were similar between the POAG and NTG groups, and no differences in optic disc perfusion were observed between POAG and NTG. Univariate analysis revealed significant correlation between optic disc perfusion and VF MD, VF PSD, and rim area in both POAG and NTG groups (p≤0.0288). However, normalized optic disc perfusion was correlated with some structural measures (retinal nerve fiber layer thickness and ONH cup/disc ratio) only in POAG eyes. Conclusions Optic disc perfusion detected with OMAG was significantly reduced in POAG and NTG groups compared to normal controls, but no difference was seen between POAG and NTG groups with similar levels of VF damage. Disc perfusion was significantly correlated with VF MD, VF PSD, and rim area in glaucomatous eyes. Vascular changes at the optic disc as measured using OMAG may provide useful information for

  8. Crouzon syndrome with primary optic nerve atrophy and normal brain functions: A case report

    PubMed Central

    Pal, Uma Shankar; Gupta, Chandan; Chellappa, Arul A.L.

    2012-01-01

    Background This report and review of literature aimed to assess an unusual case of Crouzon syndrome characterized by distinctive disfigurement of craniofacial skeletal and soft tissue structures with primary optic nerve atropy. Methods We present a case of a 12-year-old girl with Crouzon syndrome displaying classic facial abnormalities with reduced vision and hearing loss. Conclusion Crouzon syndrome should be managed as early as possible as it results in airway obstruction, decreased vision, mental retardation and poor cosmetic appearance. PMID:25737846

  9. 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

  10. Brevican-containing perineuronal nets of extracellular matrix in dissociated hippocampal primary cultures.

    PubMed

    John, Nora; Krügel, Hans; Frischknecht, Renato; Smalla, Karl-Heinz; Schultz, Christian; Kreutz, Michael R; Gundelfinger, Eckart D; Seidenbecher, Constanze I

    2006-04-01

    Perineuronal nets (PNN) are specialized extracellular matrix structures enwrapping CNS neurons, which are important regulators for neuronal and synaptic functions. Brevican, a chondroitin sulfate proteoglycan, is an integral component of PNN. Here, we have investigated the appearance of these structures in hippocampal primary cultures. The expression profile of brevican in mixed cultures resembles the in vivo pattern with a strong upregulation of all isoforms during the second and 3rd weeks in culture. Brevican is primarily synthesized by co-cultured glial fibrillary acidic protein (GFAP-)-positive astrocytes and co-assembles with its interaction partners in PNN-like structures on neuronal somata and neurites as identified by counterstaining with the PNN marker Vicia villosa lectin. Both excitatory and inhibitory synapses are embedded into PNN. Furthermore, axon initial segments are strongly covered by a dense brevican coat. Altogether, we show that mature primary cultures can form PNN, and that basic features of these extracellular matrix structures may be studied in vitro. PMID:16503162

  11. Investigation of the optical properties of normal fibroblasts and fibroblasts cultured with cancer cells in terahertz frequency range

    NASA Astrophysics Data System (ADS)

    Strepitov, E. A.; Liakhov, E. P.; Balbekin, N. S.; Khodzitsky, M. K.; Smolyanskaya, O. A.; Trulyov, A. S.; Serebryakova, M. K.

    2015-07-01

    The optical properties of normal fibroblasts and fibroblasts cultured with cancer cells were studied in the frequency range of 0.2 - 1.0 THz. The results show the possibility to distinguish healthy cells from corrupted ones using their optical parameters.

  12. 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.

  13. 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. PMID:26414012

  14. 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

  15. Raman spectroscopy analysis of differences in composition of spent culture media of in vitro cultured preimplantation embryos isolated from normal and fat mice dams.

    PubMed

    Fabian, Dušan; Kačmarová, Martina; Kubandová, Janka; Čikoš, Štefan; Koppel, Juraj

    2016-06-01

    The aim of the present study was to compare overall patterns of metabolic activity of in vitro cultured preimplantation embryos isolated from normal and fat mice dams by means of non-invasive profiling of spent culture media using Raman spectroscopy. To produce females with two different types of body condition (normal and fat), a previously established two-generation model was used, based on overfeeding of experimental mice during prenatal and early postnatal development. Embryos were isolated from spontaneously ovulating and naturally fertilized dams at the 2-cell stage of development and cultured to the blastocyst stage in synthetic oviductal medium KSOMaa. Embryos from fat mice (displaying significantly elevated body weight and fat) showed similar developmental capabilities in vitro as embryos isolated from normal control dams (displaying physiological body weight and fat). The results show that alterations in the composition of culture medium caused by the presence of developing mouse preimplantation embryos can be detected using Raman spectroscopy. Metabolic activity of embryos was reflected in evident changes in numerous band intensities in the 1620-1690cm(-1) (amide I) region and in the 1020-1140cm(-1) region of the Raman spectrum for KSOMaa. Moreover, multivariate analysis of spectral data proved that the composition of proteins and other organic compounds in spent samples obtained after the culture of embryos isolated from fat dams was different from that in spent samples obtained after the culture of embryos from control dams. This study demonstrates that metabolic activity of cultured preimplantation embryos might depend on the body condition of their donors. PMID:27288336

  16. Effects of short-chain fatty acids on primary urothelial cells in culture: implications for intravesical use in enterocystoplasties.

    PubMed

    Solomon, L Z; Jennings, A M; Sharpe, P; Cooper, A J; Malone, P S

    1998-10-01

    The presence of inflammatory changes and mucopus production in an enterocystoplasty may be similar to the condition of diversion colitis and starvation diarrhea caused by a lack of luminal short-chain fatty acids (SCFAs). We postulate a therapeutic role for intravesical SCFA. Because this treatment will also contact the urothelium, we have assessed the effect on cellular proliferation by utilizing primary urothelial cells in culture. Primary urothelial cells were grown from biopsy samples of normal urothelium obtained intraoperatively. A cocktail of SCFA used in the treatment of diversion colitis was incubated with these cells for time intervals ranging from 30 minutes to 72 hours at drug concentrations ranging from 0.04 to 20 mmol/L butyrate equivalent (BE). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to measure the residual viable biomass to assess growth inhibition. These experiments were repeated on cells grown on matrigel substrate. The human urothelial cancer line RT112 was likewise exposed to SCFAs to assess selectivity between primary and transformed cells. Primary urothelial cells in culture undergo growth inhibition when exposed to SCFAs. The concentration of SCFAs required to reduce the general biomass by 50% or more (IC> or =50) was 20 mmol/L BE when exposure was for 2 hours or less. When drug exposure was prolonged for 72 hours, the IC> or =50 was 2.5 mmol/L BE. Cells grown on matrigel had their growth similarly inhibited. The IC > or = 50 for the RT112 cell line was 2.5 mmol/L BE after 72 hours of drug incubation. Primary urothelial cells in culture undergo a time- and dose-dependent growth inhibition when exposed to SCFAs. This inhibition is particularly apparent at the higher doses similar to those in use in clinical practice. Cells grown on a matrigel substrate suffer growth attenuation similar to that affecting cells grown on polystyrene plates. In vivo assessment in a rodent intravesical model is

  17. 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…

  18. 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

  19. Primary Endodermal Epithelial Cell Culture from the Yolk Sac Membrane of Japanese Quail Embryos.

    PubMed

    Lin, Han Jen; Wang, Siou Huei; Pan, Yu Hui; Ding, Shih-Torng

    2016-01-01

    We established an endodermal epithelial cell culture model (EEC) for studying the function of certain enzymes and proteins in mediating nutrient utilization by avian embryos during development. Fertilized Japanese quail eggs were incubated at 37 °C for 5 days and then yolk sac membranes (YSM) were collected to establish the EEC culture system. We isolated the embryonic endoderm layer from YSM, and sliced the membrane into 2 - 3 mm pieces and partially digested with collagenase before seeding in 24-well culture plates. The EECs proliferate out of the tissue and are ready for cell culture studies. We found that the EECs had typical characteristics of YSM in vivo, for example, accumulation of lipid droplets, expression of sterol O-acyltransferase and lipoprotein lipase. The partial digestion treatment significantly increased the successful rate of EEC culture. Utilizing the EECs, we demonstrated that the expression of SOAT1 was regulated by the cAMP dependent protein kinase A related pathway. This primary Japanese quail EEC culture system is a useful tool to study embryonic lipid transportation and to clarify the role of genes involved in mediating nutrient utilization in YSM during avian embryonic development. PMID:27022687

  20. 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.

  1. Generating Primary Cultures of Murine Cardiac Myocytes and Cardiac Fibroblasts to Study Viral Myocarditis

    PubMed Central

    Sherry, Barbara

    2016-01-01

    Viruses can induce direct damage to cardiac myocytes and cardiac fibroblasts resulting in myocarditis and impaired cardiac function. Cardiac myocytes and cardiac fibroblasts display different capacities to support viral infection and generate a protective antiviral response. This chapter provides detailed protocols for generation and characterization of primary cultures of murine cardiac myocytes and cardiac fibroblasts, offering a powerful tool to probe cell type-specific responses that determine protection against viral myocarditis. PMID:25836571

  2. Cultural aspects of primary healthcare in india: A case- based analysis

    PubMed Central

    2011-01-01

    Delivering quality primary care to large populations is always challenging, and that is certainly the case in India. While the sheer magnitude of patients can create difficulties, not all challenges are about logistics. Sometimes patient health-seeking behaviour leads to delays in obtaining medical help for reasons that have more to do with culture, social practice and religious belief. When primary care is accessed via busy state-run outpatient departments there is often little time for the physician to investigate causes behind a patient's condition, and these factors can adversely affect patient outcomes. We consider the case of a woman with somatic symptoms seemingly triggered by psychological stresses associated with social norms and familial cultural expectations. These expectations conflict with her personal and professional aspirations, and although she eventually receives psychiatric help and her problems are addressed, initially, psycho-social factors underlying her condition posed a hurdle in terms of accessing appropriate medical care. While for many people culture, belief and social norms exert a stabilising, positive influence, in situations where someone's personal expectations differ significantly from accepted social norms, individual autonomy can be directly challenged, and in which case, something has to give. The result of such challenges can negatively impact on health and well-being, and for patients with immature defence mechanisms for dealing with inner conflict, such an experience can be damaging and ensuing somatic disturbances are often difficult to treat. Patients with culture-bound symptoms are not uncommon within primary care in India or in other Asian countries and communities. We argue that such cases need to be properly understood if satisfactory patient outcomes are to be achieved. While some causes are structural, having to do with how healthcare is accessed and delivered, others are about cultural values, social practices and

  3. 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

  4. Characterization of abalone Haliotis tuberculata-Vibrio harveyi interactions in gill primary cultures.

    PubMed

    Pichon, Delphine; Cudennec, Benoit; Huchette, Sylvain; Djediat, Chakib; Renault, Tristan; Paillard, Christine; Auzoux-Bordenave, Stéphanie

    2013-10-01

    The decline of European abalone Haliotis tuberculata populations has been associated with various pathogens including bacteria of the genus Vibrio. Following the summer mortality outbreaks reported in France between 1998 and 2000, Vibrio harveyi strains were isolated from moribund abalones, allowing in vivo and in vitro studies on the interactions between abalone H. tuberculata and V. harveyi. This work reports the development of primary cell cultures from abalone gill tissue, a target tissue for bacterial colonisation, and their use for in vitro study of host cell-V. harveyi interactions. Gill cells originated from four-day-old explant primary cultures were successfully sub-cultured in multi-well plates and maintained in vitro for up to 24 days. Cytological parameters, cell morphology and viability were monitored over time using flow cytometry analysis and semi-quantitative assay (XTT). Then, gill cell cultures were used to investigate in vitro the interactions with V. harveyi. The effects of two bacterial strains were evaluated on gill cells: a pathogenic bacterial strain ORM4 which is responsible for abalone mortalities and LMG7890 which is a non-pathogenic strain. Cellular responses of gill cells exposed to increasing concentrations of bacteria were evaluated by measuring mitochondrial activity (XTT assay) and phenoloxidase activity, an enzyme which is strongly involved in immune response. The ability of gill cells to phagocyte GFP-tagged V. harveyi was evaluated by flow cytometry and gill cells-V. harveyi interactions were characterized using fluorescence microscopy and transmission electron microscopy. During phagocytosis process we evidenced that V. harveyi bacteria induced significant changes in gill cells metabolism and immune response. Together, the results showed that primary cell cultures from abalone gills are suitable for in vitro study of host-pathogen interactions, providing complementary assays to in vivo experiments. PMID:23756730

  5. 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.

  6. Immunohistochemical characterisation of epithelial cells of rodent harderian glands in primary culture

    PubMed Central

    DJERIDANE, YASMINA; SIMONNEAUX, VALERIE; KLOSEN, PAUL; VIVIEN-ROELS, BERTHE; PEVET, PAUL

    1999-01-01

    The aims of the current investigation were (1) to establish an efficient procedure for the isolation of rodent harderian gland cells and to define conditions for maintenance of viable differentiated cells; (2) to compare the in vitro growth pattern of cultured epithelial cells; and (3) to characterise the cultured epithelial cells from 3 rodent species: Wistar rats, Syrian hamsters and Djungarian hamsters. We have established primary culture conditions that permit the maintenance of viable and differentiated secretory cells from adult rodent harderian gland. This study demonstrates that the cell growth pattern is faster in hamsters than in rats and despite morphological changes, epithelial cells reestablish their distinctive (biochemical/metabolic) phenotype as indicated by lipid-containing vacuoles, porphyrin pigment and serotonin and tryptophan hydroxylase labelling. PMID:10634691

  7. 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

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

    PubMed Central

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

    2015-01-01

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

  9. Recent Developments in Understanding the Role of Aqueous Humor Outflow in Normal and Primary Open Angle Glaucoma

    PubMed Central

    Hann, Cheryl R.; Fautsch, Michael P.

    2015-01-01

    Primary open angle glaucoma (POAG) is the second leading cause of blindness in the world's rapidly aging population. POAG is characterized by progressive degeneration of neural structures in the posterior segment, often associated with a concomitant elevation of intraocular pressure. Changes in IOP are believed to be caused by a disruption in the normal outflow of aqueous humor. This article reviews recent research associated with normal and POAG aqueous humor outflow. Novel findings elucidating biochemical and pathological changes in the ocular tissues affected in POAG are presented. Stem cell research, identification of lymphatic markers, and increased use of mouse models give researchers exciting new tools to understand aqueous humor outflow, changes associated with POAG and identify underlying causes of the disease. PMID:26236568

  10. 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.

  11. 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.

  12. 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

  13. Comparison of Schlemm's canal's biological parameters in primary open-angle glaucoma and normal human eyes with swept source optical

    NASA Astrophysics Data System (ADS)

    Wang, Fei; Shi, Guohua; Li, Xiqi; Lu, Jing; Ding, Zhihua; Sun, Xinghuai; Jiang, Chunhui; Zhang, Yudong

    2012-11-01

    Thirty-seven normal and primary open angle glaucoma (POAG) subjects were noninvasively imaged by a tailor-made real-time anterior segment swept source optical coherence tomography (SS-OCT) to demonstrate the differences of the Schlemm's canal (SC) between POAG and normal eyes. After the cross-section images of the anterior chamber angle were acquired by SS-OCT, SC was confirmed by two independent masked observers and the average area, long diameter, and perimeter of the SC were measured. In normal subjects the circumference, area, and long diameter is 580.34±87.81 μm, 8023.89±1486.10 μ, and 272.83±49.39 μm, respectively, and these parameters were 393.25±98.04 μm, 3941.50±1210.69 μ, and 190.91±46.47 μm in the POAG subjects. The area of SC in the normal ones was significantly larger than that in POAG eyes (p<0.001), so as the long diameter and the perimeter (p<0.001 p<0.001).

  14. Effect of serum proteins on haem uptake and metabolism in primary cultures of liver cells.

    PubMed Central

    Sinclair, P R; Bement, W J; Gorman, N; Liem, H H; Wolkoff, A W; Muller-Eberhard, U

    1988-01-01

    A role of haemopexin in transporting haem to hepatocytes for degradation has been inferred from the high affinity of haemopexin for haem. We have examined this question in primary cultures of chick-embryo and adult rat liver cells. We present here the results of four sets of experiments which indicate that haemopexin retarded haem uptake by hepatocytes in culture. (1) Haem bound to bovine serum albumin is known to repress the activity of delta-aminolaevulinate synthase in chick cultures as indicated by decreased porphyrin accumulation. When haem-albumin was added in the presence of excess purified or freshly secreted chicken haemopexin, no haem-mediated repression of porphyrin production was observed. The haem-mediated repression of porphyrin accumulation was partially prevented when human, but not chicken, albumin was added to cultures. This finding reflected the higher affinity of human albumin for haem compared with that of chicken albumin. (2) Haemopexin inhibited the ability of haem to be incorporated into cytochrome P-450 induced in the chick cultures in the presence of the iron chelator desferrioxamine. (3) The rate of association of [55Fe]haem with cultured rat hepatocytes when [55Fe]haem-haemopexin was added was one-eighth of the rate observed when [55Fe]haem-bovine serum albumin was used as the haem donor. (4) The presence of haemopexin also diminished the catabolism of haem by both rat and chick-embryo liver cell cultures. It is concluded that the uptake and subsequent metabolic effects of haem are inhibited in cultured hepatocytes by proteins such as haemopexin which have a high affinity for haem. PMID:3223898

  15. Identification of Metabolites in the Normal Ovary and Their Transformation in Primary and Metastatic Ovarian Cancer

    PubMed Central

    Fong, Miranda Y.; McDunn, Jonathan; Kakar, Sham S.

    2011-01-01

    In this study, we characterized the metabolome of the human ovary and identified metabolic alternations that coincide with primary epithelial ovarian cancer (EOC) and metastatic tumors resulting from primary ovarian cancer (MOC) using three analytical platforms: gas chromatography mass spectrometry (GC/MS) and liquid chromatography tandem mass spectrometry (LC/MS/MS) using buffer systems and instrument settings to catalog positive or negative ions. The human ovarian metabolome was found to contain 364 biochemicals and upon transformation of the ovary caused changes in energy utilization, altering metabolites associated with glycolysis and β-oxidation of fatty acids—such as carnitine (1.79 fold in EOC, p<0.001; 1.88 fold in MOC, p<0.001), acetylcarnitine (1.75 fold in EOC, p<0.001; 2.39 fold in MOC, p<0.001), and butyrylcarnitine (3.62 fold, p<0.0094 in EOC; 7.88 fold, p<0.001 in MOC). There were also significant changes in phenylalanine catabolism marked by increases in phenylpyruvate (4.21 fold; p = 0.0098) and phenyllactate (195.45 fold; p<0.0023) in EOC. Ovarian cancer also displayed an enhanced oxidative stress response as indicated by increases in 2-aminobutyrate in EOC (1.46 fold, p = 0.0316) and in MOC (2.25 fold, p<0.001) and several isoforms of tocopherols. We have also identified novel metabolites in the ovary, specifically N-acetylasparate and N-acetyl-aspartyl-glutamate, whose role in ovarian physiology has yet to be determined. These data enhance our understanding of the diverse biochemistry of the human ovary and demonstrate metabolic alterations upon transformation. Furthermore, metabolites with significant changes between groups provide insight into biochemical consequences of transformation and are candidate biomarkers of ovarian oncogenesis. Validation studies are warranted to determine whether these compounds have clinical utility in the diagnosis or clinical management of ovarian cancer patients. PMID:21625518

  16. Establishment and characterization of two cell lines derived from primary cultures of Gekko japonicus cerebral cortex.

    PubMed

    Liu, Mei; Gu, Yun; Liu, Yan; Li, Jing; He, Jianghong; Lin, Sheyu; Gu, Xiaosong

    2010-02-01

    Adult Gekko japonicus is one of those vertebrates that are able to regenerate their missing or amputated tail. The most interesting feature of this animal lies in the ability of its spinal cord to regrow a functional tail. A fundamental question is whether the neuroglial cells play a different role compared with high vertebrates. Since in vitro studies using primary neuroglial cells are hampered by the limited lifespan and miscellaneous genetic background of these cells, we generated neuroglial cell lines from primary cell cultures of cerebral cortex of G. japonicus. The SV40 (simian-virus-40) T antigen gene was introduced into primary cell cultures. Cell cycle analysis, cell growth and proliferation, cell colony formation and contact inhibition, as well as karyotype assays were investigated. Two cell colonies, Gsn-1 and Gsn-3, were immunochemically characterized as glial fibrillary acidic protein and galactocerebroside-positive respectively. Compared with parental primary cells, the Gsn cells displayed shorter population doubling time, decreased percentage of cells in the G0/G1 phase, higher cell proliferation index, and increased cell activity. In assays of colony characteristics, Gsn cells showed increased cell activity at the lower cell densities or FBS (fetal bovine serum) supplement. The karyotype of immortalized Gsn cells exhibited transformational characteristics with hyperdiploid and polyploid chromosomes. The cell lines will provide a useful in vitro model for gecko neuroglial cells and facilitate systematic studies investigating the biological functions of specific gene products related to regeneration of the central nervous system. PMID:19947933

  17. Inhibition of collagen production in scleroderma fibroblast cultures by a connective tissue glycoprotein extracted from normal dermis

    SciTech Connect

    Maquart, F.X.; Bellon, G.; Cornillet-Stoupy, J.; Randoux, A.; Triller, R.; Kalis, B.; Borel, J.P.

    1985-08-01

    It was shown in a previous paper that a connective tissue glycoprotein (CTGP) extracted from normal rabbit dermis was able to inhibit total protein and collagen syntheses by normal dermis fibroblast cultures. In the present study, the effects of CTGP on scleroderma fibroblasts were investigated. (/sup 14/C)Proline incorporation into total proteins of the supernatant was not significantly different from that found in controls. By contrast, the amount of collagen, expressed as percentage of total secreted protein, was far higher in scleroderma cultures than in normal ones (14.4% +/- 6.0% vs 4.6% +/- 0.9%). Addition of CTGP to the medium induced a concentration-dependent inhibition of (/sup 14/C)proline incorporation into proteins from both control and scleroderma cells. In control cultures, no significant decrease of the percentage of collagen was observed, but over 60 micrograms/ml, both cytotoxic effects and inhibition of protein synthesis occurred. In scleroderma cultures, the inhibition was twice as effective on collagen as on noncollagen protein synthesis. The inhibition of collagen secretion was not related either to changes in collagen hydroxylation or to the intracellular catabolism of newly synthesized procollagen.

  18. E-Cigarette Affects the Metabolome of Primary Normal Human Bronchial Epithelial Cells

    PubMed Central

    Aug, Argo; Altraja, Siiri; Kilk, Kalle; Porosk, Rando; Soomets, Ursel; Altraja, Alan

    2015-01-01

    E-cigarettes are widely believed to be safer than conventional cigarettes and have been even suggested as aids for smoking cessation. However, while reasonable with some regards, this judgment is not yet supported by adequate biomedical research data. Since bronchial epithelial cells are the immediate target of inhaled toxicants, we hypothesized that exposure to e-cigarettes may affect the metabolome of human bronchial epithelial cells (HBEC) and that the changes are, at least in part, induced by oxidant-driven mechanisms. Therefore, we evaluated the effect of e-cigarette liquid (ECL) on the metabolome of HBEC and examined the potency of antioxidants to protect the cells. We assessed the changes of the intracellular metabolome upon treatment with ECL in comparison of the effect of cigarette smoke condensate (CSC) with mass spectrometry and principal component analysis on air-liquid interface model of normal HBEC. Thereafter, we evaluated the capability of the novel antioxidant tetrapeptide O-methyl-l-tyrosinyl-γ-l-glutamyl-l-cysteinylglycine (UPF1) to attenuate the effect of ECL. ECL caused a significant shift in the metabolome that gradually gained its maximum by the 5th hour and receded by the 7th hour. A second alteration followed at the 13th hour. Treatment with CSC caused a significant initial shift already by the 1st hour. ECL, but not CSC, significantly increased the concentrations of arginine, histidine, and xanthine. ECL, in parallel with CSC, increased the content of adenosine diphosphate and decreased that of three lipid species from the phosphatidylcholine family. UPF1 partially counteracted the ECL-induced deviations, UPF1’s maximum effect occurred at the 5th hour. The data support our hypothesis that ECL profoundly alters the metabolome of HBEC in a manner, which is comparable and partially overlapping with the effect of CSC. Hence, our results do not support the concept of harmlessness of e-cigarettes. PMID:26536230

  19. A Defined, Controlled Culture System for Primary Bovine Chromaffin Progenitors Reveals Novel Biomarkers and Modulators

    PubMed Central

    Masjkur, Jimmy; Levenfus, Ian; Lange, Sven; Arps-Forker, Carina; Poser, Steve; Qin, Nan; Vukicevic, Vladimir; Chavakis, Triantafyllos; Eisenhofer, Graeme; Bornstein, Stefan R.; Ehrhart-Bornstein, Monika

    2014-01-01

    We present a method to efficiently culture primary chromaffin progenitors from the adult bovine adrenal medulla in a defined, serum-free monolayer system. Tissue is dissociated and plated for expansion under support by the mitogen basic fibroblast growth factor (bFGF). The cultures, although not homogenous, contain a subpopulation of cells expressing the neural stem cell marker Hes3 that also propagate. In addition, Hes3 is also expressed in the adult adrenal medulla from where the tissue is taken. Differentiation is induced by bFGF withdrawal and switching to Neurobasal medium containing B27. Following differentiation, Hes3 expression is lost, and cells acquire morphologies and biomarker expression patterns of chromaffin cells and dopaminergic neurons. We tested the effect of different treatments that we previously showed regulate Hes3 expression and cell number in cultures of fetal and adult rodent neural stem cells. Treatment of the cultures with a combination of Delta4, Angiopoietin2, and a Janus kinase inhibitor increases cell number during the expansion phase without significantly affecting catecholamine content levels. Treatment with cholera toxin does not significantly affect cell number but reduces the ratio of epinephrine to norepinephrine content and increases the dopamine content relative to total catecholamines. These data suggest that this defined culture system can be used for target identification in drug discovery programs and that the transcription factor Hes3 may serve as a new biomarker of putative adrenomedullary chromaffin progenitor cells. PMID:24855275

  20. 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

  1. Primary possum macrophage cultures support the growth of a nidovirus associated with wobbly possum disease.

    PubMed

    Giles, Julia C; Perrott, Matthew R; Dunowska, Magdalena

    2015-09-15

    The objective of the study was to establish a system for isolation of a recently described, thus far uncultured, marsupial nidovirus associated with a neurological disease of possums, termed wobbly possum disease (WPD). Primary cultures of possum macrophages were established from livers of adult Australian brushtail possums (Trichosurus vulpecula). High viral copy numbers (up to 6.9×10(8)/mL of cell lysate) were detected in infected cell culture lysates from up to the 5th passage of the virus, indicating that the putative WPD virus (WPDV) was replicating in cultured cells. A purified virus stock with a density of 1.09 g/mL was prepared using iodixanol density gradient ultracentrifugation. Virus-like particles approximately 60 nm in diameter were observed using electron microscopy in negatively stained preparations of the purified virus. The one-step growth curve of WPDV in macrophage cultures showed the highest increase in intracellular viral RNA between 6 and 12h post-infection. Maximum levels of cell-associated viral RNA were detected at 24h post-infection, followed by a decline. Levels of extracellular RNA increased starting at 9h post-infection, with maximum levels detected at 48 h post-infection. The establishment of the in vitro system to culture WPDV will facilitate further characterisation of this novel nidovirus. PMID:26028426

  2. 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.

  3. Histomorphological and biochemical differentiation capacity in organotypic co-cultures of primary gingival cells.

    PubMed

    Tomakidi, P; Fusenig, N E; Kohl, A; Komposch, G

    1997-05-01

    To establish a three-dimensional in vitro test system mimicking the physiological situation of the oral cavity, organotypic co-cultures consisting of primary gingival cells on a collagen matrix with fibroblasts were generated. The histomorphological development after 7 and 14 d revealed close similarity with the non-keratinized gingiva epithelium. Furthermore, as epithelial specific markers synthesis and localization of keratins as well as the deposition of basement membrane components were assessed on frozen sections by immunofluorescence and keratin expression by in situ hybridization. Primary keratinocytes in conventional culture strained positive for keratin K14 and the mucosal differentiation-specific keratins K4 and K13, while primary fibroblasts, isolated from the same tissue source, and also some keratinocytes, were positive for vimentin. In organotypic co-cultures the keratinocytes formed a multilayered epithelium within 14 d containing basal cells and flattened cells in the uppermost layers. Comparable to native non-keratinized gingiva keratin 14 gene expression was clearly detectable in the basal cell compartment but showed extending immunolocalization. In addition, particularly at the early stage (7 d), basally located keratinocytes were also vimentin positive. According to morphological differentiation K4 and K13 were detectable in suprabasal position a the RNA and protein level. The major basement membrane constituents collagen type IV and laminin increased with time revealing first an interrupted and later a fully extended staining underneath the basal cells. Maintenance of basal cell function was further demonstrated by cell proliferation (BrdU incorporation) which was initially high (7 d) but declined towards the later stages (14-21 d). The results demonstrate i) that this co-culture system leads to a stratified surface epithelium with morphological and biochemical characteristics of the non-keratinized gingiva epithelium and ii) that a state of

  4. 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

  5. Hypertonic conditions enhance cartilage formation in scaffold-free primary chondrocyte cultures.

    PubMed

    Ylärinne, Janne H; Qu, Chengjuan; Lammi, Mikko J

    2014-11-01

    The potential of hypertonic conditions at in vivo levels to promote cartilage extracellular matrix accumulation in scaffold-free primary chondrocyte cultures was investigated. Six million bovine primary chondrocytes were cultured in transwell inserts in low glucose (LG), high glucose (HG), or hypertonic high glucose (HHG) DMEM supplemented with fetal bovine serum, antibiotics, and ascorbate under 5 % or 20 % O2 tension with and without transforming growth factor (TGF)-β3 for 6 weeks. Samples were collected for histological staining of proteoglycans (PGs) and type II collagen, analysis by quantitative reverse transcription plus the polymerase chain reaction (RT-PCR) of mRNA expression of aggrecan and procollagen α1 (II) and of Sox9 and procollagen α2 (I), and quantitation of PGs and PG separation in agarose gels. Cartilage tissues produced at 20 % O2 tension were larger than those formed at 5 % O2 tension. Compared with LG, the tissues grew to larger sizes in HG or HHG medium. Histological staining showed the strongest PG and type II collagen staining in cartilage generated in HG or HHG medium at 20 % O2 tension. Quantitative RT-PCR results indicated significantly higher expression of procollagen α1 (II) mRNA in cartilage generated in HHG medium at 20 % O2 tension compared with that in the other samples. TGF-β3 supplements in the culture medium provided no advantage for cartilage formation. Thus, HHG medium used at 20 % O2 tension is the most beneficial combination of the tested culture conditions for scaffold-free cartilage production in vitro and should improve cell culture for research into cartilage repair or tissue engineering. PMID:25107609

  6. Loss of estrogen receptor beta expression in malignant human prostate cells in primary cultures and in prostate cancer tissues.

    PubMed

    Pasquali, D; Rossi, V; Esposito, D; Abbondanza, C; Puca, G A; Bellastella, A; Sinisi, A A

    2001-05-01

    The aim of this study was to investigate the expression of estrogen receptor (ER) beta and alpha genes in normal (N) and malignant (C) primary cultures of human prostate epithelial cells (PEC) and fibroblasts (PFC) and in the prostate tissue donors. Both ERbeta and ERalpha messenger ribonucleic acids were found by RT-PCR analysis in six NPECs and normal prostate tissues and in only one of six CPECs and in the respective cancer tissue donor. The other five CPECs and related cancer tissue donors and all normal and cancer PFCs expressed ERalpha messenger ribonucleic acid alone. Immunoblot analysis, using a polyclonal anti-ERbeta (C-terminal) antibody, demonstrated ERbeta protein in all NPEC lysates and in one of the six CPECS: ERalpha protein was expressed in both NPECs and CPECs when a polyclonal antibody directed against the ERalpha N-terminal domain was used. In contrast, ERalpha protein was not detected in two of the six CPEC lysates when ERalpha C-terminal monoclonal antibodies were used. Using a set of primers designed to amplify the region from exons 6-8, RT-PCR analysis demonstrated the absence of the expected transcript in these cells. The present study shows that the ERbeta gene is expressed together with ERalpha in normal prostates and NPECs, whereas it is barely detectable in prostate cancer and CPECS: Moreover, in some CPECs, the ERalpha gene may be transcribed in a changed protein, resulting from the expression of a deletion variant. Together, these data suggest that prostate malignancy is associated with a potential disorder of ER-mediated pathways. PMID:11344205

  7. Distinct angiotensin II receptor in primary cultures of glial cells from rat brain

    SciTech Connect

    Raizada, M.K.; Phillips, M.I.; Crews, F.T.; Sumners, C.

    1987-07-01

    Angiotensin II (Ang-II) has profound effects on the brain. Receptors for Ang-II have been demonstrated on neurons, but no relationship between glial cells and Agn-II has been established. Glial cells (from the hypothalamus and brain stem of 1-day-old rat brains) in primary culture have been used to demonstrate the presence of specific Ang-II receptors. Binding of /sup 125/I-Ang-II to glial cultures was rapid, reversible, saturable, and specific for Ang-II. The rank order of potency of /sup 125/I-Ang-II binding was determined. Scatchard analysis revealed a homogeneous population of high-affinity binding sites with a B/sub max/ of 110 fmol/mg of protein. Light-microscopic autoradiography of /sup 125/I-Ang-II binding supported the kinetic data, documenting specific Ang-II receptors on the glial cells. Ang-II stimulated a dose-dependent hydrolysis of phosphatidylinositols in glial cells, an effect mediated by Ang-II receptors. However, Ang-II failed to influence (/sup 3/H) norepinephrine uptake, and catecholamines failed to regulate Ang-II receptors, effects that occur in neurons. These observations demonstrate the presence of specific Ang-II receptors on the glial cells in primary cultures derived from normotensive rat brain. The receptors are kinetically similar to, but functionally distinct from, the neuronal Ang-II receptors.

  8. 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.

  9. 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

  10. Environmental scanning electron microscopy of the surface of normal and vitrified leaves of Gypsophila paniculata (Babies Breath) cultured in vitro.

    PubMed

    Gribble, K; Sarafis, V; Nailon, J; Holford, P; Uwins, P

    1996-06-01

    Leaf surfaces of non-tissue-cultured, vitrified and non-vitrified plantlets of Gypsophila paniculata (Babies Breath) were examined using an environmental scanning electron microscope. Non-tissue-cultured plants had a complete epidermal surface, recessed stomata and wax present on the leaf surface. The surface of tissue-cultured plantlets appeared similar to non-tissue-cultured plants excepting stomata were slightly protruding and less wax appeared to be present. In both non-tissue-cultured and tissue-cultured plants stomata were found both opened and closed and were observed closing. In contrast vitrified plantlets had abnormal, malformed stomata which appeared non-functional. The ventral surfaces of leaves seemed more normal than the dorsal, this may be due to the former receiving more light. Additionally, discontinuities were found in the epidermis. Often epidermal holes were found in association with stomatal apertures. It is suggested that the main cause of desiccation of vitrified G. paniculata plantlets ex vitro is due to loss of water from the discontinuity in epidermis and not because of non-functional stomata. Liquid water could be seen through the epidermal holes indicating that at least some of the extra water in vitrified plantlets is contained in the intercellular spaces. PMID:24178169

  11. Comparison of outcomes between patients with idiopathic normal pressure hydrocephalus who received a primary versus a salvage shunt.

    PubMed

    Moran, Dane; Hung, Alice; Vakili, Sharif; Fialho, Hugo; Jeon, Lee; Sankey, Eric W; Jusué-Torres, Ignacio; Lu, Jennifer; Goodwin, C Rory; Elder, Benjamin D; Rigamonti, Daniele

    2016-07-01

    Placement of a ventriculoperitoneal (VP) shunt is the treatment of choice for communicating hydrocephalus; however, the extent to which VP shunting is able to relieve symptoms in patients who had previously been treated with cerebrospinal fluid diverting therapy at an outside institution remains unclear. A retrospective review of patients with idiopathic normal pressure hydrocephalus treated with VP shunts at a single institution between 1993 and 2013 was conducted. Patients were classified as having received a primary VP shunt if they had not been previously treated with a VP shunt, ventriculoatrial shunt, lumboperitoneal shunt, or endoscopic third ventriculostomy. Patients were classified as having received a salvage VP shunt if they had been previously treated by one of these four modalities at an outside institution prior to their presentation to our institution. There were 357 patients who received a primary shunt and 33 patients who received a salvage shunt. Patients who had a salvage shunt placed had significantly higher odds of requiring a future revision (54% versus 41%; odds ratio=2.85; 95% confidence interval [CI]: 1.24-6.57; p=0.014). Patients who received a salvage shunt had statistically significantly lower rates of gait improvement at 6months in comparison to patients who received a primary shunt (relative risk=0.35; 95% CI: 0.14-0.87; p=0.025). Despite these findings, there was no significant difference at last follow-up in improvement in gait, continence, and cognition, indicating that outcomes for patients requiring a salvage shunt were comparable to patients receiving a primary shunt. PMID:26898583

  12. 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

  13. 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.

  14. 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.

  15. Long term cultures of primary human hepatocytes as an alternative to drug testing in animals.

    PubMed

    Ullrich, Anett; Stolz, Donna B; Ellis, Ewa C; Strom, Stephen C; Michalopoulos, George K; Hengstler, Jan G; Runge, Dieter

    2009-01-01

    Due to species differences, primary human hepatocytes are still the in vitro system of choice to analyse liver specific processes and functions. Human hepatocytes were cultured for several weeks in a serum-free two-dimensional culture system, which was used to study the effects of acetaminophen (APAP) on hepatocellular functions and vitality. Non-invasive determinations of albumin, urea and lactate dehydrogenase concentrations in cell culture supernatants allowed continuous monitoring for at least two weeks. APAP was applied every 4 days for 24 h. Each application reduced urea production by 25% and albumin synthesis by approximately 70% without any effects on cellular viability. After removal of the substance, hepatocellular functions returned to control levels within one (urea) to three (albumin) days. The repetitive analyses of APAP-mediated effects on cellular metabolism led to identical results for up to five cycles. The drug also caused reversible and repetitive ultrastructural modifications, in particular an almost complete replacement of rough endoplasmic reticulum by smooth endoplasmic reticulum and a massive degradation of glycogen stores. The data demonstrate the suitability of the culture system to serve as a model for repetitive testing of drug-mediated changes on hepatocellular functions, thereby reducing animal studies during drug development. PMID:20383475

  16. Gentamicin inhibits degradation of phosphatidylinositol in primary culture of rabbit proximal tubular cells

    SciTech Connect

    Josepovitz, C.; Ramsammy, L.; Kalovanides, G.J.

    1986-03-01

    Gentamicin (G) induces a phosphatidylinositol (PI) enriched phospholipidosis in renal proximal tubular cells, the cause of which has been attributed to inhibition of degradation by lysosomal phospholipases. To test this hypothesis the authors measured the effect of G on phospholipid (PL) metabolism in primary cultures of rabbit proximal tubular cells. Cells incubated in medium containing G (10/sup -5/-10/sup -3/M) accumulated G and PL in a dose and time dependent manner. At the end of 6 days the total PL of cells incubated in G (10/sup -3/M) was 413 +/- 39 nmol/mg protein compared to 288 +/- 13 nmol/mg protein in control cells. The cell content of PI increased 335% above baseline. To assess the role of impaired degradation in the accumulation of PI, cells were incubated in medium containing (/sup 3/H)myoinositol for two days to label the PI pool after which cells were exposed to G (10/sup -3/M) for 2,4 or 6 days and the decline of (/sup 3/H)PI was determined. In control cultures the time for (/sup 3/H)PI to decline 50% was 1.17 days. In cultures exposed to G the t 1/2 was 2.88 days. The authors conclude that rabbit proximal tubular cells grown in primary culture accumulate G and develop a PI-enriched phospholipidosis which is due at least in part to decreased degradation of PI. The results lend strong support to the hypothesis that G-induced phospholipidosis reflects inhibition of lysosomal phospholipases.

  17. 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

  18. Process cost and facility considerations in the selection of primary cell culture clarification technology.

    PubMed

    Felo, Michael; Christensen, Brandon; Higgins, John

    2013-01-01

    The bioreactor volume delineating the selection of primary clarification technology is not always easily defined. Development of a commercial scale process for the manufacture of therapeutic proteins requires scale-up from a few liters to thousands of liters. While the separation techniques used for protein purification are largely conserved across scales, the separation techniques for primary cell culture clarification vary with scale. Process models were developed to compare monoclonal antibody production costs using two cell culture clarification technologies. One process model was created for cell culture clarification by disc stack centrifugation with depth filtration. A second process model was created for clarification by multi-stage depth filtration. Analyses were performed to examine the influence of bioreactor volume, product titer, depth filter capacity, and facility utilization on overall operating costs. At bioreactor volumes <1,000 L, clarification using multi-stage depth filtration offers cost savings compared to clarification using centrifugation. For bioreactor volumes >5,000 L, clarification using centrifugation followed by depth filtration offers significant cost savings. For bioreactor volumes of ∼ 2,000 L, clarification costs are similar between depth filtration and centrifugation. At this scale, factors including facility utilization, available capital, ease of process development, implementation timelines, and process performance characterization play an important role in clarification technology selection. In the case study presented, a multi-product facility selected multi-stage depth filtration for cell culture clarification at the 500 and 2,000 L scales of operation. Facility implementation timelines, process development activities, equipment commissioning and validation, scale-up effects, and process robustness are examined. PMID:23847160

  19. Primary Air-Liquid Interface Culture of Nasal Epithelium for Nasal Drug Delivery.

    PubMed

    Ong, Hui Xin; Jackson, Claire L; Cole, Janice L; Lackie, Peter M; Traini, Daniela; Young, Paul M; Lucas, Jane; Conway, Joy

    2016-07-01

    Nasal drug administration is a promising alternative to oral and parenteral administration for both local and systemic delivery of drugs. The benefits include its noninvasive nature, rapid absorption, and circumvention of first pass metabolism. Hence, the use of an in vitro model using human primary nasal epithelial cells could be key to understanding important functions and parameters of the respiratory epithelium. This model will enable investigators to address important and original research questions using a biologically relevant in vitro platform that mimics the in vivo nasal epithelial physiology. The purpose of this study was to establish, systematically characterize, and validate the use of a primary human nasal epithelium model cultured at the air-liquid interface for the study of inflammatory responses and drug transport and to simultaneously quantify drug effects on ciliary activity. PMID:27223825

  20. Tissue Factor Activity in Lymphocyte Cultures from Normal Individuals and Patients with Hemophilia A

    PubMed Central

    Rickles, Frederick R.; Hardin, John A.; Pitlick, Frances A.; Hoyer, Leon W.; Conrad, Marcel E.

    1973-01-01

    The procoagulant material of lymphocytes has been characterized as tissue factor. Lymphocytes stimulated with phytohemagglutinin or the purified protein derivative of the tubercle bacillus developed procoagulant activity with incubation in tissue culture. While this material corrected the prolonged clotting time of factor VIII (AHF) deficient plasma, we have shown, utilizing a sensitive radioimmunoassay, that no AHF antigen was present in the cell cultures. Further, we have demonstrated this material to be tissue factor by coagulation techniques and immunological cross-reactivity. The published data regarding factor VIII synthesis is reviewed in light of these observations and comments are made regarding the role of the lymphocyte procoagulant. PMID:4634046

  1. Synthesis of even C/sub 24/-C/sub 30/ primary normal aliphatic alcohols from cyclododecanone

    SciTech Connect

    Zakharkin, L.I.; Churilova, I.M.; Anikina, E.V.

    1988-06-10

    A simple method has been developed for the oxidation of cyclododecanone by sodium persulfate in an aqueous methanol solution of sulfuric acid to give 12-hydroxydodecanoic acid, which upon treatment with HBr in acetic acid gives 12-bromododecanoic acid with RMgX, where R = C/sub 12/H/sub 25/, C/sub 14/H/sub 29/, C/sub 16/H/sub 33/, and C/sub 18/H/sub 37/ in THF in the presence of Li/sub 2/CuCl/sub 4/ and subsequent reduction of the salts of the alkanecarboxylic acids by LiAlH/sub 4/ give primary even normal C/sub 24/-C/sub 30/ alcohols.

  2. Cytotoxicity evaluation using cryopreserved primary human hepatocytes in various culture formats.

    PubMed

    Richert, Lysiane; Baze, Audrey; Parmentier, Céline; Gerets, Helga H J; Sison-Young, Rowena; Dorau, Martina; Lovatt, Cerys; Czich, Andreas; Goldring, Christopher; Park, B Kevin; Juhila, Satu; Foster, Alison J; Williams, Dominic P

    2016-09-01

    Sixteen training compounds selected in the IMI MIP-DILI consortium, 12 drug-induced liver injury (DILI) positive compounds and 4 non-DILI compounds, were assessed in cryopreserved primary human hepatocytes. When a ten-fold safety margin threshold was applied, the non-DILI-compounds were correctly identified 2h following a single exposure to pooled human hepatocytes (n=13 donors) in suspension and 14-days following repeat dose exposure (3 treatments) to an established 3D-microtissue co-culture (3D-MT co-culture, n=1 donor) consisting of human hepatocytes co-cultured with non-parenchymal cells (NPC). In contrast, only 5/12 DILI-compounds were correctly identified 2h following a single exposure to pooled human hepatocytes in suspension. Exposure of the 2D-sandwich culture human hepatocyte monocultures (2D-sw) for 3days resulted in the correct identification of 11/12 DILI-positive compounds, whereas exposure of the human 3D-MT co-cultures for 14days resulted in identification of 9/12 DILI-compounds; in addition to ximelagatran (also not identified by 2D-sw monocultures, Sison-Young et al., 2016), the 3D-MT co-cultures failed to detect amiodarone and bosentan. The sensitivity of the 2D human hepatocytes co-cultured with NPC to ximelagatran was increased in the presence of lipopolysaccharide (LPS), but only at high concentrations, therefore preventing its classification as a DILI positive compound. In conclusion (1) despite suspension human hepatocytes having the greatest metabolic capacity in the short term, they are the least predictive of clinical DILI across the MIP-DILI test compounds, (2) longer exposure periods than 72h of human hepatocytes do not allow to increase DILI-prediction rate, (3) co-cultures of human hepatocytes with NPC, in the presence of LPS during the 72h exposure period allow the assessment of innate immune system involvement of a given drug. PMID:27363785

  3. Cultural Reciprocity in Sociocultural Perspective: Adapting the Normalization Principle for Family Collaboration.

    ERIC Educational Resources Information Center

    Harry, Beth; Rueda, Robert; Kalyanpur, Maya

    1999-01-01

    Findings from a collaborative action research project involving seven culturally diverse families with children with disabilities are used to illustrate how professionals can provide assistance in a family's zone of proximal development, rather than targeting goals that are normative for the mainstream, but not for the family. (Author/CR)

  4. 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…

  5. 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 ...

  6. The Influence of Cultural Bias on Motivation to Learn English: The Case of Khoe Primary School Students in Eastern Botswana

    ERIC Educational Resources Information Center

    Magogwe, Joel Mokuedi

    2009-01-01

    This study investigated the influence of cultural bias in the teaching of English and in the books used to teach English in primary schools attended by Khoe students in eastern Botswana. The study also explored the link between cultural bias and the attitudes and motivation of Khoe students learning English. One hundred and thirty-seven students…

  7. Brief Report: Enhanced Normalization of CD4/CD8 Ratio With Earlier Antiretroviral Therapy at Primary HIV Infection

    PubMed Central

    Inshaw, Jamie; Kaleebu, Pontiano; Cooper, David; Ramjee, Gita; Schechter, Mauro; Tambussi, Giuseppe; Fox, Julie; Samuel, Miriam; Miro, Jose M.; Weber, Jonathan; Porter, Kholoud; Fidler, Sarah

    2016-01-01

    Background: Total CD4+ T-cell counts predict HIV disease progression but do not necessarily reflect normalization of immune function. CD4/CD8 ratio is a marker of immune dysfunction, a prognostic indicator for non-AIDS mortality, and reflects viral reservoir size. Despite antiretroviral therapy (ART), recovery of CD4/CD8 ratio in chronic HIV infection is incomplete; we hypothesize enhanced CD4/CD8 ratio recovery with earlier treatment initiation in recently infected individuals. Methods: CD4+ count and CD4/CD8 ratio were analyzed using data from 2 cohorts: SPARTAC trial and the UK HIV Seroconverters Cohort where primary HIV infection (PHI) was defined as within 6 months from estimated date of infection. Using time-to-event methods and Cox proportional hazard models, we examined the effect of CD4/CD8 ratio at seroconversion on disease progression (CD4 <350 cells per cubic millimeter/ART initiation) and factors associated with time from ART initiation to CD4/CD8 normalization (ratio >1.0). Findings: Of 573 seroconverters, 482 (84%) had abnormal CD4/CD8 ratios at HIV seroconversion. Individuals with higher CD4/CD8 ratio at seroconversion were significantly less likely to reach the disease progression endpoint [adjusted hazard ratio (aHR) (95% CI) = 0.52 (0.32 to 0.82), P = 0.005]. The longer the interval between seroconversion and ART initiation [HR (95% CI) = 0.98 per month increase (0.97, 0.99), P < 0.001], the less likely the CD4/CD8 ratio normalization. ART initiation within 6 months from seroconversion was significantly more likely to normalize [HR (95% CI) = 2.47 (1.67 to 3.67), P < 0.001] than those initiating later. Interpretation: Most individuals presenting in PHI have abnormal CD4/CD8 ratios. The sooner the ART is initiated in PHI, the greater the probability of achieving normal CD4/CD8 ratio. PMID:27070122

  8. Impact of intense pulsed light irradiation on cultured primary fibroblasts and a vascular endothelial cell line

    PubMed Central

    WU, DI; ZHOU, BINGRONG; XU, YANG; YIN, ZHIQIANG; LUO, DAN

    2012-01-01

    The aim of this study was to determine the effects of intense pulsed light (IPL) on cell proliferation and the secretion of vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMPs) in human fibroblasts and vascular endothelial cell lines, and to investigate the effects of IPL on the mRNA expression levels of type I and III procollagens in cultured human fibroblasts. Foreskin fibroblasts and a vascular endothelial cell line (ECV034) were cultured and treated with various wavelengths and doses of IPL irradiation. After culture for 1, 12, 24 and 48 h following IPL irradiation, fibroblasts and the vascular endothelial cell line were harvested for investigation of morphological changes by light microscopy, cell proliferation viability by MTT assay, and VEGF and MMP secretions by ELISA. The mRNA expression levels of type I and III procollagens in the fibroblasts were detected by RT-PCR. No marked morphological changes were observed in the cultured fibroblasts compared with the control. Cell growth and cellular viability were increased in fibroblasts 24 and 48 h after IPL irradiation. The levels of type I and III procollagen mRNA expression in fibroblasts increased in a time-dependent manner. However, the IPL management had no impact on VEGF and MMP secretion levels in fibroblasts and the ECV034 cell line at any time-point after irradiation as well as cell morphology and cellular proliferation. IPL irradiation may induce cellular proliferation and promote the expression of procollagen mRNAs directly in cultured primary fibroblasts, which may primarily contribute to photorejuvenation. PMID:23170124

  9. Mitochondrial activity assessed by cytofluorescence after in-vitro-irradiation of primary rat brain cultures

    SciTech Connect

    Cervos-Navarro, J.; Hamdorf, G. )

    1993-05-01

    Mitochondria play a key role in cell homeostasis and are the first cell organells affected by ionizing irradiation, as it was proved by previous electron microscopic investigations. In order to observe functional parameters of mitochondria after low-dose irradiation, primary rat brain cultures (prepared from 15-day-old rat fetuses) were irradiated from a [sup 60]Co-source with 0.5 and 1 Gy at the age of 2 or 7 days in vitro (div). Cytofluorescence measurement was made by a Cytofluor[sup [trademark]2350] using Rhodamine 123. This fluorescent dye is positively charged and accumulates specifically in the mitochondria of living cells without cytotoxic effect. Since its retention depends on the negative membrane potential as well as the proton gradient that exists across the inner mitochondrial membrane, Rhodamine 123 accumulation reflects the status of mitochondrial activity as a whole. After irradiation with 0.5 and 1 Gy on day 2 in culture there was a decrease in Rhodamine uptake in the irradiated cultures during the first week after the irradiation insult which reached minimum values after 3 days. Rhodamine uptake increased during the following period and finally reached the values of the control cultures. In the second experiment with irradiated cultures on day 7 and the same doses of 0.5 and 1 Gy the accumulation of Rhodamine decreased only initially then increased tremendously. After both doses values of Rhodamine-accumulation were higher than the control level. The results demonstrated that irradiation caused a change in mitochondrial activity depending on the time of irradiation. The dramatic increase over the control levels after irradiation on day 7 in vitro is attributed to the fact that at this time synapses have already developed. Deficiency of mitochondrial activity as well as hyperactivity and the consequent change in energy production may lead to changes in neuronal metabolism including an increase in production of free radicals.

  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. Osmotic shock of cultured primary mammary cells amplifies the hormonal induction of casein gene expression.

    PubMed

    Malienou-Ngassa, R; Puissant, C; Houdebine, L M

    1990-10-01

    Primary cells from rabbit mammary gland cultured on floating collagen were transfected with various plasmids in different conditions. Conventional transfection methods using DEAE-dextran or calcium phosphate followed by an osmotic shock with dimethyl sulphoxide (DMSO), polyethylene glycol (PEG) or glycerol did not prevent lactogenic hormones to induce casein synthesis. On the contrary and unexpectedly, casein synthesis was markedly stimulated by transfection. This amplification was obtained as well with DMSO, PEG and glycerol alone or in the presence of DEAE-dextran, calcium phosphate or DNA. None of these compounds induced casein synthesis in the absence of prolactin. A shock by DMSO also amplified the accumulation of beta-casein mRNA in the presence of prolactin. These results show for the first time that primary cultured mammary cells can be efficiently transfected and still keep their capacity to respond to lactogenic hormones. They also indicate that the short osmotic shocks conventionally used in transfection have a potent long-term stimulatory effect on casein gene expression, which is mediated through an unknown mechanism. PMID:2292339

  13. Using Photobleaching to Measure Spindle Microtubule Dynamics in Primary Cultures of Dividing Drosophila Meiotic Spermatocytes.

    PubMed

    Savoian, Matthew S

    2015-07-01

    In dividing animal cells, a microtubule (MT)-based bipolar spindle governs chromosome movement. Current models propose that the spindle facilitates and/or generates translocating forces by regionally depolymerizing the kinetochore fibers (k-fibers) that bind each chromosome. It is unclear how conserved these sites and the resultant chromosome-moving mechanisms are between different dividing cell types because of the technical challenges of quantitatively studying MTs in many specimens. In particular, our knowledge of MT kinetics during the sperm-producing male meiotic divisions remains in its infancy. In this study, I use an easy-to-implement photobleaching-based assay for measuring spindle MT dynamics in primary cultures of meiotic spermatocytes isolated from the fruit fly Drosophila melanogaster. By use of standard scanning confocal microscopy features, fiducial marks were photobleached on fluorescent protein (FP)-tagged MTs. These were followed by time-lapse imaging during different division stages, and their displacement rates were calculated using public domain software. I find that k-fibers continually shorten at their poles during metaphase and anaphase A through the process of MT flux. Anaphase chromosome movement is complemented by Pac-Man, the shortening of the k-fiber at its chromosomal interface. Thus, Drosophila spermatocytes share the sites of spindle dynamism and mechanisms of chromosome movement with mitotic cells. The data reveal the applicability of the photobleaching assay for measuring MT dynamics in primary cultures. This approach can be readily applied to other systems. PMID:25802491

  14. 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...

  15. Isolated primary blast alters neuronal function with minimal cell death in organotypic hippocampal slice cultures.

    PubMed

    Effgen, Gwen B; Vogel, Edward W; Lynch, Kimberly A; Lobel, Ayelet; Hue, Christopher D; Meaney, David F; Bass, Cameron R Dale; Morrison, Barclay

    2014-07-01

    An increasing number of U.S. soldiers are diagnosed with traumatic brain injury (TBI) subsequent to exposure to blast. In the field, blast injury biomechanics are highly complex and multi-phasic. The pathobiology caused by exposure to some of these phases in isolation, such as penetrating or inertially driven injuries, has been investigated extensively. However, it is unclear whether the primary component of blast, a shock wave, is capable of causing pathology on its own. Previous in vivo studies in the rodent and pig have demonstrated that it is difficult to deliver a primary blast (i.e., shock wave only) without rapid head accelerations and potentially confounding effects of inertially driven TBI. We have previously developed a well-characterized shock tube and custom in vitro receiver for exposing organotypic hippocampal slice cultures to pure primary blast. In this study, isolated primary blast induced minimal hippocampal cell death (on average, below 14% in any region of interest), even for the most severe blasts tested (424 kPa peak pressure, 2.3 ms overpressure duration, and 248 kPa*ms impulse). In contrast, measures of neuronal function were significantly altered at much lower exposures (336 kPa, 0.84 ms, and 86.5 kPa*ms), indicating that functional changes occur at exposures below the threshold for cell death. This is the first study to investigate a tolerance for primary blast-induced brain cell death in response to a range of blast parameters and demonstrate functional deficits at subthreshold exposures for cell death. PMID:24558968

  16. U. v. -enhanced reactivation of u. v. -irradiated herpes virus by primary cultures of rat hepatocytes

    SciTech Connect

    Zurlo, J.; Yager, J.D. )

    1984-04-01

    Carcinogen treatment of cultured mammalian cells prior to infection with u.v.-irradiated virus results in enhanced virus survival and mutagenesis suggesting the induction of SOS-type processes. The development of a primary rat hepatocyte culture system is reported to investigate cellular responses to DNA damage which may be relevant to hepatocarcinogenesis in vivo. Enhanced reactivation of u.v.-irradiated Herpes simplex virus type 1 (HSV-1) occurred in hepatocytes irradiated with u.v. Cultured hepatocytes were pretreated with u.v. at the time of enhanced DNA synthesis. These treatments caused an inhibition followed by a recovery of DNA synthesis. At various times after pretreatment, the hepatocytes were infected with control or u.v.-irradiated HSV-1 at low multiplicity, and virus survival was measured. U.v.-irradiated HSV-1 exhibited the expected two-component survival curve in control or u.v. pretreated hepatocytes. The magnitude of enhanced reactivation of HSV-1 was dependent on the u.v. dose to the hepatocytes, the time of infection following u.v. pretreatment, and the level of DNA synthesis at the time of pretreatment. These results suggest that u.v. treatment of rat hepatocytes causes the induction of SOS-type functions tht may have a role in the initiation of hepatocarcinogenesis.

  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. PMID:25384208

  18. Calcium-activated chloride currents in primary cultures of rabbit distal convoluted tubule.

    PubMed

    Bidet, M; Tauc, M; Rubera, I; de Renzis, G; Poujeol, C; Bohn, M T; Poujeol, P

    1996-10-01

    Chloride (Cl-) conductances were studied in primary cultures of rabbit distal convoluted tubule (very early distal "bright" convoluted tubule, DCTb) by the whole cell patch-clamp technique. We identified a Cl- current activated by 2 microM extracellular ionomycin. The kinetics of the macroscopic current were time dependent for depolarizing potentials with a slow developing component. The steady state current presented outward rectification, and the ion selectivity sequence was I- > Br- > > Cl > glutamate. The current was inhibited by 0.1 mM 5-nitro-2-(3-phenylpropyl-amino)benzoic acid, 1 mM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, and 1 mM diphenylamine-2-carboxylate. To identify the location of the Cl- conductance, 6-methoxy-N-(3-sulfopropyl)quinolinium fluorescence experiments were carried out in confluent cultures developed on collagen-coated permeable filters. Cl- removal from the apical solution induced a Cl- efflux that was stimulated by 10 microM forskolin. Forskolin had no effect on the basolateral Cl- permeability Cl- substitution in the basolateral solution induced an efflux stimulated by 2 microM ionomycin or 50 microM extracellular ATP Ionomycin had no effect on the apical Cl- fluxes. Thus cultured DCTb cells exhibit Ca(2+)-activated Cl- channels located in the basolateral membrane. This Cl- permeability was active at a resting membrane potential and could participate in the Cl- reabsorption across the DCTb in control conditions. PMID:8898026

  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. In vitro culture of oocytes and granulosa cells collected from normal, obese, emaciated and metabolically stressed ewes.

    PubMed

    Tripathi, S K; Farman, M; Nandi, S; Mondal, S; Gupta, Psp; Kumar, V Girish

    2016-07-01

    The present study was undertaken to investigate the oocyte morphology, its fertilizing capacity and granulosa cell functions in ewes (obese, normal, metabolic stressed and emaciated). Ewes (Ovis aries) of approximately 3 years of age (Bellary breed) from a local village were screened, chosen and categorized into a) normal b) obese but not metabolically stressed, c) Emaciated but not metabolically stressed d) Metabolically stressed based on body condition scoring and blood markers. Oocytes and granulosa cells were collected from ovaries of the ewes of all categories after slaughter and were classified into good (oocytes with more than three layers of cumulus cells and homogenous ooplasm), fair (oocytes one or two layers of cumulus cells and homogenous ooplasm) and poor (denuded oocytes or with dark ooplasm). The good and fair quality oocytes were in vitro matured and cultured with fresh semen present and the fertilization, cleavage and blastocyst development were observed. The granulosa cells were cultured for evaluation of metabolic activity by use of the MTT assay, and cell viability, cell number as well as estrogen and progesterone production were assessed. It was observed that the good and fair quality oocytes had greater metabolic activity when collected from normal and obese ewes compared with those from emaciated and metabolically stressed ewes. No significant difference was observed in oocyte quality and maturation amongst the oocytes collected from normal and obese ewes. The cleavage and blastocyst production rates were different for the various body condition classifications and when ranked were: normal>obese>metabolically stressed>emaciated. Lesser metabolic activity was observed in granulosa cells obtained from ovaries of emaciated ewes. However, no changes were observed in viability and cell number of granulosa cells obtained from ewes with the different body condition categories. Estrogen and progesterone production from cultured granulosa cells were

  1. Improving Normal Tissue Complication Probability Models: The Need to Adopt a 'Data-Pooling' Culture

    SciTech Connect

    Deasy, Joseph O.; Bentzen, Soren M.; Jackson, Andrew; Ten Haken, Randall K.; Yorke, Ellen D.; Constine, Louis S.; Sharma, Ashish; Marks, Lawrence B.

    2010-03-01

    Clinical studies of the dependence of normal tissue response on dose-volume factors are often confusingly inconsistent, as the QUANTEC reviews demonstrate. A key opportunity to accelerate progress is to begin storing high-quality datasets in repositories. Using available technology, multiple repositories could be conveniently queried, without divulging protected health information, to identify relevant sources of data for further analysis. After obtaining institutional approvals, data could then be pooled, greatly enhancing the capability to construct predictive models that are more widely applicable and better powered to accurately identify key predictive factors (whether dosimetric, image-based, clinical, socioeconomic, or biological). Data pooling has already been carried out effectively in a few normal tissue complication probability studies and should become a common strategy.

  2. Improving normal tissue complication probability models: the need to adopt a "data-pooling" culture.

    PubMed

    Deasy, Joseph O; Bentzen, Søren M; Jackson, Andrew; Ten Haken, Randall K; Yorke, Ellen D; Constine, Louis S; Sharma, Ashish; Marks, Lawrence B

    2010-03-01

    Clinical studies of the dependence of normal tissue response on dose-volume factors are often confusingly inconsistent, as the QUANTEC reviews demonstrate. A key opportunity to accelerate progress is to begin storing high-quality datasets in repositories. Using available technology, multiple repositories could be conveniently queried, without divulging protected health information, to identify relevant sources of data for further analysis. After obtaining institutional approvals, data could then be pooled, greatly enhancing the capability to construct predictive models that are more widely applicable and better powered to accurately identify key predictive factors (whether dosimetric, image-based, clinical, socioeconomic, or biological). Data pooling has already been carried out effectively in a few normal tissue complication probability studies and should become a common strategy. PMID:20171511

  3. IMPROVING NORMAL TISSUE COMPLICATION PROBABILITY MODELS: THE NEED TO ADOPT A “DATA-POOLING” CULTURE

    PubMed Central

    Deasy, Joseph O.; Bentzen, Søren M.; Jackson, Andrew; Ten Haken, Randall K.; Yorke, Ellen D.; Constine, Louis S.; Sharma, Ashish; Marks, Lawrence B.

    2010-01-01

    Clinical studies of the dependence of normal tissue response on dose-volume factors are often confusingly inconsistent, as the QUANTEC reviews demonstrate. A key opportunity to accelerate progress is to begin storing high-quality datasets in repositories. Using available technology, multiple repositories could be conveniently queried, without divulging protected health information, to identify relevant sources of data for further analysis. After obtaining institutional approvals, data could then be pooled, greatly enhancing the capability to construct predictive models that are more widely applicable and better powered to accurately identify key predictive factors (whether dosimetric, image-based, clinical, socioeconomic, or biological). Data pooling has already been carried out effectively in a few normal tissue complication probability studies and should become a common strategy. PMID:20171511

  4. Nontargeted Stressful Effects in Normal Human Fibroblast Cultures Exposed to Low Fluences of High Charge, High Energy (HZE) Particles: Kinetics of Biologic Responses and Significance of Secondary Radiations

    PubMed Central

    Gonon, Géraldine; Groetz, Jean-Emmanuel; de Toledo, Sonia M.; Howell, Roger W.; Fromm, Michel; Azzam, Edouard I.

    2014-01-01

    The induction of nontargeted stressful effects in cell populations exposed to low fluences of high charge (Z) and high energy (E) particles is relevant to estimates of the health risks of space radiation. We investigated the up-regulation of stress markers in confluent normal human fibroblast cultures exposed to 1,000 MeV/u iron ions [linear energy transfer (LET) ~151 keV/μm] or 600 MeV/u silicon ions (LET ~50 keV/μm) at mean absorbed doses as low as 0.2 cGy, wherein 1–3% of the cells were targeted through the nucleus by a primary particle. Within 24 h postirradiation, significant increases in the levels of phospho-TP53 (serine 15), p21Waf1 (CDKN1A), HDM2, phospho-ERK1/2, protein carbonylation and lipid peroxidation were detected, which suggested participation in the stress response of cells not targeted by primary particles. This was supported by in situ studies that indicated greater increases in 53BP1 foci formation, a marker of DNA damage. than expected from the number of primary particle traversals. The effect was expressed as early as 15 min after exposure, peaked at 1 h and decreased by 24 h. A similar tendency occurred after exposure of the cell cultures to 0.2 cGy of 3.7 MeV α particles (LET ~109 keV/μm) that targets ~1.6% of nuclei, but not after 0.2 cGy from 290 MeV/u carbon ions (LET ~13 keV/μm) by which, on average, ~13% of the nuclei were hit, which highlights the importance of radiation quality in the induced effect. Simulations with the FLUKA multi-particle transport code revealed that fragmentation products, other than electrons, in cell cultures exposed to HZE particles comprise <1% of the absorbed dose. Further, the radial spread of dose due to secondary heavy ion fragments is confined to approximately 10–20 μm. Thus, the latter are unlikely to significantly contribute to stressful effects in cells not targeted by primary HZE particles. PMID:23465079

  5. Effects of culture substrates and normal hepatic sinusoidal cells on in vitro hepatocyte synthesis of Apo-SAA.

    PubMed

    Subrahmanyan, L; Kisilevsky, R

    1988-03-01

    Primary hepatocyte cultures synthesize apo-SAA upon stimulation with supernatant from lipopolysaccharide (LPS)-treated macrophages. The matrices on which the hepatocytes were grown influence their basal apo-SAA synthetic capability. Fibronectin was superior. Coculturing hepatocytes with hepatic sinusoidal cells did not adversely affect the ability of hepatocytes to synthesize and secrete apo-SAA into the culture medium. In 72 h, clear islands of endothelial cells nestled in layers of hepatocytes. Both apo-SAA and apo-SAA were made in considerable quantities but no evidence could be obtained that the apo-SAA were free of apo-A-1. The coculturing of hepatocytes with liver sinusoidal cells, the site of ultimate AA deposition, is a first step in establishing an in vitro system for AA amyloidogenesis. PMID:3353686

  6. Effects of interferon-gamma on primary cultures of human brain microvessel endothelial cells.

    PubMed Central

    Huynh, H. K.; Dorovini-Zis, K.

    1993-01-01

    Primary cultures of human brain microvessel endothelial cells were used to study the effects of human recombinant interferon-gamma (IFN-gamma) on cerebral endothelium in vitro. Incubation of monolayers with various concentrations of IFN-gamma (10 to 200 U/ml) for 12 to 96 hours induced surface expression of class II major histocompatibility complex (Ia) antigen in a time- and concentration-dependent manner. In immunogold-stained cultures, labeling was observed as early as 12 hours, was maximal after 48 hours, and persisted at plateau levels in the continuous presence of the cytokine. Expression was blocked by coincubation with anti-IFN-gamma antibody and was reversed 4 days following removal of IFN-gamma from the culture media. Endothelial cells treated with IFN-gamma for 3 to 4 days became spindle-shaped, extensively overlapped, and frequently formed cellular whorls. These changes did not occur in the presence of anti-IFN-gamma antibody and reversed upon removal of IFN-gamma from the media. The morphological alterations were associated with increased permeability of confluent monolayers to macromolecules as compared with untreated cultures. The results of these studies indicate that human brain microvessel endothelial cells respond to in vitro cytokine stimulation by undergoing profound morphological, functional, and permeability changes. We conclude that cerebral endothelium may play an important role in the initiation and regulation of lymphocyte traffic across the blood-brain barrier in inflammatory disorders of the human central nervous system. Images Figure 3 Figure 4 Figure 5 Figure 6 Figure 8 PMID:8475997

  7. Normal reproductive capacity of heifers that originated from in vitro fertilized embryos cultured with an antiviral compound.

    PubMed

    Givens, M Daniel; Marley, Mylissa S D; Riddell, Kay P; Galik, Patricia K; Stringfellow, David A

    2009-07-01

    Bovine viral diarrhea virus (BVDV) can associate with in vitro fertilized (IVF) bovine embryos despite washing and trypsin treatment. An antiviral compound, DB606 (2-(4-[2-imidazolinyl]phenyl)-5-(4-methoxyphenyl)furan), inhibits the replication of BVDV in bovine uterine tubal epithelial cells, Madin Darby bovine kidney cells, and fetal fibroblast cells. As well, DB606 in in vitro culture medium does not affect embryonic development. Antiviral-treated-IVF embryos placed into recipients developed into clinically normal calves. The objective of this project was to determine if these resultant heifer calves were capable of reproducing. Seven heifers from each of the treatment groups (natural breeding, IVF embryo, and IVF embryo cultured in DB606) of the previous study were used. At 20-27 months of age, the heifers were exposed to a fertile bull in a single pasture during a 63 d breeding season. Five of the seven heifers originating from natural breeding were pregnant 35 d after removal of the bull and calved. All of the heifers resulting from transfer of untreated IVF embryos were pregnant at 35 d; however, one aborted the fetus at 5-7 months of gestation. All of the heifers derived from transfer of IVF embryos cultured in DB606 were pregnant and calved. Offspring from dams of all treatment groups were clinically normal at birth. Adjusted 205 d weaning weights were not significantly different among the offspring of the treated and untreated dams. These results indicate that culture of bovine-IVF embryos in DB606 does not impair future reproductive capacity of resulting heifers. PMID:18691836

  8. Putative Nanobacteria Represent Physiological Remnants and Culture By-Products of Normal Calcium Homeostasis

    PubMed Central

    Young, John D.; Young, Lena; Wu, Cheng-Yeu; Young, Andrew

    2009-01-01

    Putative living entities called nanobacteria (NB) are unusual for their small sizes (50–500 nm), pleomorphic nature, and accumulation of hydroxyapatite (HAP), and have been implicated in numerous diseases involving extraskeletal calcification. By adding precipitating ions to cell culture medium containing serum, mineral nanoparticles are generated that are morphologically and chemically identical to the so-called NB. These nanoparticles are shown here to be formed of amorphous mineral complexes containing calcium as well as other ions like carbonate, which then rapidly acquire phosphate, forming HAP. The main constituent proteins of serum-derived NB are albumin, fetuin-A, and apolipoprotein A1, but their involvement appears circumstantial since so-called NB from different body fluids harbor other proteins. Accordingly, by passage through various culture media, the protein composition of these particles can be modulated. Immunoblotting experiments reveal that antibodies deemed specific for NB react in fact with either albumin, fetuin-A, or both, indicating that previous studies using these reagents may have detected these serum proteins from the same as well as different species, with human tissue nanoparticles presumably absorbing bovine serum antigens from the culture medium. Both fetal bovine serum and human serum, used earlier by other investigators as sources of NB, paradoxically inhibit the formation of these entities, and this inhibition is trypsin-sensitive, indicating a role for proteins in this inhibitory process. Fetuin-A, and to a lesser degree albumin, inhibit nanoparticle formation, an inhibition that is overcome with time, ending with formation of the so-called NB. Together, these data demonstrate that NB are most likely formed by calcium or apatite crystallization inhibitors that are somehow overwhelmed by excess calcium or calcium phosphate found in culture medium or in body fluids, thereby becoming seeds for calcification. The structures

  9. Putative nanobacteria represent physiological remnants and culture by-products of normal calcium homeostasis.

    PubMed

    Young, John D; Martel, Jan; Young, Lena; Wu, Cheng-Yeu; Young, Andrew; Young, David

    2009-01-01

    Putative living entities called nanobacteria (NB) are unusual for their small sizes (50-500 nm), pleomorphic nature, and accumulation of hydroxyapatite (HAP), and have been implicated in numerous diseases involving extraskeletal calcification. By adding precipitating ions to cell culture medium containing serum, mineral nanoparticles are generated that are morphologically and chemically identical to the so-called NB. These nanoparticles are shown here to be formed of amorphous mineral complexes containing calcium as well as other ions like carbonate, which then rapidly acquire phosphate, forming HAP. The main constituent proteins of serum-derived NB are albumin, fetuin-A, and apolipoprotein A1, but their involvement appears circumstantial since so-called NB from different body fluids harbor other proteins. Accordingly, by passage through various culture media, the protein composition of these particles can be modulated. Immunoblotting experiments reveal that antibodies deemed specific for NB react in fact with either albumin, fetuin-A, or both, indicating that previous studies using these reagents may have detected these serum proteins from the same as well as different species, with human tissue nanoparticles presumably absorbing bovine serum antigens from the culture medium. Both fetal bovine serum and human serum, used earlier by other investigators as sources of NB, paradoxically inhibit the formation of these entities, and this inhibition is trypsin-sensitive, indicating a role for proteins in this inhibitory process. Fetuin-A, and to a lesser degree albumin, inhibit nanoparticle formation, an inhibition that is overcome with time, ending with formation of the so-called NB. Together, these data demonstrate that NB are most likely formed by calcium or apatite crystallization inhibitors that are somehow overwhelmed by excess calcium or calcium phosphate found in culture medium or in body fluids, thereby becoming seeds for calcification. The structures described

  10. Age-changes in gene expression in primary mixed glia cultures from young vs old rat cerebral cortex are modified by interactions with neurons

    PubMed Central

    Kremsky, Isaac; Morgan, Todd E.; Hou, Xiaogang; Li, Lei; Finch, Caleb E.

    2013-01-01

    Astrocytic GFAP expression increases during normal aging in many brain regions and in primary astrocyte cultures derived from aging rodent brains. As shown below, we unexpectedly found that the age-related increase of GFAP expression was suppressed in mixed glia (astrocytes + microglia). However, the age-related increase of GFAP was observed when E18 neurons were co-cultured with mixed glia. Thus, the presence of microglia can suppress the age-related increase of GFAP, in primary cultures of astrocytes. To more broadly characterize how aging and co-culture with neurons alters glial gene expression, we profiled gene expression in mixed glia from young (3 mo) and old (24 mo) male rat cerebral cortex by Affymetrix microarray (Rat230 2.0). The majority of age changes were independent of the presence of neurons. Overall, the expression of 2-fold more genes increased with age than decreased with age. The minority of age changes that were either suppressed or revealed by the presence of neurons may be useful to analyze glial-neuron interaction during aging. Some in vitro changes are shared with those of aging rat hippocampus in studies from the Landfield group (Rowe et al., 2007; Kadish et al., 2009). PMID:22226781

  11. Improved protocols for protein and RNA isolation from three-dimensional collagen sandwich cultures of primary hepatocytes.

    PubMed

    Heidebrecht, F; Schulz, I; Keller, M; Behrens, S-E; Bader, A

    2009-10-01

    The sandwich culture is the most widely used long-term culture system for functional primary hepatocytes. Despite its advantages, the currently available protocols for protein and RNA extraction are either time-consuming or contain steps that may skewer the results. This paper describes improved protocols for RNA and protein extraction from sandwich cultures that are easy to perform, require short working time, and use no additional enzymatic reactions that could change the expression profile of the cells. The quality of the RNA is excellent, allowing also applications requiring high purity such as microarrays. In general, the protocols are suited for any cells in 3D collagen culture. PMID:19539596

  12. Evaluation of a carp primary hepatocyte culture system for screening chemicals for oestrogenic activity.

    PubMed

    Bickley, L K; Lange, A; Winter, M J; Tyler, C R

    2009-09-14

    The presence of endocrine disrupting chemicals (EDCs) in the environment has driven the development of screening and testing assays to both identify chemicals with hormonal activity and evaluate their potential to cause adverse effects. As the number of animals used for research and regulatory purposes rises, and set against a desire to reduce animal testing, there is increased emphasis on the development and application of in vitro techniques to evaluate chemical risks to the environment. Induction of vitellogenin (VTG) in isolated fish liver cells has been used successfully to identify a wide range of EDCs, including both natural and synthetic oestrogens and a variety of other xenoestrogens. However, the vitellogenic response reported for hepatocytes in culture has been shown to vary widely, making comparisons between studies difficult. The work presented in this paper explored the variability of the vitellogenic response in primary cultures of common carp (Cyprinus carpio) hepatocytes following exposure to the model oestrogenic compound, 17beta-oestradiol (E2). As expected, variability in the vitellogenic response was observed, both in terms of the sensitivity and magnitude of VTG induction, for hepatocytes isolated from different fish. An apparent difference was observed in the response of isolated hepatocytes based on the sex of the donor fish; maximum levels of E2-stimulated VTG synthesis in hepatocytes derived from females appeared higher (1962 ng mL(-1)+/-487 [n=9] compared with 1194 ng mL(-1)+/-223 for hepatocytes from males [n=9]) and EC(50) values lower (1.61+/-0.4 microM E2 for females and 2.12+/-0.2 microM E2 for males). However, these differences were not statistically significant, likely in part due to the variation observed in the vitellogenic response. In particular, hepatocytes derived from female fish showed more variation than their male counterparts (the co-efficient of variation for females was 77% compared to 28% for males). Despite the

  13. 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

  14. Isolation and characterization of membranes from normal and transformed tissue-culture cells

    PubMed Central

    Graham, John M.

    1972-01-01

    Homogenates of baby-hamster kidney cells and rat embryo fibroblasts prepared by nitrogen cavitation contain a small population of slowly sedimenting mitochondria or mitochondrial fragments, which contaminate the microsomal fraction. This appears to limit the resolution of surface membrane and endoplasmic reticulum on magnesium-containing dextran gradients. The microsomal material and mitochondria can, however, be completely separated on a 10–60% (w/w) sucrose zonal gradient containing a 30% sucrose plateau. On magnesium-containing dextran gradients this mitochondria-free microsomal material can be resolved into at least two surface membrane fractions and at least two endoplasmic reticulum fractions. Comparison of polyoma virus-transformed and normal baby-hamster kidney cells reveals some interesting differences in their microsomal fractionation patterns and the characteristics of the Na+/K+-Mg2+ adenosine triphosphatase of their surface membranes, in particular a tenfold lower Km in the virus-transformed cells. The fractionation patterns of normal and spontaneously transformed rat embryo fibroblasts are also briefly discussed, particularly in relation to the significance of the observation that both the surface membrane and endoplasmic reticulum from these cells can be subfractionated. PMID:4348259

  15. Tissue culture of normal and cystic fibrosis sweat gland duct cells. I. Alterations in dome formation.

    PubMed

    Hazen-Martin, D J; Spicer, S S; Sens, M A; Jenkins, M Q; Westphal, M C; Sens, D A

    1987-01-01

    The elucidation of the underlying defect in fluid secretion by cystic fibrosis (CF) sweat glands is hindered by the unavailability of an experimental model for investigating this disease. As a potential model system, a serum-free growth medium was developed that supports the explant growth of epithelial cells from fragments of human skin. Immunohistochemical analysis demonstrated that these epithelial cell outgrowths originated from the duct of the sweat gland. By electron microscopy, the cells were demonstrated to possess keratinocyte-like morphology as noted by the presence of a multilayered outgrowth of cells containing well-defined keratin bundles. Identical outgrowths from skin biopsies of CF patients were compared to normal outgrowths and alterations were noted to occur in dome formation and in the number of intercellular spaces between cells. Doming alterations were also noted to occur in the CF heterozygous state. No differences in cell fine structure or in growth factor requirements for cell proliferation were noted between normal and CF cells. The potential use of this system as a model for CF research is discussed. PMID:2432458

  16. Primary transitions between the yrast superdeformed band and low-lying normal deformed states in {sup 194}Pb

    SciTech Connect

    Hauschild, K.; Bernstein, L.A.; Becker, J.A.

    1996-12-31

    The observation of one-step `primary` gamma-ray transitions directly linking the superdeformed (SD) states to the normal deformed (ND) low-lying states of known excitation energies (E{sub x}), spins and parities (J{sup {pi}}) is crucial to determining the E{sub x} and J{sup {pi}} of the SD states. With this knowledge one can begin to address some of the outstanding problems associated with SD nuclei, such as the identical band issue, and one can also place more stringent restrictions on theoretical calculations which predict SD states and their properties. Brinkman, et al., used the early implementation of the GAMMASPHERE spectrometer array (32 detectors) and proposed a single, candidate {gamma} ray linking the {sup 194}Pb yrast SD band to the low-lying ND states in {sup 194}Pb. Using 55 detectors in the GAMMASPHERE array Khoo, et al., observed multiple links between the yrast SD band in {sup 194}Hg and the low-lying level scheme and conclusively determined E{sub x} and J of the yrast SD states. Here the authors report on an experiment in which Gammasphere with 88 detectors was used and the E{sub x} and J{sup {pi}} values of the yrast SD states in {sup 194}Pb were uniquely determined. Twelve one-step linking transitions between the yrast SD band and low-lying states in {sup 194}Pb have been identified, including the transition proposed by Brinkman. These transitions have been placed in the level scheme of {sup 194}Pb using coincidence relationships and agreements between the energies of the primary transitions and the energy differences in level spacings. Furthermore, measurements of angular asymmetries have yielded the multipolarities of the primaries which have allowed J{sup {pi}} assignments of the {sup 194}Pb SD states to be unambiguously determined for the first time without a priori assumptions about the character of SD bands. A study performed in parallel to this work using the EUROGAM-II array reports similar, but somewhat less extensive, results.

  17. Primary culture of intestinal epithelial cells as a potential model for Toxoplasma gondii enteric cycle studies.

    PubMed

    Moura, Marcos de Assis; Amendoeira, Maria Regina Reis; Barbosa, Helene Santos

    2009-09-01

    The primary culture of intestinal epithelial cells from domestic cats is an efficient cellular model to study the enteric cycle of Toxoplasma gondii in a definitive host. The parasite-host cell ratio can be pointed out as a decisive factor that determines the intracellular fate of bradyzoites forms. The development of the syncytial-like forms of T. gondii was observed using the 1:20 bradyzoite-host cell ratio, resulting in similar forms described in in vivo systems. This alternative study potentially opens up the field for investigation into the molecular aspects of this interaction. This can contribute to the development of new strategies for intervention of a main route by which toxoplasmosis spreads. PMID:19876557

  18. In vitro metabolism and toxicity assessment of N-methylcarbazole in primary cultured rat hepatocytes.

    PubMed

    Yang, W; Jiang, T R; Davis, P J; Acosta, D

    1991-01-01

    N-Methycarbazole (NMC), a carcinogen and mutagen in tobacco smoke, was converted to two major metabolites by primary cultured rat hepatocytes as measured by high performance liquid chromatography (HPLC): N-hydroxymethylcarbazole (NHMC) and carbazole. These two metabolites had comparable retention times and identical ultraviolet spectra as those of reference standards. Identical retention times and mass spectra were also observed as detected by gas chromatography-mass spectroscopy (GC-MS) for NHMC and its reference standard. The toxicities of NMC and its two metabolites were assessed by lactate dehydrogenase (LDH) leakage and neutral red (NR) uptake. The rank order of cytotoxicity of NMC and its metabolites was found to be: NHMC greater than NMC greater than carbazole. Thus, we conclude that the hydroxylation of NMC to NHMC may represent a toxification step, while the further dealkylation to carbazole is most likely a detoxication process. PMID:1896996

  19. 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

  20. 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.

  1. Differential Proteomic Analysis of Human Placenta-Derived Mesenchymal Stem Cells Cultured on Normal Tissue Culture Surface and Hyaluronan-Coated Surface

    PubMed Central

    Wong, Tzyy Yue; Chen, Ying-Hui; Liu, Szu-Heng; Solis, Mairim Alexandra; Yu, Chen-Hsiang; Chang, Chiung-Hsin; Huang, Lynn L. H.

    2016-01-01

    Our previous results showed that hyaluronan (HA) preserved human placenta-derived mesenchymal stem cells (PDMSC) in a slow cell cycling mode similar to quiescence, the pristine state of stem cells in vivo, and HA was found to prevent murine adipose-derived mesenchymal stem cells from senescence. Here, stable isotope labeling by amino acid in cell culture (SILAC) proteomic profiling was used to evaluate the effects of HA on aging phenomenon in stem cells, comparing (1) old and young passage PDMSC cultured on normal tissue culture surface (TCS); (2) old passage on HA-coated surface (CHA) compared to TCS; (3) old and young passage on CHA. The results indicated that senescence-associated protein transgelin (TAGLN) was upregulated in old TCS. Protein CYR61, reportedly senescence-related, was downregulated in old CHA compared to old TCS. The SIRT1-interacting Nicotinamide phosphoribosyltransferase (NAMPT) increased by 2.23-fold in old CHA compared to old TCS, and is 0.48-fold lower in old TCS compared to young TCS. Results also indicated that components of endoplasmic reticulum associated degradation (ERAD) pathway were upregulated in old CHA compared to old TCS cells, potentially for overcoming stress to maintain cell function and suppress senescence. Our data points to pathways that may be targeted by HA to maintain stem cells youth. PMID:27057169

  2. 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.

  3. Drug Metabolism Enzyme Expression and Activity in Primary Cultures of Human Proximal Tubular Cells

    PubMed Central

    Lash, Lawrence H.; Putt, David A.; Cai, Hongliang

    2008-01-01

    We previously catalogued expression and activity of organic anion and cation, amino acid, and peptide transporters in primary cultures of human proximal tubular (hPT) cells to establish them as a cellular model to study drug transport in the human kidney [Toxicology 228, 200–218 (2006)]. Here, we extend our analysis to drug metabolism enzymes. Expression of 11 cytochrome P450 (CYP) enzymes was determined with specific antibodies. CYP1B1, CYP3A4, and CYP4A11 were the only CYP enzymes readily detected in total cell extracts. These same CYP enzymes, as well as CYP3A5 and possibly CYP2D6, were detected in microsomes from confluent hPT cells, although expression levels varied among kidney samples. In agreement with Western blot data, only activity of CYP3A4/5 was detected among the enzyme activities measured. Expression of all three glutathione S-transferases (GSTs) known to be found in hPT cells, GSTA, GSTP, and GSTT, was readily detected. Variable expression of three sulfotransferases (SULTs), SULT1A3, SULT1E, and SULT2A1, and three UDP-glucuronosyltransferases (UGTs), UGT1A1, UGT1A6, and UGT2B7, was also detected. When examined over the course of cell growth to confluence, expression of all enzymes was generally maintained at readily measurable levels, although they were often lower than in fresh tissue. These results indicate that primary cultures of hPT cells possess significant capacity to metabolize many classes of drugs, and can be used as an effective model to study drug metabolism. PMID:18055091

  4. A primary culture of distal convoluted tubules expressing functional thiazide-sensitive NaCl transport.

    PubMed

    Markadieu, Nicolas; San-Cristobal, Pedro; Nair, Anil V; Verkaart, Sjoerd; Lenssen, Ellen; Tudpor, Kukiat; van Zeeland, Femke; Loffing, Johannes; Bindels, René J M; Hoenderop, Joost G J

    2012-09-15

    Studying the molecular regulation of the thiazide-sensitive Na(+)-Cl(-) cotransporter (NCC) is important for understanding how the kidney contributes to blood pressure regulation. Until now, a native mammalian cell model to investigate this transporter remained unknown. Our aim here is to establish, for the first time, a primary distal convoluted tubule (DCT) cell culture exhibiting transcellular thiazide-sensitive Na(+) transport. Because parvalbumin (PV) is primarily expressed in the DCT, where it colocalizes with NCC, kidneys from mice expressing enhanced green-fluorescent protein (eGFP) under the PV gene promoter (PV-eGFP-mice) were employed. The Complex Object Parametric Analyzer and Sorter (COPAS) was used to sort fluorescent PV-positive tubules from these kidneys, which were then seeded onto permeable supports. After 6 days, DCT cell monolayers developed transepithelial resistance values of 630 ± 33 Ω·cm(2). The monolayers also established opposing transcellular concentration gradients of Na(+) and K(+). Radioactive (22)Na(+) flux experiments showed a net apical-to-basolateral thiazide-sensitive Na(+) transport across the monolayers. Both hypotonic low-chloride medium and 1 μM angiotensin II increased this (22)Na(+) transport significantly by four times, which could be totally blocked by 100 μM hydrochlorothiazide. Angiotensin II-stimulated (22)Na(+) transport was also inhibited by 1 μM losartan. Furthermore, NCC present in the DCT monolayers was detected by immunoblot and immunocytochemistry studies. In conclusion, a murine primary DCT culture was established which expresses functional thiazide-sensitive Na(+)-Cl(-) transport. PMID:22759396

  5. Excitatory amino acid-stimulated uptake of /sup 22/Na+ in primary astrocyte cultures

    SciTech Connect

    Kimelberg, H.K.; Pang, S.; Treble, D.H.

    1989-04-01

    In this study we have found that L-glutamic acid, as well as being taken up by a Na+-dependent mechanism, will stimulate the uptake of 22Na+ by primary astrocyte cultures from rat brain in the presence of ouabain. By simultaneously measuring the uptake of 22Na+ and L-3H-glutamate a stoichiometry of 2-3 Na+ per glutamate was measured, implying electrogenic uptake. Increasing the medium K+ concentration to depolarize the cells inhibited L-3H-glutamate uptake, while calculations of the energetics of the observed L-3H-glutamate accumulation also supported an electrogenic mechanism of at least 2 Na+:1 glutamate. In contrast, kinetic analysis of the Na+ dependence of L-3H-glutamate uptake indicated a stoichiometry of Na+ to glutamate of 1:1, but further analysis showed that the stoichiometry cannot be resolved by purely kinetic studies. Studies with glutamate analogs, however, showed that kainic acid was a very effective stimulant of 22Na+ uptake, but 3H-kainic acid showed no Na+ -dependent uptake. Furthermore, while L-3H-glutamate uptake was very sensitive to lowered temperatures, glutamate-stimulated 22Na+ uptake was relatively insensitive. These results indicate that glutamate-stimulated uptake of 22Na+ in primary astrocytes cultures cannot be explained solely by cotransport of Na+ with glutamate, and they suggest that direct kainic acid-type receptor induced stimulation of Na+ uptake also occurs. Since both receptor and uptake effects involve transport of Na+, accurate measurements of the Na+ :glutamate stoichiometry for uptake can only be done using completely specific inhibitors of these 2 systems.

  6. Electrotransfer of gene encoding endostatin into normal and neoplastic mouse tissues: inhibition of primary tumor growth and metastatic spread.

    PubMed

    Cichoń, Tomasz; Jamrozy, Laura; Glogowska, Joanna; Missol-Kolka, Ewa; Szala, Stanisław

    2002-09-01

    Electroporation-mediated gene transfer relies upon direct delivery of plasmids into cells permeabilized by electric fields, a method more efficient than transfer using nonviral vectors, although neither approaches the transfer efficiency of viral vectors. Here we studied electrotransfer of a gene encoding an angiogenesis inhibitor (endostatin) into primary tumors and muscle tissues, which would serve as a site of synthesis and secretion into the bloodstream of a therapeutic antimetastatic protein with systemic effects. Optimum electroporation conditions (voltage, number and duration of impulses, separation of caliper electrodes) were first established to maximize expression of a reporter gene transferred into murine Renca kidney carcinoma, B16(F10) melanoma, or skeletal muscle tissues. In neoplastic tissues, electrotransfer of plasmid DNA was far more efficient than electroporation with lipoplexes, but no differences between naked DNA and lipoplexes were found in case of electroporated muscles. We then studied the electrotransfer of plasmid DNA carrying the endostatin gene into pre-established experimental Renca tumors. A significant inhibition of tumor growth was observed in animals electroporated with this construct. Electrotransfer of the endostatin gene into muscle tissues resulted in reduced numbers of experimental B16(F10) metastases in the lungs. This study clearly shows that electroporation may be used to efficiently transfer antiangiogenic genes into both normal and neoplastic tissues. PMID:12189527

  7. Cerebellum-dependent associative learning deficits in primary dystonia are normalized by rTMS and practice.

    PubMed

    Hoffland, B S; Kassavetis, P; Bologna, M; Teo, J T H; Bhatia, K P; Rothwell, J C; Edwards, M J; van de Warrenburg, B P

    2013-07-01

    Eyeblink classical conditioning (EBCC) is a cerebellum-dependent paradigm of associative motor learning, and abnormal EBCC is a neurophysiological indicator of cerebellar dysfunction. We have previously demonstrated impaired EBCC in patients with primary dystonia, but it remains uncertain if this represents actual cerebellar pathology or reflects a functional cerebellar disruption. We examined this further by: (1) studying acquisition and retention of EBCC in a second session in eight patients with cervical dystonia (CD) who had a first session 7-10 days earlier; and (2) by investigating the potential of continuous theta burst stimulation (cTBS) over the right cerebellar hemisphere to modify a first-ever EBCC session in 11 patients with CD. EBCC data of eight healthy controls previously studied were used for additional between-group comparisons. We observed an improvement of EBCC in a second session in patients with CD, which is in contrast to patients with proven cerebellar pathology who do not show further improvement of EBCC in additional sessions. We also found that cerebellar cTBS paradoxically normalized EBCC in patients with CD, while we previously showed that it disrupts EBCC in healthy volunteers. Combined, these two experiments are in keeping with a functional and reversible disruption of the cerebellum in dystonia, a phenomenon that is probably secondary to either cerebellar compensation or to cerebellar recruitment in the abnormal sensorimotor network. PMID:23551802

  8. Cellular responses in primary epidermal cultures from rainbow trout exposed to zinc chloride.

    PubMed

    Ní Shúilleabháin, Sharon; Mothersill, Carmel; Sheehan, David; O'Brien, Nora M; O' Halloran, John; van Pelt, Frank N A M; Kilemade, Michael; Davoren, Maria

    2006-11-01

    In this study, we examined the effects of zinc chloride (ZnCl(2)) (0-200mg/L) on primary epidermal cultures from Oncorhynchus mykiss. Increases in the rate and amount of mucus released were detected post-exposure, as was a dose-dependent increase in the synthesis of acidic glycoproteins. The cytotoxicity of ZnCl(2) to the cultures was significantly increased (P< or =0.05) when exposures were conducted in serum-free medium as opposed to medium containing serum. Significant increases in the levels of apoptosis and necrosis were observed with increasing exposure concentration, but these were seen to decrease over time. The in vitro cytological and pathological changes observed in this study were found to be in accordance with previously published in vivo studies on the effects of heavy metals on the integument. This model system may help to further elucidate the effects of ecotoxicants on the external innate immune system of fish. PMID:16223524

  9. Harvest and primary culture of the murine aldosterone-sensitive distal nephron

    PubMed Central

    Labarca, Mariana; Nizar, Jonathan M.; Walczak, Elisabeth M.; Dong, Wuxing; Pao, Alan C.

    2015-01-01

    The aldosterone-sensitive distal nephron (ASDN) exhibits axial heterogeneity in structure and function from the distal convoluted tubule to the medullary collecting duct. Ion and water transport is primarily divided between the cortex and medulla of the ASDN, respectively. Transcellular transport in this segment is highly regulated in health and disease and is integrated across different cell types. We currently lack an inexpensive, high-yield, and tractable technique to harvest and culture cells for the study of gene expression and physiological properties of mouse cortical ASDN. To address this need, we harvested tubules bound to Dolichos biflorus agglutinin lectin-coated magnetic beads from the kidney cortex and characterized these cell preparations. We determined that these cells are enriched for markers of distal convoluted tubule, connecting tubule, and cortical collecting duct, including principal and intercalated cells. In primary culture, these cells develop polarized monolayers with high resistance (1,000-1,500 Ω * cm2) and maintain expression and activity of key channels. These cells demonstrate an amiloride-sensitive short-circuit current that can be enhanced with aldosterone and maintain measurable potassium and anion secretion. Our method can be easily adopted to study the biology of the ASDN and to investigate phenotypic differences between wild-type and transgenic mouse models. PMID:25810438

  10. Adult human arterial smooth muscle cells in primary culture. Modulation from contractile to synthetic phenotype.

    PubMed

    Thyberg, J; Nilsson, J; Palmberg, L; Sjölund, M

    1985-01-01

    Smooth muscle cells were isolated enzymatically from adult human arteries, grown in primary culture in medium containing 10% whole blood serum, and studied by transmission electron microscopy and [3H]thymidine autoradiography. In the intact arterial wall and directly after isolation, each smooth muscle cell had a nucleus with a wide peripheral zone of condensed chromatin and a cytoplasm dominated by myofilament bundles with associated dense bodies. After 1-2 days of culture, the cells had attached to the substrate and started to spread out. At the same time, a characteristic fine-structural modification took place. It included nuclear enlargement, dispersion of the chromatin and formation of large nucleoli. Moreover, myofilament bundles disappeared and an extensive rough endoplasmic reticulum and a large Golgi complex were organized in the cytoplasm. This morphological transformation of the cells was completed in 3-4 days. It was accompanied by initiation of DNA replication and mitosis. The observations demonstrate that adult human arterial smooth muscle cells, when cultivated in vitro, pass through a phenotypic modulation of the same type as arterial smooth muscle cells from experimental animals. This modulation gives the cells morphological and functional properties resembling those of the modified smooth muscle cells found in fibroproliferative lesions of atherosclerosis. Further studies of the regulation of smooth muscle phenotype and growth may provide important clues for a better understanding of the pathogenesis of atherosclerosis. PMID:3967287

  11. 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

  12. 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

  13. Mitochondrial bioenergetics and neuronal survival modelled in primary neuronal culture and isolated nerve terminals.

    PubMed

    Nicholls, David G; Brand, Martin D; Gerencser, Akos A

    2015-04-01

    Mitochondria play multiple roles in the maintenance of neuronal function under physiological and pathological conditions. In addition to ATP generation, they can act as major short-term calcium sinks and can both generate, and be damaged by, reactive oxygen species. Two complementary preparations have been extensively employed to investigate in situ neuronal mitochondrial bioenergetics, primary neuronal cultures and acutely isolated nerve terminals, synaptosomes. A major focus of the cell culture preparation has been the investigation of glutamate excitotoxicity. Oxidative phosphorylation, calcium transport and reactive oxygen species play complex interlocking roles in the life and death of the glutamate exposed neuron. Synaptosomes may be isolated from specific brain regions at any developmental stage and therefore provide a valuable ex vivo approach in studying mouse models. Recent advances have allowed synaptosomal bioenergetics to be studied on a microgram scale, and, in combination with approaches to correct for functional and transmitter heterogeneity, have allowed hypotheses concerning presynaptic mitochondrial dysfunction to be tested on a variety of genetic models of neurodegenerative disorders. PMID:25172197

  14. Detection of reactive oxygen species in primary cultures of cerebellar granule cells.

    PubMed

    Atlante, A; Passarella, S

    1999-12-01

    The aim of this work was to develop a novel procedure useful to detect the formation of two reactive oxygen species, i.e. superoxide and singlet oxygen, in neuron monolayer primary cultures, thus, making possible the investigation of the effect of certain compounds on reactive oxygen species formation. Thus, use was made of two reactive oxygen species detecting systems consisting of ferricytochrome c (Fe-cyt c) and imidazole-RNO (N, N-dimethyl-4-nitrosoaniline) which allow for the photometric detection of superoxide anion and singlet oxygen, respectively. Both of them were used to assess the formation of reactive oxygen species in cerebellar granule cells exposed to glutamate: both superoxide anion and singlet oxygen proved to be generated in glutamate neurotoxicity in a way sensitive to glutamate NMDA-receptor inhibitor, MK-801 ((+)-5-methyl-10,11-dihydro-5H-dibenzo(a, d)cyclohepten-5,10-imine hydrogen maleate), to Ca(2+) complexing agent, EGTA, and to certain antioxidants. In principle, the reported protocol can be applied to any cell type in culture. PMID:10592334

  15. 3-bromopyruvate inhibits glycolysis, depletes cellular glutathione, and compromises the viability of cultured primary rat astrocytes.

    PubMed

    Ehrke, Eric; Arend, Christian; Dringen, Ralf

    2015-07-01

    The pyruvate analogue 3-bromopyruvate (3-BP) is an electrophilic alkylator that is considered a promising anticancer drug because it has been shown to kill cancer cells efficiently while having little toxic effect on nontumor cells. To test for potential adverse effects of 3-BP on brain cells, we exposed cultured primary rat astrocytes to 3-BP and investigated the effects of this compound on cell viability, glucose metabolism, and glutathione (GSH) content. The presence of 3-BP severely compromised cell viability and slowed cellular glucose consumption and lactate production in a time- and concentration-dependent manner, with half-maximal effects observed at about 100 µM 3-BP after 4 hr of incubation. The cellular hexokinase activity was not affected in 3-BP-treated astrocytes, whereas within 30 min after application of 3-BP the activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was inhibited, and cellular GSH content was depleted in a concentration-dependent manner, with half-maximal effects observed at about 30 µM 3-BP. The depletion of cellular GSH after exposure to 100 µM 3-BP was not prevented by the presence of 10 mM of the monocarboxylates lactate or pyruvate, suggesting that 3-BP is not taken up into astrocytes predominantly by monocarboxylate transporters. The data suggest that inhibition of glycolysis by inactivation of GAPDH and GSH depletion contributes to the toxicity that was observed for 3-BP-treated cultured astrocytes. PMID:25196479

  16. 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

  17. Association of ICAM-1 with the cytoskeleton in rat alveolar epithelial cells in primary culture.

    PubMed

    Barton, W W; Wilcoxen, S E; Christensen, P J; Paine, R

    1996-11-01

    Intercellular adhesion molecule-1 ICAM-1) is a transmembrane adhesion protein that is expressed constitutively on the apical surface of type I cells in vivo and on type II cells in vitro as they spread in culture, assuming type I cell-like characteristics. To investigate the possible interaction of ICAM-1 with the alveolar epithelial cell cytoskeleton, rat type II cells in primary culture were extracted with nonionic detergent, and residual ICAM-1 associated with the cytoskeletal remnants was determined using immunofluorescence microscopy, immunoprecipitation, and cell-based enzyme-linked immunosorbent assay. A large fraction of alveolar epithelial cell ICAM-1 remained associated with the cytoskeleton after detergent extraction, whereas two other transmembrane molecules, transferrin receptor and class II major histocompatibility complex, were completely removed. ICAM-1 was redistributed on the cell surface after the disruption of actin filaments with cytochalasin B, suggesting interaction with the actin cytoskeleton. In contrast, ICAM-1 was completely detergent soluble in rat pulmonary artery endothelial cells, human umbilical vein endothelial cells, and rat alveolar macrophages. The association of ICAM-1 with the alveolar epithelial cell cytoskeleton was not altered after stimulation with inflammatory cytokines. However, detergent resistant ICAM-1 was significantly increased after crosslinking of ICAM-1 on the cell surface, suggesting that this cytoskeletal association may be modulated by interactions of alveolar epithelial cells with inflammatory cells. The association of ICAM-1 with the cytoskeleton in alveolar epithelial cells may provide a fixed intermediary between mobile inflammatory cells and the alveolar surface. PMID:8944713

  18. 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

  19. 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

  20. 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

  1. 15N labeled brain enables quantification of proteome and phosphoproteome in cultured primary neurons

    PubMed Central

    Liao, Lujian; Sando, Richard C.; Farnum, John B.; Vanderklish, Peter W.; Maximov, Anton; Yates, John R.

    2011-01-01

    Terminally differentiated primary cells represent a valuable in vitro model to study signaling events associated within a specific tissue. Quantitative proteomic methods using metabolic labeling in primary cells encounter labeling efficiency issues hindering the use of these cells. Here we developed a method to quantify the proteome and phosphoproteome of cultured neurons using 15N labeled brain tissue as an internal standard, and applied this method to determine how an inhibitor of an excitatory neural transmitter receptor, phencyclidine (PCP), affects the global phosphoproteome of cortical neurons. We identified over 10,000 phosphopeptides and made accurate quantitative measurements of the neuronal phosphoproteome after neuronal inhibition. We show that short PCP treatments lead to changes in phosphorylation for 7% of neuronal phosphopeptides and that prolonged PCP treatment alters the total levels of several proteins essential for synaptic transmission and plasticity and leads to a massive reduction in the synaptic strength of inhibitory synapses. The results provide valuable insights into the dynamics of molecular networks implicated in PCP-mediated NMDA receptor inhibition and sensorimotor deficits. PMID:22070516

  2. Application of retinoic acid to obtain osteocytes cultures from primary mouse osteoblasts.

    PubMed

    Mattinzoli, Deborah; Messa, Piergiorgio; Corbelli, Alessandro; Ikehata, Masami; Mondini, Anna; Zennaro, Cristina; Armelloni, Silvia; Li, Min; Giardino, Laura; Rastaldi, Maria Pia

    2014-01-01

    The need for osteocyte cultures is well known to the community of bone researchers; isolation of primary osteocytes is difficult and produces low cell numbers. Therefore, the most widely used cellular system is the osteocyte-like MLO-Y4 cell line. The method here described refers to the use of retinoic acid to generate a homogeneous population of ramified cells with morphological and molecular osteocyte features. After isolation of osteoblasts from mouse calvaria, all-trans retinoic acid (ATRA) is added to cell medium, and cell monitoring is conducted daily under an inverted microscope. First morphological changes are detectable after 2 days of treatment and differentiation is generally complete in 5 days, with progressive development of dendrites, loss of the ability to produce extracellular matrix, down-regulation of osteoblast markers and up-regulation of osteocyte-specific molecules. Daily cell monitoring is needed because of the inherent variability of primary cells, and the protocol can be adapted with minimal variation to cells obtained from different mouse strains and applied to transgenic models. The method is easy to perform and does not require special instrumentation, it is highly reproducible, and rapidly generates a mature osteocyte population in complete absence of extracellular matrix, allowing the use of these cells for unlimited biological applications. PMID:24894124

  3. 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

  4. Determining the Long-term Effect of Antibiotic Administration on the Human Normal Intestinal Microbiota Using Culture and Pyrosequencing Methods.

    PubMed

    Rashid, Mamun-Ur; Zaura, Egijia; Buijs, Mark J; Keijser, Bart J F; Crielaard, Wim; Nord, Carl Erik; Weintraub, Andrej

    2015-05-15

    The purpose of the study was to assess the effect of ciprofloxacin (500 mg twice daily for 10 days) or clindamycin (150 mg 4 times daily for 10 days) on the fecal microbiota of healthy humans for a period of 1 year as compared to placebo. Two different methods, culture and microbiome analysis, were used. Fecal samples were collected for analyses at 6 time-points. The interval needed for the normal microbiota to be normalized after ciprofloxacin or clindamycin treatment differed for various bacterial species. It took 1-12 months to normalize the human microbiota after antibiotic administration, with the most pronounced effect on day 11. Exposure to ciprofloxacin or clindamycin had a strong effect on the diversity of the microbiome, and changes in microbial composition were observed until the 12th month, with the most pronounced microbial shift at month 1. No Clostridium difficile colonization or C. difficile infections were reported. Based on the pyrosequencing results, it appears that clindamycin has more impact than ciprofloxacin on the intestinal microbiota. PMID:25922405

  5. Propagation of normal human epithelial cell populations using an in vivo culture system. Description and applications.

    PubMed

    Klein-Szanto, A J; Terzaghi, M; Mirkin, L D; Martin, D; Shiba, M

    1982-08-01

    A new model using xenotransplanted human epithelia was developed for the study of toxic and carcinogenic effects of chemicals. Epithelial cells from the respiratory tract of 4 male and 3 female premature and fullterm fetuses were enzymatically removed and inoculated into deepithelialized rat tracheas. These were sealed at both ends and transplanted subcutaneously into nude mice. After 3-4 weeks, a normal mucociliary epithelium covered the tracheal lumen. At this stage the epithelial cells could be isolated again and transplanted into new denuded rat tracheas. This passaging could be repeated up to six times, each permitting an amplification factor of approximately 3. Tracheal transplants containing cells of human origin (in vivo Passages 2-4) were treated with 7,12-dimethylbenz(a)anthracene. Hyperplasias, squamous metaplasias, and dysplasias were seen 1-8 weeks after initiation of treatment, indicating that the responses of human and rodent epithelial cells to polycyclic aromatic hydrocarbons are similar. Initial experiments with skin and esophageal epithelia suggest that other covering epithelia could also be used in this fashion for evaluation of toxicants and carcinogens that are likely to come into contact with these tissues. PMID:6821529

  6. 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

  7. 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

  8. 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

  9. Synergistic Effect of IGF-1 and OP-1 on Matrix Formation by Normal and OA Chondrocytes Cultured in Alginate Beads.

    PubMed Central

    Chubinskaya, Susan; Hakimiyan, Arnavaz; Pacione, Carol; Yanke, Adam; Rappoport, Lev; Aigner, Thomas; Rueger, David C.; Loeser, Richard F.

    2007-01-01

    Summary Objective Growth factor therapy may be useful for stimulation of cartilage matrix synthesis and repair. Thus, the purpose of our study was to further understand the effect of combined insulin-like growth factor 1 (IGF-1) and osteogenic protein 1 (OP-1) treatment on the matrix synthesized by human adult normal and osteoarthritic (OA) chondrocytes. Design Chondrocytes were isolated post-mortem from articular cartilage from tali of normal human donors and femoral condyles of OA patients undergoing knee replacement surgery. Cells were cultured in alginate beads for 21 days in four experimental groups: 1) “mini-ITS”-control; 2) 100ng/ml IGF-1; 3) 100ng/ml OP-1; 4) IGF-1 + OP-1, each at 100 ng/ml. Beads were processed for histological (Safranin O and fast green), morphometrical and immunohistochemical (aggrecan, decorin, type I, II, VI, and X collagens, and fibronectin accumulation) analyses. Results Histology showed that IGF-1 alone did not induce substantial matrix production. OP-1 alone caused a considerable matrix formation, but the highest matrix accumulation by normal and OA chondrocytes was found when OP-1 and IGF-1 were added together. Morphometrical analysis indicated larger matrices produced by OA chondrocytes than by normal cells under the combined treatment. All tested matrix proteins were more abundant in the combination group. Type X collagen was detected only under the combined OP-1 and IGF-1 treatment and was present at very low levels. Type I collagen was found only in OA chondrocytes. Conclusions The results obtained in the current study suggest that combined therapy with IGF-1 and OP-1 may have a greater potential in treating cartilage defects seen in OA than use of either growth factor alone. PMID:17126570

  10. 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

  11. Dopaminergic neurotoxicity of homocysteine and its derivatives in primary mesencephalic cultures.

    PubMed

    Heider, I; Lehmensiek, V; Lenk, Th; Müller, Th; Storch, A

    2004-01-01

    Levodopa and dopamine are metabolized to 3-O-methyldopa and 3-methoxytyramine, respectively, by the enzyme catechol-O-methyltransferase (COMT) leading to the production of the demethylated cofactor S-adenosylhomo-cysteine (SAH) and subsequently homocysteine (HC). Indeed, treatment of Parkinson's disease (PD) patients with levodopa leads to increased HC blood levels. Therefore, HC is discussed to be involved in the pathogenesis of PD as well as in enhanced progression of PD in patients treated with levodopa. Here we investigated the toxicity of HC and its derivatives SAH, homocysteic acid (HCA) and cysteic acid (CA) on tyrosine hydroxylase (TH)-positive neurons in primary mesencephalic cultures from rat in vitro. Furthermore, we evaluated the toxicity of HC on cultures stressed with the dopaminergic neurotoxin 1-methyl-4-phenylpyridinium (MPP+). Incubation with HC or HCA did not result in significant effects on TH-positive neuron survival with concentrations up to 1 mM, but led to morphological changes of TH-positive cells with significantly fewer and shorter neurites at concentrations of > or = 100 microM after 48 h. In contrast, SAH and CA were toxic at concentrations of >100 microM after 48h. Furthermore, MPP+ showed strong toxicity towards TH-positive cells after 48 h (half-maximal toxic concentration: 20 microM), whereas co-incubation with HC for 24 or 48 h did not further alter TH-positive cell survival. Taken together, our results do not demonstrate relevant dopaminergic toxicity of HC in vitro, and therefore HC is most likely not involved in the pathogenesis of PD or in accelerating the progression of PD by levodopa. PMID:15354384

  12. Biological effects of inorganic arsenic on primary cultures of rat astrocytes.

    PubMed

    Catanzaro, Irene; Schiera, Gabriella; Sciandrello, Giulia; Barbata, Giusi; Caradonna, Fabio; Proia, Patrizia; Di Liegro, Italia

    2010-10-01

    It is well established that inorganic arsenic induces neurotoxic effects and neurological defects in humans and laboratory animals. The cellular and molecular mechanisms of its actions, however, remain elusive. Herein we report the effects of arsenite (NaAsO2) on primary cultures of rat astrocytes. Cells underwent induction of heat shock protein 70 only at the highest doses of inorganic arsenic (30 and 60 microM), suggesting a high threshold to respond to stress. We also investigated arsenic genotoxicity with the comet assay. Interestingly, although cells treated with 10 microM arsenite for 24 h maintained >70% viability, with respect to untreated cells, high DNA damage was already observed. Since arsenic is not known to be a direct-acting genotoxic agent, we investigated the possibility that its effects are due, in astrocytes as well, to ROS formation, as already described for other cell types. However, FACS analysis after CM-H2DCFDA staining did not evidence any significant increase of ROS production while, on the contrary, at the highest arsenite concentrations used, ROS production decreased. Concordantly, we found that, if most cells in the culture are still alive (i.e. up to 10 microM arsenite), they show a treatment-dependent increase in the concentration of SOD1. On the other hand, SOD2 concentration did not change. Finally, we found that astrocytes also synthesize PIPPin, an RNA-binding protein, the concentration of which was recently reported to change in response to stress induced by cadmium. Here we also report that, in cells exposed to high doses of arsenite, an anti-PIPPin antibody-positive faster migrating protein appears. PMID:20818482

  13. 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

  14. 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

  15. 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. PMID:27338714

  16. Primary isolation of Mycobacterium bovis from bovine tissues: conditions for maximising the number of positive cultures.

    PubMed

    Corner, L A L; Gormley, E; Pfeiffer, D U

    2012-04-23

    In studies of Mycobacterium bovis infection in animals a definitive diagnosis requires the isolation of the organism. However, the optimum conditions for the primary isolation of M. bovis have not been determined. The aim of this study was to determine for primary isolation of M. bovis, (a) the incubation time required to achieve maximum sensitivity (i.e., the number of positive samples identified), (b) the effect of decontaminants on bacterial growth rates, and (c) the influence of media and the number of slopes of media on the number of positive samples detected. Two agar-based media, modified Middlebrook 7H11 (7H11) and tuberculosis blood agar (B83), and an egg-based medium, Stonebrink's (SB) were compared. Three decontaminants, 2% (w/v) sodium hydroxide (NaOH), 0.75% (w/v) and 0.075% (w/v) cetylpyridinium chloride (CPC, also called hexadecylpyridinium chloride, HPC) and 0.5% (w/v) benzalkonium chloride (BC) were evaluated against treatment with sterile distilled water. The inoculated media slopes were incubated for up to 15 weeks. Colonies first appeared after 2 weeks on all media types and 75% of positive slopes were identified by 8 weeks. An incubation time of 15 weeks was required to identify all positive samples. The slowest growth was associated with inocula that contained the fewest viable bacilli. The time to the appearance of colonies was influenced by medium type: the median time to detection of colonies was 28 days on 7H11 and B83, and 36 days on SB. However, SB returned the greatest number of positive samples. Decontamination procedures increased the minimum incubation time required to detect positive cultures, probably due to the toxic effect of the decontaminants. Increasing the number of inoculated slopes resulted in an increased number of positive samples and a decreased time to the detection of colonies. Overall, the detection of M. bovis was significantly influenced by the choice of media, the decontaminant and the duration of incubation of

  17. 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

  18. Structure of the Primary Cell Walls of Suspension-Cultured Rosa glauca Cells

    PubMed Central

    Joseleau, Jean-Paul; Chambat, Gérard

    1984-01-01

    Xyloglucans, characteristic hemicellulosic polysaccharides of plant primary walls, have been isolated from Rosa glauca suspension-cultured cells. The cell wall material was fractionated by two sequences of extraction based on solubilization of the hemicelluloses in alkaline and organic solvent systems, respectively. In both cases, only a part (about 50%) of the total xyloglucan could be extracted, the rest remaining tightly associated with cellulose and necessitating the use of acid to be solubilized. Purification of xyloglucans was effected by formation of a gel in appropriate mixtures of dimethyl sulfoxide and water. Further fractionation could be achieved on a cellulose column eluted with chaotropic solvents. This demonstrated the heterogeneity of xyloglucans in the primary cell walls. Analytical data show that all fractions are constituted with the same sugars: l-arabinose, l-fucose, d-galactose, d-xylose, and d-glucose, but their relative proportions differ, particularly the ratio of glucose to xylose which varies from 1.2 to 2 within the different xyloglucans. The structure of these hemicelluloses was established by methylation analysis and shown to consist of a (1 → 4)-linked glucan backbone which carries substituents on the O-6 of glucose. Here again, the multiple forms of xyloglucans was suggested by the various patterns of substitutions found on the different fractions. The configuration of the linkages were established by 13C nuclear magnetic resonance spectroscopy and shown to be β for the glucan backbone, α for the xylosyl and fucosyl substituents, and β for the galactosyl substituents. These configurations agree with the specific rotation of the xyloglucan. PMID:16663483

  19. 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

  20. 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

  1. Copper Nanoparticles and Copper Sulphate Induced Cytotoxicity in Hepatocyte Primary Cultures of Epinephelus coioides

    PubMed Central

    Wang, Tao; Chen, Xiaoyan; Long, Xiaohua; Liu, Zhaopu; Yan, Shaohua

    2016-01-01

    Copper nanoparticles (Cu-NPs) were widely used in various industrial and commercial applications. The aim of this study was to analyze the cytotoxicity of Cu-NPs on primary hepatocytes of E.coioides compared with copper sulphate (CuSO4). Cultured cells were exposed to 0 or 2.4 mg Cu L-1 as CuSO4or Cu-NPs for 24-h. Results showed either form of Cu caused a dramatic loss in cell viability, more so in the CuSO4 than Cu-NPs treatment. Compared to control, either CuSO4 or Cu-NPs significantly increased reactive oxygen species(ROS) and malondialdehyde(MDA) concentration in hepatocytes by overwhelming total superoxide dismutase (T-SOD) activity, catalase(CAT) activity and glutathione(GSH) concentration. In addition, the antioxidative-related genes [SOD (Cu/Zn), SOD (Mn), CAT, GPx4] were also down-regulated. The apoptosis and necrosis percentage was significantly higher after the CuSO4 or Cu-NPs treatment than the control. The apoptosis was induced by the increased cytochrome c concentration in the cytosol and elevated caspase-3, caspase-8 and caspase-9 activities. Additionally, the apoptosis-related genes (p53, p38β and TNF-α) and protein (p53 protein) were up-regulated after the CuSO4 or Cu-NPs treatment, with CuSO4 exposure having a greater effect than Cu-NPs. In conclusion, Cu-NPs had similar types of toxic effects as CuSO4 on primary hepatocytes of E.coioides, but toxicity of CuSO4 was more severe than that of Cu-NPs. PMID:26890000

  2. Copper Nanoparticles and Copper Sulphate Induced Cytotoxicity in Hepatocyte Primary Cultures of Epinephelus coioides.

    PubMed

    Wang, Tao; Chen, Xiaoyan; Long, Xiaohua; Liu, Zhaopu; Yan, Shaohua

    2016-01-01

    Copper nanoparticles (Cu-NPs) were widely used in various industrial and commercial applications. The aim of this study was to analyze the cytotoxicity of Cu-NPs on primary hepatocytes of E.coioides compared with copper sulphate (CuSO4). Cultured cells were exposed to 0 or 2.4 mg Cu L-1 as CuSO4or Cu-NPs for 24-h. Results showed either form of Cu caused a dramatic loss in cell viability, more so in the CuSO4 than Cu-NPs treatment. Compared to control, either CuSO4 or Cu-NPs significantly increased reactive oxygen species(ROS) and malondialdehyde(MDA) concentration in hepatocytes by overwhelming total superoxide dismutase (T-SOD) activity, catalase(CAT) activity and glutathione(GSH) concentration. In addition, the antioxidative-related genes [SOD (Cu/Zn), SOD (Mn), CAT, GPx4] were also down-regulated. The apoptosis and necrosis percentage was significantly higher after the CuSO4 or Cu-NPs treatment than the control. The apoptosis was induced by the increased cytochrome c concentration in the cytosol and elevated caspase-3, caspase-8 and caspase-9 activities. Additionally, the apoptosis-related genes (p53, p38β and TNF-α) and protein (p53 protein) were up-regulated after the CuSO4 or Cu-NPs treatment, with CuSO4 exposure having a greater effect than Cu-NPs. In conclusion, Cu-NPs had similar types of toxic effects as CuSO4 on primary hepatocytes of E.coioides, but toxicity of CuSO4 was more severe than that of Cu-NPs. PMID:26890000

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

    PubMed Central

    Cao, Zhengyu; Shafer, Timothy J.; Crofton, Kevin M.; Gennings, Chris

    2011-01-01

    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 target using a functional measure. Objectives: We investigated the potency and efficacy of 11 structurally diverse food-use pyrethroids to evoke sodium (Na+) influx in neurons and tested the hypothesis of dose additivity for a mixture of these same 11 compounds. Methods: We determined pyrethroid-induced increases in Na+ influx in primary cultures of cerebrocortical neurons using the Na+-sensitive dye sodium-binding benzofuran isophthalate (SBFI). Concentration-dependent responses for 11 pyrethroids were determined, and the response to dilutions of a mixture of all 11 compounds at an equimolar mixing ratio was assessed. Additivity was tested assuming a dose-additive model. Results: Seven pyrethroids produced concentration-dependent, tetrodotoxin-sensitive Na+ influx. The rank order of potency was deltamethrin > S-bioallethrin > β-cyfluthrin > λ-cyhalothrin > esfenvalerate > tefluthrin > fenpropathrin. Cypermethrin and bifenthrin produced modest increases in Na+ influx, whereas permethrin and resmethrin were inactive. When all 11 pyrethroids were present at an equimolar mixing ratio, their actions on Na+ influx were consistent with a dose-additive model. Conclusions: These data provide in vitro relative potency and efficacy measurements for 7 pyrethroid compounds in intact mammalian neurons. Despite differences in individual compound potencies, we found the action of a mixture of all 11 pyrethroids to be additive when we used an appropriate statistical model. These results are consistent with a previous report of the additivity of pyrethroids in vivo. PMID:21665567

  4. 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

  5. Isolation and characterization of rheumatoid arthritis synovial fibroblasts from primary cultureprimary culture cells markedly differ from fourth-passage cells

    PubMed Central

    Zimmermann, Thomas; Kunisch, Elke; Pfeiffer, Robert; Hirth, Astrid; Stahl, Hans-Detlev; Sack, Ulrich; Laube, Anke; Liesaus, Eckehard; Roth, Andreas; Palombo-Kinne, Ernesta; Emmrich, Frank; Kinne, Raimund W

    2001-01-01

    To reduce culture artifacts by conventional repeated passaging and long-term culture in vitro, the isolation of synovial fibroblasts (SFB) was attempted from rheumatoid arthritis (RA) synovial membranes by trypsin/collagenase digest, short-term in vitro adherence (7 days), and negative isolation using magnetobead-coupled anti-CD14 monoclonal antibodies. This method yielded highly enriched SFB (85% prolyl-4-hydroxylase+/74% Thy-1/CD90+ cells; <2% contaminating macrophages; <1% leukocytes/endothelial cells) that, in comparison with conventional fourth-passage RA-SFB, showed a markedly different phenotype and significantly lower proliferation rates upon stimulation with platelet-derived growth factor and IL-1β. This isolation method is simple and reliable, and may yield cells with features closer to the in vivo configuration of RA-SFB by avoiding extended in vitro culture. PMID:11178129

  6. Distribution and origin of the basement membrane component perlecan in rat liver and primary hepatocyte culture.

    PubMed Central

    Rescan, P. Y.; Loréal, O.; Hassell, J. R.; Yamada, Y.; Guillouzo, A.; Clément, B.

    1993-01-01

    Basement membranes contain three major components (ie collagen IV, laminin, and the heparan sulfate proteoglycan termed perlecan). Although the distribution and origin of both collagen IV and laminin have been well documented in the liver, perlecan has been poorly investigated, so far. We have studied the distribution and cellular origin of perlecan in rat livers in various conditions as well as in hepatocyte primary culture. By immunolocalization in both adult and 18-day-old fetal liver, perlecan was found in portal spaces, around central veins, and throughout the lobule. Immunoelectron microscopy revealed its presence at the level of basement membranes surrounding bile ducts and blood vessels, and in the space of Disse discontinuously interacting with hepatocyte microvilli. Precursors of perlecan were detected in the rough endoplasmic reticulum of bile duct cells and both vascular and sinusoidal endothelial cells. Both hepatocytes and Ito cells were negative. Northern-blot analysis confirmed the lack of appreciable expression of perlecan in hepatocytes isolated from either fetal or adult livers. In 18-month-diethylnitrosamine-treated rat liver, perlecan was abundant in neoplastic nodules. Electron microscopic investigation revealed an almost continuous layer of perlecan in the space of Disse and intracellular staining in sinusoidal endothelial cells, only. Perlecan mRNAs were detectable in malignant nodules, and absent in hepatocytes from nontumorous areas. Hepatocytes expressed high levels of perlecan mRNAs only when put in culture. This expression was reduced in conditions that allow improvement of hepatocyte survival and function (ie addition of corticoids, dimethylsulfoxide or nicotinamide to the medium, or in coculture with liver epithelial cells from biliary origin). Immunolocalization by light and electron microscopy showed that deposition of the proteoglycan occurred in coculture, in basement membranelike structures located around hepatocyte cords. In

  7. 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

    2012-01-01

    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

  8. Primary cultures of prostate cells and their ability to activate carcinogens.

    PubMed

    Martin, F L; Cole, K J; Muir, G H; Kooiman, G G; Williams, J A; Sherwood, R A; Grover, P L; Phillips, D H

    2002-01-01

    Differences in the incidence of prostate cancer (CaP) amongst different migrant populations point to causative agents of dietary and/or environmental origin. Prostate tissues were obtained following transurethral resection of the prostate (TURP) or radical retropubic prostatectomy. After surgery, TURP-derived or tumour-adjacent tissue fragments were minced in warm PFMR-4A medium (37 degrees C) and suspensions pipetted into collagen-coated petri dishes. Non-adherent material was removed by washing with fresh medium after 12 h. Adhered cells subsequently reacted positively with monoclonal antibodies to prostate specific antigen (PSA). PSA was also detected in the medium. The genotoxicities of the chemical carcinogens 2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine (PhIP), its N-hydroxy metabolite (N-OH-PhIP) and benzo[a]pyrene (B[a]P) in adherent cell populations from different donors (n=8) were examined. Cells were treated in suspension for 30 min at 37 degrees C in the presence of the DNA repair inhibitors hydroxyurea (HU) and cytosine arabinoside (ara-C). DNA single-strand breaks were detected in cells by the alkaline single cell-gel electrophoresis ('Comet') assay and quantified by measuring comet tail length (CTL) in microm. All three carcinogens induced dose-related increases in CTLs (P<0.0001) in cells from four donors 24 h post-seeding. However, in cells from a further two donors the genotoxic effects of PhIP, N-OH-PhIP and B[a]P were much less apparent after 48 h than after 24 h in culture. After 96 h in culture, cells from these donors appeared to be resistant to the comet-forming activity of the compounds. However, B[a]P-DNA adducts were still measurable by (32)P-postlabelling for up to 14 days following a 24-h exposure to 50 microM B[a]P in adhered cells from another two donors. This study shows that primary cultures of cells derived from the prostate can activate members of two classes of chemical carcinogens. Further development may provide a robust

  9. 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

  10. A Primary Neuron Culture System for the Study of Herpes Simplex Virus Latency and Reactivation

    PubMed Central

    Kobayashi, Mariko; Kim, Ju-Youn; Camarena, Vladimir; Roehm, Pamela C.; Chao, Moses V.; Wilson, Angus C.; Mohr, Ian

    2012-01-01

    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

  11. Human Peripheral Blood Mononuclear Cells Cultured in Normal and Hyperglycemic Media in Simulated Microgravity Using NASA Bioreactors

    NASA Technical Reports Server (NTRS)

    Lawless, DeSales

    2003-01-01

    We sought answers to several questions this summer at NASA Johnson Space Center. Initial studies involved the in vitro culture of human peripheral blood mononuclear in cells in different conditioned culture media. Several human cancer clones were similarly studied to determine responses to aberrant glycosylation by the argon laser. The cells were grown at unit gravity in flasks and in simulated microgravity using NASA bioreactors. The cells in each instance were analyzed by flow cytometry. Cell cycle analysis was acquired by staining nuclear DNA with propidium iodide. Responses to the laser stimulation was measured by observing autofluorescence emitted in the green and red spectra after stimulation. Extent of glycosylation correlated with the intensity of the laser stimulated auto-fluorescence. Our particular study was to detect and monitor aberrant glycosylation and its role in etiopathogenesis. Comparisons were made between cells known to be neoplastic and normal cell controls using the same Laser Induced Autofluorescence technique. Studies were begun after extensive literature searches on using the antigen presenting potential of dendritic cells to induce proliferation of antigen specific cytotoxic T-cells. The Sendai virus served as the antigen. Our goal is to generate sufficient numbers of such cells in the simulated microgravity environment for use in autologous transplants of virally infected individuals including those positive for hepatitis and HIV.

  12. Comparative growth, cross stress resistance, transcriptomics of Streptococcus pyogenes cultured under low shear modeled microgravity and normal gravity

    PubMed Central

    Kalpana, Duraisamy; Im, Chanki; Lee, Yang Soo

    2015-01-01

    Streptococcus pyogenes is commonly found on pharynx, mouth and rarely on skin, lower gastrointestinal tract. It is a potential pathogen causing tonsillitis, pneumonia, endocarditis. The present study was undertaken to study the effects of low shear modeled microgravity on growth, morphology, antibiotic resistance, cross-stress resistance to various stresses and alteration in gene expression of S. pyogenes. The growth analysis performed using UV–Visible spectroscopy indicated decrease in growth of S. pyogenes under low shear modeled microgravity. Morphological analysis by Bio-transmission electron microscopy (TEM), Bio-scanning electron microscopy (SEM) did not reveal much difference between normal and low shear modeled microgravity grown S. pyogenes. The sensitivity of S. pyogenes to antibiotics ampicillin, penicillin, streptomycin, kanamycin, hygromycin, rifampicin indicates that the bacterium is resistant to hygromycin. Further S. pyogenes cultured under low shear modeled microgravity was found to be more sensitive to ampicillin and rifampicin as compared with normal gravity grown S. pyogenes. The bacteria were tested for the acid, osmotic, temperature and oxidative cross stress resistances. The gene expression of S. pyogenes under low shear modeled microgravity analyzed by microarray revealed upregulation of 26 genes and down regulation of 22 genes by a fold change of 1.5. PMID:26858535

  13. Fibrochondrogenic potential of synoviocytes from osteoarthritic and normal joints cultured as tensioned bioscaffolds for meniscal tissue engineering in dogs

    PubMed Central

    Bobe, Gerd; Duesterdieck-Zellmer, Katja F.

    2014-01-01

    Meniscal tears are a common cause of stifle lameness in dogs. Use of autologous synoviocytes from the affected stifle is an attractive cell source for tissue engineering replacement fibrocartilage. However, the diseased state of these cells may impede in vitro fibrocartilage formation. Synoviocytes from 12 osteoarthritic (“oaTSB”) and 6 normal joints (“nTSB”) were cultured as tensioned bioscaffolds and compared for their ability to synthesize fibrocartilage sheets. Gene expression of collagens type I and II were higher and expression of interleukin-6 was lower in oaTSB versus nTSB. Compared with nTSB, oaTSB had more glycosaminoglycan and alpha smooth muscle staining and less collagen I and II staining on histologic analysis, whereas collagen and glycosaminoglycan quantities were similar. In conclusion, osteoarthritic joint—origin synoviocytes can produce extracellular matrix components of meniscal fibrocartilage at similar levels to normal joint—origin synoviocytes, which makes them a potential cell source for canine meniscal tissue engineering. PMID:25289180

  14. 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…

  15. 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...

  16. 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

  17. Astrocytes Enhance Streptococcus suis-Glial Cell Interaction in Primary Astrocyte-Microglial Cell Co-Cultures.

    PubMed

    Seele, Jana; Nau, Roland; Prajeeth, Chittappen K; Stangel, Martin; Valentin-Weigand, Peter; Seitz, Maren

    2016-01-01

    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

  18. Genotoxic effects of alpha-hexachlorocyclohexane in primary cultures of rodent and human hepatocytes.

    PubMed

    Mattioli, F; Robbiano, L; Adamo, D; Federa, R; Martelli, A; Brambilla, G

    1996-01-01

    The genotoxicity of alpha-hexachlorocyclohexane (alpha-HCH) was evaluated in primary cultures of mouse, rat and human hepatocytes. DNA fragmentation was measured by the alkaline elution technique and DNA repair synthesis by quantitative autoradiography. A 20 h exposure to subtoxic concentrations ranging from 0.056 to 0.32 mM produced a dose-dependent frequency of DNA breaks in rat hepatocytes and in hepatocytes from four of five human donors, but not in mouse hepatocytes, DNA repair induction was absent in hepatocytes from all three species. The reduction in the frequency of DNA breaks observed in rat hepatocytes simultaneously exposed to metyrapone suggests that alpha-HCH is transformed into reactive species by a cytochrome P450-dependent reaction. The detection of DNA fragmentation but not of DNA repair synthesis may be tentatively explained by assuming that alpha-HCH behaves as a chemical eliciting short patch DNA repair, which is more easily revealed as genotoxic by the occurrence of DNA single-strand breaks. PMID:8671720

  19. 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.

  20. Primary human osteoblast culture on 3D porous collagen-hydroxyapatite scaffolds.

    PubMed

    Jones, Gemma L; Walton, Robin; Czernuszka, Jan; Griffiths, Sarah L; El Haj, Alicia J; Cartmell, Sarah H

    2010-09-15

    There is a need in tissue-engineering for 3D scaffolds that mimic the natural extracellular matrix of bone to enhance cell adhesion, proliferation, and differentiation. The scaffold is also required to be degradable. A highly porous scaffold has been developed to incorporate two of the extracellular components found in bone-collagen and hydroxyapatite (HA). The scaffold's collagen component is an afibrillar monomeric type I atelocollagen extracted from foetal calf's skin. This provided a novel environment for the inclusion of HA powder. Five hundred thousand primary human osteoblasts were seeded onto 4 mm cubed scaffolds that varied in ratio of HA to collagen. Weight ratios of 1:99, 25:75, 50:50, and 75:25 hydroxyapatite:collagen (HA:Collagen) were analysed. The scaffolds plus cells were cultured for 21 days. DNA assays and live/dead viability staining demonstrated that all of the scaffolds supported cell proliferation and viability. An alkaline phosphatase assay showed similar osteoblast phenotype maintenance on all of the 3D scaffolds analysed at 21 days. MicroCT analysis demonstrated an increase in total sample volume (correlating to increase in unmineralised matrix production). An even distribution of HA throughout the collagen matrix was observed using this technique. Also at 3 weeks, reductions in the percentage of the mineralised phase of the constructs were seen. These results indicate that each of the ratios of HA/collagen scaffolds have great potential for bone tissue engineering. PMID:20694991

  1. Nucleoside transport at the blood-testis barrier studied with primary-cultured sertoli cells.

    PubMed

    Kato, Ryo; Maeda, Tomoji; Akaike, Toshihiro; Tamai, Ikumi

    2005-02-01

    Nucleosides are essential for nucleotide synthesis in testicular spermatogenesis. In the present study, the mechanism of the supply of nucleosides to the testicular system across the blood-testis barrier was studied using primary-cultured Sertoli cells from rats and TM4 cells from mice. Uptake of uridine by these cells was time- and concentration-dependent. Uridine uptake was decreased under Na(+)-free conditions, and the system was presumed to be high affinity, indicating an Na(+)-dependent concentrative nucleoside transporter (CNT) is involved. On the other hand, nitrobenzylthioinosine, a potent inhibitor of Na(+)-independent equilibrative nucleoside transporters (ENTs), inhibited uridine uptake by the Sertoli cells in a concentration-dependent manner. Expression of nucleoside transporters ENT1, ENT2, ENT3, CNT1, CNT2, and CNT3 was detected in Sertoli cells by reverse transcriptase-polymerase chain reaction analysis. Inhibition studies of the uptake of uridine by various nucleosides both in the presence and absence of Na(+) indicated that the most of those expressed nucleoside transporters, ENTs and CNTs, are involved functionally. These results demonstrated that Sertoli cells are equipped with multiple nucleoside transport systems, including ENT1, ENT2, and CNTs, to provide nucleosides for spermatogenesis. PMID:15547112

  2. Transport mechanisms for adenosine and uridine in primary-cultured rat cortical neurons and astrocytes.

    PubMed

    Nagai, Katsuhito; Nagasawa, Kazuki; Fujimoto, Sadaki

    2005-09-01

    Endogenous adenosine and uridine are important modulators of neural survival and activity. In the present study, we examined transport mechanisms of adenosine and uridine in primary-cultured rat cortical neurons, and compared the results for neurons with those for astrocytes. Reverse transcription-polymerase chain reaction identified the mRNAs for ENT1, ENT2, and CNT2, but not CNT1 and CNT3, in neurons and astrocytes. [3H]Adenosine and [3H]uridine were time-, temperature-, and concentration-dependently taken up into neurons and astrocytes. In kinetic analyses, the uptake of both substrates by neurons and astrocytes consisted of two and one, respectively, saturable transport components. The uptake clearance for both substrates by neurons was greater than that by astrocytes. The relative contribution of the high-affinity major component of both substrates to total uptake was estimated to be approximately 80% in neurons. The uptake of [3H]adenosine and [3H]uridine by both neurons and astrocytes was almost entirely Na+-independent, and sensitive to micro, but not nano, molar concentrations of nitrobenzylmercaptopurine riboside, which are transport characteristics of ENT2. Therefore, it was indicated that adenosine and uridine are more efficiently taken up into neurons than into astrocytes, and ENT2 may predominantly contribute to the transport of the nucleosides as a high-affinity transport system in neurons, as in the case of astrocytes. PMID:16043124

  3. Gene expression in primary cultured astrocytes affected by aluminum: alteration of chaperons involved in protein folding

    PubMed Central

    Aremu, David A.; Ezomo, Ojeiru F.

    2010-01-01

    Objectives Aluminum is notorious as a neurotoxic metal. The aim of our study was to determine whether endoplasmic reticulum (ER) stress is involved in aluminum-induced apoptosis in astrocytes. Methods Mitochondrial RNA (mRNA) was analyzed by reverse transcription (RT)-PCR following pulse exposure of aluminum glycinate to primary cultured astrocytes. Tunicamycin was used as a positive control. Results Gene expression analysis revealed that Ire1β was up-regulated in astrocytes exposed to aluminum while Ire1α was up-regulated by tunicamycin. Exposure to aluminum glycinate, in contrast to tunicamycin, seemed to down-regulate mRNA expression of many genes, including the ER resident molecular chaperone BiP/Grp78 and Ca2+-binding chaperones (calnexin and calreticulin), as well as stanniocalcin 2 and OASIS. The down-regulation or non-activation of the molecular chaperons, whose expressions are known to be protective by increasing protein folding, may spell doom for the adaptive response. Exposure to aluminum did not have any significant effects on the expression of Bax and Bcl2 in astrocytes. Conclusions The results of this study demonstrate that aluminum may induce apoptosis in astrocytes via ER stress by impairing the protein-folding machinery. PMID:21432213

  4. 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

  5. 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. PMID:23991687

  6. 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

  7. 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

  8. Lipogenesis from lactate in rat neurons and astrocytes in primary culture.

    PubMed Central

    Tabernero, A; Bolaños, J P; Medina, J M

    1993-01-01

    The rate of synthesis of phospholipid and sterol species from L-lactate in neurons and astrocytes in primary culture was studied. Both types of cells actively utilized lactate as lipid precursor, although the rate of lipogenesis was about 2-fold greater in astrocytes than in neurons. The incorporation of lactate into phospholipids was significantly higher than that into sterols in both types of cells, but the ratio of phospholipid/sterol synthesis was much higher in astrocytes than in neurons. Phosphatidylcholine (PC) was the main phospholipid synthesized in both types of cells, followed by phosphatidylethanolamine (PE), phosphatidylserine and phosphatidylinositol. No detectable synthesis of sphingomyelins was observed. The ratio of PC/PE synthesis was about 2-fold higher in astrocytes than in neurons. The main sterol synthesized in neurons was lanosterol, followed by desmosterol. However, the main sterol synthesized in astrocytes was desmosterol, followed by lanosterol and cholesterol. The different ratios of phospholipid/sterol and PC/PE synthesis found in neurons and astrocytes may result in different membrane fluidity being higher in astrocytes than in neurons. This may be associated with differences in the functionality of both types of cells. PMID:8379917

  9. Peptidomic Analyses of Mouse Astrocytic Cell Lines and Rat Primary Cultured Astrocytes

    PubMed Central

    Yin, Ping; Knolhoff, Ann M.; Rosenberg, Harry J.; Millet, Larry J.; Gillette, Martha U.; Sweedler, Jonathan V.

    2012-01-01

    Astrocytes play an active role in the modulation of synaptic transmission by releasing cell-cell signaling molecules in response to various stimuli that evoke a Ca2+ increase. We expand on recent studies of astrocyte intracellular and secreted proteins by examining the astrocyte peptidome in mouse astrocytic cell lines and rat primary cultured astrocytes, as well as those peptides secreted from mouse astrocytic cell lines in response to Ca2+-dependent stimulations. We identified 57 peptides derived from 24 proteins with LC–MS/MS and CE–MS/MS in the astrocytes. Among the secreted peptides, four peptides derived from elongation factor 1, macrophage migration inhibitory factor, peroxiredoxin-5, and galectin-1, were putatively identified by mass-matching to peptides confirmed to be found in astrocytes. Other peptides in the secretion study were mass-matched to those found in prior peptidomics analyses on mouse brain tissue. Complex peptide profiles were observed after stimulation, suggesting that astrocytes are actively involved in peptide secretion. Twenty-six peptides were observed in multiple stimulation experiments but not in controls and thus appear to be released in a Ca2+-dependent manner. These results can be used in future investigations to better understand stimulus-dependent mechanisms of astrocyte peptide secretion. PMID:22742998

  10. Effects of anti-inflammatory drugs in primary kidney cell culture of a freshwater fish.

    PubMed

    Ribas, João Luiz Coelho; da Silva, Cesar A; de Andrade, Lucas; Galvan, Gabrieli Limberger; Cestari, Marta Margarete; Trindade, Edvaldo S; Zampronio, Aleksander R; de Assis, Helena C Silva

    2014-09-01

    The non-steroidal anti-inflammatory drugs are emerging contaminants in aquatic ecosystems. This study aimed to evaluate toxic effects of some representative drugs of this pharmaceutical group on primary culture of monocytic lineage of Hoplias malabaricus anterior kidney. The effects of diclofenac, acetaminophen and ibuprofen in cell viability, lipopolysaccharide (LPS)-induced NO production and genotoxicity were evaluated. Cytometry analysis CD11b(+) cells showed 71.5% of stem cells, 19.5% of macrophages and 9% of monocytes. Cell viability was lower in the ficoll compared to percoll separation. LPS-induced NO production by these cells was blocked after treatment with dexamethasone and NG-Methyl-L-Arginine (L-NMMA). Exposure of the cells to diclofenac (0.2-200 ng/mL), acetaminophen (0.025-250 ng/mL) ibuprofen (10-1000 ng/mL) reduced basal NO production and inhibited LPS-induced NO production at all concentrations after 24 h of exposure. Genotoxicity occurred at the highest concentration of diclofenac and at the intermediary concentration of acetaminophen. Genotoxicity was also observed by ibuprofen. In summary, the pharmaceuticals influenced NO production and caused DNA damage in monocytic cells suggesting that these drugs can induce immunosuppression and genotoxicity in fish. PMID:25038277

  11. 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.

  12. Interleukin-13 induces goblet cell differentiation in primary cell culture from Guinea pig tracheal epithelium.

    PubMed

    Kondo, Mitsuko; Tamaoki, Jun; Takeyama, Kiyoshi; Nakata, Junko; Nagai, Atsushi

    2002-11-01

    The Th2 cytokines, interleukin (IL)-4 and IL-13, bind to IL-4Ralpha, and cause goblet cell metaplasia/hyperplasia with increased mucin expression in vivo. However, there is not enough evidence that these cytokines directly induce mucin production in vitro. In this study, primary epithelial cells from guinea pig trachea were cultured at an air-liquid interface, and immediately after achieving confluence at Day 7 they were treated with human recombinant IL-4 or IL-13 for 14 d. IL-13-treated cells consisted of a large number of fully mature goblet cells with a smaller number of ciliated cells. Secretory granules of the goblet cells were positive for both periodic acid-Schiff and toluidine blue, and showed exocytosis. By contrast, IL-4 failed to induce goblet cell differentiation. The electric resistances of IL-13-treated cells were lower than those of IL-4-treated cells and nontreated cells, suggesting leaky epithelia. MUC5AC protein level in cell lysates measured by ELISA was several-fold higher in IL-13-treated cells than in nontreated cells, whereas the level in IL-4-treated cells was not changed. These data suggest that human recombinant IL-13, but not IL-4, can induce differentiation into mature goblet cells that produce MUC5AC protein in guinea pig tracheal epithelial cells in vitro. PMID:12397012

  13. 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

  14. 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

  15. 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

  16. 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

  17. PRIMARY CULTURE OF CHOROIDAL EPITHELIAL CELLS: CHARACTERIZATION OF AN IN VITRO MODEL OF BLOOD-CSF BARRIER

    PubMed Central

    ZHENG, WEI; ZHAO, QIUQU; GRAZIANO, JOSEPH H.

    2016-01-01

    Summary A primary rat choroidal epithelial cell culture system was developed to investigate mechanisms of heavy metal toxicity on the blood-cerebrospinal fluid (CSF) barrier. Epithelial cells were dissociated from choroidal tissue by pronase digestion and cultured in standard DMEM culture media supplemented with 10% fetal bovine serum and 10 ng epithelial growth factor per ml. The procedure yielded 2–5 × 104 cells from pooled plexuses of three to four rats, and a viability of 77–85%. The cultures displayed a dominant polygonal type of epithelial cells, with a population doubling time of 2–3 d. The cultures were of distinct choroidal epithelial origins. For example, immunocytochemical studies using monospecific rabbit anti-rat TTR polyclonal antibody revealed a strong positive stain of transthyretin (TTR), a thyroxine transport protein exclusively produced by the choroidal epithelia. Also, reverse-transcriptase polymerase chain reaction (PCR) confirmed the presence of specific TTR mRNA in the cultures. The cultures were further adapted to grow on a freely permeable membrane sandwiched between two culture chambers. The formation of an impermeable confluent monolayer occurred within 5 d after seeding and was verified by the presence of a steady electrical resistance across the membrane (80 ± 10 ohm per cm2). The epithelial barriers appeared to actively transport [125I]-thyroxine from the basal to apical chamber. These results suggest that this primary cell culture system possesses typical choroidal epithelial characteristics and appears to be a suitable model for in vitro mechanistic investigations of blood–CSF barrier. PMID:9542634

  18. 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

  19. 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

  20. 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…

  1. 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…

  2. Inhibitory effects of polyoxyethylene stearate, PANTA, and neutral pH on growth of Mycobacterium genavense in BACTEC primary cultures.

    PubMed

    Realini, L; Van Der Stuyft, P; De Ridder, K; Hirschel, B; Portaels, F

    1997-11-01

    We report on the influences of polyoxyethylene stearate (POES), PANTA, and pH on primary cultures of Mycobacterium genavense in BACTEC vials. As a model for primary cultures from tissue, seven different strains first isolated from AIDS patients (five from Switzerland and two from the United States) were inoculated into nude mice in order to obtain large amounts of bacilli to test different conditions simultaneously. Our results demonstrate that the size of the inoculum (10[6] acid-fast bacilli/vial), an acid pH (pH 6.0), and the absence of additives (POES and PANTA) significantly (P < 0.001) increased the probability of a successful culture in 1 month, considering growth index (GI) of > or =100 or a GI of > or =999 as criterion of success. In logistic regression analysis, all factors maintained a significant (P < 0.001) independent effect, and no interactions were observed between them. The best conditions for the primary cultures of M. genavense were the use of Middlebrook 7H12 medium at pH 6.0 without any additives. PMID:9350735

  3. The effects of BmNPV on biochemical changes in primary cultures of Bombyx mori embryonic tissue.

    PubMed

    Matindoost, Leila; Sendi, Jalal Jalali; Soleimanjahi, Hoorieh; Etebari, Kayvan; Rahbarizade, Fateme

    2008-01-01

    The effect of Bombyx mori nuclear polyhedrosis virus (BmNPV) on biochemical changes of TC-100 medium containing 10% fetal bovine serum (FBS) in embryonic primary cultures of silkworm was investigated. The primary cultures that reached 60% confluence were infected by 0.5, 1, and 2-ml viral inoculums (diluted with TC-100 medium representing multiplicity of infection (MOI) of 0.25, 0.5, and 1). Glucose, uric acid, urea, total protein, cholesterol, and alkaline phosphatase were measured in the medium of BmNPV-infected primary cultures. All biochemical compounds showed significant changes. Glucose decreased considerably by about 55 mg/ml, while different concentrations of the virus inoculums did not demonstrate significant differences among them. Total protein had only increased in 2 ml concentration and there were no changes in other concentrations. Uric acid as a by-product accumulated dramatically in all concentrations, while the amount of urea reduced in all treatments and this reduction was more evident in lower concentrations. Cholesterol consumption was high in cultures postinfection, while alkaline phosphatase (ALP) activity decreased in infected cells. PMID:18288542

  4. 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.

  5. Modulation of endocytic trafficking and apical stability of CFTR in primary human airway epithelial cultures.

    PubMed

    Cholon, Deborah M; O'Neal, Wanda K; Randell, Scott H; Riordan, John R; Gentzsch, Martina

    2010-03-01

    CFTR is a highly regulated apical chloride channel of epithelial cells that is mutated in cystic fibrosis (CF). In this study, we characterized the apical stability and intracellular trafficking of wild-type and mutant CFTR in its native environment, i.e., highly differentiated primary human airway epithelial (HAE) cultures. We labeled the apical pool of CFTR and subsequently visualized the protein in intracellular compartments. CFTR moved from the apical surface to endosomes and then efficiently recycled back to the surface. CFTR endocytosis occurred more slowly in polarized than in nonpolarized HAE cells or in a polarized epithelial cell line. The most common mutation in CF, DeltaF508 CFTR, was rescued from endoplasmic reticulum retention by low-temperature incubation but transited from the apical membrane to endocytic compartments more rapidly and recycled less efficiently than wild-type CFTR. Incubation with small-molecule correctors resulted in DeltaF508 CFTR at the apical membrane but did not restore apical stability. To stabilize the mutant protein at the apical membrane, we found that the dynamin inhibitor Dynasore and the cholesterol-extracting agent cyclodextrin dramatically reduced internalization of DeltaF508, whereas the proteasomal inhibitor MG-132 completely blocked endocytosis of DeltaF508. On examination of intrinsic properties of CFTR that may affect its apical stability, we found that N-linked oligosaccharides were not necessary for transport to the apical membrane but were required for efficient apical recycling and, therefore, influenced the turnover of surface CFTR. Thus apical stability of CFTR in its native environment is affected by properties of the protein and modulation of endocytic trafficking. PMID:20008117

  6. Modulation of endocytic trafficking and apical stability of CFTR in primary human airway epithelial cultures

    PubMed Central

    Cholon, Deborah M.; O'Neal, Wanda K.; Randell, Scott H.; Riordan, John R.

    2010-01-01

    CFTR is a highly regulated apical chloride channel of epithelial cells that is mutated in cystic fibrosis (CF). In this study, we characterized the apical stability and intracellular trafficking of wild-type and mutant CFTR in its native environment, i.e., highly differentiated primary human airway epithelial (HAE) cultures. We labeled the apical pool of CFTR and subsequently visualized the protein in intracellular compartments. CFTR moved from the apical surface to endosomes and then efficiently recycled back to the surface. CFTR endocytosis occurred more slowly in polarized than in nonpolarized HAE cells or in a polarized epithelial cell line. The most common mutation in CF, ΔF508 CFTR, was rescued from endoplasmic reticulum retention by low-temperature incubation but transited from the apical membrane to endocytic compartments more rapidly and recycled less efficiently than wild-type CFTR. Incubation with small-molecule correctors resulted in ΔF508 CFTR at the apical membrane but did not restore apical stability. To stabilize the mutant protein at the apical membrane, we found that the dynamin inhibitor Dynasore and the cholesterol-extracting agent cyclodextrin dramatically reduced internalization of ΔF508, whereas the proteasomal inhibitor MG-132 completely blocked endocytosis of ΔF508. On examination of intrinsic properties of CFTR that may affect its apical stability, we found that N-linked oligosaccharides were not necessary for transport to the apical membrane but were required for efficient apical recycling and, therefore, influenced the turnover of surface CFTR. Thus apical stability of CFTR in its native environment is affected by properties of the protein and modulation of endocytic trafficking. PMID:20008117

  7. Neurogenic and Neurotrophic Effects of BDNF Peptides in Mouse Hippocampal Primary Neuronal Cell Cultures

    PubMed Central

    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 H2O2-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

  8. 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. PMID:26093110

  9. Primary airway epithelial cell culture and asthma in children-lessons learnt and yet to come.

    PubMed

    McLellan, Kirsty; Shields, Mike; Power, Ultan; Turner, Steve

    2015-12-01

    Until recently the airway epithelial cell (AEC) was considered a simple barrier that prevented entry of inhaled matter into the lung parenchyma. The AEC is now recognized as having an important role in the inflammatory response of the respiratory system to inhaled exposures, and abnormalities of these responses are thought to be important to asthma pathogenesis. This review first explores how the challenges of studying nasal and bronchial AECs in children have been addressed and then summarizes the results of studies of primary AEC function in children with and without asthma. There is good evidence that nasal AECs may be a suitable surrogate for the study of certain aspects of bronchial AEC function, although bronchial AECs remain the gold standard for asthma research. There are consistent differences between children with and without asthma for nasal and bronchial AEC mediator release following exposure to a range of pro-inflammatory stimulants including interleukins (IL)-1β, IL-4, and IL-13. However, there are inconsistencies between studies, e.g., release of IL-6, an important pro-inflammatory cytokine, is not increased in children with asthma relative to controls in all studies. Future work should expand current understanding of the "upstream" signalling pathways in AEC, study AEC from children before the onset of asthma symptoms and in vitro models should be developed that replicate the in vivo status more completely, e.g., co-culture with dendritic cells. AECs are difficult to obtain from children and collaboration between centers is expected to yield meaningful advances in asthma understanding and ultimately help deliver novel therapies. PMID:26178976

  10. Phosphoinositides metabolism in primary culture of dog thyroid cells: Effects of thyrotropin and carbachol

    SciTech Connect

    Taguchi, M.; Field, J.B. )

    1990-04-01

    Thyrotropin (TSH) and carbachol stimulated in a dose-dependent manner the accumulation of 3H-glycerophosphoinositol (GPI), 3H-inositol monophosphate (IP1), 3H-inositol bisphosphate (IP2) and 3H-inositol trisphosphate (IP3) in primary cultures of dog thyroid cells prelabeled with myo-(2-3H)inositol. TSH, 250 mU/mL, stimulated 3H-IP3 level after a 10-minute incubation while 10 mU/mL TSH increased it during a 60-minute incubation. The effect of carbachol was more rapid and greater than that of TSH. Carbachol, 100 mumol/L, elevated 3H-IP3 after a 2-minute incubation and 3H-IP3 formation was increased by as little as 1 mumol/L carbachol. TSH stimulation was observed only if the cells were deprived of TSH for 5 days before being labeled with 3H-inositol. Prolongation of the labeling period or addition of TSH, (Bu)2cAMP or carbachol during the labeling increased 3H-inositol incorporation into polyphoinositides (PIPs). When the cells were labeled without any other addition, control and TSH-stimulated 3H-IP3 levels increased in parallel with 3H-PIP levels. However, TSH or carbachol-stimulated 3H-IP3 levels did not increase in proportion to 3H-PIPs level when the cells were labeled with TSH or (Bu)2cAMP. Thus, the ratio of 3H-IP3/3H-PIPs (both control and TSH or carbachol-stimulated) decreased in the cells labeled with TSH or (Bu)2cAMP, which might reflect TSH stimulation of 3H-inositol incorporation into PIPs pool(s) that do not participate in hormone-induced hydrolysis of PIPs.

  11. Effect of glutamate on lysosomal membrane permeabilization in primary cultured cortical neurons

    PubMed Central

    YAN, MIN; ZHU, WENBO; ZHENG, XIAOKE; LI, YUAN; TANG, LIPENG; LU, BINGZHENG; CHEN, WENLI; QIU, PENGXIN; LENG, TIANDONG; LIN, SUIZHEN; YAN, GUANGMEI; YIN, WEI

    2016-01-01

    Glutamate is the principal neurotransmitter in the central nervous system. Glutamate-mediated excitotoxicity is the predominant cause of cerebral damage. Recent studies have shown that lysosomal membrane permeabilization (LMP) is involved in ischemia-associated neuronal death in non-human primates. This study was designed to investigate the effect of glutamate on lysosomal stability in primary cultured cortical neurons. Glutamate treatment for 30 min induced the permeabilization of lysosomal membranes as assessed by acridine orange redistribution and immunofluorescence of cathepsin B in the cytoplasm. Inhibition of glutamate excitotoxicity by the NMDA receptor antagonist MK-801 and the calcium chelator ethylene glycolbis (2-aminoethylether)-N, N, N′, N′-tetraacetic acid, rescued lysosomes from permeabilization. The role of calpain and reactive oxygen species (ROS) in inducing LMP was also investigated. Ca2+ overload following glutamate treatment induced the activation of calpain and the production of ROS, which are two major contributors to neuronal death. It has been reported that lysosomal-associated membrane protein 2 (LAMP2) and heat shock protein (HSP)70 are two calpain substrates that promote LMP in cancer cells; however, it was found that calpains were activated by glutamate, but only LAMP2 was subsequently degraded. Furthermore, LMP was not alleviated by treatment with the calpain inhibitors calpeptin and SJA6017, which blocked the cleavage of the calpain substrate α-fodrin. It was demonstrated that LMP was significantly alleviated by treatment with the antioxidant N-Acetyl-L-cysteine, indicating that LMP involvement in early glutamate excitotoxicity may be mediated partly by ROS rather than calpain activation. Overall, these data shed light on the role of ROS-mediated LMP in early glutamate excitotoxicity. PMID:26821268

  12. 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

  13. Overexpression of glucose transporters in rat mesangial cells cultured in a normal glucose milieu mimics the diabetic phenotype.

    PubMed

    Heilig, C W; Concepcion, L A; Riser, B L; Freytag, S O; Zhu, M; Cortes, P

    1995-10-01

    An environment of high glucose concentration stimulates the synthesis of extracellular matrix (ECM) in mesangial cell (MC) cultures. This may result from a similar increase in intracellular glucose concentration. We theorized that increased uptake, rather than glucose concentration per se is the major determinant of exaggerated ECM formation. To test this, we compared the effects of 35 mM glucose on ECM synthesis in normal MCs with those of 8 mM glucose in the same cells overexpressing the glucose transporter GLUT1 (MCGT1). Increasing medium glucose from 8 to 35 mM caused normal MCs to increase total collagen synthesis and catabolism, with a net 81-90% increase in accumulation. MCs transduced with the human GLUT1 gene (MCGT1) grown in 8 mM glucose had a 10-fold greater GLUT1 protein expression and a 1.9, 2.1, and 2.5-fold increase in cell myo-inositol, lactate production, and cell sorbitol content, respectively, as compared to control MCs transduced with bacterial beta-galactosidase (MCLacZ). MCGT1 also demonstrated increased glucose uptake (5-fold) and increased net utilization (43-fold), and greater synthesis of individual ECM components than MCLacZ. In addition, total collagen synthesis and catabolism were also enhanced with a net collagen accumulation 111-118% greater than controls. Thus, glucose transport activity is an important modulator of ECM formation by MCs; the presence of high extracellular glucose concentrations is not necessarily required for the stimulation of matrix synthesis. PMID:7560072

  14. Representative mammalian cell culture test materials for assessment of primary recovery technologies: A rapid method with industrial applicability

    PubMed Central

    Popova, Daria; Stonier, Adam; Pain, David; Titchener-Hooker, Nigel J; Farid, Suzanne S

    2015-01-01

    Mammalian cell culture material is often difficult to produce accurately and reproducibly for downstream studies. This article presents a methodology for the creation of a set of cell culture test materials where key variables including cell density, cell viability, product, and the host cell protein (HCP) load can be manipulated individually. The methodology was developed using a glutamine synthetase Chinese hamster ovary cell line cultured at 5-L and 70-L scales. Cell concentration post-cell growth was manipulated using tangential flow filtration to generate a range of target cell densities of up to 100 × 106 cells/mL. A method to prepare an apoptotic cell stock to achieve target viabilities of 40–90% is also described. In addition, a range of IgG1 and HCP concentrations was achieved. The results illustrate that the proposed methodology is able to mimic different cell culture profiles by decoupling the control of the key variables. The cell culture test materials were shown to be representative of typical cell culture feed material in terms of particle size distribution and HCP population. This provides a rapid method to create the required feeds for assessing the feasibility of primary recovery technologies designed to cope with higher cell density cultures. PMID:25377169

  15. Representative mammalian cell culture test materials for assessment of primary recovery technologies: a rapid method with industrial applicability.

    PubMed

    Popova, Daria; Stonier, Adam; Pain, David; Titchener-Hooker, Nigel J; Farid, Suzanne S

    2015-01-01

    Mammalian cell culture material is often difficult to produce accurately and reproducibly for downstream studies. This article presents a methodology for the creation of a set of cell culture test materials where key variables including cell density, cell viability, product, and the host cell protein (HCP) load can be manipulated individually. The methodology was developed using a glutamine synthetase Chinese hamster ovary cell line cultured at 5-L and 70-L scales. Cell concentration post-cell growth was manipulated using tangential flow filtration to generate a range of target cell densities of up to 100 × 10(6) cells/mL. A method to prepare an apoptotic cell stock to achieve target viabilities of 40-90% is also described. In addition, a range of IgG1 and HCP concentrations was achieved. The results illustrate that the proposed methodology is able to mimic different cell culture profiles by decoupling the control of the key variables. The cell culture test materials were shown to be representative of typical cell culture feed material in terms of particle size distribution and HCP population. This provides a rapid method to create the required feeds for assessing the feasibility of primary recovery technologies designed to cope with higher cell density cultures. PMID:25377169

  16. Cell Culture Made Easy.

    ERIC Educational Resources Information Center

    Dye, Frank J.

    1985-01-01

    Outlines steps to generate cell samples for observation and experimentation. The procedures (which use ordinary laboratory equipment) will establish a short-term primary culture of normal mammalian cells. Information on culture vessels and cell division and a list of questions to generate student interest and involvement in the topics are…

  17. 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.

  18. Temporal expression of transporters and receptors in a rat primary co-culture blood-brain barrier model.

    PubMed

    Liu, Houfu; Li, Yang; Lu, Sijie; Wu, Yiwen; Sahi, Jasminder

    2014-10-01

    1. The more relevant primary co-cultures of brain microvessel endothelial cells and astrocytes (BMEC) are less utilized for screening of potential CNS uptake when compared to intestinal and renal cell lines. 2. In this study, we characterized the temporal mRNA expression of major CNS transporters and receptors, including the transporter regulators Pxr, Ahr and Car in a rat BMEC co-cultured model. Permeability was compared with the Madin-Darby canine kidney (MDCKII)-MDR1 cell line and rat brain in situ perfusion model. 3. Our data demonstrated differential changes in expression of individual transporters and receptors over the culture period. Expression of ATP-binding cassette transporters was better retained than that of solute carrier transporters. The insulin receptor (IR) was best maintained among investigated receptors. AhR demonstrated high mRNA expression in rat brain capillaries and expression was better retained than Pxr or Car in culture. Mdr1b expression was up-regulated during primary culture, albeit Mdr1a mRNA levels were much higher. P-gp and Bcrp-1 were highly expressed and functional in this in vitro system. 4. Permeability measurements with 18 CNS marketed drugs demonstrated weak correlation between rBMEC model and rat in situ permeability and moderate correlation with MDCKII-MDR1 cells. 5. We have provided appropriate methodologies, as well as detailed and quantitative characterization data to facilitate improved understanding and rational use of this in vitro rat BBB model. PMID:24827375

  19. Occurrence of caldesmon (a calmodulin-binding protein) in cultured cells: comparison of normal and transformed cells.

    PubMed Central

    Owada, M K; Hakura, A; Iida, K; Yahara, I; Sobue, K; Kakiuchi, S

    1984-01-01

    Caldesmon is a calmodulin-binding and F-actin-binding protein originally purified from chicken gizzard smooth muscle. This protein binds to F-actin filaments in a Ca2+- and calmodulin-dependent "flip-flop" fashion, thereby regulating the function of actin filaments. Here we report that various lines of cultured cells contain a Mr 77,000 protein that specifically reacts with the affinity-purified caldesmon antibody raised against chicken gizzard caldesmon . Among the fibroblast proteins that had been pulse-labeled with [35S]methionine, the Mr 77,000 protein was the only protein band detected on the NaDodSO4 gel that reacted with the anticaldesmon . The subcellular distribution of the Mr 77,000 protein was investigated by the indirect immunofluorescence technique using the anticaldesmon . In all fibroblast cell lines examined, the immunofluorescence localized along the cellular stress fibers and in leading edges of the cell. In Rous sarcoma virus-transformed cells (S7-1), however, the distribution of the fluorescence changed to a diffuse and blurred appearance. These staining patterns of anticaldesmon obtained with the normal and transformed cells coincided with those of antiactin in the corresponding states, strongly suggesting the functional linkage between the Mr 77,000 protein and actin filaments. We propose to refer to this Mr 77,000 protein as caldesmon 77. The cellular level of caldesmon 77 in transformed S7-1 cells decreased to about one-third of that in their normal counterparts (cell line no. 7). Essentially the same result was obtained with normal rat kidney cells infected with the temperature-sensitive transformation mutant Schmidt-Ruppin strain of Rous sarcoma virus (68 N2 clone). The cellular level of caldesmon 77 observed at a permissive temperature (35 degrees C) was about one-third of that at a nonpermissive temperature (38.5 degrees C). These changes of caldesmon 77 in transformed cells may correlate with the loss of Ca2+ regulation in the

  20. 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

  1. Transfer of beta-amyloid precursor protein gene using adenovirus vector causes mitochondrial abnormalities in cultured normal human muscle.

    PubMed Central

    Askanas, V; McFerrin, J; Baqué, S; Alvarez, R B; Sarkozi, E; Engel, W K

    1996-01-01

    As in Alzheimer-disease (AD) brain, vacuolated muscle fibers of inclusion-body myositis (IBM) contain abnormally accumulated beta-amyloid precursor protein (beta APP), including its beta-amyloid protein epitope, and increased beta APP-751 mRNA. Other similarities between IBM muscle and AD brain phenotypes include paired helical filaments, hyperphosphorylated tau protein, apolipoprotein E, and mitochondrial abnormalities, including decreased cytochrome-c oxidase (COX) activity. The pathogenesis of these abnormalities in IBM muscle and AD brain is not known. We now report that direct transfer of the beta APP gene, using adenovirus vector, into cultured normal human muscle fibers causes structural abnormalities of mitochondria and decreased COX activity. In this adenovirus-mediated beta APP gene transfer, we demonstrated that beta APP overproduction can induce mitochondrial abnormalities. The data suggest that excessive beta APP may be responsible for mitochondrial and COX abnormalities in IBM muscle and perhaps AD brain. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 PMID:8577761

  2. Macro-micro imaging of cardiac–neural circuits in co-cultures from normal and diseased hearts

    PubMed Central

    Bub, Gil; Burton, Rebecca-Ann B

    2015-01-01

    The autonomic nervous system plays an important role in the modulation of normal cardiac rhythm, but is also implicated in modulating the heart’s susceptibility to re-entrant ventricular and atrial arrhythmias. The mechanisms by which the autonomic nervous system is pro-arrhythmic or anti-arrhythmic is multifaceted and varies for different types of arrhythmia and their cardiac substrates. Despite decades of research in this area, fundamental questions related to how neuron density and spatial organization modulate cardiac wave dynamics remain unanswered. These questions may be ill-posed in intact tissues where the activity of individual cells is often experimentally inaccessible. Development of simplified biological models that would allow us to better understand the influence of neural activation on cardiac activity can be beneficial. This Symposium Review summarizes the development of in vitro cardiomyocyte cell culture models of re-entrant activity, as well as challenges associated with extending these models to include the effects of neural activation. PMID:25398529

  3. Low doses of nanodiamonds and silica nanoparticles have beneficial hormetic effects in normal human skin fibroblasts in culture.

    PubMed

    Mytych, Jennifer; Wnuk, Maciej; Rattan, Suresh I S

    2016-04-01

    Nanodiamonds (ND) and silica nanoparticles (SiO2-NP) have been much investigated for their toxicity at high doses, little is known about their biological activity at low concentrations. Here we report the biphasic dose response of ND and SiO2-NP in modulating normal human facial skin fibroblasts (FSF1) in culture. ND and SiO2-NP at low concentration (up to 0.5 μg/ml) had beneficial effects on FSF1 in terms of increasing their proliferation and metabolic activity. Exposure of FSF1 cells to low levels of NP enhanced their wound healing ability in vitro and slowed down aging during serial passaging as measured by maintenance of youthful morphology, reduction in the rate of loss of telomeres, and the over all proliferative characteristics. Furthermore, NP treatment induced the activation of Nrf2- and FOXO3A-mediated cellular stress responses, including an increased expression of heme oxygenease (HO-1), sirtuin (SIRT1), and DNA methyltransferase II (DNMT2). These results imply that ND and SiO2-NP at low doses are potential hormetins, which exert mild stress-induced beneficial hormetic effects through improved survival, longevity, maintenance, repair and function of human cells. PMID:26814705

  4. Uneven distribution of nucleoside transporters and intracellular enzymatic degradation prevent transport of intact [14C] adenosine across the sheep choroid plexus epithelium as a monolayer in primary culture

    PubMed Central

    Redzic, Zoran B; Isakovic, Aleksandra J; Misirlic Dencic, Sonja T; Popadic, Dusan; Segal, Malcolm B

    2006-01-01

    Background Efflux transport of adenosine across the choroid plexus (CP) epithelium might contribute to the homeostasis of this neuromodulator in the extracellular fluids of the brain. The aim of this study was to explore adenosine transport across sheep CP epithelial cell monolayers in primary culture. Methods To explore transport of adenosine across the CP epithelium, we have developed a method for primary culture of the sheep choroid plexus epithelial cells (CPEC) on plastic permeable supports and analysed [14C] adenosine transport across this cellular layer, [14C] adenosine metabolism inside the cells, and cellular uptake of [14C] adenosine from either of the chambers. The primary cell culture consisted of an enriched epithelial cell fraction from the sheep fourth ventricle CP and was grown on laminin-precoated filter inserts. Results and conclusion CPEC grew as monolayers forming typical polygonal islands, reaching optical confluence on the third day after the seeding. Transepithelial electrical resistance increased over the time after seeding up to 85 ± 9 Ω cm2 at day 8, while permeability towards [14C] sucrose, a marker of paracellular diffusion, simultaneously decreased. These cells expressed some features typical of the CPEC in situ, including three nucleoside transporters at the transcript level that normally mediate adenosine transport across cellular membranes. The estimated permeability of these monolayers towards [14C] adenosine was low and the same order of magnitude as for the markers of paracellular diffusion. However, inhibition of the intracellular enzymes, adenosine kinase and adenosine deaminase, led to a significant increase in transcellular permeability, indicating that intracellular phosphorylation into nucleotides might be a reason for the low transcellular permeability. HPLC analysis with simultaneous detection of radioactivity revealed that [14C] radioactivity which appeared in the acceptor chamber after the incubation of CPEC monolayers

  5. 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.

  6. (/sup 3/H) spiperone binding sites in rat primary cultures, C6 glioma, and B104 neuroblastoma

    SciTech Connect

    Severson, J.A.; de Vellis, J.S.; Finch, C.E.

    1980-01-01

    Binding sites for (/sup 3/H) spiperone were detected on membranes from primary glial cultures from neonatal rat cortex and striatum and from the C6 glioma cells. (/sup 3/H) spiperone binding was displacable by d-butaclamol. However, competition studies suggest that (/sup 3/H) spiperone binding to primary glial cultures was mainly to serotonergic sites, and binding to the C6 glioma was alpha-adrenergic. No specific binding was detected to membranes from B104 neuroblastoma cells. Although (/sup 3/H) spiperone binding to glial sites in whole striatum under generally used conditions is small (about 10%), the striatal glial hyperactivity which is often associated with neuronal degeneration could lead to an overestimation of presumed neuronal binding sites for dopaminergic ligands.

  7. 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

  8. 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. PMID:26503557

  9. Primary skeletal muscle cells cultured on gelatin bead microcarriers develop structural and biochemical features characteristic of adult skeletal muscle.

    PubMed

    Kubis, Hans-Peter; Scheibe, Renate J; Decker, Brigitte; Hufendiek, Karsten; Hanke, Nina; Gros, Gerolf; Meissner, Joachim D

    2016-04-01

    A primary skeletal muscle cell culture, in which myoblasts derived from newborn rabbit hindlimb muscles grow on gelatin bead microcarriers in suspension and differentiate into myotubes, has been established previously. In the course of differentiation and beginning spontaneous contractions, these multinucleated myotubes do not detach from their support. Here, we describe the development of the primary myotubes with respect to their ultrastructural differentiation. Scanning electron microscopy reveals that myotubes not only grow around the surface of one carrier bead but also attach themselves to neighboring carriers, forming bridges between carriers. Transmission electron microscopy demonstrates highly ordered myofibrils, T-tubules, and sarcoplasmic reticulum. The functionality of the contractile apparatus is evidenced by contractile activity that occurs spontaneously or can be elicited by electrostimulation. Creatine kinase activity increases steadily until day 20 of culture. Regarding the expression of isoforms of myosin heavy chains (MHC), we could demonstrate that from day 16 on, no non-adult MHC isoform mRNAs are present. Instead, on day 28 the myotubes express predominantly adult fast MHCIId/x mRNA and protein. This MHC pattern resembles that of fast muscles of adult rabbits. In contrast, primary myotubes grown on matrigel-covered culture dishes express substantial amounts of non-adult MHC protein even on day 21. To conclude, primary myotubes grown on microcarriers in their later stages exhibit many features of adult skeletal muscle and characteristics of fast type II fibers. Thus, the culture represents an excellent model of adult fast skeletal muscle, for example, when investigating molecular mechanisms of fast-to-slow fiber-type transformation. PMID:26610066

  10. The myofibroblast markers α-SM actin and β-actin are differentially expressed in 2 and 3-D culture models of fibrotic and normal skin.

    PubMed

    Vozenin, M C; Lefaix, J L; Ridi, R; Biard, D S; Daburon, F; Martin, M

    1998-01-01

    To characterize the differences between fibrotic myofibroblasts and normal fibroblasts, we studied two differentiation markers: α-smooth muscle (SM) actin, a specific marker of myofibroblast differentiation, and β-actin, which is overexpressed in the fibrotic tissue. Experiments were performed on fibroblasts isolated from normal pig skin and on subcutaneous myofibroblasts isolated from pig radiation-induced fibrosis. Three culture models were used: cells in monolayers, equivalent dermis, consisting of fibroblasts embedded into a matrix composed of type I collagen, and in vitro reconstituted skin, in which the matrix and containing life fibroblasts were overlaid with keratinocytes. Samples were studied using immunofluorescence and western-blotting. In monolayers cultures, both fibrosis and normal cells expressed α-SM actin. Furthermore, similar amounts of β-actin protein were found. In these conditions, the resulting alterations in the phenotypes of cells made comparison of cultured fibrotic and normal cells irrelevant. Under the two 3-D culture models, normal fibroblasts no longer expressed α-SM actin. They expressed β-actin at the basal level. Moreover, the fibrotic myofibroblasts in both 3-D models retained their differentiation features, expressing α-SM actin and overexpressing β-actin. We found that this normalization was mainly related to the genomic programmation acquired by the cells in the tissue. Cellular motility and microenvironment were also involved, whereas cellular proliferation was not a major factor. Consequently, both three-dimensional models allowed the study of radiation-induced fibrosis in vitro, provided good extrapolations to in vivo conditions and avoided certain of culture artefacts. PMID:22359004

  11. 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. PMID:21501697

  12. 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. PMID:23559662

  13. Gene expression profiling and differentiation assessment in primary human hepatocyte cultures, established hepatoma cell lines, and human liver tissues

    SciTech Connect

    Olsavsky, Katy M.; Page, Jeanine L.; Johnson, Mary C.; Zarbl, Helmut; Strom, Stephen C.; Omiecinski, Curtis J. . E-mail: cjo10@psu.edu

    2007-07-01

    Frequently, primary hepatocytes are used as an in vitro model for the liver in vivo. However, the culture conditions reported vary considerably, with associated variability in performance. In this study, we characterized the differentiation character of primary human hepatocytes cultured using a highly defined, serum-free two-dimensional sandwich system, one that configures hepatocytes with collagen I as the substratum together with a dilute extracellular matrix (Matrigel{sup TM}) overlay combined with a defined serum-free medium containing nanomolar levels of dexamethasone. Gap junctional communication, indicated by immunochemical detection of connexin 32 protein, was markedly enhanced in hepatocytes cultured in the Matrigel sandwich configuration. Whole genome expression profiling enabled direct comparison of liver tissues to hepatocytes and to the hepatoma-derived cell lines, HepG2 and Huh7. PANTHER database analyses were used to identify biological processes that were comparatively over-represented among probe sets expressed in the in vitro systems. The robustness of the primary hepatocyte cultures was reflected by the extent of unchanged expression character when compared directly to liver, with more than 77% of the probe sets unchanged in each of the over-represented categories, representing such genes as C/EBP{alpha}, HNF4{alpha}, CYP2D6, and ABCB1. In contrast, HepG2 and Huh7 cells were unchanged from the liver tissues for fewer than 48% and 55% of these probe sets, respectively. Further, hierarchical clustering of the hepatocytes, but not the cell lines, shifted from donor-specific to treatment-specific when the probe sets were filtered to focus on phenobarbital-inducible genes, indicative of the highly differentiated nature of the hepatocytes when cultured in a highly defined two-dimensional sandwich system.

  14. 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

  15. Transporters involved in regulation of intracellular pH in primary cultured rat brain endothelial cells

    PubMed Central

    Taylor, Caroline J; Nicola, Pieris A; Wang, Shanshan; Barrand, Margery A; Hladky, Stephen B

    2006-01-01

    Fluid secretion across the blood–brain barrier, critical for maintaining the correct fluid balance in the brain, entails net secretion of HCO3−, which is brought about by the combined activities of ion transporters situated in brain microvessels. These same transporters will concomitantly influence intracellular pH (pHi). To analyse the transporters that may be involved in the maintenance of pHi and hence secretion of HCO3−, we have loaded primary cultured endothelial cells derived from rat brain microvessels with the pH indicator BCECF and suspended them in standard NaCl solutions buffered with Hepes or Hepes plus 5% CO2/HCO3−. pHi in the standard solutions showed a slow acidification over at least 30 min, the rate being less in the presence of HCO3− than in its absence. However, after accounting for the difference in buffering, the net rates of acid loading with and without HCO3− were similar. In the nominal absence of HCO3− the rate of acid loading was increased equally by removal of external Na+ or by inhibition of Na+/H+ exchange by ethylisopropylamiloride (EIPA). By contrast, in the presence of HCO3− the increase in the rate of acid loading when Na+ was removed was much larger and the rate was then also significantly greater than the rate observed in the absence of both Na+ and HCO3−. Removal of Cl− in the presence of HCO3− produced an alkalinization followed by a resumption of the slow acid gain. Removal of Na+ following removal of Cl− increased the rate of acid gain. In the presence of HCO3− and initial presence of Na+ and Cl−, DIDS inhibited the changes in pHi produced by removal of either Na+ or Cl−. These are the expected results if these cells possess an AE-like Cl−/HCO3− exchanger, a ‘channel-like’ permeability allowing slow influx of acid (or efflux of HCO3−), a NBC-like Cl−-independent Na+−HCO3− cotransporter, and a NHE-like Na+/H+ exchanger. The in vitro rates of HCO3− loading via the Na+−HCO3

  16. Three-dimensional cultures of normal human osteoblasts: proliferation and differentiation potential in vitro and upon ectopic implantation in nude mice.

    PubMed

    Ferrera, D; Poggi, S; Biassoni, C; Dickson, G R; Astigiano, S; Barbieri, O; Favre, A; Franzi, A T; Strangio, A; Federici, A; Manduca, P

    2002-05-01

    We report the establishment in vitro of three-dimensional (3D) cultures of human osteoblasts (hOB) derived from normal adults and supported uniquely by the extracellular matrix (ECM) they deposit. Osteoblasts were cultured in 3D cultures in vitro for up to 120 days. The 3D cultures, examined at 25, 31, and 48 days, expressed protein markers of osteoblastic cells, namely osteonectin, collagen type I, fibronectin, osteopontin, bone sialoprotein, biglycan, and decorin. Sequentially, alkaline phosphatase (AP) and then Ca incorporation, mineralization of matrix (monitored by histochemistry and transmission electron microscopy), and finally osteocalcin expression, were detected in the 3D cultures. Ultrastructurally, morphology progressed from early to mature osteoblast and to osteocyte-like. Cells were embedded in a matrix with organized collagen type I fibers containing, increasingly with time of culture, needle-shaped crystals, often associated with matrix vesicles, characteristic of those in bone. During the culture (up to 120 days) there was an outgrowth of proliferating osteogenic cells from the 3D structure. Subcutaneous implantation in nude mice for 20 days of osteoblasts cultured in 3D culture for different lengths of time in vitro, showed progression of mineralization from the inner region of the implant outward, with peripheral cells being embedded in nonmineralized, collagen-rich matrix. The 3D implants were invaded by vessels derived from the host. PMID:11996910

  17. Primary amenorrhea in adolescent girls: normal coitus or not? Always take a look in the physician's office

    PubMed Central

    2014-01-01

    Background Primary care physicians are frequently faced with the challenge of evaluating primary amenorrhea in adolescent girls. Approximately 15% of these women have abnormal genital examination, with Müllerian agenesis being the second most frequent cause. We report two cases of adolescents with Müllerian agenesis that presented to a tertiary adolescent medicine center with primary amenorrhea and the very rare sexual phenomenon of urethral coitus. The aim of this report is to emphasize the importance of performing a genital examination in girls who present with amenorrhea in the primary care setting, even if ‘normal’ vaginal sexual activity is assumed. Case presentations A 19-year-old Caucasian and a 16-year-old Filipino girl presented to a tertiary adolescent medicine center with primary amenorrhea and a history of ‘normal’ vaginal coitus. Investigation revealed Müllerian agenesis in association with urethral coitus in both cases; neither patient suffered significant urethral damage to require urethra reconstruction. However, the first adolescent had recurrent pyelonephritis and renal scarring and the second had dysuria. To the best of our knowledge, Case 1 also represents the second reported case of pituitary prolactinoma in association with Müllerian agenesis. The first adolescent underwent a hernia repair and vaginoplasty, whereas the second had vaginal dilatations. Conclusion Our cases highlight the need for careful assessment of the external genitalia and vagina patency in all girls with amenorrhea, even if they report ‘normal’ vaginal sexual activity. Early identification of anatomic disorders such as Müllerian agenesis, will allow provision of proper care according to the patient’s needs and the existing abnormalities, and prevention of rare, unintentional but potentially physically and emotionally harmful, patterns of sexual intercourse. PMID:24507015

  18. 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

  19. 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

  20. Exploring the Culture of Reading among Primary School Teachers in Botswana

    ERIC Educational Resources Information Center

    Commeyras, Michelle; Mazile, Bontshetse Mosadimotho

    2011-01-01

    In Botswana, it is common to hear the lament that there is no culture of reading. We aim to contribute to the development of more specific understanding of a culture of reading by reporting on information gathered from teachers who attended workshops we offered titled "Teachers as Readers AND Readers as Teachers: Creating Young Readers and…

  1. 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

  2. In vitro synthesis of primary specific anti-breast cancer antibodies by normal human peripheral blood mononuclear cells

    PubMed Central

    Norkina, Oxana; Lum, Lawrence G.

    2013-01-01

    In this study, we developed a unique in vitro model to mimic the endogenous tumor microenvironment to understand the effect of immunotherapy with activated T-cells (ATC) armed with anti-CD3 × anti-Her2 bispecific antibody (aATC) on antibody response by naive immune cells. This model contained a co-culture of naïve peripheral blood mononuclear cells (PBMC), breast cancer cells (SK-BR-3), ATC or aATC and CpG ODNs. Culture supernatants were tested at various time points for anti-SK-BR-3 antibodies by ELISA, Western blot and flow cytometry. PBMC cocultured with non-irradiated aATC or irradiated (*) aATC showed significant increases in anti-tumor antibody production at day 14 (P < 0.0001) in the presence of CpG-ODN compared to unstimulated PBMC cultures (n = 9). Antibody specificity was confirmed by ELISA, Western blot and flow cytometry. Co-cultures containing *aATC and CpG showed significantly enhanced levels of IgG2 (P < 0.001) and cytokines that promote IgG2 synthesis including IL-13 (P < 0.02), IFNγ (P < 0.01) and GM-CSF (P < 0.05) compared to unstimulated PBMC control (n = 3). We show that aATC targeting and lysis of tumor cells induces an anti-tumor antibody response in our in vitro model. This model provides a unique opportunity to evaluate the interactions of T-cells, B-cells, and antigen-presenting cells leading to specific anti-tumor antibody responses. PMID:21713642

  3. Use of Primary Rat and Human Hepatocyte Sandwich Cultures for Activation of Indirect Carcinogens: Monitoring of DNA Strand Breaks and Gene Mutations in Co-cultured Cells.

    PubMed

    Fahrig, R; Rupp, M; Steinkamp-Zucht, A; Bader, A

    1998-08-01

    Loss of cytochrome P-450 content is a common feature in conventional culture systems of primary hepatocytes. In contrast to the standard in vitro situation, in vivo each hepatocyte is exposed to an extracellular matrix (space of Disse) at two opposing basolateral surfaces. This in vivo symmetry has been reconstructed in vitro by culturing rat or human hepatocytes within two layers of collagen, thus forming a sandwich configuration. Activation of dimethylbenzanthracene (DMBA) or benzo[a]pyrene (BaP) was studied in rat and human hepatocytes. Genotoxic effects were studied in a three-dimensional co-culture model between sandwich hepatocytes and mammalian cells using the comet assay for detection of DNA strand breaks, and the HPRT test for detection of gene mutations. Sandwich hepatocytes generated active metabolites. The maintenance of metabolic properties in hepatocytes was dependent on extracellular matrix geometry. The number of DMBA- or BaP-induced genotoxic effects tended to be higher than in standard S-9 mix assays. While the ability to activate indirect carcinogens disappears within hours in primary hepatocytes, hepatocyte sandwich cultures enhance their ability to activate indirect carcinogens within 1 wk and retain this activity for up to 2 wk. This is the main advantage of the sandwich method over the more simple and conventional assays. While freshly isolated hepatocytes, regardless of whether in sandwich culture or in conventional assays, are injured by the isolation procedure and possess a corresponding reduced activation ability, hepatocytes in sandwich cultures recover over the course of a few days, and acquire a much higher ability to activate indirect carcinogens. Consequently, the indirect carcinogens BaP and DMBA, which were ineffective (BaP) or exhibited only weak effects (DMBA) at a concentration of 160nmol/ml in 1-2-day-old hepatocytes, were clearly effective (BaP) or showed about a threefold increase in genotoxicity (DMBA) in 8-day

  4. Maintenance of Hepatic Functions in Primary Human Hepatocytes Cultured on Xeno-Free and Chemical Defined Human Recombinant Laminins.

    PubMed

    Watanabe, Masaaki; Zemack, Helen; Johansson, Helene; Hagbard, Louise; Jorns, Carl; Li, Meng; Ellis, Ewa

    2016-01-01

    Refined methods for maintaining specific functions of isolated hepatocytes under xeno-free and chemical defined conditions is of great importance for the development of hepatocyte research and regenerative therapy. Laminins, a large family of heterotrimeric basement membrane adhesion proteins, are highly cell and tissue type specific components of the extracellular matrix and strongly influence the behavior and function of associated cells and/or tissues. However, detailed biological functions of many laminin isoforms are still to be evaluated. In this study, we determined the distribution of laminin isoforms in human liver tissue and isolated primary human hepatocytes by western blot analysis, and investigated the efficacy of different human recombinant laminin isoforms on hepatic functions during culture. Protein expressions of laminin-chain α2, α3, α4, β1, β3, γ1, and γ2 were detected in both isolated human hepatocytes and liver tissue. No α1 and α5 expression could be detected in liver tissue or hepatocytes. Hepatocytes were isolated from five different individual livers, and cultured on human recombinant laminin isoforms -111, -211, -221, -332, -411, -421, -511, and -521 (Biolamina AB), matrigel (extracted from Engelbreth-Holm-Swarm sarcoma), or collagen type IV (Collagen). Hepatocytes cultured on laminin showed characteristic hexagonal shape in a flat cell monolayer. Viability, double stranded DNA concentration, and Ki67 expression for hepatocytes cultured for six days on laminin were comparable to those cultured on EHS and Collagen. Hepatocytes cultured on laminin also displayed production of human albumin, alpha-1-antitrypsin, bile acids, and gene expression of liver-enriched factors, such as hepatocyte nuclear factor 4 alpha, glucose-6-phosphate, cytochrome P450 3A4, and multidrug resistance-associated protein 2. We conclude that all forms of human recombinant laminin tested maintain cell viability and liver-specific functions of primary human

  5. Determination of Radiation Absorbed Dose to Primary Liver Tumors and Normal Liver Tissue Using Post-Radioembolization 90Y PET

    PubMed Central

    Srinivas, Shyam M.; Natarajan, Navin; Kuroiwa, Joshua; Gallagher, Sean; Nasr, Elie; Shah, Shetal N.; DiFilippo, Frank P.; Obuchowski, Nancy; Bazerbashi, Bana; Yu, Naichang; McLennan, Gordon

    2014-01-01

    Background: Radioembolization with Yttrium-90 (90 Y) microspheres is becoming a more widely used transcatheter treatment for unresectable hepatocellular carcinoma (HCC). Using post-treatment 90 Y positron emission tomography/computerized tomography (PET/CT) scans, the distribution of microspheres within the liver can be determined and quantitatively assessed. We studied the radiation dose of 90 Y delivered to liver and treated tumors. Methods: This retrospective study of 56 patients with HCC, including analysis of 98 liver tumors, measured and correlated the dose of radiation delivered to liver tumors and normal liver tissue using glass microspheres (TheraSpheres®) to the frequency of complications with modified response evaluation criteria in solid tumors (mRECIST). 90 Y PET/CT and triphasic liver CT scans were used to contour treated tumor and normal liver regions and determine their respective activity concentrations. An absorbed dose factor was used to convert the measured activity concentration (Bq/mL) to an absorbed dose (Gy). Results: The 98 studied tumors received a mean dose of 169 Gy (mode 90–120 Gy; range 0–570 Gy). Tumor response by mRECIST criteria was performed for 48 tumors that had follow-up scans. There were 21 responders (mean dose 215 Gy) and 27 non-responders (mean dose 167 Gy). The association between mean tumor absorbed dose and response suggests a trend but did not reach statistical significance (p = 0.099). Normal liver tissue received a mean dose of 67 Gy (mode 60–70 Gy; range 10–120 Gy). There was a statistically significant association between absorbed dose to normal liver and the presence of two or more severe complications (p = 0.036). Conclusion: Our cohort of patients showed a possible dose–response trend for the tumors. Collateral dose to normal liver is non-trivial and can have clinical implications. These methods help us understand whether patient adverse events, treatment success, or

  6. Recombinant human FIZZ3/resistin stimulates lipolysis in cultured human adipocytes, mouse adipose explants, and normal mice.

    PubMed

    Ort, Tatiana; Arjona, Anibal A; MacDougall, John R; Nelson, Pam J; Rothenberg, Mark E; Wu, Frank; Eisen, Andrew; Halvorsen, Yuan-Di C

    2005-05-01

    Human FIZZ3 (hFIZZ3) was identified as an ortholog of mouse resistin (mResistin), an adipocyte-specific secreted factor linked to insulin resistance in rodents. Unlike mResistin, hFIZZ3 is expressed in macrophages and monocytes, but is undetectable in adipose tissue. The profound macrophage infiltration of adipose that occurs during obesity suggests that hFIZZ3 may play an important role in adipocyte biology. Using a recombinant protein produced in Escherichia coli, we report here that chronic treatment of cultured human adipocytes with hFIZZ3 results in hypotropic cells with smaller lipid droplets. Recombinant hFIZZ3 facilitates preadipocyte proliferation and stimulates adipocyte triglyceride lipolysis, whereas recombinant mResistin inhibits adipocyte differentiation, with no detectable effect on proliferation or lipolysis. In addition, insulin-stimulated glucose uptake and Akt phosphorylation are not altered in hFIZZ3-treated adipocytes, indicating an intact insulin response. In mouse adipose explants, hFIZZ3 accelerates simultaneously triglyceride lipolysis and fatty acid reesterification, as assessed by measurement of glycerol and fatty acid release. Consistent with the in vitro findings, acute administration of recombinant hFIZZ3 into normal mice caused a significant increase in serum glycerol concentration with no elevation in free fatty acid at 45 min post injection. Taken together, the data suggest that recombinant hFIZZ3 can influence adipose metabolism by regulating preadipocyte cell number, adipocyte lipid content, and energy expenditure via accelerating the fatty acid/triglyceride futile cycle. PMID:15705777

  7. Isolation, purification, culture and characterisation of myoepithelial cells from normal and neoplastic canine mammary glands using a magnetic-activated cell sorting separation system.

    PubMed

    Sánchez-Céspedes, R; Maniscalco, L; Iussich, S; Martignani, E; Guil-Luna, S; De Maria, R; Martín de Las Mulas, J; Millán, Y

    2013-08-01

    Mammary gland tumours, the most common malignant neoplasm in bitches, often display myoepithelial (ME) cell proliferation. The aim of this study was to isolate, purify, culture and characterise ME cells from normal and neoplastic canine mammary glands. Monodispersed cells from three normal canine mammary glands and five canine mammary tumours were incubated with an anti-Thy1 antibody and isolated by magnetic-activated cell sorting (MACS). Cells isolated from two normal glands (cell lines CmME-N1 and CmME-N2) and four tumours (cell lines CmME-K1 from a complex carcinoma, CmME-K2 from a simple tubulopapillary carcinoma, and CmME-K3 and CmME-K4 from two carcinomas within benign tumours) were cultured in supplemented DMEM/F12 media for 40days. Cell purity was >90%. Tumour-derived ME cell lines exhibited heterogeneous morphology, growth patterns and immunocytochemical expression of cytokeratins, whereas cell lines from normal glands retained their morphology and levels of cytokeratin expression during culture. Cell lines from normal glands and carcinomas within benign tumours grew more slowly than those from simple and complex carcinomas. This methodology has the potential to be used for in vitro analysis of the role of ME cells in the growth and progression of canine mammary tumours. PMID:23583698

  8. 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. PMID:16959333

  9. IL-1 receptor antagonist (IL-1Ra) does not inhibit the production of C-reactive protein or serum amyloid A protein by human primary hepatocytes. Differential regulation in normal and tumour cells.

    PubMed Central

    Gabay, C; Genin, B; Mentha, G; Iynedjian, P B; Roux-Lombard, P; Guerne, P A

    1995-01-01

    The synthesis of some class 1 acute-phase proteins (APP), including C-reactive protein (CRP) and serum amyloid A (SAA) protein is completely blocked by the IL-1 receptor antagonist (IL-1Ra), whereas the production of fibrinogen, a class 2 APP, is increased by IL-1Ra in hepatoma cells, but this has never been tested in human hepatocytes in primary culture. Since previous studies on the contributions of cytokine inhibitors in connective tissues diseases suggested that IL-1 and tumour necrosis factor-alpha (TNF-alpha) might play an important role in the regulation of CRP, we decided to examine in more detail the respective roles of IL-1 beta, IL-6, and TNF-alpha and their inhibitors in the production of APP by human primary hepatocytes versus the hepatoma cell line PLC/PRF/5. In the hepatoma cell line, IL-1 beta and/or TNF-alpha had synergistic effects with IL-6 on the production of CRP and SAA. In contrast, these cytokines were devoid of effect in normal hepatocytes. The production of fibrinogen was increased by IL-6 and decreased by IL-1 (and TNF-alpha) in both cell types. The secretion of CRP and SAA by primary hepatocytes incubated with a cytokine-rich mononuclear cell-conditioned medium was totally unaffected by IL-1Ra or anti-TNF-alpha antibodies. In contrast, the addition of IL-1Ra increased the production of fibrinogen by both hepatoma cells and primary hepatocytes incubated with the mononuclear cell-conditioned medium. We therefore conclude that IL-1 beta and TNF-alpha do not exert any significant effect on the synthesis of CRP and SAA by human primary hepatocytes. Images Fig. 6 PMID:7743670

  10. 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.

  11. 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

  12. 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

  13. Intraeye retinal nerve fiber layer and macular thickness asymmetry measurements for the discrimination of primary open-angle glaucoma and normal tension glaucoma

    PubMed Central

    Khanal, Safal; Davey, Pinakin Gunvant; Racette, Lyne; Thapa, Madhu

    2015-01-01

    Purpose The aim of this study was to evaluate the diagnostic capability of intraeye retinal nerve fiber layer (RNFL) thickness and macular thickness (MT) asymmetry measurements for the discrimination of normal tension glaucoma (NTG) and primary open-angle glaucoma (POAG) using spectral domain optical coherence tomography (SD-OCT). Methods A total of 90 subjects were enrolled including 30 consecutive healthy subjects, 30 consecutive subjects with POAG, and 30 consecutive subjects with NTG. RNFL thicknesses around the optic disc as well as MT measurements were taken with circular and radial SD-OCT scans. Intraeye retinal and MT asymmetry were calculated as the absolute difference between superior and inferior hemispheres of the eye using posterior pole asymmetry analysis protocol. Analysis of variance was used for comparison and areas under the receiver operating characteristic (AROC) were obtained for different parameters among the three diagnostic groups. Results There was a significant difference in MT asymmetry for all comparison groups (normal-NTG, p < 0.05; normal-POAG, p < 0.001; and NTG-POAG, p < 0.001). Intraeye retinal nerve fiber thickness asymmetry measurements were not different between the groups (normal-NTG, p < 0.187; normal-POAG, p < 0.056; and NTG-POAG, p < 0.837). The area under ROC curves exceeded 0.800 for all the studied parameters, including the MT asymmetry except for intraeye RNFL thickness asymmetry which had the lowest AROC as well as the least sensitivity for identifying subjects with NTG from normal (AROC = 0.626, sensitivity = 30%); POAG from normal (AROC = 0.644, sensitivity = 37%) and NTG from POAG (AROC = 0.533, sensitivity = 13%). Conclusion The intraeye MT asymmetry holds significant potential as a distinguishing parameter for NTG and POAG. PMID:26652244

  14. 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

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

    PubMed

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

    2015-12-01

    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

  16. Effect of PITX2 knockdown on transcriptome of primary human trabecular meshwork cell cultures

    PubMed Central

    Paylakhi, Seyed Hassan; Fan, Jian-Bing; Mehrabian, Mohadeseh; Sadeghizadeh, Majid; Yazdani, Shahin; Katanforoush, Ali; Kanavi, Mozhgan Rezaei; Ronaghi, Mostafa

    2011-01-01

    Purpose To identify genes whose expressions in primary human trabecular meshwork (TM) cell cultures are affected by the transcription factor pituitary homeobox 2 (PITX2) and to identify genes that may have roles in glaucoma. Known glaucoma causing genes account for disease in a small fraction of patients, and we aimed at identification of other genes that may have subtle and accumulative effects not easily identifiable by a genetic approach. Methods Expression profiles derived using microarrays were compared between TM control cells and cells treated with PITX2 siRNAs using three protocols so as to minimize false positive and negative results. The first protocol was based on the commonly used B statistic. The second and third protocols were based on fold change in expression. The second protocol used a threshold of at least 2 fold change in expression, whereas the third protocol used ranking in fold change without setting a threshold. The likelihood of a selected gene being a true positive was considered to correlate with the number of protocols by which it was selected. By considering all genes that were selected by at least one protocol, the likelihood of false negatives was expected to decrease. Effects on a subset of selected genes were verified by real time PCR, western blots, and immunocytochemistry. Effects on ALDH1A1, were further pursued because its protein product, aldehyde dehydrogenase 1 family, member A1, has roles in oxidative stress and because oxidative stress is known to be relevant to the etiology of glaucoma. Results The expression level of 41 genes was assessed by to be possibly affected by PITX2 knockdown. Twenty one genes were down-regulated and twenty were upregulated. The expression of five genes was assessed to be altered by all three analysis protocols. The five genes were DIRAS3 (DIRAS family, GTP-binding RAS-like 3), CXCL6 (chemokine (C-X-C motif) ligand 6), SAMD5 (sterile alpha motif domain containing 5), CBFB (core-binding factor, beta

  17. Permeability properties and occludin expression in a primary cultured model gill epithelium from the stenohaline freshwater goldfish.

    PubMed

    Chasiotis, Helen; Kelly, Scott P

    2011-05-01

    Techniques for the primary culture of fish gill epithelia on permeable supports have provided 'reconstructed' gill models appropriate for the study of gill permeability characteristics in vitro. Models developed thus far have been derived from euryhaline fish species that can tolerate a wide range of environmental salinity. This study reports on procedures for the primary culture of a model gill epithelium derived from goldfish, a stenohaline freshwater (FW) fish that cannot tolerate high environmental salt concentrations. The reconstructed goldfish gill epithelium was cultured on permeable filter inserts and using electron microscopy and immunocytochemical techniques, was determined to be composed exclusively of gill pavement cells. When cultured under symmetrical conditions (i.e. with culture medium bathing both apical and basolateral surfaces), epithelial preparations generated appreciable transepithelial resistance (TER) (e.g. 1,150 ± 46 Ωcm(2)) within 36-42 h post-seeding in inserts. When apical medium was replaced with FW (asymmetrical conditions to mimic conditions that occur in vivo), epithelia exhibited increased TER and elevated paracellular permeability. Changes in permeability occurred in association with altered occludin-immunoreactive band position by western blot and no change in occludin mRNA abundance. We contend that the goldfish gill model will provide a useful in vitro tool for examining the molecular components of a stenohaline fish gill epithelium that participate in the regulation of gill permeability. The model will allow molecular observations to be made together with assessment of changing physiological properties that relate to permeability. Together, this will allow further insight into mechanisms that regulate gill permeability in fishes. PMID:21085969

  18. 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. PMID:23501253

  19. 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 ...

  20. Steroid metabolism by purified adult rat Leydig cells in primary culture

    SciTech Connect

    Browning, J.Y.; Tcholakian, R.K.; Kessler, M.J.; Grotjan, H.E. Jr.

    1982-11-01

    To characterize Leydig cell steroidogensis, we examined the metabolism of (3H)pregnenolone (3 beta-hydroxy-5-pregnen-20-one) to androgens in the presence and absence of human chorionic gonadotropin (hCG) as a function of culture duration. Approximately 20-30% of the (3H)pregnenolone was converted to testosterone (17 beta-hydroxy-4-androsten-3-one) by purified Leydig cells at 0, 3 and 5 days (d) of culture. Androstenedione (4-androstene-3,17-dione) and dihydrotestosterone (17 beta-hydroxy-5 alpha-androstan-3-one) were also produced while on day 5 of culture, significant amounts of progesterone (4-pregnene-3,20-dione) were isolated. The delta 5 intermediates, 17-hydroxypregnenolone (3 beta, 17-dihydroxy-5-pregnen-20-one) and dehydroepiandrosterone (3 beta-hydroxy-5-androsten-17-one), accounted for less than 1% of substrate conversion, indicating a clear preference for Leydig cells to metabolize (3H)pregnenolone via the delta 4 pathway. On day 0 of culture, unidentified metabolites considered of predominately polar steroids while on day 5 of culture, the unidentified metabolites consisted of predominately nonpolar steroids. In the presence of hCG, (3H-pregnenolone metabolism did not differ from basal on day 0 or 3 of culture. HCG increased the conversion of pregnenolone to progesterone and 17-hydroxyprogesterone (17-hydroxy-4-pregnene-3,20-dione) on 5d. This suggests that Leydig cells cultured for 5d have decreased C17-20 desmolase activity or that hCG acutely stimulates 3 beta-hydroxysteroid dehydrogenase and delta 5-delta 5 isomerase activities.

  1. 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

  2. The Culture of Primary Motor and Sensory Neurons in Defined Media on Electrospun Poly-L-lactide Nanofiber Scaffolds

    PubMed Central

    Leach, Michelle K.; Feng, Zhang-Qi; Gertz, Caitlyn C.; Tuck, Samuel J.; Regan, Tara M.; Naim, Youssef; Vincent, Andrea M.; Corey, Joseph M.

    2011-01-01

    Electrospinning is a technique for producing micro- to nano-scale fibers. Fibers can be electrospun with varying degrees of alignment, from highly aligned to completely random. In addition, fibers can be spun from a variety of materials, including biodegradable polymers such as poly-L-lactic acid (PLLA). These characteristics make electrospun fibers suitable for a variety of scaffolding applications in tissue engineering. Our focus is on the use of aligned electrospun fibers for nerve regeneration. We have previously shown that aligned electrospun PLLA fibers direct the outgrowth of both primary sensory and motor neurons in vitro. We maintain that the use of a primary cell culture system is essential when evaluating biomaterials to model real neurons found in vivo as closely as possible. Here, we describe techniques used in our laboratory to electrospin fibrous scaffolds and culture dorsal root ganglia explants, as well as dissociated sensory and motor neurons, on electrospun scaffolds. However, the electrospinning and/or culture techniques presented here are easily adapted for use in other applications. PMID:21372783

  3. 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

  4. 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

  5. Induction of gamma-globin gene transcription by hydroxycarbamide in primary erythroid cell cultures from Lepore patients.

    PubMed

    Calzolari, Roberta; Pecoraro, Alice; Borruso, Vito; Troia, Antonio; Acuto, Santina; Maggio, Aurelio; Di Marzo, Rosalba

    2008-05-01

    Increased expression of fetal haemoglobin (HbF) may ameliorate the clinical course of beta-thalassemia and sickle cell disease. Some pharmacological agents, such as hydroxycarbamide (HC), can increase fetal haemoglobin synthesis during adult life. Cellular selection and/or molecular mechanisms have been proposed to account for this increase. To explore the mechanism of action of HC we focused on homozygous Hb-Lepore patients that presented with high fetal haemoglobin levels and were good responders to HC treatment "in vivo". We performed primary erythroid cultures from peripheral blood of four homozygous Lepore patients. The increase in HBG (gamma-globin) transcription levels and HbF content in these cultures, after HC treatment, were detected by quantitative real time polymerase chain reaction analysis and flow cytometric analysis. Primary transcript "in-situ" hybridization analysis showed a 2-fold increase in the number of cells expressing both HBG alleles in HC-treated erythroid cultures. These studies, demonstrating the larger number of biallelic HBG expressing cells, suggest that HC is able to stimulate the activation of HBG transcription. These observations provide evidences that the molecular mechanism of action is involved in the increase of fetal haemoglobin production by HC. PMID:18422777

  6. 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

  7. 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

  8. Shibire mutations reveal distinct dynamin-independent and -dependent endocytic pathways in primary cultures of Drosophila hemocytes.

    PubMed

    Guha, A; Sriram, V; Krishnan, K S; Mayor, S

    2003-08-15

    We have developed a primary cell culture system derived from embryonic and larval stages of Drosophila. This allows for high-resolution imaging and genetic analyses of endocytic processes. Here, we have investigated endocytic pathways of three types of molecules: an endogenous receptor that binds anionic ligands (ALs), glycosylphosphatidylinositol (GPI)-anchored protein (GPI-AP), and markers of the fluid phase in primary hemocytes. We find that the endogenous AL-binding receptor (ALBR) is internalized into Rab5-positive endosomes, whereas the major portion of the fluid phase is taken up into Rab5-negative endosomes; GPI-APs are endocytosed into both classes of endosomes. ALBR and fluid-phase-containing early endosomes subsequently fuse to yield a population of Rab7-positive late endosomes. In primary culture, the endocytic phenotype of ALBR internalization in cells carrying mutations in Drosophila Dynamin (dDyn) at the shibire locus (shits) parallels the temperature-sensitive behavior of shits animals. At the restrictive temperature in shits cells, receptor-bound ALs remain completely surface accessible, localized to clathrin and alpha-adaptin-positive structures. On lowering the temperature, ALs are rapidly sequestered, suggesting a reversible block at a late step in dDyn-dependent endocytosis. By contrast, GPI-AP and fluid-phase endocytosis are quantitatively unaffected at the restrictive temperature in shits hemocytes, demonstrating a constitutive dDyn and Rab5-independent endocytic pathway in Drosophila. PMID:12857788

  9. COMMUNICATION: Folate and S-adenosylmethionine modulate synaptic activity in cultured cortical neurons: acute differential impact on normal and apolipoprotein-deficient mice

    NASA Astrophysics Data System (ADS)

    Serra, Michael; Chan, Amy; Dubey, Maya; Gilman, Vladimir; Shea, Thomas B.

    2008-12-01

    Folate deficiency is accompanied by a decline in the cognitive neurotransmitter acetylcholine and a decline in cognitive performance in mice lacking apolipoprotein E (ApoE-/- mice), a low-density lipoprotein that regulates aspects of lipid metabolism. One direct consequence of folate deficiency is a decline in S-adenosylmethionine (SAM). Since dietary SAM supplementation maintains acetylcholine levels and cognitive performance in the absence of folate, we examined herein the impact of folate and SAM on neuronal synaptic activity. Embryonic cortical neurons from mice expressing or lacking ApoE (ApoE+/+ or -/-, respectively) were cultured for 1 month on multi-electrode arrays, and signaling was recorded. ApoE+/+ cultures displayed significantly more frequent spontaneous signals than ApoE-/- cultures. Supplementation with 166 µm SAM (not normally present in culture medium) increased signal frequency and decreased signal amplitude in ApoE+/+ cultures. SAM also increased the frequency of tightly clustered signal bursts. Folate deprivation reversibly reduced signal frequency in ApoE+/+ cultures; SAM supplementation maintained signal frequency despite folate deprivation. These findings support the importance of dietary supplementation with folate and SAM on neuronal health. Supplementation with 166 µm SAM did not alter signaling in ApoE-/- cultures, which may be a reflection of the reduced SAM levels in ApoE-/- mice. The differential impact of SAM on ApoE+/+ and -/- neurons underscores the combined impact of nutritional and genetic deficiencies on neuronal homeostasis.

  10. Procedures for the reconstruction, primary culture and experimental use of rainbow trout gill epithelia.

    PubMed

    Schnell, Sabine; Stott, Lucy C; Hogstrand, Christer; Wood, Chris M; Kelly, Scott P; Pärt, Peter; Owen, Stewart F; Bury, Nic R

    2016-03-01

    This protocol describes how to reconstruct and culture the freshwater rainbow trout gill epithelium on flat permeable membrane supports within cell culture inserts. The protocol describes gill cell isolation, cultured gill epithelium formation, maintenance, monitoring and preparation for use in experimental procedures. To produce a heterogeneous gill epithelium, as seen in vivo, seeding of isolated gill cells twice over a 2-d period is required. As a consequence, this is termed the double-seeded insert technique. Approximately 5-12 d after cell isolation and seeding, preparations develop electrically tight gill epithelia that can withstand freshwater on the apical cell surface. The system can be used to study freshwater gill physiology, and it is a humane alternative for toxicity testing, bioaccumulation studies and environmental water quality monitoring. PMID:26866792

  11. 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. PMID:26854253

  12. 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

  13. Regulation of transgenes in three-dimensional cultures of primary mouse mammary cells demonstrates oncogene dependence and identifies cells that survive deinduction

    PubMed Central

    Jechlinger, Martin; Podsypanina, Katrina; Varmus, Harold

    2009-01-01

    The advent of targeted therapies for cancer has provoked interest in experimental models for the systematic study of oncogene dependence. To that end, we developed a three-dimensional (3D) culture system to analyze the responses of primary mouse mammary epithelial cells to the induction and deinduction of oncogenes. Mammary cells derived from normal virgin mice, or from tritransgenic mice (TetO-MYC;TetO-KrasG12D;MMTV-rtTA) in which MYC and mutant Kras can be regulated by doxycycline, develop from single cells into polarized acini. Lumen formation occurs without apparent apoptosis, and the hollow spheres of cells enlarge by division, with metaphase plates oriented perpendicularly to the apical surface. When MYC and KrasG12D are induced, the acini enlarge and form solid, depolarized spheres. Upon deinduction of MYC and KrasG12D the solid structures regress, leaving a repolarized monolayer of viable cells. These cells display a phenotype consistent with progenitors of mammary epithelium: They exclude Hoechst dye 33342, and reform acini in 3D cultures and repopulate mammary fat pads more efficiently than cells harvested from uninduced acini. Moreover, cells in the surviving spheres retain the ability to respond to reinduction and thus may represent the type of cells that give rise to recurrent tumors. PMID:19605689

  14. Ionizing Irradiation Not Only Inactivates Clonogenic Potential in Primary Normal Human Diploid Lens Epithelial Cells but Also Stimulates Cell Proliferation in a Subset of This Population

    PubMed Central

    Fujimichi, Yuki; Hamada, Nobuyuki

    2014-01-01

    Over the past century, ionizing radiation has been known to induce cataracts in the crystalline lens of the eye, but its mechanistic underpinnings remain incompletely understood. This study is the first to report the clonogenic survival of irradiated primary normal human lens epithelial cells and stimulation of its proliferation. Here we used two primary normal human cell strains: HLEC1 lens epithelial cells and WI-38 lung fibroblasts. Both strains were diploid, and a replicative lifespan was shorter in HLEC1 cells. The colony formation assay demonstrated that the clonogenic survival of both strains decreases similarly with increasing doses of X-rays. A difference in the survival between two strains was actually insignificant, although HLEC1 cells had the lower plating efficiency. This indicates that the same dose inactivates the same fraction of clonogenic cells in both strains. Intriguingly, irradiation enlarged the size of clonogenic colonies arising from HLEC1 cells in marked contrast to those from WI-38 cells. Such enhanced proliferation of clonogenic HLEC1 cells was significant at ≥2 Gy, and manifested as increments of ≤2.6 population doublings besides sham-irradiated controls. These results suggest that irradiation of HLEC1 cells not only inactivates clonogenic potential but also stimulates proliferation of surviving uniactivated clonogenic cells. Given that the lens is a closed system, the stimulated proliferation of lens epithelial cells may not be a homeostatic mechanism to compensate for their cell loss, but rather should be regarded as abnormal. This is because these findings are consistent with the early in vivo evidence documenting that irradiation induces excessive proliferation of rabbit lens epithelial cells and that suppression of lens epithelial cell divisions inhibits radiation cataractogenesis in frogs and rats. Thus, our in vitro model will be useful to evaluate the excessive proliferation of primary normal human lens epithelial cells that

  15. 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

  16. Gal4-gene-dependent alterations of embryo development and cell growth in primary culture of sea urchins.

    PubMed

    Bulgakov, V P; Odintsova, N A; Plotnikov, S V; Kiselev, K V; Zacharov, E V; Zhuravlev, Y N

    2002-10-01

    Primary cell cultures from sea urchins have a low proliferative level that prevents the establishment of long-term cultures. To increase expression levels of the genes regulating cell growth in sea urchins, and thus enhance cell growth, we used the transcriptional activator gene Gal4 found earlier in yeast. Sea urchin embryos were treated with plasmid DNA containing the Gal4 gene. Expression of the transgene was confirmed by reverse transcriptase polymerase chain reaction. When the fully functional gene was used, embryos effectively formed teratoma-like structures after 50 to 55 hours of cultivation. In contrast, the Gal4 gene, devoid of acidic activating regions, possessed little activity as a teratogen. The Gal4-treated cells in blastula-derived culture showed higher DNA synthesis and higher proliferative activity than control cells. We suggest that formation of the teratoma-like structures in embryos, activation of DNA synthesis, and significant increase of cell number in embryo-derived cell cultures could be attributed to Gal4 gene action. PMID:14961241

  17. A novel isolation method for macrophage-like cells from mixed primary cultures of adult rat liver cells.

    PubMed

    Kitani, Hiroshi; Takenouchi, Takato; Sato, Mitsuru; Yoshioka, Miyako; Yamanaka, Noriko

    2010-08-31

    We report a simple and efficient method to obtain macrophage-like cells from the mixed primary cultures of adult rat liver cells. A parenchymal hepatocyte enriched fraction was prepared from adult rat livers and seeded into culture flasks. After 7 to 10 days of culture, when most hepatocytes were degenerated or transformed into fibroblastic cells, macrophage-like cells vigorously proliferated on the cell sheet. By shaking the flasks, macrophage-like cells were readily detached. Subsequent transfer and incubation in plastic dishes resulted in quick and selective adhesion of macrophage-like cells, while other contaminating cells remained suspended in the medium. After rinsing with saline, attached macrophage-like cells were harvested with 95 to 99% purity, as evaluated by flow cytometry or immunocytochemistry. These cells showed typical macrophage morphology and were strongly positive for markers of rat macrophages, such as ED-1, ED-3, and OX-41, but negative for cytokeratins and alpha-smooth muscle actin. They possessed functional properties of typical macrophages, including active phagocytosis of latex beads, proliferative response to recombinant GM-CSF, secretion of inflammatory and anti-inflammatory cytokines upon stimulation with LPS, and formation of multinucleated giant cells. As more than 10(6) cells can be recovered repeatedly from a T75 culture flask at two to three day intervals for more than two weeks, our procedure might implicate a novel alternative to obtain Kupffer cells in sufficient number and purity without complex equipment and skills. PMID:20600081

  18. 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

  19. 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

  20. 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

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

    EPA Science Inventory

    Cell culture models are often used in mechanistic studies of toxicant action. However, one area of uncertainty is the extrapolation of dose from the in vitro model to the in vivo tissue. A common assumption of in vitro studies is that media concentration is a predictive marker of...

  2. 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…

  3. 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...

  4. US: A Cultural Mosaic. Teacher Handbook for a Primary-Grade Multidiscipline, Multicultural Program.

    ERIC Educational Resources Information Center

    Martinez, Jimmie; Watters, Arlene

    Activities and objectives for helping elementary school pupils develop a multiethnic and multicultural orientation toward American history and culture are presented in this teacher's guide. The major objective was to develop an interdisciplinary educational program which would influence young children in a positive way as they developed life-long…

  5. 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…

  6. 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

  7. 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...

  8. World Culture and Experienced Primary School Teachers' Understandings of Educational Changes in Taiwan

    ERIC Educational Resources Information Center

    Ke, I-Chung

    2008-01-01

    Studies by neo-institutionalists in sociology suggest that the institutions, organizations and governing rules in the environment are becoming more similar worldwide as the process of globalization continues. A so-called "world culture" based on science was hypothesized to explain the phenomena related to standardization and convergence. While…

  9. 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…

  10. Primary-Grade Students' Knowledge and Thinking about Food as a Cultural Universal.

    ERIC Educational Resources Information Center

    Brophy, Jere; Alleman, Janet; O'Mahony, Carolyn

    The traditional K-3 social studies curriculum has focused on food, clothing, shelter, communication, transportation, and other cultural universals, but 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…

  11. 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…

  12. Cytotoxicity of Shiga toxin for primary cultures of human colonic and ileal epithelial cells.

    PubMed Central

    Moyer, M P; Dixon, P S; Rothman, S W; Brown, J E

    1987-01-01

    Shiga toxin purified from Shigella dysenteriae 1 was cytotoxic to cultured epithelial cells from human colon and ileum. The cytotoxicity, which affected only about 50% of treated cells, was neutralized by rabbit antiserum monospecific for Shiga toxin and mediated by protein synthesis inhibition. PMID:3570477

  13. 5-HT7 Receptors Are Not Involved in Neuropeptide Release in Primary Cultured Rat Trigeminal Ganglion Neurons.

    PubMed

    Wang, Xiaojuan; Hu, Rong; Liang, Jianbo; Li, Ze; Sun, Weiwen; Pan, Xiaoping

    2016-06-01

    Migraine is a common but complex neurological disorder. Its precise mechanisms are not fully understood. Increasing indirect evidence indicates that 5-HT7 receptors may be involved; however, their role remains unknown. Our previous in vivo study showed that selective blockade of 5-HT7 receptors caused decreased serum levels of calcitonin gene-related peptide (CGRP) in the external jugular vein following electrical stimulation of the trigeminal ganglion (TG) in an animal model of migraine. In the present study, we used an in vitro model of cultured TG cells to further investigate whether 5-HT7 receptors are directly responsible for the release of CGRP and substance P from TG neurons. We stimulated rat primary cultured TG neurons with capsaicin or potassium chloride (KCl) to mimic neurogenic inflammation, resulting in release of CGRP and substance P. 5-HT7 receptors were abundantly expressed in TG neurons. Greater than 93 % of 5-HT7 receptor-positive neurons co-expressed CGRP and 56 % co-expressed substance P. Both the capsaicin- and KCl-induced release of CGRP and substance P were unaffected by pretreatment of cultured TG cells with the selective 5-HT7 receptor agonist AS19 and antagonist SB269970. This study demonstrates for the first time that 5-HT7 receptors are abundantly co-expressed with CGRP and substance P in rat primary TG neurons and suggests that they are not responsible for the release of CGRP and substance P from cultured TG neurons evoked by capsaicin or KCl. PMID:26892478

  14. 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]. PMID:26759825

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

    PubMed Central

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

    2015-01-01

    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]. PMID:26759825

  16. Tumor necrosis factor-α impairs adiponectin signalling, mitochondrial biogenesis, and myogenesis in primary human myotubes cultures.

    PubMed

    Sente, Tahnee; Van Berendoncks, An M; Fransen, Erik; Vrints, Christiaan J; Hoymans, Vicky Y

    2016-05-01

    Skeletal muscle metabolic changes are common in patients with chronic heart failure (HF). Previously, we demonstrated a functional skeletal muscle adiponectin resistance in HF patients with reduced left ventricular ejection fraction (HFrEF). We aimed to examine the impact of adiponectin receptor 1 (AdipoR1) deficiency and TNF-α treatment on adiponectin signaling, proliferative capacity, myogenic differentiation, and mitochondrial biogenesis in primary human skeletal muscle cells. Primary cultures of myoblasts and myotubes were initiated from the musculus vastus lateralis of 10 HFrEF patients (left ventricular ejection fraction; 31.30 ± 2.89%) and 10 age- and gender-matched healthy controls. Healthy control cultures were transfected with siAdipoR1 and/or exposed to TNF-α (10 ng/ml; 72 h). Primary cultures from HFrEF patients preserved the features of adiponectin resistance in vivo. AdipoR1 mRNA was negatively correlated with time to reach maximal cell index (r = -0.7319, P = 0.003). SiRNA-mediated AdipoR1 silencing reduced pAMPK (P < 0.01), AMPK activation (P = 0.046), and myoblast proliferation rate (xCELLigence Real-Time Cellular Analysis; P < 0.0001). Moreover, TNF-α decreased the mRNA expression of genes involved in glucose (APPL1, P = 0.0002; AMPK, P = 0.021), lipid (PPARα, P = 0.025; ACADM, P = 0.003), and mitochondrial (FOXO3, P = 0.018) metabolism, impaired myogenesis (MyoD1, P = 0.053; myogenin, P = 0.048) and polarized cytokine secretion toward a growth-promoting phenotype (IL-10, IL-1β, IFN-γ, P < 0.05 for all; Meso Scale Discovery Technology). Major features of adiponectin resistance are retained in primary cultures from the skeletal muscle of HFrEF patients. In addition, our results suggest that an increased inflammatory constitution contributes to adiponectin resistance and confers alterations in skeletal muscle differentiation, growth, and function. PMID:26921438

  17. 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

  18. 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.

  19. 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

  20. Dual PI3K/AKT/mTOR inhibitor BEZ235 synergistically enhances the activity of JAK2 inhibitor against cultured and primary human myeloproliferative neoplasm cells.

    PubMed

    Fiskus, Warren; Verstovsek, Srdan; Manshouri, Taghi; Smith, Jacqueline E; Peth, Karissa; Abhyankar, Sunil; McGuirk, Joseph; Bhalla, Kapil N

    2013-05-01

    Hemopoietic progenitor cells (HPC) from myeloproliferative neoplasms (MPN) such as myelofibrosis commonly express mutant JAK2-V617F or other mutations that are associated with increased activities of JAK-STAT5/3, RAS/RAF/MAPK, and PI3K/AKT/mTOR pathways. This confers proliferative and survival advantage on the MPN HPCs. Treatment with JAK tyrosine kinase inhibitor (TKI), for example, TG101209, TG101348 (SAR302503), or INCB018424 (ruxolitinib), inhibits mutant JAK2-mediated signaling. Although effective in reducing constitutional symptoms and splenomegaly, treatment with JAK-TKI does not ameliorate myelofibrosis or significantly improve survival of patients with advanced myelofibrosis. Here, we show that treatment with the dual phosphoinositide-3-kinase (PI3K)/AKT and mTOR inhibitor BEZ235 attenuated PI3K/AKT and mTOR signaling, as well as induced cell-cycle growth arrest and apoptosis of the cultured human JAK2-V617F-expressing HEL92.1.7 (HEL), UKE1 cells, and primary CD34+ myelofibrosis (MF)-MPN cells. Treatment with BEZ235 also induced significant apoptosis of the JAK2-TKI resistant HEL/TGR cells that were selected for resistance against JAK-TKI. Cotreatment with BEZ235 and JAK2-TKI (TG101209 and SAR302503) synergistically induced lethal activity against the cultured and primary CD34+ MPN cells while relatively sparing the normal CD34+ HPCs. These findings create a compelling rationale to determine the in vivo activity of dual PI3K/mTOR inhibitors in combination with JAK inhibitors against myelofibrosis HPCs. PMID:23445613

  1. Neuroendocrine aspects of primary endogenous depression. XV: Mathematical modeling of nocturnal melatonin secretion in major depressives and normal controls.

    PubMed

    Sekula, L K; Lucke, J F; Heist, E K; Czambel, R K; Rubin, R T

    1997-03-24

    We previously reported a trend toward a higher mean nocturnal serum melatonin (MEL) concentration, based on 30-min blood sampling over 24 h, in 23 female definite endogenous depressive compared to 23 matched normal female control subjects, and no significant difference in 15 male depressives compared to their controls (Rubin et al., 1992). In both groups of patients vs. their controls, there also were trends toward an earlier MEL rise time, by about 30 min, and a later MEL peak time, by about 90 min. Because the offset of MEL secretion was not estimated in that study, the total duration of MEL secretion could not be determined. To further delineate the nocturnal MEL secretion curve, we modeled the MEL data by a linear-Beta model, a four-parameter adaptation of the Beta function. One parameter accounted-for baseline (diurnal) MEL concentration, two accounted for the shapes of the ascending and descending phases of the nocturnal secretion curve, and the fourth accounted for the area under the curve. The model permitted estimation of the start, peak, and end times of nocturnal MEL secretion. There again was a trend toward a higher mean nocturnal MEL concentration in the female depressives compared to their matched controls. There were no significant patient-control differences in secretion onset or peak times in either the women or the men except for nocturnal MEL offset time: the female patients had a trend toward a later offset time, by about 40 min, than their controls; this difference was not present in the men. With women and men analyzed together, the difference in nocturnal MEL offset time between patients and controls just reached significance (P < 0.05). The linear-Beta model appears to satisfactorily fit the MEL data and provides estimators of the onset, peak, and offset times of the activation phase of MEL secretion. This model may be applicable to more severely skewed 24-h hormone secretion curves, such as ACTH and cortisol. PMID:9109182

  2. 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...

  3. 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…

  4. 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…

  5. The Influence of School Leadership Styles and Culture on Students' Achievement in Cyprus Primary Schools

    ERIC Educational Resources Information Center

    Kythreotis, Andreas; Pashiardis, Petros; Kyriakides, Leonidas

    2010-01-01

    Purpose: This study aims to examine the validation of both the model of direct effects and the model of indirect effects of principals' leadership on student academic achievement. Design/methodology/approach: A longitudinal study was conducted in which 22 schools, 55 classes and 1,224 Cypriot primary students participated. Specifically,…

  6. 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

    . Intercellular contact, communication, and transmission of contractile forces between myocytes appears to play a primary role in remodeling the 2-dimensional cell layer into a parallel alignment of elongated myocytes with highly developed intercalated disk-like junctions. This highly differentiated state is very stable, and cultures which achieve this state exhibit significantly greater longevity than more sparsely plated myocytes. These myocytes typically continue beating, and survive from 6 to more than 12 weeks in culture. When this level of contact and differentiation are not achieved, even among beta-adrenergic stimulated myocytes, contractile activity is not sustained, myofibrils atrophy, there is little or no development of junctional complexes, and the period of myocyte viability is typically no more than 5 weeks in vitro.

  7. Analysis of neuronal cells of dissociated primary culture on high-density CMOS electrode array.

    PubMed

    Matsuda, Eiko; Mita, Takeshi; Hubert, Julien; Bakkum, Douglas; Frey, Urs; Hierlemann, Andreas; Takahashi, Hirokazu; Ikegami, Takashi

    2013-01-01

    Spontaneous development of neuronal cells was recorded around 4-34 days in vitro (DIV) with high-density CMOS array, which enables detailed study of the spatio-temporal activity of neuronal culture. We used the CMOS array to characterize the evolution of the inter-spike interval (ISI) distribution from putative single neurons, and estimate the network structure based on transfer entropy analysis, where each node corresponds to a single neuron. We observed that the ISI distributions gradually obeyed the power law with maturation of the network. The amount of information transferred between neurons increased at the early stage of development, but decreased as the network matured. These results suggest that both ISI and transfer entropy were very useful for characterizing the dynamic development of cultured neural cells over a few weeks. PMID:24109870

  8. 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.

  9. 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

  10. Interactions of the neurotoxin apamin with a Ca2+-activated K+ channel in primary neuronal cultures.

    PubMed

    Seagar, M J; Granier, C; Couraud, F

    1984-02-10

    Mono[125I]iodoapamin bound to specific sites on cultured rat embryonic neurons. The dissociation constant for the receptor-neurotoxin complex measured at equilibrium was 60-120 pM at pH 7.2 and 4 degrees C, with a maximal binding capacity of 3-8 fmol/mg of cell protein. Apamin inhibited calcium ionophore-induced 86Rb+ release from cell cultures. The dose effect curve for this pharmacological test corresponded closely to the displacement of 125I-apamin by native apamin in binding experiments. Formation of the 125I-apamin receptor complex requires exogenous K+. Reduced binding in the absence of K+ was due to diminished binding capacity rather than a lower affinity. The apamin receptor seems to be associated with a cell surface K+ site which shows 50% occupancy at 1.6 mM, and which could be involved in the regulation of channel activity. Apamin sites were present at the earliest developmental stage tested and their number did not evolve during 8 days in culture. In the same period, however, alpha-scorpion toxin binding increased by a factor of 10. The ontogenesis of Ca2+-activated K+ channels does not seem to occur in parallel with that of voltage-sensitive Na+ channels. PMID:6319399

  11. 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.

  12. Key electrophysiological, molecular, and metabolic signatures of sleep and wakefulness revealed in primary cortical cultures.

    PubMed

    Hinard, Valérie; Mikhail, Cyril; Pradervand, Sylvain; Curie, Thomas; Houtkooper, Riekelt H; Auwerx, Johan; Franken, Paul; Tafti, Mehdi

    2012-09-01

    Although sleep is defined as a behavioral state, at the cortical level sleep has local and use-dependent features suggesting that it is a property of neuronal assemblies requiring sleep in function of the activation experienced during prior wakefulness. Here we show that mature cortical cultured neurons display a default state characterized by synchronized burst-pause firing activity reminiscent of sleep. This default sleep-like state can be changed to transient tonic firing reminiscent of wakefulness when cultures are stimulated with a mixture of waking neurotransmitters and spontaneously returns to sleep-like state. In addition to electrophysiological similarities, the transcriptome of stimulated cultures strikingly resembles the cortical transcriptome of sleep-deprived mice, and plastic changes as reflected by AMPA receptors phosphorylation are also similar. We used our in vitro model and sleep-deprived animals to map the metabolic pathways activated by waking. Only a few metabolic pathways were identified, including glycolysis, aminoacid, and lipids. Unexpectedly large increases in lysolipids were found both in vivo after sleep deprivation and in vitro after stimulation, strongly suggesting that sleep might play a major role in reestablishing the neuronal membrane homeostasis. With our in vitro model, the cellular and molecular consequences of sleep and wakefulness can now be investigated in a dish. PMID:22956841

  13. 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...

  14. 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 600µg/ml and vitamin E at 2500µmol/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 121.7ppm 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

  15. Cell structure and proliferative activity of organ cultures of normal embryonic lung tissue of mice resistant (C57BL) and predisposed (A) to lung tumors

    SciTech Connect

    Kolesnichenko, T.S.; Gor'kova, T.G.

    1985-08-01

    Local factors such as proliferative activity and the numerical ratio between epithelial and mesenchymal cells, and also the character of interaction between the tissue components in ontogeny may play an important role in the realization of sensitivity of mice of a particular line to the development of lung tumors. These characteristics of lung tissue in mice of lines A and C57BL are investigated under normal conditions and during induced carcinogenesis. Results are given of a comparative study of the relative numbers of epithelial and mesenchymal cells in organ cultures of embryonic lungs. /sup 3/H-thymidine was added to the cultures on the 14th day of the experiment in a concentration of 1 microCi/m1 medium. An autoradiographic study of the cultures was performed.

  16. The mitogenic effect of KGF and the expression of its cell surface receptor on cultured normal and malignant human oral keratinocytes and on contiguous fibroblasts.

    PubMed

    Drugan, C S; Stone, A; Game, S M; Prime, S S

    1997-08-01

    This study examined the mitogenic response to keratinocyte growth factor (KGF) of normal and tumour-derived human oral keratinocytes in which the degree of cellular differentiation was known and in contiguous fibroblast cultures derived from the malignant epithelial cultures. Keratinocytes, but not fibroblasts, were stimulated by KGF, thereby demonstrating epithelial target cell specificity of the ligand. KGF-induced stimulation of the tumour-derived keratinocytes cultured in the absence of the 3T3 fibroblast support broadly correlated with the degree of cellular differentiation; well-differentiated keratinocytes were stimulated more by KGF than their less differentiated counterparts. Malignant oral keratinocytes expressed KGF cell surface receptors (KD 451-709 pM; receptors/cell 2306-13645), but KGF receptor mRNA did not correlate with either KGF-induced mitogenesis or the degree of epithelial cell differentiation. When the tumour-derived keratinocytes were cultured in the presence of 3T3 fibroblasts, the mitogenic response to KGF was comparable to normal epithelial cells. The results suggest that KGF-mediated growth stimulation may not be significant in providing a selective advantage for the growth of malignant keratinocytes. PMID:9250933

  17. 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.

  18. Lack of a correlation between micronucleus formation and radiosensitivity in established and primary cultures of human tumours.

    PubMed Central

    Villa, R.; Zaffaroni, N.; Gornati, D.; Costa, A.; Silvestrini, R.

    1994-01-01

    The radiation-induced genotoxic damage in three established cell lines and 15 primary cultures of human malignant melanoma and ovarian carcinoma showing different radiosensitivity was tested by the cytokinesis-block micronucleus assay. A dose-related increase in micronucleus frequency was observed in all the cell systems. The mean number of micronuclei per Gy of ionising radiation per binucleated cell was respectively 0.44 +/- 0.0075 and 0.43 +/- 0.04 for M14 and JR8 malignant melanoma cell lines and 0.19 +/- 0.013 for the A2780 ovarian cancer cell line. The number of micronuclei did not rank the cell lines in the same order of radiosensitivity as clonogenic cell survival, which showed a surviving fraction at 2 Gy of 0.38 +/- 0.02 for JR8, 0.34 +/- 0.05 for M14 and 0.22 +/- 0.007 for A2780. As regards primary tumour cultures, no correlation was observed between micronucleus induction and surviving fraction at 2 Gy. In conclusion, the discrepancy we observed between micronucleus formation and cell death raises doubts about the potential of the micronucleus assay as a preclinical means to predict radiosensitivity. Images Figure 1 PMID:7981062

  19. Effect of the PGD2-DP signaling pathway on primary cultured rat hippocampal neuron injury caused by aluminum overload

    PubMed Central

    Ma, Jie; Yang, Qunfang; Wei, Yuling; Yang, Yang; Ji, Chaonan; Hu, Xinyue; Mai, Shaoshan; Kuang, Shengnan; Tian, Xiaoyan; Luo, Ying; Liang, Guojuan; Yang, Junqing

    2016-01-01

    In the present study, the agonists and antagonists of DP receptor were used to examine whether the PGD2-DP signaling pathway affects neuronal function. Primary cultured hippocampal neuron was prepared and treated with aluminum maltolate (100 μM) to establish the neuronal damage model. PGD2 and cAMP content was detected by ELISA. L-PGDS and DPs mRNA and protein expression were measured by RT-PCR and Western blotting, respectively. The aluminium-load neuron was treated with the DP1 agonist BW245C, the DP1 antagonist BWA868C, the DP2 agonist DK-PGD2, and the DP2 antagonist CAY10471, respectively. Neuronal pathomorphology was observed using H-E staining. The cell viability and the lactate dehydrogenase leakage rates of neurons were measured with MTT and LDH kit, respectively. Ca2+ level was detected by Fluo-3/AM. In the model group, the MTT values obviously decreased; LDH leakage rates and PGD2 content increased significantly; L-PGDS, DP1 mRNA and protein expressions increased, and DP2 level decreased. BW245C reduced the Ca2+ fluorescence intensity and protected the neurons. DK-PGD2 increased the intensity of Ca2+ fluorescence, while CAY10471 had the opposite effect. In conclusion, contrary to the effect of DP2, the PGD2-DP1 signaling pathway protects against the primary cultured rat hippocampal neuronal injury caused by aluminum overload. PMID:27089935

  20. 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…

  1. The relevance of traditional medical cultures to modern primary health care.

    PubMed

    Young, A

    1983-01-01

    This paper attempts to identify forms of traditional medicine which have the greatest potential for advancing primary health care goals. It begins by differentiating traditional medical systems into types, according to the kinds of medical knowledge which they depend on for preventing, diagnosing, and treating sickness. Emphasis is given to the facts that some traditional systems concentrate on producing varieties of pathophysiological knowledge, while others focus on forms of etiological knowledge; and that some traditional systems accumulate the medical knowledge which they produce, while others diffuse and fragment it. These differences give clues to a medical tradition's abstract potential for achieving three distinct, and only sometimes linked, ends: curing disease, healing illness, and enhancing the productivity of official primary health care programs. To make these clues concrete, it is also necessary to know something about the different ways in which traditional medical beliefs and practices are embedded, together with modern (cosmopolitan) medicine, in actual patterns of resort. The remainder of the paper assesses the relevance, for advancing primary health care goals, of particular classes of traditional healers--e.g. herbalists, midwives, bonesetters--and technologies within different types of medical systems. Four possibilities are described; integration, complementarity, rivalry and intercalation. PMID:6623126

  2. Idiopathic primary hyperaldosteronism: normalization of plasma aldosterone after one month withdrawal of long-term therapy with aldosterone-receptor antagonist potassium canrenoate.

    PubMed

    Armanini, D; Scaroni, C; Mattarello, M J; Fiore, C; Albiger, N; Sartorato, P

    2005-03-01

    We have re-evaluated 15 patients with idiopathic primary aldosteronism one month after withdrawal of therapy with aldosterone-receptor antagonist potassium canrenoate. Therapy had lasted for 3 to 24 yr. Median blood pressure (BP) in the sitting position at the time of diagnosis was 160/100 (ranges 150-200/95-110 mmHg); while 1 month after withdrawal of therapy median BP was 145/90 (ranges 125-160/80-100 mmHg). One month after withdrawal, the ratio aldosterone (ng/dl)/plasma renin activity (ng/ml/h) in the upright position was increased only in 3 cases (median 18, range 6.1-125). We found a significant inverse correlation between the upright aldosterone/plasma renin activity (aldo/PRA) ratio, 1 month after withdrawal, and the number of years of therapy with potassium canrenoate. We conclude that long-term therapy with the aldosterone-receptor blocker, potassium canrenoate, can normalize the aldo/PRA ratio in many cases of idiopathic primary hyperaldosteronism after one-month withdrawal of the drug. These data are consistent with possible regression of idiopathic primary hyperaldosteronism after long-term therapy with potassium canrenoate, or in alternative to a persistent effect of potassium canrenoate, on aldosterone synthesis. PMID:15952408

  3. Monomeric adiponectin increases cell viability in porcine aortic endothelial cells cultured in normal and high glucose conditions: Data on kinases activation.

    PubMed

    Grossini, Elena; Farruggio, Serena; Qoqaiche, Fatima; Raina, Giulia; Camillo, Lara; Sigaudo, Lorenzo; Mary, David; Surico, Nicola; Surico, Daniela

    2016-09-01

    We found that monomeric adiponectin was able to increase cell viability in porcine aortic endothelial cells (PAE) cultured both in normal and high glucose condition. Moreover, in normal glucose condition monomeric adiponectin increased p38MAPK, Akt, ERK1/2 and eNOS phosphorylation in a dose- and time-dependent way. Also in high glucose condition monomeric adiponectin increased eNOS and above kinases phosphorylation with similar patterns but at lower extent. For interpretation of the data presented in this article, please see the research article "Monomeric adiponectin modulates nitric oxide release and calcium movements in porcine aortic endothelial cells in normal/high glucose conditions" (Grossini et al., in press) [1]. PMID:27583345

  4. Nitric oxide-induced cytotoxicity attenuation by thiopentone sodium but not pentobarbitone sodium in primary brain cultures

    PubMed Central

    Shibuta, Satoshi; Kosaka, Jun; Mashimo, Takashi; Fukuda, Yutaka; Yoshiya, Ikuto

    1998-01-01

    We describe the effects of barbiturates on the neurotoxicity induced by nitric oxide (NO) on foetal rat cultured cortical and hippocampal neurones. Cessation of cerebral blood flow leads to an initiation of a neurotoxic cascade including NO and peroxynitrite. Barbiturates are often used to protect neurones against cerebrovascular disorders clinically. However, its neuroprotective mechanism remains unclear.In the present experiment, we established a new in vitro model of brain injury mediated by NO with an NO-donor, 1-hydroxy-2-oxo-3-(3-aminopropyl)-3-isopropyl-1-triazene (NOC-5) on grid tissue culture wells. We also investigated the mechanisms of protection of CNS neurones from NO-induced neurotoxicity by thiopentone sodium, which contains a sulphhydryl group (SH-) in the medium, and pentobarbitone sodium, which does not contain SH-.Primary cultures of cortical and hippocampal neurones (prepared from 16-day gestational rat foetuses) were used after 13–14 days in culture. The cells were exposed to NOC-5 at the various concentrations for 24 h in the culture to evaluate a dose-dependent effect of NOC-5.To evaluate the role of the barbiturates, neurones were exposed to 4, 40 and 400 μM of thiopentone sodium or pentobarbitone sodium with or without 30 μM NOC-5. In addition, superoxide dismutase (SOD) at 1000 u ml−1 and 30 μM NOC-5 were co-administered for 24 h to evaluate the role of SOD.Exposure to NOC-5 induced neural cell death in a dose-dependent manner in both cortical and hippocampal cultured neurones. Approximately 90% of the cultured neurones were killed by 100 μM NOC-5.This NOC-5-induced neurotoxicity was significantly attenuated by high concentrations of thiopentone sodium (40 and 400 μM) as well as SOD, but not by pentobarbitone sodium. The survival rates of the cortical neurones and hippocampal neurones that were exposed to 30 μM NOC-5 were 11.2±4.2% and 37.2±3.0%, respectively, and in the presence of 400 μM thiopentone

  5. 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.

  6. Xenobiotic-metabolizing enzyme and transporter gene expression in primary cultures of human hepatocytes modulated by ToxCast chemicals.

    PubMed

    Rotroff, Daniel M; Beam, Andrew L; Dix, David J; Farmer, Adam; Freeman, Kimberly M; Houck, Keith A; Judson, Richard S; LeCluyse, Edward L; Martin, Matthew T; Reif, David M; Ferguson, Stephen S

    2010-02-01

    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 concentration- and time-response of the 320 ToxCast chemicals for changes in expression of genes regulated by nuclear receptors. Fourteen gene targets were monitored in quantitative nuclease protection assays: six representative cytochromes P-450, four hepatic transporters, three Phase II conjugating enzymes, and one endogenous metabolism gene involved in cholesterol synthesis. These gene targets are sentinels of five major signaling pathways: AhR, CAR, PXR, FXR, and PPARalpha. Besides gene expression, the relative potency and efficacy for these chemicals to modulate cellular health and enzymatic activity were assessed. Results demonstrated that the culture system was an effective model of chemical-induced responses by prototypical inducers such as phenobarbital and rifampicin. Gene expression results identified various ToxCast chemicals that were potent or efficacious inducers of one or more of the 14 genes, and by inference the 5 nuclear receptor signaling pathways. Significant relative risk associations with rodent in vivo chronic toxicity effects are reported for the five major receptor pathways. These gene expression data are being incorporated into the larger ToxCast predictive modeling effort. PMID:20574906

  7. 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.

  8. 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.

  9. Contribution of aquaporin 9 and multidrug resistance-associated protein 2 to differential sensitivity to arsenite between primary cultured chorion and amnion cells prepared from human fetal membranes

    SciTech Connect

    Yoshino, Yuta; Yuan, Bo; Kaise, Toshikazu; Takeichi, Makoto; Tanaka, Sachiko; Hirano, Toshihiko; Kroetz, Deanna L.; Toyoda, Hiroo

    2011-12-15

    Arsenic trioxide (arsenite, As{sup III}) has shown a remarkable clinical efficacy, whereas its side effects are still a serious concern. Therefore, it is critical to understand the effects of As{sup III} on human-derived normal cells for revealing the mechanisms underlying these side effects. We examined the effects of As{sup III} on primary cultured chorion (C) and amnion (A) cells prepared from human fetal membranes. A significant dose-dependent As{sup III}-mediated cytotoxicity was observed in the C-cells accompanied with an increase of lactate dehydrogenase (LDH) release. Higher concentrations of As{sup III} were required for the A-cells to show cytotoxicity and LDH release, suggesting that the C-cells were more sensitive to As{sup III} than the A-cells. The expression levels of aquaporin 9 (AQP9) were approximately 2 times higher in the C-cells than those in the A-cells. Both intracellular arsenic accumulation and its cytotoxicity in the C-cells were significantly abrogated by sorbitol, a competitive AQP9 inhibitor, in a dose-dependent manner. The protein expression levels of multidrug resistance-associated protein (MRP) 2 were downregulated by As{sup III} in the C-cells, but not in the A-cells. No significant differences in the expression levels of MRP1 were observed between C- and A-cells. The protein expression of P-glycoprotein (P-gp) was hardly detected in both cells, although a detectable amount of its mRNA was observed. Cyclosporine A, a broad-spectrum inhibitor for ABC transporters, and MK571, a MRP inhibitor, but not PGP-4008, a P-gp specific inhibitor, potently sensitized both cells to As{sup III}-mediated cytotoxicity. These results suggest that AQP9 and MRP2 are involved in controlling arsenic accumulation in these normal cells, which then contribute to differential sensitivity to As{sup III} cytotoxicity between these cells. -- Highlights: Black-Right-Pointing-Pointer Examination of effect of As{sup III} on primary cultured chorion (C) and amnion

  10. Toxicity Effect of Silver Nanoparticles on Mice Liver Primary Cell Culture and HepG2 Cell Line

    PubMed Central

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

    2014-01-01

    Nano-silver (AgNP) has biological properties which are significant for consumer products, food technology, textiles and medical applications (e.g. wound care products, implantable medical devices, in diagnosis, drug delivery, and imaging). For their antibacterial activity, silver nanoparticles are largely used in various commercially available products. Thus, the use of nano-silver is becoming more and more widespread in medicine. In this study we investigated the cytotoxic effects of AgNPs on liver primary cells of mice, as well as the human liver HepG2 cell. Cell viability was examined with MTT assay after HepG2 cells exposure to AgNPs at 1, 2, 3, 4, 5, 7.5, 10 ppm compared to mice primary liver cells at 1, 10, 50, 100, 150, 200, 400 ppm for 24h. AgNPs caused a concentration-dependent decrease of cell viability in both cells. IC50 value of 2.764 ppm (µg/mL) was calculated in HepG2 cell line and IC50 value of 121.7 ppm (µg/mL) was calculated in primary liver cells of mice. The results of this experiment indicated that silver nanoparticles had cytotoxic effects on HepG2 cell line and primary liver cells of mice. The results illustrated that nano-silver had 44 times stronger inhibitory effect on the growth of cancerous cells (HepG2 cell line) compared to the normal cells (primary liver cells of mice). which might further justify AgNPs as a cytotoxic agents and a potential anticancer candidate which needs further studies in this regard. PMID:24734076

  11. Pigment cell differentiation in sea urchin blastula-derived primary cell cultures.

    PubMed

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

    2014-07-01

    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

  12. 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.

  13. 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

  14. A sensitive method to assay the xanthine oxidase activity in primary cultures of cerebellar granule cells.

    PubMed

    Atlante, A; Valenti, D; Gagliardi, S; Passarella, S

    2000-11-01

    Since xanthine oxidase (XO, Xanthine:oxidoreductase, E.C.1.2.3.22) is a key enzyme in reactive oxygen specie formation which plays a major role in cell oxidative stress, the availability of a sensitive and simple assay useful to detect its activity in monolayer cell cultures is worthwhile. In order to achieve this, we developed a method in which the conversion of pterine into isoxanthopterin is monitored fluorimetrically. Temperature assay was 50 degrees C. The activity of XO was detected in cerebellar granule cells exposed to glutamate. Since XO is formed from protease-dependent xanthine dehydrogenase processing, its activity appearance was found to be prevented by the protease inhibitor, leupeptin, as well as the glutamate NMDA-receptor inhibitor, MK-801, and the Ca(++) complexing agent, EGTA. The reported novel protocol, at variance with a conventional method, is shown to be a simple, fast, sensitive and relatively cheap method to assay XO activity. In addition, the reported assay can be applied to any cell type in culture. PMID:11086257

  15. siRNA delivery into cultured primary human myoblasts--optimization of electroporation parameters and theoretical analysis.

    PubMed

    Lojk, Jasna; Mis, Katarina; Pirkmajer, Sergej; Pavlin, Mojca