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Sample records for cultured primary rat

  1. RNA synthesis in primary cultures of adult rat hepatocytes

    International Nuclear Information System (INIS)

    Fugassa, E.; Gallo, G.; Voci, A.; Cordone, A.

    1983-01-01

    The ability of hepatocyte monolayers to synthesize RNA was investigated by measuring [3H]orotic acid incorporation into RNA and the total nuclear RNA polymerase activity as a function of the time in culture. The results demonstrate that primary cultures of hepatocytes maintained in a chemically defined serum- and hormone-free medium are able to synthesize RNA actively. This ability increases within the first 2 d of culture, despite the concomitant decrease in [3H]orotic acid uptake, and decreases only after 3 d. Factors such as serum, insulin, and dexamethasone, known to improve maintenance of functional hepatocytes, markedly stimulate the uptake of labeled precursor without apparently affecting the rate of RNA synthesis by cultured cells. It is suggested that the culture of adult rat hepatocytes provides a useful experimental model for the studies of hormonal regulation of transcription in liver

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

    Science.gov (United States)

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

    2008-02-01

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

  3. A modified technique for culturing primary fetal rat cortical neurons.

    Science.gov (United States)

    Xu, Sui-Yi; Wu, Yong-Min; Ji, Zhong; Gao, Xiao-Ya; Pan, Su-Yue

    2012-01-01

    The study explored a modified primary culture system for fetal rat cortical neurons. Day E18 embryos from pregnant Sprague Dawley rats were microdissected under a stereoscope. To minimize enzymatic damage to the cultured neurons, we applied a sequential digestion protocol using papain and Dnase I. The resulting sifted cell suspension was seeded at a density of 50,000 cells per cm(2) onto 0.1 mg/mL L-PLL-covered vessels. After a four-hour incubation in high-glucose Dulbecco's Modified Eagle's Medium (HG-DMEM) to allow the neurons to adhere, the media was changed to neurobasal medium that was refreshed by changing half of the volume after three days followed by a complete medium change every week. The cells displayed progressively robust neurite extension, and nonneuronal-like cells could barely be detected by five days in vitro (DIV); cell growth was still substantial at 14 DIV. Neurons were identified by β-tubulin III immunofluorescence, and neuronal purity within the cultures was assessed at over 95% by both flow cytometry and by dark-field counting of β-tubulin III-positive cells. These results suggest that the protocol was successful and that the high purity of neurons in this system could be used as the basis for generating various cell models of neurological disease.

  4. A Modified Technique for Culturing Primary Fetal Rat Cortical Neurons

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    Sui-Yi Xu

    2012-01-01

    Full Text Available The study explored a modified primary culture system for fetal rat cortical neurons. Day E18 embryos from pregnant Sprague Dawley rats were microdissected under a stereoscope. To minimize enzymatic damage to the cultured neurons, we applied a sequential digestion protocol using papain and Dnase I. The resulting sifted cell suspension was seeded at a density of 50,000 cells per cm2 onto 0.1 mg/mL L-PLL-covered vessels. After a four-hour incubation in high-glucose Dulbecco’s Modified Eagle’s Medium (HG-DMEM to allow the neurons to adhere, the media was changed to neurobasal medium that was refreshed by changing half of the volume after three days followed by a complete medium change every week. The cells displayed progressively robust neurite extension, and nonneuronal-like cells could barely be detected by five days in vitro (DIV; cell growth was still substantial at 14 DIV. Neurons were identified by β-tubulin III immunofluorescence, and neuronal purity within the cultures was assessed at over 95% by both flow cytometry and by dark-field counting of β-tubulin III-positive cells. These results suggest that the protocol was successful and that the high purity of neurons in this system could be used as the basis for generating various cell models of neurological disease.

  5. Ethanol-induced swelling in neonatal rat primary astrocyte cultures.

    Science.gov (United States)

    Aschner, M; Allen, J W; Mutkus, L A; Cao, C

    2001-05-11

    We tested the hypothesis that astrocytes swell in response to ethanol (EtOH) exposure. The experimental approach consisted of an electrical impedance method designed to measure cell volume. In chronic experiments, EtOH (100 mM) was added to the culture media for 1, 3, or 7 days. The cells were subsequently exposed for 15 min to isotonic buffer (122 mM NaCl) also containing 100 mM EtOH. Subsequently, the cells were washed and exposed to hypotonic buffer (112 mM NaCl) containing 100 mM mannitol. Chronic exposure to EtOH led to a marked increase in cell volume compared with control cells. Specific anion cotransport blockers, such as SITS, DIDS, furosemide, or bumetanide, when simultaneously added with EtOH to hyponatremic buffer, failed to reverse the EtOH-induced effect on swelling. In acute experiments, confluent neonatal rat primary astrocyte cultures were exposed to isotonic media (122 mM NaCl) for 15 min, followed by 45-min exposure to hypotonic media (112 mM NaCl, mimicking in vivo hyponatremic conditions associated with EtOH withdrawal) in the presence of 0-100 mM EtOH. This exposure led to a concentration-dependent increase in cell volume. Combined, these studies suggest that astrocytes exposed to EtOH accumulate compensatory organic solutes to maintain cell volume, and that in response to hyponatremia and EtOH withdrawal their volume increases to a greater extent than in cells exposed to hyponatremia alone. Furthermore, the changes associated with EtOH are osmotic in nature, and they are not reversed by anion cotransport blockers.

  6. Generation of primary cultures of bovine brain endothelial cells and setup of cocultures with rat astrocytes

    DEFF Research Database (Denmark)

    Helms, Hans C; Brodin, Birger

    2014-01-01

    -brain barrier. The present protocol describes the setup of an in vitro coculture model based on primary cultures of endothelial cells from bovine brain microvessels and primary cultures of rat astrocytes. The model displays a high electrical tightness and expresses blood-brain barrier marker proteins....

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

    International Nuclear Information System (INIS)

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

    2009-01-01

    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.

  8. Adenosine formation in contracting primary rat skeletal muscle cells and endothelial cells in culture

    DEFF Research Database (Denmark)

    Hellsten, Ylva; Frandsen, Ulrik

    1997-01-01

    1. The present study examined the capacity for adenosine formation, uptake and metabolism in contracting primary rat muscle cells and in microvascular endothelial cells in culture. 2. Strong and moderate electrical simulation of skeletal muscle cells led to a significantly greater increase...

  9. Chronic 14-day exposure to insecticides or methylmercury modulates neuronal activity in primary rat cortical cultures

    NARCIS (Netherlands)

    Dingemans, Milou; Schütte, Marijke G; Wiersma, Daphne M M; de Groot, Aart; van Kleef, Gina; Wijnolts, Fiona; Westerink, Remco

    2016-01-01

    There is an increasing demand for in vitro test systems to detect neurotoxicity for use in chemical risk assessment. In this study, we evaluated the applicability of rat primary cortical cultures grown on multi-well micro-electrode arrays (mwMEAs) to detect effects of chronic 14-day exposure to

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

    International Nuclear Information System (INIS)

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

    1987-01-01

    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 125 I-Ang-II to glial cultures was rapid, reversible, saturable, and specific for Ang-II. The rank order of potency of 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 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 [ 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

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

    International Nuclear Information System (INIS)

    Cervos-Navarro, J.; Hamdorf, G.

    1993-01-01

    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 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 trademark2350 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

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

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    Masoumeh Emamghoreishi

    2015-02-01

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

  13. Differential feedback regulation of cholesterol 7α-hydroxylase mRNA and transcriptional activity by rat bile acids in primary monolayer cultures of rat hepatocytes

    NARCIS (Netherlands)

    Twisk, J.; Lehmann, E.M.; Princen, H.M.G.

    1993-01-01

    We have used primary monolayer cultures of rat hepatocytes to study the effects of physiological concentrations of various bile acids, commonly found in bile of normal rats, on the mechanism of regulation of cholesterol 7α-hydroxylase and bile acid synthesis. Addition of taurocholic acid, the most

  14. Culturing of primary rat neurons and glia on ultra-thin parylene-C

    International Nuclear Information System (INIS)

    Unsworth, C.P.; Delivopoulos, E.; Murray, A.F.

    2010-01-01

    Full text: In this article, we will describe how we have successfully cultured dissociated embryonic cortical neurons and glia from the postnatal rat hippocampus on extremely thin layers (up to 10 nm) of Parylene-C on a silicon dioxide substrate. Silicon wafers were oxidised, deposited with the biomaterial, Parylene-C, photo-lithographically patterned and plasma etched to produce chips that consisted of lines of Paryl ene-C with varying widths, thickness and lengths. The chips produced were then immersed in Horse Serum and plated with the cells. Ratios of Neurons; Glia; Cell Body were measured on, adjacent to and away from the Parylene-C. Our initial results show how these ratios remained roughly constant for ultra-thin Parylene-C thicknesses of 10 nm as compared to a benchmark thickness of 100 nm (where such cells are known to grow well). Thus, our findings demonstrate that it is possible to culture primary rat neurons and glia to practically cell membrane thicknesses of Parylene-C. Being able to culture cells on such ultra thin levels of Parylene-C will open up the possibility to develop Multi-Electrode Arrays (MEA) that can capacitively couple embedded electrodes through the parylene to the cells on its surface. Thus, providing a neat, insulated passive electrode. Only the ultra-thin thicknesses of Parylene demonstrated here would allow for the rea isation of such a technology. Hence, the outcome of this work, will be of great interest to the Neuroengineering and the Multi-Electrode Array (MEA) community, as an alternative material for the fabric tion of passive electrodes, used in capacitive coupling mode.

  15. Primary Culture of Choroid Plexuses from Neonate Rats Containing Progenitor Cells Capable of Differentiation

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    Sheng-Li Huang

    2013-12-01

    Full Text Available Background: The choroid plexuses, which could secrete a number of neurotrophins, have recently been used in transplantation in central nervous system diseases. Aims: To study the mechanism of nerve regeneration in the central nervous system by grafting choroid plexus tissues. Study Design: Animal experimentation. Methods: The choroid plexuses from the lateral ventricles of neonatal rats were cultured in adherent culture, and immunocytochemical methods were used to analyse the progenitor cells on days 2, 6, and 10 after seeding. Results: Expression of both nestin and glial fibrillary acidic protein was observed in small cell aggregates on day 2 in primary culture. Most of the nestin-positive cells on day 6 were immunoreactive to glial fibrillary acidic protein antibody. No cells expressing nestin or glial fibrillary acidic protein were seen on day 10. Conclusion: These experimental results indicate that the choroid plexus contains a specific cell population – progenitor cells. Under in vitro experimental conditions, the progenitor cells differentiated into choroid plexus epithelial cells but did not form neurons or astrocytes.

  16. Role of carnitine palmitoyltransferase I in the control of ketogenesis in primary cultures of rat astrocytes.

    Science.gov (United States)

    Blázquez, C; Sánchez, C; Velasco, G; Guzmán, M

    1998-10-01

    The role of carnitine palmitoyltransferase I (CPT-I) in the control of ketogenesis was studied in primary cultures of rat astrocytes. Ketone bodies were the major product of [14C]palmitate oxidation by cultured astrocytes, whereas CO2 made a minor contribution to the total oxidation products. Using tetradecylglycidate as a specific, cell-permeable inhibitor of CPT-I, a flux control coefficient of 0.77 +/- 0.07 was calculated for CPT-I over the flux of [14C]palmitate to ketone bodies. CPT-I from astrocytes was sensitive to malonyl-CoA (IC50 = 3.4 +/- 0.8 microM) and cross-reacted on western blots with an antibody raised against liver CPT-I. On the other hand, astrocytes expressed significant acetyl-CoA carboxylase (ACC) activity, and consequently they contained considerable amounts of malonyl-CoA. Western blot analysis of ACC isoforms showed that ACC in astrocytes--like in neurons, liver, and white adipose tissue--mostly comprised the 265-kDa isoform, whereas the 280-kDa isoform--which was highly expressed in skeletal muscle--showed much lower abundance. Forskolin was used as a tool to study the modulation of the ketogenic pathway in astrocytes. Thus, forskolin decreased in parallel ACC activity and intracellular malonyl-CoA levels, whereas it stimulated CPT-I activity and [14C]palmitate oxidation to both ketone bodies and CO2. Results show that in cultured astrocytes (a) CPT-I exerts a very high degree of control over ketogenesis from palmitate, (b) the ACC/malonyl-CoA/CPT-I system is similar to that of liver, and (c) the ACC/malonyl-CoA/CPT-I system is subject to regulation by cyclic AMP.

  17. Anandamide, Acting via CB2 Receptors, Alleviates LPS-Induced Neuroinflammation in Rat Primary Microglial Cultures

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    Natalia Malek

    2015-01-01

    Full Text Available Microglial activation is a polarized process divided into potentially neuroprotective phenotype M2 and neurotoxic phenotype M1, predominant during chronic neuroinflammation. Endocannabinoid system provides an attractive target to control the balance between microglial phenotypes. Anandamide as an immune modulator in the central nervous system acts via not only cannabinoid receptors (CB1 and CB2 but also other targets (e.g., GPR18/GPR55. We studied the effect of anandamide on lipopolysaccharide-induced changes in rat primary microglial cultures. Microglial activation was assessed based on nitric oxide (NO production. Analysis of mRNA was conducted for M1 and M2 phenotype markers possibly affected by the treatment. Our results showed that lipopolysaccharide-induced NO release in microglia was significantly attenuated, with concomitant downregulation of M1 phenotypic markers, after pretreatment with anandamide. This effect was not sensitive to CB1 or GPR18/GPR55 antagonism. Administration of CB2 antagonist partially abolished the effects of anandamide on microglia. Interestingly, administration of a GPR18/GPR55 antagonist by itself suppressed NO release. In summary, we showed that the endocannabinoid system plays a crucial role in the management of neuroinflammation by dampening the activation of an M1 phenotype. This effect was primarily controlled by the CB2 receptor, although functional cross talk with GPR18/GPR55 may occur.

  18. Hormonal regulation of rat foetal lipogenesis in brown-adipocyte primary cultures.

    OpenAIRE

    Lorenzo, M; Roncero, C; Fabregat, I; Benito, M

    1988-01-01

    Insulin stimulates lipogenesis by 100% for 5 h by a covalent modulation of acetyl-CoA carboxylase, and by 200% for 24 h by increasing malic enzyme and fatty acid synthase enzymic activities in brown-adipocyte primary cultures. At short times, noradrenaline and isoprenaline decrease lipogenesis. However, phenylephrine and glucagon have no effect. At long times, dexamethasone inhibits lipogenesis. This effect is precluded in the presence of insulin. Progesterone and tri-iodothyronine, alone or ...

  19. Receptor-mediated endocytosis and degradative processing of growth hormone by rat adipocytes in primary culture.

    Science.gov (United States)

    Roupas, P; Herington, A C

    1987-05-01

    At 37 degrees C, cultured rat adipocytes bound [125I]human GH ([125I]hGH) rapidly, with binding being detectable within 1 min of incubation. The bound [125I]hGH was then internalized (within 10 min) and accumulated in the cell interior until a steady state was reached (by 60 min). At this time, where the rates of GH internalization, processing, and release are equivalent, 55% of total cell-associated [125I]hGH was intracellular. Internalization of [125I]hGH by acutely isolated (noncultured) adipocytes was preceded by a 20-min lag phase indicative of a temporary postbinding defect. The lag phase was not seen with cultured adipocytes. After preloading of [125I]hGH into the cell interior, cultured cells rapidly released [125I]hGH (t1/2 = 20-30 min) into the extracellular medium as both intact (25%) and degraded (75%) GH. The release of intact vs. degraded GH was distinguishable on the basis of kinetics and temperature dependence. In order to determine when internalized [125I]hGH entered a catabolic compartment, cultured adipocytes were incubated with [125I]hGH and the composition of intracellular GH was determined as a function of time. All [125I]hGH internalized during the first 20 min was intact. Between 20 and 30 min some of the internalized [125I]hGH entered a catabolic compartment and degradation products began accumulating within the adipocytes. Release of degraded [125I]hGH from cultured adipocytes began at 60 min. The processing of GH through the complete degradative pathway (binding, internalization, degradation, release) required a period of 1 h at 37 degrees C.

  20. Hepatoprotective effects of Poly-[hemoglobin-superoxide dismutase-catalase-carbonic anhydrase] on alcohol-damaged primary rat hepatocyte culture in vitro.

    Science.gov (United States)

    Jiang, Wenhua; Bian, Yuzhu; Wang, Zhenghui; Chang, Thomas Ming Swi

    2017-02-01

    We have prepared a novel nanobiotherapeutic, Poly-[hemoglobin-superoxide dismutase-catalase-carbonic anhydrase], which not only transports both oxygen and carbon dioxide but also a therapeutic antioxidant. Our previous study in a severe sustained 90 min hemorrhagic shock rat model shows that it has a hepatoprotective effect. We investigate its hepatoprotective effect further in this present report using an alcohol-damaged primary hepatocyte culture model. Results show that it significantly reduced ethanol-induced AST release, lipid peroxidation, and ROS production in rat primary hepatocytes culture. It also significantly enhanced the viability of ethanol-treated hepatocytes. Thus, the result shows that Poly-[hemoglobin-superoxide dismutase-catalase-carbonic anhydrase] also has some hepatoprotective effects against alcohol-induced injury in in vitro rat primary hepatocytes cell culture. This collaborate our previous observation of its hepatoprotective effect in a severe sustained 90-min hemorrhagic shock rat model.

  1. Modeling chronic brain exposure to amphetamines using primary rat neuronal cortical cultures.

    Science.gov (United States)

    Nogueira, T B; da Costa Araújo, S; Carvalho, F; Pereira, F C; Fernandes, E; Bastos, M L; Costa, V M; Capela, J P

    2014-09-26

    Amphetamine-type psychostimulants (ATS) are used worldwide by millions of patients for several psychiatric disorders. Amphetamine (AMPH) and "ecstasy" (3,4-methylenedioxymethamphetamine or MDMA) are common drugs of abuse. The impact of chronic ATS exposure to neurons and brain aging is still undisclosed. Current neuronal culture paradigms are designed to access acute ATS toxicity. We report for the first time a model of chronic exposure to AMPH and MDMA using long-term rat cortical cultures. In two paradigms, ATS were applied to neurons at day 1 in vitro (DIV) (0, 1, 10 and 100 μM of each drug) up to 28 days (200 μM was applied to cultures up to 14 DIV). Our reincubation protocol assured no decrease in the neuronal media's drug concentration. Chronic exposure of neurons to concentrations equal to or above 100 μM of ATS up to 28 DIV promoted significant mitochondrial dysfunction and elicited neuronal death, which was not prevented by glutamate receptor antagonists at 14 DIV. ATS failed to promote accelerated senescence as no increase in β-galactosidase activity at 21 DIV was found. In younger cultures (4 or 8 DIV), AMPH promoted mitochondrial dysfunction and neuronal death earlier than MDMA. Overall, AMPH proved more toxic and was the only drug that decreased intraneuronal glutathione levels. Meanwhile, caspase 3 activity increased for either drug at 200 μM in younger cultures at 8 DIV, but not at 14 DIV. At 8 DIV, ATS promoted a significant change in the percentage of neurons and astroglia present in culture, promoting a global decrease in the number of both cells. Importantly, concentrations equal to or below 10 μM of either drug did not promote neuronal death or oxidative stress. Our paradigm of neuronal cultures long-term exposure to low micromolar concentrations of ATS closely reproduces the in vivo scenario, being valuable to study the chronic impact of ATS. Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.

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

    International Nuclear Information System (INIS)

    Zurlo, J.; Yager, J.D.

    1984-01-01

    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. In this paper, we report the development of a primary rat hepatocyte culture system to investigate cellular responses to DNA damage which may be relevant to hepatocarcinogenesis in vivo. We have obtained data demonstrating that enhanced reactivation of u.v.-irradiated Herpes simplex virus type 1 (HSV-1) occurs 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 by direct plaque assay. 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 that may have a role in the initiation of hepatocarcinogenesis

  3. Wheat extracts as an efficient cryoprotective agent for primary cultures of rat hepatocytes.

    Science.gov (United States)

    Hamel, Francine; Grondin, Mélanie; Denizeau, Francine; Averill-Bates, Diana A; Sarhan, Fathey

    2006-11-05

    Hepatocytes are an important physiological model for evaluation of metabolic and biological effects of xenobiotics. They do not proliferate in culture and are extremely sensitive to damage during freezing and thawing, even after the addition of classical cryoprotectants. Thus improved cryopreservation techniques are needed to reduce cell injury and functional impairment. Here, we describe a new and efficient cryopreservation method, which permits long-term storage and recovery of large quantities of healthy cells that maintain high hepatospecific functions. In culture, the morphology of hepatocytes cryopreserved with wheat protein extracts (WPE) was similar to that of fresh cells. Furthermore, hepatospecific functions such as albumin secretion and biotransformation of ammonium to urea were well maintained during 4 days in culture. Inductions of CYP1A1 and CYP2B in hepatocytes cryopreserved with WPEs were similar to those in fresh hepatocytes. These findings clearly show that WPEs are an excellent cryopreservant for primary hepatocytes. The extract was also found to cryopreserve other human and animal cell types such as lung carcinoma, colorectal adenocarcinoma, Chinese hamster ovary transfected with TGF-b1 cDNA, cervical cancer taken from Henrietta Lacks, intestinal epithelium, and T cell leukemia. WPEs have potential as a universal cryopreservant agent of mammalian cells. It is an economic, efficient and non-toxic agent. (c) 2006 Wiley Periodicals, Inc.

  4. Are cerebral prostanoids of astroglial origin? Studies on the prostanoid forming system in developing rat brain and primary cultures of rat astrocytes.

    Science.gov (United States)

    Seregi, A; Keller, M; Hertting, G

    1987-02-24

    Prostanoid forming capacity in vitro and convulsion-induced prostanoid formation in vivo were studied in the developing rat brain. For comparison, prostanoid synthesis in homogenates of primary astrocyte cultures of different ages was also examined. There was no significant prostanoid production in homogenates from primary astrocyte cultures prepared one week after cultivation. Two-week-old astrocyte cultures possessed a prostanoid synthesizing system of high specific activity. The relative proportions of the products were similar to those obtained in brain homogenates of adult rats, prostaglandin D2 (PGD2) being the major product. Prostanoid forming capacity of brain homogenates was low at birth, increased during development and nearly reached adult values by day 21. Generalized convulsions could be evoked by pentylenetetrazol (PTZ) irrespective of age, but convulsion-induced prostanoid formation characteristic of adult rodents did not take place before the third week of postnatal life. The close similarities between the characteristic features of prostanoid synthesis in both brain and astroglial homogenates, together with the coincidence during brain development of the expression of cerebral prostanoid synthesis with the appearance of mature astrocytes suggest that astrocytes are an important source of brain prostanoids.

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

    Energy Technology Data Exchange (ETDEWEB)

    Sarkar, D.K.; Minami, S. (Washington State Univ., Pullman (USA))

    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.

  6. Characterisation of an in vitro blood-brain barrier model based on primary porcine capillary endothelial cells in monoculture or co-culture with primary rat or porcine astrocytes and pericytes

    DEFF Research Database (Denmark)

    Thomsen, Louiza Bohn; Larsen, Annette Burkhart; Moos, Torben

    to in vivo such as efflux transporters, tight junction proteins, and high transendothelial electric resistance (TEER). Primary BCECs are isolated from a variety of mammals such as rats, mice, cattle and pigs. Often bovine and porcine BCECs are cultured in monoculture or in co-culture with rat astrocytes......In vitro blood-brain barrier (BBB) models based on primary brain capillary endothelial cells (BCECs) in monoculture or in co-culture with primary astrocytes and pericytes are often applied for studying physiology of the BBB. Primary BCECs retain many morphological and biochemical properties similar...... obtained from neonatal rats which have been shown to strengthen the barrier properties of the BCECs. In this study, brain endothelial cells (PBECs), astrocytes and pericytes are isolated from pig brains donated by the local abattoir. The brains are from 6 month old domestic pigs. The availability and high...

  7. Oxidative inactivation of mitochondrial aconitase results in iron and H2O2-mediated neurotoxicity in rat primary mesencephalic cultures.

    Directory of Open Access Journals (Sweden)

    David Cantu

    2009-09-01

    Full Text Available Mitochondrial oxidative stress is a contributing factor in the etiology of numerous neuronal disorders. However, the precise mechanism(s by which mitochondrial reactive oxygen species (ROS modify cellular targets to induce the death of neurons remains unknown. The goal of this study was to determine if oxidative inactivation of mitochondrial aconitase (m-aconitase resulted in the release of redox-active iron (Fe2+ and hydrogen peroxide (H2O2 and whether this contributes to cell death.Incubation of rat primary mesencephalic cultures with the redox cycling herbicide paraquat (PQ2+ resulted in increased production of H2O2 and Fe2+ at times preceding cell death. To confirm the role of m-aconitase as a source of Fenton reagents and death, we overexpressed m-aconitase using an adenoviral construct thereby increasing the target available for inactivation by ROS. Co-labeling studies identified astrocytes as the predominant cell type expressing transduced m-aconitase although neurons were identified as the primary cell type dying. Oxidative inactivation of m-aconitase overexpressing cultures resulted in exacerbation of H2O2 production, Fe2+ accumulation and increased neuronal death. Increased cell death in m-aconitase overexpressing cultures was attenuated by addition of catalase and/or a cell permeable iron chelator suggesting that neuronal death occurred in part via astrocyte-derived H2O2.These results suggest a role of ROS-sensitive m-aconitase as a source of Fe2+ and H2O2 and as a contributing factor to neurotoxicity.

  8. Pharmacological modification of endogenous antioxidant enzymes by ursolic acid on tetrachloride-induced liver damage in rats and primary cultures of rat hepatocytes.

    Science.gov (United States)

    Martin-Aragón, S; de las Heras, B; Sanchez-Reus, M I; Benedi, J

    2001-06-01

    The purpose of this study was to investigate possible protective effects of ursolic acid against CCl4-induced alterations of antioxidant defence enzymes in vivo as well as its effects against CCl4-intoxication in vitro. Pre-treatment of rats with ursolic acid significantly reduced serum levels of glutamate-oxalate-transaminase and glutamate-pyruvate-transaminase previously increased by administration of CCl4. Treatment with ursolic acid also significantly reversed the decreased superoxide dismutase, catalase, glutathione reductase, glutathione peroxidase activities and glutathione levels in the liver, as the concentration of reduced glutathione was increased and the content of oxidized glutathione decreased in ursolic acid treated groups. Levels of lipid peroxidation were higher in the CCl4 group but the increase was also reduced after drug treatment (p ursolic acid (p Ursolic acid also ameliorated lipid peroxidation in primary cultured rat hepatocytes exposed to CCl4, as demonstrated by a reduction in malondialdehyde production. Moreover, ursolic acid (50-500 microM) showed radical scavenging properties in terms of hydroxyl formation. The results obtained suggest that ursolic acid treatment can normalize the disturbed antioxidant status of rats intoxicated with CCl4 by maintaining the levels of glutathione and by inhibiting the production of malondialdehyde due to its radical scavenging properties.

  9. Sendai virosomal infusion of an adeno-associated virus-derived construct containing neuropeptide Y into primary rat brain cultures.

    Science.gov (United States)

    Wu, P; de Fiebre, C M; Millard, W J; Elmstrom, K; Gao, Y; Meyer, E M

    1995-05-05

    A novel neuronal gene-delivery system was investigated in primary neuron-enriched cultures with respect to driving the expression of neuropeptide Y (NPY). This delivery system consists of an adeno-associated virus-derived (AAV) plasmid, pJDT95npy, encapsulated in reconstituted Sendai virosomes. pJDT95npy contains full length rat NPY cDNA inserted downstream from the P40 promoter in a cap-gene deleted AAV-derived construct. The rep-sequences under control of the P5 and P19 promoters are intact. Virosomally encapsulated pJDT95npy drove the expression of NPY mRNAs, predominantly by P40. Total cellular NPY immunoreactivity and release in the presence of depolarization increased following pJDT95npy-transfection. Neither empty virosomes nor virosomes containing pJDT95 affected NPY mRNA expression or immunoreactivity. This study demonstrates that an AAV-derived plasmid can drive exogenous gene expression in intact neurons after infusion by Sendai virosomes.

  10. Processing of growth hormone by rat adipocytes in primary culture: differentiation between release of intact hormone and degradative processing.

    Science.gov (United States)

    Roupas, P; Herington, A C

    1987-10-01

    Rat adipocytes in primary culture have been used to study the intracellular processing of GH. These classic target cells for GH have been shown to process GH through two pathways: a nondegradative pathway which resulted in the rapid release of intact GH, and a slower, degradative pathway which involved the degradation of GH and release of degraded ligand. Differentiation between the two pathways was on the basis of differences in their kinetics and temperature dependence. The present study has investigated the relative characteristics of the two pathways further. Incubation of [125I]human GH ([125I]hGH)-preloaded adipocytes with extracellular unlabeled hGH (400 ng/ml) resulted in an increase in the absolute amount of [125I]hGH released. The increased amount of [125I]hGH released was all intact. Extracellular, unlabeled hGH had no effect on the rate or amount of degraded [125I]hGH released. This suggests that the nondegradative pathway is sensitive to the number of internalized hormone-receptor complexes and that GH which is not immediately degraded or stored in the degradative pathway, is redirected and processed via the faster non-degradative pathway. Ammonium chloride (known to inhibit the lysosomal degradation of many polypeptide hormones) markedly inhibited the absolute amount of [125I]hGH released from preloaded adipocytes. This inhibition was due to an effect on the release of degraded [125I]hGH. NH4Cl had no effect on the rate or amount of intact [125I]hGH released. Finally, it was found that dinitrophenol and sodium fluoride (agents known to deplete cellular energy) inhibited the release of degraded GH but not intact GH suggesting that the degradative pathway involves an energy-dependent step, most likely the fusion of hormone-containing vesicles with the lysosomal membrane. The mechanism of release of intact hormone by energy-independent means is not yet known. These data indicate that the processing of GH by cultured rat adipocytes is complex and involves

  11. Cadmium induces Ca2+ mediated, calpain-1/caspase-3-dependent apoptosis in primary cultured rat proximal tubular cells.

    Science.gov (United States)

    Wang, Hong; Zhai, Nianhui; Chen, Ying; Xu, Haibin; Huang, Kehe

    2017-07-01

    Calcium, as a ubiquitous second messenger, governs a large array of cellular processes and is necessary for cell survival. More recently, it was observed that the cytosolic Ca 2+ concentration ([Ca 2+ ] c ) elevation could induce apoptosis in primary cultured rat proximal tubular (rPT) cells exposed to cadmium (Cd), but the concrete mechanism is still unclear. This study was designed to investigate the signal pathway involved in [Ca 2+ ] c elevation-mediated apoptosis. The results confirmed the elevation of [Ca 2+ ] c by confocal microscopy and enhancement of the apoptosis by Hoechst 33258 staining and flow cytometer when rPT cells were exposed to Cd for 12h. Then we demonstrated that Cd enhanced the protein levels of active calpain-1 and caspase-3 in rPT cells. Pretreatment with a cytosolic Ca 2+ chelator, 1,2-Bis (2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM), markedly blocked the up-regulation of active calpain-1 and caspase-3 and inhibited the apoptosis induced by Cd. Further, rPT cells were pretreated with a cell-permeable selective calpain-1 inhibitor, 3-(4-iodophenyl)-2-mercapto-(Z)-2-propenoic acid (PD150606) and caspase-3 inhibitor, N-Acetyl-Asp-Glu-Val-Asp-CHO (Ac-DEVD-CHO), respectively. PD150606 significantly attenuated the up-regulation of active caspase-3 and the apoptosis induced by Cd. As expected, inhibition of active caspase-3 by Ac-DEVD-CHO decreased the apoptosis induced by Cd. Taken together, it could be concluded that [Ca 2+ ] c elevation did act as a pro-apoptotic signal in Cd-induced cytotoxicity of rPT cells, triggered calpain-1 and caspase-3 activation in turn, and induced apoptosis of rPT cells. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. Effect of epidermal growth factor on the labeling of the various RNA species and of nuclear proteins in primary rat astroglial cell cultures

    Energy Technology Data Exchange (ETDEWEB)

    Avola, R.; Condorelli, D.F.; Turpeenoja, L.; Ingrao, F.; Reale, S.; Ragusa, N.; Giuffrida Stella, A.M.

    1988-05-01

    This study investigated the effects of epidermal growth factor (EGF) on the labeling of various RNA species and of nuclear proteins in primary rat astroglial cell cultures. After 12 hours of EGF treatment in serum-free medium or chemically defined medium, significant increase in RNA labeling, and also in acid-soluble radioactivity and RNA content, was observed. The ratio RNA/DNA was significantly higher in EGF-treated cultures compared with controls. Ribosomal RNAs (28S and 18S), polyadenylated, and nonpolyadenylated RNAs showed a higher specific radioactivity in EGF-treated cultures. Among the nuclear proteins, the labeling of basic proteins was enhanced by EGF treatment, whereas that of total nuclear acidic protein (NHPs) was less modified, except for some NHPs separated by gel electrophoresis with a molecular weight (MW) approximately 95-83 and 44 kd, which were significantly more labeled in EGF-treated cultures.

  13. Naringenin regulates production of matrix metalloproteinases in the knee-joint and primary cultured articular chondrocytes and alleviates pain in rat osteoarthritis model.

    Science.gov (United States)

    Wang, C C; Guo, L; Tian, F D; An, N; Luo, L; Hao, R H; Wang, B; Zhou, Z H

    2017-03-23

    Inflammation of cartilage is a primary symptom for knee-joint osteoarthritis. Matrix metalloproteinases (MMPs) are known to play an important role in the articular cartilage destruction related to osteoarthritis. Naringenin is a plant-derived flavonoid known for its anti-inflammatory properties. We studied the effect of naringenin on the transcriptional expression, secretion and enzymatic activity of MMP-3 in vivo in the murine monosodium iodoacetate (MIA) osteoarthritis model. The assessment of pain behavior was also performed in the MIA rats. The destruction of knee-joint tissues was analyzed microscopically. Moreover, the effect of naringenin was also studied in vitro in IL-1β activated articular chondrocytes. The transcriptional expression of MMP-3, MMP-1, MMP-13, thrombospondin motifs (ADAMTS-4) and ADAMTS-5 was also studied in primary cultured chondrocytes of rats. Naringenin caused significant reduction in pain behavior and showed marked improvement in the tissue morphology of MIA rats. Moreover, a significant inhibition of MMP-3 expression in MIA rats was observed upon treatment with naringenin. In the in vitro tests, naringenin caused a significant reduction in the transcriptional expression, secretion and enzymatic activity of the studied degradative enzymes. The NF-κB pathway was also found to be inhibited upon treatment with naringenin in vitro. Overall, the study suggests that naringenin alleviated pain and regulated the production of matrix-metalloproteinases via regulation of NF-κB pathway. Thus, naringenin could be a potent therapeutic option for the treatment of osteoarthritis.

  14. Naringenin regulates production of matrix metalloproteinases in the knee-joint and primary cultured articular chondrocytes and alleviates pain in rat osteoarthritis model

    Directory of Open Access Journals (Sweden)

    C.C. Wang

    Full Text Available Inflammation of cartilage is a primary symptom for knee-joint osteoarthritis. Matrix metalloproteinases (MMPs are known to play an important role in the articular cartilage destruction related to osteoarthritis. Naringenin is a plant-derived flavonoid known for its anti-inflammatory properties. We studied the effect of naringenin on the transcriptional expression, secretion and enzymatic activity of MMP-3 in vivo in the murine monosodium iodoacetate (MIA osteoarthritis model. The assessment of pain behavior was also performed in the MIA rats. The destruction of knee-joint tissues was analyzed microscopically. Moreover, the effect of naringenin was also studied in vitro in IL-1β activated articular chondrocytes. The transcriptional expression of MMP-3, MMP-1, MMP-13, thrombospondin motifs (ADAMTS-4 and ADAMTS-5 was also studied in primary cultured chondrocytes of rats. Naringenin caused significant reduction in pain behavior and showed marked improvement in the tissue morphology of MIA rats. Moreover, a significant inhibition of MMP-3 expression in MIA rats was observed upon treatment with naringenin. In the in vitro tests, naringenin caused a significant reduction in the transcriptional expression, secretion and enzymatic activity of the studied degradative enzymes. The NF-κB pathway was also found to be inhibited upon treatment with naringenin in vitro. Overall, the study suggests that naringenin alleviated pain and regulated the production of matrix-metalloproteinases via regulation of NF-κB pathway. Thus, naringenin could be a potent therapeutic option for the treatment of osteoarthritis.

  15. Comparative effects on rat primary astrocytes and C6 rat glioma cells cultures after 24-h exposure to silver nanoparticles (AgNPs)

    Energy Technology Data Exchange (ETDEWEB)

    Salazar-García, Samuel; Silva-Ramírez, Ana Sonia; Ramirez-Lee, Manuel A.; Rosas-Hernandez, Hector [Universidad Autonoma de San Luis Potosi, Facultad de Ciencias Quimicas (Mexico); Rangel-López, Edgar [Instituto Nacional de Neurologia y Neurocirugia Manuel Velasco Suárez, Laboratorio de Aminoacidos Excitadores (Mexico); Castillo, Claudia G. [Facultad de Medicina, Universidad Autonoma de San Luis Potosi (Mexico); Santamaría, Abel [Instituto Nacional de Neurologia y Neurocirugia Manuel Velasco Suárez, Laboratorio de Aminoacidos Excitadores (Mexico); Martinez-Castañon, Gabriel A. [Universidad Autonoma de San Luis Potosi, Facultad de Estomatologia (Mexico); Gonzalez, Carmen, E-mail: cgonzalez.uaslp@gmail.com, E-mail: gonzalez.castillocarmen@fcq.uaslp.mx [Universidad Autonoma de San Luis Potosi, Facultad de Ciencias Quimicas (Mexico)

    2015-11-15

    The aim of this work was to compare the effects of 24-h exposure of rat primary astrocytes and C6 rat glioma cells to 7.8 nm AgNPs. Glioblastoma multiforme (GBM) is the most aggressive primary brain tumor and current treatments lead to diverse side-effects; for this reason, it is imperative to investigate new approaches, including those alternatives provided by nanotechnology, like nanomaterials (NMs) such as silver nanoparticles. Herein, we found that C6 rat glioma cells, but no primary astrocytes, decreased cell viability after AgNPs treatment; however, both cell types diminished their proliferation. The decrease of glioma C6 cells proliferation was related with necrosis, while in primary astrocytes, the decreased proliferation was associated with the induction of apoptosis. The ionic control (AgNO{sub 3}) exerted a different profile than AgNPs; the bulk form did not modify the basal effect in each determination, whereas cisplatin, a well-known antitumoral drug used as a comparative control, promoted cytotoxicity in both cell types at specific concentrations. Our findings prompt the need to determine the fine molecular and cellular mechanisms involved in the differential biological responses to AgNPs in order to develop new tools or alternatives based on nanotechnology that may contribute to the understanding, impact and use of NMs in specific targets, like glioblastoma cells.

  16. Comparative effects on rat primary astrocytes and C6 rat glioma cells cultures after 24-h exposure to silver nanoparticles (AgNPs)

    International Nuclear Information System (INIS)

    Salazar-García, Samuel; Silva-Ramírez, Ana Sonia; Ramirez-Lee, Manuel A.; Rosas-Hernandez, Hector; Rangel-López, Edgar; Castillo, Claudia G.; Santamaría, Abel; Martinez-Castañon, Gabriel A.; Gonzalez, Carmen

    2015-01-01

    The aim of this work was to compare the effects of 24-h exposure of rat primary astrocytes and C6 rat glioma cells to 7.8 nm AgNPs. Glioblastoma multiforme (GBM) is the most aggressive primary brain tumor and current treatments lead to diverse side-effects; for this reason, it is imperative to investigate new approaches, including those alternatives provided by nanotechnology, like nanomaterials (NMs) such as silver nanoparticles. Herein, we found that C6 rat glioma cells, but no primary astrocytes, decreased cell viability after AgNPs treatment; however, both cell types diminished their proliferation. The decrease of glioma C6 cells proliferation was related with necrosis, while in primary astrocytes, the decreased proliferation was associated with the induction of apoptosis. The ionic control (AgNO 3 ) exerted a different profile than AgNPs; the bulk form did not modify the basal effect in each determination, whereas cisplatin, a well-known antitumoral drug used as a comparative control, promoted cytotoxicity in both cell types at specific concentrations. Our findings prompt the need to determine the fine molecular and cellular mechanisms involved in the differential biological responses to AgNPs in order to develop new tools or alternatives based on nanotechnology that may contribute to the understanding, impact and use of NMs in specific targets, like glioblastoma cells

  17. Effect of Tissue-Culture Substratum and Extracellular Matrix Overlay on Liver-Selective and Xenobiotic Inducible Gene Expression in Primary Rat Hepatocytes

    Science.gov (United States)

    SIDHU, J.S.; FARIN, F.M.; KAVANAGH, T.J.; OMIECINSKI, C.J.

    2012-01-01

    In a previous study (Sidhu et al., 1993), we demonstrated that a combination of certain cell culture media, hormone addition, and extracellular matrix (ECM) overlay coordinately modulated the expression of certain liver-selective genes in primary rat hepatocyte cultures, including the responsiveness of genes to phenobarbital. However, little is known about the interactions between the type of substratum upon which hepatocytes are adhered and the ECM overlay, as codeterminants of liver-selective gene expression. The present study was undertaken to compare specific substrata, including tissue culture-grade plastic, Primaria, and type 1 collagen-coated plastic, in combination with the presence or absence of standard ECM or a growth-factor-reduced ECM overlay. Hepatocyte cultures were assessed either as control cultures or subsequent to treatment for 24 h with phenobarbital (0.1 or 1 mM), or beta-naphthoflavone (22 μM), to monitor responses of hepatocytes to two prototypic gene-inducing agents. Analyses of maintenance and induction of cytochrome P450 and liver-selective gene expression included measures of mRNA levels using Northern blot and slot-blot hybridization and single cell immunofluorescence assays to measure levels of specific cytochrome P450 proteins. The results of these experiments demonstrated that hepatocyte-selective expression, including the absolute level of induction response (relative to those observed in the rat liver in vivo) was highly dependent on the presence of ECM overlay but independent of the substratum employed. As studied herein, the establishment of optimal conditions for primary hepatocyte culture, enabling reproduction of responses observed in vivo, is important to further prospects for in vitro toxicity testing and for investigating molecular mechanisms of phenobarbital-mediated gene regulation. PMID:24817786

  18. Lack of insulin results in reduced seladin-1 expression in primary cultured neurons and in cerebral cortex of STZ-induced diabetic rats.

    Science.gov (United States)

    Kazkayasi, Inci; Ismail, Muhammad-Al-Mustafa; Parrado-Fernandez, Cristina; Björkhem, Ingemar; Pekiner, Can; Uma, Serdar; Cedazo-Minguez, Angel; Burul-Bozkurt, Nihan

    2016-10-28

    Several studies demonstrated that Diabetes mellitus (DM) enhances the risk for Alzheimer's disease (AD). Although hyperglycemia and perturbed function of insulin signaling have been proposed to contribute to AD pathogenesis, the molecular mechanisms behind this association is not clear yet. Seladin-1 is an enzyme catalyzing the last step in cholesterol biosynthesis converting desmosterol to cholesterol. The neuroprotective function of seladin-1 has gained interest in AD research recently. Seladin-1 has anti-apoptotic properties and regulates the expression of β-secretase (BACE-1). Here we measured seladin-1 mRNA and protein expressions in rat primary cultured neurons under diabetic conditions and also in the brains of rats with streptozotocine (STZ)-induced diabetes. We show that constant lack of insulin for 5days decreased seladin-1 levels in cultured rat primary neurons. Similarly, a decrease in seladin-1 was found in the brains of rats with STZ-induced diabetes. However, if the lack of insulin and/or high glucose treatment was intermittent, neuronal seladin-1 levels were not affected in vitro. On the other hand, treatment of neurons with metformin resulted in a significant increase in seladin-1. Constant lack of insulin for 5days, as well as high glucose treatment, increased the neuronal expression of BACE-1 in vitro, but not in the in vivo model. Our study defines insulin as a regulator of seladin-1 expression for the first time. The relevance of these findings for the association of DM with AD is discussed. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  19. Ginkgolide A contributes to the potentiation of acetaminophen toxicity by Ginkgo biloba extract in primary cultures of rat hepatocytes

    International Nuclear Information System (INIS)

    Rajaraman, Ganesh; Chen, Jie; Chang, Thomas K.H.

    2006-01-01

    The present cell culture study investigated the effect of Ginkgo biloba extract pretreatment on acetaminophen toxicity and assessed the role of ginkgolide A and cytochrome P450 3A (CYP3A) in hepatocytes isolated from adult male Long-Evans rats provided ad libitum with a standard diet. Acetaminophen (7.5-25 mM for 24 h) conferred hepatocyte toxicity, as determined by the lactate dehydrogenase (LDH) assay. G. biloba extract alone increased LDH leakage in hepatocytes at concentrations ≥ 75 μg/ml and ≥ 750 μg/ml after a 72 h and 24 h treatment period, respectively. G. biloba extract (25 or 50 μg/ml once every 24 h for 72 h) potentiated LDH leakage by acetaminophen (10 mM for 24 h; added at 48 h after initiation of extract pretreatment). The effect was confirmed by a decrease in [ 14 C]-leucine incorporation. At the level present in a modulating concentration (50 μg/ml) of the extract, ginkgolide A (0.55 μg/ml), which increased CYP3A23 mRNA levels and CYP3A-mediated enzyme activity, accounted for part but not all of the potentiating effect of the extract on acetaminophen toxicity. This occurred as a result of CYP3A induction by ginkgolide A because triacetyloleandomycin (TAO), a specific inhibitor of CYP3A catalytic activity, completely blocked the effect of ginkgolide A. Ginkgolide B, ginkgolide C, ginkgolide J, quercetin, kaempferol, isorhamnetin, and isorhamnetin-3-O-rutinoside did not alter the extent of LDH leakage by acetaminophen. In summary, G. biloba pretreatment potentiated acetaminophen toxicity in cultured rat hepatocytes and ginkgolide A contributed to this novel effect of the extract by inducing CYP3A

  20. Study of rat hepatocytes in primary culture submitted to hypoxia and reoxygenation: action of the cytoprotectors prostaglandin E1, superoxide dismutase, allopurinol and verapamil.

    Science.gov (United States)

    Andrade, Dahir Ramos de; Andrade, Dahir Ramos de; Santos, Sânia Alves Dos

    2009-01-01

    Exposure of hepatocytes to pathological conditions in a microenvironment of hypoxia and reoxygenation is very frequent in hepatic diseases. Several substances present perspectives for cytoprotective action on hepatocyte submitted to reoxygenation after hypoxia and simple hypoxia. We research therapeutic options for hepatocytes submitted to hypoxia and hypoxia + reoxygenation injury. Primary culture of rat hepatocytes was submitted to hypoxia (2 hours) plus reoxygenation (2 hours) and simple hypoxia (4 hours) in the presence or the absence of cytoprotectors. The hepatocyte lesion was evaluated by functional criteria through percentage of lactate dehydrogenase released and cell viability. The effects of the cytoprotectors prostaglandin E1 3 etag/mL, superoxide dismutase 80 microg/mL, allopurinol 20 microM and verapamil 10-4 M were studied in this model of injury. Reoxygenation after hypoxia induced more significant lesion in cultured hepatocytes compared to simple hypoxia, detected by analysis of functional criteria. There was a significant reduction of percentage of lactate dehydrogenase released and a significant increase of percentage of cell viability in the hypoxia + reoxygenation + cytoprotectors groups compared to hypoxia + reoxygenation groups. Prostaglandin E1, superoxide dismutase and verapamil also protected the group submitted to simple hypoxia, when evaluated by functional criteria. We conclude that reoxygenation after hypoxia significantly increased the lesion of cultured rat hepatocytes when compared to simple hypoxia. Prostaglandin E1, superoxide dismutase, allopurinol and verapamil acted as cytoprotectors to the rat cultured hepatocytes submitted to hypoxia + reoxygenation in vitro. The substances prostaglandin E1, superoxide dismutase and verapamil protected hepatocytes submitted to simple hypoxia on the basis of all the criteria studied in this experimental model.

  1. Protective effects of water extract of clam on normal and CCl₄-induced damage in primary cultured rat hepatocytes.

    Science.gov (United States)

    Chi, H M; Chou, S T; Lin, S C; Su, Z Y; Sheen, Lee-Yan

    2010-01-01

    The objective of this study was to investigate the effects of various concentrations and incubation times of water extract of clam (WEC) on glutathione, its antioxidant and the detoxification defense systems in normal and CCl₄-induced oxidative damaged primary rat hepatocytes. This study showed that when the hepatocytes were treated with WEC (0.14 ~ 1.68 mg/ml), the intracellular glutathione (GSH) levels, GSH/GSSG ratio, and the activities of GSH-related enzymes (GPx, GRd, and GST) were higher than those in the control at 24 or 48 hour treatments. However, the lactate dehydrogenase (LDH) leakage and microscopic observations did not differ from those of the control. Yet, when the hepatocytes were pretreated with various concentrations of WEC for 24 hours and then exposed to 5 mM carbon tetrachloride (CCl₄) for 1 hour, at concentrations of WEC between 0.42 ~ 1.68 mg/ml, the viabilities, intracellular GSH level, and activities of GST and GPx were significantly increased compared to those of the CCl₄-treated control group (p WEC could improve the viability and the capabilities of detoxification and antioxidation in hepatocytes by increasing the GSH level and the activities of GSH-related enzymes.

  2. Constant light suppresses production of Met-enkephalin-containing peptides in cultured splenic macrophages and impairs primary immune response in rats.

    Science.gov (United States)

    Valdés-Tovar, Marcela; Escobar, Carolina; Solís-Chagoyán, Héctor; Asai, Miguel; Benítez-King, Gloria

    2015-03-01

    The light-dark cycle is an environmental factor that influences immune physiology, and so, variations of the photoperiod length result in altered immune responsivity. Macrophage physiology comprises a spectrum of functions that goes from host defense to immune down-regulation, in addition to their homeostatic activities. Macrophages also play a key role in the transition from innate to adaptive immune responses. Met-enkephalin (MEnk) has been recognized as a modulator of macrophage physiology acting in an autocrine or paracrine fashion to influence macrophage activation, phenotype polarization and production of cytokines that would enhance lymphocyte activation at early stages of an immune response. Previously it was shown that splenic MEnk tissue content is reduced in rats exposed to constant light. In this work, we explored whether production of Met-enkephalin-containing peptides (MECPs) in cultured splenic macrophages is affected by exposure of rats to a constant light regime. In addition, we explored whether primary immune response was impaired under this condition. We found that in rats, 15 days in constant light was sufficient to disrupt their general activity rhythm. Splenic MEnk content oscillations and levels were also blunted throughout a 24-h period in animals subjected to constant light. In agreement, de novo synthesis of MECPs evaluated through incorporation of (35)S-methionine was reduced in splenic macrophages from rats exposed to constant light. Moreover, MECPs immunocytochemistry showed a decrease in the intracellular content and lack of granule-like deposits in this condition. Furthermore, we found that primary T-dependent antibody response was compromised in rats exposed to constant light. In those animals, pharmacologic treatment with MEnk increased IFN-γ-secreting cells. Also, IL-2 secretion from antigen-stimulated splenocytes was reduced after incubation with naloxone, suggesting that immune-derived opioid peptides and stimulation of opioid

  3. Changes in Expression of Connexin 32, Bile Canaliculus-Like Structures, and Localization of Alkaline Phosphatase in Primary Cultures of Fetal Rat Hepatocytes

    International Nuclear Information System (INIS)

    Fukazawa, Shoko; Chida, Kohsuke; Taguchi, Meiko; Takeuchi, Akihiro; Ikeda, Noriaki

    2013-01-01

    We devised an experimental design in primary cultures of fetal rat hepatocytes for studying hepatocyte differentiation over a short period. In the present study, hepatocytes were first cultured for 3 days in dexamethasone-supplemented medium and then for an additional 3 days in dexamethasone- or epidermal growth factor-supplemented medium. In hepatocytes cultured continuously in dexamethasone-supplemented medium, the expression of connexin 32 increased and bile canaliculus-like structures and localization of alkaline phosphatase in the plasma membrane around bile canaliculus-like structures were maintained. Few cells incorporated bromodeoxyuridine. On the other hand, in most of the hepatocytes cultured in epidermal growth factor-supplemented medium, the expression of connexin 32 was minimally recognized, bile canaliculus-like structures were shortened or eliminated, and alkaline phosphatase was localized as numerous fine spots throughout the cytoplasm. More than 20% of all hepatocytes incorporated bromodeoxyuridine. The present study suggests that in hepatocytes, there is a close relationship among connexin 32 expression, the maintenance of bile canaliculus-like structures, and the localization of alkaline phosphatase to the plasma membrane around the bile canaliculus-like structures, and this indicates that the present experimental model is useful for studying hepatocyte differentiation over a short period

  4. Effect of diphenyl ether herbicides and oxadiazon on porphyrin biosynthesis in mouse liver, rat primary hepatocyte culture and HepG2 cells.

    Science.gov (United States)

    Krijt, J; van Holsteijn, I; Hassing, I; Vokurka, M; Blaauboer, B J

    1993-01-01

    The effects of the herbicides fomesafen, oxyfluorfen, oxadiazon and fluazifop-butyl on porphyrin accumulation in mouse liver, rat primary hepatocyte culture and HepG2 cells were investigated. Ten days of herbicide feeding (0.25% in the diet) increased the liver porphyrins in male C57B1/6J mice from 1.4 +/- 0.6 to 4.8 +/- 2.1 (fomesafen) 16.9 +2- 2.9 (oxyfluorfen) and 25.9 +/- 3.1 (oxadiazon) nmol/g wet weight, respectively. Fluazifop-butyl had no effect on liver porphyrin metabolism. Fomesafen, oxyfluorfen and oxadiazon increased the cellular porphyrin content of rat hepatocytes after 24 h of incubation (control, 3.2 pmol/mg protein, fomesafen, oxyfluorfen and oxadiazon at 0.125 mM concentration 51.5, 54.3 and 44.0 pmol/mg protein, respectively). The porphyrin content of HepG2 cells increased from 1.6 to 18.2, 10.6 and 9.2 pmol/mg protein after 24 h incubation with the three herbicides. Fluazifop-butyl increased hepatic cytochrome P450 levels and ethoxy- and pentoxyresorufin O-dealkylase (EROD and PROD) activity, oxyfluorfen increased PROD activity. Peroxisomal palmitoyl CoA oxidation increased after fomesafen and fluazifop treatment to about 500% of control values both in mouse liver and rat hepatocytes. Both rat hepatocytes and HepG2 cells can be used as a test system for the porphyrogenic potential of photobleaching herbicides.

  5. Cadmium-induced apoptosis in primary rat cerebral cortical neurons culture is mediated by a calcium signaling pathway.

    Directory of Open Access Journals (Sweden)

    Yan Yuan

    Full Text Available Cadmium (Cd is an extremely toxic metal, capable of severely damaging several organs, including the brain. Studies have shown that Cd disrupts intracellular free calcium ([Ca(2+]i homeostasis, leading to apoptosis in a variety of cells including primary murine neurons. Calcium is a ubiquitous intracellular ion which acts as a signaling mediator in numerous cellular processes including cell proliferation, differentiation, and survival/death. However, little is known about the role of calcium signaling in Cd-induced apoptosis in neuronal cells. Thus we investigated the role of calcium signaling in Cd-induced apoptosis in primary rat cerebral cortical neurons. Consistent with known toxic properties of Cd, exposure of cerebral cortical neurons to Cd caused morphological changes indicative of apoptosis and cell death. It also induced elevation of [Ca(2+]i and inhibition of Na(+/K(+-ATPase and Ca(2+/Mg(2+-ATPase activities. This Cd-induced elevation of [Ca(2+]i was suppressed by an IP3R inhibitor, 2-APB, suggesting that ER-regulated Ca(2+ is involved. In addition, we observed elevation of reactive oxygen species (ROS levels, dysfunction of cytochrome oxidase subunits (COX-I/II/III, depletion of mitochondrial membrane potential (ΔΨm, and cleavage of caspase-9, caspase-3 and poly (ADP-ribose polymerase (PARP during Cd exposure. Z-VAD-fmk, a pan caspase inhibitor, partially prevented Cd-induced apoptosis and cell death. Interestingly, apoptosis, cell death and these cellular events induced by Cd were blocked by BAPTA-AM, a specific intracellular Ca(2+ chelator. Furthermore, western blot analysis revealed an up-regulated expression of Bcl-2 and down-regulated expression of Bax. However, these were not blocked by BAPTA-AM. Thus Cd toxicity is in part due to its disruption of intracellular Ca(2+ homeostasis, by compromising ATPases activities and ER-regulated Ca(2+, and this elevation in Ca(2+ triggers the activation of the Ca(2+-mitochondria apoptotic

  6. Neuroprotective effects of orientin on oxygen-glucose deprivation/reperfusion-induced cell injury in primary culture of rat cortical neurons.

    Science.gov (United States)

    Tian, Tian; Zeng, Junan; Zhao, Guangyu; Zhao, Wenjing; Gao, Songyi; Liu, Li

    2018-01-01

    Orientin (luteolin-8-C-glucoside) is a phenolic compound found abundantly in millet, juice, and peel of passion fruit and has been shown to have antioxidant properties. In the present study, we explored the effects of orientin on oxygen-glucose deprivation/reperfusion (OGD/RP)-induced cell injury in primary culture of rat cortical neurons using an in vitro model of neonatal ischemic brain injury. The reduced cell viability and elevated lactate dehydrogenase leakage were observed after OGD/RP exposure, which were then reversed by orientin (10, 20, and 30 µM) pretreatment in a dose-dependent manner. Additionally, OGD/RP treatment resulted in significant oxidative stress, accompanied by enhanced intracellular reactive oxygen species (ROS) generation, and obvious depletion in the activities of intracellular Mn-superoxide dismutase, catalase, and glutathione peroxidase antioxidases. However, these effects were dose dependently restored by orientin pretreatment. We also found that orientin pretreatment dose dependently suppressed [Ca 2+ ] i increase and mitochondrial membrane potential dissipation caused by OGD/RP in primary culture of rat cortical neurons. Western blot analysis showed that OGD/RP exposure induced a distinct decrease of Bcl-2 protein and a marked elevation of Bax, caspase-3, and cleaved caspase-3 proteins; whereas these effects were dose dependently reversed by orientin incubation. Both the caspase-3 activity and the apoptosis rate were increased under OGD/RP treatment, but was then dose dependently down-regulated by orientin (10, 20, and 30 µM) incubation. Moreover, orientin pretreatment dose dependently inhibited OGD/RP-induced phosphorylation of JNK and ERK1/2. Notably, JNK inhibitor SP600125 and ERK1/2 inhibitor PD98059 also dramatically attenuated OGD/RP-induced cell viability loss and ROS generation, and further, orientin failed to protect cortical neurons with the interference of JNK activator anisomycin or ERK1/2 activator FGF-2. Taken

  7. Neuroprotective effects of triterpene glycosides from glycine max against glutamate induced toxicity in primary cultured rat cortical cells.

    Science.gov (United States)

    Moon, Hyung-In; Lee, Jai-Heon

    2012-01-01

    To examine the neuroprotective effects of Glycine max, we tested its protection against the glutamate-induced toxicity in primary cortical cultured neurons. In order to clarify the neuroprotective mechanism(s) of this observed effect, isolation was performed to seek and identify active fractions and components. From such fractionation, two triterpene glycosides, 3-O-[α-l-rhamnopyranosyl(1-2)-β-d-glucopyranosyl(1-2)-β-d-glucuronopyranosyl]olean-12-en-3β,22β,24-triol (1) and 3-O-[β-d-glucopyranosyl(1-2)-β-d-galactopyranosyl(1-2)-β-d-glucuronopyranosyl]olean-12-en-3β,22β,24-triol (2) were isolated with the methanol extracts with of air-dried Glycine max. Among these compounds, compound 2 exhibited significant neuroprotective activities against glutamate-induced toxicity, exhibiting cell viability of about 50% at concentrations ranging from 0.1 μM to 10 μM. Therefore, the neuroprotective effect of Glycine max might be due to the inhibition of glutamate-induced toxicity by triterpene glycosides.

  8. Use of 5-Bromodeoxyuridine and irradiation for the estimation of the myoblast and myocyte content of primary rat heart cell cultures

    International Nuclear Information System (INIS)

    Masse, M.J.O.; Harary, I.

    1980-01-01

    A method for killing dividing cells was adapted for the elimination of dividing heart muscle cells (myoblasts) in cultures. We have used this method to demonstrate their presence and to estimate their number as well as the number of nondividing heart muscle cells (myocytes) in the neo-natal rat heart. Cells were cultivated in BUdR (5-bromodeoxyuridine) 10 -4 M for 3 days and then irradiated with long uv light. The selective elimination of dividing cells led to a loss of myosin Ca 2+ -activated ATPase in the cultures. The percent of ATPase left after irradiation was 32% of the control in cultures derived from 1-day postnatal rats and 48% in cultures from 4-day postnatal rats. This reflects an in vivo shift of myoblasts to myocytes in the muscle cell population as the rat ages

  9. Differential adenoassociated virus vector-driven expression of a neuropeptide Y gene in primary rat brain astroglial cultures after transfection with Sendai virosomes versus Lipofectin.

    Science.gov (United States)

    de Fiebre, C M; Wu, P; Notabartolo, D; Millard, W J; Meyer, E M

    1994-06-01

    The ability of Sendai virosomes or Lipofectin to introduce an AAV vector into primary rat brain astroglial cultures was characterized. The pJDT95npy vector was constructed by inserting rat NPY cDNA downstream from the indigenous AAV p5, p19 and p40 promoters in pJDT95. Lipofectin-mediated transfection with pJDT95npy (10 micrograms) resulted in pronounced expression of several NPY mRNA species: p5-driven (3.3 kb), p19-driven (2.7 kb) and p40-driven (0.6, 0.8, 1.1, and 1.8 kb). Exposure to virosomally encapsulated pJDT95npy (50 or 100 ng) resulted in transient expression of some p40-driven mRNA species (0.8 and 1.8 kb). Neither method produced astroglia cells which synthesized mature NPY immunoreactivity. This demonstrates that an AAV-derived vector can drive gene expression in astroglia, that Sendai virosomes can infuse vectors into astroglia, but that the amount of DNA infused in this manner may limit long term expression.

  10. Pellet culture model for human primary osteoblasts.

    Science.gov (United States)

    Jähn, K; Richards, R G; Archer, C W; Stoddart, M J

    2010-09-06

    In vitro monolayer culture of human primary osteoblasts (hOBs) often shows unsatisfactory results for extracellular matrix deposition, maturation and calcification. Nevertheless, monolayer culture is still the method of choice for in vitro differentiation of primary osteoblasts. We believe that the delay in mature ECM production by the monolayer cultured osteoblasts is determined by their state of cell maturation. A functional relationship between the inhibition of osteoblast proliferation and the induction of genes associated with matrix maturation was suggested within a monolayer culture model for rat calvarial osteoblasts. We hypothesize, that a pellet culture model could be utilized to decrease initial proliferation and increase the transformation of osteoblasts into a more mature phenotype. We performed pellet cultures using hOBs and compared their differentiation potential to 2D monolayer cultures. Using the pellet culture model, we were able to generate a population of cuboidal shaped central osteoblastic cells. Increased proliferation, as seen during low-density monolayer culture, was absent in pellet cultures and monolayers seeded at 40,000 cells/cm2. Moreover, the expression pattern of phenotypic markers Runx2, osterix, osteocalcin, col I and E11 mRNA was significantly different depending on whether the cells were cultured in low density monolayer, high density monolayer or pellet culture. We conclude that the transformation of the osteoblast phenotype in vitro to a more mature stage can be achieved more rapidly in 3D culture. Moreover, that dense monolayer leads to the formation of more mature osteoblasts than low-density seeded monolayer, while hOB cells in pellets seem to have transformed even further along the osteoblast phenotype.

  11. Pellet culture model for human primary osteoblasts

    Directory of Open Access Journals (Sweden)

    K Jähn

    2010-09-01

    Full Text Available In vitro monolayer culture of human primary osteoblasts (hOBs often shows unsatisfactory results for extracellular matrix deposition, maturation and calcification. Nevertheless, monolayer culture is still the method of choice for in vitro differentiation of primary osteoblasts. We believe that the delay in mature ECM production by the monolayer cultured osteoblasts is determined by their state of cell maturation. A functional relationship between the inhibition of osteoblast proliferation and the induction of genes associated with matrix maturation was suggested within a monolayer culture model for rat calvarial osteoblasts. We hypothesize, that a pellet culture model could be utilized to decrease initial proliferation and increase the transformation of osteoblasts into a more mature phenotype. We performed pellet cultures using hOBs and compared their differentiation potential to 2D monolayer cultures. Using the pellet culture model, we were able to generate a population of cuboidal shaped central osteoblastic cells. Increased proliferation, as seen during low-density monolayer culture, was absent in pellet cultures and monolayers seeded at 40,000 cells/cm2. Moreover, the expression pattern of phenotypic markers Runx2, osterix, osteocalcin, col I and E11 mRNA was significantly different depending on whether the cells were cultured in low density monolayer, high density monolayer or pellet culture. We conclude that the transformation of the osteoblast phenotype in vitro to a more mature stage can be achieved more rapidly in 3D culture. Moreover, that dense monolayer leads to the formation of more mature osteoblasts than low-density seeded monolayer, while hOB cells in pellets seem to have transformed even further along the osteoblast phenotype.

  12. Functional expression of P2 purinoceptors in a primary neuroglial cell culture of the rat arcuate nucleus.

    Science.gov (United States)

    Pollatzek, Eric; Hitzel, Norma; Ott, Daniela; Raisl, Katrin; Reuter, Bärbel; Gerstberger, Rüdiger

    2016-07-07

    The arcuate nucleus (ARC) plays an important role in the hypothalamic control of energy homeostasis. Expression of various purinoceptor subtypes in the rat ARC and physiological studies suggest a modulatory function of P2 receptors within the neuroglial ARC circuitry. A differentiated mixed neuronal and glial microculture was therefore established from postnatal rat ARC, revealing neuronal expression of ARC-specific transmitters involved in food intake regulation (neuropeptide Y (NPY), proopiomelanocortin (POMC), tyrosine hydroxylase (TH)). Some NPYergic neurons cosynthesized TH, while POMC and TH expression proved to be mutually exclusive. Stimulation with the general purinoceptor agonists 2-methylthioadenosine-5'triphosphate (2-MeSATP) and ATP but not the P2X1/P2X3 receptor subtype agonist α,β-methyleneadenosine-5'triphosphate (α,β-meATP) induced intracellular calcium signals in ARC neurons and astrocytes. Some 5-10% each of 2-MeSATP responsive neurons expressed POMC, NYP or TH. Supporting the calcium imaging data, radioligand binding studies to hypothalamic membranes showed high affinity for 2-MeSATP, ATP but not α,β-meATP to displace [α-(35)S]deoxyadenosine-5'thiotriphosphate ([(35)S]dATPαS) from P2 receptors. Repetitive superfusion with equimolar 2-MeSATP allowed categorization of ARC cells into groups with a high or low (LDD) degree of purinoceptor desensitization, the latter allowing further receptor characterization. Calcium imaging experiments performed at 37°C vs. room temperature showed further reduction of desensitization. Agonist-mediated intracellular calcium signals were suppressed in all LDD neurons but only 25% of astrocytes in the absence of extracellular calcium, suggestive of metabotropic P2Y receptor expression in the majority of ARC astrocytes. The highly P2Y1-selective receptor agonists MRS2365 and 2-methylthioadenosine-5'diphosphate (2-MeSADP) activated 75-85% of all 2-MeSATP-responsive ARC astrocytes. Taking into consideration the

  13. Comparison of the protective effects of ferulic acid and its drug-containing plasma on primary cultured neonatal rat cardiomyocytes with hypoxia/reoxygenation injury

    Science.gov (United States)

    Ren, Cong; Bao, Yong-rui; Meng, Xian-sheng; Diao, Yun-peng; Kang, Ting-guo

    2013-01-01

    Backgroud: To simulate the ischemia-reperfusion injury in vivo, hypoxia/reoxygenation injury model was established in vitro and primary cultured neonatal rat cardiomyocytes were underwent hypoxia with hydrosulfite (Na2S2O4) for 1 h followed by 1 h reoxygenation. Materials and Methods: Determination the cell viability by MTT colorimetric assay. We use kit to detect the activity of lactate dehydrogenase (LDH), Na+-K+-ATPase and Ca2+-ATPase. Do research on the effect which ferulic acid and its drug-containing plasma have to self-discipline, conductivity, action potential duration and other electrophysiological phenomena of myocardial cells by direct observation using a microscope and recording method of intracellular action potential. Results: The experimental datum showed that both can reduce the damage hydrosulfite to myocardial cell damage and improve myocardial viability, reduce the amount of LDH leak, increase activity of Na+-K+-ATPase, Ca2+-ATPase, and increase APA (Action potential amplitude), Vmax (Maximum rate of depolarization) and MPD (Maximum potential diastolic). Conclusion: Taken together, therefore, we can get the conclusion that ferulic acid drug-containing plasma has better protective effect injured myocardial cell than ferulic acid. PMID:23930002

  14. An evolutionary conserved region (ECR in the human dopamine receptor D4 gene supports reporter gene expression in primary cultures derived from the rat cortex

    Directory of Open Access Journals (Sweden)

    Haddley Kate

    2011-05-01

    Full Text Available Abstract Background Detecting functional variants contributing to diversity of behaviour is crucial for dissecting genetics of complex behaviours. At a molecular level, characterisation of variation in exons has been studied as they are easily identified in the current genome annotation although the functional consequences are less well understood; however, it has been difficult to prioritise regions of non-coding DNA in which genetic variation could also have significant functional consequences. Comparison of multiple vertebrate genomes has allowed the identification of non-coding evolutionary conserved regions (ECRs, in which the degree of conservation can be comparable with exonic regions suggesting functional significance. Results We identified ECRs at the dopamine receptor D4 gene locus, an important gene for human behaviours. The most conserved non-coding ECR (D4ECR1 supported high reporter gene expression in primary cultures derived from neonate rat frontal cortex. Computer aided analysis of the sequence of the D4ECR1 indicated the potential transcription factors that could modulate its function. D4ECR1 contained multiple consensus sequences for binding the transcription factor Sp1, a factor previously implicated in DRD4 expression. Co-transfection experiments demonstrated that overexpression of Sp1 significantly decreased the activity of the D4ECR1 in vitro. Conclusion Bioinformatic analysis complemented by functional analysis of the DRD4 gene locus has identified a a strong enhancer that functions in neurons and b a transcription factor that may modulate the function of that enhancer.

  15. The antidiabetic drug metformin decreases mitochondrial respiration and tricarboxylic acid cycle activity in cultured primary rat astrocytes

    DEFF Research Database (Denmark)

    Hohnholt, Michaela C; Blumrich, Eva-Maria; Waagepetersen, Helle S

    2017-01-01

    cycling ratio in metformin-treated astrocytes. Finally, the oxygen consumption of mitochondria isolated from metformin-treated astrocytes was drastically reduced in the presence of complex I substrates, but not of complex II substrates. These data demonstrate that exposure to metformin strongly impairs......Metformin is an antidiabetic drug that is used daily by millions of patients worldwide. Metformin is able to cross the blood-brain barrier and has recently been shown to increase glucose consumption and lactate release in cultured astrocytes. However, potential effects of metformin on mitochondrial...

  16. Transfection of rat brain endothelium in a primary culture model of the blood-brain barrier at different states of barrier maturity

    DEFF Research Database (Denmark)

    Larsen, Annette Burkhart; Thomsen, Louiza Bohn; Lichota, Jacek

    as possible, an in-vitro BBB model was established by isolating primary BCECs, pericytes, and astrocytes from rats. The model was subsequently characterized by immunostaining, RT-qPCR, and measurements of trans-endothelial electrical resistance (TEER). Previous studies have stated that efficient gene...

  17. Curcumin Pretreatment Induces Nrf2 and an Antioxidant Response and Prevents Hemin-Induced Toxicity in Primary Cultures of Cerebellar Granule Neurons of Rats

    Directory of Open Access Journals (Sweden)

    Susana González-Reyes

    2013-01-01

    Full Text Available Curcumin is a bifunctional antioxidant derived from Curcuma longa. This study identifies curcumin as a neuroprotectant against hemin-induced damage in primary cultures of cerebellar granule neurons (CGNs of rats. Hemin, the oxidized form of heme, is a highly reactive compound that induces cellular injury. Pretreatment of CGNs with 5–30 μM curcumin effectively increased by 2.3–4.9 fold heme oxygenase-1 (HO-1 expression and by 5.6–14.3-fold glutathione (GSH levels. Moreover, 15 μM curcumin attenuated by 55% the increase in reactive oxygen species (ROS production, by 94% the reduction of GSH/glutathione disulfide (GSSG ratio, and by 49% the cell death induced by hemin. The inhibition of heme oxygenase system or GSH synthesis with tin mesoporphyrin and buthionine sulfoximine, respectively, suppressed the protective effect of curcumin against hemin-induced toxicity. These data strongly suggest that HO-1 and GSH play a major role in the protective effect of curcumin. Furthermore, it was found that 24 h of incubation with curcumin increases by 1.4-, 2.3-, and 5.2-fold the activity of glutathione reductase, glutathione S-transferase and superoxide dismutase, respectively. Additionally, it was found that curcumin was capable of inducing nuclear factor (erythroid-derived 2-like 2 (Nrf2 translocation into the nucleus. These data suggest that the pretreatment with curcumin induces Nrf2 and an antioxidant response that may play an important role in the protective effect of this antioxidant against hemin-induced neuronal death.

  18. Role of Ryanodine and NMDA Receptors in Tetrabromobisphenol A-Induced Calcium Imbalance and Cytotoxicity in Primary Cultures of Rat Cerebellar Granule Cells.

    Science.gov (United States)

    Zieminska, Elzbieta; Stafiej, Aleksandra; Toczylowska, Beata; Albrecht, Jan; Lazarewicz, Jerzy W

    2015-10-01

    The study assessed the role of ryanodine receptors (RyRs) and NMDA receptors (NMDARs) in the Ca(2+) transients and cytotoxicity induced in neurons by the brominated flame retardant tetrabromobisphenol A (TBBPA). Primary cultures of rat cerebellar granule cells (CGC) were exposed to 7.5, 10, or 25 µM TBBPA for 30 min, and cell viability was assessed after 24 h. Moreover, (45)Ca uptake was measured, and changes in the intracellular Ca(2+) concentration ([Ca(2+)]i) were studied using the fluo-3 probe. The involvement of NMDARs and RyRs was verified using the pertinent receptor antagonists, 0.5 µM MK-801 and 2.5 µM bastadin 12, which was co-applied with 200 µM ryanodine, respectively. The results show that TBBPA concentration-dependently induces an increase in [Ca(2+)]i. This effect was partly suppressed by the inhibitors of RyRs and NMDARs when administered separately, and completely abrogated by their combined application. A concentration-dependent activation of (45)Ca uptake by TBBPA was prevented by MK-801 but not by RyR inhibitors. Application of ≥ 10 µM TBBPA concentration-dependently reduced neuronal viability, and this effect was only partially and to an equal degree reduced by NMDAR and RyR antagonists given either separately or in combination. Our results directly demonstrate that both the RyR-mediated release of intracellular Ca(2+) and the NMDAR-mediated influx of Ca(2+) into neurons participate in the mechanism of TBBPA-induced Ca(2+) imbalance in CGC and play a significant, albeit not exclusive, role in the mechanisms of TBBPA cytotoxicity.

  19. Clozapine, but not haloperidol, enhances glial D-serine and L-glutamate release in rat frontal cortex and primary cultured astrocytes.

    Science.gov (United States)

    Tanahashi, Shunske; Yamamura, Satoshi; Nakagawa, Masanori; Motomura, Eishi; Okada, Motohiro

    2012-03-01

    Deficient transmission at the glutamate NMDA receptor is considered a key component of the pathophysiology of schizophrenia. However, the effects of antipsychotic drugs on the release of the endogenous NMDA receptor partial agonist, D-serine, remain to be clarified. We determined the interaction between antipsychotic drugs (clozapine and haloperidol) and transmission-modulating toxins (tetanus toxin, fluorocitrate, tetrodotoxin) on the release of L-glutamate and D-serine in the medial prefrontal cortex (mPFC) of freely moving rats, using microdialysis, and primary cultures of astrocytes using extreme high-pressure liquid chromatography. Release of L-glutamate and D-serine in the mPFC and in cultured astrocytes was inhibited by tetanus toxin (a synaptobrevin inhibitor) and fluorocitrate (a glial toxin), whereas tetrodotoxin (a voltage-sensitive Na(+) blocker) inhibited depolarization-induced L-glutamate release in the mPFC without affecting that of D-serine. Clozapine (1 and 5 mg·kg(-1)), but not haloperidol (0.5 and 1 mg·kg(-1)), dose-dependently increased L-glutamate and D-serine release from both astrocytes and mPFC. Clozapine-induced release of L-glutamate and D-serine was also reduced by tetanus toxin and fluorocitrate. Tetrodotoxin reduced clozapine-induced mPFC L-glutamate release but not that of D-serine. Clozapine-induced L-glutamate release preceded clozapine-induced D-serine release. MK-801 (a NMDA receptor antagonist) inhibited the delayed clozapine-induced L-glutamate release without affecting that of D-serine. Clozapine predominantly activated glial exocytosis of D-serine, and this clozapine-induced D-serine release subsequently enhances neuronal L-glutamate release via NMDA receptor activation. The enhanced D-serine associated glial transmission seems a novel mechanism of action of clozapine but not haloperidol. © 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.

  20. Pellet culture model for human primary osteoblasts

    OpenAIRE

    K Jähn; RG Richards; CW Archer; MJ Stoddart

    2010-01-01

    In vitro monolayer culture of human primary osteoblasts (hOBs) often shows unsatisfactory results for extracellular matrix deposition, maturation and calcification. Nevertheless, monolayer culture is still the method of choice for in vitro differentiation of primary osteoblasts. We believe that the delay in mature ECM production by the monolayer cultured osteoblasts is determined by their state of cell maturation. A functional relationship between the inhibition of osteoblast proliferation an...

  1. Primary cilia in rat mature Müller glia: downregulation of IFT20 expression reduces sonic hedgehog-mediated proliferation and dedifferentiation potential of Müller glia primary cultures.

    Science.gov (United States)

    Ferraro, Silene; Gomez-Montalvo, Ana I; Olmos, Ruth; Ramirez, Monica; Lamas, Monica

    2015-05-01

    Primary cilia are specialized organelles that extend from the cell surface and concentrate signal transduction components. In the nervous system, primary cilia-associated signals, such as sonic hedgehog (Shh), regulate cell proliferation and neuronal fate. Primary cilia assembly and maintenance require a multi-subunit intraflagellar transport (IFT) protein complex. Defects in primary cilia and IFT proteins are associated to severe pathological phenotypes. In the retina, the study of primary cilia has been mainly restricted to the specialized photoreceptor outer segment. The presence and physiological role of primary cilia in other retinal cells have not been clearly elucidated. Müller cells are the main glia of the retina where they exert distinct functions to maintain homeostasis. In pathological conditions, Müller cells mount a unique regenerative response through the processes of dedifferentiation, proliferation, and differentiation into neuronal lineages. The involvement of IFT proteins or a primary cilium in these processes has not been explored. In this study, we used mature Müller glia primary cultures to reveal the presence of the primary cilia by immunoreactivity to acetylated α-tubulin and γ-tubulin, which localize to the axoneme and ciliar basal body, respectively. We demonstrate that si-RNA-mediated downregulation of IFT20 gene expression, a main component of the IFT machinery, blocks Shh-induced Müller cell proliferation. We present evidence that IFT20 ablation impairs the dedifferentiation capacity of Müller cells induced by Shh and by glutamate. Our demonstration that Müller glia expresses IFT20 and harbors primary cilia, and opens new venues of research on the role of primary cilia in the retina.

  2. Patient safety culture in primary care

    NARCIS (Netherlands)

    Verbakel, N.J.

    2015-01-01

    Background A constructive patient safety culture is a main prerequisite for patient safety and improvement initiatives. Until now, patient safety culture (PSC) research was mainly focused on hospital care, however, it is of equal importance in primary care. Measuring PSC informs practices on their

  3. Characterization of adult rat astrocyte cultures.

    Directory of Open Access Journals (Sweden)

    Débora Guerini Souza

    Full Text Available Astrocytes, a major class of glial cells, regulate neurotransmitter systems, synaptic processing, ion homeostasis, antioxidant defenses and energy metabolism. Astrocyte cultures derived from rodent brains have been extensively used to characterize astrocytes' biochemical, pharmacological and morphological properties. The aims of this study were to develop a protocol for routine preparation and to characterize a primary astrocyte culture from the brains of adult (90 days old Wistar rats. For this we used enzymatic digestion (trypsin and papain and mechanical dissociation. Medium exchange occurred from 24 h after obtaining a culture and after, twice a week up to reach the confluence (around the 4(th to 5(th week. Under basal conditions, adult astrocytes presented a polygonal to fusiform and flat morphology. Furthermore, approximately 95% the cells were positive for the main glial markers, including GFAP, glutamate transporters, glutamine synthetase and S100B. Moreover, the astrocytes were able to take up glucose and glutamate. Adult astrocytes were also able to respond to acute H2O2 exposure, which led to an increase in reactive oxygen species (ROS levels and a decrease in glutamate uptake. The antioxidant compound resveratrol was able to protect adult astrocytes from oxidative damage. A response of adult astrocytes to an inflammatory stimulus with LPS was also observed. Changes in the actin cytoskeleton were induced in stimulated astrocytes, most likely by a mechanism dependent on MAPK and Rho A signaling pathways. Taken together, these findings indicate that the culture model described in this study exhibits the biochemical and physiological properties of astrocytes and may be useful for elucidating the mechanisms related to the adult brain, exploring changes between neonatal and adult astrocytes, as well as investigating compounds involved in cytotoxicity and cytoprotection.

  4. Exploring patient safety culture in primary care

    NARCIS (Netherlands)

    Verbakel, N.J.; van Melle, M.; Langelaan, M.; Verheij, T.J.; Wagner, C.; Zwart, D.L.

    2014-01-01

    Objective. To explore perceptions of safety culture in nine different types of primary care professions and to study possible differences. Design. Cross-sectional survey. Setting. Three hundred and thirteen practices from nine types of primary care profession groups in the Netherlands. Participants.

  5. Effect of 1,25(OH)2 vitamin D3 and ionized Ca2+ on 45Ca uptake by primary cultures of aortic myocytes of spontaneously hypertensive and Wistar Kyoto normotensive rats

    International Nuclear Information System (INIS)

    Bukoski, R.D.; Xue, H.; McCarron, D.A.

    1987-01-01

    The effect of several regulators of whole animal Ca 2+ homeostasis on 45 Ca uptake by primary cultures of aortic myocytes isolated from spontaneously hypertensive (SHR) and normotensive Wistar Kyoto (WKY) rats was examined. Exposure of confluent cells to 1.0, 1.25 or 1.50 mM ionized Ca 2+ in serum-free medium for seven days resulted in increased 45 Ca uptake at the higher concentrations of Ca 2+ in cells of the SHR but not the WKY. 1,25 (OH)2 vitamin D3 (1 ng/ml) for 7 days caused enhanced influx in cells from both the SHR and WKY while parathyroid hormone (1-34) (1 ng/ml) was without effect. The data indicate that humoral factors that serve to regulate whole animal Ca 2+ homeostasis may also play a role in the regulation of Ca 2+ metabolism of the vascular smooth muscle cell

  6. The blood-brain barrier in vitro using primary culture

    DEFF Research Database (Denmark)

    Larsen, Annette Burkhart

    of the thesis involves the establishment and characterization of an in vitro BBB models based on primary cells isolated from the rat brain. Co-culture and triple culture models with astrocytes and pericytes were found to be the superior to mono cultured BCECs with respect to many important BBB characteristics...... obstacle for the treatment of central nervous system (CNS) diseases, as many potentially CNS active drugs are unable to reach their site of action within the brain. In vitro BBB models are, therefore, being developed to investigate the BBB permeability of a drug early in its development. The first part....... In the second part of the thesis, the ability of turning BCECs into protein factories is investigated using a non-viral gene carrier. Transfection and protein synthesis of BCECs cultured with confined BBB properties were found to be feasible without disrupting the BBB properties, although it was not possible...

  7. Effects of Ghrelin on Triglyceride Accumulation and Glucose Uptake in Primary Cultured Rat Myoblasts under Palmitic Acid-Induced High Fat Conditions

    Directory of Open Access Journals (Sweden)

    Lingling Han

    2015-01-01

    Full Text Available This study aimed to study the effects of acylated ghrelin on glucose and triglyceride metabolism in rat myoblasts under palmitic acid- (PA- induced high fat conditions. Rat myoblasts were treated with 0, 10−11, 10−9, or 10−7 M acylated ghrelin and 0.3 mM PA for 12 h. Triglyceride accumulation was determined by Oil-Red-O staining and the glycerol phosphate dehydrogenase-peroxidase enzymatic method, and glucose uptake was determined by isotope tracer. The glucose transporter 4 (GLUT4, AMP-activated protein kinase (AMPK, acetyl-CoA carboxylase (ACC, and uncoupling protein 3 (UCP3 were assessed by RT-PCR and western blot. Compared to 0.3 mM PA, ghrelin at 10−9 and 10−7 M reduced triglyceride content (5.855 ± 0.352 versus 5.030 ± 0.129 and 4.158 ± 0.254 mM, P<0.05 and prevented PA-induced reduction of glucose uptake (1.717 ± 0.264 versus 2.233 ± 0.333 and 2.333 ± 0.273 10−2 pmol/g/min, P<0.05. The relative protein expression of p-AMPKα/AMPKα, UCP3, and p-ACC under 0.3 mM PA was significantly reduced compared to controls (all P<0.05, but those in the 10−9 and 10−7 M ghrelin groups were significantly protected from 0.3 mM PA (all P<0.05. In conclusion, acylated ghrelin reduced PA-induced triglyceride accumulation and prevented the PA-induced decrease in glucose uptake in rat myoblasts. These effects may involve fatty acid oxidation.

  8. Effects of two medicinal plants Psidium guajava L. (Myrtaceae) and Diospyros mespiliformis L. (Ebenaceae) leaf extracts on rat skeletal muscle cells in primary culture

    Science.gov (United States)

    Belemtougri, R.G.; Constantin, B.; Cognard, C.; Raymond, G.; Sawadogo, L.

    2006-01-01

    Crude decoction, aqueous and ethanolic extracts of two medicinal plants (Psidium guajava and Diospyros mespiliformis), widely used in the central plateau of Burkina Faso to treat many diseases were evaluated for their antagonistic effects on caffeine induced calcium release from sarcoplasmic reticulum of rat skeletal muscle cells. These different extracts showed a decrease of caffeine induced calcium release in a dose dependent manner. Comparison of the results showed that Psidium guajava leaf extracts are more active than extracts of Diospyros mespiliformis and that crude decoctions show better inhibitory activity. The observed results could explaine their use as antihypertensive and antidiarrhoeal agents in traditional medicine, by inhibiting intracellular calcium release. PMID:16365927

  9. Inhibition of Primary Clinical Isolates of Human Parainfluenza Virus by DAS181 in Cell Culture and in a Cotton Rat Model

    OpenAIRE

    Jones, B. G.; Hayden, R.T.; Hurwitz, J. L.

    2013-01-01

    DAS181 is a novel drug in development for the treatment of influenza as well as human parainfluenza viruses (hPIV). Previous studies demonstrated that DAS181 inhibited laboratory strains of hPIV, but no tests were conducted with primary clinical isolates of hPIV. To fill this gap, we studied six primary isolates including hPIV-2 and hPIV-3. First tests showed that the amplification of all viruses in vitro was reproducibly inhibited with DAS181 drug concentrations ranging between 0.1 and 1 nM....

  10. Comparative sensitivity of human and rat neural cultures to chemical-induced inhibition of neurite outgrowth

    International Nuclear Information System (INIS)

    Harrill, Joshua A.; Freudenrich, Theresa M.; Robinette, Brian L.; Mundy, William R.

    2011-01-01

    There is a need for rapid, efficient and cost-effective alternatives to traditional in vivo developmental neurotoxicity testing. In vitro cell culture models can recapitulate many of the key cellular processes of nervous system development, including neurite outgrowth, and may be used as screening tools to identify potential developmental neurotoxicants. The present study compared primary rat cortical cultures and human embryonic stem cell-derived neural cultures in terms of: 1) reproducibility of high content image analysis based neurite outgrowth measurements, 2) dynamic range of neurite outgrowth measurements and 3) sensitivity to chemicals which have been shown to inhibit neurite outgrowth. There was a large increase in neurite outgrowth between 2 and 24 h in both rat and human cultures. Image analysis data collected across multiple cultures demonstrated that neurite outgrowth measurements in rat cortical cultures were more reproducible and had higher dynamic range as compared to human neural cultures. Human neural cultures were more sensitive than rat cortical cultures to chemicals previously shown to inhibit neurite outgrowth. Parallel analysis of morphological (neurite count, neurite length) and cytotoxicity (neurons per field) measurements were used to detect selective effects on neurite outgrowth. All chemicals which inhibited neurite outgrowth in rat cortical cultures did so at concentrations which did not concurrently affect the number of neurons per field, indicating selective effects on neurite outgrowth. In contrast, more than half the chemicals which inhibited neurite outgrowth in human neural cultures did so at concentrations which concurrently decreased the number of neurons per field, indicating that effects on neurite outgrowth were secondary to cytotoxicity. Overall, these data demonstrate that the culture models performed differently in terms of reproducibility, dynamic range and sensitivity to neurite outgrowth inhibitors. While human neural

  11. Calcium ionophore A23187 specifically decreases the secretion of beta-secretase cleaved amyloid precursor protein during apoptosis in primary rat cortical cultures

    DEFF Research Database (Denmark)

    Sennvik, K; Benedikz, Eirikur; Fastbom, J

    2001-01-01

    - and gamma-secretase cleavage. Alternatively, APP may be cleaved within the A beta region by alpha-secretase, preventing A beta formation. Here we investigated APP processing and secretion in primary neurons, using either colchicine or the calcium ionophore A23187 to induce apoptosis. Cell viability...... beta sAPP) did not change. In addition, caspase inhibition restored cell viability to control levels. Exposure to colchicine did not change the amount of either secreted beta-sAPP or total sAPP and caspase inhibition was only partially able to restore cell viability. We conclude that calcium...

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

    Directory of Open Access Journals (Sweden)

    Martin Aldasoro

    Full Text Available 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.

  13. Reciprocal effects of epidermal growth factor on key lipogenic enzymes in primary cultures of adult rat hepatocytes. Induction of glucose-6-phosphate dehydrogenase and suppression of malic enzyme and lipogenesis.

    Science.gov (United States)

    Yoshimoto, K; Nakamura, T; Ichihara, A

    1983-10-25

    In primary cultured hepatocytes of adult rats epidermal growth factor (EGF) caused 2- to 3-fold induction of glucose-6-phosphate dehydrogenase (EC 1.1.1.49, G6P dehydrogenase) within 2 days. The effect of EGF was additive with a similar effect of insulin. The half-maximum dose of EGF for the induction was 1 ng/ml. Induction of this enzyme by these hormones was shown by immunotitration to be due to increase of the amount of enzyme. Furthermore, this increase in the amount of enzyme was found to result from increase of syntheses of mRNA and enzyme protein. In contrast, the induction of malic enzyme (EC 1.1.1.40, L-malate:NADP+) oxidoreductase) by insulin plus triiodothyronine was strongly suppressed by the concomitant addition of EGF. Induction of G6P dehydrogenase by EGF, like that by insulin, was not suppressed by either glucagon or dibutyryl cAMP, whereas that of malic enzyme was suppressed additively by EGF and dibutyryl cAMP. EGF also suppressed stimulation of lipogenesis by insulin, measured as incorporation of [1-14C]acetate into triglycerides and phospholipids. Another difference between the inductions of G6P dehydrogenase and malic enzyme was in their dependence on cell density; G6P dehydrogenase induction by insulin and EGF was high at low cell density (3 X 10(4) cells/cm2) and less at higher cell density (13 X 10(4) cells/cm2), whereas induction of malic enzyme was high at higher cell density and less at lower cell density. These results are consistent with the dual role of G6P dehydrogenase in lipogenesis in resting cells and in synthesis of nucleic acid in growing cells. Malic enzyme plays a role only for lipogenesis in mature hepatocytes.

  14. Culture technique of rabbit primary epidermal keratinocytes

    Directory of Open Access Journals (Sweden)

    Marini M

    2012-10-01

    Full Text Available The epidermis is the protective covering outer layer of the mammalian skin. The epidermal cells are stratified squamous epithelia which undergo continuous differentiation of loss and replacement of cells. Ninety per cent of epidermal cells consist of keratinocytes that are found in the basal layer of the stratified epithelium called epidermis. Keratinocytes are responsible for forming tight junctions with the nerves of the skin as well as in the process of wound healing. This article highlights the method of isolation and culture of rabbit primary epidermal keratinocytes in vitro. Approximately 2cm x 2cm oval shaped line was drawn on the dorsum of the rabbit to mark the surgical area. Then, the skin was carefully excised using a surgical blade and the target skin specimens harvested from the rabbits were placed in transport medium comprising of Dulbecco’s Modified Eagle Medium (DMEM and 1% of antibiotic-antimycotic solution. The specimens were transferred into a petri dish containing 70% ethanol and washed for 5 min followed by a wash in 1 x Dulbecco’s Phosphate Buffered Saline (DBPS. Then, the skin specimens were placed in DMEM and minced into small pieces using a scalpel. The minced pieces were placed in a centrifuge tube containing 0.6% Dispase and 1% antibiotic-antimycotic solution overnight at 4°C in a horizontal orientation. The epidermis layer (whitish, semi-transparent was separated from the dermis (pink, opaque, gooey with the aid of curved forceps by fixing the dermis with one pair of forceps while detaching the epidermis with the second pair. The cells were cultured at a density of 4 x 104 cells/cm2 in culture flask at 37°C and 5% CO2. The cell morphology of the keratinocytes was analyzed using inverted microscope.

  15. Long-term organ culture of adult rat colon

    DEFF Research Database (Denmark)

    Shamsuddin, A.K.M.; Barrett, L.A.; Autrup, Herman

    1978-01-01

    Colon explants from adult rats were maintained in culture for over 3 months in our laboratories with good epithelial preservation and cellular differentiation. The light and transmission electron microscopic features of rat colon mucosa during the culture period are described. In all the explants...

  16. Stimulation of DNA synthesis in cultured rat alveolar type II cells

    International Nuclear Information System (INIS)

    Leslie, C.C.; McCormick-Shannon, K.; Robinson, P.C.; Mason, R.J.

    1985-01-01

    Restoration of the alveolar epithelium after injury is thought to be dependent on the proliferation of alveolar type II cells. To understand the factors that may be involved in promoting type II cell proliferation in vivo, we determined the effect of potential mitogens and culture substrata on DNA synthesis in rat alveolar type II cells in primary culture. Type II cells cultured in basal medium containing 10% fetal bovine serum (FBS) exhibited essentially no DNA synthesis. Factors that stimulated 3 H-thymidine incorporation included cholera toxin, epidermal growth factor, and rat serum. The greatest degree of stimulation was achieved by plating type II cells on an extracellular matrix prepared from bovine corneal endothelial cells and then by culturing the pneumocytes in medium containing rat serum, cholera toxin, insulin, and epidermal growth factor. Under conditions of stimulation of 3 H-thymidine incorporation there was an increased DNA content per culture dish but no increase in cell number. The ability of various culture conditions to promote DNA synthesis in type II cells was verified by autoradiography. Type II cells were identified by the presence of cytoplasmic inclusions, which were visualized by tannic acid staining before autoradiography. These results demonstrate the importance of soluble factors and culture substratum in stimulating DNA synthesis in rat alveolar type II cells in primary culture

  17. MS-377, a selective sigma receptor ligand, indirectly blocks the action of PCP in the N-methyl-D-aspartate receptor ion-channel complex in primary cultured rat neuronal cells.

    Science.gov (United States)

    Karasawa, Jun-ichi; Yamamoto, Hideko; Yamamoto, Toshifumi; Sagi, Naoki; Horikomi, Kazutoshi; Sora, Ichiro

    2002-02-22

    MS-377 ((R)-(+)-1-(4-chlorophenyl)-3-[4-(2-methoxyethyl)piperazin-1-yl]methyl-2-pyrrolidinone L-tartrate) is a antipsychotic agent that binds to sigma-1 receptor. MS-377 showed anti-dopaminergic and anti-serotonergic activities and antagonistic action against phencyclidine (PCP)-induced behaviors in an animal model. These anti-psychotic activities of MS-377 are attributable to association with sigma-1 receptor. However, the mechanism by which the sigma-1 receptor ligands exact those numerous effects remains to be elucidated. In the present study, we evaluated the effect of MS-377 on N-methyl-D-aspartate (NMDA) receptor ion-channel complex in primary cultured rat neuronal cells. First, we examined the effect of MS-377 on NMDA-induced Ca2+ influx with fura-2/ AM loaded cells. MS-377 showed no effects on the basal Ca2+ concentration and NMDA-induced Ca2+ influx by itself PCP and SKF-10047 reduced the NMDA-induced increase in intracellular Ca2+ concentration. Pre-incubation of 1 microM MS-377 was found to significantly block the reduction by PCP or SKF-10047 of the NMDA-induced Ca2+ influx. Second, the effect of MS-377 on [3H]MK-801 intact cell binding was examined. PCP, haloperidol and (+)-pentazocine inhibited [3H]MK-801 binding, although MS-377 showed no effect by itself Pre-treatment of MS-377 markedly reversed the inhibition of [3H]MK-801 binding by PCP in a dose-dependent manner. These effects of MS-377 may depend on its affinity for the sigma-1 receptor, because MS-377 is a selective sigma-1 receptor ligand without any affinity for NMDA receptor ion-channel complex. These observations suggest that the MS-377 indirectly modulated the NMDA receptor ion-channel complex, and the anti-psychotic activities of MS-377, in part, are attributable to such on action via sigma-1 receptor.

  18. ATP stimulates calcium influx in primary astrocyte cultures

    International Nuclear Information System (INIS)

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

    1988-01-01

    The effect of ATP and other purines on 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 45 Ca. Stimulation of calcium influx by ATP was investigated using a 90 sec exposure to 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 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 45 Ca influx. These findings indicate that one pathway for calcium entry in astrocytes involves purinergic receptor-operated, calcium channels

  19. Rhynchophylline Protects Cultured Rat Neurons against Methamphetamine Cytotoxicity

    Directory of Open Access Journals (Sweden)

    Dan Dan Xu

    2012-01-01

    Full Text Available Rhynchophylline (Rhy is an active component isolated from species of the genus Uncaria which has been used for the treatment of ailments to the central nervous system in traditional Chinese medicine. Besides acting as a calcium channel blocker, Rhy was also reported to be able to protect against glutamate-induced neuronal death. We thus hypothesize that Rhy may have neuroprotective activity against methamphetamine (MA. The primary neurons were cultured directly from the cerebral cortex of neonatal rats, acting as in vitro model in the present study. The neurotoxicity of MA and the protective effect of Rhy were evaluated by MTT assay. The effects of MA, Rhy or their combination on intracellular free calcium concentration ([Ca2+]i were determined in individual neocortical neurons by the Fluo-3/AM tracing method. The MTT assay demonstrated that MA has a dose-dependent neurotoxicity in neuronal cultures. The addition of Rhy prior to the exposure to MA prevented neuronal death. Time course studies with the Fluo-3/AM probe showed that Rhy significantly decreased neuronal [Ca2+]i which was elevated by the exposure to MA. Our results suggested that Rhy can protect the neuronal cultures against MA exposure and promptly attenuate intracellular calcium overload triggered by MA challenge. This is the first report demonstrating an inhibitory effect of Rhy against MA impairment in cultured neurons in vitro.

  20. Coumestrol Counteracts Interleukin-1β-Induced Catabolic Effects by Suppressing Inflammation in Primary Rat Chondrocytes.

    Science.gov (United States)

    You, Jae-Seek; Cho, In-A; Kang, Kyeong-Rok; Oh, Ji-Su; Yu, Sang-Joun; Lee, Gyeong-Je; Seo, Yo-Seob; Kim, Su-Gwan; Kim, Chun Sung; Kim, Do Kyung; Im, Hee-Jeong; Kim, Jae-Sung

    2017-02-01

    In the present study, we investigated the anti-catabolic effects of coumestrol, a phytoestrogen derived from herbal plants, against interleukin-1β-induced cartilage degeneration in primary rat chondrocytes and articular cartilage. Coumestrol did not affect the viability of human normal oral keratinocytes and primary rat chondrocytes treated for 24 h and 21 days, respectively. Although coumestrol did not significantly increase the proteoglycan contents in long-term culture, it abolished the interleukin-1β-induced loss of proteoglycans in primary rat chondrocytes and knee articular cartilage. Furthermore, coumestrol suppressed the expression of matrix-degrading enzymes such as matrix metalloproteinase-13, -3, and -1 in primary rat chondrocytes stimulated with interleukin-1β. Moreover, the expression of catabolic factors such as nitric oxide synthase, cyclooxygenase-2, prostaglandin E 2 , and inflammatory cytokines in interleukin-1β-stimulated primary rat chondrocytes was suppressed by coumestrol. In summary, these results indicate that coumestrol counteracts the catabolic effects induced by interleukin-1β through the suppression of inflammation. Therefore, based on its biological activity and safety profile, coumestrol could be used as a potential anti-catabolic biomaterial for osteoarthritis.

  1. Primary culture and growth characteristics of four different species of lens epithelial cells

    Directory of Open Access Journals (Sweden)

    Li-Xia Ji

    2015-07-01

    Full Text Available AIM: To explore the primary culture conditions for four kinds of lens epithelial cells(LECsof rat, rabbit, dog, and human, and measure their growth characteristics. METHODS: The lens capsule or anterior capsular tissue of rat, rabbit, dog and patient were removed by different methods, and they were cut into tiny pieces for primary culture by modified tissue adherent method. The morphological features of four kinds of LECs were observed under an inverted microscope.RESULTS: Four kinds of LECs of rat, rabbit, dog and human could be cultured primarily by tissue adherent method. With the evolution of tissue source, the adherent capacity of LECs gradually strengthened, cells form were changed from irregular polygon to oval, nucleus rounded and cytoplasm enriched gradually. Four kinds of LECs had fibrotic changes after several passages.CONCLUSION: LECs of rat, rabbit, dog and human can be primarily cultured. This method lays the foundation for the mechanism research of caratact and related fields on the cellular and molecular levels.

  2. Teaching Cultural History from Primary Events

    Science.gov (United States)

    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…

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

    Science.gov (United States)

    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…

  4. Preparation of rat serum suitable for mammalian whole embryo culture.

    Science.gov (United States)

    Takahashi, Masanori; Makino, Sayaka; Kikkawa, Takako; Osumi, Noriko

    2014-08-03

    Mammalian whole embryo culture (WEC) is a widely used technique for examining pharmacological toxicity in developing mouse and rat embryos and for investigating the mechanisms of developmental processes. Immediately centrifuged (IC) rat serum is commonly used for WEC and is essential for the growth and development of cultured mouse and rat embryos ex vivo. For the culture of midgestation embryos (i.e., E8.0-12.5 for the mouse, and E10.0-14.5 for the rat), 100% rat serum is the best media for supporting the growth of the embryo ex vivo. To prepare rat serum suitable for WEC, the collected blood should be centrifuged immediately to separate the blood cells from the plasma fraction. After centrifugation, the fibrin clot forms in the upper layer; this clot should be squeezed gently using a pair of sterile forceps and subsequently centrifuged to completely separate the blood cells from the serum. In this video article, we demonstrate our standard protocol for the preparation of optimal IC rat serum, including blood collection from the abdominal aorta of male rats and extraction of the serum by centrifugation.

  5. Punicalagin inhibits neuroinflammation in LPS-activated rat primary microglia.

    Science.gov (United States)

    Olajide, Olumayokun A; Kumar, Asit; Velagapudi, Ravikanth; Okorji, Uchechukwu P; Fiebich, Bernd L

    2014-09-01

    In this study, the effects of punicalagin on neuroinflammation in LPS-activated microglia were investigated. The ability of punicalagin to reduce the production of TNF-α, IL-6 and prostaglandin E2 was measured in culture medium using enzyme immunoassay. TNF-α and IL-6 gene expression in mouse hippocampal slices was measured with PCR. cyclooxygenase-2 and microsomal prostaglandin E synthase 1 protein and mRNA were evaluated with Western blotting and PCR, respectively. Further experiments to investigate effects of punicalagin on protein expressions of inflammatory targets were also determined with Western blotting. Pretreatment of rat primary microglia with punicalagin (5-40 μM) prior to LPS (10 ng/mL) stimulation produced a significant (p microglia. These results suggest that punicalagin inhibits neuroinflammation in LPS-activated microglia through interference with NF-κB signalling, suggesting its potential as a nutritional preventive strategy in neurodegenerative disorders. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Rat intermediate lobe in culture: a histological and biochemical characterization.

    Science.gov (United States)

    Chronwall, B M; Bishop, J F; Gehlert, D R

    1988-01-01

    The histology, immunohistochemistry, peptide synthesis and secretion as well as proliferation rate of rat intermediate lobe (IL) were studied in primary cultures. The cultures contained two populations of cells: melanotrophs either organized in free floating lobules or in lobules which attached to the dishes and formed a monolayer. Both populations retained their in vivo morphology: polyhedral cells with smooth, ovoid nuclei and a large number of cytoplasmic secretory coated vesicles, a well developed Golgi apparatus, abundant mitochondria and extensive areas of rough endoplasmic reticulum. The melanotrophs stained with varying intensity for alpha-MSH and in situ hybridization showed the presence of pro-opiomelanocortin (POMC) mRNA. 35S-methionine incorporation combined with 2-D gel electrophoresis demonstrated POMC peptide synthesis and radioimmunoassay confirmed its secretion into the medium. 3H-thymidine uptake in the attached melanotrophs was considerably higher than that in the free-floating melanotrophs, demonstrating the dependency of proliferation rate on the cytoarchitecture of the explant. The retention of melanotroph morphology, biosynthetic and proliferative capacity in vitro affords a valid model system for studying POMC gene expression.

  7. Radiosensitivity of primary cultured fish cells with different ploidy

    International Nuclear Information System (INIS)

    Mitani, Hiroshi; Egami, Nobuo; Kobayashi, Hiromu.

    1986-01-01

    The radiosensitivity of primary cultured goldfish cells (Carassius auratus) was investigated by colony formation assay. The radiosensitivity of cells from two varieties of goldfish, which show different sensitivity to lethal effect of ionizing radiation in vivo, was almost identical. Primary cultured cells from diploid, triploid and tetraploid fish retained their DNA content as measured by microfluorometry, and the nuclear size increases as ploidy increases. However, radiosensitivity was not related to ploidy. (author)

  8. 3D hepatic cultures simultaneously maintain primary hepatocyte and liver sinusoidal endothelial cell phenotypes.

    Directory of Open Access Journals (Sweden)

    Yeonhee Kim

    Full Text Available Developing in vitro engineered hepatic tissues that exhibit stable phenotype is a major challenge in the field of hepatic tissue engineering. However, the rapid dedifferentiation of hepatic parenchymal (hepatocytes and non-parenchymal (liver sinusoidal endothelial, LSEC cell types when removed from their natural environment in vivo remains a major obstacle. The primary goal of this study was to demonstrate that hepatic cells cultured in layered architectures could preserve or potentially enhance liver-specific behavior of both cell types. Primary rat hepatocytes and rat LSECs (rLSECs were cultured in a layered three-dimensional (3D configuration. The cell layers were separated by a chitosan-hyaluronic acid polyelectrolyte multilayer (PEM, which served to mimic the Space of Disse. Hepatocytes and rLSECs exhibited several key phenotypic characteristics over a twelve day culture period. Immunostaining for the sinusoidal endothelial 1 antibody (SE-1 demonstrated that rLSECs cultured in the 3D hepatic model maintained this unique feature over twelve days. In contrast, rLSECs cultured in monolayers lost their phenotype within three days. The unique stratified structure of the 3D culture resulted in enhanced heterotypic cell-cell interactions, which led to improvements in hepatocyte functions. Albumin production increased three to six fold in the rLSEC-PEM-Hepatocyte cultures. Only rLSEC-PEM-Hepatocyte cultures exhibited increasing CYP1A1/2 and CYP3A activity. Well-defined bile canaliculi were observed only in the rLSEC-PEM-Hepatocyte cultures. Together, these data suggest that rLSEC-PEM-Hepatocyte cultures are highly suitable models to monitor the transformation of toxins in the liver and their transport out of this organ. In summary, these results indicate that the layered rLSEC-PEM-hepatocyte model, which recapitulates key features of hepatic sinusoids, is a potentially powerful medium for obtaining comprehensive knowledge on liver metabolism

  9. Comparative Analysis of Human and Rodent Brain Primary Neuronal Culture Spontaneous Activity Using Micro-Electrode Array Technology.

    Science.gov (United States)

    Napoli, Alessandro; Obeid, Iyad

    2016-03-01

    Electrical activity in embryonic brain tissue has typically been studied using Micro Electrode Array (MEA) technology to make dozens of simultaneous recordings from dissociated neuronal cultures, brain stem cell progenitors, or brain slices from fetal rodents. Although these rodent neuronal primary culture electrical properties are mostly investigated, it has not been yet established to what extent the electrical characteristics of rodent brain neuronal cultures can be generalized to those of humans. A direct comparison of spontaneous spiking activity between rodent and human primary neurons grown under the same in vitro conditions using MEA technology has never been carried out before and will be described in the present study. Human and rodent dissociated fetal brain neuronal cultures were established in-vitro by culturing on a glass grid of 60 planar microelectrodes neurons under identical conditions. Three different cultures of human neurons were produced from tissue sourced from a single aborted fetus (at 16-18 gestational weeks) and these were compared with seven different cultures of embryonic rat neurons (at 18 gestational days) originally isolated from a single rat. The results show that the human and rodent cultures behaved significantly differently. Whereas the rodent cultures demonstrated robust spontaneous activation and network activity after only 10 days, the human cultures required nearly 40 days to achieve a substantially weaker level of electrical function. These results suggest that rat neuron preparations may yield inferences that do not necessarily transfer to humans. © 2015 Wiley Periodicals, Inc.

  10. Exploring patient safety culture in Dutch primary care.

    NARCIS (Netherlands)

    Verbakel, N.J.; Melle, M. van; Langelaan, M.; Verheij, T.J.M.; Wagner, C.; Zwart, D.L.M.

    2014-01-01

    Objective: To explore perceptions of safety culture in nine different types of primary care professions and to study possible differences. Design Cross-sectional survey: Setting: Three hundred and thirteen practices from nine types of primary care profession groups in the Netherlands. Participants:

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

    Science.gov (United States)

    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. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. Gene expression as a sensitive endpoint to evaluate cell differentiation and maturation of the developing central nervous system in primary cultures of rat cerebellar granule cells (CGCs) exposed to pesticides

    International Nuclear Information System (INIS)

    Hogberg, Helena T.; Kinsner-Ovaskainen, Agnieszka; Hartung, Thomas; Coecke, Sandra; Bal-Price, Anna K.

    2009-01-01

    The major advantage of primary neuronal cultures for developmental neurotoxicity (DNT) testing is their ability to replicate the crucial stages of neurodevelopment. In our studies using primary culture of cerebellar granule cells (CGCs) we have evaluated whether the gene expression relevant to the most critical developmental processes such as neuronal differentiation (NF-68 and NF-200) and functional maturation (NMDA and GABA A receptors), proliferation and differentiation of astrocytes (GFAP and S100β) as well as the presence of neural precursor cells (nestin and Sox10) could be used as an endpoint for in vitro DNT. The expression of these genes was assessed after exposure to various pesticides (paraquat parathion, dichlorvos, pentachlorophenol and cycloheximide) that could induce developmental neurotoxicity through different mechanisms. All studied pesticides significantly modified the expression of selected genes, related to the different stages of neuronal and/or glial cell development and maturation. The most significant changes were observed after exposure to paraquat and parathion (i.e. down-regulation of mRNA expression of NF-68 and NF-200, NMDA and GABA A receptors). Similarly, dichlorvos affected mainly neurons (decreased mRNA expression of NF-68 and GABA A receptors) whereas cycloheximide had an effect on neurons and astrocytes, as significant decreases in the mRNA expression of both neurofilaments (NF-68 and NF-200) and the astrocyte marker (S100β) were observed. Our results suggest that toxicity induced by pesticides that target multiple pathways of neurodevelopment can be identified by studying expression of genes that are involved in different stages of cell development and maturation, and that gene expression could be used as a sensitive endpoint for initial screening to identify the compounds with the potential to cause developmental neurotoxicity

  13. Effect of hypertensive rat plasma on ion transport of cultured vascular smooth muscle

    International Nuclear Information System (INIS)

    Magargal, W.W.; Overbeck, H.W.

    1986-01-01

    We layered fresh, unprocessed plasma from healthy rats with early (less than or equal to 7 days) or benign, chronic (greater than 3 wk) one-kidney, one-clip hypertension and from paired one-kidney normotensive control rats over confluent primary-cultured rat aortic smooth muscle cells. Plasma from all rats increased cellular ouabain-sensitive 86 Rb + uptake and sodium content and decreased ouabain-insensitive 86 Rb + uptake compared with uptakes and content in the presence of balanced salt solution (P less than 0.01). Cells incubated in the presence of plasma from rats with early (P less than 0.02) or chronic hypertension (P less than 0.01) had significantly reduced ouabain-sensitive 86 Rb + uptake when compared with cells incubated in normotensive plasma, but their intracellular Na+ contents were not lower. We no longer detected this uptake difference when chronic hypertensives drank 0.9% NaCl instead of water. Plasma from hypertensive rats also altered ouabain-insensitive 86 Rb + uptake by the cultured cells. These findings of this new, reproducible, and specific assay system support the hypothesis that plasma factors inhibit the membrane sodium-potassium pump in vascular smooth muscle cells in this form of hypertension. The abnormality occurs in both early and chronic stages, but may not be related to sodium intake. The data also provide evidence for plasma factors in hypertension altering membrane K+ permeability

  14. Regulation of Schwann cell proliferation in cultured segments of the adult rat sciatic nerve

    DEFF Research Database (Denmark)

    Svenningsen, Åsa Fex; Kanje, M

    1998-01-01

    Schwann cell proliferation was studied in cultured segments of the rat sciatic nerve by measurement of [3H] thymidine incorporation or through bromodeoxyuridine-(BrdU)-labelling and immunocytochemistry. The aim was to delineate mechanisms involved in the injury-induced proliferative response...... through the cAMP system. Together, these results show that Schwann cells regulate proliferation differently in an integrated environment, e.g. the nerve structure, than in isolation as primary monocultures....

  15. Lipogenesis in maintenance cultures of rat hepatocytes

    NARCIS (Netherlands)

    Geelen, Math J.H.; Gibson, David M.

    1975-01-01

    Induction of the several enzymes in the liver cytosol catalyzing de novo synthesis of fatty acids from glucose has been demonstrated in intact animals. When carbohydrate is provided to previously starved rats the metabolism of liver switches from a gluconeogenic-ketogenic economy to a

  16. Cultural activities in primary school students' spare time

    Directory of Open Access Journals (Sweden)

    Mikanović Brane

    2014-01-01

    Full Text Available Culture is a form of creative expression of a human being through which he reshapes the world, acts on it adding it value and creating new, cultural values. A human being is able to create a product of culture only when he is free and able to express himself. A contemporary man can incorporate various cultural activities into his spare time. They are especially important when they concern children and young people: regardless of whether they are used in institutional settings or in spare time. The authors conducted an empirical research of students' assumptions and beliefs concerning cultural activities in their free time. The sample comprised 233 fifth grade students. The findings show that in their spare time fifth graders: engage in various cultural activities; that students who live in urban areas attend more cultural events; that students have the opportunity to engage in extra-curricular activities in the area of culture - join cultural and artistic groups and associations and engage in various creative pursuits at different levels of participation (as consumers, full participants; and that students' attitudes concerning the influence of parents and teachers on the selection of cultural activities to be pursued do not vary greatly by gender, location or school achievement. Cultural activities do play a significant part in the free time of primary school students. This is why it is important that guidance provided in school and in spare time should be brought in greaer harmony.

  17. STUDY OF FUTURE PRIMARY SCHOOL TEACHERS’ CULTURAL TRAINING WITHIN THE INFORMATION CULTURE OF SOCIETY

    Directory of Open Access Journals (Sweden)

    Tatiana Vinnyk

    2016-05-01

    Full Text Available The article presents the results of scientific studies and experimental approbation of pedagogical conditions of future primary school teachers’ cultural training taking into account the information culture of society. The nature and structure of the notion «future primary school teachers’ cultural training» are clarified. The indicated phenomenon is considered as the structure of four levels, the core of which is personality’s humanistic orientation, the totality of psychological-pedagogical and cultural knowledge and skills, the complex of professionally significant personal qualities. The author pointed out the criteria and related indicators of cultural proficiency, they are: value-motivational (vocational and humanistic orientation; the presence of values and professional motives; motivation for success; substantial and procedural (knowledge and skills in psycho-pedagogical disciplines; the body of knowledge regarding the content and components of cultural training, cultural skills; assessment and behavioral (the existence of communicative qualities, ability to empathy, tolerance. Levels of future primary school teachers’ cultural readiness: high, average and low are characterized. The experience of ICT using in students’ cultural training is presented. Pedagogical conditions of future primary school teachers’ cultural training in University are identified, their effectiveness is proved by experimental testing

  18. Comparison of prostanoid forming capacity of neuronal and astroglial cells in primary cultures.

    Science.gov (United States)

    Keller, M; Jackisch, R; Seregi, A; Hertting, G

    1985-01-01

    Prostaglandin (PG) and thromboxane (TX) biosynthesis in primary neuronal and astroglial cell cultures was studied. Cultures obtained from fetal (15-16 days old) and neonatal rat brain hemispheres were characterized by chemical and immunocytochemical staining techniques as predominantly neurons or mature and immature astrocytes, respectively. Six-day old neuronal cell cultures grown in the presence of cytosine arabinoside (2 ?M) from the day 3 onwards were contaminated up to 10% with glioblasts. In astroglial cultures up to 3% of the cells were postively stained with a marker for oligodendroglial cells. Fibroblast contamination was below 1% in both cultures. Prostanoid formation (measured by specific radioimmunoassays) in 6-day old neuronal cell cultures was low (sum of the amount of PGs and TX formed: 1.16 +/- 0.17 (ng/mg protein/15 min) as compared to 14-day old cultured astroglial cells: 21.27 +/- 2.53 (ng/mg protein/15 min). Also the pattern of prostanoids formed was different in neuronal (PGD(2) ? PGF(2?) > TXB(2) ? PGE(2)) and astroglial cells (PGD(2) > TXB(2) ? PGF(2?) ? PGE(2) ? 6-ketoPGF(1?)). Preincubation with arachidonic acid (1 ?g/ml) did not affect prostanoid formation in both cultures, whereas it was stimulated 4-6-fold by addition of the calcium ionophore A23187 (1 ?M). These results, although found on cultured neuronal and glial cells of different stages of development, support the view that astroglial cells might play a crucial role in brain prostanoid synthesis.

  19. Enhancing Access to Primary Cultural Heritage Materials of India

    Science.gov (United States)

    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.

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

    OpenAIRE

    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 interviews. Results indicated that although cultural games count as instructional tools in four knowledge domains, actualizing their value in the clas...

  1. A 3-month age difference profoundly alters the primary rat stromal vascular fraction phenotype

    DEFF Research Database (Denmark)

    Quaade, Marlene Louise; Jensen, Charlotte Harken; Andersen, Ditte Caroline

    2016-01-01

    The stromal vascular fraction (SVF) is a heterogeneous population obtained from collagenase digestion of adipose tissue. When cultured the population becomes more homogeneous and the cells are then termed adipose stromal/stem cells (ASCs). Both the freshly isolated primary SVF population...... such as age is demanded. Here we report that even a short age difference has an impact on the phenotype of primary SVF cells. We observed that a 3-month difference in relatively young adult rats affects the expression pattern of several mesenchymal stem cell markers in their primary SVF. The younger animals...... is a very critical parameter that should be taken into account in future stem cell studies, especially when using primary cells....

  2. Achieving primary prevention program objectives through culture change systems.

    Science.gov (United States)

    Allen, J

    1986-12-01

    The appropriate goal of reducing the onset of mental disorder through modifying organic factors, stress, exploitation, coping skills, self-esteem and group support, will require cultural change. Group, organizational, and community norms can stand in the way of prevention efforts. In order to create a more supportive cultural environment for primary prevention, culture change principles must be adopted and a systematic change strategy must be employed. A four phase, Normative Systems model is proposed. The model has shown its utility over its 30 year history. A description of a Normative Systems application with migrant farm workers helps to illustrate Normative Systems project development.

  3. ISOLATION AND PRIMARY CULTURES OF HUMAN INTRAHEPATIC BILE DUCTULAR EPITHELIUM

    Science.gov (United States)

    Demetris, A. J.; Markus, B. H.; Saidman, S.; Fung, J. J.; Makowka, L.; Graner, S.; Duquesnoy, R.; Starzl, T. E.

    2010-01-01

    SUMMARY A technique for the isolation of human intrahepatic bile ductular epithelium, and the establishment of primary cultures using a serum- and growth-factor-supplemented medium combined with a connective tissue substrata is described. Initial cell isolates and monolayer cultures display phenotypic characteristics of biliary epithelial cells (low molecular weight prekeratin positive; albumin, alphafetoprotein, and Factor VIII-related antigen negative). Ultrastructural features of the cultured cells show cell polarization with surface microvilli, numerous interepithelial junctional complexes and cytoplasmic intermediate prekeratin filaments. PMID:3131298

  4. Changes in adipose tissue stromal-vascular cells in primary culture due to porcine sera

    International Nuclear Information System (INIS)

    Jewell, D.E.; Hausman, G.J.

    1986-01-01

    This study was conducted to determine the response of rat stromal-vascular cells to pig sea. Sera were collected from unselected contemporary (lean) and high backfat thickness selected (obese) pigs. Sera from obese pigs were collected either by exsanguination or cannulation. sera from lean pigs during the growing phase (45 kg) and the fattening phase (100-110 kg) were collected. Stromal-vascular cells derived rom rat inguinal tissue were cultured on either 25 cm 2 flasks, collagen-coated coverslips or petri dishes. Cell proliferation was measured by [ 3 H]-thymidine incorporation during the fourth day of culture. Coverslip cultures were used for histochemical analysis. Petri dish cultures were used for analysis of Sn-glycerol-3-phosphate dehydrogenase (GPDH) activity. All cells were plated for 24 hours in media containing 10 fetal bovine sera. Test media contained 2.5, 5.0, 10.0% sera. Sera from obese pigs increased GPDH activity and fat cell production when compared to the lean controls. The increased concentration of sera increased esterase activity and lipid as measured with oil red O. The sera from obese pigs collected at slaughter stimulated more fat cell production than obese sera collected by cannulation. These studies show there are adipogenic factors in obese pigs sera which promote fat cell development in primary cell culture

  5. Changes of muscarinic cholinergic receptors during aging process of primary cultured neutrons

    International Nuclear Information System (INIS)

    Fan Guohuang; Yi Ningyu; Xia Zongqin

    1996-01-01

    The dynamic changes of muscarinic receptor density and its reactivity during aging process in primary cultured neutrons were studied. Muscarinic receptor density was measured by 3 H-QNB binding assay, and muscarinic receptor reactivity was assessed by carbachol stimulation of cGMP formation, the latter was measured by RIA. After 2 weeks' incubation of neonatal rat brain cells, the nutrients began to rupture and the cell bodies shrank markedly showing senescent feature. The muscarinic receptor density reached peak at the 12th day in vitro (12 DIV), but the muscarinic receptor reactivity reached peak at 9 DIV and declined significantly at 12 DIV. The results demonstrated that during aging process of primary cultured neutrons, the decline of muscarinic receptor reactivity is likely prior to the decrease of receptor density

  6. Rat embryonic palatal shelves respond to TCDD in organ culture

    International Nuclear Information System (INIS)

    Abbott, B.D.; Birnbaum, L.S.

    1990-01-01

    TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin), a highly toxic environmental contaminant, is teratogenic in mice, inducing cleft palate (CP) and hydronephrosis at doses which are not overtly maternally or embryo toxic. Palatal shelves of embryonic mice respond to TCDD, both in vivo and in organ culture, with altered differentiation of medial epithelial cells. By contrast, in the rat TCDD produces substantial maternal, embryonic, and fetal toxicity, including fetal lethality, with few malformations. In this study the possible effects of maternal toxicity on induction of cleft palate were eliminated by exposure of embryonic rat palatal shelves in organ culture. The shelves were examined for specific TCDD-induced alterations in differentiation of the medial cells. On Gestation Day (GD) 14 or 15 palatal shelves from embryonic F344 rats were placed in organ culture for 2 to 3 days (IMEM:F12 medium, 5% FBS, 0.1% DMSO) containing 0, 1 x 10(-8), 1 x 10(-9), 1 x 10(-10), or 5 x 10(-11) M TCDD. The medial epithelial peridermal cells degenerated on shelves exposed to control media or 5 x 10(-11) M TCDD. Exposure to 10(-10), 10(-9), and 10(-8) M TCDD inhibited this degeneration in 20, 36, and 60% of the shelves, respectively, and was statistically significant at the two highest doses. A normally occurring decrease in [3H]TdR incorporation was inhibited in some GD 15 shelves cultured with 10(-10) and 10(-9) M TCDD. The medial cells of TCDD-exposed shelves continued to express high levels of immunohistochemically detected EGF receptors. The altered differentiation of rat medial epithelium is similar to that reported for TCDD-exposed mouse medial cells in vivo and in vitro. However, in order to obtain these responses, the cultured rat shelves require much higher concentrations of TCDD than the mouse shelves

  7. Formation of neuromuscular junctions in rat embryo cell cultures

    International Nuclear Information System (INIS)

    Koenig, Jeanine

    1978-01-01

    The morphological evidence of the primary nerve muscle contacts are described. They consist of areas of cholinesterase activity (detected histochemically) localized on the myotube membranes and of multiple clusters of ACh receptors whose 125 I-α-bungarotoxin binding sites are revealed by radioautography. After the stage of the primary nerve muscle contacts, some of which seem transient, characteristic neuromuscular junctions appear. These neuromuscular junctions which possess subneural infoldings are similar to the end-plates of the rat in vivo [fr

  8. Endogenous bile acid disposition in rat and human sandwich-cultured hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Marion, Tracy L., E-mail: tracylmarion@qualyst.com [Curriculum in Toxicology, UNC School of Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7270 (United States); Perry, Cassandra H., E-mail: cassandraperry@qualyst.com [Qualyst, Inc., Durham, NC 27713 (United States); St Claire, Robert L., E-mail: bobstclaire@qualyst.com [Qualyst, Inc., Durham, NC 27713 (United States); Brouwer, Kim L.R., E-mail: kbrouwer@unc.edu [Division of Pharmacotherapy and Experimental Therapeutics, UNC Eshelman School of Pharmacy, The University of North Carolina at Chapel Hill, CB 7569 Kerr Hall, Chapel Hill, NC 27599-7569 (United States)

    2012-05-15

    Sandwich-cultured hepatocytes (SCH) are used commonly to investigate hepatic transport protein-mediated uptake and biliary excretion of substrates. However, little is known about the disposition of endogenous bile acids (BAs) in SCH. In this study, four endogenous conjugated BAs common to rats and humans [taurocholic acid (TCA), glycocholic acid (GCA), taurochenodeoxycholic acid (TCDCA), and glycochenodeoxycholic acid (GCDCA)], as well as two BA species specific to rodents (α- and β-tauromuricholic acid; α/β TMCA), were profiled in primary rat and human SCH. Using B-CLEAR{sup ®} technology, BAs were measured in cells + bile canaliculi, cells, and medium of SCH by LC-MS/MS. Results indicated that, just as in vivo, taurine-conjugated BA species were predominant in rat SCH, while glycine-conjugated BAs were predominant in human SCH. Total intracellular BAs remained relatively constant over days in culture in rat SCH. Total BAs in control (CTL) cells + bile, cells, and medium were approximately 3.4, 2.9, and 8.3-fold greater in human than in rat. The estimated intracellular concentrations of the measured total BAs were 64.3 ± 5.9 μM in CTL rat and 183 ± 56 μM in CTL human SCH, while medium concentrations of the total BAs measured were 1.16 ± 0.21 μM in CTL rat SCH and 9.61 ± 6.36 μM in CTL human SCH. Treatment of cells for 24 h with 10 μM troglitazone (TRO), an inhibitor of the bile salt export pump (BSEP) and the Na{sup +}-taurocholate cotransporting polypeptide (NTCP), had no significant effect on endogenous BAs measured at the end of the 24-h culture period, potentially due to compensatory mechanisms that maintain BA homeostasis. These data demonstrate that BAs in SCH are similar to in vivo, and that SCH may be a useful in vitro model to study alterations in BA disposition if species differences are taken into account. -- Highlights: ► Bile acids (BAs) were measured in rat and human sandwich-cultured hepatocytes (SCH). ► Cell and medium BA

  9. Endogenous bile acid disposition in rat and human sandwich-cultured hepatocytes

    International Nuclear Information System (INIS)

    Marion, Tracy L.; Perry, Cassandra H.; St Claire, Robert L.; Brouwer, Kim L.R.

    2012-01-01

    Sandwich-cultured hepatocytes (SCH) are used commonly to investigate hepatic transport protein-mediated uptake and biliary excretion of substrates. However, little is known about the disposition of endogenous bile acids (BAs) in SCH. In this study, four endogenous conjugated BAs common to rats and humans [taurocholic acid (TCA), glycocholic acid (GCA), taurochenodeoxycholic acid (TCDCA), and glycochenodeoxycholic acid (GCDCA)], as well as two BA species specific to rodents (α- and β-tauromuricholic acid; α/β TMCA), were profiled in primary rat and human SCH. Using B-CLEAR ® technology, BAs were measured in cells + bile canaliculi, cells, and medium of SCH by LC-MS/MS. Results indicated that, just as in vivo, taurine-conjugated BA species were predominant in rat SCH, while glycine-conjugated BAs were predominant in human SCH. Total intracellular BAs remained relatively constant over days in culture in rat SCH. Total BAs in control (CTL) cells + bile, cells, and medium were approximately 3.4, 2.9, and 8.3-fold greater in human than in rat. The estimated intracellular concentrations of the measured total BAs were 64.3 ± 5.9 μM in CTL rat and 183 ± 56 μM in CTL human SCH, while medium concentrations of the total BAs measured were 1.16 ± 0.21 μM in CTL rat SCH and 9.61 ± 6.36 μM in CTL human SCH. Treatment of cells for 24 h with 10 μM troglitazone (TRO), an inhibitor of the bile salt export pump (BSEP) and the Na + -taurocholate cotransporting polypeptide (NTCP), had no significant effect on endogenous BAs measured at the end of the 24-h culture period, potentially due to compensatory mechanisms that maintain BA homeostasis. These data demonstrate that BAs in SCH are similar to in vivo, and that SCH may be a useful in vitro model to study alterations in BA disposition if species differences are taken into account. -- Highlights: ► Bile acids (BAs) were measured in rat and human sandwich-cultured hepatocytes (SCH). ► Cell and medium BA concentrations

  10. PROBLEMS OF PROPEDEVTIKA OF INFORMATION CULTURE IN THE PRIMARY SCHOOL

    Directory of Open Access Journals (Sweden)

    Svitlana M. Ivanova

    2010-08-01

    Full Text Available The article is devoted to the analysis of experience of countries in which propedevtika of informative culture is conducted in the primary classes of general school. Basic problems are selected and the ways of their decision are offered.

  11. Improving Patient Safety Culture in Primary Care: A Systematic Review

    NARCIS (Netherlands)

    Verbakel, Natasha J.; Langelaan, Maaike; Verheij, Theo J. M.; Wagner, Cordula; Zwart, Dorien L. M.

    Background: Patient safety culture, described as shared values, attitudes and behavior of staff in a health-care organization, gained attention as a subject of study as it is believed to be related to the impact of patient safety improvements. However, in primary care, it is yet unknown, which

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

    Science.gov (United States)

    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…

  13. Eyewitness Culture and History: Primary Written Sources. The Iconoclast.

    Science.gov (United States)

    McMurtry, John

    1995-01-01

    Asserts that contemporary history and historiography is "official" history that ignores the daily struggles of people for their continued survival. Argues that, while public illiteracy has nearly disappeared, individuals are ignorant of the wealth of primary-source materials of other cultures' histories. (CFR)

  14. Transcriptome analysis of primary bovine extra-embryonic cultured cells

    Directory of Open Access Journals (Sweden)

    Séverine A. Degrelle

    2015-12-01

    Full Text Available The dataset described in this article pertains to the article by Hue et al. (2015 entitled “Primary bovine extra-embryonic cultured cells: A new resource for the study of in vivo peri-implanting phenotypes and mesoderm formation” [1]. In mammals, extra-embryonic tissues are essential to support not only embryo patterning but also embryo survival, especially in late implanting species. These tissues are composed of three cell types: trophoblast (bTCs, endoderm (bXECs and mesoderm (bXMCs. Until now, it is unclear how these cells interact. In this study, we have established primary cell cultures of extra-embryonic tissues from bovine embryos collected at day-18 after artificial insemination. We used our homemade bovine 10K array (GPL7417 to analyze the gene expression profiles of these primary extra-embryonic cultured cells compared to the corresponding cells from in vivo micro-dissected embryos. Here, we described the experimental design, the isolation of bovine extra-embryonic cell types as well as the microarray expression analysis. The dataset has been deposited in Gene Expression Omnibus (GEO (accession number GSE52967. Finally, these primary cell cultures were a powerful tool to start studying their cellular properties, and will further allow in vitro studies on cellular interactions among extra-embryonic tissues, and potentially between extra-embryonic vs embryonic tissues.

  15. Lack of direct mitogenic activity of dichloroacetate and trichloroacetate in cultured rat hepatocytes

    International Nuclear Information System (INIS)

    Walgren, Jennie L.; Kurtz, David T.; McMillan, JoEllyn M.

    2005-01-01

    Dichloroacetate (DCA) and trichloroacetate (TCA) are hepatocarcinogenic metabolites of the common groundwater contaminant, 1,1,2-trichloroethylene. DCA and TCA have been shown to induce hepatocyte proliferation in vivo, but it is not known if this response is the result of direct mitogenic activity or whether cell replication occurs indirectly in response to tissue injury or inflammation. In this study we used primary cultures of rat hepatocytes, a species susceptible to DCA- but not TCA-induced hepatocarcinogenesis, to determine whether DCA and TCA are direct hepatocyte mitogens. Rat hepatocytes, cultured in growth factor-free medium, were treated with 0.01-1.0 mM DCA or TCA for 10-40 h; cell replication was then assessed by measuring incorporation of 3 H-thymidine into DNA and by cell counts. DCA or TCA treatment did not alter 3 H-thymidine incorporation in the cultured hepatocytes. Although an increase in cell number was not observed, DCA treatment significantly abrogated the normal background cell loss, suggesting an ability to inhibit apoptotic cell death in primary hepatocyte cultures. Furthermore, treatment with DCA synergistically enhanced the mitogenic response to epidermal growth factor. The data indicate that DCA and TCA are not direct mitogens in hepatocyte cultures, which is of interest in view of their ability to stimulate hepatocyte replication in vivo. Nevertheless, the synergistic enhancement of epidermal growth factor-induced hepatocyte replication by DCA is of particular interest and warrants further study

  16. Regulation of Taurine transporter activity in cultured rat retinal ganglion cells and rat retinal Muller Cells

    International Nuclear Information System (INIS)

    Eissa, Laila A.; Smith, Sylvia B.; El-sherbeny, Amira A.

    2006-01-01

    Diabetic retinopathy is one of the most common complications of diabetes. The amino acid taurine is believed to play an antioxidant protective role in diabetic retinopathy through the scavenging of the reactive species. It is not well established whether taurine uptake is altered in retina cells during diabetic conditions. Thus, the present study was designed to investigate the changes in taurine transport in cultures of rat retinal Muller cells and rat retinal ganglion cells under conditions associated with diabetes. Taurine was abundantly taken up by retinal Muller cells and rat retinal ganglion cells under normal glycemic condition. Taurine was actively transported to rat Muller cells and rat retinal ganglion cells in a Na and Cl dependant manner. Taurine uptake further significantly elevated in both type of cells after the incubation with high glucose concentration. This effect could be attributed to the increase in osmolarity. Because Nitric Oxide (NO) is a molecule implicated in the pathogenesis of diabetes, we also determined the activity of taurine transporter in cultured rat retinal Muller cells and rat retinal ganglion cells in the presence of the NO donors, SIN-1 and SNAP. Taurine uptake was elevated above control value after 24-h incubation with low concentration of NO donors. We finally investigated the ability of neurotoxic glutamate to change taurine transporter activity in both types of cells. Uptake of taurine was significantly increased in rat retinal ganglion cells when only incubated with high concentration of glutamate. Our data provide evidence that taurine transporter is present in cultured rat retinal ganglion and Muller cells and is regulated by hyperosmolarity. The data are relevant to disease such as diabetes and neuronal degeneration where retinal cell volume may dramatically change. (author)

  17. Isolation, culture and intraportal transplantation of rat marrow stromal cell

    International Nuclear Information System (INIS)

    Wang Ping; Wang Jianhua; Yan Zhiping; Li Wentao; Lin Genlai; Hu Meiyu; Wang Yanhong

    2004-01-01

    Objective: To observe the tracing and evolution of marrow stromal cell (MSC) after intraportal transplantation into the liver of homogenous rats, and to provide experimental data for MSC differentiation to hepatocyte in vivo. Methods: The MSC was isolated from the leg bone marrow of adult SD rats, and purified by culture-expanded in vitro. Before transplantation, MSC was labeled with DAPI. Then 10 5 MSC were intraportally transplanted into the homogenous rat liver. Rats were killed at 2 hours and 1, 2, 3 and 4 weeks after transplantation. The cryosection samples of liver and lung were observed under fluorescence microscopy. Results: MSC in vitro culture had high ability of proliferation. Except 4 rats were dead because of abdominal bleeding or infection, other recipients were healthy until sacrificed. The implantation cells were detected by identifying the DAPI labeled MSC in the host livers, but not in the host lungs. Conclusion: Intraportal transplanted MSC could immigrate and survive in the host livers at least for 4 weeks. They could immigrate from the small branches of portal veins to hepatic parenchyma

  18. [Characterization of epithelial primary culture from human conjunctiva].

    Science.gov (United States)

    Rivas, L; Blázquez, A; Muñoz-Negrete, F J; López, S; Rebolleda, G; Domínguez, F; Pérez-Esteban, A

    2014-01-01

    To evaluate primary cultures from human conjunctiva supplemented with fetal bovine serum, autologous serum, and platelet-rich autologous serum, over human amniotic membrane and lens anterior capsules. One-hundred and forty-eight human conjunctiva explants were cultured in CnT50(®) supplemented with 1, 2.5, 5 and 10% fetal bovine serum, autologous serum and platelet-rich autologous serum. Conjunctival samples were incubated at 37°C, 5% CO2 and 95% HR, for 3 weeks. The typical phenotype corresponding to conjunctival epithelial cells was present in all primary cultures. Conjunctival cultures had MUC5AC-positive secretory cells, K19-positive conjunctival cells, and MUC4-positive non-secretory conjunctival cells, but were not corneal phenotype (cytokeratin K3-negative) and fibroblasts (CD90-negative). Conjunctiva epithelial progenitor cells were preserved in all cultures; thus, a cell culture in CnT50(®) supplemented with 1 to 5% autologous serum over human amniotic membrane can provide better information of epithelial cell differentiation for the conjunctival surface reconstruction. Copyright © 2013 Sociedad Española de Oftalmología. Published by Elsevier Espana. All rights reserved.

  19. Control of fibronectin synthesis by rat granulosa cells in culture

    International Nuclear Information System (INIS)

    Skinner, M.K.; Dorrington, J.H.

    1984-01-01

    The secreted and cellular [ 35 S]methionine-radiolabeled proteins of cultured rat granulosa cells were separated by electrophoresis on sodium dodecylsulfate (SDS) polyacrylamide gradient slab gels. From 24 to 72 h of culture FSH increased the intensity of labeling of most of the secreted proteins. A 220,000-dalton protein, however, increased in intensity only in control cultures and became the major secreted protein after 72 h, comprising 20% of the total radiolabeled proteins. This protein was identified as fibronectin by immunoprecipitation. There was no increase in the secreted or cellular fibronectin in FSH- or testosterone- and insulin-treated cultures. These studies indicate that a component of extracellular matrix is a major secretory product of unstimulated immature granulosa cells. As hormones induce the differentiated functions of granulosa cells in culture, the secretion of fibronectin is inhibited

  20. Culture-bound syndromes in Hispanic primary care patients.

    Science.gov (United States)

    Bayles, Bryan P; Katerndahl, David A

    2009-01-01

    We sought to document Hispanic primary care patients' knowledge and experience of five culture-bound syndromes (CBS), as well as the basic socio-cultural correlates of these disorders. A convenience sample of 100 adult Hispanic patients presenting in an urban South Texas primary care clinic was recruited to complete a brief cross-sectional survey, presented in an oral format. Interviews sought information concerning five culture-bound syndromes--susto, empacho, nervios, mal de ojo, and ataques de nervios. Additional demographic, socio-economic, and acculturation data was collected. Descriptive and bivariate statistics (chi square, Fisher's) were used to assess relationships among variables and experience with each CBS. A multivariate logistic analysis was conducted to determine the possible contributions of age, gender, acculturation, and education to the personal experience of a culture-bound syndrome. Results indicate that 77% of respondents had knowledge of all five syndromes, with 42% reporting having personally experienced at least one CBS. Nervios was the most commonly suffered disorder, being reported by 30 respondents. This was followed, in declining order ofprevalence, by susto, mal de ojo, empacho, and ataques de nervios. Multivariate logistic regression analysis found that higher education beyond high school was associated with a slightly decreased likelihood of reporting having suffered from any culture-bound syndrome. While co-occurrence among these disorders occurred, the patterns of predictors suggest that the co-occurrence is not a reflection of mislabeling of one common syndrome. Knowledge of and experience with culture-bound syndromes is common among Hispanic primary care patients in South Texas. Healthcare providers ought to consider discussing these illnesses in a non-judgmental manner with patients who present with symptoms that are consistent with these syndromes. Future studies, with larger sample sizes, are warranted to elucidate the nature

  1. Primary cultures from the marine sponge Xestospongia muta (Petrosiidae, Haplosclerida).

    Science.gov (United States)

    Richelle-Maurer, Evelyn; Gomez, Raquel; Braekman, Jean-Claude; Van de Vyver, Gisèle; Van Soest, Rob W M; Devijver, Christine

    2003-01-23

    In the context of the investigations on the origin and in vitro production of bioactive compounds, primary cultures were developed from ectosomal and choanosomal cell suspensions from the sponge Xestospongia muta. Dissociated cells aggregated and reorganized into a striking reticulated network of cells, typical for X. muta. Moreover, in some cultures an isotropic reticulation of small spicules, very similar to that found in the ectosome of adult sponges, was observed. Phytohaemagglutinin promoted aggregation and the reorganization of the cells. HPLC analyses revealed that straight-chain acetylenic compounds were recovered from short-term cultures and that they were synthesized during culture. Heterotrophic bacteria were assumed to be involved in the process. Together our results established that X. muta would be an excellent experimental model to study, in laboratory conditions, the differentiation of the skeleton and the in vitro biosynthesis of straight-chain acetylenic compounds.

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

    Science.gov (United States)

    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 cloned embryos with donor cells cultured in RCMEp had a higher birth rate than that of embryos derived from DMEM (P cloned embryos. Copyright © 2015 Elsevier Inc. All rights reserved.

  3. [Colorectal cancer: tissutal explantation and primary cell culture].

    Science.gov (United States)

    Spisni, Roberto; Failli, Alessandra; Orsini, Giulia; Kastsiuchenka, Olga; Natale, Gianfranco; Castagna, Maura; Legitimo, Annalisa; Aghasbabyan, Alekandr; Ambrosini, Carlo Enrico; Consolini, Rita; Miccoli, Paolo

    2009-01-01

    Setting of cellular cultures extracted from colorectal cancer tissue represents a valid model for in vitro study of biological and molecular characteristics of each single tumor finalized to obtain a tailored chemiotherapy. The end point of this study is to create primary cellular cultures from "fresh" cancer tissue in different stages of evolution. Cancer tissue samples are obtained by means of surgical excisional biopsy or by means of semi-automatic biopsy instrument (Sprig-Cut). After having compared different approaches, two experimental protocols have been selected to have the highest number or intact cells: enzimatic digestion with trypsin and explantation. Primary cell culture free of microbic contamination, obtained mainly by means of Spring-Cut methods, underwent immunohistochemical analysis to evaluate what kind of cell have been grown in vitro by measuring the expression of CK20 and GFAP both resulted positive. The possibility of setting a primary cell culture which represents the cancer of each patient allows a pharmacologic and biomolecular study which can contribute to the development of a tailored adjuvant therapy with many advantages for the patient in terms of positive answer to the treatment and reduced toxicity.

  4. A cultural congruence test for primary school students

    Directory of Open Access Journals (Sweden)

    Bayanova L. F.

    2016-12-01

    Full Text Available The study presented in this article relies on the principles of the cultural-historical theory, which defines cultural impact as the main driving force behind psychological development. Based on the assumption that culture is a set of normative situations, the study identifies rules that are typical for primary school students in big Russian cities. These rules are grouped into what we refer to as factors of cultural compliance, which ultimately can be seen as indicators of pupils’ cultural congruence. In specifying the cultural congruence of primary school students, we take into account not only the rules of school life but also the whole range of stable rules for children 7- to 10-years-old. Researchers at the Psychology Institute of the Higher University of the Chinese Academy of Science (Wang, Zhu, & Shi, 2011 call such rules usual or contextually usual. We include rules that govern the behavior of children who have cultural differences, so in this article we are talking about the rules that are typical for children of this age in Russia. The goal of the study was to develop a test to diagnose the level of cultural congruence. The test was exposed to psychometric evaluation for validity, reliability, and discriminatory power. Factor analysis by means of varimax rotation provided for calibration of the rules by consolidating them into factors. These factors underpin the test and include the categories social interaction, academic competence, regulation, obedience, self-service, and self-control. In accordance with the principles employed in psychology, the factors confirm the construct validity of the test in relation to children’s development when they are between 7 and 10 years old. The study confirms that learning is the main activity at this age by introducing a factor that brings together rules inherent in normative situations in the education process. The social setting for psychological development, viewed as a specific

  5. Cultural significance of primary teeth for caregivers in Northeast Brazil.

    Science.gov (United States)

    Nations, Marilyn K; Calvasina, Paola Gondim; Martin, Michele N; Dias, Hilma Fontenele

    2008-04-01

    This anthropological study critically evaluates Brazilian caregivers' symbolic production and significance of their malnourished offspring's primary teeth, as well as their own, and describes popular dental practices. From January to June 2004, ethnographic interviews of 27 poor, low-literacy mothers were conducted at a public Malnutrition Treatment Center in Fortaleza, Ceará State. Participant observation of clinical pathways and home environments supplemented the data. Content analysis was performed. Results confirm that primary teeth are imbued with cultural significance in Northeast Brazil. Mothers examine mouths, perceive signs of decay, associate primary with permanent tooth healthiness, identify ethnodental illnesses, seek assistance, and perform rituals with exfoliated teeth. The mother's motivation to care for primary teeth is sparked by her memories of past toothache and attempts to avoid stigma and discrimination. Social determinants, not mothers' beliefs or behaviors, are the most critical obstacles to effective dental care. Legitimizing lay knowledge and empowering caregivers and children can improve oral health in Northeast Brazil.

  6. Liposomal clodronate selectively eliminates microglia from primary astrocyte cultures

    Directory of Open Access Journals (Sweden)

    Kumamaru Hiromi

    2012-05-01

    Full Text Available Abstract Background There is increasing interest in astrocyte biology because astrocytes have been demonstrated to play prominent roles in physiological and pathological conditions of the central nervous system, including neuroinflammation. To understand astrocyte biology, primary astrocyte cultures are most commonly used because of the direct accessibility of astrocytes in this system. However, this advantage can be hindered by microglial contamination. Although several authors have warned regarding microglial contamination in this system, complete microglial elimination has never been achieved. Methods The number and proliferative potential of contaminating microglia in primary astrocyte cultures were quantitatively assessed by immunocytologic and flow cytometric analyses. To examine the utility of clodronate for microglial elimination, primary astrocyte cultures or MG-5 cells were exposed to liposomal or free clodronate, and then immunocytologic, flow cytometric, and gene expression analyses were performed. The gene expression profiles of microglia-eliminated and microglia-contaminated cultures were compared after interleukin-6 (IL-6 stimulation. Results The percentage of contaminating microglia exceeded 15% and continued to increase because of their high proliferative activity in conventional primary astrocyte cultures. These contaminating microglia were selectively eliminated low concentration of liposomal clodronate. Although primary microglia and MG-5 cells were killed by both liposomal and free clodronate, free clodronate significantly affected the viability of astrocytes. In contrast, liposomal clodronate selectively eliminated microglia without affecting the viability, proliferation or activation of astrocytes. The efficacy of liposomal clodronate was much higher than that of previously reported methods used for decreasing microglial contamination. Furthermore, we observed rapid tumor necrosis factor-α and IL-1b gene induction in

  7. Cross-cultural aspects of depression management in primary care.

    Science.gov (United States)

    Hails, Katherine; Brill, Charlotte D; Chang, Trina; Yeung, Albert; Fava, Maurizio; Trinh, Nhi-Ha

    2012-08-01

    Major depressive disorder (MDD) is a prevalent illness in minority populations. Minority patients with MDD are often unrecognized and untreated. This review examines promising interventions to address MDD in primary care settings, where minority groups are more likely to seek care. Since 2010, eleven interventions have been developed to address patient-specific and provider-specific barriers, many of which are adaptations of the collaborative care model. Other promising interventions include cultural tailoring of the collaborative care model, as well as the addition of telepsychiatry, motivational interviewing, cultural consultation, and innovations in interpreting. Overall, collaborative care was found feasible and improved satisfaction and treatment engagement of depressed minority patients in primary care. It remains inconclusive whether these newer intervention models improve MDD treatment outcomes. Future research will be needed to establish the effectiveness of these intervention models in improving the treatment outcomes of minority populations with MDD.

  8. Organizational culture in the primary healthcare setting of Cyprus.

    Science.gov (United States)

    Zachariadou, Theodora; Zannetos, Savvas; Pavlakis, Andreas

    2013-03-24

    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. 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. 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). This was the first study performed in Cyprus assessing organizational culture in the PHC setting. In the forthcoming health system reform

  9. Sphingosine-1-phosphate stimulates rat primary chondrocyte proliferation

    International Nuclear Information System (INIS)

    Kim, Mi-Kyoung; Lee, Ha Young; Kwak, Jong-Young; Park, Joo-In; Yun, Jeanho; Bae, Yoe-Sik

    2006-01-01

    Rat primary chondrocytes express the sphingosine-1-phosphate (S1P) receptor, S1P 2 , S1P 3 , S1P 4 , but not S1P 1 . When chondrocytes were stimulated with S1P or phytosphingosine-1-phosphate (PhS1P, an S1P 1 - and S1P 4 -selective agonist), phospholipase C-mediated cytosolic calcium increase was dramatically induced. S1P and PhS1P also stimulated two kinds of mitogen-activated protein kinases, extracellular signal-regulated kinase (ERK) and p38 kinase in chondrocytes. In terms of the two phospholipids-mediated functional modulation of chondrocytes, S1P and PhS1P stimulated cellular proliferation. The two phospholipids-induced chondrocyte proliferations were almost completely blocked by PD98059 but not by SB203580, suggesting that ERK but not p38 kinase is essentially required for the proliferation. Pertussis toxin almost completely inhibited the two phospholipids-induced cellular proliferation and ERK activation, indicating the crucial role of G i protein. This study demonstrates the physiological role of two important phospholipids (S1P and PhS1P) on the modulation of rat primary chondrocyte proliferation, and the crucial role played by ERK in the process

  10. The Risk Evaluation of Tungsten Oxide Nanoparticles in Cultured Rat Liver Cells for Its Safe Applications in Nanotechnology

    Directory of Open Access Journals (Sweden)

    Hasan Turkez

    2014-08-01

    Full Text Available Tungsten (VI oxide (WO3 nanoparticles (NPs are used for many industrial purposes in everyday life. However, their effects on human health have not been sufficiently evaluated. Therefore, the present study was designed to investigate the toxicity potentials of various concentrations (0 to 1000 ppm of WO3NPs (<100 nm particle size in cultured primary rat hepatocytes. The results of cell viability assay showed that the higher concentrations of dispersed WO3 NPs (300, 500 and 1000 ppm caused significant (p<0.05 decreases of cell viability. Also, dose dependent negative alterations were observed in oxidative status and antioxidant capacity levels after the application of WO3 in cultured rat primary hepatocytes. The results of genotoxicity tests revealed that these NPs did not cause significant increases of micronucleated hepatocytes (MNHEPs but increased 8-oxo-2-deoxyguanosine (8-OH-dG levels as compared to the control culture.

  11. Thermoprotection of a functional epithelium: heat stress effects on transepithelial transport by flounder renal tubule in primary monolayer culture.

    OpenAIRE

    Brown, M A; Upender, R P; Hightower, L E; Renfro, J L

    1992-01-01

    Primary monolayer cultures of winter flounder renal proximal-tubule cells were used to determine whether transepithelial transport could be protected from the damaging effects of extreme temperature by previous mild heat shock. Renal tubule epithelial cells were enzymatically dispersed and reorganized as confluent monolayer sheets on native rat tail collagen. Transepithelial electrical properties (potential difference, resistance, short-circuit current, and Na(+)-dependent glucose current) an...

  12. Effect of D-valine and cytosine arabinoside on [3H]thymidine incorporation in rat and rabbit epididymal epithelial cell cultures

    International Nuclear Information System (INIS)

    Orgebin-Crist, M.C.; Jonas-Davies, J.; Storey, P.; Olson, G.E.

    1984-01-01

    Epithelial cell enriched primary cultures were established from the rat and the rabbit epididymis. Epithelial cell aggregates, obtained after pronase digestion of minced epididymis, attached to the culture dish and after 72 h in vitro spread out to form discrete patches of cells. These cells have an epithelioid morphology and form a monolayer of closely apposed polygonal cells where DNA synthesis, as judged by [ 3 H]thymidine uptake, is very low. In L-valine medium the nonepithelial cell contamination was no more than 10% in rat and rabbit epididymal primary cultures. The labeling index of rat epididymal cells cultured in D-valine medium was significantly lower than that of cells cultured in L-valine medium. In contrast, the labeling index of rabbit epididymal cells cultured in D-valine medium was significantly higher than that of cells cultured in L-valine medium. Cytosine arabinoside decreased the number of labeled cells in both L-valine and D-valine cultures. From these results, it appears that D-valine is a selective agent for rat epididymal epithelial cells, but not for rabbit epithelial cells, and that cytosine arabinoside is a simple and effective means to control the proliferation of fibroblast-like cells in both rat and rabbit epididymal cell cultures

  13. Feeding Frequency Affects Cultured Rat Pituitary Cells in Low Gravity

    Science.gov (United States)

    Hymer, W. C.; Grindeland, R. E.; Salada, T.; Cenci, R.; Krishnan, K.; Mukai, C.; Nagaoka, S.

    1996-01-01

    In this report, we describe the results of a rat pituitary cell culture experiment done on STS-65 in which the effect of cell feeding on the release of the six anterior pituitary hormones was studied. We found complex microgravity related interactions between the frequency of cell feeding and the quantity and quality (i.e. biological activity) of some of the six hormones released in flight. Analyses of growth hormone (GH) released from cells into culture media on different mission days using gel filtration and ion exchange chromatography yielded qualitatively similar results between ground and flight samples. Lack of cell feeding resulted in extensive cell clumping in flight (but not ground) cultures. Vigorous fibroblast growth occurred in both ground and flight cultures fed 4 times. These results are interpreted within the context of autocrine and or paracrine feedback interactions. Finally the payload specialist successfully prepared a fresh trypsin solution in microgravity, detached the cells from their surface and reinserted them back into the culture chamber. These cells reattached and continued to release hormone in microgravity. In summary, this experiment shows that pituitary cells are microgravity sensitive and that coupled operations routinely associated with laboratory cel1 culture can also be accomplished in low gravity.

  14. Characterization of primary osteocyte-like cells from rat mandibles.

    Science.gov (United States)

    El Deeb Zakhary, Ibrahim; Wenger, Karl; Elsalanty, Mohammed; Cray, James; Sharawy, Mohamed; Messer, Regina

    2017-01-01

    The mandible is continuously undergoing remodeling as a result of mechanobiologic factors, such as chewing forces, tooth loss, orthodontic forces, and periodontitis. The effects of mechanical stress and biologic signals in bone homeostasis have been the focus of many investigations. However, much of this research utilized osteocytes derived from long bones, but little is known about the mandible-derived osteocytes. This study tests a protocol to isolate and grow osteocytes from rat mandible. Rat mandibles were harvested, sectioned into small pieces, and subjected to a sequence chemical treatment and enzymatic digestion. The treated tissues were cultured for a few weeks while cells emerged. Cells were sorted by using the osteocyte marker podoplanin, an early marker for osteocyte differentiation. The cells were then characterized according to morphology, biochemical markers (osteocalcin, podoplanin, and sclerostin), and alkaline phosphatase activity and compared with an isotype cell line MLO-Y4 cells. The mandibular osteocytic cells had stellate shape and were positive for osteocalcin, podoplanin, and sclerostin and lower alkaline phosphatase activity compared with MLO-Y4 osteocyte-like cells. The protocol to isolate osteocyte-like cells will allow the investigators to investigate the mechanobiologic differences in biomechanical response between these mandibular and long bone osteocyte-like cells under various conditions. Copyright © 2016 Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

    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

  16. Hepatocellular localisation of biosynthesis of vitronectin. Characterisation of the primary structure of rat vitronectin

    NARCIS (Netherlands)

    Otter, M.; Kuiper, J.; Rijken, D.; Zonneveld, A.-J. van

    1995-01-01

    To characterize the primary structure of rat Vn, a cDNA library constructed from freshly isolated rat hepatocytes, was screened with a human Vn cDNA probe. Comparison of the sequence of the obtained rat cDNA clone with the sequences of human, mouse and rabbit Vn cDNA's showed predominantly consensus

  17. Tumor necrosis factor (cachetin) decreases adipose cell differentiation in primary cell culture

    International Nuclear Information System (INIS)

    Martin, R.J.; Jones, D.D.; Jewell, D.E.; Hausman, G.J.

    1986-01-01

    Cachetin has been shown to effect gene product expression in the established adipose cell line 3T3-L1. Expression of messenger RNA for lipoprotein lipase is suppressed in cultured adipocytes. The purpose of this study was to determine the effect of Cachetin on adipose cell differentiation in primary cell culture. Stromalvascular cells obtained from the inguinal fat pad of 4-5 week old Sprague-Dawley rats were grown in culture for two weeks. During the proliferative growth phase all cells were grown on the same medium and labelled with 3 H-thymidine. Cachetin treatment (10 -6 to 10 -10 M) was initiated on day 5, the initial phase of preadipocyte differentiation. Adipocytes and stromal cells were separated using density gradient, and 3 H-thymidine was determined for both cell types. Thymidine incorporation into adipose cells was decreased maximally (∼ 50%) at 10 -10 M. Stromalvascular cells were not influenced at any of the doses tested. Adipose cell lipid content as indicated by oil red-O staining was decreased by Cachetin. Esterase staining by adipose cells treated with Cachetin was increased indicating an increase in intracellular lipase. These studies show that Cachetin has specific effects on primary adipose cell differentiation

  18. Transfection of brain capillary endothelial cells in primary culture with defined blood-brain barrier properties.

    Science.gov (United States)

    Burkhart, Annette; Thomsen, Louiza Bohn; Thomsen, Maj Schneider; Lichota, Jacek; Fazakas, Csilla; Krizbai, István; Moos, Torben

    2015-08-07

    Primary brain capillary endothelial cells (BCECs) are a promising tool to study the blood-brain barrier (BBB) in vitro, as they maintain many important characteristics of the BBB in vivo, especially when co-cultured with pericytes and/or astrocytes. A novel strategy for drug delivery to the brain is to transform BCECs into protein factories by genetic modifications leading to secretion of otherwise BBB impermeable proteins into the central nervous system. However, a huge challenge underlying this strategy is to enable transfection of non-mitotic BCECs, taking a non-viral approach. We therefore aimed to study transfection in primary, non-mitotic BCECs cultured with defined BBB properties without disrupting the cells' integrity. Primary cultures of BCECs, pericytes and astrocytes were generated from rat brains and used in three different in vitro BBB experimental arrangements, which were characterised based on a their expression of tight junction proteins and other BBB specific proteins, high trans-endothelial electrical resistance (TEER), and low passive permeability to radiolabeled mannitol. Recombinant gene expression and protein synthesis were examined in primary BCECs. The BCECs were transfected using a commercially available transfection agent Turbofect™ to express the red fluorescent protein HcRed1-C1. The BCECs were transfected at different time points to monitor transfection in relation to mitotic or non-mitotic cells, as indicated by fluorescence-activated cell sorting analysis after 5-and 6-carboxylfluorescein diacetate succinidyl ester incorporation. The cell cultures exhibited important BBB characteristics judged from their expression of BBB specific proteins, high TEER values, and low passive permeability. Among the three in vitro BBB models, co-culturing with BCECs and astrocytes was well suited for the transfection studies. Transfection was independent of cell division and with equal efficacy between the mitotic and non-mitotic BCECs. Importantly

  19. Purine nucleotide synthesis in cultured rat embryos undergoing organogenesis

    International Nuclear Information System (INIS)

    Rowe, P.B.; Kalaizis, A.; McEwen, S.E.

    1986-01-01

    The authors show that de n ovo synthesis is the sole source of the purine nucleotides required for in vitro rat embryonic growth during organogenesis. The presence of high levels of activity of purine catabolic enzymes in the homologous serum essential for culture prohibits the salvage of purine. While the 3-carbon atom of serine is the major source of one carbon units for purine ring synthesis there is a significant contribution from the 2-ring carbon atom of tryptophan. The paper describes in detail the incorporation of (1-14C)glycine into the acid soluble phase and other processes connected with de novo purine synthesis

  20. Characterization and long-term maintenance of rat taste cells in culture.

    Science.gov (United States)

    Ozdener, Hakan; Yee, Karen K; Cao, Jie; Brand, Joseph G; Teeter, John H; Rawson, Nancy E

    2006-03-01

    Taste cells have a limited life span and are replaced from a basal cell population, although the specific factors involved in this process are not well known. Short- and long-term cultures of other sensory cells have facilitated efforts to understand the signals involved in proliferation, differentiation, and senescence, yet few studies have reported successful primary culture protocols for taste cells. Furthermore, no studies have demonstrated both proliferation and differentiation in vitro. In this study, we have developed an in vitro culture system to maintain and utilize rat primary taste cells for more than 2 months without losing key molecular and biochemical features. Gustducin, phospholipase C-beta2 (PLC-beta2), T1R3, and T2R5 mRNA were detected in the cultured cells by reverse transcriptase-polymerase chain reaction. Western blot analysis demonstrated gustducin and PLC-beta2 expression in the same samples, which was confirmed by immunocytochemistry. Labeling with bromo-2-deoxyuridine (BrdU) demonstrated proliferation, and a subset of BrdU-labeled cells were also immunoreactive for either gustducin or PLC-beta2, indicating differentiation of newly generated cells in vitro. Cultured cells also exhibited increases in intracellular calcium in response to several taste stimuli. These results indicate that taste cells from adult rats can be generated and maintained under the described conditions for at least 2 months. This system will enable further studies of the processes involved in proliferation, differentiation, and function of mammalian taste receptor cells in an in vitro preparation.

  1. Insulin-like growth factor-II receptors in cultured rat hepatocytes: regulation by cell density

    International Nuclear Information System (INIS)

    Scott, C.D.; Baxter, R.C.

    1987-01-01

    Insulin-like growth factor-II (IGF-II) receptors in primary cultures of adult rat hepatocytes were characterized and their regulation by cell density examined. In hepatocytes cultured at 5 X 10(5) cells per 3.8 cm2 plate [ 125 I]IGF-II bound to specific, high affinity receptors (Ka = 4.4 +/- 0.5 X 10(9) l/mol). Less than 1% cross-reactivity by IGF-I and no cross-reactivity by insulin were observed. IGF-II binding increased when cells were permeabilized with 0.01% digitonin, suggesting the presence of an intracellular receptor pool. Determined by Scatchard analysis and by polyacrylamide gel electrophoresis after affinity labeling, the higher binding was due solely to an increase in binding sites present on 220 kDa type II IGF receptors. In hepatocytes cultured at low densities, the number of cell surface receptors increased markedly, from 10-20,000 receptors per cell at a culture density of 6 X 10(5) cells/well to 70-80,000 receptors per cell at 0.38 X 10(5) cells/well. The increase was not due simply to the exposure of receptors from the intracellular pool, as a density-related increase in receptors was also seen in cells permeabilized with digitonin. There was no evidence that IGF binding proteins, either secreted by hepatocytes or present in fetal calf serum, had any effect on the measurement of receptor concentration or affinity. We conclude that rat hepatocytes in primary culture contain specific IGF-II receptors and that both cell surface and intracellular receptors are regulated by cell density

  2. alpha-Amanitin induced apoptosis in primary cultured dog hepatocytes.

    Directory of Open Access Journals (Sweden)

    Adam Szelag

    2010-06-01

    Full Text Available Amatoxin poisoning is caused by mushroom species belonging to the genera Amanita, Galerina and Lepiota with the majority of lethal mushroom exposures attributable to Amanita phalloides. High mortality rate in intoxications with these mushrooms is principally a result of the acute liver failure following significant hepatocyte damage due to hepatocellular uptake of amatoxins. A wide variety of amatoxins have been isolated; however, alpha-amanitin (alpha-AMA appears to be the primary toxin. Studies in vitro and in vivo suggest that alpha-AMA does not only cause hepatocyte necrosis, but also may lead to apoptotic cell death. The objective of this study was to evaluate the complex hepatocyte apoptosis in alpha-AMA cytotoxicity. All experiments were performed on primary cultured canine hepatocytes. The cells were incubated for 12 h with alpha-AMA at a final concentration of 1, 5, 10 and 20 microM. Viability test (MTT assay, apoptosis evaluation (TUNEL reaction, detection of DNA laddering and electron microscopy were performed at 6 and 12 h of exposure to alpha-AMA. There was a clear correlation between hepatocyte viability, concentration of alpha-AMA and time of exposure to this toxin. The decline in cultured dog hepatocyte viability during the exposure to alpha-AMA is most likely preceded by enhanced cellular apoptosis. Our results demonstrate that apoptosis might contribute to pathogenesis of the severe liver injury in the course of amanitin intoxication, particularly during the early phase of poisoning.

  3. Comparative effects of sodium channel blockers in short term rat whole embryo culture

    Energy Technology Data Exchange (ETDEWEB)

    Nilsson, Mats F, E-mail: Mats.Nilsson@farmbio.uu.se [Department of Pharmaceutical Biosciences, Uppsala University (Sweden); Sköld, Anna-Carin; Ericson, Ann-Christin; Annas, Anita; Villar, Rodrigo Palma [AstraZeneca R and D Södertälje (Sweden); Cebers, Gvido [AstraZeneca R and D, iMed, 141 Portland Street, Cambridge, MA 02139 (United States); Hellmold, Heike; Gustafson, Anne-Lee [AstraZeneca R and D Södertälje (Sweden); Webster, William S [Department of Anatomy and Histology, University of Sydney (Australia)

    2013-10-15

    This study was undertaken to examine the effect on the rat embryonic heart of two experimental drugs (AZA and AZB) which are known to block the sodium channel Nav1.5, the hERG potassium channel and the L-type calcium channel. The sodium channel blockers bupivacaine, lidocaine, and the L-type calcium channel blocker nifedipine were used as reference substances. The experimental model was the gestational day (GD) 13 rat embryo cultured in vitro. In this model the embryonic heart activity can be directly observed, recorded and analyzed using computer assisted image analysis as it responds to the addition of test drugs. The effect on the heart was studied for a range of concentrations and for a duration up to 3 h. The results showed that AZA and AZB caused a concentration-dependent bradycardia of the embryonic heart and at high concentrations heart block. These effects were reversible on washout. In terms of potency to cause bradycardia the compounds were ranked AZB > bupivacaine > AZA > lidocaine > nifedipine. Comparison with results from previous studies with more specific ion channel blockers suggests that the primary effect of AZA and AZB was sodium channel blockage. The study shows that the short-term rat whole embryo culture (WEC) is a suitable system to detect substances hazardous to the embryonic heart. - Highlights: • Study of the effect of sodium channel blocking drugs on embryonic heart function • We used a modified method rat whole embryo culture with image analysis. • The drugs tested caused a concentration dependent bradycardia and heart block. • The effect of drugs acting on multiple ion channels is difficult to predict. • This method may be used to detect cardiotoxicity in prenatal development.

  4. Pharmacological Activation Gi/o Protein Increases Glial Cell Line-Derived Neurotrophic Factor Production through Fibroblast Growth Factor Receptor and Extracellular Signal-Regulated Kinase Pathway in Primary Cultured Rat Cortical Astrocytes.

    Science.gov (United States)

    Hisaoka-Nakashima, Kazue; Matsumoto, Chie; Azuma, Honami; Taki, Sayaka; Takebayashi, Minoru; Nakata, Yoshihiro; Morioka, Norimitsu

    2017-01-01

    A significant reduction of glial cell line-derived neurotrophic factor (GDNF) has been identified in the pathophysiology of neurodegenerative and neuropsychiatric disorders. Thus, clarification of the mechanism of GDNF production, and modulating brain GDNF levels could be a novel therapeutic approach. A previous study demonstrated that antidepressant amitriptyline-induced GDNF production was significantly inhibited by pertussis toxin (PTX), a Gi/o protein inhibitor in astrocytes, the main source of GDNF in the brain. However, it is not known whether direct activation of Gi/o protein might induce GDNF expression, and what mechanisms might be involved after Gi/o protein activation. The current study investigated Gi/o protein-initiated GDNF production in rat cortical astrocytes using activators that directly activate Gi/o protein, mastoparan and compound48/80. Treatment of astrocytes with either mastoparan or compound48/80 increased GDNF mRNA expression at 3 and 6 h, and GDNF protein release at 24 h. Treatment of astrocyte with either mastoparan or compound48/80 increased brain-derived neurotrophic factor (BDNF) mRNA expression as well as GDNF. Mastoparan and compound48/80-induced GDNF mRNA expression were significantly inhibited by not only PTX, but also fibroblast growth factor receptor (FGFR) inhibitors, and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor. In fact, both FGFR substrate2α (FRS2α) and ERK phosphorylation were increased by treatment with either mastoparan or compound48/80, and these were significantly blocked by PTX. Thus, direct, receptor-independent Gi/o protein activation increases GDNF production through FGFR/ERK signaling pathway. The current results indicate a critical role of Gi/o signaling in the regulation of GDNF expression in astrocytes.

  5. Acute exposure of primary rat soleus muscle to zilpaterol HCl (β2 adrenergic agonist), TNFα, or IL-6 in culture increases glucose oxidation rates independent of the impact on insulin signaling or glucose uptake.

    Science.gov (United States)

    Cadaret, Caitlin N; Beede, Kristin A; Riley, Hannah E; Yates, Dustin T

    2017-08-01

    Recent studies show that adrenergic agonists and inflammatory cytokines can stimulate skeletal muscle glucose uptake, but it is unclear if glucose oxidation is similarly increased. Thus, the objective of this study was to determine the effects of ractopamine HCl (β1 agonist), zilpaterol HCl (β2 agonist), TNFα, and IL-6 on glucose uptake and oxidation rates in unstimulated and insulin-stimulated soleus muscle strips from adult Sprague-Dawley rats. Effects on phosphorylation of Akt (phospho-Akt), p38 MAPK (phospho-p38), and p44/42 MAPK (phospho-p44/42) was also determined. Incubation with insulin increased (Pglucose uptake by ∼47%, glucose oxidation by ∼32%, and phospho-Akt by ∼238%. Insulin also increased (Pglucose uptake but ∼32% greater (Pglucose oxidation than unstimulated muscle. Moreover, co-incubation with insulin+β2 agonist increased (Pglucose oxidation and phospho-Akt compared to insulin alone. Conversely, β1 agonist did not appear to affect basal or insulin-stimulated glucose metabolism, and neither β agonist affected phospho-p44/42. TNFα and IL-6 increased (Pglucose oxidation by ∼23% and ∼33%, respectively, in the absence of insulin. This coincided with increased (Pglucose oxidation rates that were similar to insulin alone, despite lower (Pglucose oxidation rates were not concomitant with greater glucose uptake. These results show that acute β2 adrenergic stimulation, but not β1 stimulation, directly increases fractional glucose oxidation in the absence of insulin and synergistically increases glucose oxidation when combined with insulin. The cytokines, TNFα and IL-6, likewise directly increased glucose oxidation in the absence of insulin, but were not additive in combination with insulin and in fact appeared to disrupt Akt-mediated insulin signaling. Rather, cytokines appear to be acting through MAPKs to elicit effects on glucose oxidation. Regardless, stimulation of glucose oxidation by these key stress factors did not rely upon

  6. [Influence of electroporation on the biological activities of primary rat hepatocytes].

    Science.gov (United States)

    Yao, Y; Zhang, D; Luo, Y; Zhang, D; Huang, A; Zhou, W; Ren, H

    2001-06-01

    To investigate the influence of electroporation on the biological activities of primary rat hepatocyte and to optimize the electroporation conditions introducing foreign genes into hepatocytes. A single-pulse procedure was performed at low voltage (220-400 V) but high capacitance (500-950 microF). Its influence on hepatocyte activities was detected by Trypan blue exclusion (TBE) and MTT analysis. Besides, ALB, ALT and LDH in the supernatants of hepatocytes were tested by biochemical assay. Little hepatocyte damage and high survival rate (>90%) was found from 36 hours till 9th day of culture. At 36th hour after electroporation, ALB, ALT and LDH in the supernatants of Group B (220V, 950 microF) and C (400 V, 950 microF) were higher than those of control group. Whereas TBE and MTT analysis failed to indicate the significant difference of cell viability between electroporation groups and control group. This electroporation procedure is one of the optimal choices to introduce foreign genes into primary rat hepatocytes.

  7. Dose-response analysis of phthalate effects on gene expression in rat whole embryo culture

    NARCIS (Netherlands)

    Robinson, J.F.; Verhoef, A.; van Beelen, V.A.; Pennings, J.L.A.; Piersma, A.H.

    2012-01-01

    The rat postimplantation whole embryo culture (WEC) model serves as a potential screening tool for developmental toxicity. In this model, cultured rat embryos are exposed during early embryogenesis and evaluated for morphological effects. The integration of molecular-based markers may lead to

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

    International Nuclear Information System (INIS)

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

    1990-01-01

    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

  9. Characterization of cysteinyl-leukotriene formation in primary astroglial cell cultures.

    Science.gov (United States)

    Seregi, A; Simmet, T; Schobert, A; Hertting, G

    1990-03-01

    The formation and composition of cysteinyl-leukotrienes (LT) in primary astroglial cell cultures prepared from newborn rat brain has been studied. Small amounts of cysteinyl-LT determined in terms of LTC4-like material in the supernatants of the cultures, became detectable after stimulation of the cells with 10(-5) M ionophore A23187. Cysteinyl-LT formation increased with time, reaching about 600 pg (mg protein)-1 after 60 min incubation. In contrast, considerable thromboxane (TX) B2 synthesis was found at 5 min following A23187-stimulation (about 30 ng TXB2 (mg protein)-1). The synthesis of cysteinyl-LT was abolished by 5 x 10(-5) M nordihydroguaiaretic acid (NDGA). Irrespective of the duration of incubation, blockage of prostanoid synthesis by 10(-6) M indomethacin did not result in increased cysteinyl-LT production. Reversed phase HPLC combined with radioimmunological detection showed that, after 60 min incubation in the presence of A23187, LTC4 and LTD4 accounted for practically all the LTC4-like immunoreactive material in the supernatants of cell cultures. No significant amounts of LTE4 could be detected. The results show that astrocytes may contribute to brain LTC4 and LTD4 synthesis. However, the cellular site of cerebral LTE4 formation seems to be other than the astroglia.

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

    Science.gov (United States)

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

    2013-12-01

    This article explores various contributing factors to explain differences in the strength of the primary care (PC) structure and services delivery across Europe. Data on the strength of primary care in 31 European countries in 2009/10 were used. The results showed that the national political agenda, economy, prevailing values, and type of healthcare system are all important factors that influence the development of strong PC. Wealthier countries are associated with a weaker PC structure and lower PC accessibility, while Eastern European countries seemed to have used their growth in national income to strengthen the accessibility and continuity of PC. Countries governed by left-wing governments are associated with a stronger PC structure, accessibility and coordination of PC. Countries with a social-security based system are associated with a lower accessibility and continuity of PC; the opposite is true for transitional systems. Cultural values seemed to affect all aspects of PC. It can be concluded that strengthening PC means mobilising multiple leverage points, policy options, and political will in line with prevailing values in a country. Copyright © 2013 Elsevier Ltd. All rights reserved.

  11. Evaluation of the developmental toxicity of citrinin using Hydra attenuata and postimplantation rat whole embryo culture.

    Science.gov (United States)

    Yang, Y G; Mayura, K; Spainhour, C B; Edwards, J F; Phillips, T D

    1993-12-31

    Citrinin (a mycotoxin produced as a frequent contaminant of food and feed by numerous species of Aspergillus and Penicillium fungi) is embryo/fetotoxic and embryocidal in mice and rats. The present study was designed to examine whether the in vivo observed developmental toxicity of citrinin could be recapitulated using the Hydra attenuata (HA) bioassay and then be confirmed in rat whole embryo culture (WEC). Results from the HA assay indicated that the minimal affective concentrations of citrinin required to elicit a toxic response in the adult hydra (MACA) and in the regenerating hydra (MACD) were 30 mg/l and 20 mg/l, respectively. The Hydra developmental hazard index (A/D ratio) was equal to 1.5, classifying citrinin as a coaffective developmental toxin. In WEC, rat embryos were cultured in homologous (rat) serum containing citrinin at various concentrations ranging from 0.0 and 300 micrograms/ml for a period of 45 h. The results indicated a concentration-dependent reduction in yolk sac diameter, crown-rump length, somite number, protein and DNA contents. No embryonic dysmorphogenesis was observed in any treatment group. Histological examination revealed severe diffuse mesodermal and ectodermal necrosis in embryos treated with 250 micrograms/ml citrinin. At lower concentrations of citrinin, embryos were neither grossly nor histologically different from controls. Both the HA and WEC bioassays demonstrated that citrinin is not a primary developmental toxin. The use of HA and WEC bioassays in tandem may facilitate the rapid detection and ranking of the developmental hazards of food and feedborne mycotoxins.

  12. Statins induce apoptosis in rat and human myotube cultures by inhibiting protein geranylgeranylation but not ubiquinone

    International Nuclear Information System (INIS)

    Johnson, Timothy E.; Zhang, Xiaohua; Bleicher, Kimberly B.; Dysart, Gary; Loughlin, Amy F.; Schaefer, William H.; Umbenhauer, Diane R.

    2004-01-01

    Statins are widely used to treat lipid disorders. These drugs are safe and well tolerated; however, in <1% of patients, myopathy and/or rhabdomyolysis can develop. To better understand the mechanism of statin-induced myopathy, we examined the ability of structurally distinct statins to induce apoptosis in an optimized rat myotube model. Compound A (a lactone) and Cerivastatin (an open acid) induced apoptosis, as measured by TUNEL and active caspase 3 staining, in a concentration- and time-dependent manner. In contrast, an epimer of Compound A (Compound B) exhibited a much weaker apoptotic response. Statin-induced apoptosis was completely prevented by mevalonate or geranylgeraniol, but not by farnesol. Zaragozic acid A, a squalene synthase inhibitor, caused no apoptosis on its own and had no effect on Compound-A-induced myotoxicity, suggesting the apoptosis was not a result of cholesterol synthesis inhibition. The geranylgeranyl transferase inhibitors GGTI-2133 and GGTI-2147 caused apoptosis in myotubes; the farnesyl transferase inhibitor FTI-277 exhibited a much weaker effect. In addition, the prenylation of rap1a, a geranylgeranylated protein, was inhibited by Compound A in myotubes at concentrations that induced apoptosis. A similar statin-induced apoptosis profile was seen in human myotube cultures but primary rat hepatocytes were about 200-fold more resistant to statin-induced apoptosis. Although the statin-induced hepatotoxicity could be attenuated with mevalonate, no effect was found with either geranylgeraniol or farnesol. In studies assessing ubiquinone levels after statin treatment in rat and human myotubes, there was no correlation between ubiquinone levels and apoptosis. Taken together, these observations suggest that statins cause apoptosis in myotube cultures in part by inhibiting the geranylgeranylation of proteins, but not by suppressing ubiquinone concentration. Furthermore, the data from primary hepatocytes suggests a cell-type differential

  13. Metabolism of 4-nitrobiphenyl (NBP) by cultured rat urothelial cells

    International Nuclear Information System (INIS)

    Swaminathan, S.; Lang, D.B.; Reznikoff, C.A.

    1986-01-01

    The potential of rat urothelial cells to metabolize NBP was evaluated by incubating 4.3 x 10 7 viable cells with 20 μM [ 3 H]NBP in a serum free medium for 48 hours. The culture medium was examined for metabolites of NBP by extraction with ethyl acetate and subsequent chromatographic analysis. High pressure liquid chromatography of the solvent extract using a Whatman ODS-3, C-18 column in 70% methanol-water at a flow rate of 1 ml/min revealed two major peaks at retention times of approximately 8 and 13 min. Thin layer chromatography showed two regions of radioactivity at Rf values of 0.35 and 0.83, the latter corresponding with NBP. Based on the chromatographic data the metabolite with the retention time of 8.0 min in HPLC and an Rf of 0.35 in TLC has been tentatively identified as 4-acetylaminobiphenyl. Analysis of binding to proteins and nucleic acids following exposure to [ 3 H]NBP revealed a significant amount (0.03% of initially applied radioactivity) in the protein fractions. Control samples of NBP incubated in medium, without the urothelial cells revealed only the parent compound. These data suggest that rat bladder cells possess the metabolic capability to reduce NBP and to generate reactive metabolites that bind to cellular macromolecules

  14. Thyroid hormone uptake by rat hepatocytes in primary culture

    NARCIS (Netherlands)

    E.P. Krenning (Eric)

    1983-01-01

    textabstractIodide is taken up by the thyroid follicular cell, oxydized and bound to thyroglobulin at the apical membrane facing the colloid in the follicular lumen. Iodinated colloid is subsequently engulfed by the follicular cell and hydrolysed, liberating thyroxine and triiodothyronine from their

  15. Vitamin C inhibits leptin secretion and some glucose/lipid metabolic pathways in primary rat adipocytes.

    Science.gov (United States)

    Garcia-Diaz, D F; Campion, J; Milagro, F I; Boque, N; Moreno-Aliaga, M J; Martinez, J A

    2010-07-01

    Antioxidant-based treatments are emerging as an interesting approach to possibly counteract obesity fat accumulation complications, since this is accompanied by an increased systemic oxidative stress. The aim of this study was to analyze specific metabolic effects of vitamin C (VC) on epididymal primary rat adipocytes. Cells were isolated and incubated for 72 h in culture medium, in the absence or presence of 1.6 nM insulin, within a range of VC concentrations (5-1000 microM). Glucose- and lipid-related variables as well as the secretion/expression patterns of several obesity-related genes were assessed. It was observed that VC dose dependently inhibited glucose uptake and lactate production, and also reduced glycerol release in both control and insulin-treated cells. Also, VC caused a dramatic concentration-dependent fall in leptin secretion especially in insulin-stimulated cells. In addition, VC (200 microM) induced Cdkn1a and Casp8, partially inhibited Irs3, and together with insulin drastically reduced Gpdh (listed as Gpd1 in the MGI database) gene expressions. Finally, VC and insulin down-regulatory effects were observed on extracellular and intracellular reactive oxygen species production respectively. In summary, this experimental assay describes a specific effect of VC in isolated rat adipocytes on glucose and fat metabolism, and on the secretion/expression of important obesity-related proteins.

  16. Development of Eimeria nieschulzi (Coccidia, Apicomplexa Gamonts and Oocysts in Primary Fetal Rat Cells

    Directory of Open Access Journals (Sweden)

    Hong Chen

    2013-01-01

    Full Text Available The in vitro production of gametocytes and oocysts of the apicomplexan parasite genus Eimeria is still a challenge in coccidiosis research. Until today, an in vitro development of gametocytes or oocysts had only been shown in some Eimeria species. For several mammalian Eimeria species, partial developments could be achieved in different cell types, but a development up to gametocytes or oocysts is still lacking. This study compares several permanent cell lines with primary fetal cells of the black rat (Rattus norvegicus concerning the qualitative in vitro development of the rat parasite Eimeria nieschulzi. With the help of transgenic parasites, the developmental progress was documented. The selected Eimeria nieschulzi strain constitutively expresses the yellow fluorescent protein and a macrogamont specific upregulated red tandem dimer tomato. In the majority of all investigated host cells the development stopped at the second merozoite stage. In a mixed culture of cells derived from inner fetal organs the development of schizont generations I-IV, macrogamonts, and oocysts were observed in crypt-like organoid structures. Microgamonts and microgametes could not be observed and oocysts did not sporulate under air supply. By immunohistology, we could confirm that wild-type E. nieschulzi stages can be found in the crypts of the small intestine. The results of this study may be helpful for characterization of native host cells and for development of an in vitro cultivation system for Eimeria species.

  17. Polyethyleneimine-mediated transfection of cultured postmitotic neurons from rat sympathetic ganglia and adult human retina

    Directory of Open Access Journals (Sweden)

    Higgins Dennis

    2001-02-01

    Full Text Available Abstract Background Chemical methods of transfection that have proven successful with cell lines often do not work with primary cultures of neurons. Recent data, however, suggest that linear polymers of the cation polyethyleneimine (PEI can facilitate the uptake of nucleic acids by neurons. Consequently, we examined the ability of a commercial PEI preparation to allow the introduction of foreign genes into postmitotic mammalian neurons. Sympathetic neurons were obtained from perinatal rat pups and maintained for 5 days in vitro in the absence of nonneuronal cells. Cultures were then transfected with varying amounts of a plasmid encoding either E. coli β-galactosidase or enhanced green fluorescence protein (EGFP using PEI. Results Optimal transfection efficiency was observed with 1 μg/ml of plasmid DNA and 5 μg/ml PEI. Expression of β-galactosidase was both rapid and stable, beginning within 6 hours and lasting for at least 21 days. A maximum yield was obtained within 72 hours with ∼ 9% of the neurons expressing β-galactosidase, as assessed by both histochemistry and antibody staining. Cotransfection of two plasmids encoding reporter genes was achieved. Postmitotic neurons from adult human retinal cultures also demonstrated an ability to take up and express foreign DNA using PEI as a vector. Conclusions These data suggest that PEI is a useful agent for the stable expression of plasmid-encoded genes in neuronal cultures.

  18. Design of a social constructivism-based curriculum for primary science education in Confucian heritage culture

    NARCIS (Netherlands)

    Vu Thu Hang, N.

    2014-01-01

    This study is about the application of social constructivism in primary science curriculum in Confucian heritage culture. It was found that the implementation of social constructivism in Confucian heritage culture was low and influenced by cultural divergences between Confucian cultural philosophy

  19. Aniline Induces Oxidative Stress and Apoptosis of Primary Cultured Hepatocytes

    Directory of Open Access Journals (Sweden)

    Yue Wang

    2016-11-01

    Full Text Available The toxicity and carcinogenicity of aniline in humans and animals have been well documented. However, the molecular mechanism involved in aniline-induced liver toxicity and carcinogenesis remains unclear. In our research, primary cultured hepatocytes were exposed to aniline (0, 1.25, 2.50, 5.0 and 10.0 μg/mL for 24 h in the presence or absence of N-acetyl-l-cysteine (NAC. Levels of reactive oxygen species (ROS, malondialdehyde (MDA, and glutathione (GSH, activities of superoxide dismutase (SOD and catalase (CAT, mitochondrial membrane potential, DNA damage, cell viability, and apoptosis were detected. Levels of ROS and MDA were significantly increased and levels of GSH and CAT, activity of SOD, and mitochondrial membrane potential in hepatocytes were significantly decreased by aniline compared with the negative control group. The tail moment and DNA content of the tail in exposed groups were significantly higher than those in the negative control group. Cell viability was reduced and apoptotic death was induced by aniline in a concentration-dependent manner. The phenomena of ROS generation, oxidative damage, loss of mitochondrial membrane potential, DNA damage and apoptosis could be prevented if ROS inhibitor NAC was added. ROS generation is involved in the loss of mitochondrial membrane potential and DNA injury, which may play a role in aniline-induced apoptosis in hepatocytes. Our study provides insight into the mechanism of aniline-induced toxicity and apoptosis of hepatocytes.

  20. Poliovirus replication and spread in primary neuron cultures.

    Science.gov (United States)

    Daley, John K; Gechman, Lisa A; Skipworth, Jason; Rall, Glenn F

    2005-09-15

    While some neurotropic viruses cause rapid central nervous system (CNS) disease upon entry into the brain parenchyma, other viruses that are cytolytic in the periphery either result in little neuropathology or are associated with a protracted course of CNS disease consistent with persistent infection. One such virus, poliovirus (PV), is an extremely lytic RNA virus that requires the expression of CD155, the poliovirus receptor (PVR), for infection. To compare the kinetics of PV infection in neuronal and non-neuronal cell types, primary hippocampal neurons and fibroblasts were isolated from CD155+ transgenic embryos and infected with the Mahoney and Sabin strains of PV. Despite similar levels of infection in these ex vivo cultures, PV-infected neurons produced 100-fold fewer infectious particles as compared to fibroblasts throughout infection, and death of PV-infected neurons was delayed approximately 48 h. Spread in neurons occurred primarily by trans-synaptic transmission and was CD155-dependent. Together, these results demonstrate that the magnitude and speed with which PV replication, spread, and subsequent cell death occur in neurons is decreased as compared to non-neuronal cells, implicating cell-specific effects on replication that may then influence viral pathogenesis.

  1. Protocol for culturing low density pure rat hippocampal neurons supported by mature mixed neuron cultures.

    Science.gov (United States)

    Yang, Qian; Ke, Yini; Luo, Jianhong; Tang, Yang

    2017-02-01

    primary hippocampal neuron cultures allow for subcellular morphological dissection, easy access to drug treatment and electrophysiology analysis of individual neurons, and is therefore an ideal model for the study of neuron physiology. While neuron and glia mixed cultures are relatively easy to prepare, pure neurons are particular hard to culture at low densities which are suitable for morphology studies. This may be due to a lack of neurotrophic factors such as brain derived neurotrophic factor (BDNF), neurotrophin-3 (NT3) and Glial cell line-derived neurotrophic factor (GDNF). In this study we used a two step protocol in which neuron-glia mixed cultures were initially prepared for maturation to support the growth of young neurons plated at very low densities. Our protocol showed that neurotrophic support resulted in physiologically functional hippocampal neurons with larger cell body, increased neurite length and decreased branching and complexity compared to cultures prepared using a conventional method. Our protocol provides a novel way to culture highly uniformed hippocampal neurons for acquiring high quality, neuron based data. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Effects on DHEA levels by estrogen in rat astrocytes and CNS co-cultures via the regulation of CYP7B1-mediated metabolism

    DEFF Research Database (Denmark)

    Fex Svenningsen, Åsa; Wicher, Grzegorz; Lundqvist, Johan

    2011-01-01

    in primary cultures of rat astrocytes. Interestingly, diarylpropionitrile, a well-known agonist of estrogen receptor β, also suppressed CYP7B1-mediated hydroxylation of DHEA. Several previous studies have reported neuroprotective effects of estrogens. The current data indicate that one of the mechanisms...

  3. A method for isolating identifying and culturing of rat trachea-bronchia epithelial cells

    International Nuclear Information System (INIS)

    Cui Fengmei; Su Shibiao; Nie Jihua; Li Bingyan; Tong Jian

    2005-01-01

    Objective: To explore a method for isolating identifying and culturing the rat trachea-bronchia epithelial cells. Methods: The rat trachea-bronchia epithelial cells were isolated by digestion with pronase and brushing with cell brush, identified using confocul and cultured in entire F12 media with no serum. Results: With this method, cells in high purity and high viability could be obtained, and about 10 6 cells per rat. The cells grow well in entire F12 media with no serum. Conclusion: The method is useful for isolating rate trachea-bronchia epithelial cells and the entire F12 media with no serum is effective for culturing. (authors)

  4. An investigation into the feasibility of culturing rat embryos in media ...

    African Journals Online (AJOL)

    in the media during 48-h rat embryo culture. Embryos are very sensitive to changes in the culture medium environment and will, as a result, develop abnormally when conditions are less than optimal. Little is known about the influence of culture envi- ronment on the medium parameters of pH, PCOr, pOr, bicarbo- nate levels ...

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

    International Nuclear Information System (INIS)

    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

  6. Regulation of Schwann cell proliferation in cultured segments of the adult rat sciatic nerve

    DEFF Research Database (Denmark)

    Svenningsen, Åsa Fex; Kanje, M

    1998-01-01

    Schwann cell proliferation was studied in cultured segments of the rat sciatic nerve by measurement of [3H] thymidine incorporation or through bromodeoxyuridine-(BrdU)-labelling and immunocytochemistry. The aim was to delineate mechanisms involved in the injury-induced proliferative response...... together with morphological evaluation of myelin association showed that proliferation occurred in Schwann cells. The results are consistent with a model in which Schwann cell proliferation is enhanced by Ca2+ through activation of calmodulin-dependent and/or PKCdependent mechanisms. Inhibition is achieved...... through the cAMP system. Together, these results show that Schwann cells regulate proliferation differently in an integrated environment, e.g. the nerve structure, than in isolation as primary monocultures....

  7. Degradation of high density lipoprotein in cultured rat luteal cells

    International Nuclear Information System (INIS)

    Rajan, V.P.; Menon, K.M.J.

    1986-01-01

    In rat ovary luteal cells, degradation of high density lipoprotein (HDL) to tricholoracetic acid (TCA)-soluble products accounts for only a fraction of the HDL-derived cholesterol used for steroidogenesis. In this study the authors have investigated the fate of 125 I]HDL bound to cultured luteal cells using pulse-chase technique. Luteal cell cultures were pulse labeled with [ 125 I]HDL 3 and reincubated in the absence of HDL. By 24 h about 50% of the initallay bound radioactivity was released into the medium, of which 60-65% could be precipitated with 10% TCA. Gel filtration of the chase incubation medium on 10% agarose showed that the amount of TCA-soluble radioactivity was nearly completely accounted for by a sharp peak in the low molecular weight region which was identified as 96% monoiodotyrosine by paper chromatography. The TCA-precipitable radioactivity was nearly completely accounted for by a sharp peak in the low molecular weight region which was identified as 96% monoiodotyrosine by paper chromatography. The TCA-precipitable radioactivity eluted over a wide range of molecular weights (15,000-80,000), and there was very little intact HDL present. Electrophoresis of the chase medium showed that component of the TCA-precipitable portion had mobility similar to apo AI. Lysosomal inhibitors of receptor-mediated endocytosis had no effect on the composition or quantity of radioactivity released during chase incubation. The results show that HDL 3 binding to luteal cells is followed by complete degradation of the lipoprotein, although the TCA-soluble part does not reflect the extent of degradation

  8. Effect of various chemicals on the metabolism of benzo(a)pyrene by cultured rat colon

    DEFF Research Database (Denmark)

    Autrup, Herman; Harris, Curtis C.; Fugaro, Steven

    1977-01-01

    The effect of various co- and anti-carcinogens of colon carcinogenesis on the metabolism of benzo(a)pyrene (BP) in cultured rat colon is reported. Rat colon enzymatically converted BP into metabolites which bind to cellular macromolecules i.e., DNA and protein. Activity of aryl hydrocarbon...

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

    International Nuclear Information System (INIS)

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

    1987-01-01

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

  10. Role of Organizational Culture on the Performance Primary School Teachers

    Science.gov (United States)

    Suharningsih; Murtedjo

    2017-01-01

    This study aims to identify and examine the role of organizational culture on teacher performance. In the present study examined the role of organizational culture with teacher performance. In accordance with the study design, namely the survey, the data collected in this research is quantitative data. The data is extracted and obtained through…

  11. Culturally Responsive Dance Pedagogy in the Primary Classroom

    Science.gov (United States)

    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…

  12. Exploring Culture in Locally Published English Textbooks for Primary Education in Turkey

    Science.gov (United States)

    Kirkgöz, Yasemin; Agçam, Reyhan

    2011-01-01

    Since language and culture are closely interwoven, the integration of culture into textbooks used for teaching English as a second/foreign language has become a widely accepted phenomenon. This study investigates the cultural elements in locally published English textbooks used for Turkish primary schools following two major curriculum innovations…

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

    Science.gov (United States)

    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. Primary Motor Neuron Culture to Promote Cellular Viability and Myelination.

    Science.gov (United States)

    Suh, Jun-Kyo Francis; Hyung, Sujin

    2018-01-01

    A culture system that can recapitulate myelination in vitro will not only help us to better understand the mechanism of myelination and demyelination but also identify possible therapeutic interventions for treating demyelinating diseases. Here, we introduce a simple and reproducible myelination culture system using mouse motor neurons (MNs) and Schwann cells (SCs). Dissociated motor neurons are plated on a feeder layer of SCs, which interact with and wrap around the axons of MNs as they differentiate in culture. In our MN-SC co-culture system, MNs survive over 3 weeks and extend long axons. Both viability and axon growth of MNs in the co-culture are markedly enhanced as compared to those of MN monocultures. Co-labeling of myelin basic proteins and neuronal cell microtubules reveals that SCs form myelin sheaths by wrapping around the axons of MNs.

  15. GDNF family ligands display distinct action profiles on cultured GABAergic and serotonergic neurons of rat ventral mesencephalon

    DEFF Research Database (Denmark)

    Ducray, Angélique; Krebs, Sandra H:; Schaller, Benoft

    2006-01-01

    the effects of GFLs on other neuronal populations in the VM is essential for their potential application as therapeutic molecules for Parkinson's disease. Hence, in a comparative study, we investigated the effects of GFLs on cell densities and morphological differentiation of gamma-aminobutyric acid......Glial-cell-line-derived neurotrophic factor (GDNF), neurturin (NRTN), artemin (ARTN) and persephin (PSPN), known as the GDNF family ligands (GFLs), influence the development, survival and differentiation of cultured dopaminergic neurons from ventral mesencephalon (VM). Detailed knowledge about......-immunoreactive (GABA-ir) and serotonin-ir (5-HT-ir) neurons in primary cultures of E14 rat VM. We observed that all GFLs [10 ng/ml] significantly increased GABA-ir cell densities (1.6-fold) as well as neurite length/neuron. However, only GDNF significantly increased the number of primary neurites/neuron, and none...

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

    NARCIS (Netherlands)

    Kringos, D.S.; Boerma, W.G.W.; Zee, J. van der; Groenewegen, P.P.

    2013-01-01

    This article explores various contributing factors to explain differences in the strength of the primary care (PC) structure and services delivery across Europe. Data on the strength of primary care in 31 European countries in 2009/10 were used. The results showed that the national political agenda,

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

    NARCIS (Netherlands)

    Kringos, D.S.; Boerma, W.G.W.; Zee, J. van der; Groenewegen, P.P.

    2013-01-01

    This article explores various contributing factors to explain differences in the strength of the primary care (PC) structure and services delivery across Europe. Data on the strength of primary care in 31 European countries in 2009/10 were used. The results showed that the national political

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

    NARCIS (Netherlands)

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

    2013-01-01

    This article explores various contributing factors to explain differences in the strength of the primary care (PC) structure and services delivery across Europe. Data on the strength of primary care in 31 European countries in 2009/10 were used. The results showed that the national political agenda,

  19. Ototoxicity of paclitaxel in rat cochlear organotypic cultures

    Energy Technology Data Exchange (ETDEWEB)

    Dong, Yang [Shanghai University of Traditional Chinese Medicine, Shanghai 201203 (China); Center for Hearing and Deafness, University at Buffalo, NY 14214 (United States); Ding, Dalian; Jiang, Haiyan [Center for Hearing and Deafness, University at Buffalo, NY 14214 (United States); Shi, Jian-rong [Shanghai University of Traditional Chinese Medicine, Shanghai 201203 (China); Salvi, Richard [Center for Hearing and Deafness, University at Buffalo, NY 14214 (United States); Roth, Jerome A., E-mail: jaroth@buffalo.edu [Department of Pharmacology and Toxicology, University at Buffalo, NY 14214 (United States)

    2014-11-01

    Paclitaxel (taxol) is a widely used antineoplastic drug employed alone or in combination to treat many forms of cancer. Paclitaxel blocks microtubule depolymerization thereby stabilizing microtubules and suppressing cell proliferation and other cellular processes. Previous reports indicate that paclitaxel can cause mild to moderate sensorineural hearing loss and some histopathologic changes in the mouse cochlea; however, damage to the neurons and the underlying cell death mechanisms are poorly understood. To evaluate the ototoxicity of paclitaxel in more detail, cochlear organotypic cultures from postnatal day 3 rats were treated with paclitaxel for 24 or 48 h with doses ranging from 1 to 30 μM. No obvious histopathologies were observed after 24 h treatment with any of the paclitaxel doses employed, but with 48 h treatment, paclitaxel damaged cochlear hair cells in a dose-dependent manner and also damaged auditory nerve fibers and spiral ganglion neurons (SGN) near the base of the cochlea. TUNEL labeling was negative in the organ of Corti, but positive in SGN with karyorrhexis 48 h after 30 μM paclitaxel treatment. In addition, caspase-6, caspase-8 and caspase-9 labeling was present in SGN treated with 30 μM paclitaxel for 48 h. These results suggest that caspase-dependent apoptotic pathways are involved in paclitaxel-induced damage of SGN, but not hair cells in cochlea. - Highlights: • Paclitaxel was toxic to cochlear hair cells and spiral ganglion neurons. • Paclitaxel-induced spiral ganglion degeneration was apoptotic. • Paclitaxel activated caspase-6, -8 and -8 in spiral ganglion neurons.

  20. Kinetics of thallium exchange in cultured rat myocardial cells.

    Science.gov (United States)

    McCall, D; Zimmer, L J; Katz, A M

    1985-03-01

    The kinetics of thallium exchange in cultured rat myocardial cells were studied and compared to those of potassium in the same tissue. Studies were carried out using low concentrations (10 nM to 5 microM) of thallium-204, approximating those likely to be encountered during clinical myocardial scintigraphy. Both thallium uptake and release could be described by a single exponential with a half-time of exchange which was approximately half that of potassium and which was largely independent of extracellular thallium concentration. Some 60% of thallium uptake occurred via an "active" or ouabain-inhibitable mechanism which, in the absence of extracellular potassium, could be activated by low concentrations (10 nM to 5 microM) of thallium. The apparent Km for thallium on this active transport mechanism was 2-7 microM. Increasing extracellular potassium from 0-10 mM caused significant, concentration-dependent decreases in both the total and the active component of the thallium influx. Similarly nonradioactive thallium (0.10 microM to 0.10 mM) caused a concentration-dependent decrease in active potassium influx. Analysis of these results by both Lineweaver-Burk plots and Dixon plots confirmed competitive inhibition, potassium on thallium influx and vice versa, for the active component of the fluxes, and noncompetitive in the remainder. These findings indicate that active transport accounts for the greater portion of the influx of thallium and potassium, and that this active transport occurs via a common mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)

  1. Effects of olanzapine on LPS-induced inflammation in rat primary glia cells.

    Science.gov (United States)

    Faour-Nmarne, Caroline; Azab, Abed N

    2016-01-01

    Olanzapine (OLZ) is an atypical antipsychotic drug that also has mood-stabilizing effects. The mechanism of action of OLZ is not fully understood. Accumulating data suggest that inflammation plays a role in the pathophysiology of mental disorders and that psychotropic drugs exhibit some anti-inflammatory properties. This study was undertaken to examine the effects of OLZ on LPS-induced inflammation in rat primary glia cells. Glia cells were extracted from newborn rat brains. OLZ (1 or 50 µM) was added to culture medium at 6 or 72 h before addition of LPS for another 18 h, and levels of IL-10, prostaglandin (PG) E2, NO and TNF-α, and expression of cyclo-oxygensase (COX)-2 and inducible NO synthase (iNOS) were determined. Treatment with 50 µM OLZ (but not 1 µM) significantly decreased LPS-induced secretion of IL-10, PGE2 and TNF-α. In contrast, 50 µM OLZ significantly increased NO levels. OLZ did not alter the expression of COX-2 or iNOS in LPS-treated cells. These results suggest that OLZ differently affects the secretion of inflammatory mediators. Most of the significant effects of OLZ were obtained when 50 µM was used, which is a high and probably therapeutically irrelevant concentration. Therefore, under the conditions used in the present study OLZ seemed to lack a potent anti-inflammatory effect. © The Author(s) 2015.

  2. Methods to induce primary and secondary traumatic damage in organotypic hippocampal slice cultures.

    Science.gov (United States)

    Adamchik, Y; Frantseva, M V; Weisspapir, M; Carlen, P L; Perez Velazquez, J L

    2000-04-01

    Organotypic brain slice cultures have been used in a variety of studies on neurodegenerative processes [K.M. Abdel-Hamid, M. Tymianski, Mechanisms and effects of intracellular calcium buffering on neuronal survival in organotypic hippocampal cultures exposed to anoxia/aglycemia or to excitotoxins, J. Neurosci. 17, 1997, pp. 3538-3553; D.W. Newell, A. Barth, V. Papermaster, A.T. Malouf, Glutamate and non-glutamate receptor mediated toxicity caused by oxygen and glucose deprivation in organotypic hippocampal cultures, J. Neurosci. 15, 1995, pp. 7702-7711; J.L. Perez Velazquez, M.V. Frantseva, P.L. Carlen, In vitro ischemia promotes glutamate mediated free radical generation and intracellular calcium accumulation in pyramidal neurons of cultured hippocampal slices, J. Neurosci. 23, 1997, pp. 9085-9094; L. Stoppini, L.A. Buchs, D. Muller, A simple method for organotypic cultures of nervous tissue, J. Neurosci. Methods 37, 1991, pp. 173-182; R.C. Tasker, J.T. Coyle, J.J. Vornov, The regional vulnerability to hypoglycemia induced neurotoxicity in organotypic hippocampal culture: protection by early tetrodotoxin or delayed MK 801, J. Neurosci. 12, 1992, pp. 4298-4308.]. We describe two methods to induce traumatic cell damage in hippocampal organotypic cultures. Primary trauma injury was achieved by rolling a stainless steel cylinder (0.9 g) on the organotypic slices. Secondary injury was followed after dropping a weight (0.137 g) on a localised area of the organotypic slice, from a height of 2 mm. The time course and extent of cell death were determined by measuring the fluorescence of the viability indicator propidium iodide (PI) at several time points after the injury. The initial localised impact damage spread 24 and 67 h after injury, cell death being 25% and 54%, respectively, when slices were kept at 37 degrees C. To validate these methods as models to assess neuroprotective strategies, similar insults were applied to slices at relatively low temperatures (30

  3. A meta-ethnography of organisational culture in primary care medical practice.

    Science.gov (United States)

    Grant, Suzanne; Guthrie, Bruce; Entwistle, Vikki; Williams, Brian

    2014-01-01

    Over the past decade, there has been growing international interest in shaping local organisational cultures in primary healthcare. However, the contextual relevance of extant culture assessment instruments to the primary care context has been questioned. The aim of this paper is to derive a new contextually appropriate understanding of the key dimensions of primary care medical practice organisational culture and their inter-relationship through a synthesis of published qualitative research. A systematic search of six electronic databases followed by a synthesis using techniques of meta-ethnography involving translation and re-interpretation. A total of 16 papers were included in the meta-ethnography from the UK, the USA, Canada, Australia and New Zealand that fell into two related groups: those focused on practice organisational characteristics and narratives of practice individuality; and those focused on sub-practice variation across professional, managerial and administrative lines. It was found that primary care organisational culture was characterised by four key dimensions, i.e. responsiveness, team hierarchy, care philosophy and communication. These dimensions are multi-level and inter-professional in nature, spanning both practice and sub-practice levels. The research contributes to organisational culture theory development. The four new cultural dimensions provide a synthesized conceptual framework for researchers to evaluate and understand primary care cultural and sub-cultural levels. The synthesised cultural dimensions present a framework for practitioners to understand and change organisational culture in primary care teams. The research uses an innovative research methodology to synthesise the existing qualitative research and is one of the first to develop systematically a qualitative conceptual framing of primary care organisational culture.

  4. Hydrogen sulfide is expressed in the human and the rat cultured nucleus pulposus cells and suppresses apoptosis induced by hypoxia.

    Directory of Open Access Journals (Sweden)

    Haolin Sun

    Full Text Available Apoptosis plays pivotal role in the pathogenesis of degenerative disc diseases, which is the primary contributor to low back pain. Although the role of hydrogen sulfide (H2S in cell apoptosis is well appreciated, the effects and mechanism that H2S regulates the program death of intervertebral disc cell are not yet elucidated. In this study, we utilized the nucleus pulposus (NP from patients with lumbar disc herniation to investigate the relationship between endogenous H2S and NP cells apoptosis in human. Furthermore, we analyzed primary rat NP cells to study the effects of exogenous H2S on hypoxia induced cell apoptosis. Human NP samples were obtained from patients with lumbar disc herniation and were divided into uncontained and contained herniation groups. Using immunohistochemistry staining and sulphur-sensitive electrode, we detected the expression of cystathionine-β-synthase (CBS and cystathionine γ-lyase (CSE, as well as the production of endogenous H2S in human NP. Tunel staining showed increased apoptosis in NP from herniated disc; and there was significant correlation between H2S generation and apoptosis in human NP. CoCl2 was then used to induce hypoxia in cultured primary rat NP cells. Annexin V staining indicated that exogenous NaHS attenuated hypoxia induced apoptosis in rat NP cells. Furthermore, hypoxia significantly increased the levels of multiple apoptosis associated proteins (Fas, Cytochromes C, Caspase 9 and cleaved-Caspase-3 in cells, which were eliminated by NaHS. Our study demonstrates the presence of endogenous H2S in human intervertebral disc; and the endogenous H2S generation rate is associated with NP apoptosis in herniated disc. In vitro study showes exogenous H2S donor attenuates hypoxia induced apoptosis in primary rat NP cells. Thus, our work provides insights that H2S may have beneficial effects in treating degenerative disc diseases.

  5. Fermented wheat powder induces the antioxidant and detoxifying system in primary rat hepatocytes.

    Directory of Open Access Journals (Sweden)

    Margherita La Marca

    Full Text Available Many plants exhibit antioxidant properties which may be useful in the prevention of oxidative stress reactions, such as those mediated by the formation of free radical species in different pathological situations. In recent years a number of studies have shown that whole grain products in particular have strong antioxidant activity. Primary cultures of rat hepatocytes were used to investigate whether and how a fermented powder of wheat (Lisosan G is able to modulate antioxidant and detoxifying enzymes, and whether or not it can activate Nrf2 transcription factor or inhibit NF-kB activation. All of the antioxidant and detoxifying enzymes studied were significantly up-regulated by 0.7 mg/ml Lisosan G treatment. In particular,quinone oxidoreductase and heme oxygenase-1 were induced, although to different degrees, at the transcriptional, protein and/or activity levels by the treatment. As for the Nrf2 transcription factor, a partial translocation of its protein from the cytosol to the nucleus after 1 h of Lisosan G treatment was revealed by immunoblotting. Lisosan G was also observed to decrease H2O2-induced toxicity Taken together, these results show that this powder of wheat is an effective inducer of ARE/Nrf2-regulated antioxidant and detoxifying genes and has the potential to inhibit the translocation of NF-kB into the nucleus.

  6. Apelin Protects Primary Rat Retinal Pericytes from Chemical Hypoxia-Induced Apoptosis

    Directory of Open Access Journals (Sweden)

    Li Chen

    2015-01-01

    Full Text Available Pericytes are a population of cells that participate in normal vessel architecture and regulate permeability. Apelin, as the endogenous ligand of G protein-coupled receptor APJ, participates in a number of physiological and pathological processes. To date, the effect of apelin on pericyte is not clear. Our study aimed to investigate the potential protection mechanisms of apelin, with regard to primary rat retinal pericytes under hypoxia. Immunofluorescence staining revealed that pericytes colocalized with APJ in the fibrovascular membranes dissected from proliferative diabetic retinopathy patients. In the in vitro studies, we first demonstrated that the expression of apelin/APJ was upregulated in pericytes under hypoxia, and apelin increased pericytes proliferation and migration. Moreover, knockdown of apelin in pericyte was achieved via lentivirus-mediated RNA interference. After the inhibition of apelin, pericytes proliferation was inhibited significantly in hypoxia culture condition. Furthermore, exogenous recombinant apelin effectively prevented hypoxia-induced apoptosis through downregulating active-caspase 3 expression and increasing the ratio of B cell lymphoma-2 (Bcl-2/Bcl-2 associated X protein (Bax in pericytes. These results suggest that apelin suppressed hypoxia-induced pericytes injury, which indicated that apelin could be a potential therapeutic target for retinal angiogenic diseases.

  7. Postimplantation Whole Embryo Culture Assay for Hamsters: An Alternative to Rat and Mouse

    Directory of Open Access Journals (Sweden)

    Bogdan Wlodarczyk

    2001-01-01

    Full Text Available Postimplantation whole embryo culture (WEC assay for rats and mice has been well established and introduced to many laboratories. Recently WEC technique for rabbits has been developed; however, information on culture of other species is very limited. Knowing the usefulness of hamsters in classical embryotoxicology, we reasoned that hamster WEC could be an alternative model for the most frequently used rat and mouse WEC. Previously we have optimized culture conditions for postimplantation hamster embryos. The aim of this study was to test the susceptibility of hamster embryos cultured in vitro to embryotoxic compounds and to compare our results with those reported by others on rat or mouse embryo culture. For that purpose we choose three known embryotoxic compounds�valproic acid, cadmium chloride, and diethylstilbestrol�and tested them using a postimplantation hamster whole embryo culture assay. Hamster embryos were cultured from 7.5 days gestation for 24 h in a medium consisting of 35% hamster serum and 65% synthetic culture medium (Iscove�s or McCoy 5A. At the end of the culture period, the embryos were examined morphologically, measured with the aid of a computer image analysis system, and total protein content was assessed. All three compounds exhibited dose-related embryotoxic and teratogenic effects in hamster embryos. The malformations observed were similar to those reported on rat and mouse embryos. Comparison of the results with data reported by other authors indicates that hamster embryos cultured in vitromight be more susceptible to embryotoxic stimuli than rat and mouse embryos.

  8. Examining the Culturally Responsive Practices of Urban Primary Physical Educators

    Science.gov (United States)

    Culp, Brian; Chepyator-Thomson, J. Rose

    2011-01-01

    Recent changes in the demographics of urban public schools have presented an opportunity to assess the instructional strategies of teachers of culturally and linguistically diverse (CLD) learners. Given that ethnic minorities represent more than 75% of the student population in 50 of America's largest public school systems, research on teachers of…

  9. The role of culture in primary health care

    African Journals Online (AJOL)

    1994-12-12

    Dec 12, 1994 ... cause of septicemia following dog bite. J CUn Microbial 1989; 27: 231-235. 3. Butler T. Weaver RE, ... laboratory identification and report of the incidence in dogs in the Cape peninsula. Medical Technology SA 1989; .... Traditional healers are very popular because they provide culturally familiar ways of ...

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

    Science.gov (United States)

    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…

  11. Effects of cholesterol transport inhibitor U18666A on APP metabolism in rat primary astrocytes.

    Science.gov (United States)

    Yang, Hongyan; Wang, Yanlin; Kar, Satyabrata

    2017-11-01

    Amyloid β (Aβ) peptides generated from the amyloid precursor protein (APP) play an important role in the degeneration of neurons and development of Alzheimer's disease (AD). Current evidence indicates that high levels of cholesterol-which increase the risk of developing AD-can influence Aβ production in neurons. However, it remains unclear how altered level/subcellular distribution of cholesterol in astrocytes can influence APP metabolism. In this study, we evaluated the effects of cholesterol transport inhibitor U18666A-a class II amphiphile that triggers redistribution of cholesterol within the endosomal-lysosomal (EL) system-on APP levels and metabolism in rat primary cultured astrocytes. Our results revealed that U18666A increased the levels of the APP holoprotein and its cleaved products (α-/β-/η-CTFs) in cultured astrocytes, without altering the total levels of cholesterol or cell viability. The cellular levels of Aβ 1-40 were also found to be markedly increased, while secretory levels of Aβ 1-40 were decreased in U18666A-treated astrocytes. We further report a corresponding increase in the activity of the enzymes regulating APP processing, such as α-secretase, β-secretase, and γ-secretase as a consequence of U18666A treatment. Additionally, APP-cleaved products are partly accumulated in the lysosomes following cholesterol sequestration within EL system possibly due to decreased clearance. Interestingly, serum delipidation attenuated enhanced levels of APP and its cleaved products following U18666A treatment. Collectively, these results suggest that cholesterol sequestration within the EL system in astrocytes can influence APP metabolism and the accumulation of APP-cleaved products including Aβ peptides, which can contribute to the development of AD pathology. © 2017 Wiley Periodicals, Inc.

  12. Arsenite stimulates glutathione export and glycolytic flux in viable primary rat brain astrocytes.

    Science.gov (United States)

    Tadepalle, Nimesha; Koehler, Yvonne; Brandmann, Maria; Meyer, Nils; Dringen, Ralf

    2014-10-01

    Intoxication with inorganic arsenicals leads to neuropathies and impaired cognitive functions. However, little is known so far on the cellular targets that are involved in the adverse effects of arsenite to brain cells. To test whether arsenite may affect neural glucose and glutathione (GSH) metabolism, primary astrocyte cultures from rat brain were used as a model system. Exposure of cultured astrocytes to arsenite in concentrations of up to 0.3mM did not compromise cell viability during incubations for up to 6h, while 1mM arsenite damaged the cells already within 2h after application. Determination of cellular arsenic contents of astrocytes that had been incubated for 2h with arsenite revealed an almost linear concentration-dependent increase in the specific cellular arsenic content. Exposure of astrocytes to arsenite stimulated the export of GSH and accelerated the cellular glucose consumption and lactate production in a time- and concentration-dependent manner. Half-maximal stimulation of GSH export and glycolytic flux were observed for arsenite in concentrations of 0.1mM and 0.3mM, respectively. The arsenite-induced stimulation of both processes was abolished upon removal of extracellular arsenite. The strong stimulation of GSH export by arsenite was prevented by MK571, an inhibitor of the multidrug resistance protein 1, suggesting that this transporter mediates the accelerated GSH export. In addition, presence of MK571 significantly increased the specific cellular arsenic content, suggesting that Mrp1 may also be involved in arsenic export from astrocytes. The data observed suggest that alterations in glucose and GSH metabolism may contribute to the reported adverse neural consequences of intoxication with arsenite. Copyright © 2014 Elsevier Ltd. All rights reserved.

  13. Immunocytochemical characterization of primary cell culture in canine transmissible venereal tumor

    Directory of Open Access Journals (Sweden)

    Luis M.M. Flórez

    Full Text Available Abstract: Immunochemistry with anti-vimentin, anti-lysozyme, anti-alpha 1 antitrypsin, anti-CD3 and anti-CD79α antibodies has been used for characterization of primary cell culture in the transmissible venereal tumor (TVT. Samples for primary cell culture and immunohistochemistry assays were taken from eight dogs with cytological and clinical diagnosis of TVT. To validate the immunochemical results in the primary cell culture of TVT, a chromosome count was performed. For the statistical analysis, the Mann-Whitney test with p<0.05 was used. TVT tissues and culture cells showed intense anti-vimentin immunoreactivity, lightly to moderate immunoreactivity for anti-lysozyme, and mild for anti-alpha-antitrypsin. No marking was achieved for CD3 and CD79α. All culture cells showed chromosomes variable number of 56 to 68. This is the first report on the use of immunocytochemical characterization in cell culture of TVT. Significant statistic difference between immunochemistry in tissue and culture cell was not established, what suggests that the use of this technique may provide greater certainty for the confirmation of tumors in the primary culture. This fact is particularly important because in vitro culture of tumor tissues has been increasingly used to provide quick access to drug efficacy and presents relevant information to identify potential response to anticancer medicine; so it is possible to understand the behavior of the tumor.

  14. Cultural influences on Ghanaian primary school pupils' conceptions ...

    African Journals Online (AJOL)

    This paper reports on a study which investigated how pupils' notions of measuring and sharing in out-of-school setting reflect their conceptions and practices in school. Two focus group interviews were carried out with 16 primary school pupils, eight each from grade 4 and grade 6 in two average achieving schools (one ...

  15. A primary preventative mental health intervention in a culturally ...

    African Journals Online (AJOL)

    The Ububele Baby Mat Service is a community-based, parent–infant mental health intervention offered at five primary health care clinics in Alexandra Township, in Johannesburg. The aim of the intervention is to promote healthy caregiver-infant attachments. There has been a steady increase in the number of mother-baby ...

  16. Ouabain binding to cultured vascular smooth muscle cells of the spontaneously hypertensive rat

    International Nuclear Information System (INIS)

    Hopp, L.; Khalil, F.; Tamura, H.; Kino, M.; Searle, B.M.; Tokushige, A.; Aviv, A.

    1986-01-01

    The binding of ouabain and K + to the Na + pump were analyzed in serially passed cultured vascular smooth muscle cells (VSMCs) originating from spontaneously hypertensive (SH) Wistar-Kyoto (WKY), and American Wistar (W) rats. The techniques have utilized analyses of displacement of [ 3 H]ouabain by both unlabeled ouabain and K + from specific binding sites on the VSMCs. The authors have found that 1) each of the VSMC preparations from the three rat strains appeared to demonstrate one population of specific ouabain receptors (Na + pumps); 2) the number of Na + pump units of both the SH and WKY rats was significantly lower than the number of Na + pump units of W rat VSMCs; 3) the equilibrium dissociation constant values (μM) for ouabain in VSMCs of SH and WKY rats were similar but were significantly higher than that of VSMCs derived from W rats; and 4) among the VSMCs originating from the three rat strains, the apparent equilibrium dissociation constant value for K + (mM) was the lowest in those of the SH rat compared with VSMCs of the WKY rat and W rat. Previous studies have demonstrated increased passive Na + and K + transport rate constants of SH rat VSMCs compared with either W or WKY rat cells. These findings suggest the possibility of higher permeabilities of the SH cells. They propose that the combined effect of a low number of Na + pump units with higher permeabilities to Na + and K + predisposes VSMCs of the SH rat to disturbances in their cellular ionic regulation. These genetic defects, if they occur in vivo, may lead to an increase in the vascular tone

  17. Organizational culture predicts job satisfaction and perceived clinical effectiveness in pediatric primary care practices.

    Science.gov (United States)

    Brazil, Kevin; Wakefield, Dorothy B; Cloutier, Michelle M; Tennen, Howard; Hall, Charles B

    2010-01-01

    In recent years, there has been a growing understanding that organizational culture is related to an organization's performance. However, few studies have examined organizational culture in medical group practices. The purpose of this study was to examine the relationship of organizational culture on provider job satisfaction and perceived clinical effectiveness in primary care pediatric practices. This cross-sectional study included 36 primary care pediatric practices located in Connecticut. There were 374 participants in this study, which included 127 clinicians and 247 nonclinicians. Office managers completed a questionnaire that recorded staff and practice characteristics; all participants completed the Organizational Culture Scale, a questionnaire that assessed the practice on four cultural domains (i.e., group, developmental, rational, and hierarchical), and the Primary Care Organizational Questionnaire that evaluated perceived effectiveness and job satisfaction. Hierarchical linear models using a restricted maximum likelihood estimation method were used to evaluate whether the practice culture types predicted job satisfaction and perceived effectiveness. Group culture was positively associated with both satisfaction and perceived effectiveness. In contrast, hierarchical and rational culture were negatively associated with both job satisfaction and perceived effectiveness. These relationships were true for clinicians, nonclinicians, and the practice as a whole. Our study demonstrates that practice culture is associated with job satisfaction and perceived clinical effectiveness and that a group culture was associated with high job satisfaction and perceived effectiveness.

  18. In vitro Analysis of Neurospheres Derived from Glioblastoma Primary Culture: A Novel Methodology Paradigm.

    Science.gov (United States)

    Pavon, Lorena Favaro; Marti, Luciana C; Sibov, Tatiana Tais; Malheiros, Suzana M F; Brandt, Reynaldo Andre; Cavalheiro, Sergio; Gamarra, Lionel F

    2014-01-07

    Glioblastomas are the most lethal primary brain tumor that frequently relapse or progress as focal masses after radiation, suggesting that a fraction of tumor cells are responsible for the tumor regrowth. The identification of a brain tumor cell subpopulation with potent tumorigenic activity supports the cancer stem cell hypothesis in solid tumors. The goal of this study is to determine a methodology for the establishment of primary human glioblastoma cell lines. Our aim is achieved by taking the following approaches: (i) the establishment of primary glioblastoma cell culture; (ii) isolation of neurospheres derived from glioblastoma primary cultures; (iii) selection of CD133 cells from neurospheres, (iv) formation of subspheres in the CD133-positive population, (v) study of the expression level of GFAP, CD133, Nestin, Nanog, CD34, Sox2, CD44, and CD90 markers on tumor subspheres. Hence, we described a successful method for isolation of CD133-positive cell population and establishment of glioblastoma neurospheres from this primary culture, which are more robust than the ones derived straight from the tumor. Pointed out that the neurospheres derived from glioblastoma primary culture showed 29% more cells expressing CD133 then the ones straight tumor-derived, denoting a higher concentration of CD133-positive cells in the neurospheres derived from glioblastoma primary culture. These CD133-positive fractions were able to further generate subspheres. The subspheres derived from glioblastoma primary culture presented a well-defined morphology while the ones derived from the fresh tumor were sparce and less robust. And the negative fraction of CD133 cells was unable to generate subspheres. The tumor subspheres expressed GFAP, CD133, Nestin, Nanog, CD44, and CD90. Also, the present study describes an optimization of neurospheres/subspheres isolation from glioblastoma primary culture by selection of CD133-positive adherent stem cell.

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

    Science.gov (United States)

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

    2002-08-01

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

  20. Characterization of Amino Acid Profile and Enzymatic Activity in Adult Rat Astrocyte Cultures.

    Science.gov (United States)

    Souza, Débora Guerini; Bellaver, Bruna; Hansel, Gisele; Arús, Bernardo Assein; Bellaver, Gabriela; Longoni, Aline; Kolling, Janaina; Wyse, Angela T S; Souza, Diogo Onofre; Quincozes-Santos, André

    2016-07-01

    Astrocytes are multitasking players in brain complexity, possessing several receptors and mechanisms to detect, participate and modulate neuronal communication. The functionality of astrocytes has been mainly unraveled through the study of primary astrocyte cultures, and recently our research group characterized a model of astrocyte cultures derived from adult Wistar rats. We, herein, aim to characterize other basal functions of these cells to explore the potential of this model for studying the adult brain. To characterize the astrocytic phenotype, we determined the presence of GFAP, GLAST and GLT 1 proteins in cells by immunofluorescence. Next, we determined the concentrations of thirteen amino acids, ATP, ADP, adenosine and calcium in astrocyte cultures, as well as the activities of Na(+)/K(+)-ATPase and acetylcholine esterase. Furthermore, we assessed the presence of the GABA transporter 1 (GAT 1) and cannabinoid receptor 1 (CB 1) in the astrocytes. Cells demonstrated the presence of glutamine, consistent with their role in the glutamate-glutamine cycle, as well as glutamate and D-serine, amino acids classically known to act as gliotransmitters. ATP was produced and released by the cells and ADP was consumed. Calcium levels were in agreement with those reported in the literature, as were the enzymatic activities measured. The presence of GAT 1 was detected, but the presence of CB 1 was not, suggesting a decreased neuroprotective capacity in adult astrocytes under in vitro conditions. Taken together, our results show cellular functionality regarding the astrocytic role in gliotransmission and neurotransmitter management since they are able to produce and release gliotransmitters and to modulate the cholinergic and GABAergic systems.

  1. Novel Nuclear Protein Complexes of Dystrophin 71 Isoforms in Rat Cultured Hippocampal GABAergic and Glutamatergic Neurons.

    Directory of Open Access Journals (Sweden)

    Rafael Rodríguez-Muñoz

    Full Text Available The precise functional role of the dystrophin 71 in neurons is still elusive. Previously, we reported that dystrophin 71d and dystrophin 71f are present in nuclei from cultured neurons. In the present work, we performed a detailed analysis of the intranuclear distribution of dystrophin 71 isoforms (Dp71d and Dp71f, during the temporal course of 7-day postnatal rats hippocampal neurons culture for 1h, 2, 4, 10, 15 and 21 days in vitro (DIV. By immunofluorescence assays, we detected the highest level of nuclear expression of both dystrophin Dp71 isoforms at 10 DIV, during the temporal course of primary culture. Dp71d and Dp71f were detected mainly in bipolar GABAergic (≥60% and multipolar Glutamatergic (≤40% neurons, respectively. We also characterized the existence of two nuclear dystrophin-associated protein complexes (DAPC: dystrophin 71d or dystrophin 71f bound to β-dystroglycan, α1-, β-, α2-dystrobrevins, α-syntrophin, and syntrophin-associated protein nNOS (Dp71d-DAPC or Dp71f-DAPC, respectively, in the hippocampal neurons. Furthermore, both complexes were localized in interchromatin granule cluster structures (nuclear speckles of neuronal nucleoskeleton preparations. The present study evinces that each Dp71's complexes differ slightly in dystrobrevins composition. The results demonstrated that Dp71d-DAPC was mainly localized in bipolar GABAergic and Dp71f-DAPC in multipolar Glutamatergic hippocampal neurons. Taken together, our results show that dystrophin 71d, dystrophin 71f and DAP integrate protein complexes, and both complexes were associated to nuclear speckles structures.

  2. Novel Nuclear Protein Complexes of Dystrophin 71 Isoforms in Rat Cultured Hippocampal GABAergic and Glutamatergic Neurons.

    Science.gov (United States)

    Rodríguez-Muñoz, Rafael; Cárdenas-Aguayo, María Del Carmen; Alemán, Víctor; Osorio, Beatriz; Chávez-González, Oscar; Rendon, Alvaro; Martínez-Rojas, Dalila; Meraz-Ríos, Marco Antonio

    2015-01-01

    The precise functional role of the dystrophin 71 in neurons is still elusive. Previously, we reported that dystrophin 71d and dystrophin 71f are present in nuclei from cultured neurons. In the present work, we performed a detailed analysis of the intranuclear distribution of dystrophin 71 isoforms (Dp71d and Dp71f), during the temporal course of 7-day postnatal rats hippocampal neurons culture for 1h, 2, 4, 10, 15 and 21 days in vitro (DIV). By immunofluorescence assays, we detected the highest level of nuclear expression of both dystrophin Dp71 isoforms at 10 DIV, during the temporal course of primary culture. Dp71d and Dp71f were detected mainly in bipolar GABAergic (≥60%) and multipolar Glutamatergic (≤40%) neurons, respectively. We also characterized the existence of two nuclear dystrophin-associated protein complexes (DAPC): dystrophin 71d or dystrophin 71f bound to β-dystroglycan, α1-, β-, α2-dystrobrevins, α-syntrophin, and syntrophin-associated protein nNOS (Dp71d-DAPC or Dp71f-DAPC, respectively), in the hippocampal neurons. Furthermore, both complexes were localized in interchromatin granule cluster structures (nuclear speckles) of neuronal nucleoskeleton preparations. The present study evinces that each Dp71's complexes differ slightly in dystrobrevins composition. The results demonstrated that Dp71d-DAPC was mainly localized in bipolar GABAergic and Dp71f-DAPC in multipolar Glutamatergic hippocampal neurons. Taken together, our results show that dystrophin 71d, dystrophin 71f and DAP integrate protein complexes, and both complexes were associated to nuclear speckles structures.

  3. Contribution to Cultural Organization, Working Motivation and Job Satisfaction on the Performance of Primary School Teacher

    Science.gov (United States)

    Murtedjo; Suharningsih

    2016-01-01

    The purposes of this study are: (1) describes the performance of the teacher, organizational culture, work motivation and job satisfaction; (2) determine whether there is a significant direct relationship between organizational culture, work motivation and job satisfaction on the performance of primary school teachers. Through the study of the…

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

    Science.gov (United States)

    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…

  5. A Multi-Cultural Interaction through Video Conferencing in Primary Schools

    Science.gov (United States)

    Duygu Eristi, Suzan

    2012-01-01

    This study investigated Turkish and Canadian primary school students' ways of expressing their perception of cultural understanding through video conferencing and that of cultural interaction through video conferencing. The qualitative research data were collected in the form of interviews. The results obtained were analyzed and interpreted based…

  6. A primary neuron culture system for the study of herpes simplex virus latency and reactivation.

    Science.gov (United States)

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

    2012-04-02

    Herpes simplex virus type-1 (HSV-1) establishes a life-long latent infection in peripheral neurons. This latent reservoir is the source of recurrent reactivation events that ensure transmission and contribute to clinical disease. Current antivirals do not impact the latent reservoir and there are no vaccines. While the molecular details of lytic replication are well-characterized, mechanisms controlling latency in neurons remain elusive. Our present understanding of latency is derived from in vivo studies using small animal models, which have been indispensable for defining viral gene requirements and the role of immune responses. However, it is impossible to distinguish specific effects on the virus-neuron relationship from more general consequences of infection mediated by immune or non-neuronal support cells in live animals. In addition, animal experimentation is costly, time-consuming, and limited in terms of available options for manipulating host processes. To overcome these limitations, a neuron-only system is desperately needed that reproduces the in vivo characteristics of latency and reactivation but offers the benefits of tissue culture in terms of homogeneity and accessibility. Here we present an in vitro model utilizing cultured primary sympathetic neurons from rat superior cervical ganglia (SCG) (Figure 1) to study HSV-1 latency and reactivation that fits most if not all of the desired criteria. After eliminating non-neuronal cells, near-homogeneous TrkA(+) neuron cultures are infected with HSV-1 in the presence of acyclovir (ACV) to suppress lytic replication. Following ACV removal, non-productive HSV-1 infections that faithfully exhibit accepted hallmarks of latency are efficiently established. Notably, lytic mRNAs, proteins, and infectious virus become undetectable, even in the absence of selection, but latency-associated transcript (LAT) expression persists in neuronal nuclei. Viral genomes are maintained at an average copy number of 25 per neuron

  7. Functional analysis of ultra high information rates conveyed by rat vibrissal primary afferents

    NARCIS (Netherlands)

    A.M. Chagas (André); L. Theis (Lucas); B. Sengupta (Biswa); M.C. Stüttgen (Maik); M. Bethge (Matthias); C. Schwarz (Cornelius)

    2013-01-01

    textabstractSensory receptors determine the type and the quantity of information available for perception. Here, we quantified and characterized the information transferred by primary afferents in the rat whisker system using neural system identification. Quantification of "how much" information is

  8. Proteomic Characterization of Primary Mouse Hepatocytes in Collagen Monolayer and Sandwich Culture.

    Science.gov (United States)

    Orsini, Malina; Sperber, Saskia; Noor, Fozia; Hoffmann, Esther; Weber, Susanne N; Hall, Rabea A; Lammert, Frank; Heinzle, Elmar

    2018-01-01

    Dedifferentiation of primary hepatocytes in vitro makes their application in long-term studies difficult. Embedding hepatocytes in a sandwich of extracellular matrix is reported to delay the dedifferentiation process to some extent. In this study, we compared the intracellular proteome of primary mouse hepatocytes (PMH) in conventional monolayer cultures (ML) to collagen sandwich culture (SW) after 1 day and 5 days of cultivation. Quantitative proteome analysis of PMH showed no differences between collagen SW and ML cultures after 1 day. Glycolysis and gluconeogenesis were strongly affected by long-term cultivation in both ML and SW cultures. Interestingly, culture conditions had no effect on cellular lipid metabolism. After 5 days, PMH in collagen SW and ML cultures exhibit characteristic indications of oxidative stress. However, in the SW culture the defense system against oxidative stress is significantly up-regulated to deal with this, whereas in the ML culture a down-regulation of these important enzymes takes place. Regarding the multiple effects of ROS and oxidative stress in cells, we conclude that the down-regulation of these enzymes seem to play a role in the loss of hepatic function observed in the ML cultivation. In addition, enzymes of the urea cycle were clearly down-regulated in ML culture. Proteomics confirms lack in oxidative stress defense mechanisms as the major characteristic of hepatocytes in monolayer cultures compared to sandwich cultures. J. Cell. Biochem. 119: 447-454, 2018. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

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

    Science.gov (United States)

    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…

  10. Dopaminergic differentiation of human neural stem cells mediated by co-cultured rat striatal brain slices

    DEFF Research Database (Denmark)

    Anwar, Mohammad Raffaqat; Andreasen, Christian Maaløv; Lippert, Solvej Kølvraa

    2008-01-01

    differentiation, we co-cultured cells from a human neural forebrain-derived stem cell line (hNS1) with rat striatal brain slices. In brief, coronal slices of neonatal rat striatum were cultured on semiporous membrane inserts placed in six-well trays overlying monolayers of hNS1 cells. After 12 days of co......Properly committed neural stem cells constitute a promising source of cells for transplantation in Parkinson's disease, but a protocol for controlled dopaminergic differentiation is not yet available. To establish a setting for identification of secreted neural compounds promoting dopaminergic...

  11. TERATOGENIC EFFECTS OF TRANSPLACENTAL TRANSFUSION OF HETEROLOGOUS ANTISERA SIMULATED IN AN EXPERIMENTAL-MODEL USING INVITRO WHOLE RAT EMBRYO CULTURE

    NARCIS (Netherlands)

    VANDERZEE, DC; POELMANN, RE; ZWIERSTRA, RP; MENTINK, MMT; VERMEIJKEERS, C

    1991-01-01

    The effects of the transplacental transfusion of heterologous rabbit-anti-rat antiserum (RAR antiserum) and subsequent immunological interaction on the development of 9-10 days old rat embryos (stages 8-10 somites) were studied using an in vitro whole rat embryo culture. Transplacental transfusion

  12. Comparative genomic and proteomic analysis of high grade glioma primary cultures and matched tumor in situ.

    LENUS (Irish Health Repository)

    Howley, R

    2012-10-15

    Developing targeted therapies for high grade gliomas (HGG), the most common primary brain tumor in adults, relies largely on glioma cultures. However, it is unclear if HGG tumorigenic signaling pathways are retained under in-vitro conditions. Using array comparative genomic hybridization and immunohistochemical profiling, we contrasted the epidermal and platelet-derived growth factor receptor (EGFR\\/PDGFR) in-vitro pathway status of twenty-six primary HGG cultures with the pathway status of their original HGG biopsies. Genomic gains or amplifications were lost during culturing while genomic losses were more likely to be retained. Loss of EGFR amplification was further verified immunohistochemically when EGFR over expression was decreased in the majority of cultures. Conversely, PDGFRα and PDGFRβ were more abundantly expressed in primary cultures than in the original tumor (p<0.05). Despite these genomic and proteomic differences, primary HGG cultures retained key aspects of dysregulated tumorigenic signaling. Both in-vivo and in-vitro the presence of EGFR resulted in downstream activation of P70s6K while reduced downstream activation was associated with the presence of PDGFR and the tumor suppressor, PTEN. The preserved pathway dysregulation make this glioma model suitable for further studies of glioma tumorigenesis, however individual culture related differences must be taken into consideration when testing responsiveness to chemotherapeutic agents.

  13. The association between culture, climate and quality of care in primary health care teams.

    Science.gov (United States)

    Hann, Mark; Bower, Peter; Campbell, Stephen; Marshall, Martin; Reeves, David

    2007-09-01

    Culture and climate represent shared beliefs and values that may influence quality of care in health care teams, and which could be manipulated for quality improvement. However, there is a lack of agreement on the theoretical and empirical relationships between climate and culture, and their relative power as predictors of quality of care. This study sought to examine the association between self-report measures of climate and culture in primary care teams and comprehensive measures of quality of care. The data were derived from a cross-sectional survey of 492 professionals in 42 general practices in England. Self-report measures of culture (the Competing Values Framework) and climate (the Team Climate Inventory) were used, together with validated measures of quality of care from medical records and self-report. The majority of practices could be characterized as 'clan' culture type. Practices with a dominant clan culture scored higher on climate for participation and teamwork. There were no associations between culture and quality of care, and only limited evidence of associations between climate and quality. The current analysis would not support the hypothesis that culture and climate are important predictors of quality of care in primary care. Although larger studies are required to provide a definitive test, the results may suggest the need for a more complex model of the associations between culture, climate and outcomes, and further research may be required into the interaction between culture and climate with other determinants of behaviour such as internal and external incentives.

  14. Chlorpyrifos exerts opposing effects on axonal and dendritic growth in primary neuronal cultures

    International Nuclear Information System (INIS)

    Howard, Angela S.; Bucelli, Robert; Jett, David A.; Bruun, Donald; Yang, Dongren; Lein, Pamela J.

    2005-01-01

    Evidence that children are widely exposed to organophosphorus pesticides (OPs) and that OPs cause developmental neurotoxicity in animal models raises significant concerns about the risks these compounds pose to the developing human nervous system. Critical to assessing this risk is identifying specific neurodevelopmental events targeted by OPs. Observations that OPs alter brain morphometry in developing rodents and inhibit neurite outgrowth in neural cell lines suggest that OPs perturb neuronal morphogenesis. However, an important question yet to be answered is whether the dysmorphogenic effect of OPs reflects perturbation of axonal or dendritic growth. We addressed this question by quantifying axonal and dendritic growth in primary cultures of embryonic rat sympathetic neurons derived from superior cervical ganglia (SCG) following in vitro exposure to chlorpyrifos (CPF) or its metabolites CPF-oxon (CPFO) and trichloropyridinol (TCP). Axon outgrowth was significantly inhibited by CPF or CPFO, but not TCP, at concentrations ≥0.001 μM or 0.001 nM, respectively. In contrast, all three compounds enhanced BMP-induced dendritic growth. Acetylcholinesterase was inhibited only by the highest concentrations of CPF (≥1 μM) and CPFO (≥1 nM); TCP had no effect on this parameter. In summary, these compounds perturb neuronal morphogenesis via opposing effects on axonal and dendritic growth, and both effects are independent of acetylcholinesterase inhibition. These findings have important implications for current risk assessment practices of using acetylcholinesterase inhibition as a biomarker of OP neurotoxicity and suggest that OPs may disrupt normal patterns of neuronal connectivity in the developing nervous system

  15. Kinetics and autoradiography of high affinity uptake of serotonin by primary astrocyte cultures

    Energy Technology Data Exchange (ETDEWEB)

    Katz, D.M.; Kimelberg, H.K.

    1985-07-01

    Primary astrocyte cultures prepared from the cerebral cortices of neonatal rats showed significant accumulation of serotonin (5-hydroxytryptamine; (/sup 3/H)-5-HT). At concentrations in the range of 0.01 to 0.7 microM (/sup 3/H)-5-HT, this uptake was 50 to 85% Na+ dependent and gave a Km of 0.40 +/- 0.11 microM (/sup 3/H)-5-HT and a Vmax of 6.42 +/- 0.85 (+/- SEM) pmol of (/sup 3/H)-5-HT/mg of protein/4 min for the Na+-dependent component. In the absence of Na+ the uptake was nonsaturable. Omission of the monoamine oxidase inhibitor pargyline markedly reduced the Na+-dependent component of (/sup 3/H)-5-HT uptake but had a negligible effect on the Na+-independent component. This suggest significant oxidative deamination of serotonin after it has been taken up by the high affinity system, followed by release of its metabolite. The authors estimated that this system enabled the cells to concentrate (/sup 3/H)-5-HT up to 44-fold at an external (/sup 3/H)-5-HT concentration of 10(-7) M. Inhibition of (/sup 3/H)-5-HT uptake by a number of clinically effective antidepressants was also consistent with a specific high affinity uptake mechanism for 5-HT, the order of effectiveness of inhibition being chlorimipramine greater than fluoxetine greater than imipramine = amitriptyline greater than desmethylimipramine greater than iprindole greater than mianserin. Uptake of (/sup 3/H)-5-HT was dependent on the presence of Cl- as well as Na+ in the medium, and the effect of omission of both ions was nonadditive. Varying the concentration of K+ in the media from 1 to 50 mM had a limited effect on (/sup 3/H)-5-HT uptake.

  16. Intracellular processing of growth hormone receptors by adipocytes in primary culture.

    Science.gov (United States)

    Roupas, P; Herington, A C

    1988-05-01

    Rat adipocytes in primary culture have been used to study the intracellular processing of growth hormone (GH) receptors. Pretreatment of adipocytes with 20 micrograms/ml cycloheximide resulted in a rapid decline (t1/2 approximately 45 min) of the 125I-human growth hormone (hGH) binding capacity of the cells. This decline occurred at a faster rate in the presence of extracellular unlabeled hGH (400 ng/ml) and was not due to receptor occupancy. These data suggest that GH receptors turn over rapidly and constitutively on the plasma membrane and in the absence of protein synthesis are not replaced. Dissociation of GH-receptor complexes was shown not to occur at pH 5.5, the pH encountered in the acidic pre-lysosomal compartments (endosomes) where intracellular dissociation of many hormone-receptor complexes takes place. These data, together, suggest that the majority of GH receptors are not recycled but instead suffer the same fate as the majority of GH, i.e. degradation. To determine the rate of appearance of GH receptors at the cell surface, adipocytes were first treated with trypsin and then incubated at 37 degrees C to permit incorporation of any available GH receptors into the plasma membrane. Binding of 125I-hGH recovered to pre-trypsin levels by 2 h. This recovery was completely blocked by concomitant treatment with monensin, cytochalasin B, colchicine and 2,4-dinitrophenol. NH4Cl had no effect on receptor recovery. These data suggest that once GH receptors are synthesized in the rough endoplasmic reticulum, they travel via the Golgi apparatus to the plasma membrane (by processes involving both microfilaments and microtubules) and are then inserted into the plasma membrane in an energy-dependent step.

  17. Macromolecular metabolism of a differentiated rat keratinocyte culture system following exposure to sulfur mustard

    International Nuclear Information System (INIS)

    Vaughan, F.L.; Zaman, S.; Scavarelli, R.; Bernstein, I.A.

    1988-01-01

    A method for producing a stratified, squamous epithelium in vitro by cultivating rat keratinocytes on nylon membranes has been developed in this laboratory. This epidermal-like culture is being used to obtain a better understanding of the mechanism of skin vesication after topical exposure to the sulfur mustard bis(beta-chloroethyl) sulfide (BCES) dissolved in a selected solvent. Radiolabeled macromolecular precursors (thymidine, uridine, and leucine) have been used to study the effect of BCES on the synthesis of DNA, RNA, and protein, respectively, after topical exposure to the mustard at concentrations of 0.01-500 nmol/cm2 dissolved in 70% dimethyl sulfoxide (DMSO). From these and other studies it has been determined that exposure to even the low concentration of 0.01 nmol BCES/cm2 for 30 min results in significant inhibition of [ 3 H]thymidine incorporation, although complete recovery occurs by 24 h. Significant inhibition of [ 3 H]uridine and [ 14 C]leucine incorporation is observed only after exposure to much higher concentrations of BCES (10-500 nmol/cm2). This suggests a very early lesion in macromolecular metabolism with DNA being the primary target

  18. Explant culture of rat colon: A model system for studying metabolism of chemical carcinogens

    DEFF Research Database (Denmark)

    Autrup, Herman; Stoner, G.D.; Jackson, F.

    1978-01-01

    An explant culture system has been developed for the long-term maintenance of colonic tissue from the rat. Explants of 1 cm2 in size were placed in tissue-culture dishes to which was added 2 ml of CMRL-1066 medium supplemented with glucose, hydrocortisone, beta-retinyl acetate, and either 2.5% bo......,12-dimethylbenz[alpha]anthracene, aflatoxin B1, dimethylnitrosamine, 1,2-dimethylhydrazine, and methylazoxymethanol acetate into chemical species that bind to cellular DNA and protein....

  19. Production and degradation of AMP in cultured rat skeletal and heart muscle: a comparative study

    International Nuclear Information System (INIS)

    Zoref-Shani, E.; Kessler-Icekson, G.; Shainberg, A.; Sperling, O.

    1986-01-01

    The authors clarify the mechanisms operating in the skeletal and heart muscles to produce and degrade AMP. Rat myotube and cardiomyocyte cultures were prepared and purine synthesis was gauged by the rate of C 14-formate incorporation into purines. It is shown that the cell cultures exhibited capacity for de novo purine synthesis, indicated by incorporation of C 14-formate into purines. The rate of C 14-formate into the cardiomyocyte purines was markedly lower than that in the skeletal muscle myotubes

  20. Assessment of patient safety culture in primary care setting, Al-Mukala, Yemen.

    Science.gov (United States)

    Webair, Hana H; Al-Assani, Salwa S; Al-Haddad, Reema H; Al-Shaeeb, Wafa H; Bin Selm, Manal A; Alyamani, Abdulla S

    2015-10-13

    Patient safety culture in primary care is the first step to achieve high quality health care. This study aims to provide a baseline assessment of patient safety culture in primary care settings in Al-Mukala, Yemen as a first published study from a least developed country. A survey was conducted in primary healthcare centres and units in Al-Mukala District, Yemen. A comprehensive sample from the available 16 centres was included. An Arabic version of the Medical Office Survey on Patient Safety Culture was distributed to all health workers (110). Participants were physicians, nurses and administrative staff. The response rate from the participating centres was 71 %. (N = 78). The percent positive responses of the items is equal to the percentage of participants who answered positively. Composite scores were calculated by averaging the percent positive response on the items within a dimension. Positive safety culture was defined as 60 % or more positive responses on items or dimensions. Patient safety culture was perceived to be generally positive with the exception of the dimensions of 'Communication openness', 'Work pressure and pace' and 'Patient care tracking/follow-up', as the percent positive response of these dimensions were 58, 57, and 52 % respectively. Overall, positive rating on quality and patient safety were low (49 and 46 % respectively). Although patient safety culture in Al-Mukala primary care setting is generally positive, patient safety and quality rating were fairly low. Implementation of a safety and quality management system in Al-Mukala primary care setting are paramount. Further research is needed to confirm the applicability of the Medical Office Survey on Patient Safety Culture (MOSPSC) for Al-Mukala primary care.

  1. Neurotoxicity of coral snake phospholipases A2 in cultured rat hippocampal neurons.

    Science.gov (United States)

    de Carvalho, Nathalia Delazeri; Garcia, Raphael CaioTamborelli; Ferreira, Adilson Kleber; Batista, Daniel Rodrigo; Cassola, Antonio Carlos; Maria, Durvanei; Lebrun, Ivo; Carneiro, Sylvia Mendes; Afeche, Solange Castro; Marcourakis, Tania; Sandoval, Maria Regina Lopes

    2014-03-13

    The neurotoxicity of two secreted Phospholipases A2 from Brazilian coral snake venom in rat primary hippocampal cell culture was investigated. Following exposure to Mlx-8 or Mlx-9 toxins, an increase in free cytosolic Ca(2+) and a reduction in mitochondrial transmembrane potential (ΔΨm) became evident and occurred prior to the morphological changes and cytotoxicity. Exposure of hippocampal neurons to Mlx-8 or Mlx-9 caused a decrease in the cell viability as assessed by MTT and LDH assays. Inspection using fluorescent images and ultrastructural analysis by scanning and transmission electron microscopy showed that multiphase injury is characterized by overlapping cell death phenotypes. Shrinkage, membrane blebbing, chromatin condensation, nucleosomal DNA fragmentation and the formation of apoptotic bodies were observed. The most striking alteration observed in the electron microscopy was the fragmentation and rarefaction of the neuron processes network. Degenerated terminal synapses, cell debris and apoptotic bodies were observed among the fragmented fibers. Numerous large vacuoles as well as swollen mitochondria and dilated Golgi were noted. Necrotic signs such as a large amount of cellular debris and membrane fragmentation were observed mainly when the cells were exposed to highest concentration of the PLA2-neurotoxins. PLA2s exposed cultures showed cytoplasmic vacuoles filled with cell debris, clusters of mitochondria presented mitophagy-like structures that are in accordance to patterns of programmed cell death by autophagy. Finally, we demonstrated that the sPLA2s, Mlx-8 and Mlx-9, isolated from the Micrurus lemniscatus snake venom induce a hybrid cell death with apoptotic, autophagic and necrotic features. Furthermore, this study suggests that the augment in free cytosolic Ca(2+) and mitochondrial dysfunction are involved in the neurotoxicity of Elapid coral snake venom sPLA2s. Copyright © 2014 Elsevier B.V. All rights reserved.

  2. Plate reader-based cell viability assays for glioprotection using primary rat optic nerve head astrocytes.

    Science.gov (United States)

    Kaja, Simon; Payne, Andrew J; Naumchuk, Yuliya; Levy, Deborah; Zaidi, Danish H; Altman, Alexa M; Nawazish, Saba; Ghuman, Jasleen K; Gerdes, Bryan C; Moore, Mark A; Koulen, Peter

    2015-09-01

    Optic nerve head astrocytes (ONHAs) are the major glia cell type in the non-myelinated optic nerve head where they contribute critically to extracellular matrix synthesis during development and throughout life. In glaucoma, and in related disorders affecting the optic nerve and the optic nerve head, pathological changes include altered astrocyte gene and protein expression resulting in their activation and extracellular matrix remodeling. ONHAs are highly sensitive to mechanical and oxidative stress resulting in the initiation of axon damage early during pathogenesis. Furthermore, ONHAs are crucial for the maintenance of retinal ganglion cell physiology and function. Therefore, glioprotective strategies with the goal to preserve and/or restore the structural and functional viability of ONHA in order to slow glaucoma and related pathologies are of high clinical relevance. Herein, we describe the development of standardized methods that will allow for the systematic advancement of such glioprotective strategies. These include isolation, purification and culture of primary adult rat ONHAs, optimized immunocytochemical protocols for cell type validation, as well as plate reader-based assays determining cellular viability, proliferation and the intracellular redox state. We validated and standardized our protocols by performing a glioprotection study using primary ONHAs. Specifically, we measured protection against exogenously-applied oxidative stress using tert-butylhydroperoxide (tBHP) as a model of disease-mediated oxidative stress in the retina and optic nerve head by the prototypic antioxidant, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox). Levels of oxidative stress were increased in the response to exogenously applied tBHP and were assessed by 6-carboxy-2', 7' dichlorodihydrofluorescein diacetate (DCFDA) fluorescence. Normalized DCFDA fluorescence showed a maximal 5.1-fold increase; the half-maximal effect (EC50) for tBHP was 212 ± 25

  3. Down-regulation of protein kinase C protects cerebellar granule neurons in primary culture from glutamate-induced neuronal death

    International Nuclear Information System (INIS)

    Favaron, M.; Manev, H.; Bertolino, M.; Szekely, A.M.; DeErausquin, G.; Guidotti, A.; Costa, E.; Siman, R.

    1990-01-01

    Exposing primary cultures of cerebellar granule neurons to 100 nM phorbol 12-myristate 13-acetate (PMA) for 24 hr decreases the Ca 2+ /phosphatidylserine/diolein-dependent protein kinase C. Immunoblot analysis of the homogenates with polyclonal antibodies raised against either the β-type PKC peptide or total rat brain PKC reveals a virtual loss of 78-kDa PKC immunoreactivity in the supernatant and marked decrease of PKC immunoreactivity in the pellet. Exposure of the cultures to 50 μM glutamate for 15 min (no Mg 2+ ) induces the translocation of supernatant PKC immunoreactivity to the pellet. PMA-induced down-regulation of PKC decreases glutamate-elicited neurotoxicity. Yet, the culture exposure to 100 nM PMA fails to decrease the high-affinity binding of [ 3 H]glutamate to neuronal membranes and does not reduce glutamate-induced activation of ionotropic or metabolotropic receptors (assayed as total membrane current measured in whole-cell voltage-clamped neurons, 45 Ca 2+ uptake in intact monolayers, inositolphospholipid hydrolysis, and transcriptional activation and translation of c-fos mRNA). On the other hand, PMA-induced PKC down-regulation reduces any increase in 45 Ca 2+ uptake or Ca 2+ -dependent proteolysis after glutamate withdrawal. These results support the view that PKC translocation is operative in glutamate-induced destabilization of cytosolic ionized Ca 2+ homeostasis and neuronal death

  4. Sodium coupled bicarbonate influx regulates intracellular and apical pH in cultured rat caput epididymal epithelium.

    Science.gov (United States)

    Zuo, Wu-Lin; Li, Sheng; Huang, Jie-Hong; Yang, Deng-Liang; Zhang, Geng; Chen, Si-Liang; Ruan, Ye-Chun; Ye, Ke-Nan; Cheng, Christopher H K; Zhou, Wen-Liang

    2011-01-01

    The epithelium lining the epididymis provides an optimal acidic fluid microenvironment in the epididymal tract that enable spermatozoa to complete the maturation process. The present study aims to investigate the functional role of Na(+)/HCO(3)(-) cotransporter in the pH regulation in rat epididymis. Immunofluorescence staining of pan cytokeratin in the primary culture of rat caput epididymal epithelium showed that the system was a suitable model for investigating the function of epididymal epithelium. Intracellular and apical pH were measured using the fluorescent pH sensitive probe carboxy-seminaphthorhodafluor-4F acetoxymethyl ester (SNARF-4F) and sparklet pH electrode respectively to explore the functional role of rat epididymal epithelium. In the HEPES buffered Krebs-Henseleit (KH) solution, the intracellular pH (pHi) recovery from NH(4)Cl induced acidification in the cultured caput epididymal epithelium was completely inhibited by amiloride, the inhibitor of Na(+)/H(+) exchanger (NHE). Immediately changing of the KH solution from HEPES buffered to HCO(3)(-) buffered would cause another pHi recovery. The pHi recovery in HCO(3)(-) buffered KH solution was inhibited by 4, 4diisothiocyanatostilbene-2,2-disulfonic acid (DIDS), the inhibitor of HCO(3)(-) transporter or by removal of extracellular Na(+). The extracellular pH measurement showed that the apical pH would increase when adding DIDS to the apical side of epididymal epithelial monolayer, however adding DIDS to the basolateral side had no effect on apical pH. The present study shows that sodium coupled bicarbonate influx regulates intracellular and apical pH in cultured caput epididymal epithelium.

  5. Metabolism of N-nitrosamines by cultured human and rat esophagus.

    Science.gov (United States)

    Autrup, H; Stoner, G D

    1982-04-01

    The metabolism of several N-nitrosamines (N-nitrosodimethylamine, N-nitrosoethylmethylamine, N-nitrosodiethylamine, N-nitrosobenzylmethylamine, and N-nitrosopyrrolidine) in cultured human and rat esophagus has been investigated by measuring (a) CO2, (b) metabolites with an oxo group, and (c) metabolites bound to DNA. Both acyclic and cyclic N-nitrosamines were metabolized by rat esophagus. The highest level of metabolite binding was seen with N-nitrosobenzylmethylamine, an organotrophic carcinogen for the rat esophagus. The binding level was about 100-fold higher than in human esophagus. This compound methylated rat esophageal DNA at positions 7 and O6 of guanine. The level of benzylation in rat was one-tenth of the level of methylation. Formation of benzaldehyde exceeded that of formaldehyde plus CO2 by a factor of six, indicating that the methylene group was preferentially oxidized. N-Nitrosoethylmethylamine, another unsymmetrical N-nitrosamine, was preferentially oxidized by rat esophagus in the ethyl group, as shown by higher formation of CO2 and acetaldehyde from the compound labeled in the ethyl group. The highest binding level to DNA from this compound was observed with the methyl group. No binding was detected to human esophagus. N-Nitrosopyrrolidine was oxidized by both rat and human esophagus in the alpha position, as measured by the formation of 2,4-dinitrophenylhydrazone derivative of 4-hydroxybutanal. Binding of metabolites of N-nitrosopyrrolidine to DNA was detected only in rat esophagus. As measured by the formation of both CO2 and formaldehyde, N-nitrosodimethylamine was metabolized by both human and rat esophagus. While most of the radioactivity associated with DNA was found to be incorporated into guanine and adenine, methylation of the guanine positions 7 and O6 was detected by chromatography of the hydrolyzed rat DNA. The results indicate significant quantitative and perhaps qualitative differences between cultured rat and human esophagus in

  6. Rat bone marrow stem cells isolation and culture as a bone formative experimental system

    Directory of Open Access Journals (Sweden)

    Amer Smajilagić

    2013-02-01

    Full Text Available Bone marrow mesenchymal cells have been identified as a source of pluripotent stem cells with multipotential potential and differentiation in to the different cells types such as are osteoblast, chondroblast, adipoblast. In this research we describe pioneering experiment of tissue engineering in Bosnia and Herzegovina, of the isolation and differentiation rat bone marrow stromal cells in to the osteoblast cells lineages. Rat bone marrow stromal cells were isolated by method described by Maniatopulos using their plastic adherence capatibility. The cells obtained by plastic adherence were cultured and serially passaged in the osteoinductive medium to differentiate into the osteocytes. Bone marrow samples from rats long bones used for isolation of stromal cells (BMSCs. Under determinate culture conditions BMSCs were differentiated in osteogenic cell lines detected by Alizarin red staining three weeks after isolation. BMSCs as autologue cells model showed high osteogenetic potential and calcification capatibility in vitro. In future should be used as alternative method for bone transplantation in Regenerative Medicine.

  7. Antidiabetic effect of taurine in cultured rat skeletal l6 myotubes.

    Science.gov (United States)

    Cheong, Sun Hee; Chang, Kyung Ja

    2013-01-01

    Taurine (2-aminoethanesulfonic acid), a sulfur-containing β-amino acid, is found in all animal cells at millimolar concentrations and has been reported to show various health promoting activities including antidiabetic properties. The beneficial effects of taurine in diabetes mellitus have been known. However, the exact mechanism of hypoglycemic action of taurine is not properly defined. In this study, we investigated antidiabetic effect of taurine in the cell culture system using rat skeletal muscle cells. In cultured rat skeletal L6 myotubes, we studied the effect of taurine (0-100 μM) on glucose uptake to plasma membrane from the aspects of AMP-activated protein kinase (AMPK) signaling. Taurine stimulated glucose uptake in a dose-dependent manner by activating AMPK signaling. From these results, it may suggest that taurine show antidiabetic effect by stimulating insulin-independent glucose uptake in rat skeletal muscle.

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

    Science.gov (United States)

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

    2015-01-01

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

  9. Embryotoxicant-specific transcriptomic responses in rat postimplantation whole-embryo culture

    NARCIS (Netherlands)

    Robinson, J.F.; van Beelen, V.A.; Verhoef, A.; Renkens, M.F.J.; Luijten, M.; van Herwijnen, M.; Westerman, A.; Pennings, J.L.; Piersma, A.H.

    2010-01-01

    Rat postimplantation whole-embryo culture (WEC) is a promising alternative test for the assessment of developmental toxicity. Toxicogenomic-based approaches may improve the predictive ability of the WEC model by providing a means to identify compound-specific mechanistic responses associated with

  10. Upregulation of endothelin ETB receptor-mediated vasoconstriction in rat coronary artery after organ culture

    DEFF Research Database (Denmark)

    Eskesen, Karen; Edvinsson, Lars

    2006-01-01

    descending coronary arteries isolated from hearts of rats as response to application of the selective endothelin ET(B) receptor agonist, Sarafotoxin 6c and endothelin-1. In segments cultured 1 day in serum free Dulbecco's Modified Eagle's Medium, Sarafotoxin 6c induced a concentration dependent contraction...

  11. Comparative evaluation of the teratogenicity of genistein and genistin using rat whole embryo culture and limbud micromass culture methods.

    Science.gov (United States)

    Zou, Peng; Xing, Lina; Tang, Qiuqiong; Liu, Ran; Hao, Weidong

    2012-08-01

    Genistein (GEN) is one kind of phytoestrogen. Several studies have demonstrated the teratogenic potential of GEN in vitro by postimplantation rat whole embryo culture (WEC) assay, but GEN showed no teratogenic effects in vivo even at a dose up to 1000 mg/kg bw/day. The mechanism of such discrepancy is still unclear. Because more than 80% of total genistein (free plus glycoside form) in circulation is its glycoside metabolite, genistin (GIN), we thus hypothesize that genistin is non-teratogenic. To prove this hypothesis, rat whole embryo culture (WEC) and limbud micromass culture methods were applied to compare the teratogenic effects of GEN and GIN on developing embryos in vitro. In WEC assay, we found that the development of embryos was affected by GEN treatment dose-dependently, while GIN-treated embryos displayed slight developmental defects only at the highest dose (222 μM). In micromass culture assay, the IC50 of cell proliferation and differentiation for GEN were 15.6 and 37.2 μM, respectively, while neither was influenced by GIN treatment up to 111 μM. Collectively, our study indicated that GEN showed no teratogenic effects in vivo probably due to its transformation to the non-teratogenic metabolite, GIN. Copyright © 2012 Elsevier Ltd. All rights reserved.

  12. Unilateral nasal obstruction affects motor representation development within the face primary motor cortex in growing rats.

    Science.gov (United States)

    Abe, Yasunori; Kato, Chiho; Uchima Koecklin, Karin Harumi; Okihara, Hidemasa; Ishida, Takayoshi; Fujita, Koichi; Yabushita, Tadachika; Kokai, Satoshi; Ono, Takashi

    2017-06-01

    Postnatal growth is influenced by genetic and environmental factors. Nasal obstruction during growth alters the electromyographic activity of orofacial muscles. The facial primary motor area represents muscles of the tongue and jaw, which are essential in regulating orofacial motor functions, including chewing and jaw opening. This study aimed to evaluate the effect of chronic unilateral nasal obstruction during growth on the motor representations within the face primary motor cortex (M1). Seventy-two 6-day-old male Wistar rats were randomly divided into control ( n = 36) and experimental ( n = 36) groups. Rats in the experimental group underwent unilateral nasal obstruction after cauterization of the external nostril at 8 days of age. Intracortical microstimulation (ICMS) mapping was performed when the rats were 5, 7, 9, and 11 wk old in control and experimental groups ( n = 9 per group per time point). Repeated-measures multivariate ANOVA was used for intergroup and intragroup statistical comparisons. In the control and experimental groups, the total number of positive ICMS sites for the genioglossus and anterior digastric muscles was significantly higher at 5, 7, and 9 wk, but there was no significant difference between 9 and 11 wk of age. Moreover, the total number of positive ICMS sites was significantly smaller in the experimental group than in the control at each age. It is possible that nasal obstruction induced the initial changes in orofacial motor behavior in response to the altered respiratory pattern, which eventually contributed to face-M1 neuroplasticity. NEW & NOTEWORTHY Unilateral nasal obstruction in rats during growth periods induced changes in arterial oxygen saturation (SpO 2 ) and altered development of the motor representation within the face primary cortex. Unilateral nasal obstruction occurring during growth periods may greatly affect not only respiratory function but also craniofacial function in rats. Nasal obstruction should be treated

  13. Mitochondrial ribosomal protein S18-2 evokes chromosomal instability and transforms primary rat skin fibroblasts

    KAUST Repository

    Kashuba, Elena

    2015-05-12

    We have shown earlier that overexpression of the human mitochondrial ribosomal protein MRPS18-2 (S18-2) led to immortalization of primary rat embryonic fibroblasts. The derived cells expressed the embryonic stem cell markers, and cellular pathways that control cell proliferation, oxidative phosphorylation, cellular respiration, and other redox reactions were activated in the immortalized cells. Here we report that, upon overexpression of S18-2 protein, primary rat skin fibroblasts underwent cell transformation. Cells passed more than 300 population doublings, and two out of three tested clones gave rise to tumors in experimental animals. Transformed cells showed anchorage-independent growth and loss of contact inhibition; they expressed epithelial markers, such as E-cadherin and β-catenin. Transformed cells showed increased telomerase activity, disturbance of the cell cycle, and chromosomal instability. Taken together, our data suggest that S18-2 is a newly identified oncoprotein that may be involved in cancerogenesis.

  14. The ligase chain reaction as a primary screening tool for the detection of culture positive tuberculosis.

    LENUS (Irish Health Repository)

    O'Connor, T M

    2012-02-03

    BACKGROUND: The ligase chain reaction Mycobacterium tuberculosis assay uses ligase chain reaction technology to detect tuberculous DNA sequences in clinical specimens. A study was undertaken to determine its sensitivity and specificity as a primary screening tool for the detection of culture positive tuberculosis. METHODS: The study was conducted on 2420 clinical specimens (sputum, bronchoalveolar lavage fluid, pleural fluid, urine) submitted for primary screening for Mycobacterium tuberculosis to a regional medical microbiology laboratory. Specimens were tested in parallel with smear, ligase chain reaction, and culture. RESULTS: Thirty nine patients had specimens testing positive by the ligase chain reaction assay. Thirty two patients had newly diagnosed tuberculosis, one had a tuberculosis relapse, three had tuberculosis (on antituberculous therapy when tested), and three had healed tuberculosis. In the newly diagnosed group specimens were smear positive in 21 cases (66%), ligase chain reaction positive in 30 cases (94%), and culture positive in 32 cases (100%). Using a positive culture to diagnose active tuberculosis, the ligase chain reaction assay had a sensitivity of 93.9%, a specificity of 99.8%, a positive predictive value of 83.8%, and a negative predictive value of 99.9%. CONCLUSIONS: This study is the largest clinical trial to date to report the efficacy of the ligase chain reaction as a primary screening tool to detect Mycobacterium tuberculosis infection. The authors conclude that ligase chain reaction is a useful primary screening test for tuberculosis, offering speed and discrimination in the early stages of diagnosis and complementing traditional smear and culture techniques.

  15. An improved in vitro blood-brain barrier model: rat brain endothelial cells co-cultured with astrocytes.

    Science.gov (United States)

    Abbott, N Joan; Dolman, Diana E M; Drndarski, Svetlana; Fredriksson, Sarah M

    2012-01-01

    In vitro blood-brain barrier (BBB) models using primary cultured brain endothelial cells are important for establishing cellular and molecular mechanisms of BBB function. Co-culturing with BBB-associated cells especially astrocytes to mimic more closely the in vivo condition leads to upregulation of the BBB phenotype in the brain endothelial cells. Rat brain endothelial cells (RBECs) are a valuable tool allowing ready comparison with in vivo studies in rodents; however, it has been difficult to obtain pure brain endothelial cells, and few models achieve a transendothelial electrical resistance (TEER, measure of tight junction efficacy) of >200 Ω cm(2), i.e. the models are still relatively leaky. Here, we describe methods for preparing high purity RBECs and neonatal rat astrocytes, and a co-culture method that generates a robust, stable BBB model that can achieve TEER >600 Ω cm(2). The method is based on >20 years experience with RBEC culture, together with recent improvements to kill contaminating cells and encourage BBB differentiation.Astrocytes are isolated by mechanical dissection and cell straining and are frozen for later co-culture. RBECs are isolated from 3-month-old rat cortices. The brains are cleaned of meninges and white matter and enzymatically and mechanically dissociated. Thereafter, the tissue homogenate is centrifuged in bovine serum albumin to separate vessel fragments from other cells that stick to the myelin plug. The vessel fragments undergo a second enzyme digestion to separate pericytes from vessels and break down vessels into shorter segments, after which a Percoll gradient is used to separate capillaries from venules, arterioles, and single cells. To kill remaining contaminating cells such as pericytes, the capillary fragments are plated in puromycin-containing medium and RBECs grown to 50-60% confluence. They are then passaged onto filters for co-culture with astrocytes grown in the bottom of the wells. The whole procedure takes ∼2

  16. Embryonic zebrafish primary cell culture for transfection and live cellular and subcellular imaging.

    Science.gov (United States)

    Sassen, Wiebke A; Lehne, Franziska; Russo, Giulio; Wargenau, Sven; Dübel, Stefan; Köster, Reinhard W

    2017-10-01

    Although having great potential for live cell imaging to address numerous cell biological questions with high spatial and temporal resolution, primary cell cultures of zebrafish embryos are not widely used. We present an easy-to-use protocol for preparing primary cell cultures of 2 dpf zebrafish embryos allowing for live cell imaging of fully differentiated cells such as neurons and myocytes. We demonstrate that different cell types can be identified by morphology and expression of transgenic cell type-specific fluorescent reporters and that fluorescent cells can be sorted by flow cytometry to prepare an enriched culture. To facilitate subcellular imaging in live primary cells, we successfully tested a selection of fluorescent vital dyes. Most importantly, we demonstrate that zebrafish primary cells can be transfected efficiently with expression constructs allowing for visualizing subcellular structures with fluorescent marker proteins for time lapse imaging. We propose zebrafish primary cell culture as a versatile tool to address cell biological questions in combination with a powerful in vivo model. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. DIFFERENTIAL-EFFECTS OF EICOSAPENTAENOIC ACID ON GLYCEROLIPID AND APOLIPOPROTEIN-B METABOLISM IN PRIMARY HUMAN HEPATOCYTES COMPARED TO HEPG2 CELLS AND PRIMARY RAT HEPATOCYTES

    NARCIS (Netherlands)

    LIN, YG; SMIT, MJ; HAVINGA, R; VERKADE, HJ; VONK, RJ; KUIPERS, F

    1995-01-01

    We compared the effects of eicosapentaenoic acid (EPA) and oleic acid (OA) on glycerolipid and apolipoprotein B (apoB) metabolism in primary human hepatocytes, HepG2 cells and primary rat hepatocytes. Cells were incubated for 1 to 5 h with 0.25 mM bovine serum albumin in the absence (control) or

  18. Science-Based Thematic Cultural Art Learning in Primary School (2013 Curriculum

    Directory of Open Access Journals (Sweden)

    Warih Handayaningrum

    2016-12-01

    Full Text Available This study is aimed at discussing the development result of thematic cultural art subject’s learning material based on science for primary school (2013 curriculum. This study is expected to inspire teacher to develop learning material that may explore artworks exist in our living environment (based on the context of children’s environment. This study applies steps in developmental research collaboration by Borg & Gall (1989 and Puslitjaknov (2008 to create the product. The development stages comprise observation in several primary schools in Surabaya, Gresik, and Sidoarjo that has implemented 2013 curriculum that is followed up by stages of development. Furthermore, prototype of cultural and art thematic learning material development results are verified by learning material experts, material expert, primary school teacher, and revised afterwards. The result of this research development is a set of teacher and student books. Science-based cultural art here means cultural art learning as the main medium to introduce local culture products (music, drawing, dance, and drama by integrating mathematics, sciences, Bahasa Indonesia, and local language subjects. Cultural art products in the form of dance, music, drawing, dramas will help children to understand a simple mathematical concept, such as: two-dimensional figure, geometry, comparing or estimating longer-shorter, smaller-bigger, or more-less.

  19. Preventive effect of piracetam and vinpocetine on hypoxia-reoxygenation induced injury in primary hippocampal culture.

    Science.gov (United States)

    Solanki, P; Prasad, D; Muthuraju, S; Sharma, A K; Singh, S B; Ilavzhagan, G

    2011-04-01

    The present study investigates the potential of Piracetam and Vinpocetine (nootropic drugs, known to possess neuroprotective properties) in preventing hypoxia-reoxygenation induced oxidative stress in primary hippocampal cell culture. The hippocampal culture was exposed to hypoxia (95% N(2), 5% CO(2)) for 3h and followed by 1h of reoxygenation (21% O(2) and 5% CO(2)) at 37 °C. The primary hippocampal cultures were supplemented with the optimum dose of Piracetam and Vinpocetine, independently, and the cultures were divided into six groups, viz. Control/Normoxia, Hypoxia, Hypoxia+Piracetam, Hypoxia+Vinpocetine, Normoxia + Piracetam and Normoxia+Vinpocetine. The cell-viability assays and biochemical oxidative stress parameters were evaluated for each of the six groups. Administration of 1mM Piracetam or 500 nM Vinpocetine significantly prevents the culture from hypoxia-reoxygenation injury when determined by Neutral Red assay, LDH release and Acetylcholine esterase activity. Results showed that Piracetam and Vinpocetine supplementation significantly prevented the fall of mitochondrial membrane potential, rise in ROS generation and reduction in antioxidant levels associated with the hypoxia-reoxygenation injury. In conclusion, the present study establishes that both Piracetam and Vinpocetine give neuroprotection against hypoxia-reoxygenation injury in primary hippocampal cell culture. Copyright © 2010 Elsevier Ltd. All rights reserved.

  20. Characteristics of human infant primary fibroblast cultures from Achilles tendons removed post-mortem

    DEFF Research Database (Denmark)

    Rohde, Marianne Cathrine; Corydon, Thomas Juhl; Hansen, Jakob

    2014-01-01

    Primary cell cultures were investigated as a tool for molecular diagnostics in a forensic setting. Fibroblast cultures had been established from human Achilles tendon resected at autopsies, from cases of sudden infant death syndrome and control infants who died in traumatic events (n=41). After...... into the study. High interpersonal variation in growth rates and cell doubling time was seen, but no statistically significant differences were found with increasing age of the donor (mean 19 weeks), length of post-mortem interval prior to sampling (6-100h), or increase in years of storage. Fibroblast cultures...

  1. Rat fetal ventral mesencephalon grown as solid tissue cultures

    DEFF Research Database (Denmark)

    Höglinger, G U; Sautter, J; Meyer, Morten

    1998-01-01

    in vitro (DIV) in the presence or absence (controls) of BDNF [100 ng/ml]. The dopamine content in the culture medium, analyzed by HPLC, was significantly higher (4-5 fold) in the BDNF group at DIV 8 and DIV 12 compared to the corresponding control levels (40 pg/ml). The number of tyrosine hydroxylase...

  2. Trimethyltin (TMT) neurotoxicity in organotypic rat hippocampal slice cultures

    DEFF Research Database (Denmark)

    Noraberg, J; Gramsbergen, J B; Fonnum, F

    1998-01-01

    The neurotoxic effects of trimethyltin (TMT) on the hippocampus have been extensively studied in vivo. In this study, we examined whether the toxicity of TMT to hippocampal neurons could be reproduced in organotypic brain slice cultures in order to test the potential of this model for neurotoxico...

  3. [Application of the cultural competence model in the experience of care in nursing professionals Primary Care].

    Science.gov (United States)

    Gil Estevan, María Dolores; Solano Ruíz, María Del Carmen

    2017-11-01

    To know the experiences and perceptions of nurses in providing care and health promotion, women belonging to groups at risk of social vulnerability, applying the model of cultural competence Purnell. Phenomenological qualitative study. Department of Health Elda. A total of 22 primary care professional volunteers. Semi-structured interviews and focus groups with recording and content analysis, according to the theory model of cultural competence. Socio-cultural factors influence the relationship between professionals and users of the system. The subtle racism and historical prejudices create uncomfortable situations and mistrust. The language barrier makes it difficult not only communication, but also the monitoring and control of the health-disease process. The physical appearance and stereotypes are determining factors for primary care professionals. Although perceived misuse of health services are also talking about changes. The spiritual aspects of religious beliefs alone are taken into account in the case of Muslim women, not being considered as important in the case of Gypsy women and Romanian women. To provide quality care, consistent and culturally competent, it is necessary to develop training programs for professionals in cultural competence, to know the culture of other, and work without preconceived ideas, and ethnocentric; since the greater the knowledge of the cultural group being served, the better the quality of care provided. Copyright © 2017 Elsevier España, S.L.U. All rights reserved.

  4. Cultural respect strategies in Australian Aboriginal primary health care services: beyond education and training of practitioners.

    Science.gov (United States)

    Freeman, Toby; Edwards, Tahnia; Baum, Fran; Lawless, Angela; Jolley, Gwyn; Javanparast, Sara; Francis, Theresa

    2014-08-01

    There is little literature on health-service-level strategies for culturally respectful care to Aboriginal and Torres Strait Islander Australians. We conducted two case studies, which involved one Aboriginal community controlled health care service and one state government-managed primary health care service, to examine cultural respect strategies, client experiences and barriers to cultural respect. Data were drawn from 22 interviews with staff from both services and four community assessment workshops, with a total of 21 clients. Staff and clients at both services reported positive appraisals of the achievement of cultural respects. Strategies included: being grounded in a social view of health, including advocacy and addressing social determinants; employing Aboriginal staff; creating a welcoming service; supporting access through transport, outreach, and walk-in centres; and integrating cultural protocol. Barriers included: communication difficulties; racism and discrimination; and externally developed programs. Service-level strategies were necessary to achieving cultural respect. These strategies have the potential to improve Aboriginal and Torres Strait Islander health and wellbeing. Primary health care's social determinants of health mandate, the community controlled model, and the development of the Aboriginal and Torres Strait Islander health workforce need to be supported to ensure a culturally respectful health system. © 2014 Public Health Association of Australia.

  5. A method for establishing human primary gastric epithelial cell culture from fresh surgical gastric tissues.

    Science.gov (United States)

    Aziz, Faisal; Yang, Xuesong; Wen, Qingping; Yan, Qiu

    2015-08-01

    At present, biopsy specimens, cancer cell lines and tissues obtained by gastric surgery are used in the study and analysis of gastric cancer, including the molecular mechanisms and proteomics. However, fibroblasts and other tissue components may interfere with these techniques. Therefore, the present study aimed to develop a procedure for the isolation of viable human gastric epithelial cells from gastric surgical tissues. A method was developed to culture human gastric epithelial cells using fresh, surgically excised tissues and was evaluated using immunocytochemistry, periodic acid-Schiff (PAS) staining and cell viability assays. Low cell growth was observed surrounding the gastric tissue on the seventh day of tissue explant culture. Cell growth subsequently increased, and at 12 days post-explant a high number of pure epithelial cells were detected. The gastric cancer cells exhibited rapid growth with a doubling time of 13-52 h, as compared to normal cells, which had a doubling time of 20-53 h. Immunocytochemical analyses of primary gastric cells revealed positive staining for cytokeratin 18 and 19, which indicated that the culture was comprised of pure epithelial cells and contained no fibroblasts. Furthermore, PAS staining demonstrated that the cultured gastric cells produced neutral mucin. Granulin and carbohydrate antigen 724 staining confirmed the purity of gastric cancer and normal cells in culture. This method of cell culture indicated that the gastric cells in primary culture consisted of mucin-secreting gastric epithelial cells, which may be useful for the study of gastric infection with Helicobacter pylori and gastric cancer.

  6. Isolation and culture of primary adult skin fibroblasts from the Asian elephant (Elephas maximus

    Directory of Open Access Journals (Sweden)

    Puntita Siengdee

    2018-01-01

    Full Text Available Background Primary cultures from Asian elephants (Elephas maximus allow scientists to obtain representative cells that have conserved most of their original characteristics, function, physiology and biochemistry. This technique has thus gained significant importance as a foundation for further cellular, cell biology and molecular research. Therefore, the aim of this study was to describe conditions for the successful establishment of primary adult fibroblasts from Asian elephant carcasses. Methods Ear tissue sample collection from Asian elephant carcasses and our recommendations are given. We describe here a simple modified protocol for successful isolation and maintenance of primary adult fibroblasts from elephant ear skin. Ear samples from each individual (five 3 × 3 cm2 pieces were brought to the laboratory within 3 h after collection, kept in transportation medium at 0–4 °C. The ear tissues were prepared by a combination of 10% collagenase type II digestion procedure together with a simple explant procedure. Primary fibroblasts were cultured at 37 °C in Dulbecco’s modified Eagle’s medium (DMEM with 20% fetal calf serum (FCS in a humidified atmosphere containing 5% CO2. After the third passage, fibroblasts were routinely trypsinized with 0.25% trypsin/EDTA and cultured in DMEM with 10% FCS at 37 °C and 5% CO2. Traditional cell counting method was used to measure cell viability and growth curve. Long-term storage of cells used freezing medium consisting of 40% FCS (v/v. Results We explored the most suitable conditions during sample collection (post-mortem storage time and sample storage temperature, which is the most important step in determining primary outgrowth. Our study successfully established and cultured primary adult skin fibroblasts obtained from post-mortem E. maximus ear skin tissues from six carcasses, with a success rate of around 83.3%. Outgrowth could be seen 4–12 days after explantation, and epithelial

  7. The First Study of Patient Safety Culture in Iranian Primary Health Centers

    Directory of Open Access Journals (Sweden)

    Mojtaba Sedaghat

    2012-07-01

    Full Text Available Although the error in health care has received attention recently, patient safety culture in health centers has been relatively neglected. To measure the patient safety culture in primary health centers. A cross-sectional study, utilizing the modified version of the Hospital Survey on Patient Safety Culture (HSOPSC developed by the Agency for Healthcare Research and Quality (AHRQ and a demographic questionnaire. Healthcare staffs from health centers were participated in the survey. The patient safety culture score including subscores on 11 dimensions and 39 items; patient safety grade and number of events reported. The overall positive response rate of patient safety culture was 57 ±16.8 (CI 9555%-59%. The dimensions that received higher positive response rate were "Teamwork across units of health center", "Teamwork within units", "Head of center support for patient safety". The lowest percentage of positive responses was "Non punitive response to error". There were no relationship between working years and patients safely culture score. Similarly, no relationship was found between professional, gender and total patients safely culture score. Statistical analysis showed discrepancies between Iranian health centers and the US hospitals in three dimensions. For improving patient safety culture in health centers, it is necessary to have enough staff and establish an environment to be open and fair with staff which helps report errors spontaneously and without any fear. The findings of this study could be used to measure changes in patient safety culture over the time.

  8. Nurses' perceptions of workplace culture in primary health care in Finland.

    Science.gov (United States)

    Hahtela, N; Paavilainen, E; McCormack, B; Helminen, M; Slater, P; Suominen, T

    2015-12-01

    This study aimed to describe nurses' perceptions of workplace culture, especially in regard to stress levels, job satisfaction and the practice environment in primary health care. Health care is facing many challenges related to its attractiveness as a place of employment and the maintenance of a sufficient workforce supply. Previous studies report increasing rates of nurse job dissatisfaction and intentions to leave their current positions both in Finland and also globally. Improving workplace culture is thus vital in meeting the challenges related to recruitment and retention. A cross-sectional descriptive design was used to describe nurses' perceptions of workplace culture. Data were collected by questionnaire from 22 units in nine primary healthcare organizations in Finland, and analysed using descriptive and inferential statistics. Most of the respondents indicated that they were not certain whether their workplace culture was either positive or negative. Profession, age and work shift characteristics had an effect on the respondents' perceptions of workplace culture. Younger licensed practical and registered nurses assessed their workplace culture more positively, whereas older registered nurses and those working rotating rosters viewed workplace culture more negatively. The findings suggest that both unit and demographic characteristics affect workplace culture. This survey highlights that a positive workplace culture is one of the key factors in retaining and recruiting nurses, and provides an essential evidence that may be considered by other healthcare organizations. Nurse managers and healthcare leaders need to address workload management and take into account the related variables that affect a unit's workplace culture. © 2015 International Council of Nurses.

  9. CD90 Expression on human primary cells and elimination of contaminating fibroblasts from cell cultures.

    Science.gov (United States)

    Kisselbach, Lynn; Merges, Michael; Bossie, Alexis; Boyd, Ann

    2009-01-01

    Cluster Differentiation 90 (CD90) is a cell surface glycoprotein originally identified on mouse thymocytes. Although CD90 has been identified on a variety of stem cells and at varying levels in non-lymphoid tissues such as on fibroblasts, brain cells, and activated endothelial cells, the knowledge about the levels of CD90 expression on different cell types, including human primary cells, is limited. The goal of this study was to identify CD90 as a human primary cell biomarker and to develop an efficient and reliable method for eliminating unwanted or contaminating fibroblasts from human primary cell cultures suitable for research pursuant to cell based therapy technologies.

  10. Smooth muscle myosin regulation by serum and cell density in cultured rat lung connective tissue cells.

    Science.gov (United States)

    Babij, P; Zhao, J; White, S; Woodcock-Mitchell, J; Mitchell, J; Absher, M; Baldor, L; Periasamy, M; Low, R B

    1993-08-01

    RNA and protein analyses were used to detect expression of SM1 and SM2 smooth muscle myosin heavy chain (MHC) in cultured adult rat lung connective tissue cells (RL-90). Smooth muscle MHC mRNA expression in confluent cells grown in 10% serum was approximately 50% of the level in adult stomach. Similar results were obtained in cells cultured at low density (25% confluency) in 1% serum. However, in low-density cultures transferred to 10% serum for 24 h, the level of MHC mRNA decreased to approximately 20% of that in adult stomach. Smooth muscle alpha-actin showed a pattern of expression similar to that for smooth muscle MHC. Expression of nonmuscle MHC-A mRNA was higher in all culture conditions compared to stomach. MHC-A mRNA expression was less in low-density cultures in low serum and increased when low-density cultures were transferred to 10% serum for 24 h. MHC-B mRNA expression was less in low- vs. high-density cultures. In contrast to MHC-A, however, MHC-B mRNA expression in low-density cultures was higher in low serum. Immunofluorescence and immunoblotting with SM1-specific antibody demonstrated the presence of the SM1 protein isoform as well as reactivity to a protein band migrating slightly faster than SM2. These results demonstrate that cultured rat lung connective tissue cells express smooth muscle MHC and that expression is modulated by culture conditions.

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

    Directory of Open Access Journals (Sweden)

    McPhee Sarah

    2007-02-01

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

  12. Transfection of myoblasts in primary culture with isomeric cationic cholesterol derivatives

    NARCIS (Netherlands)

    Bischoff, Rainer; Cordier, Y.; Perraud, F.; Thioudellet, C.; Braun, S.; Pavirani, A.

    1997-01-01

    Transfection of satellite cells from dog muscle (myoblasts) in primary culture has been optimized with respect to the position of the cholesteryl moiety along the polyamine chain of spermidine or spermine. Spermidine or spermine were derivatized with cholesterylchloroformate giving rise to three

  13. Primary cultures of glomerular parietal epithelial cells or podocytes with proven origin.

    NARCIS (Netherlands)

    Kabgani, N.; Grigoleit, T.; Schulte, K.; Sechi, A.; Sauer-Lehnen, S.; Tag, C.; Boor, P.; Kuppe, C.; Warsow, G.; Schordan, S.; Mostertz, J.; Chilukoti, R.K.; Homuth, G.; Endlich, N.; Tacke, F.; Weiskirchen, R.; Fuellen, G.; Endlich, K.; Floege, J.; Smeets, B.; Moeller, M.J.

    2012-01-01

    Parietal epithelial cells (PECs) are crucially involved in the pathogenesis of rapidly progressive glomerulonephritis (RPGN) as well as in focal and segmental glomerulosclerosis (FSGS). In this study, transgenic mouse lines were used to isolate pure, genetically tagged primary cultures of PECs or

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

    Science.gov (United States)

    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…

  15. Incorporating Cultural and Linguistic Diversity into Policy and Practice: Case Studies from an English Primary School

    Science.gov (United States)

    Dakin, Justine

    2017-01-01

    Global migration has increased the number of non-English speaking pupils in UK schools, challenging a system which is politically and ideologically monolingual. This article examines how staff at a UK primary school positioned newly arrived pupils and their families, both culturally and linguistically, in terms of Cummins' educator role…

  16. The Role of Visual Culture Studies on Primary School Students' Interpretation of Visual World

    Science.gov (United States)

    Turkcan, Burcin; Yasar, Sefik

    2011-01-01

    Visual culture studies have become the focus of research in recent years mainly due to the frequent use of art applications in Visual Arts courses in primary schools and the inadequate content of discipline based art education for the interpretation of today's multiple stimulants. By interrogating students' semantic stratums related to visuals,…

  17. Meaningful cultural learning by imitative participation: the case of abstract thinking in primary school

    NARCIS (Netherlands)

    van Oers, B.

    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

  18. Aboriginal Community Engagement in Primary Schooling: Promoting Learning through a Cross-Cultural Lens

    Science.gov (United States)

    Turner, Angela; Wilson, Katie; Wilks, Judith L.

    2017-01-01

    This article reports on action research conducted at a primary school in rural New South Wales, Australia. The research responded to an expressed school aspiration to foster greater understanding of local Aboriginal culture, historical perspectives and knowledge systems within the school. An exploratory model was developed using a mixed methods…

  19. Detection of genotoxicity in the marine environment: A preliminary feasibility study using primary mussel tissue culture

    Energy Technology Data Exchange (ETDEWEB)

    Cornet, Michel [UMR 5805 EPOC ' Environnements et Paleoenvironnements Oceaniques' , Universite Bordeaux 1, CNRS, Avenue des Facultes, 33405 Talence Cedex (France)]. E-mail: m.cornet@epoc.u-bordeaux1.fr

    2007-08-15

    The purpose of this study was to evaluate the feasibility and potential usefulness of primary cultures of somatic tissues from adult mussel by means of sister chromatid exchange induction (SCE). This research is an initial pilot study carried out with mussel mantle tissue using seawater artificially contaminated with cadmium and polluted seawater from the port of Arcachon. With regard to cadmium concentration, mean SCE numbers showed a progressive increase from 1.07 {+-} 0.18 per diploid cell in controls (i.e. cultures without contaminant) to 2.91 {+-} 0.42 per diploid cell for the highest concentration, 10{sup -4} M. With regard to the medium prepared with seawater from the port of Arcachon, the mean SCE number reached a value of 5.85 {+-} 0.85 per diploid cell. The analysis of SCEs induced by cadmium showed DNA responses even at the lowest concentration (i.e. 10{sup -7} M). The study demonstrates the feasibility of the sister chromatid exchange (SCE) approach based upon primary mussel tissue culture, for the genotoxicity testing of contaminated seawater. Highlights from this procedure are (1) the presence of an active cell proliferation, (2) the use whole-water samples, (3) the possibility of culturing without serum, (4) the absence of cell dissociation before culturing and (5) a cellular proliferation which can be obtained in cultures carried out in a medium containing seawater whose salinity is comprise between 28 and 35 per mille.

  20. Circadian rhythms of Per2::Luc in individual primary mouse hepatocytes and cultures.

    Directory of Open Access Journals (Sweden)

    Casey J Guenthner

    Full Text Available BACKGROUND: Hepatocytes, the parenchymal cells of the liver, express core clock genes, such as Period2 and Cryptochrome2, which are involved in the transcriptional/translational feedback loop of the circadian clock. Whether or not the liver is capable of sustaining rhythms independent of a central pacemaker is controversial. Whether and how circadian information may be shared among cells in the liver in order to sustain oscillations is currently unknown. RESULTS: In this study we isolated primary hepatocytes from transgenic Per2(Luc mice and used bioluminescence as a read-out of the state of the circadian clock. Hepatocytes cultured in a collagen gel sandwich configuration exhibited persistent circadian rhythms for several weeks. The amplitude of the rhythms damped, but medium changes consistently reset the phase and amplitude of the cultures. Cry2(-/- Per2(Luc cells oscillated robustly and expressed a longer period. Co-culturing with wildtype cells did not significantly shorten the period, indicating that coupling among hepatocytes is insufficient to synchronize cells with significantly differing periods. However, spatial patterns revealed by cellular imaging of wildtype cultures provided evidence of weak local coupling among the hepatocytes. CONCLUSIONS: Our results with primary hepatocyte cultures demonstrate that cultured hepatocytes are weakly coupled. While this coupling is not sufficient to sustain global synchrony, it does increase local synchrony, which may stabilize the circadian rhythms of peripheral oscillators, such as the liver, against noise in the entraining signals.

  1. Attenuation of excitatory amino acid toxicity by metabotropic glutamate receptor agonists and aniracetam in primary cultures of cerebellar granule cells.

    Science.gov (United States)

    Pizzi, M; Fallacara, C; Arrighi, V; Memo, M; Spano, P F

    1993-08-01

    Activation of glutamate ionotropic receptors represents the primary event in the neurotoxicity process triggered by excitatory amino acids. We demonstrate here that the concentration-dependent stimulation of metabotropic glutamate receptor (mGluR) by the selective agonist trans-1-aminocyclopentane-1,3-dicarboxylate or by quisqualate counteracts both glutamate- and kainate-induced neurotoxicity in primary cultures of rat cerebellar granule cells. The mGluR-evoked responses are potentiated by aniracetam, which per se also elicits neuroprotection. Aniracetam concentration-dependently counteracted glutamate-, kainate-, or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-induced cell death and greatly facilitated neuroprotective response achieved by different concentrations of both quisqualate and trans-1-aminocyclopentane-1,3-dicarboxylate. In addition, aniracetam potentiated the mGluR-coupled stimulation of phospholipase C, as revealed by the measurement of 3H-inositol phosphate formation. Thus, mGluRs could be a suitable target for novel pharmacological strategies pointing to the treatment of neurodegenerative diseases.

  2. The metabolism of malate by cultured rat brain astrocytes

    Energy Technology Data Exchange (ETDEWEB)

    McKenna, M.C.; Tildon, J.T.; Couto, R.; Stevenson, J.H.; Caprio, F.J. (Department of Pediatrics, University of Maryland School of Medicine, Baltimore (USA))

    1990-12-01

    Since malate is known to play an important role in a variety of functions in the brain including energy metabolism, the transfer of reducing equivalents and possibly metabolic trafficking between different cell types; a series of biochemical determinations were initiated to evaluate the rate of 14CO2 production from L-(U-14C)malate in rat brain astrocytes. The 14CO2 production from labeled malate was almost totally suppressed by the metabolic inhibitors rotenone and antimycin A suggesting that most of malate metabolism was coupled to the electron transport system. A double reciprocal plot of the 14CO2 production from the metabolism of labeled malate revealed biphasic kinetics with two apparent Km and Vmax values suggesting the presence of more than one mechanism of malate metabolism in these cells. Subsequent experiments were carried out using 0.01 mM and 0.5 mM malate to determine whether the addition of effectors would differentially alter the metabolism of high and low concentrations of malate. Effectors studied included compounds which could be endogenous regulators of malate metabolism and metabolic inhibitors which would provide information regarding the mechanisms regulating malate metabolism. Both lactate and aspartate decreased 14CO2 production from malate equally. However, a number of effectors were identified which selectively altered the metabolism of 0.01 mM malate including aminooxyacetate, furosemide, N-acetylaspartate, oxaloacetate, pyruvate and glucose, but had little or no effect on the metabolism of 0.5 mM malate. In addition, alpha-ketoglutarate and succinate decreased 14CO2 production from 0.01 mM malate much more than from 0.5 mM malate. In contrast, a number of effectors altered the metabolism of 0.5 mM malate more than 0.01 mM. These included methionine sulfoximine, glutamate, malonate, alpha-cyano-4-hydroxycinnamate and ouabain.

  3. Establishment and Characterization of Primary Cultures from Iranian Oral Squamous Cell Carcinoma Patients by Enzymatic Method and Explant Culture

    Directory of Open Access Journals (Sweden)

    Meysam Ganjibakhsh

    2017-10-01

    Full Text Available Objectives: Oral Squamous Cell Carcinoma (OSCC is the most frequent oral cancer worldwide. It is known as the eighth most common cancer in men and as the fifth most common cancer in women. Cytogenetic and biochemical studies in recent decades have emphasized the necessity of providing an appropriate tool for such researches. Cancer cell culture is a useful tool for investigations on biochemical, genetic, molecular and immunological characteristics of different cancers, including oral cancer. Here, we explain the establishment process of five primary oral cancer cells derived from an Iranian population.Materials and Methods: The specimens were obtained from five oral cancer patients. Enzymatic, explant culture and magnetic-activated cell sorting (MACS methods were used for cell isolation. After quality control tests, characterization and authentication of primary oral cancer cells were performed by short tandem repeats (STR profiling, chromosome analysis, species identification, and monitoring the growth, morphology and the expression of CD326 and CD133 markers.Results: Five primary oral cancer cells were established from an Iranian population. The flow cytometry results showed that the isolated cells were positive for CD326 and CD133 markers. Furthermore, the cells were free from mycoplasma, bacterial and fungal contamination. No misidentified or cross-contaminated cells were detected by STR analysis.Conclusions: Human primary oral cancer cells provide an extremely useful platform for studying carcinogenesis pathways of oral cancer in Iranian population. They may be helpful in explaining the ethnic differences in cancer biology and the individuality in anticancer drug response in future studies.

  4. Although it is rapidly metabolized in cultured rat hepatocytes, lauric acid is used for protein acylation

    OpenAIRE

    Rioux, Vincent,; Daval, Stéphanie; Guillou, Hervé; Jan, Sophie; Legrand, Philippe,

    2003-01-01

    International audience; This study was designed to examine the metabolic fate of exogenous lauric acid in cultured rat hepatocytes, in terms of both lipid metabolism and acylation of proteins. Radiolabeled [ 1-$^{14}$C] -lauric acid at 0.1 mM in the culture medium was rapidly taken up by the cells ($94.8 \\pm 2.2\\%$ of the initial radioactivity was cleared from the medium after a 4 h incubation) but its incorporation into cellular lipids was low ($24.6 \\pm 4.2\\%$ of initial radioactivity after...

  5. Comparison of mesencephalic free-floating tissue culture grafts and cell suspension grafts in the 6-hydroxydopamine-lesioned rat

    DEFF Research Database (Denmark)

    Meyer, Morten; Widmer, H R; Wagner, B

    1998-01-01

    days in culture or directly as dissociated cell suspensions, and compared with regard to neuronal survival and ability to normalize rotational behavior in adult rats with unilateral 6-hydroxydopamine (6-OHDA) lesions. Other lesioned rats received injections of cell-free medium and served as controls...... of grafted dopaminergic neurons and to correlate that with the behavioral effects. Additional cultures and acutely prepared explants were also fixed and stored for histological investigation in order to estimate the loss of dopaminergic neurons in culture and after transplantation. Similar behavioral...... improvements in terms of significant reductions in amphetamine-induced rotations were observed in rats grafted with FFRT cultures (127%) and rats grafted with cell suspensions (122%), while control animals showed no normalization of rotational behavior. At 84 days after transplantation, there were similar...

  6. The transfilter transmission of [3H]-proline labelled material in cultured rat tooth germs

    International Nuclear Information System (INIS)

    Woltiers, J.M.L.

    1978-01-01

    Mesenchyme and epithelium from 20-day old embryonic rat tooth germs were cultured for 2 days with Millipore filter separating the two tissues. Either the mesenchyme or the epithelium was labelled with [ 3 H]-proline. Differentiation of the tissues proceeded as in unseparated controls. Collagenase-digestible tritiated material produced by the mesenchyme passed through the filter and accumulated at the interface between filter and epithelium. Findings show that collagen could have a stabilizing influence on the differentiation of the ameloblasts. (author)

  7. Toxicity of Polychlorinated Diphenyl Ethers in Hydra Attenuata and in Rat Whole Embryo Culture

    Science.gov (United States)

    1991-05-01

    teratogenicity using the Hydra attenuata system, a screening test for the rapid detection and prioritization of developmental toxins (Johnson et al...validate other delivery systems for use with the hydra assay. 67 Rat whole embryo culture provides the opportunity to study the effects of a toxin on a...DATE 3. REPORT TYPE AND DATES COVERED May 1991 THESIS/ &I 4. TITLE AND SUBTITLE 5. FUNDING NUMBERS Toxicity of Polychlorinated Diphenyl Ethers in Hydra

  8. Human primary liver cancer-derived organoid cultures for disease modeling and drug screening.

    Science.gov (United States)

    Broutier, Laura; Mastrogiovanni, Gianmarco; Verstegen, Monique Ma; Francies, Hayley E; Gavarró, Lena Morrill; Bradshaw, Charles R; Allen, George E; Arnes-Benito, Robert; Sidorova, Olga; Gaspersz, Marcia P; Georgakopoulos, Nikitas; Koo, Bon-Kyoung; Dietmann, Sabine; Davies, Susan E; Praseedom, Raaj K; Lieshout, Ruby; IJzermans, Jan N M; Wigmore, Stephen J; Saeb-Parsy, Kourosh; Garnett, Mathew J; van der Laan, Luc Jw; Huch, Meritxell

    2017-12-01

    Human liver cancer research currently lacks in vitro models that can faithfully recapitulate the pathophysiology of the original tumor. We recently described a novel, near-physiological organoid culture system, wherein primary human healthy liver cells form long-term expanding organoids that retain liver tissue function and genetic stability. Here we extend this culture system to the propagation of primary liver cancer (PLC) organoids from three of the most common PLC subtypes: hepatocellular carcinoma (HCC), cholangiocarcinoma (CC) and combined HCC/CC (CHC) tumors. PLC-derived organoid cultures preserve the histological architecture, gene expression and genomic landscape of the original tumor, allowing for discrimination between different tumor tissues and subtypes, even after long-term expansion in culture in the same medium conditions. Xenograft studies demonstrate that the tumorogenic potential, histological features and metastatic properties of PLC-derived organoids are preserved in vivo. PLC-derived organoids are amenable for biomarker identification and drug-screening testing and led to the identification of the ERK inhibitor SCH772984 as a potential therapeutic agent for primary liver cancer. We thus demonstrate the wide-ranging biomedical utilities of PLC-derived organoid models in furthering the understanding of liver cancer biology and in developing personalized-medicine approaches for the disease.

  9. Primary cell culture of Echinococcus granulosus developed from the cystic germinal layer: biological and functional characterization.

    Science.gov (United States)

    Albani, Clara M; Elissondo, María Celina; Cumino, Andrea C; Chisari, Andrea; Denegri, Guillermo M

    2010-09-01

    Cell cultures of parasitic helminths are an invaluable tool for investigations of basic biological processes, as well as for development of improved chemotherapeutic agents and molecular interactions between host and parasite. We carried out a simple and efficient methodology to isolate Echinococcus granulosus germinal cells which were maintained for at least 4 months while cultivated in the presence of reducing agents and hormones. Microscopic analysis of the primary cell culture revealed the presence of cells with similar Echinococcus germinal cell morphology and behaviour. Population doubling time was estimated at 48 h, showing a rapid division rate. To discard possible host contamination, the specificity of the primary culture was tested by nested PCR, analyzing mdh gene expression and obtaining only one product with the expected size. We also studied the expression of specific E. granulosus proteins in primary cell culture. The novel and systematized method described here constitutes a powerful tool for investigations in cystic echinococcosis on biochemical and biological aspects related to the life cycle of the parasite and mechanisms of host-parasite interactions. This method also constitutes a powerful tool for the design of more efficient therapeutic alternatives. Copyright (c) 2010 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

  10. Neuron-oligodendrocyte myelination co-culture derived from embryonic rat spinal cord and cerebral cortex.

    Science.gov (United States)

    Pang, Yi; Zheng, Baoying; Kimberly, Simpson L; Cai, Zhengwei; Rhodes, Philip G; Lin, Rick C S

    2012-01-01

    An in vitro myelination model derived from rat central nervous system (CNS) remains to be established. Here, we describe a simple and reproducible myelination culture method using dissociated neuron-oligodendrocyte (OL) co-cultures from either the embryonic day 16 (E16) rat spinal cord or cerebral cortex. The dissociated cells are plated directly on poly-L-lysine-coated cover slips and maintained in a modified myelination medium that supports both OL and neuron differentiation. The spinal cord derived OL progenitor cells develop quickly into myelin basic protein (MBP)+ mature OLs and start to myelinate axons around 17 days in vitro (DIV17). Myelination reaches its peak around six weeks (DIV40) and the typical nodes of Ranvier are revealed by paranodal proteins Caspr and juxaparanodal protein Kv1.2 immunoreactivity. Electron microscopy (EM) shows typical myelination cytoarchitecture and synaptic organization. In contrast, the cortical-derived co-culture requires triiodothyronine (T3) in the culture medium for myelination. Finally, either hypomyelination and/or demyelination can be induced by exposing proinflammatory cytokines or demyelinating agents to the co-culture, suggesting the feasibility of this modified in vitro myelination model for myelin-deficit investigation.

  11. Changes in expression of a functional Gi protein in cultured rat heart cells

    International Nuclear Information System (INIS)

    Allen, I.S.; Gaa, S.T.; Rogers, T.B.

    1988-01-01

    The muscarinic cholinergic agonist, carbachol, and pertussis toxin were used to examine the functional status of the guanine nucleotide-binding protein that inhibits adenylate cyclase (G i ) in cultured neonatal rat heart myocytes. The isoproterenol stimulation of adenylate cyclase activity in myocyte membranes and adenosine 3',5'-cyclic monophosphate (cAMP) accumulation in intact cells (4 days in culture) were insensitive to carbachol. However, in cells cultured for 11 days, carbachol inhibited isoproterenol-stimulated cAMP accumulation by 30%. Angiotensin II (ANG II) was also found to inhibit isoproterenol-stimulated cAMP accumulation in day 11 cells in a dose-dependent manner. Pertussis toxin treatment reversed the inhibitory effects of both ANG II and carbachol, suggesting a role for G i in the process. Carbachol binding to membranes from day 4 cells was relatively insensitive to guanine nucleotides when compared with binding to membranes from day 11 or adult cells. Furthermore, pertussis toxin-mediated 32 P incorporation into a 39- to 41-kDa substrate in day 11 membranes was increased 3.2-fold over that measured in day 4 membranes. These findings support the view that, although G i is expressed, it is nonfunctional in 4-day-old cultured neonatal rat heart myocytes and acquisition of functional G i is dependent on culture conditions. Furthermore, the ANG II receptor can couple to G i in heart

  12. Study of rat hepatocytes in primary culture submitted to hypoxia and reoxygenation: action of the cytoprotectors prostaglandin E1, superoxide dismutase, allopurinol and verapamil Estudo de hepatócitos de rato em cultura primária submetidos a hipóxia e reoxigenação: ação dos citoprotetores prostaglandina E1, superóxido dismutase, alopurinol e verapamil

    Directory of Open Access Journals (Sweden)

    Dahir Ramos de Andrade Jr.

    2009-12-01

    Full Text Available CONTEXT: Exposure of hepatocytes to pathological conditions in a microenvironment of hypoxia and reoxygenation is very frequent in hepatic diseases. Several substances present perspectives for cytoprotective action on hepatocyte submitted to reoxygenation after hypoxia and simple hypoxia. OBJECTIVE: We research therapeutic options for hepatocytes submitted to hypoxia and hypoxia + reoxygenation injury. METHODS: Primary culture of rat hepatocytes was submitted to hypoxia (2 hours plus reoxygenation (2 hours and simple hypoxia (4 hours in the presence or the absence of cytoprotectors. The hepatocyte lesion was evaluated by functional criteria through percentage of lactate dehydrogenase released and cell viability. The effects of the cytoprotectors prostaglandin E1 3 ηg/mL, superoxide dismutase 80 μg/mL, allopurinol 20 μM and verapamil 10-4 M were studied in this model of injury. RESULTS: Reoxygenation after hypoxia induced more significant lesion in cultured hepatocytes compared to simple hypoxia, detected by analysis of functional criteria. There was a significant reduction of percentage of lactate dehydrogenase released and a significant increase of percentage of cell viability in the hypoxia + reoxygenation + cytoprotectors groups compared to hypoxia + reoxygenation groups. Prostaglandin E1, superoxide dismutase and verapamil also protected the group submitted to simple hypoxia, when evaluated by functional criteria. CONCLUSIONS: We conclude that reoxygenation after hypoxia significantly increased the lesion of cultured rat hepatocytes when compared to simple hypoxia. Prostaglandin E1, superoxide dismutase, allopurinol and verapamil acted as cytoprotectors to the rat cultured hepatocytes submitted to hypoxia + reoxygenation in vitro. The substances prostaglandin E1, superoxide dismutase and verapamil protected hepatocytes submitted to simple hypoxia on the basis of all the criteria studied in this experimental model.CONTEXTO: A exposi

  13. Bacterial infection affects protein synthesis in primary lymphoid tissues and circulating lymphocytes of rats.

    Science.gov (United States)

    Papet, Isabelle; Ruot, Benoît; Breuillé, Denis; Walrand, Stéphane; Farges, Marie-Chantal; Vasson, Marie-Paule; Obled, Christiane

    2002-07-01

    Bacterial infection alters whole-body protein homeostasis. Although immune cells are of prime importance for host defense, the effect of sepsis on their protein synthesis rates is poorly documented. We analyzed protein synthesis rates in rat primary lymphoid tissues and circulating lymphocytes after infection. Male Sprague-Dawley rats were studied 1, 2, 6 or 10 d after an intravenous injection of live Escherichia coli. Control healthy rats consumed food ad libitum (d 0) or were pair-fed to infected rats. Protein synthesis was quantified using a flooding dose of L-(4,4,4-(2)H(3))valine. Sepsis induced a delayed increase in total blood leukocytes and a rapid and persistent inversion of the counts. Basal fractional rates of protein synthesis (ks) were 117, 73 and 29%/d in bone marrow, thymus and circulating lymphocytes, respectively. Pair-feeding strongly depressed the absolute protein synthesis rates (ASR) of bone marrow (d 2 and 10) and thymus (d 2-10). The infection per se increased bone marrow, thymus and circulating lymphocyte ks but at various postinfection times. It decreased bone marrow (d 1) and thymus (d 1 and 2) ASR but increased lymphocyte (d 2 and 10) and bone marrow (d 10) ASR. Our results reflect the deleterious effect of anorexia on primary lymphoid tissues. The host defense against bacterial infection exhibited time- and tissue-dependent modifications of protein synthesis, indicating that blood lymphocyte protein data are not representative of the immune system as a whole. Optimization of nutritional supports would be facilitated by including protein synthesis measurements of the immune system.

  14. A cell culture model using rat coronary artery adventitial fibroblasts to measure collagen production

    Directory of Open Access Journals (Sweden)

    Meszaros Gary

    2007-05-01

    Full Text Available Abstract Background We have developed a rat cell model for studying collagen type I production in coronary artery adventitial fibroblasts. Increased deposition of adventitial collagen type I leads to stiffening of the blood vessel, increased blood pressure, arteriosclerosis and coronary heart disease. Although the source and mechanism of collagen deposition is yet unknown, the adventitia appears to play a significant role. To demonstrate the application of our cell model, cultured adventitial fibroblasts were treated with sex hormones and the effect on collagen production measured. Methods Hearts (10–12 weeks were harvested and the left anterior descending coronary artery (LAD was isolated and removed. Tissue explants were cultured and cells (passages 2–4 were confirmed as fibroblasts using immunohistochemistry. Optimal conditions were determined for cell tissue harvest, timing, proliferation and culture conditions. Fibroblasts were exposed to 10-7 M testosterone or 10-7 M estrogen for 24 hours and either immunostained for collagen type I or subjected to ELISA. Results Results showed increased collagen staining in fibroblasts treated with testosterone compared to control and decreased staining with estrogen. ELISA results showed that testosterone increased collagen I by 20% whereas estrogen decreased collagen I by 15%. Conclusion Data demonstrates the usefulness of our cell model in studying the specific role of the adventitia apart from other blood vessel tissue in rat coronary arteries. Results suggest opposite effects of testosterone and estrogen on collagen synthesis in the rat coronary artery adventitial fibroblasts.

  15. Evaluation of castor oil-based polyurethane membranes in rat bone-marrow cell culture.

    Science.gov (United States)

    Cerejo, Sofia de Amorim; Rahal, Sheila Canevese; Lima Neto, João Ferreira de; Voorwald, Fabiana Azevedo; Alvarenga, Fernanda da Cruz Landim e

    2011-10-01

    To evaluate three methods to isolate rats MSCs and to analyze the potential of a castor oil polyurethane base membrane as a scaffold for MSCs. Four male Wistar rats, aged 20-30 days were used. Bone marrow aspirates from femur and tibia were harvested using DMEM high glucose and heparin. The cell culture was performed in three different ways: direct culture and two types of density gradients. After 15 days, was made the 1st passage and analyzed cell viability with markers Hoerscht 33342 and propidium iodide. The MSCs were characterized by surface markers with the aid of flow cytometry. After this, three types of castor oil polyurethane membranes associated with the MSCs were kept on the 6-well plate for 5 days and were analyzed by optical microscopy to confirm cell aggregation and growth. Separation procedures 1 and 2 allowed adequate isolation of MSCs and favored cell growth with the passage being carried out at 70% confluence after 15 days in culture. The cells could not be isolated using procedure 3. When the 3 castor oil polyurethane membrane types were compared it was possible to observe that the growth of MSCs was around 80% in membrane type 3, 20% in type 2, and 10% in type 1. Both Ficoll-Hypaque densities allow isolation of rat MSCs, and especially castor oil-based membrane type 3 may be used as a scaffold for MSCs.

  16. Establishment of primary bovine intestinal epithelial cell culture and clone method.

    Science.gov (United States)

    Zhan, Kang; Lin, Miao; Liu, Ming-Mei; Sui, Yang-Nan; Zhao, Guo-Qi

    2017-01-01

    The aim of this study was to establish bovine intestinal epithelial cell (BIEC) line and provide a novel clone cell method. Although various strategies of bovine cell culture and clone techniques have been reported, these methods remain not established. Here, we culture successfully primary BIECs and establish a novel clone cell method. Our result showed that BIECs could be successfully cultured and passaged about generation 5. These cellular aggregates and clusters were adherent loosely at day 2 of culture. Cell aggregates and clusters start to proliferate after approximately 4 d. The BIECs showed positive reaction against cytokeratin 18, E-cadherin, and characteristics of epithelial-like morphology. In addition, the fatty acid-binding proteins (FABPs), villin, and intestinal peptidase (IP) band were positive in BIECs. Our results suggest that the establishment of culturing and clone BIEC methods will apply to isolate and clone other primary cells. These BIECs could therefore contribute to the study of bovine intestinal nutrient absorption and regulation, immune regulation, and the pathogenesis of the bovine intestinal disease, which will provide intestinal cell model in vitro.

  17. Falcarindiol allosterically modulates GABAergic currents in cultured rat hippocampal neurons.

    Science.gov (United States)

    Wyrembek, Paulina; Negri, Roberto; Kaczor, Przemysław; Czyżewska, Marta; Appendino, Giovanni; Mozrzymas, Jerzy Wladyslaw

    2012-04-27

    Falcarindiol (1), a C-17 polyacetylenic diol, shows a pleiotropic profile of bioactivity, but the mechanism(s) underlying its actions are largely unknown. Large amounts of 1 co-occur in water hemlock (Oenanthe crocata) along with the convulsant polyacetylenic toxin oenanthotoxin (2), a potent GABA(A) receptor (GABA(A)R) inhibitor. Since these compounds are structurally and biogenetically related, it was considered of interest to evaluate whether 1 could affect GABAergic activity, and for this purpose a model of hippocampal cultured neurons was used. Compound 1 significantly increased the amplitude of miniature inhibitory postsynaptic currents, accelerated their onset, and prolonged the decay kinetics. This compound enhanced also the amplitude of currents elicited by 3 μM GABA and accelerated their fading, reducing, however, currents evoked by a saturating (10 mM) GABA concentration. Moreover, kinetic analysis of responses to 10 mM GABA revealed that 1 upregulated the rate and extent of desensitization and slowed the current onset and deactivation. Taken together, these data show that 1 exerts a potent modulatory action on GABA(A)Rs, possibly by modulating agonist binding and desensitization, overall potentially decreasing the toxicity of co-occurring GABA-inhibiting convulsant toxins. © 2012 American Chemical Society and American Society of Pharmacognosy

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

    Energy Technology Data Exchange (ETDEWEB)

    Miyoshi, Ko, E-mail: miyoshi@cc.okayama-u.ac.jp [Department of Brain Science, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 2-5-1 Shikatacho, Okayama 700-8558 (Japan); Kasahara, Kyosuke; Miyazaki, Ikuko; Asanuma, Masato [Department of Brain Science, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 2-5-1 Shikatacho, Okayama 700-8558 (Japan)

    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.

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

    International Nuclear Information System (INIS)

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

    2009-01-01

    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 2 CO 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.

  20. MicroRNA signature characterizes primary tumors that metastasize in an esophageal adenocarcinoma rat model.

    Directory of Open Access Journals (Sweden)

    Ali H Zaidi

    Full Text Available To establish a miRNA signature for metastasis in an animal model of esophageal adenocarcinoma (EAC.The incidence of esophageal adenocarcinoma (EAC has dramatically increased and esophageal cancer is now the sixth leading cause of cancer deaths worldwide. Mortality rates remain high among patients with advanced stage disease and esophagectomy is associated with high complication rates. Hence, early identification of potentially metastatic disease would better guide treatment strategies.The modified Levrat's surgery was performed to induce EAC in Sprague-Dawley rats. Primary EAC and distant metastatic sites were confirmed via histology and immunofluorescence. miRNA profiling was performed on primary tumors with or without metastasis. A unique subset of miRNAs expressed in primary tumors and metastases was identified with Ingenuity Pathway Analysis (IPA along with upstream and downstream targets. miRNA-linked gene expression analysis was performed on a secondary cohort of metastasis positive (n=5 and metastasis negative (n=28 primary tumors.The epithelial origin of distant metastasis was established by IF using villin (VIL1 and mucin 5AC (MUC5AC antibodies. miRNome analysis identified four down-regulated miRNAs in metastasis positive primary tumors compared to metastasis negative tumors: miR-92a-3p (p=0.0001, miR-141-3p (p=0.0022, miR-451-1a (p=0.0181 and miR133a-3p (p=0.0304. Six target genes identified in the top scoring networks by IPA were validated as significantly, differentially expressed in metastasis positive primary tumors: Ago2, Akt1, Kras, Bcl2L11, CDKN1B and Zeb2.In vivo metastasis was confirmed in the modified Levrat's model. Analysis of the primary tumor identified a distinctive miRNA signature for primary tumors that metastasized.

  1. In vitro differentiation of rat spermatogonia into round spermatids in tissue culture.

    Science.gov (United States)

    Reda, A; Hou, M; Winton, T R; Chapin, R E; Söder, O; Stukenborg, J-B

    2016-09-01

    Do the organ culture conditions, previously defined for in vitro murine male germ cell differentiation, also result in differentiation of rat spermatogonia into post-meiotic germ cells exhibiting specific markers for haploid germ cells? We demonstrated the differentiation of rat spermatogonia into post-meiotic cells in vitro, with emphasis on exhibiting, protein markers described for round spermatids. Full spermatogenesis in vitro from immature germ cells using an organ culture technique in mice was first reported 5 years ago. However, no studies reporting the differentiation of rat spermatogonia into post-meiotic germ cells exhibiting the characteristic protein expression profile or into functional sperm have been reported. Organ culture of testicular fragments of 5 days postpartum (dpp) neonatal rats was performed for up to 52 days. Evaluation of microscopic morphology, testosterone levels, mRNA and protein expression as measured by RT-qPCR and immunostaining were conducted to monitor germ cell differentiation in vitro. Potential effects of melatonin, Glutamax® medium, retinoic acid and the presence of epidydimal fat tissue on the spermatogenic process were evaluated. A minimum of three biological replicates were performed for all experiments presented in this study. One-way ANOVA, ANOVA on ranks and student's t-test were applied to perform the statistical analysis. Male germ cells, present in testicular tissue pieces grown from 5 dpp rats, exhibited positive protein expression for Acrosin and Crem (cAMP (cyclic adenosine mono phosphate) response element modulator) after 52 days of culture in vitro. Intra-testicular testosterone production could be observed after 3 days of culture, while when epididymal fat tissue was added, spontaneous contractility of cultured seminiferous tubules could be observed after 21 days. However, no supportive effect of the supplementation with any factor or the co-culturing with epididymal fat tissue on germ cell differentiation in

  2. Decreased proliferative, migrative and neuro-differentiative potential of postnatal rat enteric neural crest-derived cells during culture in vitro

    International Nuclear Information System (INIS)

    Yu, Hui; Pan, Wei-Kang; Zheng, Bai-Jun; Wang, Huai-Jie; Chen, Xin-Lin; Liu, Yong; Gao, Ya

    2016-01-01

    A growing body of evidence supports the potential use of enteric neural crest-derived cells (ENCCs) as a cell replacement therapy for Hirschsprung's disease. Based on previous observations of robust propagation of primary ENCCs, as opposed to their progeny, it is suggested that their therapeutic potential after in vitro expansion may be restricted. We therefore examined the growth and differentiation activities and phenotypic characteristics of continuous ENCC cultures. ENCCs were isolated from the intestines of postnatal rats and were identified using an immunocytochemical approach. During continuous ENCC culture expansion, proliferation, migration, apoptosis, and differentiation potentials were monitored. The Cell Counting Kit-8 was used for assessment of ENCC vitality, Transwell inserts for cell migration, immunocytochemistry for cell counts and identification, and flow cytometry for apoptosis. Over six continuous generations, ENCC proliferation potency was reduced and with prolonged culture, the ratio of migratory ENCCs was decreased. The percentage of apoptosis showed an upward trend with prolonged intragenerational culture, but showed a downward trend with prolonged culture of combined generations. Furthermore, the percentage of peripherin + cells decreased whilst the percentage of GFAP + cells increased with age. The results demonstrated that alterations in ENCC growth characteristics occur with increased culture time, which may partially account for the poor results of proposed cell therapies. - Highlights: • Differences were identified between primary and daughter ENCCs. • Daughter ENCCs had reduced proliferation, migration and differentiation. • Daughter ENCCs also had increased apoptosis. • These altered characteristics warrant further investigation.

  3. Decreased proliferative, migrative and neuro-differentiative potential of postnatal rat enteric neural crest-derived cells during culture in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Hui [Department of Pediatric Surgery, the Second Affiliated Hospital, Xi’an Jiaotong University, No 157, Xi Wu Road, Xi’an 710004, Shaanxi (China); Institute of Neurobiology, Environment and Genes Related to Diseases Key Laboratory of Chinese Ministry of Education, Xi’an Jiaotong University, No 96, Yan Ta Xi Road, Xi’an 710061, Shaanxi (China); Pan, Wei-Kang; Zheng, Bai-Jun; Wang, Huai-Jie [Department of Pediatric Surgery, the Second Affiliated Hospital, Xi’an Jiaotong University, No 157, Xi Wu Road, Xi’an 710004, Shaanxi (China); Chen, Xin-Lin; Liu, Yong [Institute of Neurobiology, Environment and Genes Related to Diseases Key Laboratory of Chinese Ministry of Education, Xi’an Jiaotong University, No 96, Yan Ta Xi Road, Xi’an 710061, Shaanxi (China); Gao, Ya, E-mail: ygao@mail.xjtu.edu.cn [Department of Pediatric Surgery, the Second Affiliated Hospital, Xi’an Jiaotong University, No 157, Xi Wu Road, Xi’an 710004, Shaanxi (China)

    2016-05-01

    A growing body of evidence supports the potential use of enteric neural crest-derived cells (ENCCs) as a cell replacement therapy for Hirschsprung's disease. Based on previous observations of robust propagation of primary ENCCs, as opposed to their progeny, it is suggested that their therapeutic potential after in vitro expansion may be restricted. We therefore examined the growth and differentiation activities and phenotypic characteristics of continuous ENCC cultures. ENCCs were isolated from the intestines of postnatal rats and were identified using an immunocytochemical approach. During continuous ENCC culture expansion, proliferation, migration, apoptosis, and differentiation potentials were monitored. The Cell Counting Kit-8 was used for assessment of ENCC vitality, Transwell inserts for cell migration, immunocytochemistry for cell counts and identification, and flow cytometry for apoptosis. Over six continuous generations, ENCC proliferation potency was reduced and with prolonged culture, the ratio of migratory ENCCs was decreased. The percentage of apoptosis showed an upward trend with prolonged intragenerational culture, but showed a downward trend with prolonged culture of combined generations. Furthermore, the percentage of peripherin{sup +} cells decreased whilst the percentage of GFAP{sup +} cells increased with age. The results demonstrated that alterations in ENCC growth characteristics occur with increased culture time, which may partially account for the poor results of proposed cell therapies. - Highlights: • Differences were identified between primary and daughter ENCCs. • Daughter ENCCs had reduced proliferation, migration and differentiation. • Daughter ENCCs also had increased apoptosis. • These altered characteristics warrant further investigation.

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

    Science.gov (United States)

    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 to…

  5. Differential heat shock response of primary human cell cultures and established cell lines

    DEFF Research Database (Denmark)

    Richter, W W; Issinger, O G

    1986-01-01

    degrees C treatment, whereas in immortalized cell lines usually 90% of the cells were found in suspension. Enhanced expression of the major heat shock protein (hsp 70) was found in all heat-treated cells. In contrast to the primary cell cultures, established and transformed cell lines synthesized...... a protein with an apparent molecular mass of 70 kDa and an isoelectric pH of 7.0 as early as 3 h after the initial hyperthermal treatment....

  6. Students' perceptions of primary teachers' interpersonal behaviour and of cultural dimensions in the classroom environment.

    OpenAIRE

    Fisher, D.; Waldrip, B.; den Brok, P.

    2006-01-01

    Within the domain of learning environments research many studies have investigated students’ perceptions of their teachers’ interpersonal behavior. The present study adds to this line of research by (a) focusing on primary education, rather than secondary education, (b) establishing associations between perceptions of teacher interpersonal behavior and perceptions of cultural elements of the learning environment rather than uniquely focusing on interpersonal behavior, (c) linking perceptions ...

  7. Comparison of radiometric and conventional culture systems in detecting Haemophilus influenzae type b bacteremia in rats

    International Nuclear Information System (INIS)

    Mitchell, M.J.; Zwahlen, A.; Elliott, H.L.; Ford, N.K.; Charache, F.P.; Moxon, E.R.

    1985-01-01

    To compare the efficiency of detecting Haemophilus influenzae type b bacteremia by the BACTEC radiometric system and a conventional Trypticase soy broth blood culture system, the authors developed an in vivo model of bacteremia in rats. After intravenous injection of 50 to 200 CFU into adult rats, there was a linear logarithmic increase in CFU per milliliter of rat blood during the first 10 h (r = 0.98), allowing accurate prediction of the level of bacteremia with time. Culture bottles were inoculated with 0.5 ml of blood obtained by cardiac puncture and processed as clinical samples in the microbiology laboratory with RS and conventional protocols. They found the following. (i) The first detection of bacteremia by RS was similar to that by TSB if a Gram stain of the TSB was done on day 1 and was superior if that smear was omitted (P less than 0.01). (ii) The detection times in both systems were comparable at different magnitudes of bacteremia (10(1) to 10(4) CFU/ml). (iii) Supplementation of inoculated bottles with 2 ml of sterile rat blood interfered with Gram stain detection in TSB but resulted in increased 14 CO 2 production in RS. (iv) No difference in detection time was found between RS and TSB for four different clinical isolates. These studies show that, in a biologically relevant model, the detection of positive blood cultures for H. influenzae type b by RS was comparable to or better than detection by TSB when blood was processed analogously to clinical specimens

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

    Directory of Open Access Journals (Sweden)

    Worthington Roger P

    2011-06-01

    Full Text Available Abstract 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

  9. Cannabidiol inhibits synaptic transmission in rat hippocampal cultures and slices via multiple receptor pathways

    Science.gov (United States)

    Ledgerwood, CJ; Greenwood, SM; Brett, RR; Pratt, JA; Bushell, TJ

    2011-01-01

    BACKGROUND AND PURPOSE Cannabidiol (CBD) has emerged as an interesting compound with therapeutic potential in several CNS disorders. However, whether it can modulate synaptic activity in the CNS remains unclear. Here, we have investigated whether CBD modulates synaptic transmission in rat hippocampal cultures and acute slices. EXPERIMENTAL APPROACH The effect of CBD on synaptic transmission was examined in rat hippocampal cultures and acute slices using whole cell patch clamp and standard extracellular recordings respectively. KEY RESULTS Cannabidiol decreased synaptic activity in hippocampal cultures in a concentration-dependent and Pertussis toxin-sensitive manner. The effects of CBD in culture were significantly reduced in the presence of the cannabinoid receptor (CB1) inverse agonist, LY320135 but were unaffected by the 5-HT1A receptor antagonist, WAY100135. In hippocampal slices, CBD inhibited basal synaptic transmission, an effect that was abolished by the proposed CB1 receptor antagonist, AM251, in addition to LY320135 and WAY100135. CONCLUSIONS AND IMPLICATIONS Cannabidiol reduces synaptic transmission in hippocampal in vitro preparations and we propose a role for both 5-HT1A and CB1 receptors in these CBD-mediated effects. These data offer some mechanistic insights into the effects of CBD and emphasize that further investigations into the actions of CBD in the CNS are required in order to elucidate the full therapeutic potential of CBD. PMID:20825410

  10. Effect of galactose on binding and endocytosis of asialoglycoprotein in cultured rat hepatocytes

    International Nuclear Information System (INIS)

    Hata, Soichiro; Ishii, Koji

    1998-01-01

    99m Tc-diethylenetriaminepentaacetic acid-galactosyl-human serum albumin ( 99m Tc-GSA) has been applied clinically in scintigraphy to estimate functioning liver mass, but it is not so sensitive in differentiating mild liver injury from normal liver. 99m Tc-GSA is thought to bind to the asialoglycoprotein receptor (ASGP-R) and is then internalized and degraded in the hepatocytes. The aim of this study is to know whether D-galactose inhibits GSA binding or internalization to hepatocytes because ASGP-R recognizes galactose residues of ligands. Isolated rat hepatocytes were obtained by collagenase perfusion, pre-cultured for 2 h after plating, and then cultured for 16 to 18 h until use. The effect of galactose on GSA binding and internalization into cells was investigated by using cultured hepatocytes. Galactose non-competitively inhibited GSA binding to cultured hepatocytes, but its Ki value was quite high (505±38 mM). Galactose significantly inhibited GSA internalization into hepatocytes at 27 mM. It was clarified that D-galactose inhibited GSA internalization rather than binding at a low concentration. Further in vivo studies in rats are needed to know whether an administration of galactose prior to performing 99m Tc-GSA scintigraphy can brake it possible to estimate the functioning mass in mild liver injury. (author)

  11. Long-term culture of rat hippocampal neurons at low density in serum-free medium: combination of the sandwich culture technique with the three-dimensional nanofibrous hydrogel PuraMatrix.

    Science.gov (United States)

    Kaneko, Ai; Sankai, Yoshiyuki

    2014-01-01

    The primary culture of neuronal cells plays an important role in neuroscience. There has long been a need for methods enabling the long-term culture of primary neurons at low density, in defined serum-free medium. However, the lower the cell density, the more difficult it is to maintain the cells in culture. Therefore, we aimed to develop a method for long-term culture of neurons at low density, in serum-free medium, without the need for a glial feeder layer. Here, we describe the work leading to our determination of a protocol for long-term (>2 months) primary culture of rat hippocampal neurons in serum-free medium at the low density of 3×10(4) cells/mL (8.9×10(3) cells/cm2) without a glial feeder layer. Neurons were cultured on a three-dimensional nanofibrous hydrogel, PuraMatrix, and sandwiched under a coverslip to reproduce the in vivo environment, including the three-dimensional extracellular matrix, low-oxygen conditions, and exposure to concentrated paracrine factors. We examined the effects of varying PuraMatrix concentrations, the timing and presence or absence of a coverslip, the timing of neuronal isolation from embryos, cell density at plating, medium components, and changing the medium or not on parameters such as developmental pattern, cell viability, neuronal ratio, and neurite length. Using our method of combining the sandwich culture technique with PuraMatrix in Neurobasal medium/B27/L-glutamine for primary neuron culture, we achieved longer neurites (≥3,000 µm), greater cell viability (≥30%) for 2 months, and uniform culture across the wells. We also achieved an average neuronal ratio of 97%, showing a nearly pure culture of neurons without astrocytes. Our method is considerably better than techniques for the primary culture of neurons, and eliminates the need for a glial feeder layer. It also exhibits continued support for axonal elongation and synaptic activity for long periods (>6 weeks).

  12. Extra collagen overlay prolongs the differentiated phenotype in sandwich-cultured rat hepatocytes.

    Science.gov (United States)

    Oorts, Marlies; Keemink, Janneke; Deferm, Neel; Adriaensen, Robin; Richert, Lysiane; Augustijns, Patrick; Annaert, Pieter

    2017-10-25

    Sandwich-cultured rat hepatocytes (SCRH) have become an invaluable in vitro model to study hepatic drug disposition. SCRH are maintained between two layers of extracellular matrix. In this configuration, culture periods of 4days are typically applicable. The aim of the present study was to modify conventional SCRH by applying an additional collagen overlay to prolong the hepatic phenotype in SCRH and thus to extend the applicability of the model. The cultures receiving an extra top layer ('SCRH-plus' cultures) were compared with the conventional SCRH by testing the morphology, cell functionality, metabolic capacity and Mrp2-activity. In the SCRH-plus cultures, cell functionality, evaluated by measuring urea production, was increased from day 5 onwards, compared to conventional cultures. Furthermore, these cells retained the appearance of typical hepatocytes, in contrast with conventional sandwich cultures which showed rapid dedifferentiation. SCRH-plus exhibited significantly improved metabolic clearance mediated by cytochrome P450 3A compared to conventional SCRH whereas UDP-glucuronosyltransferase-mediated metabolism was unaffected. Both conventional SCRH and SCRH-plus showed extensive biliary networks at day 4 of culture. However, from day 4 onwards, a decline in biliary excretion index (BEI) was observed in the conventional SCRH, while BEI values in SCRH-plus cultures did not decrease until day 7. The application of an extra top layer of collagen on the SCRH prolongs their useful life-span to 7days. Therefore, SCRH-plus cultures will broaden the applications of SCRH in terms of long-term toxicity evaluation and when determining metabolism of low turnover compounds. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. Comparison of gene expression profile in embryonic mesencephalon and neuronal primary cultures.

    Directory of Open Access Journals (Sweden)

    Dario Greco

    Full Text Available In the mammalian central nervous system (CNS an important contingent of dopaminergic neurons are localized in the substantia nigra and in the ventral tegmental area of the ventral midbrain. They constitute an anatomically and functionally heterogeneous group of cells involved in a variety of regulatory mechanisms, from locomotion to emotional/motivational behavior. Midbrain dopaminergic neuron (mDA primary cultures represent a useful tool to study molecular mechanisms involved in their development and maintenance. Considerable information has been gathered on the mDA neurons development and maturation in vivo, as well as on the molecular features of mDA primary cultures. Here we investigated in detail the gene expression differences between the tissue of origin and ventral midbrain primary cultures enriched in mDA neurons, using microarray technique. We integrated the results based on different re-annotations of the microarray probes. By using knowledge-based gene network techniques and promoter sequence analysis, we also uncovered mechanisms that might regulate the expression of CNS genes involved in the definition of the identity of specific cell types in the ventral midbrain. We integrate bioinformatics and functional genomics, together with developmental neurobiology. Moreover, we propose guidelines for the computational analysis of microarray gene expression data. Our findings help to clarify some molecular aspects of the development and differentiation of DA neurons within the midbrain.

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

    Science.gov (United States)

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

    2016-01-01

    This study examined how aspects of quality improvement (QI) culture changed during the introduction of the Veterans Health Administration (VHA) patient-centered medical home initiative and how they were influenced by existing organizational factors, including VHA facility complexity and practice location. A voluntary survey, measuring primary care providers' (PCPs') perspectives on QI culture at their primary care clinics, was administered in 2010 and 2012. Participants were 320 PCPs from hospital- and community-based primary care practices in Pennsylvania, West Virginia, Delaware, New Jersey, New York, and Ohio. PCPs in community-based outpatient clinics reported an improvement in established processes for QI, and communication and cooperation from 2010 to 2012. However, their peers in hospital-based clinics did not report any significant improvements in QI culture. In both years, compared with high-complexity facilities, medium- and low-complexity facilities had better scores on the scales assessing established processes for QI, and communication and cooperation. © The Author(s) 2014.

  15. Determination of radiosensitivity in established and primary squamous cell carcinoma cultures using the micronucleus assay

    Energy Technology Data Exchange (ETDEWEB)

    Champion, A.R.; Hanson, J.A.; Venables, S.E.; Gaffney, C.C. [Velindre Hospital NHS Trust, Whitchurch, Cardiff (United Kingdom). Cellular and Molecular Radiation Research Unit; McGregor, A.D. [Morriston Hospital NHS Trust, Swansea (United Kingdom). Welsh Regional Burns and Plastic Surgery Unit

    1997-03-01

    In this study, the cytokinesis-block micronucleus assay (CBMN) was used to measure radiosensitivity in three established cell lines (SCC-61, V175 and V134) and 10 primary cell cultures of squamous cell carcinoma (SCC) of the head and neck. Assessment involved optimisation of the assay to determine cytochalasin-B (CB) concentration and sampling time postirradiation. A much closer correlation between dose-response data measured in the clonogenic and micronucleus assays was found when the micronucleus assay was performed under standardised conditions for each cell line (2 {mu}g/ml CB: 48 h postirradiation) instead of predetermined optimised assay conditions. This indicates that, for these SCC cell lines, the CBMN assay may be able to predict in vitro radiosensitivity. To be of clinical use in predicting radiosensitivity, the CBMN assay also needs to be evaluated with primary cell cultures. In this study, no relationship between micronucleus frequency at 2 or 6 Gy and patient clinical outcome 12 months following surgery and radiotherapy was seen. Similarly, no association between patient outcome and tumour stage, nodal stage and histology was observed. These CBMN assay data from the primary cell cultures are presently inconclusive as a measure of patient tumour radiosensitivity. (Author).

  16. Chemical Profiling of Primary Mesothelioma Cultures Defines Subtypes with Different Expression Profiles and Clinical Responses.

    Science.gov (United States)

    Schunselaar, Laurel M; Quispel-Janssen, Josine M M F; Kim, Yongsoo; Alifrangis, Constantine; Zwart, Wilbert; Baas, Paul; Neefjes, Jacques

    2018-04-01

    Purpose: Finding new treatment options for patients with malignant pleural mesothelioma is challenging due to the rarity and heterogeneity of this cancer type. The absence of druggable targets further complicates the development of new therapies. Current treatment options are therefore limited, and prognosis remains poor. Experimental Design: We performed drug screening on primary mesothelioma cultures to guide treatment decisions of corresponding patients that were progressive after first- or second-line treatment. Results: We observed a high concordance between in vitro results and clinical outcomes. We defined three subgroups responding differently to the anticancer drugs tested. In addition, gene expression profiling yielded distinct signatures that segregated the differently responding subgroups. These genes signatures involved various pathways, most prominently the fibroblast growth factor pathway. Conclusions: Our primary mesothelioma culture system has proved to be suitable to test novel drugs. Chemical profiling of primary mesothelioma cultures allows personalizing treatment for a group of patients with a rare tumor type where clinical trials are notoriously difficult. This personalized treatment strategy is expected to improve the poor prospects of patients with mesothelioma. Clin Cancer Res; 24(7); 1761-70. ©2017 AACR See related commentary by John and Chia, p. 1513 . ©2017 American Association for Cancer Research.

  17. Real-time monitoring of circadian clock oscillations in primary cultures of mammalian cells using Tol2 transposon-mediated gene transfer strategy

    Directory of Open Access Journals (Sweden)

    Yamanaka Iori

    2010-01-01

    Full Text Available Abstract Background The circadian rhythm in mammals is orchestrated by a central pacemaker in the brain, but most peripheral tissues contain their own intrinsic circadian oscillators. The circadian rhythm is a fundamental biological system in mammals involved in the regulation of various physiological functions such as behavior, cardiovascular functions and energy metabolism. Thus, it is important to understand the correlation between circadian oscillator and physiological functions in peripheral tissues. However, it is still difficult to investigate the molecular oscillator in primary culture cells. Results In this study, we used a novel Tol2 transposon based Dbp promoter or Bmal1 promoter driven luciferase reporter vector system to detect and analyze the intrinsic molecular oscillator in primary culture cells (mouse embryonic fibroblasts, fetal bovine heart endothelial cells and rat astrocytes. The results showed circadian molecular oscillations in all examined primary culture cells. Moreover, the phase relationship between Dbp promoter driven and Bmal1 promoter driven molecular rhythms were almost anti-phase, which suggested that these reporters appropriately read-out the intrinsic cellular circadian clock. Conclusions Our results indicate that gene transfer strategy using the Tol2 transposon system of a useful and safe non-viral vector is a powerful tool for investigating circadian rhythms in peripheral tissues.

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

    NARCIS (Netherlands)

    Parker, D.; Wensing, M.; Esmail, A.; Valderas, J.M.

    2015-01-01

    BACKGROUND: There is little guidance available to healthcare practitioners about what tools they might use to assess the patient safety culture. OBJECTIVE: To identify useful tools for assessing patient safety culture in primary care organizations in Europe; to identify those aspects of performance

  19. Individualized medicine for renal cell carcinoma: establishment of primary cell line culture from surgical specimens.

    Science.gov (United States)

    Kim, Fernando J; Campagna, Adriano; Khandrika, Lakshmipathi; Koul, Sweaty; Byun, Seok-Soo; vanBokhoven, Adrie; Moore, Ernest E; Koul, Hari

    2008-10-01

    The lack of effective "in vivo" and "in vitro" models to predict success of pharmacological therapy for patients with renal cell carcinoma, as well as, the variety of cancer cell types demands the development of better experimental models to understand the pathophysiology of the disease and evaluate drug sensitivity in vitro. To develop primary renal cancer cell culture irrespective of tumor grade and tumor type, harvested from the patient's pathological specimen immediately after the laparoscopic radical nephrectomy to study potential "in vivo" pharmacological sensitivity. A total of 24 patients (17 males and 7 females). Mean age of 63.1+/-3.1 y.o. The mean size of the renal masses was 7.56+/-3.1 cm. Normal and pathological renal tissue was collected immediately after the specimen was extracted and submitted to enzymatic digestion for 16-24 hours. Clear cell carcinoma cells were selected through multiple passages in DMEM medium supplemented with glucose and antibiotics. Establishment of cell line culture from all the patients' specimens irrespective of tumor grade and tumor type was achieved successfully. In addition to the tumor cell line culture, normal parenchyma tissue yielded primary cell lines to allow testing the response of tumor types to various pharmacological therapeutic agents and toxicity of such treatments to healthy tissue. From the initial collection of the specimens obtained after the removal of the kidney to the development of cell lines took occurred in average 32+6 hrs. The cells in culture showed characteristics of epithelial cells; like expression on cytokeratin and were maintained in culture for more than 20 passages. The development of renal cancer cell cultures in vitro is labor intense but may yield a more realistic model to tailor pharmacological therapies and predict therapeutic success prior to "in vivo" application-a step in the direction of individualized medicine for RCC.

  20. Primary culture of secretagogue-responsive parietal cells from rabbit gastric mucosa

    International Nuclear Information System (INIS)

    Chew, C.S.; Ljungstroem, M.S.; Smolka, A.; Brown, M.R.

    1989-01-01

    A new procedure for isolation and primary culture of gastric parietal cells is described. Parietal cells from rabbit gastric mucosa are enriched to greater than 95% purity by combining a Nycodenz gradient separation with centrifugal elutriation. Cells are plated on the basement membrane matrix, Matrigel, and maintained in culture for at least 1 wk. Parietal cells cultured in this manner remain differentiated, cross-react with monoclonal H+-K+-ATPase antibodies, and respond to histamine, gastrin, and cholinergic stimulation with increased acid production as measured by accumulation of the weak base, [ 14 C]aminopyrine. When stimulated, cultured cells undergo ultrastructural changes in which intracellular canaliculi expand and numerous microvilli are observed. These ultrastructural changes are similar to those previously found to occur in vivo and in acutely isolated parietal cells. Morphological transformations in living cells can also be observed with differential interference contrast optics in the light microscope. After histamine stimulation, intracellular canaliculi gradually expand to form large vacuolar spaces. When the H2 receptor antagonist, cimetidine, is added to histamine-stimulated cells, these vacuoles gradually disappear. The ability to maintain hormonally responsive parietal cells in primary culture should make it possible to study direct, long-term effects of a variety of agonists and antagonists on parietal cell secretory-related activity. These cultured cells should also prove to be useful for the study of calcium transients, ion fluxes, and intracellular pH as related to acid secretion in single cells, particularly since morphological transformations can be used to monitor physiological responses at the same time within the same cell

  1. Enhanced expression of contractile endothelin ET(B) receptors in rat coronary artery after organ culture

    DEFF Research Database (Denmark)

    Johnsson, E.; Maddahi, A.; Wackenfors, A.

    2008-01-01

    Endothelin-1 is a potent vasoconstrictor mediating its effects via two receptor subtypes, the endothelin type A (ET(A)) preferentially situated on smooth muscle cells, mediating vasoconstriction and endothelin type B (ET(B)) mainly located on endothelial cells, mediating vasodilatation....... In cardiovascular disease and in organ culture in vitro, endothelin ET(B) receptors are up-regulated on smooth muscle cells. The objectives of the present study were to characterise the endothelin receptor-induced vasoconstriction and quantify the endothelin receptor mRNA levels and immunoreactivity in fresh...... and cultured rat coronary arteries. We demonstrate that endothelin-1 induces strong and equal concentration-dependent contractions in fresh and cultured segments from the left anterior descending coronary artery. Sarafotoxin 6c, an endothelin ET(B) receptor agonist, had negligible effect in fresh arteries...

  2. Functional analysis of ultra high information rates conveyed by rat vibrissal primary afferents

    Directory of Open Access Journals (Sweden)

    André eMaia Chagas

    2013-12-01

    Full Text Available Sensory receptors determine the type and the quantity of information available for perception. Here, we quantified and characterized the information transferred by primary afferents in the rat whisker system using neural system identification. Quantification of ‘how much’ information is conveyed by primary afferents, using the direct method, a classical information theoretic tool, revealed that primary afferents transfer huge amounts of information (up to 529 bits/s. Information theoretic analysis of instantaneous spike-triggered kinematic stimulus features was used to gain functional insight on ‘what’ is coded by primary afferents. Amongst the kinematic variables tested - position, velocity, and acceleration - primary afferent spikes encoded velocity best. The other two variables contribute to information transfer, but only if combined with velocity. We further revealed three additional characteristics that play a role in information transfer by primary afferents. Firstly, primary afferent spikes show preference for well separated multiple stimuli (i.e. well separated sets of combinations of the three instantaneous kinematic variables. Secondly, spikes are sensitive to short strips of the stimulus trajectory (up to 10 ms pre-spike time, and thirdly, they show spike patterns (precise doublet and triplet spiking. In order to deal with these complexities, we used a flexible probabilistic neuron model fitting mixtures of Gaussians to the spike triggered stimulus distributions, which quantitatively captured the contribution of the mentioned features and allowed us to achieve a full functional analysis of the total information rate indicated by the direct method. We found that instantaneous position, velocity, and acceleration explained about 50% of the total information rate. Adding a 10 ms pre-spike interval of stimulus trajectory achieved 80-90%. The final 10-20% were found to be due to non-linear coding by spike bursts.

  3. Regulation of pro-adrenocorticotropin-endorphin synthesis and secretion in cultured neonatal rat anterior pituitary

    Energy Technology Data Exchange (ETDEWEB)

    Sato, S.M.; Mains, R.E. (Johns Hopkins Univ. School of Medicine, Baltimore, MD (USA))

    1987-08-01

    Previous work demonstrated that newborn rat anterior pituitary corticotropes display processing patterns for pro-ACTH/endorphin that are different from the adult. The synthesis and release of beta-endorphin-related peptides was examined in dispersed cell and explant cultures of newborn anterior pituitary to investigate corticotrope development further. The temporal pattern of pro-ACTH/endorphin processing differed significantly from adult rat melanotropes and AtT-20 cells. While pro-ACTH/endorphin processing begins within 30 min of synthesis in adult melanotropes and AtT-20 cells, pulse-labeling of newborn corticotropes in culture indicated that pro-ACTH/endorphin remained uncleaved for at least 90 min after synthesis. With further incubation, there was a decrease in radioactivity associated with the precursor and an equivalent rise in the radioactivity associated with beta-endorphin and beta-lipotropin. However, unprocessed precursor still remained in the cultured newborn anterior pituitary cells after a 25-h chase. Although intact pro-ACTH/endorphin from newborn corticotropes was very long-lived, the precursor did undergo oligosaccharide maturation and became endoglycosidase H resistant within 1 h after synthesis. Similar to the adult, pro-ACTH/endorphin synthesis was doubled in cultures of newborn anterior pituitary chronically treated with 10 nM CRF resulting in a 3- to 4-fold stimulation of secretion over the basal rate. However, unlike the AtT-20 cell or adult rat corticotrope, the proteolytic processing of pro-ACTH/endorphin in the newborn corticotrope was altered by chronic secretagogue treatment; less pro-ACTH/endorphin was converted to beta-endorphin in secretagogue-treated corticotropes than in controls. Thus processing of pro-ACTH/endorphin in the corticotrope is not mature by birth and can be regulated by chronic CRF treatment.

  4. Manganese influences the expression of fatty acid synthase and malic enzyme in cultured primary chicken hepatocytes.

    Science.gov (United States)

    Lu, Lin; Wang, Meiling; Liao, Xiudong; Zhang, Liyang; Luo, Xugang

    2017-12-01

    Two experiments were designed to investigate the effects of Mn source and concentration on the mRNA expression and enzymatic activities of fatty acid synthase (FAS) and malic enzyme (ME) in cultured primary broiler hepatocytes. In Expt 1, primary broiler hepatocytes were treated with 0 (control), 0·25, 0·50 or 0·75 mmol/l of Mn as inorganic manganese chloride (MnCl2.4H2O) for 24 and 48 h. In Expt 2, primary broiler hepatocytes were incubated with 0 (control), 0·25 or 0·50 mmol/l of Mn as either manganese chloride or Mn-amino acid chelate for 48 h. The mRNA levels and activities of FAS and ME in the hepatocytes were measured in Expts 1 and 2. The results in Expt 1 showed that only at 48 h mRNA expression levels of FAS and ME in the hepatocytes decreased linearly (P0·33) on any of the measured cellular parameters. The results suggested that Mn might reduce cell damage and regulate FAS and ME expression at a transcriptional level in primary cultured broiler hepatocytes.

  5. Hepatoprotective Flavonoids in Opuntia ficus-indica Fruits by Reducing Oxidative Stress in Primary Rat Hepatocytes

    Science.gov (United States)

    Kim, Jung Wha; Kim, Tae Bum; Kim, Hyun Woo; Park, Sang Wook; Kim, Hong Pyo; Sung, Sang Hyun

    2017-01-01

    Background: Liver disorder was associated with alcohol consumption caused by hepatic cellular damages. Opuntia ficus-indica fruit extracts (OFIEs), which contain betalain pigments and polyphenols including flavonoids, have been introduced as reducing hangover symptoms and liver protective activity. Objective: To evaluate hepatoprotective activity of OFIEs and isolated compounds by high-speed countercurrent chromatography (HSCCC). Materials and Methods: The extract of O. ficus-indica fruits was fractionated into methylene chloride and n-butanol. The n-butanol fraction was isolated by HSCCC separation (methylene chloride-methanol-n-butanol-water, 5:4:3:5, v/v/v/v). The hepatoprotective activity of OFIEs and isolated compounds was evaluated on rat primary hepatocytes against ethanol-induced toxicity. Antioxidative parameters such as glutathione reductase and glutathione peroxidase (GSH-Px) enzymes and the GSH content were measured. Results: Two flavonoids, quercetin 3-O-methyl ester (1) and (+)-taxifolin, and two flavonoid glycosides, isorhamnetin 3-O-β-d-glucoside (3) and narcissin (4), were isolated from the n-butanol fraction by HSCCC separation. Among them, compound 2 significantly protected rat primary hepatocytes against ethanol exposure by preserving antioxidative properties of GR and GSH-Px. Conclusions: OFIEs and (+)-taxifolin were suggested to reduce hepatic damage by alcoholic oxidative stress. SUMMARY Hepatoprotective Flavonoids were isolated from Opuntia ficus-indica by high -speed countercurrent chromatography (HSCCC). PMID:28839374

  6. Effects of aluminum on immune functions of cultured splenic T and B lymphocytes in rats.

    Science.gov (United States)

    She, Yue; Wang, Nan; Chen, Chongxiao; Zhu, Yanzhu; Xia, Shiliang; Hu, Chongwei; Li, Yanfei

    2012-06-01

    The effects of Aluminum (Al) exposure on immune functions of cultured splenic T and B lymphocytes of rats were studied. The lymphocytes were isolated from spleen of healthy male Wistar rats weighing 110-120 g. The cultured cells in RPMI-1640 medium were exposed to 0 (control group), 0.035 (low-dose group), 0.07 (medial-dose group), and 0.14 (high-dose group) mg/mL Al(3+) as aluminum trichloride (AlCl(3)) in an incubator under 5% CO(2) at 37°C for 24 h. The T and B lymphocyte proliferation was measured with a tetrazolium dye colorimetric assay. The levels of interleukin (IL)-2, IL-6, and tumor necrosis factor (TNF)-α were determined by iodine [(125)I] IL-2, IL-6, and TNF-α radioimmunoassay kits, respectively. The proportions of CD3(+), CD4(+), and CD8(+) T lymphocytes were measured with a flow cytometer. The results showed that the T and B lymphocyte proliferation, the levels of IL-2, IL-6, TNF-α, the proportions of CD3(+) and CD4(+) T lymphocytes, and the ratio of CD4(+)/CD8(+) T lymphocytes were lowered by Al treatments, while the proportion of CD8(+) T lymphocytes was increased. These findings indicate that Al exposure can inhibit the immune functions of splenic T and B lymphocytes of rats in vitro.

  7. Characterization of the Relationship of CDKL5 with MeCP2 and Dnmt1 in PrimaryRat Cortical Neurons

    Directory of Open Access Journals (Sweden)

    Zhi Yi

    Full Text Available ABSTRACT Cyclin-dependent kinase-like 5 (CDKL5 is a protein kinase that is homologous to mitogen-activated protein kinases (MAPKs and cyclin-dependent kinases (CDKs. Mutations in the CDKL5 gene cause X-linked infantile spasms and phenotypes that overlap with that of Rett syndrome, which is a neurodevelopmental disorder caused primarily by mutations in the methyl CpG binding protein 2 gene (MECP2. Previous studies in transfected cell lines showed that CDKL5 interacts with MeCP2 and DNA (cytosine-5-methyltransferase 1 (Dnmt1. However, little is known about the relationships of CDKL5 with interacting proteins in primary neuronal cultures. In this study, we investigated the expression patterns of CDKL5, MeCP2 and Dnmt1, and their interaction in cultured rat cortical neurons. Using real-time PCR analysis, we found that CDKL5, MeCP2 and Dnmt1 have similar expression patterns at the mRNA level. In contrast, the expression patterns of those proteins at the protein level are different and could be inversely correlated, as shown by western blotting. Using co-immunoprecipitation, we further demonstrated that CDKL5 interacts with MeCP2 and Dnmt1 in primary rat cortical neurons. These data suggest that a functional link exists among CDKL5, MeCP2 and Dnmt1 during neuronal development and may provide further insight into the pathogenesis of Rett syndrome.

  8. Culturated rat cerebral cortex explants and their application in the study of SPECT scan radiopharaceuticals

    International Nuclear Information System (INIS)

    Jong, B.M. de.

    1989-01-01

    In this thesis mechanics that result in the distinct localization of radiopharmaceuticals within the brain have been investigated. In order to 'get more insight' in uptake and binding of radiopharmaceuticals bu brain tissue, use has been made of the tissue culture technique. Tissue culture privides the opportunity of doing experiments with brain tissue under stable conditions, in the absence of a blood-brain barrier, and without interference by cerebral blood flow. The present thesis is presented in two sections. The first part focusses on longterm culture of 'organotypic' cerebral neocortex tissue, obtained from neonatal rat brain and explanted into a chemically defined medium. Procedures were developed which enabled culturing of this tissue without the occurence of central necrosis and with the preservation of a characteristic histiotypic organization. Morphological characteristics of the cultures were described and measured at various ages in vitro. In the second part, the cultures were used to study mechanisms that might contribute to the tissue uptake of radiopharmaceuticals which are in clinical use for SPECT brain imaging. (author). 369 refs.; 50 figs.; 13 tabs

  9. Expression of IsK protein mRNA in cultured rat strial marginal cells.

    Science.gov (United States)

    Tu, T Y; Chiu, J H; Chang, T J; Yang, A H; Lien, C F

    1999-01-01

    A cell culture system of marginal cells (MC) of the rat stria vascularis was established by the explant method. When grown on plastic dishes, cultured MC showed a polygonal "cobblestone-like" appearance. Dome formation, composed of several hundreds to thousands of cells, occurring after confluence suggested that vectorial transport of ion(s) with accompanying fluid developed in the cultured MC. Transmission electron microscopy demonstrated junctional complexes formed of tight junctions and desmosomes at the upper lateral membranes. The polymerase chain reaction (PCR) product, amplified with primers made from the cDNA reverse transcribed from cultured MC, yielded a distinct band compatible with the expected size of the PCR products amplified from cDNA of positive control groups containing IsK protein, indicating that cultured MC expressed the IsK protein mRNA. The results show that cultured MC can form large domes and express the most characteristic IsK protein, indicating that they maintain their vectorial electrolyte transport function and, possibly, the ability to secrete K+ in this condition.

  10. Electron microscopy of primary cell cultures in solution and correlative optical microscopy using ASEM

    Energy Technology Data Exchange (ETDEWEB)

    Hirano, Kazumi; Kinoshita, Takaaki [Laboratory of Cell Biology, Department of Bioinformatics, Faculty of Engineering, Soka University, 1-236 Tangi-machi, Hachioji, Tokyo 192-8577 (Japan); Uemura, Takeshi [Department of Molecular Neurobiology and Pharmacology, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan); Department of Molecular and Cellular Physiology, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto, Nagano 390-8621 (Japan); Motohashi, Hozumi [Department of Gene Expression Regulation, Institute of Development, Aging and Cancer, Tohoku University, 4-1 Seiryo-cho, Aoba-ku, Sendai 980-8575 (Japan); Watanabe, Yohei; Ebihara, Tatsuhiko [Biomedical Research Institute, National Institute of Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba 305-8566 (Japan); Nishiyama, Hidetoshi [JEOL Ltd., 1-2 Musashino 3-chome, Akishima, Tokyo 196-8558 (Japan); Sato, Mari [Biomedical Research Institute, National Institute of Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba 305-8566 (Japan); Suga, Mitsuo [JEOL Ltd., 1-2 Musashino 3-chome, Akishima, Tokyo 196-8558 (Japan); Maruyama, Yuusuke; Tsuji, Noriko M. [Biomedical Research Institute, National Institute of Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba 305-8566 (Japan); Yamamoto, Masayuki [Department of Medical Biochemistry, Tohoku University Graduate School of Medicine, 2-1 Seiryo-cho, Aoba-ku, Sendai 980-8575 (Japan); Nishihara, Shoko, E-mail: shoko@soka.ac.jp [Laboratory of Cell Biology, Department of Bioinformatics, Faculty of Engineering, Soka University, 1-236 Tangi-machi, Hachioji, Tokyo 192-8577 (Japan); Sato, Chikara, E-mail: ti-sato@aist.go.jp [Biomedical Research Institute, National Institute of Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba 305-8566 (Japan)

    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. - Highlights: • In situ correlative light electron microscopy of samples in open solution by ASEM. • Primary cultures for in-solution CLEM by developing SiN-film coating methods • First visualization of fluorescent magnetic beads in aqueous solution by CLEM. • Presynaptic induction of neurons by GluRδ2-N-terminus-coated beads studied by CLEM. • Axonal partitioning, bacterial phagocytosis, platelet formation imaged by CLEM.

  11. Electron microscopy of primary cell cultures in solution and correlative optical microscopy using ASEM

    International Nuclear Information System (INIS)

    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

    2014-01-01

    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. - Highlights: • In situ correlative light electron microscopy of samples in open solution by ASEM. • Primary cultures for in-solution CLEM by developing SiN-film coating methods • First visualization of fluorescent magnetic beads in aqueous solution by CLEM. • Presynaptic induction of neurons by GluRδ2-N-terminus-coated beads studied by CLEM. • Axonal partitioning, bacterial phagocytosis, platelet formation imaged by CLEM

  12. NAP (davunetide) protects primary hippocampus culture by modulating expression profile of antioxidant genes during limiting oxygen conditions.

    Science.gov (United States)

    Arya, A; Meena, R; Sethy, N K; Das, M; Sharma, M; Bhargava, K

    2015-04-01

    Hypoxia is a well-known threat to neuronal cells and triggers the pathophysiological syndromes in extreme environments such as high altitudes and traumatic conditions such as stroke. Among several prophylactic molecules proven suitable for ameliorating free radical damage, NAP (an octapeptide with initial amino acids: asparagine/N, alanine/A, and proline/P) can be considered superlative, primarily due to its high permeability into brain through blood-brain barrier and observed activity at femtomolar concentrations. Several mechanisms of action of NAP have been hypothesized for its protective role during hypoxia, yet any distinct mechanism is unknown. Oxidative stress is advocated as the leading event in hypoxia; we, therefore, investigated the regulation of key antioxidant genes to understand the regulatory role of NAP in providing neuroprotection. Primary neuronal culture of rat was subjected to cellular hypoxia by limiting the oxygen concentration to 0.5% for 72 h and observing the prophylactic efficacies of 15fM NAP by conventional cell death assays using flow cytometry. We performed real-time quantitative polymerase chain reaction to comprehend the regulatory mechanism. Further, we validated the significantly regulated candidates by enzyme assays and immunoblotting. In the present study, we report that NAP regulates a major clad of cellular antioxidants and there is an involvement of more than one route of action in neuroprotection during hypoxia.

  13. Radiosensitivity and TP 53, EGFR amplification and LOH10 analysis of primary glioma cell cultures

    International Nuclear Information System (INIS)

    Gerlach, B.; Harder, A.H.; Slotman, B.J.; Sminia, P.; Hulsebos, T.J.M.; Leenstra, S.; Peter Vandertop, W.; Hartmann, K.A.

    2002-01-01

    Aim: Determination of in-vitro radiosensitivity and genetic alterations of cell cultures derived from human glioma biopsy tissue and established glioma cell lines. Material and Methods: Fresh brain tumor specimens of six patients were processed to early passage cell cultures. In addition the cell lines D 384 and Gli 6 were used. Cell cultures were irradiated with doses from 2 to 10 Gy. Following irradiation, cell survival was determined by clonogenic assay and survival curves were generated. The surviving fractions after 2 Gy (SF2) and 4 Gy (SF4) were used as radiosensitivity parameters. Genetic analysis included determination of the mutational and loss of heterozygosity (LOH) status of TP 53 (exons 5-8), the LOH 10- and epidermal growth factor receptor gene (EGFR) amplification status. Results: The SF2 and SF4 values ranged from 0.54 to 0.88 (mean: 0.70) and from 0.13 to 0.52 (mean: 0.32), respectively. Genetic alterations were found in the Gli 6 cell line and in two primary cell cultures. The genetic profile of Gli 6 showed LOH but no TP 53 mutation, complete LOH 10 and no EGFR amplification. The VU 15 cell culture showed TP 53 mutation but no LOH 10 or EGFR amplification, while VU 24 showed incomplete LOH 10, EGFR amplification and no TP 53 mutation. In the other four cell cultures and D 384 cell line no genetic alterations were diagnosed. Histopathological classification of glioblastoma multiforme and/or genetic alterations resulted in lower radiosensitivity. Conclusion: In this small series of early passage glioma cell cultures low radiosensitivity and alterations in cell regulatory genes were seen. Further testing of biological behavior in larger series of patient-derived material is ongoing. (orig.)

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

    Science.gov (United States)

    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.

  15. Primary rat Sertoli and interstitial cells exhibit a differential response to cadmium

    Energy Technology Data Exchange (ETDEWEB)

    Clough, S.R.; Welsh, M.J.; Payne, A.H.; Brown, C.D.; Brabec, M.J. (Eastern Michigan Univ., Ypsilanti (USA))

    1990-01-01

    Two cell types central to the support of spermatogenesis, the Sertoli cell and the interstitial (Leydig) cell, were isolated from the same cohort of young male rats and challenged with cadmium chloride to compare their susceptibility to the metal. Both cell types were cultured under similar conditions, and similar biochemical endpoints were chosen to minimize experimental variability. These endpoints include the uptake of 109Cd, reduction of the vital tetrazolium dye MTT, incorporation of 3H-leucine, change in heat-stable cadmium binding capacity, and production of lactate. Using these parameters, it was observed that the Sertoli cell cultures were adversely affected in a dose-and time-dependent manner, while the interstitial cell cultures, treated with identical concentrations of CdCl2, were less affected. The 72-hr LC50's for Sertoli cells and interstitial cells were 4.1 and 19.6 microM CdCl2, respectively. Thus, different cell populations within the same tissue may differ markedly in susceptibility to a toxicant. These in vitro data suggest that the Sertoli cell, in relation to the interstitium, is particularly sensitive to cadmium. Because the Sertoli cell provides functional support for the seminiferous epithelium, the differential sensitivity of this cell type may, in part, explain cadmium-induced testicular dysfunction, particularly at doses that leave the vascular epithelium intact.

  16. Receptor-mediated uptake of low density lipoprotein stimulates bile acid synthesis by cultured rat hepatocytes

    International Nuclear Information System (INIS)

    Junker, L.H.; Davis, R.A.

    1989-01-01

    The cellular mechanisms responsible for the lipoprotein-mediated stimulation of bile acid synthesis in cultured rat hepatocytes were investigated. Adding 280 micrograms/ml of cholesterol in the form of human or rat low density lipoprotein (LDL) to the culture medium increased bile acid synthesis by 1.8- and 1.6-fold, respectively. As a result of the uptake of LDL, the synthesis of [14C]cholesterol from [2-14C]acetate was decreased and cellular cholesteryl ester mass was increased. Further studies demonstrated that rat apoE-free LDL and apoE-rich high density lipoprotein (HDL) both stimulated bile acid synthesis 1.5-fold, as well as inhibited the formation of [14C]cholesterol from [2-14C]acetate. Reductive methylation of LDL blocked the inhibition of cholesterol synthesis, as well as the stimulation of bile acid synthesis, suggesting that these processes require receptor-mediated uptake. To identify the receptors responsible, competitive binding studies using 125I-labeled apoE-free LDL and 125I-labeled apoE-rich HDL were performed. Both apoE-free LDL and apoE-rich HDL displayed an equal ability to compete for binding of the other, suggesting that a receptor or a group of receptors that recognizes both apolipoproteins is involved. Additional studies show that hepatocytes from cholestyramine-treated rats displayed 2.2- and 3.4-fold increases in the binding of apoE-free LDL and apoE-rich HDL, respectively. These data show for the first time that receptor-mediated uptake of LDL by the liver is intimately linked to processes activating bile acid synthesis

  17. Mammalian intestinal epithelial cells in primary culture: a mini-review.

    Science.gov (United States)

    Kaeffer, Bertrand

    2002-03-01

    Epithelial cells lining the digestive tract represent a highly organized system built up by multipotent stem cells. A process of asymmetric mitosis produces a population of proliferative cells that are rapidly renewed and migrate along the crypt-villus axis, differentiating into functional mature cells before dying and exfoliating into the intestinal lumen. Isolated crypts or epithelial cells retaining high viability can be prepared within a few h after tissue sampling. After cells are cultured in serum-free media, short-term studies (16-48 h) can be conducted for endocrinology, energy metabolism, or programmed cell death. However, long-term primary culture of intestinal cells (up to 10 d) is still difficult despite progress in isolation methodologies and manipulation of the cell microenvironment. The main problem in developing primary culture is the lack of structural markers specific to the stem cell compartment. The design of a microscopic multidimensional analytic system to record the expression profiles of biomarkers all along the living intestinal crypt should improve basic knowledge of the survival and growth of adult crypt stem cells, and the selection of totipotent embryonic stem cells capable of differentiating into intestinal tissues should facilitate studies of the genomic basis of endodermal tissue differentiation.

  18. Establishment of primary cultures for mouse ameloblasts as a model of their lifetime

    International Nuclear Information System (INIS)

    Suzawa, Tetsuo; Itoh, Nao; Takahashi, Naoyuki; Katagiri, Takenobu; Morimura, Naoko; Kobayashi, Yasuna; Yamamoto, Toshinori; Kamijo, Ryutaro

    2006-01-01

    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

  19. Assessment of Patient Safety Culture in Primary Health Care Settings in Kuwait

    Directory of Open Access Journals (Sweden)

    Maha Mohamed Ghobashi

    2014-01-01

    Full Text Available Background Patient safety is critical component of health care quality. We aimed to assess the awareness of primary healthcare staff members about patient safety culture and explore the areas of deficiency and opportunities for improvement concerning this issue.Methods: This descriptive cross sectional study surveyed 369 staff members in four primary healthcare centers in Kuwait using self-administered “Hospital Survey on Patient Safety Culture” adopted questionnaire. The total number of respondents was 276 participants (response rate = 74.79%.Results: Five safety dimensions with lowest positivity (less than 50% were identified and these are; the non – punitive response to errors, frequency of event reporting, staffing, communication openness, center handoffs and transitions with the following percentages of positivity 24%, 32%, 41%, 45% and 47% respectively. The dimensions of highest positivity were teamwork within the center’s units (82% and organizational learning (75%.Conclusion: Patient safety culture in primary healthcare settings in Kuwait is not as strong as improvements for the provision of safe health care. Well-designed patient safety initiatives are needed to be integrated with organizational policies, particularly the pressing need to address the bioethical component of medical errors and their disclosure, communication openness and emotional issues related to them and investing the bright areas of skillful organizational learning and strong team working attitudes.    

  20. The toxicity of uranyl nitrate on primary brain cell culture of L. Hoevenii

    International Nuclear Information System (INIS)

    Ismail Bahari; Fauziah Mohd Noor

    1995-01-01

    In Malaysia, uranium is indirectly being concentrated by mining and petroleum industries that have no relevance to its use. Concentration of uranium and the production of TENORM may give rise to radiological risk to workers and the environment. A study was conducted to determine the toxicity of a uranium compound, uranyl nitrate. For this purpose a primary brain cell culture derived from L. hoevenii was used. The nature of uranil nitrate toxicity was determined by comparing with the effects induced by mitomycin C and gamma radiation. The toxicity of these agents were measured by observing changes in Unschedule DNA Synthesis (UDS) and the induction of micronucleus. Result from the study showed that UO sub 2 sup 2+ is UDS positive and is toxic to the primary brain cells of L. hoevenii. It gives a response profile that is almost similar to that induced by gamma radiation and mitomycin C. We believed that a low concentration, UO sub 2 sup 2+ acts as a chemo toxic agent rather than as an ionising radiation. At higher concentration the toxicity of UO sub 2 sup 2+ comes from both its chemo toxic and radiation effects. Results of this study also show the ability of the primary culture to carry out repair on its DNA damaged by the UDS positive agents

  1. Arctigenin Increases Hemeoxygenase-1 Gene Expression by Modulating PI3K/AKT Signaling Pathway in Rat Primary Astrocytes

    OpenAIRE

    Jeong, Yeon-Hui; Park, Jin-Sun; Kim, Dong-Hyun; Kim, Hee-Sun

    2014-01-01

    In the present study, we found that the natural compound arctigenin inhibited hydrogen peroxide-induced reactive oxygen species (ROS) production in rat primary astrocytes. Since hemeoxygenase-1 (HO-1) plays a critical role as an antioxidant defense factor in the brain, we examined the effect of arctigenin on HO-1 expression in rat primary astrocytes. We found that arctigenin increased HO-1 mRNA and protein levels. Arctigenin also increases the nuclear translocation and DNA binding of Nrf2/c-J...

  2. Cytokines effects on radio-induced apoptosis in cortical and hippocampal rat cells in culture

    International Nuclear Information System (INIS)

    Coffigny, H.; Briot, D.; Le Nin, I.

    2000-01-01

    In the central nervous system in development the radio-induced cell death occurs mainly by apoptosis. The effects of modulating factors like cytokines were studied on this kind of death. To handle more easily parameters implicated in nerve cell apoptosis, we studied the effects of radiation with a in vitro system. Cells were isolated from rat foetal cortex and hippocampus, two of the major structures implicated in human mental retardation observed after exposition in utero at Hiroshima and Nagasaki. Cortical or hippocampal cells were isolated from 17 day-old rat foetuses by enzymatic and mechanical treatments and irradiated with 0.50 or 1 Gy. The cells from both structures were cultured 1 or 3 days in serum free medium. Cytokines like βNGF, NT3, EGF, βTGF, α and βFGF, IGF I and II, interleukines like Il 1β, Il 2 and IL 6 were added to the medium. In 3 days cortical cell culture, only βFGF increased cell survival with as little as 10 ng/ml. This effect was dose dependent. In hippocampal cell culture, no significant increase of cell survival occurred with 10 ng/ml of any cytokines. In the same system culture with 1 Gy irradiation, the positive or negative effect of the association of βFGF with another cytokine was tested on cell survival. Only the association with EGF induced higher cell survival in cortical cell culture. In hippocampal cell culture where βFGF alone had no effect, the cell survival was not modified by the association. In the same system, the triple association of βFGF-EGF with another cytokine was tested on hippocampal and cortical cell cultures. No significant effect was observed in both cultures but cell survival trented to decrease with βTGF. In order to avoid the mitotic effect of cytokines in the 3 day-old culture, experiments were carried out on 20 hours cell culture, before the end of the first round of the cell cycle, with the selected cytokines (βFGF or βFGF-EGF). Without irradiation, the percentage of cortical cell survival

  3. Neuroprotection by biodegradable PAMAM ester (e-PAM-R)-mediated HMGB1 siRNA delivery in primary cortical cultures and in the postischemic brain.

    Science.gov (United States)

    Kim, Il-Doo; Lim, Chae-Moon; Kim, Jung-Bin; Nam, Hye Yeong; Nam, Kihoon; Kim, Seung-Woo; Park, Jong-Sang; Lee, Ja-Kyeong

    2010-03-19

    Although RNA interference (RNAi)-mediated gene silencing provides a powerful strategy for modulating specific gene functions, difficulties associated with siRNA delivery have impeded the development of efficient therapeutic applications. In particular, the efficacy of siRNA delivery into neurons has been limited by extremely low transfection efficiencies. e-PAM-R is a biodegradable arginine ester of PAMAM dendrimer, which is readily degradable under physiological conditions (pH 7.4, 37 degrees C). In the present study, we investigated the efficiency of siRNA delivery by e-PAM-R in primary cortical cultures and in rat brain. e-PAM-R/siRNA complexes showed high transfection efficiencies and low cytotoxicities in primary cortical cultures. Localization of fluorescence-tagged siRNA revealed that siRNA was delivered not only into the nucleus and cytoplasm, but also along the processes of the neuron. e-PAM-R/siRNA complex-mediated target gene reduction was observed in over 40% of cells and it was persistent for over 48 h. The potential use of e-PAM-R was demonstrated by gene knockdown after transfecting High mobility group box-1 (HMGB1, a novel cytokine-like molecule) siRNA into H(2)O(2)- or NMDA-treated primary cortical cultures. In these cells, HMGB1 siRNA delivery successfully reduced both basal and H(2)O(2)- or NMDA-induced HMGB1 levels, and as a result of that, neuronal cell death was significantly suppressed in both cases. Furthermore, we showed that e-PAM-R successfully delivered HMGB1 siRNA into the rat brain, wherein HMGB1 expression was depleted in over 40% of neurons and astrocytes of the normal brain. Moreover, e-PAM-R-mediated HMGB1 siRNA delivery notably reduced infarct volume in the postischemic rat brain, which is generated by occluding the middle cerebral artery for 60 min. These results indicate that e-PAM-R, a novel biodegradable nonviral gene carrier, offers an efficient means of transfecting siRNA into primary neuronal cells and in the brain and of

  4. Neonicotinoid Insecticides Alter the Gene Expression Profile of Neuron-Enriched Cultures from Neonatal Rat Cerebellum

    Directory of Open Access Journals (Sweden)

    Junko Kimura-Kuroda

    2016-10-01

    Full Text Available Neonicotinoids are considered safe because of their low affinities to mammalian nicotinic acetylcholine receptors (nAChRs relative to insect nAChRs. However, because of importance of nAChRs in mammalian brain development, there remains a need to establish the safety of chronic neonicotinoid exposures with regards to children’s health. Here we examined the effects of longterm (14 days and low dose (1 μM exposure of neuron-enriched cultures from neonatal rat cerebellum to nicotine and two neonicotinoids: acetamiprid and imidacloprid. Immunocytochemistry revealed no differences in the number or morphology of immature neurons or glial cells in any group versus untreated control cultures. However, a slight disturbance in Purkinje cell dendritic arborization was observed in the exposed cultures. Next we performed transcriptome analysis on total RNAs using microarrays, and identified significant differential expression (p < 0.05, q < 0.05, ≥1.5 fold between control cultures versus nicotine-, acetamiprid-, or imidacloprid-exposed cultures in 34, 48, and 67 genes, respectively. Common to all exposed groups were nine genes essential for neurodevelopment, suggesting that chronic neonicotinoid exposure alters the transcriptome of the developing mammalian brain in a similar way to nicotine exposure. Our results highlight the need for further careful investigations into the effects of neonicotinoids in the developing mammalian brain.

  5. Elastase effect on the extracellular matrix of rat aortic smooth muscle cells in culture

    International Nuclear Information System (INIS)

    Kispert, J.; Mogayzel, P.J. Jr.; Pratt, C.A.; Toselli, P.; Wolfe, B.L.; Faris, B.; Franzblau, C.

    1986-01-01

    The effect of porcine pancreatic elastase on the extracellular matrix (ECM) of neonatal rat aortic smooth muscle cell cultures was monitored both chemically and ultrastructurally. Initially, the elastin appeared as non-coalesced material closely associated with filaments, presumably microfibrils. The insoluble elastin accumulated in the ECM of cells in culture for 6 weeks accounted for 40-45% of the total protein. After exposure to elastase for 30-60 minutes, the elastin content was reduced to 14-20%. The reduction in the total protein content of the cultures after elastase treatment was due primarily to the loss of elastin. Although the amino acid compositions of the elastin isolated from cultures both before and after elastase treatment were similar, there were striking ultrastructural differences in the amorphous elastin. The elastin assumed a mottled appearance after elastase exposure, similar to that seen in in vivo emphysema models. Pulse experiments with 3 H-valine demonstrated an increase in protein synthesis by the cells 20 hours after elastase exposure, suggesting the potential for elastin repair. The use of this culture system will aid in clarifying the role of elastolysis in pulmonary and vascular injuries

  6. Cytopathology of pathogenic and nonpathogenic Naegleria species for cultured rat neuroblastoma cells.

    OpenAIRE

    Marciano-Cabral, F M; Fulford, D E

    1986-01-01

    The cytopathology for rat neuroblastoma cells (B-103) and the pathogenicity for B6C3F1 mice of four species of Naegleria have been compared. Both live amoebae and cell-free extracts of N. australiensis, N. fowleri, N. gruberi, and N. lovaniensis added to 51Cr-labeled B-103 cells caused release of radiolabel. All four species of Naegleria exhibited surface extensions termed food cups. Only N. fowleri and N. australiensis were pathogenic for mice. Electron microscopic observations of cultures o...

  7. Cytopathology of pathogenic and nonpathogenic Naegleria species for cultured rat neuroblastoma cells.

    Science.gov (United States)

    Marciano-Cabral, F M; Fulford, D E

    1986-05-01

    The cytopathology for rat neuroblastoma cells (B-103) and the pathogenicity for B6C3F1 mice of four species of Naegleria have been compared. Both live amoebae and cell-free extracts of N. australiensis, N. fowleri, N. gruberi, and N. lovaniensis added to 51Cr-labeled B-103 cells caused release of radiolabel. All four species of Naegleria exhibited surface extensions termed food cups. Only N. fowleri and N. australiensis were pathogenic for mice. Electron microscopic observations of cultures of either N. australiensis or N. lovaniensis with B-103 cells established that the cytopathology involved lysis of the B-103 target cells.

  8. ELECTROPHYSIOLOGICAL CHARACTERIZATION OF DOPAMINERGIC AND NONDOPAMINERGIC NEURONS IN ORGANOTYPIC SLICE CULTURES OF THE RAT VENTRAL MESENCEPHALON

    DEFF Research Database (Denmark)

    STEENSEN, BH; NEDERGAARD, S; OSTERGAARD, K

    1995-01-01

    -old organotypic slice cultures of the ventral mesencephalon prepared from newborn rats. Dopaminergic neurones were distinguished from non-dopaminergic neurones by staining with the autofluorescent serotonin analogue 5,7-dihydroxytryptamine and briefly viewing the preparation with short exposures to ultraviolet...... 81 M Omega), were silent or fired spontaneously at a low frequency (0-9 Hz), and no spontaneous GABA(A)-ergic inhibitory postsynaptic potentials or inward rectification were present. In contrast, non-dopaminergic neurones had fast action potentials (0.6-3.2 ms), low input resistance (mean 32 M Omega...

  9. Effect of Albumin on the Biliary Clearance of Compounds in Sandwich-Cultured Rat Hepatocytes

    OpenAIRE

    Wolf, Kristina K.; Brouwer, Kenneth R.; Pollack, Gary M.; Brouwer, Kim L.R.

    2008-01-01

    The purpose of the present study was to evaluate the effects of bovine serum albumin (BSA) and essentially fatty acid-free BSA (BSA-FAF) on the biliary clearance of compounds in sandwich-cultured rat hepatocytes. Unbound fraction (fu), biliary excretion index (BEI), and unbound intrinsic biliary clearance (intrinsic Cl’biliary) were determined for digoxin, pravastatin, and taurocholate in the absence or presence of BSA or BSA-FAF. BSA had little effect on the BEI or intrinsic Cl’biliary of th...

  10. Primary Blast-Induced Changes in Akt and GSK3β Phosphorylation in Rat Hippocampus

    Directory of Open Access Journals (Sweden)

    Yushan Wang

    2017-08-01

    Full Text Available Traumatic brain injury (TBI due to blast from improvised explosive devices has been a leading cause of morbidity and mortality in recent conflicts in Iraq and Afghanistan. However, the mechanisms of primary blast-induced TBI are not well understood. The Akt signal transduction pathway has been implicated in various brain pathologies including TBI. In the present study, the effects of simulated primary blast waves on the phosphorylation status of Akt and its downstream effector kinase, glycogen synthase kinase 3β (GSK3β, in rat hippocampus, were investigated. Male Sprague-Dawley (SD rats (350–400 g were exposed to a single pulse shock wave (25 psi; ~7 ms duration and sacrificed 1 day, 1 week, or 6 weeks after exposure. Total and phosphorylated Akt, as well as phosphorylation of its downstream effector kinase GSK3β (at serine 9, were detected with western blot analysis and immunohistochemistry. Results showed that Akt phosphorylation at both serine 473 and threonine 308 was increased 1 day after blast on the ipsilateral side of the hippocampus, and this elevation persisted until at least 6 weeks postexposure. Similarly, phosphorylation of GSK3β at serine 9, which inhibits GSK3β activity, was also increased starting at 1 day and persisted until at least 6 weeks after primary blast on the ipsilateral side. In contrast, p-Akt was increased at 1 and 6 weeks on the contralateral side, while p-GSK3β was increased 1 day and 1 week after primary blast exposure. No significant changes in total protein levels of Akt and GSK were observed on either side of the hippocampus at any time points. Immunohistochemical results showed that increased p-Akt was mainly of neuronal origin in the CA1 region of the hippocampus and once phosphorylated, the majority was translocated to the dendritic and plasma membranes. Finally, electrophysiological data showed that evoked synaptic N-methyl-d-aspartate (NMDA receptor activity was

  11. Cross cultural training in primary mental health care consultations in Moldova - The tEACH perspective.

    Science.gov (United States)

    Møller, Jane Ege; van Weel-Baumgarten, Evelyn

    2017-09-01

    This article reports experiences and challenges encountered in a cross-cultural training project in Moldova that was undertaken by tEACH, the teaching subcommittee of EACH: International Association for Communication in Healthcare, in cooperation with local and international stakeholders. As part of a major health policy reform, the aim was to equip a group of trainers with the skills to train Moldovan professionals in skills for primary mental health care, including communication skills. The project consisted of 3 weeks of training using mainly experiential teaching methods to allow participants to practice content and methods, including interactive lecturing, roleplay, feedback and video. A majority of the participants reported that they acquired key facilitation skills. They valued the opportunity to practice and receive feedback. However, some reported that there was too much focus on communication skills, which was thought to be less relevant in a Moldovan context. Furthermore our learner-centered approach was occasionally experienced as a lack of structure CONCLUSION: The tEACH expertise plays an important role in supporting trainers in cross-cultural contexts with effective communication skills methods. Teaching in a cross-cultural context is only successful through continuous dialogue with stakeholders and demands attention to cultural differences. Copyright © 2017. Published by Elsevier B.V.

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

    Science.gov (United States)

    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. © 2013 by the American College of Medical Quality.

  13. Membrane Mechanics of Primary Afferent Neurons in the Dorsal Root Ganglia of Rats.

    Science.gov (United States)

    Kanda, Hirosato; Gu, Jianguo G

    2017-04-25

    Membrane mechanics is an important biological factor regulating many cellular functions including cell motility, intercellular and intracellular signaling, gene expression, and membrane ion channel activity. Primary afferent neurons transduce sensory information about temperature, touch, and pain. These sensory functions may be profoundly affected by the states of primary afferent neuron mechanics. However, membrane mechanics of primary afferent neurons is largely unknown. In this study, we established the optical trapping technique for determining membrane mechanics of cultured primary afferent neurons of the dorsal root ganglia (DRG). We further determined the roles of cytoskeleton and membrane lipids in DRG neuron mechanics. We found that DRG neurons had a plasma membrane tension of ∼54 pN/μm, and the tension was significantly decreased to ∼29 pN/μm by cytochalasin D treatment to disrupt actin cytoskeleton and increased to ∼79 pN/μm by methyl-β-cyclodextrin treatment to sequester membrane cholesterol. DRG neuron membrane stiffness was not significantly affected by the cytoskeleton disruption but was significantly increased after cholesterol sequestration. Our findings elucidate membrane mechanical properties of primary afferent neurons, which provide, to our knowledge, a new perspective on their sensory functions. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  14. Inhibition of cell growth by EGR-1 in human primary cultures from malignant glioma

    Directory of Open Access Journals (Sweden)

    Gagliardi Franco

    2004-01-01

    Full Text Available Abstract Background The aim of this work was to investigate in vitro the putative role of EGR-1 in the growth of glioma cells. EGR-1 expression was examined during the early passages in vitro of 17 primary cell lines grown from 3 grade III and from 14 grade IV malignant astrocytoma explants. The explanted tumors were genetically characterized at the p53, MDM2 and INK4a/ARF loci, and fibronectin expression and growth characteristics were examined. A recombinant adenovirus overexpressing EGR-1 was tested in the primary cell lines. Results Low levels of EGR-1 protein were found in all primary cultures examined, with lower values present in grade IV tumors and in cultures carrying wild-type copies of p53 gene. The levels of EGR-1 protein were significantly correlated to the amount of intracellular fibronectin, but only in tumors carrying wild-type copies of the p53 gene (R = 0,78, p = 0.0082. Duplication time, plating efficiency, colony formation in agarose, and contact inhibition were also altered in the p53 mutated tumor cultures compared to those carrying wild-type p53. Growth arrest was achieved in both types of tumor within 1–2 weeks following infection with a recombinant adenovirus overexpressing EGR-1 but not with the control adenovirus. Conclusions Suppression of EGR-1 is a common event in gliomas and in most cases this is achieved through down-regulation of gene expression. Expression of EGR-1 by recombinant adenovirus infection almost completely abolishes the growth of tumor cells in vitro, regardless of the mutational status of the p53 gene.

  15. [Primary culture of cat intestinal epithelial cell and construction of its cDNA library].

    Science.gov (United States)

    Ye, L; Gui-Hua, Z; Kun, Y; Hong-Fa, W; Ting, X; Gong-Zhen, L; Wei-Xia, Z; Yong, C

    2017-04-12

    Objective To establish the primary cat intestinal epithelial cells (IECs) culture methods and construct the cDNA library for the following yeast two-hybrid experiment, so as to screen the virulence interaction factors among the final host. Methods The primary cat IECs were cultured by the tissue cultivation and combined digestion with collagenase XI and dispase I separately. Then the cat IECs cultured was identified with the morphological observation and cyto-keratin detection, by using goat anti-cyto-keratin monoclonal antibodies. The mRNA of cat IECs was isolated and used as the template to synthesize the first strand cDNA by SMART™ technology, and then the double-strand cDNAs were acquired by LD-PCR, which were subsequently cloned into the plasmid PGADT7-Rec to construct yeast two-hybrid cDNA library in the yeast strain Y187 by homologous recombination. Matchmaker™ Insert Check PCR was used to detect the size distribution of cDNA fragments after the capacity calculation of the cDNA library. Results The comparison of the two cultivation methods indicated that the combined digestion of collagenase XI and dispase I was more effective than the tissue cultivation. The cat IECs system of continuous culture was established and the cat IECs with high purity were harvested for constructing the yeast two-hybrid cDNA library. The library contained 1.1×10 6 independent clones. The titer was 2.8×10 9 cfu/ml. The size of inserted fragments was among 0.5-2.0 kb. Conclusion The yeast two-hybrid cDNA library of cat IECs meets the requirements of further screen research, and this study lays the foundation of screening the Toxoplasma gondii virulence interaction factors among the cDNA libraries of its final hosts.

  16. Primary Culture of Canine Growth Plate Chondrocytes as a Model of Biomineralization

    OpenAIRE

    Ryuji, HOSOKAWA; Kenji, KIKUZAKI; Daisuke, CHIBA; Yasumasa, AKAGAWA; Department of Removable Prosthodontics, Hiroshima University School of Dentistry; Department of Removable Prosthodontics, Hiroshima University School of Dentistry; Department of Removable Prosthodontics, Hiroshima University School of Dentistry; Department of Removable Prosthodontics, Hiroshima University School of Dentistry

    1999-01-01

    This study investigated the mineralization process in primary cultures of dog growth plate chondrocytes as a model of biomineralization. Chondrocytes were isolated from the growth plates of ribs of 1-week-old dogs. The chondrocytes were maintained at extremely high density (5x10^4 cells/well) in collagen-coated 96-well dishes in a-MEM supplemented with 10% fetal bovine serum and 50 μg/ml ascorbic acid. Mineralization was initiated between days 20 and 24; however, the addition of fibroblast gr...

  17. Ethanol affects network activity in cultured rat hippocampus: mediation by potassium channels.

    Directory of Open Access Journals (Sweden)

    Eduard Korkotian

    Full Text Available The effects of ethanol on neuronal network activity were studied in dissociated cultures of rat hippocampus. Exposure to low (0.25-0.5% ethanol concentrations caused an increase in synchronized network spikes, and a decrease in the duration of individual spikes. Ethanol also caused an increase in rate of miniature spontaneous excitatory postsynaptic currents. Higher concentrations of ethanol eliminated network spikes. These effects were reversible upon wash. The effects of the high, but not the low ethanol were blocked by the GABA antagonist bicuculline. The enhancing action of low ethanol was blocked by apamin, an SK potassium channel antagonist, and mimicked by 1-EBIO, an SK channel opener. It is proposed that in cultured hippocampal networks low concentration of ethanol is associated with SK channel activity, rather than the GABAergic receptor.

  18. Effects of static magnetic fields on bone formation in rat osteoblast cultures.

    Science.gov (United States)

    Yamamoto, Y; Ohsaki, Y; Goto, T; Nakasima, A; Iijima, T

    2003-12-01

    Although the promotional effects on osteoblasts of pulsed electromagnetic fields have been well-demonstrated, the effects of static magnetic fields (SMF) remain unclear; nevertheless, magnets have been clinically used as a 'force source' in various orthodontic treatments. We undertook the present investigation to study the effects of SMF on osteoblastic differentiation, proliferation, and bone nodule formation using a rat calvaria cell culture. During a 20-day culture, the values of the total area and the number and average size of bone nodules showed high levels in the presence of SMF. In the matrix development and mineralization stages, the calcium content in the matrix and two markers of osteoblastic phenotype (alkaline phosphatase and osteocalcin) also showed a significant increase. Accordingly, these findings suggest that SMF stimulates bone formation by promoting osteoblastic differentiation and/or activation.

  19. [3H]GABA uptake as a marker for cell type in primary cultures of cerebellum and olfactory bulb

    International Nuclear Information System (INIS)

    Currie, D.N.; Dutton, G.R.

    1980-01-01

    Uptake of [ 3 H]GABA into cell cultures of rat cerebellum and olfactory bulb was studied by autoradiography, using β-alanine and aminocyclohexane carboxylic acid to distinguish neuronal-specific and glial-specific uptake. Neurons and astrocytes were also labelled by tetanus toxin and anti-GFAP respectively. This combination of markers allowed identification and quantification of several cell types. Cerebellar cultures were found to contain 77% granule neurons, 7.5% inhibitory neurons (probably stellate and basket cells) and 15% astrocytes. Olfactory bulb cultures were over 50% in small neurons which accumulated GABA, the olfactory bulb granule neuron being GABAergic in vivo. (Auth.)

  20. Reactive oxygen species levels and DNA fragmentation on astrocytes in primary culture after acute exposure to low intensity microwave electromagnetic field.

    Science.gov (United States)

    Campisi, Agata; Gulino, Marisa; Acquaviva, Rosaria; Bellia, Paolo; Raciti, Giuseppina; Grasso, Rosaria; Musumeci, Francesco; Vanella, Angelo; Triglia, Antonio

    2010-03-31

    The exposure of primary rat neocortical astroglial cell cultures to acute electromagnetic fields (EMF) in the microwave range was studied. Differentiated astroglial cell cultures at 14 days in vitro were exposed for 5, 10, or 20min to either 900MHz continuous waves or 900MHz waves modulated in amplitude at 50Hz using a sinusoidal waveform and 100% modulation index. The strength of the electric field (rms value) at the sample position was 10V/m. No change in cellular viability evaluated by MTT test and lactate dehydrogenase release was observed. A significant increase in ROS levels and DNA fragmentation was found only after exposure of the astrocytes to modulated EMF for 20min. No evident effects were detected when shorter time intervals or continuous waves were used. The irradiation conditions allowed the exclusion of any possible thermal effect. Our data demonstrate, for the first time, that even acute exposure to low intensity EMF induces ROS production and DNA fragmentation in astrocytes in primary cultures, which also represent the principal target of modulated EMF. Our findings also suggest the hypothesis that the effects could be due to hyperstimulation of the glutamate receptors, which play a crucial role in acute and chronic brain damage. Furthermore, the results show the importance of the amplitude modulation in the interaction between EMF and neocortical astrocytes. Copyright 2010 Elsevier Ireland Ltd. All rights reserved.

  1. Substance P signalling in primary motor cortex facilitates motor learning in rats.

    Directory of Open Access Journals (Sweden)

    Benjamin Hertler

    Full Text Available Among the genes that are up-regulated in response to a reaching training in rats, Tachykinin 1 (Tac1-a gene that encodes the neuropeptide Substance P (Sub P-shows an especially strong expression. Using Real-Time RT-PCR, a detailed time-course of Tac1 expression could be defined: a significant peak occurs 7 hours after training ended at the first and second training session, whereas no up-regulation could be detected at a later time-point (sixth training session. To assess the physiological role of Sub P during movement acquisition, microinjections into the primary motor cortex (M1 contralateral to the trained paw were performed. When Sub P was injected before the first three sessions of a reaching training, effectiveness of motor learning became significantly increased. Injections at a time-point when rats already knew the task (i.e. training session ten and eleven had no effect on reaching performance. Sub P injections did not influence the improvement of performance within a single training session, but retention of performance between sessions became strengthened at a very early stage (i.e. between baseline-training and first training session. Thus, Sub P facilitates motor learning in the very early phase of skill acquisition by supporting memory consolidation. In line with these findings, learning related expression of the precursor Tac1 occurs at early but not at later time-points during reaching training.

  2. Rat glomerular epithelial cells in culture. Parietal or visceral epithelial origin

    International Nuclear Information System (INIS)

    Norgaard, J.O.

    1987-01-01

    Isolated glomeruli from rats were explanted under standard culture conditions and outgrowths were studied by light and electron microscopy in order to identify the cells. Rat glomerular samples contained 20 to 30% structurally well-preserved encapsulated glomeruli which had a large rate of attachment to the substrate and very constantly gave rise to cellular outgrowth. In order to label cells from which outgrowth originated the glomerular incorporation of [ 3 H]thymidine was studied in the preattachment phase. By light and electron microscope autoradiograph it was demonstrated that label was located only over visceral and parietal epithelial cells during the first 3 days of culture. Incorporation of [ 3 H]thymidine was seen in mesangial cells after 5 days, i.e., after the glomeruli had attached to the culture vessels and the initial outgrowth had appeared. Consequently the first cells to grow out were of epithelial origin. Glomeruli were then incubated with [ 3 H]thymidine for the first 2 1/2 days of culture in order to label the epithelial cells, then were allowed to attach to the substrate and induce cell outgrowth. By light microscope autoradiography performed with the outgrowths in situ two types of cells with labeled nuclei were seen: (a) a small, polyhedral ciliated cell which grew in colonies where the cells were joined by junctional complexes (type I), and (b) a second very large, often multinucleated cell (type II). Based on the structural resemblance with their counterparts in situ and on comparisons with positively identified visceral epithelial cells in outgrowths from other species it is suggested that type I cells are derived from the parietal epithelium of Bowman's capsule and type II cells from the visceral epithelium

  3. VARIATIVE EDUCATION IN THE CHANGING WORLD: SOCIO-CULTURAL PERSPECTIVE OF THE PRIMARY SCHOOL DEVELOPMENT

    Directory of Open Access Journals (Sweden)

    A. G. Asmolov

    2015-03-01

    Full Text Available The paper reflects the ideas of the author (one of the new educational standard developers concerning the prospects of primary school evolution, published in the Educational Policy journal in 2011, being still relevant due to the introduction of the Federal Act on Education from September 1, 2013.In author's opinion, a successful accomplishment and implementation of the given act and standards can be achieved through careful explanation of their content and essence to the interested people, and justification of the related benefit for the society and teachers alike. The alteration of educational concepts is regarded as a challenging task, education being the most conservative sphere of human activity. As a solution to the primary school problem, the paper emphasizes several referent points including teachers' motivation, strengthening and updating of their professional skills and knowledge, etc. The primary school appears to be the most important basis for children’s personal socialization. And therefore, it should transform into the school of understanding, cultural dialog, game and project didactics, variant education, and foundation for the universal learning activities and value formation. The primary school should teach productive cooperation with adults and advanced peers, help in solving complicated life tasks, and raise children’s confidence in personal strength and capability. 

  4. VARIATIVE EDUCATION IN THE CHANGING WORLD: SOCIO-CULTURAL PERSPECTIVE OF THE PRIMARY SCHOOL DEVELOPMENT

    Directory of Open Access Journals (Sweden)

    A. G. Asmolov

    2013-01-01

    Full Text Available The paper reflects the ideas of the author (one of the new educational standard developers concerning the prospects of primary school evolution, published in the Educational Policy journal in 2011, being still relevant due to the introduction of the Federal Act on Education from September 1, 2013.In author's opinion, a successful accomplishment and implementation of the given act and standards can be achieved through careful explanation of their content and essence to the interested people, and justification of the related benefit for the society and teachers alike. The alteration of educational concepts is regarded as a challenging task, education being the most conservative sphere of human activity. As a solution to the primary school problem, the paper emphasizes several referent points including teachers' motivation, strengthening and updating of their professional skills and knowledge, etc. The primary school appears to be the most important basis for children’s personal socialization. And therefore, it should transform into the school of understanding, cultural dialog, game and project didactics, variant education, and foundation for the universal learning activities and value formation. The primary school should teach productive cooperation with adults and advanced peers, help in solving complicated life tasks, and raise children’s confidence in personal strength and capability. 

  5. Cross-Cultural Validation of Urdu Version KOOS in Indian Population with Primary Knee Osteoarthritis

    Directory of Open Access Journals (Sweden)

    Mahamed Ateef

    2017-01-01

    Full Text Available Purpose. The primary aim of this study was to translate a self-reported questionnaire (KOOS from English to Urdu and then to see its internal consistency, agreement, test-retest reliability, and validity among primary OA knee patients. Methodology. First, KOOS questionnaire was translated from English language to Urdu through standardized cross-cultural protocol. This translated version of KOOS was administered to 111 radiographically diagnosed primary OA knee patients at two times with 48-hour interval in-between. Cronbach’s alpha, floor and ceiling effect, intraclass correlation coefficient (ICC, absolute agreement %, and Spearman correlation were used to fulfill our objectives. Results. Average time to administer this questionnaire was 20 minutes. There was good internal consistency with Cronbach’s alpha ranging from 0.7246 to 0.9139. The absolute agreement of each item between two tests ranged from 81.08% to 98.20%. Test-retest reliability was excellent (“r” ranged from 0.9673 to 0.9782. There was no ceiling effect; however less than 4% floor effect was seen in two subscales. There was significant difference that existed between different X-ray grades in all subscales meaning good content validity for disease prognosis. Conclusion. The present results show that KOOS Urdu version is a reliable and valid measure for primary OA knee patients.

  6. CPEB1 modulates lipopolysaccharide-mediated iNOS induction in rat primary astrocytes

    International Nuclear Information System (INIS)

    Kim, Ki Chan; Hyun Joo, So; Shin, Chan Young

    2011-01-01

    Highlights: → Expression and phosphorylation of CPEB1 is increased by LPS stimulation in rat primary astrocytes. → JNK regulates expression and phosphorylation of CPEB1 in reactive astrocytes. → Down-regulation of CPEB1 using siRNA inhibits oxidative stress and iNOS induction by LPS stimulation. → CPEB1 may play an important role in regulating inflammatory responses in reactive astrocytes induced by LPS. -- Abstract: Upon CNS damage, astrocytes undergo a series of biological changes including increased proliferation, production of inflammatory mediators and morphological changes, in a response collectively called reactive gliosis. This process is an essential part of the brains response to injury, yet much is unknown about the molecular mechanism(s) that induce these changes. In this study, we investigated the role of cytoplasmic polyadenylation element binding protein 1 (CPEB1) in the regulation of inflammatory responses in a model of reactive gliosis, lipopolysaccharide-stimulated astrocytes. CPEB1 is an mRNA-binding protein recently shown to be expressed in astrocytes that may play a role in astrocytes migration. After LPS stimulation, the expression and phosphorylation of CPEB1 was increased in rat primary astrocytes in a JNK-dependent process. siRNA-induced knockdown of CPEB1 expression inhibited the LPS-induced up-regulation of iNOS as well as NO and ROS production, a hallmark of immunological activation of astrocytes. The results from the study suggest that CPEB1 is actively involved in the regulation of inflammatory responses in astrocytes, which might provide new insights into the regulatory mechanism after brain injury.

  7. Involvement of muscarinic acetylcholine receptors in chloride secretion by cultured rat epididymal epithelium.

    Science.gov (United States)

    Du, Jian-Yang; Zuo, Wu-Lin; Chen, Min-Hui; Xiang, Hui; Zhou, Wen-Liang

    2006-09-01

    The aim of our present study was to investigate the short-circuit current response to carbachol in cultured rat cauda epididymal epithelia and the signal transduction mechanisms involved. Carbachol added basolaterally induced a concentration-dependent increase in short-circuit current (Isc) across the epididymal epithelium consisting of a rapidly rising phase and a long term sustained response. The response was almost abolished by removing Cl(-) from the extracellular medium and blockable by pretreating the tissues with DPC, indicating a substantial contribution of Cl(-) secretion to the carbachol-induced response. The muscarinic acetylcholine receptor antagonist atropine inhibited the response, but the nicotinic acetylcholine receptors antagonist curarine had no effect, suggesting that only the muscarinic acetylcholine receptors mediated the secretory response of the basolateral side of rat cauda epididymal epithelium to carbachol. Addition of carbachol to the apical side of the tissue was found not to elicit an Isc response. These results suggested that muscarinic receptors are present in the basolateral side of rat cauda epididymal epithelium. Activation of these receptors by acetylcholine released from the nerve endings regulates epididymal transepithelial Cl(-) secretion. Cholinergic stimulation therefore contributes to the formation of luminal fluid microenvironment.

  8. Cultured rat and purified human Pneumocystis carinii stimulate intra- but not extracellular free radical production in human neutrophils

    DEFF Research Database (Denmark)

    Jensen, T; Aliouat, E M; Lundgren, B

    1998-01-01

    The production of free radicals in human neutrophils was studied in both Pneumocystis carinii derived from cultures of L2 rat lung epithelial-like cells and Pneumocystis carinii purified from human lung. Using the cytochrome C technique, which selectively measured extracellular superoxide...... generation, hardly any free radical production was observed after stimulation with cultured rat-derived P. carinii. A chemiluminescence technique, which separately measured intra- and extracellular free radical production, was subsequently employed to differentiate the free radical generation....... It was established that 1) P. carinii stimulated intra- but not extracellular free radical production in human neutrophils, 2) opsonized cultured rat-derived P. carinii stimulated human neutrophils to a strong intracellular response of superoxide production, and 3) opsonized P. carinii, purified from human lung also...

  9. Isolated rat dental pulp cell culture and transplantation with an alginate scaffold.

    Science.gov (United States)

    Fujiwara, Shiro; Kumabe, Shunji; Iwai, Yasutomo

    2006-05-01

    Many studies have been conducted on tissue stem cells in the field of regenerative medicine, and cultured dental pulp mesenchymal cells have been reported to secrete dentin matrix. In the present study we used alginate as a scaffold to transplant subcultured rat dental-pulp-derived cells subcutaneously into the back of nude mice. We found that when beta-glycerophosphate was added to the culture medium, the mRNA of the dentin sialophosphoprotein (DSPP) gene coding dentin sialoprotein (DSP) and dentin phosphoprotein (DPP) was expressed, and an increase in alkaline phosphatase, an early marker of odontoblast differentiation, was also demonstrated. Six weeks after implantation, subcutaneous formation of radiopaque calcified bodies was observed in situ. Immunohistochemical and fine structure studies identified expression of type I collagen, type III collagen, and DSP in the mineralizing transplants, and isolated odontoblast-like cells began to form dentin-like hard tissue formation. Scattered autolyzing apoptotic cells were also observed in the transplants. The study showed that subcultured rat dental-pulp-derived cells actively differentiate into odontoblast-like cells and induce calcification in an alginate scaffold.

  10. Dose-response analysis of phthalate effects on gene expression in rat whole embryo culture.

    Science.gov (United States)

    Robinson, Joshua F; Verhoef, Aart; van Beelen, Vincent A; Pennings, Jeroen L A; Piersma, Aldert H

    2012-10-01

    The rat postimplantation whole embryo culture (WEC) model serves as a potential screening tool for developmental toxicity. In this model, cultured rat embryos are exposed during early embryogenesis and evaluated for morphological effects. The integration of molecular-based markers may lead to improved objectivity, sensitivity and predictability of WEC in assessing developmental toxic properties of compounds. In this study, we investigated the concentration-dependent effects of two phthalates differing in potency, mono(2-ethylhexyl) phthalate (MEHP) and monomethyl phthalate (MMP, less toxic), on the transcriptome in WEC to examine gene expression in relation with dysmorphogenesis. MEHP was more potent than MMP in inducing gene expression changes as well as changes on morphology. MEHP induced significant enrichment of cholesterol/lipid/steroid (CLS) metabolism and apoptosis pathways which was associated with developmental toxicity. Regulation of genes within CLS metabolism pathways represented the most sensitive markers of MEHP exposure, more sensitive than classical morphological endpoints. As shown in direct comparisons with toxicogenomic in vivo studies, alterations in the regulation of CLS metabolism pathways has been previously identified to be associated with developmental toxicity due to phthalate exposure in utero. Our results support the application of WEC as a model to examine relative phthalate potency through gene expression and morphological responses. Additionally, our results further define the applicability domain of the WEC model for developmental toxicological investigations. Copyright © 2012 Elsevier Inc. All rights reserved.

  11. Ghrelin-Induced Enhancement of Vasopressin and Oxytocin Secretion in Rat Neurohypophyseal Cell Cultures.

    Science.gov (United States)

    Gálfi, M; Radács, M; Molnár, Zs; Budai, I; Tóth, G; Pósa, A; Kupai, K; Szalai, Z; Szabó, R; Molnár, H A; Gardi, J; László, Ferenc A; Varga, Cs

    2016-12-01

    The effects of ghrelin on vasopressin and oxytocin secretion were studied in 13-14-day cell cultures of isolated rat neurohypophyseal tissue. The vasopressin and oxytocin contents of the supernatant were determined by radioimmunoassay after a 1- or 2-h incubation. Significantly increased levels of vasopressin and oxytocin production were detected in the cell culture media following ghrelin administration, depending on the ghrelin doses. The oxytocin level proved to be more elevated than that of vasopressin. The increase of vasopressin and oxytocin secretion could be totally blocked by previous administration of the ghrelin receptor antagonist ([D-Lys 3 ]-growth hormone-releasing peptide-6). Application of the ghrelin receptor antagonist after ghrelin administration proved ineffective. The results indicate that vasopressin and oxytocin release is influenced directly by the ghrelin system, and the effects of ghrelin on vasopressin and oxytocin secretion from the neurohypophyseal tissue in rats can occur at the level of the posterior pituitary. Our observations lend support to the view that neurohypophysis contains ghrelin receptors.

  12. Effect of oxygen deprivation on metabolism of arachidonic acid by cultures of rat heart cells

    International Nuclear Information System (INIS)

    Freyss-Beguin, M.; Millanvoye-van Brussel, E.; Duval, D.

    1989-01-01

    To investigate the mechanisms responsible for the impairment of phospholipid metabolism observed in ischemic cells, we have studied the effect of conditions simulating ischemia on the metabolism of arachidonic acid (AA) by muscle (M-) and nonmuscle (F-) cells isolated from newborn rat hearts and cultured separately. In muscle cells, oxygen deprivation induces a significant stimulation of the release of [ 14 C]AA from prelabeled cells associated with a preferential redistribution of [ 14 C]AA into cell triglycerides but not formation of radioactive prostaglandins. Moreover, the fatty acid content of phospholipids, as measured by capillary gas chromatography, appears markedly reduced in ischemic myocardial cells. This fact may be related to phospholipase stimulation during ischemia as suggested by the antagonistic effect of mepacrine or p-bromophenacyl bromide. In contrast, oxygen deprivation failed to induce any significant alteration of AA metabolism in fibroblast-like heart cells. Our results indicate that these cultures of newborn rat heart cells, which exhibit many of the features observed in intact organ during ischemia, may represent a useful experimental model to investigate the pharmacological control of the membrane phospholipid turnover

  13. [The modulating role of essential and non-essential amino acids in organotypic tissue culture in rats of different ages].

    Science.gov (United States)

    Chalisova, N I; Penniiaĭnen, V A

    2003-05-01

    The effect of amino acids L-lysin, L-asparagin, L-arginin, L-glutamate was investigated in organotypic tissue culture of spleen, liver and brain cortex of rats at different age. The amino acids in concentration 0.05 ng/ml are active inducing a less intensive growth zone, as compared to control, in 1-day and in older rats--an intensive growth zone, as compared to control in 21-day rats. The data obtained suggest a modulating role of amino acids in the tissues at different stages of maturation.

  14. Signaling by TGF-betas in tubule cultures of adult rat testis.

    Science.gov (United States)

    Chan, Kai-Hui; Galuska, Sebastian P; Kudipudi, Pradeep Kumar; Riaz, Mohammad Assad; Loveland, Kate L; Konrad, Lutz

    2017-01-01

    Although signal transduction of transforming growth factor-betas (TGF-βs) is well characterized in individual cell types, data about TGF-β signaling in a cellular context is still scarce. In this study, we used ex vivo tubule cultures from adult rat testis to investigate TGF-β signaling. We show for the first time in testicular tubules, that TGF-βs also signal via the BMP type I receptors, with ALK2 used by TGF-β1 and ALK3 and ALK6 by TGF-β2. This signal transduction is mediated via Smad3 as well as via Smad1. In contrast, BMPs (BMP2 and BMP7) do not signal via the high-affinity type I and type II TGFβ receptors, TBR1 or TBR2. Furthermore, treatment of tubule cultures with either TGF-β1 or TGF-β2 had profound significant stimulatory effects on secretion of plasminogen activator-1 (PAI-1) through utilization of TGF-β and BMP receptors. Specific inhibitors for either TBR1 or BMP receptors yielded nearly complete inhibition of TGF-β signaling. The TBR1-TBR2 signalosome was detected with Duolink upon stimulation with either TGF-β1 or TGF-β2, predominantly in spermatogenic cells of the adult rat testis, particularly in elongated spermatids. In summary, this examination of intact rat testicular tubules demonstrated for the first time that TGF-βs signal mainly through TBR1 and TBR2 but also use BMP receptors, including for secretion of PAI-1. Whereas ALK2 participates in the TGF-β1-induced TBR1-TBR2 signalosome, ALK3 and ALK6 are involved in signaling of TGF-β2. Detection of the TBR1-TBR2 signalosome in late spermiogenic cells indicates a post-meiotic activity.

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

    Science.gov (United States)

    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…

  16. Designing a Primary Science Curriculum in a Globalizing World: How Do Social Constructivism and Vietnamese Culture Meet?

    Science.gov (United States)

    H?ng, Ngô Vu Thu; Meijer, Marijn Roland; Bulte, Astrid M. W.; Pilot, Albert

    2017-01-01

    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…

  17. Implementing guidelines and training initiatives to improve cross-cultural communication in primary care consultations: a qualitative participatory European study

    NARCIS (Netherlands)

    Teunissen, E.; Gravenhorst, K.; Dowrick, C.; Weel-Baumgarten, E.M. van; Driessen Mareeuw, F.A. van den; Brun, T. de; Burns, N.; Lionis, C.; Mair, F.S.; O'Donnell, C.; O'Reilly-de Brún, M.P.; Papadakaki, M.; Saridaki, A.; Spiegel, W.; Weel, C. van; Muijsenbergh, M.E.T.C. van den; Macfarlane, A.

    2017-01-01

    BACKGROUND: Cross-cultural communication in primary care is often difficult, leading to unsatisfactory, substandard care. Supportive evidence-based guidelines and training initiatives (G/TIs) exist to enhance cross cultural communication but their use in practice is sporadic. The objective of this

  18. Designing a primary science curriculum in a globalizing world: how do social constructivism and Vietnamese culture meet?

    NARCIS (Netherlands)

    Vu Thu Hang, N.; Meijer, M.R.; Bulte, A.M.W.; Pilot, A.

    2017-01-01

    The implementation of social constructivist approaches to learning science in primary education in Vietnamese culture as an example of Confucian heritage culture (CHC) remains challenging and problematic. This theoretical paper focuses on the initial phase of a design-based research approach; that

  19. Xenobiotic-Metabolizing Enzyme and Transporter Gene Expression in Primary Cultures of Human Hepatocytes Modulated by Toxcast Chemicals

    Science.gov (United States)

    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 ...

  20. Arctigenin Increases Hemeoxygenase-1 Gene Expression by Modulating PI3K/AKT Signaling Pathway in Rat Primary Astrocytes.

    Science.gov (United States)

    Jeong, Yeon-Hui; Park, Jin-Sun; Kim, Dong-Hyun; Kim, Hee-Sun

    2014-11-01

    In the present study, we found that the natural compound arctigenin inhibited hydrogen peroxide-induced reactive oxygen species (ROS) production in rat primary astrocytes. Since hemeoxygenase-1 (HO-1) plays a critical role as an antioxidant defense factor in the brain, we examined the effect of arctigenin on HO-1 expression in rat primary astrocytes. We found that arctigenin increased HO-1 mRNA and protein levels. Arctigenin also increases the nuclear translocation and DNA binding of Nrf2/c-Jun to the antioxidant response element (ARE) on HO-1 promoter. In addition, arctigenin increased ARE-mediated transcriptional activities in rat primary astrocytes. Further mechanistic studies revealed that arctigenin increased the phosphorylation of AKT, a downstream substrate of phosphatidylinositol 3-kinase (PI3K). Treatment of cells with a PI3K-specific inhibitor, LY294002, suppressed the HO-1 expression, Nrf2 DNA binding and ARE-mediated transcriptional activities in arctigenin-treated astrocyte cells. The results collectively suggest that PI3K/AKT signaling pathway is at least partly involved in HO-1 expression by arctigenin via modulation of Nrf2/ARE axis in rat primary astrocytes.

  1. The lining of the gut in the developing rat embryo. Its relation to the hypoblast (primary endoderm) and the notochord

    NARCIS (Netherlands)

    Lamers, W. H.; Spliet, W. G.; Langemeyer, R. A.

    1987-01-01

    A light microscopical study of the morphogenesis of the gut in the rat embryo was undertaken to provide a careful map of temporal changes in the topographical relations of the (definitive) endoderm, the notochord and the hypoblast (primary endoderm). The borderline between the (definitive) endoderm

  2. Multiple pathways of sigma(1) receptor ligand uptakes into primary cultured neuronal cells.

    Science.gov (United States)

    Yamamoto, H; Karasawa, J; Sagi, N; Takahashi, S; Horikomi, K; Okuyama, S; Nukada, T; Sora, I; Yamamoto, T

    2001-08-03

    Although many antipsychotics have affinities for sigma receptors, the transportation pathway of exogenous sigma(1) receptor ligands to intracellular type-1 sigma receptors are not fully understood. In this study, sigma(1) receptor ligand uptakes were studied using primary cultured neuronal cells. [(3)H](+)-pentazocine and [(3)H](R)-(+)-1-(4-chlorophenyl)-3-[4-(2-methoxyethyl)piperazin-1-yl]methyl-2-pyrrolidinone L-tartrate (MS-377), used as a selective sigma(1) receptor ligands, were taken up in a time-, energy- and temperature-dependent manner, suggesting that active transport mechanisms were involved in their uptakes. sigma(1) receptor ligands taken up into primary cultured neuronal cells were not restricted to agonists, but also concerned antagonists. The uptakes of these ligands were mainly Na(+)-independent. Kinetic analysis of [(3)H](+)-pentazocine and [(3)H]MS-377 uptake showed K(m) values (microM) of 0.27 and 0.32, and V(max) values (pmol/mg protein/min) of 17.4 and 9.4, respectively. Although both ligands were incorporated, the pharmacological properties of these two ligands were different. Uptake of [(3)H](+)-pentazocine was inhibited in the range 0.4-7.1 microM by all the sigma(1) receptor ligands used, including N,N-dipropyl-2-[4-methoxy-3-(2-phenylethoxy)phenyl]ethylamine monohydrochloride (NE-100), a selective sigma(1) receptor ligand. In contrast, the inhibition of [(3)H]MS-377 uptake was potently inhibited by haloperidol, characterized by supersensitivity (IC(50), approximately 2 nM) and was inhibited by NE-100 with low sensitivity (IC(50), 4.5 microM). Moreover, kinetic analysis revealed that NE-100 inhibited [(3)H]MS-377 uptake in a noncompetitive manner, suggesting that NE-100 acted at a site different from the uptake sites of [(3)H]MS-377. These findings suggest that there are at least two uptake pathways for sigma(1) receptor ligands in primary cultured neuronal cells (i.e. a haloperidol-sensitive pathway and another, unclear, pathway). In

  3. The effect of supplementation of amino acids and taurine to modified KSOM culture medium on rat embryo development.

    Science.gov (United States)

    Nakamura, Kazuomi; Morimoto, Kayoko; Shima, Kaoru; Yoshimura, Yuki; Kazuki, Yasuhiro; Suzuki, Osamu; Matsuda, Junichiro; Ohbayashi, Tetsuya

    2016-11-01

    The rat is widely used as a laboratory animal for research. In particular, genetically engineered rats are essential for production of animal models of several diseases. Although embryo manipulation techniques are needed to produce them, such technology for rat preimplantation embryos is not as advanced as it is for mouse embryos. One reason is that in vitro culture systems for preimplantation embryos are limited in rats. Therefore, we intended to develop a new culture system for rat preimplantation embryos focusing on supplementation of amino acids as nutrition to the culture media. First, we found that taurine, glycine, glutamate, and alanine were abundant in the oviductal fluid of Wistar rats. The profile of taurine and these three amino acids was unchanged during the estrous cycle and from Days 0 to 3 of pregnancy (Day 0; vaginal plug was confirmed). Second, we assessed the effect of phosphate and phenol red on the development of rat zygotes and confirmed that they caused two-cell block. Third, we examined the effect of changing the medium on zygote development because addition of amino acids into culture medium causes ammonium accumulation, which is detrimental to embryo development. Blastocyst formation was suppressed in cultures with no medium change (P = 0.004; decreased to approximately one-fourth of that with medium change). Fourth, we examined the effect of supplementation of these three amino acids and taurine to modified potassium simplex optimized medium (KSOM). The zygote development rates were increased by the three amino acids and taurine in a concentration-dependent manner at 48, 72, and 96 hours (P = 0.001, 0.005, and 0.009, respectively) in culture. Finally, we confirmed that blastocysts cultured in modified KSOM had the capacity to develop to full term after implantation. These results showed that not only the supply of nutrients but also removal of wastes and toxicants is important for culture of rat preimplantation embryos. Copyright

  4. An imbalance in progenitor cell populations reflects tumour progression in breast cancer primary culture models

    LENUS (Irish Health Repository)

    Donatello, Simona

    2011-04-26

    Abstract Background Many factors influence breast cancer progression, including the ability of progenitor cells to sustain or increase net tumour cell numbers. Our aim was to define whether alterations in putative progenitor populations could predict clinicopathological factors of prognostic importance for cancer progression. Methods Primary cultures were established from human breast tumour and adjacent non-tumour tissue. Putative progenitor cell populations were isolated based on co-expression or concomitant absence of the epithelial and myoepithelial markers EPCAM and CALLA respectively. Results Significant reductions in cellular senescence were observed in tumour versus non-tumour cultures, accompanied by a stepwise increase in proliferation:senescence ratios. A novel correlation between tumour aggressiveness and an imbalance of putative progenitor subpopulations was also observed. Specifically, an increased double-negative (DN) to double-positive (DP) ratio distinguished aggressive tumours of high grade, estrogen receptor-negativity or HER2-positivity. The DN:DP ratio was also higher in malignant MDA-MB-231 cells relative to non-tumourogenic MCF-10A cells. Ultrastructural analysis of the DN subpopulation in an invasive tumour culture revealed enrichment in lipofuscin bodies, markers of ageing or senescent cells. Conclusions Our results suggest that an imbalance in tumour progenitor subpopulations imbalances the functional relationship between proliferation and senescence, creating a microenvironment favouring tumour progression.

  5. Effect of anabolics on bovine granulosa-luteal cell primary cultures.

    Directory of Open Access Journals (Sweden)

    Bartolomeo Biolatti

    2007-10-01

    Full Text Available Granulosa cell tumours are observed with increased frequency among calves slaughtered in Northern Italy. The use of illegal anabolics in breeding was taken into account as a cause of this pathology. An in vitro approach was used to detect the possible alterations of cell proliferation induced by anabolics on primary cultures of bovine granulosa-luteal cells. Cultures were treated with different concentrations of substances illegally used in cattle (17beta-estradiol, clenbuterol and boldione. Cytotoxicity was determined by means of MTT test, to exclude toxic effects induced by anabolics and to determine the highest concentration to be tested. Morphological changes were evaluated by means of routine cytology, while PCNA expression was quantified in order to estimate cell proliferation. Cytotoxic effects were revealed at the highest concentrations. The only stimulating effect on cell proliferation was detected in boldione treated cultures: after 48 h treated cells, compared to controls, showed a doubled expression of PCNA. In clenbuterol and 17beta-estradiol treated cells PCNA expression was similar to controls or even decreased. As the data suggest an alteration in cell proliferation, boldione could have a role in the early stage of pathogenesis of granulosa cell tumour in cattle.

  6. Epidermal growth factor in the rat prostate

    DEFF Research Database (Denmark)

    Tørring, Niels; Jørgensen, P E; Poulsen, Steen Seier

    1998-01-01

    Epidermal growth factor (EGF) induces proliferation in prostate epithelial and stromal cells in primary culture. This investigation was set up to characterize the time and spatial expression of EGF in the rat prostate....

  7. Epidural motor cortex stimulation suppresses somatosensory evoked potentials in the primary somatosensory cortex of the rat.

    Science.gov (United States)

    Chiou, Ruei-Jen; Lee, Hsiao-Yun; Chang, Chen-Wei; Lin, Kuan-Hung; Kuo, Chung-Chih

    2012-06-29

    Motor cortex stimulation (MCS) is a promising clinical procedure to help alleviate chronic pain. Animal models demonstrated that MCS is effective in lessening nocifensive behaviors. The present study explored the effects of MCS on cortical somatosensory evoked potentials (SEPs) recorded at the primary somatosensory cortex (SI) of the rat. SEPs were evoked by electrical stimulation applied to the contralateral forepaws. Effects of different intensities, frequencies, and durations of MCS were tested. MCS at ≥2V suppressed SEPs of the ipsilateral SI. Suppression lasted 120 min at an intensity of 5 V. The optimal frequency was 50 Hz, and the duration was 30s. In contrast, MCS did not affect SEPs recorded on the contralateral SI. Cortical stimulation out of the motor cortex did not induce a decrease in the ipsilateral SEPs. We also investigated involvement of the endogenous opioid system in this inhibition of SEPs induced by MCS. The opioid antagonist, naloxone (0.5 mg/kg), was administered 30 min before MCS. Application of naloxone completely prevented the inhibitory effect of MCS on ipsilateral SEPs. These results demonstrate that MCS blocked the transmission of somatosensory information to the primary somatosensory cortex, and this interference was mediated by the endogenous opioid system. This inhibitory effect on sensory transmission induced by MCS may reflect its antinociceptive effect. Copyright © 2012 Elsevier B.V. All rights reserved.

  8. Neuroprotective effect of chondroitinase ABC on primary and secondary brain injury after stroke in hypertensive rats.

    Science.gov (United States)

    Chen, Xin-ran; Liao, Song-jie; Ye, Lan-xiang; Gong, Qiong; Ding, Qiao; Zeng, Jin-sheng; Yu, Jian

    2014-01-16

    Focal cerebral infarction causes secondary damage in the ipsilateral ventroposterior thalamic nucleus (VPN). Chondroitin sulfate proteoglycans (CSPGs) are a family of putative inhibitory components, and its degradation by chondroitinase ABC (ChABC) promotes post-injury neurogenesis. This study investigated the role of ChABC in the primary and secondary injury post stroke in hypertension. Renovascular hypertensive Sprague-Dawley rats underwent middle cerebral artery occlusion (MCAO), and were subjected to continuous intra-infarct infusion of ChABC (0.12 U/d for 7 days) 24 h later. Neurological function was evaluated by a modified neurologic severity score. Neurons were counted in the peri-infarct region and the ipsilateral VPN 8 and 14 days after MCAO by Nissl staining and NeuN labeling. The expressions of CSPGs, growth-associated protein-43 (GAP-43) and synaptophysin (SYN) were detected with immunofluorescence or Western blotting. The intra-infarct infusion of ChABC, by degrading accumulated CSPGs, rescued neuronal loss and increased the levels of GAP-43 and SYN in both the ipsilateral cortex and VPN, indicating enhancd neuron survival as well as augmented axonal growth and synaptic plasticity, eventually improving overall neurological function. The study demonstrated that intra-infarct ChABC infusion could salvage the brain from both primary and secondary injury by the intervention on the neuroinhibitory environment post focal cerebral infarction. © 2013 Published by Elsevier B.V.

  9. Inhibition of Ras farnesylation by lovastatin leads to downregulation of proliferation and migration in primary cultured human glioblastoma cells.

    Science.gov (United States)

    Bouterfa, H L; Sattelmeyer, V; Czub, S; Vordermark, D; Roosen, K; Tonn, J C

    2000-01-01

    Malignant astrocytomas are the most common primary intracranial human tumors. All therapeutic approaches are limited due to their high proliferative capacity and their ability to diffusely invade the brain. Amplification of tyrosine kinase receptors and their signaling pathways have been implicated as contributing to the molecular pathogenesis of astrocytomas, providing possible new targets for therapeutic intervention. In particular, astrocytomas, although lacking oncogenic Ras mutations, have elevated levels of activated Ras. Lovastatin, an inhibitor of the beta-hydroxy-beta-methylglutary CoA reductase (HMG-CoA-reductase), is currently used to treat patients with hypercholesterolemia. In addition, it inhibits isoprenylation of several members of the Ras superfamily of proteins and therefore has multiple cellular effects including the reduction of proliferation. In this study, we investigated the impact of lovastatin on two human glioma cell lines and on 15 primary cell cultures established from biopsies of patients with glioblastoma multiforme (GBM,) Proliferation of glioma cell lines and primary tumor cells was determined by cell counting and by using the MTT assay. The cell morphology was analyzed by staining of actin filaments with phalloidin. Apoptosis was measured using the TUNEL assay. To investigate the influence of this drug on glioma cell motility, tumor cell migration was investigated using three dimensional spheroid disintegration assays. In addition, tumor cell invasion was analyzed with a confrontational assay between tumor spheroids and rat brain aggregates. Inhibition of farnesyl biosynthesis using lovastatin led to a block in Ras mediated signaling, indicated by lower MAPK activity. Consequently, tumor cell proliferation was reduced up to 80%. Lovastatin appeared to decrease glioma viability by inducing apoptosis, as indicated by morphological changes and increase of TUNEL positive cells. Lovastatin acts through isoprenoid depletion, because

  10. Synthesis and secretion of glucagon-like peptide-1 by fetal rat intestinal cells in culture.

    Science.gov (United States)

    Jackson Huang, T H; Brubaker, P L

    1995-07-01

    Secretion of the intestinal proglucagon-derived peptides (PGDPs) including the incretin glucagon-like peptide-1 (GLP-1) is regulated, at least in part, by the duodenal hormone glucose-dependent insulinotropic peptide (GIP) through a protein kinase (PK) A-dependent pathway. It has been demonstrated that the activation of PKA increases the synthesis of some intestinal PGDPs, particularly the glucagon-like immunoreactive (GLI) peptides glicentin and oxyntomodulin. However, the effects of GIP on GLI and GLP-1 synthesis are not known. Fetal rat intestinal cells in culture were therefore treated for up to 24 h with 5MM: dbcAMP or 10(-6) M: GIP and the changes in glicentin, oxyntomodulin, GLP-1(x-37) and GLP-1(x-36NH2) secretion and synthesis were examined by RIA and HPLC. Both dbcAMP and GIP increased the acute (2 h; to 224±21 and 256±20% of controls, respectively,P<0.001) and chronic (24 h; to 230±22 and 130±6% of controls, respectively,P<0.001) secretion of intestinal PGDPs. In contrast, the total culture content of PGDPs was increased only after 24 h of incubation (to 156±15 and 125±7% of controls for dbcAMP and GIP, respectively,P<0.01). HPLC analysis confirmed that the intestinal cultures produced the GLI peptides glicentin and oxyntomodulin, as well as the biologically active forms of GLP-1, GLP-7(7-37) and GLP-1(7-36NH2). The relative proportion of these peptides was not altered by treatment with dbcAMP or GIP. Thus, in addition to its effects on GLP-1 release from the rat intestine, GIP appears to be an important regulator of the synthesis of this insulinotropic peptide.

  11. Epidermal growth factor in the rat prostate

    DEFF Research Database (Denmark)

    Tørring, Niels; Jørgensen, P E; Poulsen, Steen Seier

    1998-01-01

    Epidermal growth factor (EGF) induces proliferation in prostate epithelial and stromal cells in primary culture. This investigation was set up to characterize the time and spatial expression of EGF in the rat prostate.......Epidermal growth factor (EGF) induces proliferation in prostate epithelial and stromal cells in primary culture. This investigation was set up to characterize the time and spatial expression of EGF in the rat prostate....

  12. Royal Jelly Prevents Osteoporosis in Rats: Beneficial Effects in Ovariectomy Model and in Bone Tissue Culture Model

    Directory of Open Access Journals (Sweden)

    Saburo Hidaka

    2006-01-01

    Full Text Available Royal jelly (RJ has been used worldwide for many years as medical products, health foods and cosmetics. Since RJ contains testosterone and has steroid hormone-type activities, we hypothesized that it may have beneficial effects on osteoporosis. We used both an ovariectomized rat model and a tissue culture model. Rats were divided into eight groups as follows: sham-operated (Sham, ovariectomized (OVX, OVX given 0.5% (w/w raw RJ, OVX given 2.0% (w/w RJ, OVX given 0.5% (w/w protease-treated RJ (pRJ, OVX given 2.0% (w/w pRJ, OVX given 17β-estradiol and OVX given its vehicle, respectively. The Ovariectomy decreased tibial bone mineral density (BMD by 24%. Administration of 17β-estradiol to OVX rats recovered the tibial BMD decrease by 100%. Administration of 2.0% (w/w RJ and 0.5–2.0% (w/w pRJ to OVX rats recovered it by 85% or more. These results indicate that both RJ and pRJ are almost as effective as 17β-estradiol in preventing the development of bone loss induced by ovariectomy in rats. In tissue culture models, both RJ and pRJ increased calcium contents in femoral-diaphyseal and femoral-metaphyseal tissue cultures obtained from normal male rats. However, in a mouse marrow culture model, they neither inhibited the parathyroid hormone (PTH-induced calcium loss nor affected the formation of osteoclast-like cells induced by PTH in mouse marrow culture system. Therefore, our results suggest that both RJ and pRJ may prevent osteoporosis by enhancing intestinal calcium absorption, but not by directly antagonizing the action of PTH.

  13. Influence of flow conditions and matrix coatings on growth and differentiation of three-dimensionally cultured rat hepatocytes.

    Science.gov (United States)

    Fiegel, Henning C; Havers, Joerg; Kneser, Ulrich; Smith, Molly K; Moeller, Tim; Kluth, Dietrich; Mooney, David J; Rogiers, Xavier; Kaufmann, Peter M

    2004-01-01

    Maintenance of liver-specific function of hepatocytes in culture is still difficult. Improved culture conditions may enhance the cell growth and function of cultured cells. We investigated the effect of three-dimensional culture under flow conditions, and the influence of surface modifications in hepatocyte cultures. Hepatocytes were harvested from Lewis rats. Cells were cultured on three-dimensional polymeric poly-lactic-co-glycolic acid (PLGA) matrices in static culture, or in a pulsatile flow-bioreactor system. Different surface modifications of matrices were investigated: coating with collagen I, collagen IV, laminin, or fibronectin; or uncoated matrix. Hepatocyte numbers, DNA content, and albumin secretion rate were assessed over the observation period. Culture under flow condition significantly enhanced cell numbers. An additional improvement of this effect was observed, when matrix coating was used. Cellular function also showed a significant increase (4- to 5-fold) under flow conditions when compared with static culture. Our data showed that culture under flow conditions improves cell number, and strongly enhances cellular function. Matrix modification by coating with extracellular matrix showed overall an additive stimulatory effect. Our conclusion is that combining three-dimensional culture under flow conditions and using matrix modification significantly improves culture conditions and is therefore attractive for the development of successful culture systems for hepatocytes.

  14. Development and validation of primary human myometrial cell culture models to study pregnancy and labour

    Directory of Open Access Journals (Sweden)

    Mosher Andrea A

    2013-01-01

    Full Text Available Abstract Background The development of the in vitro cell culture model has greatly facilitated the ability to study gene expression and regulation within human tissues. Within the human uterus, the upper (fundal segment and the lower segment may provide distinct functions throughout pregnancy and during labour. We have established primary cultured human myometrial cells, isolated from both upper and lower segment regions of the pregnant human uterus, and validated them for the purpose of studying human pregnancy and labour. The specific objectives of this study were to monitor the viability and characterize the expression profile using selected cellular, contractile and pregnancy associated markers in the primary cultured human myometrial cells. Labour has been described as an inflammatory process; therefore, the ability of these cells to respond to an inflammatory stimulus was also investigated. Methods Myometrial cells isolated from paired upper segment (US and lower segment (LS biopsies, obtained from women undergoing Caesarean section deliveries at term prior to the onset of labour, were used to identify expression of; α smooth muscle actin, calponin, caldesmon, connexin 43, cyclo-oxygenase-2 (COX-2, oxytocin receptor, tropomyosin and vimentin, by RT-PCR and/or immunocytochemistry. Interleukin (IL-1β was used to treat cells, subsequently expression of COX-2 mRNA and release of interleukin-8 (CXCL8, were measured. ANOVA followed by Bonferroni’s multiple comparisons test was performed. Results We demonstrate that US and LS human myometrial cells stably express all markers examined to at least passage ten (p10. Connexin 43, COX-2 and vimentin mRNA expression were significantly higher in LS cells compared to US cells. Both cell populations respond to IL-1β, demonstrated by a robust release of CXCL8 and increased expression of COX-2 mRNA from passage one (p1 through to p10. Conclusions Isolated primary myometrial cells maintain expression of

  15. Astrocytes contribute to the effects of etomidate on synaptic transmission in rat primary somatosensory cortex.

    Science.gov (United States)

    Yang, Hao; Wang, Yuan; Zhang, Yu; Zhang, You; Xu, Mao-Sheng; Yuan, Jie; Yu, Tian

    2016-07-01

    Little is known about the mechanisms of unconsciousness induced by general anesthetics. Previous studies have shown that the primary somatosensory cortex (S1) is a sensitive region to a variety of intravenous general anesthetics. Etomidate is a widely used intravenous anesthetic that can influence synaptic transmission. Recently, there are some evidences suggesting that astrocytes, a type of glia cell, also contribute to information transmission in the brain, and modulate synaptic function by releasing neuroactive substances. However, it is unknown whether astrocytes influence the effects of etomidate on information transmission in S1 pyramidal neurons. In the present study, the role of astrocytes in etomidate-induced unconsciousness was investigated by using the whole-cell patch clamp technique. We observed etomidate at clinically relevant concentrations inhibited the spontaneous postsynaptic currents (sPSCs) of rat S1 pyramidal neurons in a concentration-dependent manner, and the EC50 value of etomidate for inhibiting sPSCs from the concentration-effect curve was 6.9μM. Furthermore, in the presence of fluorocitrate, a glia-selective metabolism inhibitor that blocks the aconitase enzyme, both the amplitude and frequency of sPSCs in rat S1 pyramidal neurons were reduced, and the inhibitory effects of etomidate on sPSCs amplitude was strengthened without affecting the effects of etomidate on frequency. From these data, we deduce that etomidate suppresses synaptic activity via presynaptic and postsynaptic components. Furthermore, astrocytes participate in synaptic transmission and influence the effects of etomidate on postsynaptic receptors. This study provides new insight into the role of astrocytes in etomidate-induced unconsciousness. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. Oxidative stress is involved in Dasatinib-induced apoptosis in rat primary hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Xue, Tao; Luo, Peihua; Zhu, Hong; Zhao, Yuqin [Institute of Pharmacology and Toxicology, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058 (China); Wu, Honghai; Gai, Renhua; Wu, Youping [Center for Drug Safety Evaluation and Research of Zhejiang University, Hangzhou 310058 (China); Yang, Bo [Institute of Pharmacology and Toxicology, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058 (China); Yang, Xiaochun, E-mail: yangxiaochun@zju.edu.cn [Institute of Pharmacology and Toxicology, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058 (China); Center for Drug Safety Evaluation and Research of Zhejiang University, Hangzhou 310058 (China); He, Qiaojun, E-mail: qiaojunhe@zju.edu.cn [Institute of Pharmacology and Toxicology, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058 (China); Center for Drug Safety Evaluation and Research of Zhejiang University, Hangzhou 310058 (China)

    2012-06-15

    Dasatinib, a multitargeted inhibitor of BCR–ABL and SRC kinases, exhibits antitumor activity and extends the survival of patients with chronic myeloid leukemia (CML) and Philadelphia chromosome-positive acute lymphoblastic leukemia (ALL). However, some patients suffer from hepatotoxicity, which occurs through an unknown mechanism. In the present study, we found that Dasatinib could induce hepatotoxicity both in vitro and in vivo. Dasatinib reduced the cell viability of rat primary hepatocytes, induced the release of alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) in vitro, and triggered the ballooning degeneration of hepatocytes in Sprague–Dawley rats in vivo. Apoptotic markers (chromatin condensation, cleaved caspase-3 and cleaved PARP) were detected to indicate that the injury induced by Dasatinib in hepatocytes in vitro was mediated by apoptosis. This result was further validated in vivo using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assays. Here we found that Dasatinib dramatically increased the level of reactive oxygen species (ROS) in hepatocytes, reduced the intracellular glutathione (GSH) content, attenuated the activity of superoxide dismutase (SOD), generated malondialdehyde (MDA), a product of lipid peroxidation, decreased the mitochondrial membrane potential, and activated nuclear factor erythroid 2-related factor 2 (Nrf2) and mitogen-activated protein kinases (MAPK) related to oxidative stress and survival. These results confirm that oxidative stress plays a pivotal role in Dasatinib-mediated hepatotoxicity. N-acetylcysteine (NAC), a typical antioxidant, can scavenge free radicals, attenuate oxidative stress, and protect hepatocytes against Dasatinib-induced injury. Thus, relieving oxidative stress is a viable strategy for reducing Dasatinib-induced hepatotoxicity. -- Highlights: ►Dasatinib shows potential hepatotoxicity both in vitro and in vivo. ►Apoptosis plays a vital role in Dasatinib

  17. Neonicotinoid Insecticides Alter the Gene Expression Profile of Neuron-Enriched Cultures from Neonatal Rat Cerebellum.

    Science.gov (United States)

    Kimura-Kuroda, Junko; Nishito, Yasumasa; Yanagisawa, Hiroko; Kuroda, Yoichiro; Komuta, Yukari; Kawano, Hitoshi; Hayashi, Masaharu

    2016-10-04

    Neonicotinoids are considered safe because of their low affinities to mammalian nicotinic acetylcholine receptors (nAChRs) relative to insect nAChRs. However, because of importance of nAChRs in mammalian brain development, there remains a need to establish the safety of chronic neonicotinoid exposures with regards to children's health. Here we examined the effects of longterm (14 days) and low dose (1 μM) exposure of neuron-enriched cultures from neonatal rat cerebellum to nicotine and two neonicotinoids: acetamiprid and imidacloprid. Immunocytochemistry revealed no differences in the number or morphology of immature neurons or glial cells in any group versus untreated control cultures. However, a slight disturbance in Purkinje cell dendritic arborization was observed in the exposed cultures. Next we performed transcriptome analysis on total RNAs using microarrays, and identified significant differential expression (p neonicotinoid exposure alters the transcriptome of the developing mammalian brain in a similar way to nicotine exposure. Our results highlight the need for further careful investigations into the effects of neonicotinoids in the developing mammalian brain.

  18. The effects of beryllium metal particles on the viability and function of cultured rat alveolar macrophages

    International Nuclear Information System (INIS)

    Finch, G.L.; Lowther, W.T.; Hoover, M.D.; Brooks, A.L.

    1988-01-01

    Rat pulmonary alveolar macrophages (PAM) were exposed in vitro to beryllium metal particles. The particles used were relatively large (Be-II) and small (Be-V) size fractions of beryllium metal obtained from an aerosol cyclone, and a beryllium metal aerosol generated by laser vaporization of beryllium metal in an argon atmosphere (Be-L). Glass beads (GB) were used as a negative control particle. The endpoints examined included cell killing (trypan blue dye exclusion) and phagocytic ability (sheep red blood cell uptake). Phagocytic ability was inhibited by beryllium particles at concentrations that did not cause appreciable cell killing. Results based on the mass concentration of particles in culture medium were transformed by the amount of specific surface area of the particles to permit expression of toxicity on the basis of amount of surface area of particles per unit volume of culture medium. On a mass concentration basis, the order of cytotoxicity was Be-L > Be-V ∼ Be-II > GB; for inhibition of phagacytosis, the cytotoxicity order was Be-L ∼ Be-V > Be-II > GB. On a surface area concentration basis, the order of toxicity for viability was altered to Be-II > Be-L ∼ Be-V (with GB indeterminant) and to Be-V > Be-II ∼ Be-L > GB for inhibition of phagocytosis. We conclude that there are factors in addition to specific surface area that influence the expression of toxic effects in cultured PAM. (author)

  19. Ethanol-induced cell damage in cultured rat antral mucosa assessed by chromium-51 release

    International Nuclear Information System (INIS)

    Sewell, R.B.; Ling, T.S.; Yeomans, N.D.

    1986-01-01

    We have developed an in vitro method for studying ethanol-induced injury to gastric mucosa using organ culture of rat antrum. Cell damage was assessed by measurement of the release of [ 51 Cr]sodium chromate from preloaded cells, a method adapted from a standard immunologic technique. This system provided rapid and highly reproducible quantitation of tissue injury as assessed by 51 Cr release into the culture medium. The threshold concentration for ethanol-induced damage was between 10 and 15% v/v, similar to in vivo thresholds observed by others. 51 Cr release could also be induced by very short exposure to ethanol (5-15 min), and then continued despite ethanol removal. Interestingly, after continuous ethanol exposure, a plateau of maximum 51 Cr release was reached 60 min after exposure to ethanol over the concentration range 20-50%, suggesting tissue adaptation to ethanol damage. This organ culture system, which allows precise control of experimental conditions, may be useful for studying mechanisms of gastric mucosal injury and protection

  20. Endocytosis of heat-denatured albumin by cultured rat Kupffer cells

    International Nuclear Information System (INIS)

    Brouwer, A.; Knook, D.L.

    1982-01-01

    Purified Kupffer cells were obtained by centrifugal elutriation of sinusoidal cells isolated by pronase treatment of the rat liver. The endocytosis of radioactively labeled heat-aggregated colloidal albumin (CA 125 I) was investigated in maintenance cultures of the purified Kupffer cells. The endocytic capacity of the cells was studied during 4 days of culture. Maximum uptake was observed after 24 hr of culture, with a gradual decline during the following days. When the uptake was measured after incubation with increasing concentrations of CA 125 I, a saturation effect was observed. This finding and the observed high rate of uptake are strong indications that receptor sites on the cell membrane are involved in the mechanism of endocytosis. The uptake of CA 125 I by Kupffer cells was inhibited by the metabolic inhibitors fluoride and antimycin A, indicating that endocytosis of CA 125 I is dependent on energy derived from both glycolysis and mitochondrial respiration. The mechanism of internalization may also require the action of microfilaments as well as intact microtubules, since both cytochalasin B and colchicine inhibited the uptake of CA 125 I. The intracellular degradation of CA 125 I by Kupffer cells was strongly inhibited by chloroquine but not by colchicine. The degradation of ingested CA 125 I occurred within the Kupffer cell lysosomes

  1. Cultured Human Epidermis Combined With Meshed Skin Autografts Accelerates Epithelialization and Granulation Tissue Formation in a Rat Model.

    Science.gov (United States)

    Sakamoto, Michiharu; Morimoto, Naoki; Inoie, Masukazu; Takahagi, Miki; Ogino, Shuichi; Jinno, Chizuru; Suzuki, Shigehiko

    2017-06-01

    As the take rate of cultured epidermal autografts in burn wound treatment is variable, widely expanded meshed auto skin grafts are often used in combination with cultured epidermal autograft to increase the take rate and achieve definitive wound coverage. However, a long time (3-4 weeks) required to prepare a cultured epidermis sheet is a disadvantage. Allogeneic cultured epidermis can be prepared in advance and cryopreserved to be used in combination with auto meshed skin grafts for treating third-degree burns. Nevertheless, the human cultured epidermis (hCE) has not been proved to accelerate wound healing after meshed skin grafting. Here, we investigated the effect of hCE on wound healing in a rat model of meshed skin grafting. Human cultured epidermis was prepared from human neonatal foreskin and assessed by the release of growth factors into the culture medium using enzyme-linked immunosorbent assay. Skin wounds were inflicted on male F344 rats and treated by the application of widely meshed (6:1 ratio) autogenous skin grafts with or without hCE (n = 8 rats per group). Wound area, neoepithelium length, granulation tissue formation, and neovascularization were evaluated on day 7 postgrafting. Human cultured epidermis secreted IL-1α, Basic fibroblast growth factor, platelet-derived growth factor-AA, TGF-α, TGF-β1, and vascular endothelial growth factor in vitro. In rats, hCE accelerated wound closure (P = 0.003), neoepithelium growth (P = 0.019), and granulation tissue formation (P = 0.043), and increased the number of capillaries (P = 0.0003) and gross neovascularization area (P = 0.008) compared with the control group. The application of hCE with meshed grafts promoted wound closure, possibly via secretion of growth factors critical for cell proliferation and migration, suggesting that hCE can enhance the healing effect of widely expanded skin autografts.

  2. The stable prostacyclin-analogue, iloprost, unlike prostanoids and leukotrienes, potently stimulates cyclic adenosine monophosphate synthesis of primary astroglial cell cultures.

    Science.gov (United States)

    Seregi, A; Schobert, A; Hertting, G

    1988-06-01

    The effect of different eicosanoids on adenosine-3', 5'-cyclic-monophosphate (cAMP) accumulation in primary astroglial cell cultures prepared from newborn rat brain was studied. The stable prostacyclin-analogue, iloprost, effectively stimulated cAMP synthesis in a concentration-dependent, saturable manner, the EC50 being about 3 x 10(-8) M. Prostaglandin (PG) E2 was less potent, without reaching plateau even at 10(-5) M. Prostaglandins D2 and F2 alpha, and the stable thromboxane A2-analogue, U 46619, as well as leukotrienes (LT) B4, C4, D4 and E4 were not effective and did not attenuate basal or isoprenaline (10(-8) M)-stimulated astroglial cAMP formation. This is the first indication for the existence of a prostacyclin receptor coupled positively to the adenylate cyclase in astrocytes. Other eicosanoids are unlikely to be involved in receptor-mediated regulation of astroglial cAMP levels.

  3. Unimodal primary sensory cortices are directly connected by long-range horizontal projections in the rat sensory cortex

    Directory of Open Access Journals (Sweden)

    Jimmy eStehberg

    2014-09-01

    Full Text Available Research based on functional imaging and neuronal recordings in the barrel cortex subdivision of primary somatosensory cortex (SI of the adult rat has revealed novel aspects of structure-function relationships in this cortex. Specifically, it has demonstrated that single whisker stimulation evokes subthreshold neuronal activity that spreads symmetrically within gray matter from the appropriate barrel area, crosses cytoarchitectural borders of SI and reaches deeply into other unimodal primary cortices such as primary auditory (AI and primary visual (VI. It was further demonstrated that this spread is supported by a spatially matching underlying diffuse network of border-crossing, long-range projections that could also reach deeply into AI and VI. Here we seek to determine whether such a network of border-crossing, long-range projections is unique to barrel cortex or characterizes also other primary, unimodal sensory cortices and therefore could directly connect them. Using anterograde (BDA and retrograde (CTb tract-tracing techniques, we demonstrate that such diffuse horizontal networks directly and mutually connect VI, AI and SI. These findings suggest that diffuse, border-crossing axonal projections connecting directly primary cortices are an important organizational motif common to all major primary sensory cortices in the rat. Potential implications of these findings for topics including cortical structure-function relationships, multisensory integration, functional imaging and cortical parcellation are discussed.

  4. Ontogeny of the serotonergic projection to rat neocortex: transient expression of a dense innervation to primary sensory areas

    Energy Technology Data Exchange (ETDEWEB)

    D' Amato, R.J.; Blue, M.E.; Largent, B.L.; Lynch, D.R.; Ledbetter, D.J.; Molliver, M.E.; Snyder, S.H.

    1987-06-01

    The development of serotonergic innervation to rat cerebral cortex was characterized by immunohistochemical localization of serotonin combined with autoradiographic imaging of serotonin-uptake sites. In neonatal rat, a transient, dense, serotonergic innervation appears in all primary sensory areas of cortex. In somatosensory cortex, dense patches of serotonergic innervation are aligned with specialized cellular aggregates called barrels. The dense patches are not apparent after 3 weeks of age, and the serotonergic innervation becomes more uniform in adult neocortex. This precocious neonatal serotonergic innervation may play a transient physiologic role in sensory areas of cortex or may exert a trophic influence on the development of cortical circuitry and thalamocortical connections.

  5. Novel relationships of growth factors to the G1/S transition in cultured astrocytes from rat forebrain.

    Science.gov (United States)

    Langan, T J; Slater, M C; Kelly, K

    1994-01-01

    The cell cycle encompasses the sequential events regulating cell division. In mammalian brain, initiation of astrocyte cycling is critical during development and injury. To investigate the timing of growth factor requirements as they commit to passing through the G1 phase, primary and secondary rat astrocytes were stimulated to enter the cycle after serum or growth factor deprivation. Bromodeoxyuridine immunofluorescence was used to monitor S phase nuclei after growth factor re-addition (at time 0). Cycle kinetics were identical whether quiescent cultures were exposed to 10% (vol/vol) calf serum, or to a defined medium containing fibroblast growth factor, insulin, and epidermal growth factor. The control point in late G1 that represents commitment to achieving the G1/S transition was identified by cycloheximide (CHX, 0.1 microgram/ml) addition. Sensitivity to cycle arrest by CHX disappeared at 9-10 h. In contrast, shift-down to growth factor-deficient medium arrested cell cycling virtually until G1/S (12 h). With selective exposure during late G1 (9-12 h), no single agent permitted cycle progression. However, any two agents enabled cycling, and complementary or synergistic effects were apparent. These requirements were identical in astroglia from newborn and long-term cultures. Thus, temporal dissociation exists between the processes of escape from CHX sensitivity and from requirements for growth factors, two recognized hallmarks of commitment to cycle progression. Furthermore, simultaneous presence of at least two growth factors is necessary at or near G1/S. Both findings distinguish astrocytes from several other cell types.

  6. DEVELOPMENT OF PRIMARY CELL CULTURE FROM TAIL EPIDERMAL TISSUE OF KOI CARP (Cyprinus carpio koi)

    OpenAIRE

    Lila Gardenia; Isti Koesharyani

    2014-01-01

    Primary cell culture from tail epidermal tissue of koi carp (Cyprinus carpio koi) was developed. Cells were grown in Leibovits-15 medium supplemented with 20% fetal bovine serum and antibiotics (Penicillin/Streptomycin and Kanamycin). Cell growth was observed in a range of incubation temperature (17oC±2oC, 22oC±2oC, 27oC±2oC, and 32oC±2oC) in order to determine the optimum temperature. The cells were able to grow at a range of temperature between 17oC to 32oC with optimal growth at 22oC. Prim...

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

    Directory of Open Access Journals (Sweden)

    Marcos de Assis Moura

    2009-09-01

    Full Text Available 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.

  8. Primary neuron culture for nerve growth and axon guidance studies in zebrafish (Danio rerio.

    Directory of Open Access Journals (Sweden)

    Zheyan Chen

    Full Text Available Zebrafish (Danio rerio is a widely used model organism in genetics and developmental biology research. Genetic screens have proven useful for studying embryonic development of the nervous system in vivo, but in vitro studies utilizing zebrafish have been limited. Here, we introduce a robust zebrafish primary neuron culture system for functional nerve growth and guidance assays. Distinct classes of central nervous system neurons from the spinal cord, hindbrain, forebrain, and retina from wild type zebrafish, and fluorescent motor neurons from transgenic reporter zebrafish lines, were dissociated and plated onto various biological and synthetic substrates to optimize conditions for axon outgrowth. Time-lapse microscopy revealed dynamically moving growth cones at the tips of extending axons. The mean rate of axon extension in vitro was 21.4±1.2 µm hr(-1 s.e.m. for spinal cord neurons, which corresponds to the typical ∼0.5 mm day(-1 growth rate of nerves in vivo. Fluorescence labeling and confocal microscopy demonstrated that bundled microtubules project along axons to the growth cone central domain, with filamentous actin enriched in the growth cone peripheral domain. Importantly, the growth cone surface membrane expresses receptors for chemotropic factors, as detected by immunofluorescence microscopy. Live-cell functional assays of axon extension and directional guidance demonstrated mammalian brain-derived neurotrophic factor (BDNF-dependent stimulation of outgrowth and growth cone chemoattraction, whereas mammalian myelin-associated glycoprotein inhibited outgrowth. High-resolution live-cell Ca(2+-imaging revealed local elevation of cytoplasmic Ca(2+ concentration in the growth cone induced by BDNF application. Moreover, BDNF-induced axon outgrowth, but not basal outgrowth, was blocked by treatments to suppress cytoplasmic Ca(2+ signals. Thus, this primary neuron culture model system may be useful for studies of neuronal development

  9. Conditions for the primary culture of eye imaginal discs from Drosophila melanogaster.

    Science.gov (United States)

    Li, C; Meinertzhagen, I A

    1995-11-01

    We have established a primary culture system for Drosophila eye imaginal discs. With this system, we were able to obtain neurite outgrowth from intact eye discs, eye disc fragments, and dissociated eye imaginal disc cells. Immunoreactivity to antibody 24B10 indicates that these extending neurites are photoreceptor axons. Three culture media were tested for their ability to support the survival of and neurite extension from eye disc fragments in vitro at 23 degrees C. These, with supplements, were: five parts of Schneider's Drosophila medium with four parts of basal Eagle's medium ("4 + 5"); Leibovitz's L-15 medium (L-15); and Shields and Sang's M3 modified medium (MM3). We obtained the best results with MM3 supplemented with 2% fetal bovine serum (FBS). Eye disc fragments survived in this medium for at least 20 days. Pigmentation in the nonphotoreceptor pigment cells in cultures from the prepupa required the presence of 20-hydroxyecdysone (20-HE) (1 micrograms/ml), whereas neurite outgrowth was seen in the absence of 20-HE. Donor animals had to fall within a range of ages to obtain appropriate eye disc differentiation in vitro. Eye disc from 5-h pupae (P + 5) or older commenced ommachrome synthesis in vitro in a temporal sequence close to that found in vivo, whereas the in vitro synthesis of this pigment was delayed in eye discs from younger flies. Average neurite length was not affected by age among pupae younger than P + 5; but neurite outgrowth from P + 24 was scarce, probably because by this time photoreceptor axons had already grown in vivo and were severed and unable to regenerate in vitro. Eye discs taken from third instar larvae or white prepupae continued their mitotic activity in vitro. Together with the advance of the morphogenetic furrow at the leading edge of retinal development, this observation is consistent with the evidence that pattern formation continues in vitro. Morphogenetic changes were manifested in cultures. Viability tests with calcein AM

  10. Caffeine prevents bilirubin-induced cytotoxicity in cultured newborn rat astrocytes.

    Science.gov (United States)

    Deliktaş, Mehmet; Ergin, Hacer; Demiray, Aydın; Akça, Hakan; Özdemir, Özmert M A; Özdemir, Mehmet Bülent

    2018-01-02

    Unconjugated bilirubin (UCB) may cause neurotoxicity in preterm neonates due to immaturity of UGT1A1 leading to bilirubin accumulation in the brain. Caffeine used in the treatment of apnea of prematurity was reported to decrease mechanical ventilation requirement, the frequencies of bronchopulmonary dysplasia, patent ductus arteriosus, cerebral palsy and neurodevelopmental disorders in very low birth weight infants. However, the effect of caffeine on hyperbilirubinemia was not yet clarified. We used astrocyte cell cultures obtained from 2-day-old Wistar albino rats via modified Cole and de Vellis method. UCB concentration toxic to 50% of astrocytes, and caffeine concentration increasing cell viability 100% were used in experiments. While no medication was applied to the control group, UCB (50 μM) and caffeine (100 μM) were applied to the bilirubin and caffeine groups for 24 h. Prophylactic and therapeutic caffeine groups were treated with caffeine 4 h before and after UCB exposure. The effects of caffeine were investigated in rat astrocytes exposed to UCB in terms of cell viability, apoptosis, antioxidant defense, proinflammatory cytokines, and Toll-like receptor (TLR)s. Compared to the control group, UCB increased apoptosis, malondialdehyde (MDA), tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6, total nitrate/nitrite, and TLR4 levels, and decreased cell viability, catalase (CAT), glutathione peroxidase (GPx), superoxide dismutase (SOD) activities, glutathione, and TLR9 levels (for all p < .001). Conversely, prophylactic and therapeutic caffeine improved the detrimental effects of UCB. Caffeine seems encouraging for the prevention and treatment of bilirubin neurotoxicity in rats by means of its antiapoptotic, antioxidant, anti-inflammatory, anti-nitrosative, and anti-TLR-4 properties.

  11. Graft of autologous fibroblasts in gingival tissue in vivo after culture in vitro. Preliminary study on rats.

    Science.gov (United States)

    Simain-Sato, F; Lahmouzi, J; Heinen, E; Defresne, M P; De Pauw-Gillet, M C; Grisar, T; Legros, J J; Legrand, R

    1999-08-01

    Several grafting techniques and guided tissue regeneration techniques (GTR) have been well-developed in periodontal surgery. However, these techniques could induce pain and side effects, such as a gingival recession during the healing period following the therapy. The graft of a small autologous connective tissue, using non-invasive surgical techniques could yield several benefits for the patients. Our preliminary study explores the feasibility of collecting healthy gingival tissues, culturing them in vitro to amplify rat gingival fibroblasts (RGF) and inoculating the obtained cells into autologous rat gingival tissues in vivo. Gingival tissues samples were cultured as explants as described by Freshney et al. and Adolphe. Confluent cells surrounding explants were detached after 7 d of culture from Petri dishes using 0.05% trypsin and designated "first transferred cells" (T1). At the third passage (T3), cells cultured as monolayer were either examined under microscopy--phase contrast, scanning, or transmission electron--or numerated after trypan blue exclusion test. Autologous RGF labelled with fluorochrome were inoculated at the vestibular and palatine site of gingival tissue close to the superior incisors. In this preliminary study, 12 Wistar rats were used; for each, 2 biopsies were dissected and fixed for phase contrast or fluorescence microscopy. On d 1, 3 and 7 after injection in rat gingival tissues, fluorochrome-labelled cells could be detected in all these.

  12. Biosynthesis of the D2 cell adhesion molecule: pulse-chase studies in cultured fetal rat neuronal cells

    DEFF Research Database (Denmark)

    Lyles, J M; Norrild, B; Bock, E

    1984-01-01

    D2 is a membrane glycoprotein that is believed to function as a cell adhesion molecule (CAM) in neural cells. We have examined its biosynthesis in cultured fetal rat brain neurones. We found D2-CAM to be synthesized initially as two polypeptides: Mr 186,000 (A) and Mr 136,000 (B). With increasing...

  13. Na(+), K(+)-ATPase dysfunction causes cerebrovascular endothelial cell degeneration in rat prefrontal cortex slice cultures.

    Science.gov (United States)

    Kurauchi, Yuki; Hisatsune, Akinori; Seki, Takahiro; Katsuki, Hiroshi

    2016-08-01

    Cerebrovascular endothelial cell dysfunction resulting in imbalance of cerebral blood flow contributes to the onset of psychiatric disorders such as depression, schizophrenia and bipolar disorder. Although decrease in Na(+), K(+)-ATPase activity has been reported in the patients with schizophrenia and bipolar disorder, the contribution of Na(+), K(+)-ATPase to endothelial cell dysfunction remains poorly understood. Here, by using rat neonatal prefrontal cortex slice cultures, we demonstrated that pharmacological inhibition of Na(+), K(+)-ATPase by ouabain induced endothelial cell injury. Treatment with ouabain significantly decreased immunoreactive area of rat endothelial cell antigen-1 (RECA-1), a marker of endothelial cells, in a time-dependent manner. Ouabain also decreased Bcl-2/Bax ratio and phosphorylation level of glycogen synthase kinase 3β (GSK3β) (Ser9), which were prevented by lithium carbonate. On the other hand, ouabain-induced endothelial cell injury was exacerbated by concomitant treatment with LY294002, an inhibitor of phosphoinositide 3- (PI3-) kinase. We also found that xestospongin C, an inhibitor of inositol triphosphate (IP3) receptor, but not SEA0400, an inhibitor of Na(+), Ca(2+) exchanger (NCX), protected endothelial cells from cytotoxicity of ouabain. These results suggest that cerebrovascular endothelial cell degeneration induced by Na(+), K(+)-ATPase inhibition resulting in Ca(2+) release from endoplasmic reticulum (ER) and activation of GSK3β signaling underlies pathogenesis of these psychiatric disorders. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. Binding, internalization, and degradation of atrial natriuretic peptide in cultured vascular smooth muscle cells of rat

    Energy Technology Data Exchange (ETDEWEB)

    Hirata, Y.; Takata, S.; Tomita, M.; Takaichi, S.

    1985-11-15

    Binding, internalization, and degradation of /sup 125/I-labeled-rat atrial natriuretic peptide (rANP) were studied in cultured rat aortic vascular smooth muscle cells (VSMC). At 37 degrees C, /sup 125/I-labeled-rANP rapidly bound to VSMCs, but the cell-bound radioactivity rapidly decreased upon subsequent incubation, while the binding was slow at 4 degrees C, reaching to an apparent equilibrium after 6 hrs. The cell-bound /sup 125/I-labeled-rANP at 37 degrees C is rapidly dissociated from VSMC (t 1/2: approximately 40 min) with the appearance of degradaded product(s) of radioligand in the medium, whereas the degradation was minimal at 4 degrees C. This degradative process was blocked by inhibitors of metabolic energy production (azide, dinitrophenol), inhibitors of lysosomal cathepsins (leupeptin, pepstatin), and lysosomotropic agents (NH/sub 4/Cl, chloroquine, lidocaine, methylamine, dansylcadaverine), but not by inhibitors of serine or thiol proteases. /sup 125/I-labeled-rANP initially bound to the cell-surface was rapidly internalized, and delivered to lysosomal structures, which was confirmed by autoradiographic studies. These data indicate that rANP, after binding to the cell-surface receptors, is rapidly internalized into the cells through receptor-mediated endocytosis, and subsequently degradaded by lysosomal hydrolases.

  15. Triazole induced concentration-related gene signatures in rat whole embryo culture.

    Science.gov (United States)

    Robinson, Joshua F; Tonk, Elisa C M; Verhoef, Aart; Piersma, Aldert H

    2012-09-01

    Commonly used as antifungal agents in agriculture and medicine, triazoles have been shown to cause teratogenicity in a diverse set of animal models. Here, we evaluated the dose-dependent impacts of flusilazole, cyproconazole and triadimefon, on global gene expression in relation to effects on embryonic development using the rat whole embryo culture (WEC) model. After 4 h exposure, we identified changes in gene expression due to triazole exposure which preceded morphological alterations observed at 48 h. In general, across the three triazoles, we observed similar directionality of regulation in gene expression and the magnitude of effects on gene expression correlated with the degree of induced developmental toxicity. Significantly regulated genes included key members of steroid/cholesterol and retinoic acid metabolism and hindbrain developmental pathways. Direct comparisons with previous studies suggest that triazole-gene signatures identified in the WEC overlap with zebrafish and mouse, and furthermore, triazoles impact gene expression in a similar manner as retinoic acid exposures in rat embryos. In summary, we further differentiate pathways underlying triazole-developmental toxicity using WEC and demonstrate the conservation of these response-pathways across model systems. Copyright © 2012 Elsevier Inc. All rights reserved.

  16. Humanin rescues cultured rat cortical neurons from NMDA-induced toxicity not by NMDA receptor.

    Science.gov (United States)

    Cui, Ai-Ling; Li, Jian-Zhong; Feng, Zhi-Bo; Ma, Guo-Lin; Gong, Liang; Li, Chun-Ling; Zhang, Ce; Li, Kefeng

    2014-01-01

    Excitatory neurotoxicity has been implicated in many pathological situations and there is no effective treatment available. Humanin is a 24-aa peptide cloned from the brain of patients with Alzheimer's disease (AD). In the present study, excitatory toxicity was induced by N-methyl-D-aspartate (NMDA) in primarily cultured rat cortical neurons. MTT assessment, lactate dehydrogenase (LDH) release, and calcein staining were employed to evaluate the protective activity of humanin on NMDA induced toxicity. The results suggested that NMDA (100 μmol/L, 2.5 hr) triggered neuronal morphological changes, lactate dehydrogenase (LDH) release (166% of the control), reduction of cell viability (about 50% of the control), and the decrease of living cell density (about 50% of the control). When pretreated with humanin, the toxicity was suppressed. The living cells' density of humanin treated group was similar to that of control. The cell viability was attenuated dose-dependently (IC50 = 0.132 nmol/L). The LDH release was also neutralized in a dose-dependent manner. In addition, the intracellular Ca(2+) overloading triggered by NMDA reverted quickly and humanin could not inhibit it. These findings indicate that humanin can rescue cortical neurons from NMDA-induced toxicity in rat but not through interfering with NMDA receptor directly.

  17. Humanin Rescues Cultured Rat Cortical Neurons from NMDA-Induced Toxicity Not by NMDA Receptor

    Directory of Open Access Journals (Sweden)

    Ai-Ling Cui

    2014-01-01

    Full Text Available Excitatory neurotoxicity has been implicated in many pathological situations and there is no effective treatment available. Humanin is a 24-aa peptide cloned from the brain of patients with Alzheimer’s disease (AD. In the present study, excitatory toxicity was induced by N-methyl-D-aspartate (NMDA in primarily cultured rat cortical neurons. MTT assessment, lactate dehydrogenase (LDH release, and calcein staining were employed to evaluate the protective activity of humanin on NMDA induced toxicity. The results suggested that NMDA (100 μmol/L, 2.5 hr triggered neuronal morphological changes, lactate dehydrogenase (LDH release (166% of the control, reduction of cell viability (about 50% of the control, and the decrease of living cell density (about 50% of the control. When pretreated with humanin, the toxicity was suppressed. The living cells’ density of humanin treated group was similar to that of control. The cell viability was attenuated dose-dependently (IC50 = 0.132 nmol/L. The LDH release was also neutralized in a dose-dependent manner. In addition, the intracellular Ca2+ overloading triggered by NMDA reverted quickly and humanin could not inhibit it. These findings indicate that humanin can rescue cortical neurons from NMDA-induced toxicity in rat but not through interfering with NMDA receptor directly.

  18. Expression of Tau40 induces activation of cultured rat microglial cells.

    Directory of Open Access Journals (Sweden)

    Lu Wang

    Full Text Available Accumulation of microtubule-associated protein tau has been observed in the brain of aging and tauopathies. Tau was observed in microglia, but its role is not illustrated. By immunofluorescence staining and the fractal dimension value assay in the present study, we observed that microglia were activated in the brains of rats and mice during aging, simultaneously, the immunoreactivities of total tau and the phosphorylated tau were significantly enhanced in the activated microglia. Furtherly by transient transfection of tau40 (human 2N/4R tau into the cultured rat microglia, we demonstrated that expression of tau40 increased the level of Iba1, indicating activation of microglia. Moreover, expression of tau40 significantly enhanced the membranous localization of the phosphorylated tau at Ser396 in microglia possibly by a mechanism involving protein phosphatase 2A, extracellular signal-regulated kinase and glycogen synthase kinase-3β. It was also found that expression of tau40 promoted microglial migration and phagocytosis, but not proliferation. And we observed increased secretion of several cytokines, including interleukin (IL-1β, IL-6, IL-10, tumor necrosis factor-α and nitric oxide after the expression of tau40. These data suggest a novel role of human 2N/4R tau in microglial activation.

  19. Designing a primary science curriculum in a globalizing world: How do social constructivism and Vietnamese culture meet?

    Science.gov (United States)

    Hằng, Ngô Vũ Thu; Meijer, Marijn Roland; Bulte, Astrid M. W.; Pilot, Albert

    2017-09-01

    The implementation of social constructivist approaches to learning science in primary education in Vietnamese culture as an example of Confucian heritage culture remains challenging and problematic. This theoretical paper focuses on the initial phase of a design-based research approach; that is, the description of the design of a formal, written curriculum for primary science education in which features of social constructivist approaches to learning are synthesized with essential aspects of Vietnamese culture. The written design comprises learning aims, a framework that is the synthesis of learning functions, learning settings and educational expectations for learning phases, and exemplary curriculum units. Learning aims are formulated to comprehensively develop scientific knowledge, skills, and attitudes toward science for primary students. Derived from these learning aims, the designed framework consists of four learning phases respectively labeled as Engagement, Experience, Exchange, and Follow-up. The designed framework refers to knowledge of the "nature of science" education and characteristics of Vietnamese culture as an example of Confucian heritage culture. The curriculum design aims to serve as an educational product that addresses previously analyzed problems of primary science education in the Vietnamese culture in a globalizing world.

  20. Progesterone and synthetic progestin, dienogest, induce apoptosis of human primary cultures of adenomyotic stromal cells.

    Science.gov (United States)

    Yamanaka, Akiyoshi; Kimura, Fuminori; Kishi, Yohei; Takahashi, Kentaro; Suginami, Hiroshi; Shimizu, Yutaka; Murakami, Takashi

    2014-08-01

    To investigate the direct effects of progesterone receptor (PR) agonists on proliferation and apoptosis of human adenomyotic cells. Human primary cultures of adenomyotic stromal cells (ASCs) from 24 patients with adenomyosis were co-treated with estradiol (E2) plus the PR agonists, endogenous progesterone (P) or the synthetic progestin dienogest (DNG), which is used to treat endometriosis. In ASCs, anti-proliferative effects and induction of apoptosis were evaluated in the presence or absence of P (10(-8)-10(-6)M) or DNG (10(-8)-10(-6)M) in culture medium containing E2. Cellular proliferation was analyzed with bromodeoxyuridine incorporation and flow cytometry. Apoptosis was detected with annexin V/7-amino-actinomycin D (7-AAD) staining with flow cytometry and cellular caspase 3/7 activity. P and DNG significantly decreased the proportion of cells in the S phase. In addition, both P and DNG increased apoptosis as measured by annexin V-positive/7-AAD -negative cells and caspase 3/7 activity. Both endogenous P and synthetic progestin directly inhibited cellular proliferation and induced apoptosis in human ASCs. These pharmacological features of progestational compounds provide insight into the therapeutic strategy for the treatment of adenomyosis. Copyright © 2014 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

  1. Effects of parathyroid hormone on cytosolic calcium in renal proximal tubular primary cultures

    International Nuclear Information System (INIS)

    Hruska, K.A.; Goligorsky, M.; Scoble, J.; Tsutsumi, M.; Westbrook, S.; Moskowitz, D.

    1986-01-01

    The effects of parathyroid hormone on the cytoplasmic Ca 2+ concentration of canine renal proximal tubule cells grown in primary culture were determined using the fluorescent Ca 2+ indicator quin 2. The cultured cells exhibited responses to hormones, enzyme activities, transport functions, and morphology characteristic of the proximal convoluted tubule. Parathyroid hormone stimulated an immediate rise in cytoplasmic Ca 2+ , both in suspended cells and cells studies as a monolayer on Nuclepore filters. The rise in cytoplasmic Ca 2+ induced by the hormone was sustained for 15-30 min, was dose dependent, and was not mimicked by cyclic AMP. Removing Ca 2+ from the extracellular media markedly decreased cytoplasmic Ca 2+ and abolished the effects of parathyroid hormone on cytosolic Ca 2+ . 8-(N,N-diethylamino)octyl-3,4,5-trimethyoxybenzoate blocked the effects of the hormone on cytosolic Ca 2+ , but mitochondrial uncouplers failed to inhibit the effects of the hormone to increase cytoplasmic Ca 2+ . These studies support a role of Ca 2+ i the activation of proximal renal tubule cells by parathyroid hormone

  2. Language, culture and emotions: exploring ethnic minority patients' emotional expressions in primary healthcare consultations.

    Science.gov (United States)

    De Maesschalck, Stéphanie; Deveugele, Myriam; Willems, Sara

    2011-09-01

    This study explores ethnic minority patients' expression of emotional cues and concerns in primary healthcare, and examines relationships with patient, provider and consultation attributes. 191 video-recorded consultations were analyzed using the VR-CoDES. Patients were interviewed before the consultation. Generalized Estimating Equations models (GEE) were used to test for associations. Psychosocial versus bio-medically oriented encounters contained significantly more cues (p≤0.05). Patients with poor versus good language proficiency expressed significantly less cues (p≤0.001). No significant correlations were found with patients' cultural values, patients' or physicians' gender or the presence of an interpreter. Female patients express more concerns (p≤0.05), female physicians have a higher number of concerns expressed by patients (p≤0.02). This study shows that independent of physician and diagnosis, patients' language proficiency has a more important impact on the number of cues expressed by the patient than cultural difference. Medical schools and Continuing Medical Education should focus on training programs for recognizing and handling linguistic barriers between physicians and patients. Patient education programs should encourage patients who experience language barriers to open up to physicians. In situations where language is a barrier, physicians and patients should be encouraged to use interpreters to enhance the expression of emotions. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  3. Primary school as the hub of the social and cultural life in the local community

    Directory of Open Access Journals (Sweden)

    Jasna Mažgon

    2016-04-01

    Full Text Available For quite some time, Slovenian society has been preserving a specific model of social organisation rooted in the communal form. In functioning as a socio-cultural centre of the local community the school greatly surpassed its primary role of educating children. The process of urbanisation that has reached rural areas brought very interesting changes to the previously expanded function of the local school. We examined how, today, schools perceive a need to connect with and engage in their local environments. The perceptions of connections and their real modalities do differ and the ways in which schools respond to the needs of the localities (and vice versa depend on the prevalent model of social organisation. Exceptions to this are more significant in localities where the school might be one of very few public institutions or the only public institution present at the local level. Although the schools wish to motivate and engage local residents also in other environments, they often lack the time and energy to do so. The results of qualitative analysis indicated that merging or closing local schools could have negative demographic and socio-cultural consequences. At the same time, the analysis pointed to unrealised potential in the localities lacking tradition, such as new urban areas where the school could be the crucial element in the social organisation of the local community.

  4. Evaluation of Kosovo Primary Education Programs in Terms of Cultural Diversit

    Directory of Open Access Journals (Sweden)

    Serdan KERVAN

    2017-12-01

    Full Text Available Communities that have lived together for a long time have used the same language and created a number of common values. All of these common values ​​constitute the cult of that community. Culture is a complex phenomenon that reflects the ways in which a nation solves its problems, the ways in which it interacts with each other and with others, and the way they think (Naylor, 1996. This research was conducted by teachers 1-5. the structure of the class curricula and their impact on student achievement is determined by a semi-structured interview form, so it is a qualitative research based on the cause-and-effect relationship. The universe of this research is the class teachers who work in the primary schools in the cities where different mother-tongue education is provided in Kosovo. According to the teachers 'views, the influences of the programs applied for different cultures on the students' success were determined as meaningful differences in the age, education level, teaching language of the teachers and whether or not they were educated in different mother tongues in the same school.

  5. Dose–response analysis of phthalate effects on gene expression in rat whole embryo culture

    Energy Technology Data Exchange (ETDEWEB)

    Robinson, Joshua F. [National Institute for Public Health and the Environment (RIVM), Bilthoven (Netherlands); Department of Toxicogenomics, Maastricht University, Maastricht (Netherlands); Verhoef, Aart; Beelen, Vincent A. van; Pennings, Jeroen L.A. [National Institute for Public Health and the Environment (RIVM), Bilthoven (Netherlands); Piersma, Aldert H., E-mail: aldert.piersma@rivm.nl [National Institute for Public Health and the Environment (RIVM), Bilthoven (Netherlands); Institute for Risk Assessment Sciences (IRAS), Utrecht University, Utrecht (Netherlands)

    2012-10-01

    The rat postimplantation whole embryo culture (WEC) model serves as a potential screening tool for developmental toxicity. In this model, cultured rat embryos are exposed during early embryogenesis and evaluated for morphological effects. The integration of molecular-based markers may lead to improved objectivity, sensitivity and predictability of WEC in assessing developmental toxic properties of compounds. In this study, we investigated the concentration-dependent effects of two phthalates differing in potency, mono(2-ethylhexyl) phthalate (MEHP) and monomethyl phthalate (MMP, less toxic), on the transcriptome in WEC to examine gene expression in relation with dysmorphogenesis. MEHP was more potent than MMP in inducing gene expression changes as well as changes on morphology. MEHP induced significant enrichment of cholesterol/lipid/steroid (CLS) metabolism and apoptosis pathways which was associated with developmental toxicity. Regulation of genes within CLS metabolism pathways represented the most sensitive markers of MEHP exposure, more sensitive than classical morphological endpoints. As shown in direct comparisons with toxicogenomic in vivo studies, alterations in the regulation of CLS metabolism pathways has been previously identified to be associated with developmental toxicity due to phthalate exposure in utero. Our results support the application of WEC as a model to examine relative phthalate potency through gene expression and morphological responses. Additionally, our results further define the applicability domain of the WEC model for developmental toxicological investigations. -- Highlights: ► We examine the effect of two phthalates on gene expression and morphology in WEC. ► MEHP is more potent than MMP in inducing gene expression changes and dysmorphogenesis. ► MEHP significantly disrupts cholesterol metabolism pathways in a dose-dependent manner. ► Specific phthalate-related mechanisms in WEC are relevant to mechanisms in vivo.

  6. The endozepine ODN stimulates [{sup 3}H]thymidine incorporation in cultured rat astrocytes

    Energy Technology Data Exchange (ETDEWEB)

    Gandolfo, P.; Patte, C.; Thoumas, J.L.; Leprince, J.; Vaudry, H.; Tonon, M.C. [European Institute for Peptide Research (IFRMP no. 23), Laboratory of Cellular and Molecular Neuroendocrinology, INSERM U 413, UA CNRS, University of Rouen, 76821 Mont-Saint-Aignan (France)

    1999-05-15

    High concentrations of diazepam-binding inhibitor (DBI) mRNA have been detected in astrocytoma, suggesting that DBI-derived peptides may play a role in glial cell proliferation. In the present study, we have investigated the effect of a processing product of DBI, the octadecaneuropeptide ODN, on DNA synthesis in cultured rat astrocytes. At very low concentrations (10{sup -14} to 10{sup -11} M), ODN caused a dose-dependent increase of [{sup 3}H]thymidine incorporation. At higher doses (10{sup -10} to 10{sup -5} M), the effect of ODN gradually declined. The central-type benzodiazepine receptor antagonist flumazenil (10{sup -6} M) completely suppressed the stimulatory action of ODN whereas the peripheral-type benzodiazepine receptor ligand, PK11195 (10{sup -6} M) had no effect. The ODN-induced stimulation of [{sup 3}H]thymidine incorporation was mimicked by methyl 6,7-dimethoxy-4-ethyl-{beta}-carboline-3-carboxylate (DMCM). The GABA{sub A} receptor antagonist bicuculline (10{sup -4} M) suppressed the effect of both ODN and DMCM on DNA synthesis. Exposure of cultured astrocytes to the specific GABA{sub A} agonist 3APS (10{sup -10} to 10{sup -4} M) also induced a dose-related increase of [{sup 3}H]thymidine incorporation. The present study indicates that ODN, acting through central-type benzodiazepine receptors associated with the GABA{sub A} receptor complex, stimulates DNA synthesis in rat glial cells. These data provide evidence for an autocrine role of endozepines in the control of glial cell proliferation. (Copyright (c) 1999 Elsevier Science B.V., Amsterdam. All rights reserved.)

  7. Characterization of amino acid metabolism by cultured rat kidney cells: Study with 15N

    International Nuclear Information System (INIS)

    Nissim, I.; States, B.; Yudkoff, M.; Segal, S.

    1987-01-01

    The present study evaluates the metabolism of glutamine and glutamate by normal rat kidney (NRK) cells. The major aim was to evaluate the effect of acute acidosis on the metabolism of amino acid and ammonia formation by cultured NRK cells. Experiments at either pH 7.0 or 7.4 were conducted with phosphate-buffered saline supplemented with either 1 mM [5- 15 N]glutamine, [2- 15 N]glutamine, or [ 15 N]glutamate. Incubation with either glutamine or glutamate as a precursor showed that production of ammonia and glucose was increased significantly at pH 7.0 vs 7.4. In experiments with [5- 15 N]glutamine, the authors found that ∼57 and 43% of ammonia N was derived from 5-N of glutamine at pH 7.4 and 7.0, respectively. Three major metabolic pathways of [2- 15 N]glutamine or [ 15 N]glutamate disposal were identified: (1) transamination reactions involving the pH-independent formation of [ 15 N] aspartate and [ 15 N]alanine; (2) the synthesis of [6- 15 NH 2 ]adenine nucleotide, a process more active at pH 7.4 vs. 7.0; and (3) glutamine synthesis from [ 15 N]glutamate, especially at pH 7.4. The data indicate that NRK cells in culture consume glutamine and glutamate and generate ammonia and various amino acids, depending on the H + concentration in the media. The studies suggest that these cell lines may provide a useful model for studying various aspects of the effect of pH on rat renal ammoniagenesis

  8. Slow conduction in mixed cultured strands of primary ventricular cells and stem cell-derived cardiomyocytes

    Directory of Open Access Journals (Sweden)

    Jan Pavel Kucera

    2015-09-01

    Full Text Available 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.

  9. Reactive oxygen species are involved in BMP-induced dendritic growth in cultured rat sympathetic neurons.

    Science.gov (United States)

    Chandrasekaran, Vidya; Lea, Charlotte; Sosa, Jose Carlo; Higgins, Dennis; Lein, Pamela J

    2015-07-01

    Previous studies have shown that bone morphogenetic proteins (BMPs) promote dendritic growth in sympathetic neurons; however, the downstream signaling molecules that mediate the dendrite promoting activity of BMPs are not well characterized. Here we test the hypothesis that reactive oxygen species (ROS)-mediated signaling links BMP receptor activation to dendritic growth. In cultured rat sympathetic neurons, exposure to any of the three mechanistically distinct antioxidants, diphenylene iodinium (DPI), nordihydroguaiaretic acid (NGA) or desferroxamine (DFO), blocked de novo BMP-induced dendritic growth. Addition of DPI to cultures previously induced with BMP to extend dendrites caused dendritic retraction while DFO and NGA prevented further growth of dendrites. The inhibition of the dendrite promoting activity of BMPs by antioxidants was concentration-dependent and occurred without altering axonal growth or neuronal cell survival. Antioxidant treatment did not block BMP activation of SMAD 1,5 as determined by nuclear localization of these SMADs. While BMP treatment did not cause a detectable increase in intracellular ROS in cultured sympathetic neurons as assessed using fluorescent indicator dyes, BMP treatment increased the oxygen consumption rate in cultured sympathetic neurons as determined using the Seahorse XF24 Analyzer, suggesting increased mitochondrial activity. In addition, BMPs upregulated expression of NADPH oxidase 2 (NOX2) and either pharmacological inhibition or siRNA knockdown of NOX2 significantly decreased BMP-7 induced dendritic growth. Collectively, these data support the hypothesis that ROS are involved in the downstream signaling events that mediate BMP7-induced dendritic growth in sympathetic neurons, and suggest that ROS-mediated signaling positively modulates dendritic complexity in peripheral neurons. Copyright © 2015 Elsevier Inc. All rights reserved.

  10. Identification of cultural determinants of antibiotic use cited in primary care in Europe: a mixed research synthesis study of integrated design "Culture is all around us".

    Science.gov (United States)

    Touboul-Lundgren, Pia; Jensen, Siri; Drai, Johann; Lindbæk, Morten

    2015-09-17

    Inappropriate antibiotic prescribing, particularly for respiratory tract infections (RTI) in ambulatory care, has become a worldwide public health threat due to resulting antibiotic resistance. In spite of various interventions and campaigns, wide variations in antibiotic use persist between European countries. Cultural determinants are often referred to as a potential cause, but are rarely defined. To our knowledge, so far no systematic literature review has focused on cultural determinants of antibiotic use. The aim of this study was to identify cultural determinants, on a country-specific level in ambulatory care in Europe, and to describe the influence of culture on antibiotic use, using a framework of cultural dimensions. A computer-based systematic literature review was conducted by two research teams, in France and in Norway. Eligible publications included studies exploring antibiotic use in primary care in at least two European countries based on primary study results, featuring a description of cultural determinants, and published between 1997 and 2015. Quality assessment was conducted independently by two researchers, one in each team, using appropriate checklists according to study design. Each included paper was characterized according to method, countries involved, sampling and main results, and cultural determinants mentioned in each selected paper were extracted, described and categorized. Finally, the influence of Hofstede's cultural dimensions associated with antibiotic consumption within a primary care setting was described. Among 24 eligible papers, 11 were rejected according to exclusion criteria. Overall, 13 papers meeting the quality assessment criteria were included, of which 11 used quantitative methods and two qualitative or mixed methods. The study participants were patients (nine studies) and general practitioners (two studies). This literature review identified various cultural determinants either patient-related (illness perception

  11. Contrasting effects of cerebrospinal fluid from motor neuron disease patients on the survival of primary motor neurons cultured with or without glia.

    Science.gov (United States)

    Barber, Siân C; Wood-Allum, Clare A; Sargsyan, Siranush A; Walsh, Theresa; Cox, Laura E; Monk, Peter N; Shaw, Pamela J

    2011-07-01

    Motor neuronal (MN) degeneration in motor neuron disease (MND) often starts focally before spreading to neighbouring MN populations, suggesting soluble factors may contribute to disease propagation. Whether cerebrospinal fluid (CSF) from MND patients contains such factors has been difficult to prove. We aimed to determine the effect of glia on the response of MNs to CSF from MND patients. Primary rat spinal MNs grown in mono-culture or cocultured with glia were exposed to CSF from patients (MND-CSF) or controls (Con-CSF) and survival measured by cell counting. In mono-culture both MND-CSF and Con-CSF reduced MN survival with MND-CSF reducing MN survival by less than Con-CSF. In coculture MN survival was unchanged by exposure to MND-CSF while exposure to Con-CSF improved MN survival. In separate experiments, murine MNs grown in mono-culture and stressed by growth factor withdrawal were partially rescued by the application of monocyte chemoattractant protein-1 (MCP-1), a trophic factor previously found to be elevated in MND-CSF. Our results suggest that MND-CSF may contain factors harmful to MNs as well as factors protective of MNs, the interplay of which is altered by the presence of glial cells. These preliminary results further emphasize the importance of MN environment to MN health.

  12. Transcriptomic comparison of primary bovine horn core carcinoma culture and parental tissue at early stage

    Directory of Open Access Journals (Sweden)

    Sharadindu Shil

    2017-01-01

    Full Text Available Aim: Squamous cell carcinoma or SCC of horn in bovines (bovine horn core carcinoma frequently observed in Bos indicus affecting almost 1% of cattle population. Freshly isolated primary epithelial cells may be closely related to the malignant epithelial cells of the tumor. Comparison of gene expression in between horn’s SCC tissue and its early passage primary culture using next generation sequencing was the aim of this study. Materials and Methods: Whole transcriptome sequencing of horn’s SCC tissue and its early passage cells using Ion Torrent PGM were done. Comparative expression and analysis of different genes and pathways related to cancer and biological processes associated with malignancy, proliferating capacity, differentiation, apoptosis, senescence, adhesion, cohesion, migration, invasion, angiogenesis, and metabolic pathways were identified. Results: Up-regulated genes in SCC of horn’s early passage cells were involved in transporter activity, catalytic activity, nucleic acid binding transcription factor activity, biogenesis, cellular processes, biological regulation and localization and the down-regulated genes mainly were involved in focal adhesion, extracellular matrix receptor interaction and spliceosome activity. Conclusion: The experiment revealed similar transcriptomic nature of horn’s SCC tissue and its early passage cells.

  13. DEVELOPMENT OF PRIMARY CELL CULTURE FROM TAIL EPIDERMAL TISSUE OF KOI CARP (Cyprinus carpio koi

    Directory of Open Access Journals (Sweden)

    Lila Gardenia

    2014-06-01

    Full Text Available Primary cell culture from tail epidermal tissue of koi carp (Cyprinus carpio koi was developed. Cells were grown in Leibovits-15 medium supplemented with 20% fetal bovine serum and antibiotics (Penicillin/Streptomycin and Kanamycin. Cell growth was observed in a range of incubation temperature (17oC±2oC, 22oC±2oC, 27oC±2oC, and 32oC±2oC in order to determine the optimum temperature. The cells were able to grow at a range of temperature between 17oC to 32oC with optimal growth at 22oC. Primary cells infected with koi herpes virus produced typical cytopathic effects characterized by severe vacuolation and deformation of nuclei, which is consistent with those of previous reports. Artificial injection experiment by using supernatant koi herpes virus SKBM-1 isolate revealed that it could cause 90% mortality in infected fish within two weeks. PCR test with Sph I-5 specific primers carried out with DNA template from supernatant virus, pellet cell, and gills of infected fish showed positive results in all samples (molecular weight of DNA target 290 bp. The cells were found to be susceptible to koi herpes virus and can be used for virus propagation.

  14. Cytotoxicity of heavy metals on primary cultured alveolar type II cells.

    Science.gov (United States)

    Takano, Yasuo; Taguchi, Tetsuya; Suzuki, Isao; Balis, John U; Yuri, Kazunari

    2002-06-01

    The lung is the primary target organ of airborne heavy metal-induced toxicity. The aims of this study were to investigate differential acute lung cytotoxicity caused by heavy metals using a primary culture of alveolar type II cells and to establish an in vitro assessment model of lung toxicity. The cytotoxicity of heavy metals was determined by measuring the lactate dehydrogenase release and (51)chromium release from lyzed cells. With respect to the LC(50) values, drug concentrations causing a 50% loss in cell viability, the mean value of Hg was 110 microM and that of Cd was 220 to 250 microM. Cytotoxicity was graded high for Hg and Cd, moderate for Pb and Ni, and negligible for Mn. Additional morphological observations of cell membrane integrity by scanning electron microscopy were compatible with the results of biochemical measurements. In conclusion, we have presented an in vitro assessment model of lung toxicity, which can be used effectively to assess the differential effects of heavy metals on alveolar type II cells. The findings suggests that the potential mechanisms of cytotoxicity are dependent on both the nature and the concentration of the metals.

  15. Designing and Using an Organisational Culture Inquiry Tool to Glimpse the Relational Nature of Leadership and Organisational Culture within a South Australian Primary School

    Science.gov (United States)

    Giles, David; Bills, Andrew

    2017-01-01

    This case study research found that the relational leadership and organisational culture at a public primary school situated in a high poverty location in South Australia was built upon the strength of the inter-relationships between the teachers, teachers and leadership, and between teachers and students. Supported by what we called "dynamic…

  16. Millisecond-timescale local network coding in the rat primary somatosensory cortex.

    Directory of Open Access Journals (Sweden)

    Seif Eldawlatly

    Full Text Available Correlation among neocortical neurons is thought to play an indispensable role in mediating sensory processing of external stimuli. The role of temporal precision in this correlation has been hypothesized to enhance information flow along sensory pathways. Its role in mediating the integration of information at the output of these pathways, however, remains poorly understood. Here, we examined spike timing correlation between simultaneously recorded layer V neurons within and across columns of the primary somatosensory cortex of anesthetized rats during unilateral whisker stimulation. We used bayesian statistics and information theory to quantify the causal influence between the recorded cells with millisecond precision. For each stimulated whisker, we inferred stable, whisker-specific, dynamic bayesian networks over many repeated trials, with network similarity of 83.3±6% within whisker, compared to only 50.3±18% across whiskers. These networks further provided information about whisker identity that was approximately 6 times higher than what was provided by the latency to first spike and 13 times higher than what was provided by the spike count of individual neurons examined separately. Furthermore, prediction of individual neurons' precise firing conditioned on knowledge of putative pre-synaptic cell firing was 3 times higher than predictions conditioned on stimulus onset alone. Taken together, these results suggest the presence of a temporally precise network coding mechanism that integrates information across neighboring columns within layer V about vibrissa position and whisking kinetics to mediate whisker movement by motor areas innervated by layer V.

  17. Morphometric analysis of feedforward pathways from the primary somatosensory area (S1 of rats

    Directory of Open Access Journals (Sweden)

    A.L. de Sá

    2016-01-01

    Full Text Available We used biotinylated dextran amine (BDA to anterogradely label individual axons projecting from primary somatosensory cortex (S1 to four different cortical areas in rats. A major goal was to determine whether axon terminals in these target areas shared morphometric similarities based on the shape of individual terminal arbors and the density of two bouton types: en passant (Bp and terminaux (Bt. Evidence from tridimensional reconstructions of isolated axon terminal fragments (n=111 did support a degree of morphological heterogeneity establishing two broad groups of axon terminals. Morphological parameters associated with the complexity of terminal arbors and the proportion of beaded Bp vs stalked Bt were found to differ significantly in these two groups following a discriminant function statistical analysis across axon fragments. Interestingly, both groups occurred in all four target areas, possibly consistent with a commonality of presynaptic processing of tactile information. These findings lay the ground for additional work aiming to investigate synaptic function at the single bouton level and see how this might be associated with emerging properties in postsynaptic targets.

  18. Effect of 710 nm visible light irradiation on neurite outgrowth in primary rat cortical neurons following ischemic insult

    Energy Technology Data Exchange (ETDEWEB)

    Choi, Dong-Hee [Center for Neuroscience Research, SMART Institute of Advanced Biomedical Science, Konkuk University, Seoul (Korea, Republic of); Department of Medical Science, Konkuk University School of Medicine, Seoul (Korea, Republic of); Lee, Kyoung-Hee; Kim, Ji-Hye; Kim, Moon Young [Center for Neuroscience Research, SMART Institute of Advanced Biomedical Science, Konkuk University, Seoul (Korea, Republic of); Lim, Jeong Hoon [Department of Rehabilitation Medicine, Konkuk University School of Medicine, Seoul (Korea, Republic of); Rehabilitation Medicine, Division of Neurology, Department of Medicine, National University Hospital, National University Health System (Singapore); Lee, Jongmin, E-mail: leej@kuh.ac.kr [Center for Neuroscience Research, SMART Institute of Advanced Biomedical Science, Konkuk University, Seoul (Korea, Republic of); Department of Rehabilitation Medicine, Konkuk University School of Medicine, Seoul (Korea, Republic of)

    2012-06-01

    Highlights: Black-Right-Pointing-Pointer 710 nm wavelength light (LED) has a protective effect in the stroke animal model. Black-Right-Pointing-Pointer We determined the effects of LED irradiation in vitro stroke model. Black-Right-Pointing-Pointer LED treatment promotes the neurite outgrowth through MAPK activation. Black-Right-Pointing-Pointer The level of synaptic markers significantly increased with LED treatment. Black-Right-Pointing-Pointer LED treatment protects cell death in the in vitro stroke model. -- Abstract: Objective: We previously reported that 710 nm Light-emitting Diode (LED) has a protective effect through cellular immunity activation in the stroke animal model. However, whether LED directly protects neurons suffering from neurodegeneration was entirely unknown. Therefore, we sought to determine the effects of 710 nm visible light irradiation on neuronal protection and neuronal outgrowth in an in vitro stroke model. Materials and methods: Primary cultured rat cortical neurons were exposed to oxygen-glucose deprivation (OGD) and reoxygenation and normal conditions. An LED array with a peak wavelength of 710 nm was placed beneath the covered culture dishes with the room light turned off and were irradiated accordingly. LED treatments (4 min at 4 J/cm{sup 2} and 50 mW/cm{sup 2}) were given once to four times within 8 h at 2 h intervals for 7 days. Mean neurite density, mean neurite diameter, and total fiber length were also measured after microtubule associated protein 2 (MAP2) immunostaining using the Axio Vision program. Synaptic marker expression and MAPK activation were confirmed by Western blotting. Results: Images captured after MAP2 immunocytochemistry showed significant (p < 0.05) enhancement of post-ischemic neurite outgrowth with LED treatment once and twice a day. MAPK activation was enhanced by LED treatment in both OGD-exposed and normal cells. The levels of synaptic markers such as PSD 95, GAP 43, and synaptophysin significantly

  19. Effect of 710 nm visible light irradiation on neurite outgrowth in primary rat cortical neurons following ischemic insult

    International Nuclear Information System (INIS)

    Choi, Dong-Hee; Lee, Kyoung-Hee; Kim, Ji-Hye; Kim, Moon Young; Lim, Jeong Hoon; Lee, Jongmin

    2012-01-01

    Highlights: ► 710 nm wavelength light (LED) has a protective effect in the stroke animal model. ► We determined the effects of LED irradiation in vitro stroke model. ► LED treatment promotes the neurite outgrowth through MAPK activation. ► The level of synaptic markers significantly increased with LED treatment. ► LED treatment protects cell death in the in vitro stroke model. -- Abstract: Objective: We previously reported that 710 nm Light-emitting Diode (LED) has a protective effect through cellular immunity activation in the stroke animal model. However, whether LED directly protects neurons suffering from neurodegeneration was entirely unknown. Therefore, we sought to determine the effects of 710 nm visible light irradiation on neuronal protection and neuronal outgrowth in an in vitro stroke model. Materials and methods: Primary cultured rat cortical neurons were exposed to oxygen-glucose deprivation (OGD) and reoxygenation and normal conditions. An LED array with a peak wavelength of 710 nm was placed beneath the covered culture dishes with the room light turned off and were irradiated accordingly. LED treatments (4 min at 4 J/cm 2 and 50 mW/cm 2 ) were given once to four times within 8 h at 2 h intervals for 7 days. Mean neurite density, mean neurite diameter, and total fiber length were also measured after microtubule associated protein 2 (MAP2) immunostaining using the Axio Vision program. Synaptic marker expression and MAPK activation were confirmed by Western blotting. Results: Images captured after MAP2 immunocytochemistry showed significant (p < 0.05) enhancement of post-ischemic neurite outgrowth with LED treatment once and twice a day. MAPK activation was enhanced by LED treatment in both OGD-exposed and normal cells. The levels of synaptic markers such as PSD 95, GAP 43, and synaptophysin significantly increased with LED treatment in both OGD-exposed and normal cells (p < 0.05). Conclusion: Our data suggest that LED treatment may promote

  20. Application of primary cell cultures of laryngeal carcinoma and laser scanning cytometry in the evaluation of tumor reactivity to cisplatinum.

    Directory of Open Access Journals (Sweden)

    Krzysztof Kupisz

    2008-06-01

    Full Text Available Unsatisfactory effects of treatment of laryngeal carcinoma patients stimulate the clinicians as well as researchers to develop new more effective treatment models and to find new reliable prognostic factors. The aim of the present study was the evaluation of the use of primary cell cultures of the laryngeal carcinoma and laser scanning cytometry (LSC in the assessment of tumor reactivity to cisplatinum. Nineteen primary cultures of laryngeal carcinoma cells established from fragments of laryngeal carcinoma infiltrations were cultured with or without cisplatin, stained with monoclonal antibodies against P53 and BCL-2 proteins and analyzed by LSC. Cisplatin added to the culture medium leads to the significant increase of P53 expression and decrease of BCL-2 expression. Moreover, changes of P53 and BCL-2 expressions were significantly correlated. Our findings of apoptosis regulatory mechanisms could be useful in patient qualification for the chemotherapeutic follow-up treatment.

  1. Metabolic impact of overexpression of liver glycogen synthase with serine-to-alanine substitutions in rat primary hepatocytes.

    Science.gov (United States)

    Kadotani, Akito; Fujimura, Maho; Nakamura, Takao; Ohyama, Sumika; Harada, Naomoto; Maruki, Hiroko; Tamai, Yoshitaka; Kanatani, Akio; Eiki, Jun-Ichi; Nagata, Yasufumi

    2007-10-15

    To investigate the effect of elevation of liver glycogen synthase (GYS2) activity on glucose and glycogen metabolism, we performed adenoviral overexpression of the mutant GYS2 with six serine-to-alanine substitutions in rat primary hepatocytes. Cell-free assays demonstrated that the serine-to-alanine substitutions caused constitutive activity and electrophoretic mobility shift. In rat primary hepatocytes, overexpression of the mutant GYS2 significantly reduced glucose production by 40% and dramatically induced glycogen synthesis via the indirect pathway rather than the direct pathway. Thus, we conclude that elevation of glycogen synthase activity has an inhibitory effect on glucose production in hepatocytes by shunting gluconeogenic precursors into glycogen. In addition, although intracellular compartmentation of glucose-6-phosphate (G6P) remains unclear in hepatocytes, our results imply that there are at least two G6P pools via gluconeogenesis and due to glucose phosphorylation, and that G6P via gluconeogenesis is preferentially used for glycogen synthesis in hepatocytes.

  2. Transcriptomics analysis of retinoic acid embryotoxicity in rat postimplantation whole embryo culture.

    Science.gov (United States)

    Luijten, Mirjam; van Beelen, Vincent A; Verhoef, Aart; Renkens, Marc F J; van Herwijnen, Marcel H M; Westerman, Anja; van Schooten, Frederik-J; Pennings, Jeroen L A; Piersma, Aldert H

    2010-09-01

    Rodent postimplantation whole embryo culture (WEC) is a classical alternative test to study developmental toxicants. Here, we have successfully applied transcriptomics to monitor early responses in WEC after exposure to the embryotoxicant retinoic acid (RA). We demonstrated that RA exposures ranging from 2 to 24h affect RA-responsive genes in individual embryos. Furthermore, 2, 3 or 4 somite embryos gave similar responses, allowing combining embryos of these embryonic stages within the same analysis. Microarray analysis of embryonic gene expression after RA exposure revealed the regulation of many genes known to be RA responsive. Finally, use of a culture medium based on bovine serum instead of rat serum yielded similar gene expression responses after RA exposure. These findings support the robustness of the identified gene expression patterns and show the feasibility of detecting early gene expression changes in WEC after embryotoxic exposures. This approach may result in a more sensitive readout for detecting embryotoxicity in WEC. Copyright 2010 Elsevier Inc. All rights reserved.

  3. Regulation of the sodium-potassium pump in cultured rat skeletal myotubes by intracellular sodium ions

    International Nuclear Information System (INIS)

    Brodie, C.; Sampson, S.R.

    1989-01-01

    The properties of the Na-K pump and some of the factors controlling its amount and function were studied in rat myotubes in culture. The number of Na-K pump sites was quantified by measuring the amount of [ 3 H]ouabain bound to whole-cell preparations. Activity of the pump was determined by measurement of ouabain-sensitive 86 Rb-uptake and component of membrane potential. Chronic treatment of myotubes with tetrodotoxin (TTX), which lowers [Na]i, decreased the number of Na-K pumps, the ouabain-sensitive 86Rb uptake, and the size of the electrogenic pump component of Em. In contrast, chronic treatment with either ouabain or veratridine, which increases [Na+]i, resulted in an elevated level of Na-K pump sites. This effect was blocked by inhibitors of protein synthesis. Neither rates of degradation nor affinity of pump sites in cells treated with TTX, veratridine, or ouabain differred from those in control cells. The number and activity of Na-K pump sites were unaffected by chronic elevation in [Ca]i or chronic depolarization. We conclude that alterations in the level in intracellular Na ions play the major role in regulation of Na-K pump synthesis in cultured mammalian skeletal muscle

  4. Primary Human Uterine Leiomyoma Cell Culture Quality Control: Some Properties of Myometrial Cells Cultured under Serum Deprivation Conditions in the Presence of Ovarian Steroids.

    Directory of Open Access Journals (Sweden)

    Camila Bonazza

    Full Text Available 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.

  5. Multicultural Education: Learners with Diverse Linguistic and Cultural Background : A Case Study of one Primary School in Norway

    OpenAIRE

    Tosic, Milan

    2012-01-01

    Abstract This study aims to investigate how a primary school in Norway addresses learners with diverse linguistic and cultural background, in this study referred as culturally and linguistically diverse learners (CLD learners). The study is founded on the premises of multicultural education (MCE) which is considered essential to address the education of CLD learners. Therefore, the scope of the study is based on a five- category theoretical framework comprising: understanding the concept ...

  6. Feasibility of Primary Tumor Culture Models and Preclinical Prediction Assays for Head and Neck Cancer: A Narrative Review

    International Nuclear Information System (INIS)

    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

  7. Feasibility of Primary Tumor Culture Models and Preclinical Prediction Assays for Head and Neck Cancer: A Narrative Review

    Energy Technology Data Exchange (ETDEWEB)

    Dohmen, Amy J. C., E-mail: a.dohmen@nki.nl [Department of Head and Neck Surgery and Oncology, The Netherlands Cancer Institute-Antoni van Leeuwenhoek, Plesmanlaan 121, Amsterdam 1066 CX (Netherlands); Department of Cell Biology, the Netherlands Cancer Institute-Antoni van Leeuwenhoek, Plesmanlaan 121, Amsterdam 1066 CX (Netherlands); Swartz, Justin E. [Department of Otorhinolaryngology-Head and Neck Surgery, University Medical Center Utrecht, Heidelberglaan 100, Utrecht 3508 GA (Netherlands); Van Den Brekel, Michiel W. M. [Department of Head and Neck Surgery and Oncology, The Netherlands Cancer Institute-Antoni van Leeuwenhoek, Plesmanlaan 121, Amsterdam 1066 CX (Netherlands); Willems, Stefan M. [Department of Pathology, University Medical Center Utrecht, Heidelberglaan 100, Utrecht 3508 GA (Netherlands); Spijker, René [Medical library, Academic Medical Center, Amsterdam 1100 DE (Netherlands); Dutch Cochrane Centre, Julius Center for Health Sciences and Primary Care, University Medical Center Utrecht, Heidelberglaan 100, Utrecht 3508 GA (Netherlands); Neefjes, Jacques [Department of Cell Biology, the Netherlands Cancer Institute-Antoni van Leeuwenhoek, Plesmanlaan 121, Amsterdam 1066 CX (Netherlands); Zuur, Charlotte L. [Department of Head and Neck Surgery and Oncology, The Netherlands Cancer Institute-Antoni van Leeuwenhoek, Plesmanlaan 121, Amsterdam 1066 CX (Netherlands)

    2015-08-28

    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.

  8. Three-dimensional culture of rat calvarial osteoblasts in porous biodegradable polymers

    Science.gov (United States)

    Ishaug-Riley, S. L.; Crane-Kruger, G. M.; Yaszemski, M. J.; Mikos, A. G.

    1998-01-01

    Neonatal rat calvarial osteoblasts were cultured in 90% porous, 75:25 poly(DL-lactic-co-glycolic acid) (PLGA) foam scaffolds for up to 56 days to examine the effects of the cell seeding density, scaffold pore size, and foam thickness on the proliferation and function of the cells in this three-dimensional environment. Osteoblasts were seeded at either 11.1 x 10(5) or 22.1 x 10(5) cells per cm2 onto PLGA scaffolds having pore sizes in the range of 150-300 or 500-710 microm with a thickness of either 1.9 or 3.2 mm. After 1 day in culture, 75.6 and 68.6% of the seeded cells attached and proliferated on the 1.9 mm thick scaffolds of 150-300 microm pore size for the low and high seeding densities, respectively. The number of osteoblasts continued to increase throughout the study and eventually leveled off near 56 days, as indicated by a quantitative DNA assay. Osteoblast/foam constructs with a low cell seeding density achieved comparable DNA content and alkaline phosphatase (ALPase) activity after 14 days, and mineralization results after 56 days to those with a high cell seeding density. A maximum penetration depth of osseous tissue of 220+/-40 microm was reached after 56 days in the osteoblast/foam constructs of 150-300 microm pore size initially seeded with a high cell density. For constructs of 500-710 microm pore size, the penetration depth was 190+/-40 microm under the same conditions. Scaffold pore size and thickness did not significantly affect the proliferation or function of osteoblasts as demonstrated by DNA content, ALPase activity, and mineralized tissue formation. These data show that comparable bone-like tissues can be engineered in vitro over a 56 day period using different rat calvarial osteoblast seeding densities onto biodegradable polymer scaffolds with pore sizes in the range of 150-710 microm. When compared with the results of a previous study where similar polymer scaffolds were seeded and cultured with marrow stromal cells, this study

  9. Higher protein kinase C ζ in fatty rat liver and its effect on insulin actions in primary hepatocytes.

    Directory of Open Access Journals (Sweden)

    Wei Chen

    Full Text Available We previously showed the impairment of insulin-regulated gene expression in the primary hepatocytes from Zucker fatty (ZF rats, and its association with alterations of hepatic glucose and lipid metabolism. However, the molecular mechanism is unknown. A preliminary experiment shows that the expression level of protein kinase C ζ (PKCζ, a member of atypical PKC family, is higher in the liver and hepatocytes of ZF rats than that of Zucker lean (ZL rats. Herein, we intend to investigate the roles of atypical protein kinase C in the regulation of hepatic gene expression. The insulin-regulated hepatic gene expression was evaluated in ZL primary hepatocytes treated with atypical PKC recombinant adenoviruses. Recombinant adenovirus-mediated overexpression of PKCζ, or the other atypical PKC member PKCι/λ, alters the basal and impairs the insulin-regulated expressions of glucokinase, sterol regulatory element-binding protein 1c, the cytosolic form of phosphoenolpyruvate carboxykinase, the catalytic subunit of glucose 6-phosphatase, and insulin like growth factor-binding protein 1 in ZL primary hepatocytes. PKCζ or PKCι/λ overexpression also reduces the protein level of insulin receptor substrate 1, and the insulin-induced phosphorylation of AKT at Ser473 and Thr308. Additionally, PKCι/λ overexpression impairs the insulin-induced Prckz expression, indicating the crosstalk between PKCζ and PKCι/λ. We conclude that the PKCζ expression is elevated in hepatocytes of insulin resistant ZF rats. Overexpressions of aPKCs in primary hepatocytes impair insulin signal transduction, and in turn, the down-stream insulin-regulated gene expression. These data suggest that elevation of aPKC expression may contribute to the hepatic insulin resistance at gene expression level.

  10. Glucose-mediated control of ghrelin release from primary cultures of gastric mucosal cells

    Science.gov (United States)

    Sakata, Ichiro; Park, Won-Mee; Walker, Angela K.; Piper, Paul K.; Chuang, Jen-Chieh; Osborne-Lawrence, Sherri

    2012-01-01

    The peptide hormone ghrelin is released from a distinct group of gastrointestinal cells in response to caloric restriction, whereas its levels fall after eating. The mechanisms by which ghrelin secretion is regulated remain largely unknown. Here, we have used primary cultures of mouse gastric mucosal cells to investigate ghrelin secretion, with an emphasis on the role of glucose. Ghrelin secretion from these cells upon exposure to different d-glucose concentrations, the glucose antimetabolite 2-deoxy-d-glucose, and other potential secretagogues was assessed. The expression profile of proteins involved in glucose transport, metabolism, and utilization within highly enriched pools of mouse ghrelin cells and within cultured ghrelinoma cells was also determined. Ghrelin release negatively correlated with d-glucose concentration. Insulin blocked ghrelin release, but only in a low d-glucose environment. 2-Deoxy-d-glucose prevented the inhibitory effect of high d-glucose exposure on ghrelin release. mRNAs encoding several facilitative glucose transporters, hexokinases, the ATP-sensitive potassium channel subunit Kir6.2, and sulfonylurea type 1 receptor were expressed highly within ghrelin cells, although neither tolbutamide nor diazoxide exerted direct effects on ghrelin secretion. These findings suggest that direct exposure of ghrelin cells to low ambient d-glucose stimulates ghrelin release, whereas high d-glucose and glucose metabolism within ghrelin cells block ghrelin release. Also, low d-glucose sensitizes ghrelin cells to insulin. Various glucose transporters, channels, and enzymes that mediate glucose responsiveness in other cell types may contribute to the ghrelin cell machinery involved in regulating ghrelin secretion under these different glucose environments, although their exact roles in ghrelin release remain uncertain. PMID:22414807

  11. Glutamate receptor antagonists and growth factors modulate dentate granule cell neurogenesis in organotypic, rat hippocampal slice cultures

    DEFF Research Database (Denmark)

    Poulsen, Frantz Rom; Blaabjerg, Morten; Montero, Maria

    2005-01-01

    Generation of dentate granule cells and its modulation by glutamate receptor antagonists, growth factors and pilocarpine-induced seizure-like activity was investigated in rat hippocampal slice cultures derived from 1-week-old rats and grown for 2 weeks. Focussing on the dentate granule cell layer...... the number of TUC-4-positive cells, just as combining pilocarpine with the neurogenesis-stimulating compounds, prevented or reduced the increase of TUC-4-positive cells. None of the treatments were found to induce dentate granule cell death within the observed period. Labeling of dividing cells by adding 5...

  12. Analysis on the Relationship between Trust Culture and Prejudices in Primary Schools

    Science.gov (United States)

    Erdogan, Cetin

    2016-01-01

    Problem Statement: Trust is crucial for creating a positive culture in the school environment, which is called as trust culture. On the other hand, prejudice is thought to be a potential barrier for creating trust culture in schools. Thus, it is meaningful to examine the relationship between trust culture and prejudice in schools and then to…

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

    Science.gov (United States)

    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

  14. Functional differentiation and alveolar morphogenesis of primary mammary cultures on reconstituted basement membrane

    Energy Technology Data Exchange (ETDEWEB)

    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

  15. A new approach for studying GPCR dimers: drug-induced inactivation and reactivation to reveal GPCR dimer function in vitro, in primary culture, and in vivo

    Science.gov (United States)

    Teitler, Milt; Klein, Michael T.

    2011-01-01

    GPCRs are a major family of homologous proteins and are key mediators of the effects of numerous endogenous neurotransmitters, hormones, cytokines, therapeutic drugs, and drugs-of-abuse. Despite the enormous amount of research on the pharmacological and biochemical properties of GPCRs, the question as to whether they exist as monomers, dimers, or higher order structures in the body is unanswered. The GPCR dimer field has been dominated by techniques involving recombinant cell lines expressing mutant receptors, often involving the solubilization of the receptors. These techniques cannot be applied in vivo or even to primary cell cultures. This review will focus on a novel approach to exploring the functional properties of homodimers. Studies of the 5-HT7 and 5-HT2A serotonin receptors have revealed that binding of a pseudo-irreversible antagonist (“inactivator”) to one of the orthosteric sites of a homodimer abolishes all receptor activity, and subsequent binding of a competitive antagonist to the orthosteric site of the second protomer releases the inactivator, allowing the receptor to return to an active state. This approach demonstrates allosteric crosstalk between protomers of native GPCR homodimers, indicating that GPCRs do exist and function as homodimers in both recombinant cells and rat primary astrocytes. This technique can be applied universally using intact recombinant or primary cells in culture, membrane homogenate preparations and, potentially, in vivo. The data obtained using the 5-HT7 and 5-HT2A receptors are strongly supportive of a GPCR homodimer structure, with little evidence of monomer involvement in the function of these receptors. PMID:22119169

  16. Autocrine proliferative effects of hGH are maintained in primary cultures of human mammary carcinoma cells.

    Science.gov (United States)

    Chiesa, Jean; Ferrer, Catherine; Arnould, Cécile; Vouyovitch, Cécile M; Diaz, Jean-Jacques; Gonzalez, Samia; Mares, Pierre; Morel, Gérard; Wu, Zheng-Sheng; Zhu, Tao; Lobie, Peter E; Mertani, Hichem C

    2011-09-01

    Empirical evidence suggests that autocrine human GH (hGH) may possess a proliferative and oncogenic role in human mammary carcinoma. However, this concept is largely derived from studies using cultured human mammary carcinoma cell (HMCC) lines. We investigated the expression and functionality of hGH and the hGH receptor in isolated cultures of primary HMCC. Epithelial cell adhesion molecule-positive primary HMCC were isolated from surgical biopsies of patients with mammary carcinoma and cultured in vitro. Expression of hGH and hGH receptor was determined by RT-PCR, immunofluorescence microscopy, and ELISA. The proliferative response of the cultured primary HMCC to hGH stimulation or hGH inhibition with a hGH antagonist was determined. One hundred percent of cultured primary HMCC expressed the hGH receptor, and 52% expressed hGH at the mRNA level. hGH-positive primary HMCC produced hGH protein within the cell and secreted hGH to the media. Both hGH-negative and hGH-positive HMCC responded to hGH stimulation with large increases in cell number. hGH-positive HMCC responded to inhibition of hGH by a hGH antagonist with a decrease in cell number, whereas hGH-negative HMCC did not. Primary HMCC proliferate in response to hGH, and the proliferation of hGH-positive HMCC is inhibited by hGH antagonism. Inhibition of hGH in patients with mammary carcinoma may therefore limit tumor growth.

  17. Characterisation of citrate and iron citrate uptake by cultured rat hepatocytes

    International Nuclear Information System (INIS)

    Graham, R.M.; Morgan, E.H.; Baker, E.

    1998-01-01

    Background/Aims: the endogenous low molecular weight iron chelator, citrate, is considered to be an important contributor to iron transport and the liver the main site of uptake of iron citrate in subjects suffering from diseases of iron overload. Moreover, the citrate-metabolising enzyme, aconitase, is implicated in the regulation of cellular iron metabolism. This study was undertaken to determine the role of citrate and ferric citrate in the uptake of iron by rat hepatocytes. Methods: Cultured rat hepatocytes were incubated (37 deg. C, 15 min) with 100 μM [ 14 C]-citrate in the presence or absence of 1.0 μM 55 Fe. Membrane-bound and intracellular radiolabel were separated by incubation with the general protease, Pronase. Results: Our results suggest that ferric citrate uptake is mediated by a specific citrate binding site which exhibits a higher affinity for citrate in the presence of iron than in its absence. Citrate was internalised by hepatocytes, with at least 70% being oxidised to CO 2 within 15 min. Citrate uptake was pH-dependent, did not require the presence of sodium and increased with increasing iron concentration. Metabolic energy, anion channels, the Na + , K + -ATPase and vesicle acidification do not appear to play a role in uptake of ferric citrate, but functional sulphydryl groups may be involved. Conclusions: The data suggest either that ferric citrate complexes with higher molar ratios of iron to citrate relative to the incubation medium are bound preferentially to the membrane, or that once citrate has delivered its iron to the membrane, the complex dissociates and the components are internalised separately. (au)

  18. Efflux and hydrolysis of phosphorylethanolamine and phosphorylcholine in stressed cultured rat lenses.

    Science.gov (United States)

    Jernigan, H M; Desouky, M A; Geller, A M; Blum, P S; Ekambaram, M C

    1993-01-01

    Phosphorylcholine (P-choline), and phosphorylethanolamine (P-ethanolamine) are among the organic phosphate compounds that decrease in concentration in some cataracts and in lenses that have been subjected to cataractogenic osmotic or oxidative stresses. Decreases in the lenticular concentrations of these compounds could be caused by decreased synthesis, increased utilization or hydrolysis, or increased efflux, or by a combination of these mechanisms. To distinguish between these possibilities, the P-choline and P-ethanolamine pools of intact cultured rat lenses were radiolabeled with [3H]choline or [3H]ethanolamine, and the lenses were then subjected to oxidative or osmotic stress. The efflux and hydrolysis of P-[3H]choline and P-[3H]ethanolamine were then followed for up to 48 hr. Osmotic stress induced by incubation with 30 mM xylose caused increased P-choline and P-ethanolamine efflux from the lenses, but had little effect on the rate of hydrolysis of these compounds. For example, xylose-treated lenses lost 26% of their initial P-[3H]ethanolamine content into the medium during a 24 hr incubation, compared with only 6% for control lenses. Oxidative stress from singlet oxygen (generated by rose bengal and light) also increased lenticular P-choline efflux three to four-fold, with minimal effects on hydrolysis, and the increased efflux was accompanied by a decrease in the P-choline concentration. Permeability increases also were observed in lenses oxidatively stressed by H2O2 or by riboflavin and light. These results show that a variety of cataractogenic stresses, both oxidative and osmotic, cause increased permeability of rat lenses to organic phosphate compounds, including P-choline and P-ethanolamine.(ABSTRACT TRUNCATED AT 250 WORDS)

  19. Posterior Thalamic Nucleus Modulation of Tactile Stimuli Processing in Rat Motor and Primary Somatosensory Cortices

    Directory of Open Access Journals (Sweden)

    Diana Casas-Torremocha

    2017-09-01

    Full Text Available Rodents move rhythmically their facial whiskers and compute differences between signals predicted and those resulting from the movement to infer information about objects near their head. These computations are carried out by a large network of forebrain structures that includes the thalamus and the primary somatosensory (S1BF and motor (M1wk cortices. Spatially and temporally precise mechanorreceptive whisker information reaches the S1BF cortex via the ventroposterior medial thalamic nucleus (VPM. Other whisker-related information may reach both M1wk and S1BF via the axons from the posterior thalamic nucleus (Po. However, Po axons may convey, in addition to direct sensory signals, the dynamic output of computations between whisker signals and descending motor commands. It has been proposed that this input may be relevant for adjusting cortical responses to predicted vs. unpredicted whisker signals, but the effects of Po input on M1wk and S1BF function have not been directly tested or compared in vivo. Here, using electrophysiology, optogenetics and pharmacological tools, we compared in adult rats M1wk and S1BF in vivo responses in the whisker areas of the motor and primary somatosensory cortices to passive multi-whisker deflection, their dependence on Po activity, and their changes after a brief intense activation of Po axons. We report that the latencies of the first component of tactile-evoked local field potentials in M1wk and S1BF are similar. The evoked potentials decrease markedly in M1wk, but not in S1BF, by injection in Po of the GABAA agonist muscimol. A brief high-frequency electrical stimulation of Po decreases the responsivity of M1wk and S1BF cells to subsequent whisker stimulation. This effect is prevented by the local application of omega-agatoxin, suggesting that it may in part depend on GABA release by fast-spiking parvalbumin (PV-expressing cortical interneurons. Local optogenetic activation of Po synapses in different

  20. Fabrication of biocompatible free-standing nanopatterned films for primary neuronal cultures

    KAUST Repository

    Cesca, F.

    2014-09-10

    Devising and constructing biocompatible devices for nervous system regeneration is an extremely challenging task. Besides tackling the issue of biocompatibility, biomaterials for neuroscience applications should mimic the complex environment of the extracellular matrix, which in vivo provides neurons with a series of cues and signals to guide cells towards their appropriate targets. In this work, a novel nanopatterned biocompatible poly-ε-caprolactone (PCL) film is realized to assist the attachment and growth of primary hippocampal neurons. Costly and time-consuming processes can be avoided using plasma-surface nanotexturing obtained by a mixed gas SF6/Ar at −5 °C. The intrinsic composition and line topography of nanopatterned PCL ensure healthy development of the neuronal network, as shown by confocal microscopy, by analysing the expression of a range of neuronal markers typical of mature cultures, as well as by scanning electron microscopy. In addition, we show that surface nanopatterning improves differentiation of neurons compared to flat PCL films, while no neural growth was observed on either flat or nanopatterned substrates in the absence of a poly-D-lysine coating. Thus, we successfully optimized a nanofabrication protocol to obtain nanostructured PCL layers endowed with several mechanical and structural characteristics that make them a promising, versatile tool for future tissue engineering studies aimed at neural tissue regeneration.

  1. Novel approach for transient protein expression in primary cultures of human dental pulp-derived cells.

    Science.gov (United States)

    Suguro, Hisashi; Mikami, Yoshikazu; Koshi, Rieko; Ogiso, Bunnai; Watanabe, Eri; Watanabe, Nobukazu; Honda, Masaki J; Asano, Masatake; Komiyama, Kazuo

    2011-08-01

    Transfection is a powerful method for investigating variable biological functions of desired genes. However, the efficiency of transfection into primary cultures of dental pulp-derived cells (DPDC) is low. Therefore, using a recombinant vaccinia virus (vTF7-3), which contains T7 RNA polymerase, we have established a transient protein expression system in DPDCs. In this study, we used the human polymeric immunoglobulin receptor (pIgR) cDNA as a model gene. pIgR expression by the vTF7-3 expression system was confirmed by flow cytometry analysis and Western blotting. Furthermore, exogenous pIgR protein localized at the cell surface in DPDCs and formed a secretory component (SC). This suggests that exogenous pIgR protein expressed by the vTF7-3 expression system acts like endogenous pIgR protein. These results indicate the applicability of the method for cells outgrown from dental pulp tissue. In addition, as protein expression could be detected shortly after transfection (approximately 5h), this experimental system has been used intensely for experiments examining very early steps in protein exocytosis. Copyright © 2011 Elsevier Inc. All rights reserved.

  2. OPTIMIZATION OF PHYSICAL CULTURE LESSONS IN PRIMARY SCHOOL ON THE BASE OF MOBILE GAMES’ APPLICATION

    Directory of Open Access Journals (Sweden)

    L.V. Shuba

    2017-04-01

    Full Text Available Purpose: to improve methodic of mobile games’ application for physical culture lessons’ optimization in primary school that would facilitate motor fitness increase. Material: in experiment 50 boys (7-9 yrs from main health group (no health problems participated. For training strength we chose games, which required short speed-power tensions with at moderate loads. The following objects were used: filled ball, dump bells, benches or partners. In games with overcoming own weight we used: wall bars, beams, vertical rope. Results: peculiar features of the improved methodic are as follows: for preparatory part we worked out ten separate complexes of general exercises in game form; for all parts of lesson mobile games were selected so that they should facilitate maximal development of physical qualities; each training was emotionally positively colored that motivate pupils’ wish to attend the lessons. Conclusions: for effective development of physical qualities the decisive factor was consideration of sensitive periods and complex approach to physical education lessons.

  3. Emotions in primary care: Are there cultural differences in the expression of cues and concerns?

    Science.gov (United States)

    Schouten, Barbara C; Schinkel, Sanne

    2015-11-01

    This study compared native-Dutch and Turkish-Dutch patients' expressions of emotional cues/concerns and GPs' responses to these cues/concerns. Relations between patient's cues/concerns and GPs' perceptions of the patient's health complaint were examined too. 82 audiotaped encounters with native-Dutch and 38 with Turkish-Dutch GP patients were coded using the VR-CoDES and VR-CoDES-P. Patients filled out a survey before each consultation to assess their cultural identification, Dutch language proficiency and health-related variables. GPs filled out a survey after each consultation to assess their perceptions of the patient's health complaint. Turkish-Dutch patients expressed more cues than native-Dutch patients, which was explained by higher worries about their health and worse perceived general health. GPs responded more often with space-providing responses to Turkish-Dutch patients compared to native-Dutch patients. Turkish-Dutch patients' cue expression strongly influenced GPs' perceptions about the presence of psychosocial problems. Migrant patient-related factors influence the amount of emotional cue expression in primary care. GPs perceive these cues as indicating the presence of psychosocial problems and provide space for patients to elaborate on their emotional distress. GPs should be trained in using more affective communication techniques to enhance elicitation of the underlying reasons for migrant patients' enhanced emotional cue expression. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  4. Use of cultured rat embryos to evaluate the teratogenic activity of serum: cadmium and cyclophosphamide. [Serum-based culture media for growing rat embryos is used to determine the teratogenicity of cadmium and cyclophosphamide

    Energy Technology Data Exchange (ETDEWEB)

    Klein, N. W.; Vogler, M. A.; Chatot, C. L.; Pierro, L. J.

    1979-01-01

    Head fold stage rat embryos were cultured for 48 hrs in vitro on serum taken at various intervals from rats that had been injected ip with either cadmium or cyclophosphamide. Their response was compared to that of embryos cultured for the same period on control serum to which these substances were added directly. One and 4 hr sera from cadmium injected rats (2.13 mg Cd/sup + +//kg) were lethal. Eight hr serum allowed survival but embryos were exencephalic and contained reduced amounts of protein and DNA. The response to direct cadmium was characteristically different and was related to dosage and the extent to which zero-time embryos had progressed through the head fold stage. At 1.6 ..mu..M, Cd/sup + +/-susceptible embryos were hemorrhagic but not exencephalic. One hr serum from rats given cyclophosphamide (180 mg/kg) was lethal. On 4 hr serum, embryos survived but were exencephalic and contained less protein and DNA than controls. Embryos were resistant to direct cyclophosphamide up to 800 ..mu..g per ml of medium. At this concentration, embryos appeared morphologically normal but contained reduced amounts of protein.

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

    NARCIS (Netherlands)

    Vu Thu Hang, N.; Meijer, M.R.; Bulte, A.M.W.; Pilot, A.

    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

  6. The Implementation of a Social Constructivist Approach in Primary Science Education in Confucian Heritage Culture: The Case of Vietnam

    Science.gov (United States)

    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…

  7. Characterization of connective tissue growth factor expression in primary cultures of human tubular epithelial cells: modulation by hypoxia

    NARCIS (Netherlands)

    Kroening, Sven; Neubauer, Emily; Wullich, Bernd; Aten, Jan; Goppelt-Struebe, Margarete

    2010-01-01

    Kroening S, Neubauer E, Wullich B, Aten J, Goppelt-Struebe M. Characterization of connective tissue growth factor expression in primary cultures of human tubular epithelial cells: modulation by hypoxia. Am J Physiol Renal Physiol 298:F796-F806, 2010. First published December 23, 2009;

  8. Patient safety culture in out-of-hours primary care services in the Netherlands: a cross-sectional survey.

    NARCIS (Netherlands)

    Smits, M.; Keizer, E.; Giesen, P.; Deilkas, E.C.T.; Hofoss, D.; Bondevik, G.T

    2018-01-01

    Objective: To examine patient safety culture in Dutch out-of-hours primary care using the safety attitudes questionnaire (SAQ) which includes five factors: teamwork climate, safety climate, job satisfaction, perceptions of management and communication openness. Design: Cross-sectional observational

  9. Feasibility of Primary Tumor Culture Models and Preclinical Prediction Assays for Head and Neck Cancer: A Narrative Review

    NARCIS (Netherlands)

    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

  10. Further characterization of the adhesive-tumor-cell culture system for measuring the radiosensitivity of human tumor primary cultures

    International Nuclear Information System (INIS)

    Brock, W.A.; Bock, S.P.; Williams, M.; Baker, F.L.

    1987-01-01

    This study extends the use of the adhesive-tumor-cell culture system to include: over 100 sensitivity measurements at 2.0 Gy; tumorgenicity determinations in nude mice; and flow cytometry of the cells grown in the system. The malignant nature of the growing cells was proved by injecting cells into nude mice. Tumors resulted in 60% of the cases and the histology of each xenograft was similar to that of the human tumor. Flow cytometry was used to obtain DNA histograms of the original cell suspension and of cultures during the two week culture period in order to obtain quantitative information about the growth of aneuploid versus diploid populations. The results thus far demonstrate that 95% of aneuploid populations yield aneuploid growth; of the first 20 cases studied, only one suspension with an aneuploid peak resulted in diploid growth. Of further interest was the observation that it is not unusual for a minor aneuploid population to become the predominate growth fraction after two weeks in culture. These results demonstrate that the adhesive-tumor-cell culture system supports the growth of malignant cells, that multiple cell populations exist in cell suspensions derived from solid tumors, and that differences exist between the radiosensitivity of cells at 2.0 Gy in different histology types

  11. Non-specific interference of certain components of tissue culture media with the radioimmunoassay of rat alpha-foetoprotein

    International Nuclear Information System (INIS)

    Dambuyant, C.; Sizaret, Ph.

    1975-01-01

    Interferences of 'Williams' tissue culture medium used for cultivating rat hepatocytes upon rat alpha-foetoprotein (AFP) radioimmunoassay have been investigated. They are not due to foetal calf serum proteins which are added as growth factor and can be abolished by dialysis which appears to be necessary for the distinction between AFP non-producer and low-producer cell lines. Of the three major groups of non-mineral components examined, amino acid solution played a major role. When individual amino acids were examined using the double antibody technique, arginine was found to interfere predominantly; its dose-response curve was parallel to that of rat AFP which confirmed that an immunological identity between two substances cannot be established on the basis of parallelism as the only criterion

  12. Vitamin A and feeding statuses modulate the insulin-regulated gene expression in Zucker lean and fatty primary rat hepatocytes.

    Directory of Open Access Journals (Sweden)

    Wei Chen

    Full Text Available Unattended hepatic insulin resistance predisposes individuals to dyslipidemia, type 2 diabetes and many other metabolic complications. The mechanism of hepatic insulin resistance at the gene expression level remains unrevealed. To examine the effects of vitamin A (VA, total energy intake and feeding conditions on the insulin-regulated gene expression in primary hepatocytes of Zucker lean (ZL and fatty (ZF rats, we analyze the expression levels of hepatic model genes in response to the treatments of insulin and retinoic acid (RA. We report that the insulin- and RA-regulated glucokinase, sterol regulatory element-binding protein-1c and cytosolic form of phosphoenolpyruvate carboxykinase expressions are impaired in hepatocytes of ZF rats fed chow or a VA sufficient (VAS diet ad libitum. The impairments are partially corrected when ZF rats are fed a VA deficient (VAD diet ad libitum or pair-fed a VAS diet to the intake of their VAD counterparts in non-fasting conditions. Interestingly in the pair-fed ZL and ZF rats, transient overeating on the last day of pair-feeding regimen changes the expression levels of some VA catabolic genes, and impairs the insulin- and RA-regulated gene expression in hepatocytes. These results demonstrate that VA and feeding statuses modulate the hepatic insulin sensitivity at the gene expression level.

  13. Although it is rapidly metabolized in cultured rat hepatocytes, lauric acid is used for protein acylation.

    Science.gov (United States)

    Rioux, Vincent; Daval, Stéphanie; Guillou, Hervé; Jan, Sophie; Legrand, Philippe

    2003-01-01

    This study was designed to examine the metabolic fate of exogenous lauric acid in cultured rat hepatocytes, in terms of both lipid metabolism and acylation of proteins. Radiolabeled [14C]-lauric acid at 0.1 mM in the culture medium was rapidly taken up by the cells (94.8 +/- 2.2% of the initial radioactivity was cleared from the medium after a 4 h incubation) but its incorporation into cellular lipids was low (24.6 +/- 4.2% of initial radioactivity after 4 h), due to the high beta-oxidation of lauric acid in hepatocytes (38.7 +/- 4.4% after the same time). Among cellular lipids, lauric acid was preferentially incorporated into triglycerides (10.6 +/- 4.6% of initial radioactivity after 4 h). Lauric acid was also rapidly converted to palmitic acid by two successive elongations. Protein acylation was detected after metabolic labeling of the cells with [11,12-3H]-lauric acid. Two-dimensional electrophoresis separation of the cellular proteins and autoradiography evidenced the incorporation of radioactivity into 35 well-resolved proteins. Radiolabeling of several proteins resulted from covalent linkage to the precursor [11,12-3H]-lauric acid or to its elongation product, myristic acid. The covalent linkages between these proteins and lauric acid were broken by base hydrolysis, indicating that the linkage was of the thioester or ester-type. Endogenous myristic acid produced by lauric acid elongation was used for both protein N-myristoylation and protein S-acylation. Therefore, these results show for the first time that, although it is rapidly metabolized in hepatocytes, exogenous lauric acid is a substrate for the acylation of liver proteins.

  14. Full Length Bid is sufficient to induce apoptosis of cultured rat hippocampal neurons

    Directory of Open Access Journals (Sweden)

    Ward Manus W

    2007-02-01

    Full Text Available Abstract Background Bcl-2 homology domain (BH 3-only proteins are pro-apoptotic proteins of the Bcl-2 family that couple stress signals to the mitochondrial cell death pathways. The BH3-only protein Bid can be activated in response to death receptor activation via caspase 8-mediated cleavage into a truncated protein (tBid, which subsequently translocates to mitochondria and induces the release of cytochrome-C. Using a single-cell imaging approach of Bid cleavage and translocation during apoptosis, we have recently demonstrated that, in contrast to death receptor-induced apoptosis, caspase-independent excitotoxic apoptosis involves a translocation of full length Bid (FL-Bid from the cytosol to mitochondria. We induced a delayed excitotoxic cell death in cultured rat hippocampal neurons by a 5-min exposure to the glutamate receptor agonist N-methyl-D-aspartate (NMDA; 300 μM. Results Western blot experiments confirmed a translocation of FL-Bid to the mitochondria during excitotoxic apoptosis that was associated with the release of cytochrome-C from mitochondria. These results were confirmed by immunofluorescence analysis of Bid translocation during excitotoxic cell death using an antibody raised against the amino acids 1–58 of mouse Bid that is not able to detect tBid. Finally, inducible overexpression of FL-Bid or a Bid mutant that can not be cleaved by caspase-8 was sufficient to induce apoptosis in the hippocampal neuron cultures. Conclusion Our data suggest that translocation of FL-Bid is sufficient for the activation of mitochondrial cell death pathways in response to glutamate receptor overactivation.

  15. Characterization of rat primary trigeminal satellite glial cells and associated extracellular vesicles under normal and inflammatory conditions

    DEFF Research Database (Denmark)

    Vinterhøj, Hye Sook Han; Stensballe, Allan; Duroux, Meg

    2018-01-01

    that are proposed as novel biomarkers of SGCs activation under inflammatory conditions. Findings from this study highlight the utility of using fresh primary SGC cultures as a model to further investigate EVs under normal and inflammatory conditions. SIGNIFICANCE: This study demonstrated that....

  16. The occurrence of primary pulmonary neoplasms in rats after inhalation of 147Pm in fused aluminosilicate particles

    International Nuclear Information System (INIS)

    Herbert, R.A.; Scott, B.R.; Hahn, F.F.; Newton, G.J.; Snipes, M.B.; Damon, E.G.; Boecker, B.B.

    1988-01-01

    To determine the biological response following low-energy, beta irradiation of the lung, F344/Crl rats were exposed to aerosols of promethium-147 in fused aluminosilicate particles and observed for their life spans. Radiation pneumonitis and pulmonary fibrosis caused the majority of deaths during the first year after exposure with cumulative doses to the lungs of 210 to 630 Gy. Primary pulmonary neoplasms were responsible for the majority of deaths that occurred beyond 1 yr after exposure and in rats receiving lower cumulative doses to the lung. Hemangiosarcomas and squamous cell carcinomas were the most prevalent pulmonary neoplasms. Three adenocarcinomas were found. The uncorrected crude incidence of primary lung tumors increased with increasing dose to the lung for cumulative doses less than 140 Gy. With higher doses, the incidence declined. Adjusting the data for competing risks eliminated the turnover in the dose-response curve. The times of onset of pulmonary tumors and median survival times were dose-dependent. Rats with higher accumulated radiation doses developed fatal lung tumors at earlier times after exposure. (author)

  17. Degraded auditory processing in a rat model of autism limits the speech representation in non-primary auditory cortex.

    Science.gov (United States)

    Engineer, C T; Centanni, T M; Im, K W; Borland, M S; Moreno, N A; Carraway, R S; Wilson, L G; Kilgard, M P

    2014-10-01

    Although individuals with autism are known to have significant communication problems, the cellular mechanisms responsible for impaired communication are poorly understood. Valproic acid (VPA) is an anticonvulsant that is a known risk factor for autism in prenatally exposed children. Prenatal VPA exposure in rats causes numerous neural and behavioral abnormalities that mimic autism. We predicted that VPA exposure may lead to auditory processing impairments which may contribute to the deficits in communication observed in individuals with autism. In this study, we document auditory cortex responses in rats prenatally exposed to VPA. We recorded local field potentials and multiunit responses to speech sounds in primary auditory cortex, anterior auditory field, ventral auditory field. and posterior auditory field in VPA exposed and control rats. Prenatal VPA exposure severely degrades the precise spatiotemporal patterns evoked by speech sounds in secondary, but not primary auditory cortex. This result parallels findings in humans and suggests that secondary auditory fields may be more sensitive to environmental disturbances and may provide insight into possible mechanisms related to auditory deficits in individuals with autism. © 2014 Wiley Periodicals, Inc.

  18. Infection in a rat model reactivates attenuated virulence after long-term axenic culture of Acanthamoeba spp

    Directory of Open Access Journals (Sweden)

    Carolina De Marco Verissimo

    2013-11-01

    Full Text Available Prolonged culturing of many microorganisms leads to the loss of virulence and a reduction of their infective capacity. However, little is known about the changes in the pathogenic strains of Acanthamoeba after long culture periods. Our study evaluated the effect of prolonged culturing on the invasiveness of different isolates of Acanthamoeba in an in vivo rat model. ATCC strains of Acanthamoeba, isolates from the environment and clinical cases were evaluated. The in vivo model was effective in establishing the infection and differentiating the pathogenicity of the isolates and re-isolates. The amoebae cultured in the laboratory for long periods were less virulent than those that were recently isolated, confirming the importance of passing Acanthamoeba strains in animal models.

  19. African American Identity and a Theory for Primary Cultural Instructional Design

    Science.gov (United States)

    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…

  20. Exposure to low-dose bisphenol A impairs meiosis in the rat seminiferous tubule culture model: a physiotoxicogenomic approach.

    Directory of Open Access Journals (Sweden)

    Sazan Ali

    Full Text Available BACKGROUND: Bisphenol A (BPA is one of the most widespread chemicals in the world and is suspected of being responsible for male reproductive impairments. Nevertheless, its molecular mode of action on spermatogenesis is unclear. This work combines physiology and toxicogenomics to identify mechanisms by which BPA affects the timing of meiosis and induces germ-cell abnormalities. METHODS: We used a rat seminiferous tubule culture model mimicking the in vivo adult rat situation. BPA (1 nM and 10 nM was added to the culture medium. Transcriptomic and meiotic studies were performed on the same cultures at the same exposure times (days 8, 14, and 21. Transcriptomics was performed using pangenomic rat microarrays. Immunocytochemistry was conducted with an anti-SCP3 antibody. RESULTS: The gene expression analysis showed that the total number of differentially expressed transcripts was time but not dose dependent. We focused on 120 genes directly involved in the first meiotic prophase, sustaining immunocytochemistry. Sixty-two genes were directly involved in pairing and recombination, some of them with high fold changes. Immunocytochemistry indicated alteration of meiotic progression in the presence of BPA, with increased leptotene and decreased diplotene spermatocyte percentages and partial meiotic arrest at the pachytene checkpoint. Morphological abnormalities were observed at all stages of the meiotic prophase. The prevalent abnormalities were total asynapsis and apoptosis. Transcriptomic analysis sustained immunocytological observations. CONCLUSION: We showed that low doses of BPA alter numerous genes expression, especially those involved in the reproductive system, and severely impair crucial events of the meiotic prophase leading to partial arrest of meiosis in rat seminiferous tubule cultures.

  1. Influence of anatomic site and age on the replication and differentiation of rat adipocyte precursors in culture.

    OpenAIRE

    Djian, P; Roncari, A K; Hollenberg, C H

    1983-01-01

    Using a propagating cell culture system of adipocyte precursors from 70-400-g rats, we explored the possibility that regional variations in properties of adipose tissue may reflect site-specific characteristics intrinsic to the cells, rather than extracellular influences. Initially, studies were made of the nature of the fibroblastlike cells from perirenal adipose tissue stroma. Using colony-forming techniques, it was shown that these cells were adipocyte precursors; each confluent colony tha...

  2. Effects of Corticosterone on Immune Functions of Cultured Rat Splenic Lymphocytes Exposed to Aluminum Trichloride.

    Science.gov (United States)

    Yang, Xu; Xu, Feibo; Zhuang, Cuicui; Bai, Chongsheng; Huang, Wanyue; Song, Miao; Han, Yanfei; Li, Yanfei

    2016-10-01

    Aluminum (Al) exposure is toxic to immune system. Studies have implicated that glucocorticoids (GCs) exert the dual regulation effect on the immune function depending on the concentration. However, it is unknown whether a dual effect of GCs exists in the AlCl3-treated lymphocytes. Corticosterone (Cort) is one kind of GCs. To investigate the effect of different concentration Cort on AlCl3-treated lymphocytes, rat splenic lymphocyte was isolated and cultured with 0.55 mmol/L AlCl3, simultaneously administrated Cort at final concentration of 0 (control group, CG), 10(-8) (low-level group, LG), and 10(-6) (high-level group, HG) mol/L, respectively. Another group without AlCl3 and Cort served as the blank group (BG). We found that low concentration Cort increased the T and B lymphocyte proliferation rate, proportions of CD4(+) T lymphocyte subset, IgG, IL-2, and TNF-α contents, whereas high concentration Cort decreased those in AlCl3-treated lymphocytes. In conclusion, the results of this study indicated that low concentration Cort relieves the immunotoxicity of AlCl3 on the splenic lymphocytes, whereas high concentration Cort aggravates it.

  3. Application of a metabolizing system as an adjunct to the rat whole embryo culture.

    Science.gov (United States)

    Luijten, Mirjam; Verhoef, Aart; Westerman, Anja; Piersma, Aldert H

    2008-08-01

    Rodent post-implantation whole embryo culture (WEC) is a validated and widely used method in both mechanistic studies and as a screening test for developmental toxicants. Since metabolism is lacking in the WEC because of the developmental stage of the embryo, pro-teratogenic compounds are not detected. We investigated the inclusion of an in vitro metabolizing system as a pre-incubation step in the existing WEC. We started by testing cyclophosphamide, a known pro-teratogen. Without metabolic activation, cyclophosphamide is not teratogenic to rat embryos, as evidenced by a lack of effect on either embryonic growth or on morphological development. After pre-incubation with Aroclor-induced hepatic microsomes, cyclophosphamide became highly embryotoxic in the WEC. We tested five additional compounds to prevalidate this method: valpromide, 2-acetylaminofluorene, 2-methoxyethanol, retinol, and benzo[a]pyrene. Results revealed that not all of these five compounds underwent (complete) metabolic activation. This is probably due to the fact that microsomes do not contain the full spectrum of hepatic enzymes. Attempts have been made to replace microsomes by S9 liver fractions, which do contain microsomal enzymes as well as a series of cytoplasmic enzymes. However, S9 appeared to be extremely toxic in the WEC. Future experiments have to be performed to explore alternative ways of complete metabolic activation.

  4. Role of dopamine D2 receptors in ischemia/reperfusion induced apoptosis of cultured neonatal rat cardiomyocytes

    Directory of Open Access Journals (Sweden)

    Zhao Ya-jun

    2011-02-01

    Full Text Available Abstract Background Myocardial ischemia/reperfusion injury is the major cause of morbidity and mortality for cardiovascular diseases. Dopamine D2 receptors are expressed in cardiac tissues. However, the roles of dopamine D2 receptors in myocardial ischemia/reperfusion injury and cardiomyocyte apoptosis are unclear. Here we investigated the effects of both dopamine D2 receptors agonist (bromocriptine and antagonist (haloperidol on apoptosis of cultured neonatal rat ventricular myocytes induced by ischemia/reperfusion injury. Methods Myocardial ischemia/reperfusion injury was simulated by incubating primarily cultured neonatal rat cardiomyocytes in ischemic (hypoxic buffer solution for 2 h. Thereafter, these cells were incubated for 24 h in normal culture medium. Results Treatment of the cardiomyocytes with 10 μM bromocriptine significantly decreased lactate dehydrogenase activity, increased superoxide dismutase activity, and decreased malondialdehyde content in the culture medium. Bromocriptine significantly inhibited the release of cytochrome c, accumulation of [Ca2+]i, and apoptosis induced by ischemia/reperfusion injury. Bromocriptine also down-regulated the expression of caspase-3 and -9, Fas and Fas ligand, and up-regulated Bcl-2 expression. In contrast, haloperidol (10 μM had no significant effects on the apoptosis of cultured cardiomyocytes under the aforementioned conditions. Conclusions These data suggest that activation of dopamine D2 receptors can inhibit apoptosis of cardiomyocytes encountered during ischemia/reperfusion damage through various pathways.

  5. Efficacy of various durations of in vitro predegeneration on the cell count and purity of rat Schwann-cell cultures.

    Science.gov (United States)

    Kraus, Armin; Täger, Joachim; Kohler, Konrad; Manoli, Theodora; Haerle, Max; Werdin, Frank; Hoffmann, Jürgen; Schaller, Hans-Eberhard; Sinis, Nektarios

    2010-01-01

    The efficacy of Schwann-cell cultivation can be enhanced by in vitro predegeneration of the harvested cells compared to immediate culture. The aim of this study was to improve Schwann-cell culture efficacy by comparing three different durations of predegeneration. The sciatic and median nerves of 6-8-week-old Lewis rats were harvested and subjected to either 2-day, 7-day, or 14-day predegeneration in Dulbecco's Modified Eagle's Medium supplemented with 10% fetal calf serum and 1% Penicillin/Streptomycin. Afterward, tissue was enzymatically dissociated and placed in a modified melanocyte growth medium. The cell count was determined immediately after dissociation while the cell purity was determined one subculture/trypsinization cycle later after cell attachment to the culture plate by means of optical microscopy and immunocytochemistry. Particular attention was then paid to the Schwann-cell-to-fibroblast relation. The cumulative cell count in the culture was 5.8 x 10(5) for 2-day, 1.12 x 10(6) for 7-day, and 1.48 x 10(6) for 14-day predegeneration. The culture purity was approximately equal for 2- and 7-day predegeneration (88% Schwann cells, 12% fibroblasts after 2 days; 85% Schwann cells, 15% fibroblasts after 7 days). After 14 days, however, cell cultures were significantly debased by fibroblast proliferation (57% Schwann cells, 43% fibroblasts). In vitro predegeneration is a particularly suitable procedural method to increase the cultural Schwann-cell yield. The number of cultivated rat Schwann cells is doubled by 7-day in vitro predegeneration in comparison to 2-day predegeneration. After 14-day predegeneration, however, the culture is significantly debased by fibroblasts. Therefore, 7-day in vitro predegeneration is an advisable predegeneration period.

  6. Short-term spheroid culture of primary colorectal cancer cells as an in vitro model for personalizing cancer medicine

    DEFF Research Database (Denmark)

    Jeppesen, Maria; Hagel, Grith; Glenthoj, Anders

    2017-01-01

    Chemotherapy treatment of cancer remains a challenge due to the molecular and functional heterogeneity displayed by tumours originating from the same cell type. The pronounced heterogeneity makes it difficult for oncologists to devise an effective therapeutic strategy for the patient. One approach...... for increasing treatment efficacy is to test the chemosensitivity of cancer cells obtained from the patient's tumour. 3D culture represents a promising method for modelling patient tumours in vitro. The aim of this study was therefore to evaluate how closely short-term spheroid cultures of primary colorectal...... cancer cells resemble the original tumour. Colorectal cancer cells were isolated from human tumour tissue and cultured as spheroids. Spheroid cultures were established with a high success rate and remained viable for at least 10 days. The spheroids exhibited significant growth over a period of 7 days...

  7. Histamine modulates contraction and cyclic nucleotides in cultured rat mesangial cells. Differential effects mediated by histamine H1 and H2 receptors.

    OpenAIRE

    Sedor, J R; Abboud, H E

    1985-01-01

    Histamine influences the glomerular microcirculation and modulates immune-inflammatory responses. In the rat kidney, histamine is synthesized by glomeruli and stimulates cyclic nucleotide production specifically in glomeruli. We investigated the in vitro effect of histamine on cyclic nucleotide accumulation in rat cultured glomerular mesangial and epithelial cells. Histamine stimulated cyclic AMP (cAMP) accumulation in cultured mesangial cells (64.0 +/- 22.1 to 511.4 +/- 86.6 pmol/mg protein,...

  8. Regional Susceptibility to Domoic Acid in Primary Astrocyte Cells Cultured from the Brain Stem and Hippocampus

    Directory of Open Access Journals (Sweden)

    Olga M. Pulido

    2008-02-01

    Full Text Available Domoic acid is a marine biotoxin associated with harmful algal blooms and is the causative agent of amnesic shellfish poisoning in marine animals and humans. It is also an excitatory amino acid analog to glutamate and kainic acid which acts through glutamate receptors eliciting a very rapid and potent neurotoxic response. The hippocampus, among other brain regions, has been identified as a specific target site having high sensitivity to DOM toxicity. Histopathology evidence indicates that in addition to neurons, the astrocytes were also injured. Electron microscopy data reported in this study further supports the light microscopy findings. Furthermore, the effect of DOM was confirmed by culturing primary astrocytes from the hippocampus and the brain stem and subsequently exposing them to domoic acid. The RNA was extracted and used for biomarker analysis. The biomarker analysis was done for the early response genes including c-fos, c-jun, c-myc, Hsp-72; specific marker for the astrocytes- GFAP and the glutamate receptors including GluR 2, NMDAR 1, NMDAR 2A and B. Although, the astrocyte-GFAP and c-fos were not affected, c-jun and GluR 2 were down-regulated. The microarray analysis revealed that the chemokines / cytokines, tyrosine kinases (Trk, and apoptotic genes were altered. The chemokines that were up-regulated included - IL1-a, IL-1B, IL-6, the small inducible cytokine, interferon protein IP-10, CXC chemokine LIX, and IGF binding proteins. The Bax, Bcl-2, Trk A and Trk B were all downregulated. Interestingly, only the hippocampal astrocytes were affected. Our findings suggest that astrocytes may present a possible target for pharmacological interventions for the prevention and treatment of amnesic shellfish poisoning and for other brain pathologies involving excitotoxicity

  9. Hexane extract from Polygonum multiflorum attenuates glutamate-induced apoptosis in primary cultured cortical neurons.

    Science.gov (United States)

    Jang, Ji Yeon; Kim, Ha Neui; Kim, Yu Ri; Choi, Young Whan; Choi, Yung Hyun; Lee, Jun Hyuk; Shin, Hwa Kyoung; Choi, Byung Tae

    2013-01-09

    Polygonum multiflorum has traditionally had wide use as an anti-aging treatment in East Asian countries. We investigated the neuroprotective effects of Polygonum multiflorum against glutamate-induced neurotoxicity with a focus on the anti-apoptotic mechanism in primary cultured cortical neurons. Cell viability, cytotoxicity, morphological, flow cytometry, Western blot, and caspase activity assays were performed for examination of the neuroprotective effects of active hexane extract from Polygonum multiflorum (HEPM). Pretreatment with HEPM resulted in significantly decreased glutamate-induced neurotoxicity in a concentration-dependent manner and also resulted in drastically inhibited glutamate-induced apoptosis. Treatment with HEPM resulted in decreased expression of glutamate-induced death receptor (DR)4, and enhanced expression of glutamate-attenuated anti-apoptotic proteins, including Bcl-2, XIAP, and cIAP-1, and slightly reduced glutamate-induced cleavage of Bid. In addition, treatment with HEPM resulted in suppressed glutamate-induced activation of caspase-8, caspase-9, and caspase-3, and, subsequently, decreased degradation of poly(ADP-ribose) polymerase, β-catenin, and phospholipase Cγ1 protein, which are downstream targets of activated caspase-3. The results of this study demonstrated that HEPM exerts a neuroprotective effect against glutamate-induced neurotoxicity via inhibi