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Sample records for cultured mouse hepatocytes

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

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    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.

  2. Effects of clofibric acid on mRNA expression profiles in primary cultures of rat, mouse and human hepatocytes

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    Richert, Lysiane; Lamboley, Christelle; Viollon-Abadie, Catherine; Grass, Peter; Hartmann, Nicole; Laurent, Stephane; Heyd, Bruno; Mantion, Georges; Chibout, Salah-Dine; Staedtler, Frank

    2003-01-01

    The mRNA expression profile in control and clofibric acid (CLO)-treated mouse, rat, and human hepatocytes was analyzed using species-specific oligonucleotide DNA microarrays (Affymetrix). A statistical empirical Bayes procedure was applied in order to select the significantly differentially expressed genes. Treatment with the peroxisome proliferator CLO induced up-regulation of genes involved in peroxisome proliferation and in cell proliferation as well as down-regulation of genes involved in apoptosis in hepatocytes of rodent but not of human origin. CLO treatment induced up-regulation of microsomal cytochrome P450 4a genes in rodent hepatocytes and in two of six human hepatocyte cultures. In addition, genes encoding phenobarbital-inducible cytochrome P450s were also up-regulated by CLO in rodent and human hepatocyte cultures. Up-regulation of phenobarbital-inducible UDP-glucuronosyl-transferase genes by CLO was observed in both rat and human but not in mouse hepatocytes. CLO treatment induced up-regulation of L-fatty acid binding protein (L-FABP) gene in hepatocytes of both rodent and human origin. However, while genes of the cytosolic, microsomal, and mitochondrial pathways involved in fatty acid transport and metabolism were up-regulated by CLO in both rodent and human hepatocyte cultures, genes of the peroxisomal pathway of lipid metabolism were up-regulated in rodents only. An up-regulation of hepatocyte nuclear factor 1α (HNF1α) by CLO was observed only in human hepatocyte cultures, suggesting that this trans-activating factor may play a key role in the regulation of fatty acid metabolism in human liver as well as in the nonresponsiveness of human liver to CLO-induced regulation of cell proliferation and apoptosis

  3. Effects of clofibric acid on mRNA expression profiles in primary cultures of rat, mouse and human hepatocytes.

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    Richert, Lysiane; Lamboley, Christelle; Viollon-Abadie, Catherine; Grass, Peter; Hartmann, Nicole; Laurent, Stephane; Heyd, Bruno; Mantion, Georges; Chibout, Salah-Dine; Staedtler, Frank

    2003-09-01

    The mRNA expression profile in control and clofibric acid (CLO)-treated mouse, rat, and human hepatocytes was analyzed using species-specific oligonucleotide DNA microarrays (Affymetrix). A statistical empirical Bayes procedure was applied in order to select the significantly differentially expressed genes. Treatment with the peroxisome proliferator CLO induced up-regulation of genes involved in peroxisome proliferation and in cell proliferation as well as down-regulation of genes involved in apoptosis in hepatocytes of rodent but not of human origin. CLO treatment induced up-regulation of microsomal cytochrome P450 4a genes in rodent hepatocytes and in two of six human hepatocyte cultures. In addition, genes encoding phenobarbital-inducible cytochrome P450s were also up-regulated by CLO in rodent and human hepatocyte cultures. Up-regulation of phenobarbital-inducible UDP-glucuronosyl-transferase genes by CLO was observed in both rat and human but not in mouse hepatocytes. CLO treatment induced up-regulation of L-fatty acid binding protein (L-FABP) gene in hepatocytes of both rodent and human origin. However, while genes of the cytosolic, microsomal, and mitochondrial pathways involved in fatty acid transport and metabolism were up-regulated by CLO in both rodent and human hepatocyte cultures, genes of the peroxisomal pathway of lipid metabolism were up-regulated in rodents only. An up-regulation of hepatocyte nuclear factor 1alpha (HNF1alpha) by CLO was observed only in human hepatocyte cultures, suggesting that this trans-activating factor may play a key role in the regulation of fatty acid metabolism in human liver as well as in the nonresponsiveness of human liver to CLO-induced regulation of cell proliferation and apoptosis.

  4. Intracellular cholesterol-binding proteins enhance HDL-mediated cholesterol uptake in cultured primary mouse hepatocytes.

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    Storey, Stephen M; McIntosh, Avery L; Huang, Huan; Landrock, Kerstin K; Martin, Gregory G; Landrock, Danilo; Payne, H Ross; Atshaves, Barbara P; Kier, Ann B; Schroeder, Friedhelm

    2012-04-15

    A major gap in our knowledge of rapid hepatic HDL cholesterol clearance is the role of key intracellular factors that influence this process. Although the reverse cholesterol transport pathway targets HDL to the liver for net elimination of free cholesterol from the body, molecular details governing cholesterol uptake into hepatocytes are not completely understood. Therefore, the effects of sterol carrier protein (SCP)-2 and liver fatty acid-binding protein (L-FABP), high-affinity cholesterol-binding proteins present in hepatocyte cytosol, on HDL-mediated free cholesterol uptake were examined using gene-targeted mouse models, cultured primary hepatocytes, and 22-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-amino]-23,24-bisnor-5-cholen-3β-ol (NBD-cholesterol). While SCP-2 overexpression enhanced NBD-cholesterol uptake, counterintuitively, SCP-2/SCP-x gene ablation also 1) enhanced the rapid molecular phase of free sterol uptake detectable in rate and maximal uptake of HDL free cholesterol and 2) differentially enhanced free cholesterol uptake mediated by the HDL3, rather than the HDL2, subfraction. The increased HDL free cholesterol uptake was not due to increased expression or distribution of the HDL receptor [scavenger receptor B1 (SRB1)], proteins regulating SRB1 [postsynaptic density protein (PSD-95)/Drosophila disk large tumor suppressor (dlg)/tight junction protein (ZO1) and 17-kDa membrane-associated protein], or other intracellular cholesterol trafficking proteins (steroidogenic acute response protein D, Niemann Pick C, and oxysterol-binding protein-related proteins). However, expression of L-FABP, the single most prevalent hepatic cytosolic protein that binds cholesterol, was upregulated twofold in SCP-2/SCP-x null hepatocytes. Double-immunogold electron microscopy detected L-FABP sufficiently close to SRB1 for direct interaction, similar to SCP-2. These data suggest a role for L-FABP in HDL cholesterol uptake, a finding confirmed with SCP-2/SCP-x/L-FABP null

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

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    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.

  6. Repolarization of hepatocytes in culture.

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    Talamini, M A; Kappus, B; Hubbard, A

    1997-01-01

    We have evaluated the biochemical, morphological, and functional redevelopment of polarity in freshly isolated hepatocytes cultured using a double layer collagen gel sandwich technique. Western blot analysis showed increased cellular levels of the cell adhesion protein uvomorulin as cultured hepatocytes repolarized. Immunofluorescence studies using antibodies against domain-specific membrane proteins showed polarity as early as 48 hours, although the pattern of the polymeric Immunoglobulin-A receptor (pIgA-R) differed from in vivo liver. Electron microscopy showed developing bile canaliculi at 1 day. However, the functional presence of tight junctions was absent at 1 day, but present at 5 days. We further showed functional polarity to be present at 4 days by documenting the ability of cultured hepatocytes to metabolize and excrete fluorescein diacetate into visible bile canaliculi. We conclude that hepatocytes cultured appropriately develop morphological and functional polarity. Hepatocyte culture is therefore a useful tool for the study of mechanisms responsible for the development of polarized function.

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

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    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.

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

    International Nuclear Information System (INIS)

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

    2010-01-01

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

  9. Evaluation of perfluoroalkyl acid activity using primary mouse and human hepatocytes

    International Nuclear Information System (INIS)

    Rosen, Mitchell B.; Das, Kaberi P.; Wood, Carmen R.; Wolf, Cynthia J.; Abbott, Barbara D.; Lau, Christopher

    2013-01-01

    While perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) have been studied at length, less is known about the biological activity of other perfluoroalkyl acids (PFAAs) detected in the environment. Using a transient transfection assay developed in COS-1 cells, our group has previously evaluated a variety of PFAAs for activity associated with activation of peroxisome proliferator-activated receptor alpha (PPARα). Here we use primary heptatocytes to further assess the biological activity of a similar group of PFAAs using custom designed Taqman Low Density Arrays. Primary mouse and human hepatoyctes were cultured for 48 h in the presence of varying concentrations of 12 different PFAAs or Wy14,643, a known activator of PPARα. Total RNA was collected and the expression of 48 mouse or human genes evaluated. Gene selection was based on either in-house liver microarray data (mouse) or published data using primary hepatocytes (human). Gene expression in primary mouse hepatocytes was more restricted than expected. Genes typically regulated in whole tissue by PPARα agonists were not altered in mouse cells including Acox1, Me1, Acaa1a, Hmgcs1, and Slc27a1. Cyp2b10, a gene regulated by the constitutive androstane receptor and a transcript normally up-regulated by in vivo exposure to PFAAs, was also unchanged in cultured mouse hepatocytes. Cyp4a14, Ehhadh, Pdk4, Cpt1b, and Fabp1 were regulated as expected in mouse cells. A larger group of genes were differentially expressed in human primary hepatocytes, however, little consistency was observed across compounds with respect to which genes produced a significant dose response making the determination of relative biological activity difficult. This likely reflects weaker activation of PPARα in human versus rodent cells as well as variation among individual cell donors. Unlike mouse cells, CYP2B6 was up-regulated in human hepatocytes by a number of PFAAs as was PPARδ. Rankings were conducted on the limited

  10. Definition of metabolism-dependent xenobiotic toxicity with co-cultures of human hepatocytes and mouse 3T3 fibroblasts in the novel integrated discrete multiple organ co-culture (IdMOC) experimental system: results with model toxicants aflatoxin B1, cyclophosphamide and tamoxifen.

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    Li, Albert P; Uzgare, Aarti; LaForge, Yumiko S

    2012-07-30

    The integrated discrete multiple organ co-culture system (IdMOC) allows the co-culturing of multiple cell types as physically separated cells interconnected by a common overlying medium. We report here the application of IdMOC with two cell types: the metabolically competent primary human hepatocytes, and a metabolically incompetent cell line, mouse 3T3 fibroblasts, in the definition of the role of hepatic metabolism on the cytotoxicity of three model toxicants: cyclophosphamide (CPA), aflatoxin B1 (AFB) and tamoxifen (TMX). The presence of hepatic metabolism in IdMOC with human hepatocytes was demonstrated by the metabolism of the P450 isoform 3A4 substrate, luciferin-IPA. The three model toxicants showed three distinct patterns of cytotoxic profile: TMX was cytotoxic to 3T3 cells in the absence of hepatocytes, with slightly lower cytotoxicity towards both 3T3 cells and hepatocytes in the IdMOC. AFB was selective toxic towards the human hepatocytes and relatively noncytotoxic towards 3T3 cells both in the presence and absence of the hepatocytes. CPA cytotoxicity to the 3T3 cells was found to be significantly enhanced by the presence of the hepatocytes, with the cytotoxicity dependent of the number of hepatocytes, and with the cytotoxicity attenuated by the presence of a non-specific P450 inhibitor, 1-aminobenzotriazole. We propose here the following classification of toxicants based on the role of hepatic metabolism as defined by the human hepatocyte-3T3 cell IdMOC assay: type I: direct-acting cytotoxicants represented by TMX as indicated by cytotoxicity in 3T3 cells in the absence of hepatocytes; type II: metabolism-dependent cytotoxicity represented by AFB1 with effects localized within the site of metabolic activation (i. e. hepatocytes); and type III: metabolism-dependent cytotoxicity with metabolites that can diffuse out of the hepatocytes to cause toxicity in cells distal from the site of metabolism, as exemplified by CPA. Copyright © 2012 Elsevier Ireland

  11. Acquisition of lipid metabolic capability in hepatocyte-like cells directly induced from mouse fibroblasts

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    Shizuka eMiura

    2014-08-01

    Full Text Available Recently, the numbers of patients with non-alcoholic fatty liver disease (NAFLD and non-alcoholic steatohepatitis (NASH have increased worldwide. NAFLD and NASH are known as risk factors for liver cirrhosis and hepatocellular carcinoma. Because many factors can promote the progression of NAFLD and NASH, the treatment of these patients involves various strategies. Thus, it is desired that drugs for patients with NAFLD and NASH should be developed more easily and rapidly using cultures of primary hepatocytes. However, it is difficult to use hepatocytes as a tool for drug screening, because these cells cannot be functionally maintained in culture. Thus, in this study, we sought to examine whether induced hepatocyte-like (iHep cells, which were directly induced from mouse dermal fibroblasts by infection with a retrovirus expressing Hnf4α and Foxa3, possess the potential for lipid metabolism, similar to hepatocytes. Our data showed that iHep cells were capable of synthesizing lipids from a cis-unsaturated fatty acid, a trans-unsaturated fatty acid, and a saturated fatty acid, accumulating the synthesized lipids in cellular vesicles, and secreting the lipids into the culture medium. Moreover, the lipid synthesis in iHep cells was significantly inhibited in cultures with lipid metabolism improvers. These results demonstrate that iHep cells could be useful not only for screening of drugs for patients with NAFLD and NASH, but also for elucidation of the mechanisms underlying hereditary lipid metabolism disorders, as an alternative to hepatocytes.

  12. In vitro differentiation and maturation of mouse embryonic stem cells into hepatocytes

    International Nuclear Information System (INIS)

    Ishii, Takamichi; Yasuchika, Kentaro; Fujii, Hideaki; Hoppo, Toshitaka; Baba, Shinji; Naito, Masato; Machimoto, Takafumi; Kamo, Naoko; Suemori, Hirofumi; Nakatsuji, Norio; Ikai, Iwao

    2005-01-01

    It is difficult to induce the maturation of embryonic stem (ES) cells into hepatocytes in vitro. We previously reported that Thy1-positive mesenchymal cells derived from the mouse fetal liver promote the maturation of hepatic progenitor cells. Here, we isolated alpha-fetoprotein (AFP)-producing cells from mouse ES cells for subsequent differentiation into hepatocytes in vitro by coculture with Thy1-positive cells. ES cells expressing green fluorescent protein (GFP) under the control of an AFP promoter were cultured under serum- and feeder layer-free culture conditions. The proportion of GFP-positive cells plateaued at 41.6 ± 12.2% (means ± SD) by day 7. GFP-positive cells, isolated by flow cytometry, were cultured in the presence or absence of Thy1-positive cells as a feeder layer. Isolated GFP-positive cells were stained for AFP, Foxa2, and albumin. The expression of mRNAs encoding tyrosine amino transferase, tryptophan 2,3-dioxygenase, and glucose-6-phosphatase were only detected following coculture with Thy1-positive cells. Following coculture with Thy1-positive cells, the isolated cells produced and stored glycogen. Ammonia clearance activity was also enhanced following coculture. Electron microscopic analysis indicated that the cocultured cells exhibited the morphologic features of mature hepatocytes. In conclusion, coculture with Thy1-positive cells in vitro induced the maturation of AFP-producing cells isolated from ES cell cultures into hepatocytes

  13. Gender differences in methionine accumulation and metabolism in freshly isolated mouse hepatocytes: Potential roles in toxicity

    International Nuclear Information System (INIS)

    Dever, Joseph T.; Elfarra, Adnan A.

    2009-01-01

    L-Methionine (Met) is hepatotoxic at high concentrations. Because Met toxicity in freshly isolated mouse hepatocytes is gender-dependent, the goal of this study was to assess the roles of Met accumulation and metabolism in the increased sensitivity of male hepatocytes to Met toxicity compared with female hepatocytes. Male hepatocytes incubated with Met (30 mM) at 37 o C exhibited higher levels of intracellular Met at 0.5, 1.0, and 1.5 h, respectively, compared to female hepatocytes. Conversely, female hepatocytes had higher levels of S-adenosyl-L-methionine compared to male hepatocytes. Female hepatocytes also exhibited higher L-methionine-L-sulfoxide levels relative to control hepatocytes, whereas the increases in L-methionine-D-sulfoxide (Met-D-O) levels were similar in hepatocytes of both genders. Addition of aminooxyacetic acid (AOAA), an inhibitor of Met transamination, significantly increased Met levels at 1.5 h and increased Met-D-O levels at 1.0 and 1.5 h only in Met-exposed male hepatocytes. No gender differences in cytosolic Met transamination activity by glutamine transaminase K were detected. However, female mouse liver cytosol exhibited higher methionine-DL-sulfoxide (MetO) reductase activity than male mouse liver cytosol at low (0.25 and 0.5 mM) MetO concentrations. Collectively, these results suggest that increased cellular Met accumulation, decreased Met transmethylation, and increased Met and MetO transamination in male mouse hepatocytes may be contributing to the higher sensitivity of the male mouse hepatocytes to Met toxicity in comparison with female mouse hepatocytes.

  14. RNA synthesis in primary cultures of adult rat hepatocytes

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    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

  15. Impaired mitochondrial functions contribute to 3-bromopyruvate toxicity in primary rat and mouse hepatocytes.

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    Sobotka, Ondřej; Endlicher, René; Drahota, Zdeněk; Kučera, Otto; Rychtrmoc, David; Raad, Marjan; Hakeem, Khurum; Červinková, Zuzana

    2016-08-01

    A compound with promising anticancer properties, 3-bromopyruvate (3-BP) is a synthetic derivative of a pyruvate molecule; however, its toxicity in non-malignant cells has not yet been fully elucidated. Therefore, we elected to study the effects of 3-BP on primary hepatocytes in monolayer cultures, permeabilized hepatocytes and isolated mitochondria. After a 1-h treatment with 100 μM 3-BP cell viability of rat hepatocytes was decreased by 30 % as measured by the WST-1 test (p < 0.001); after 3-h exposure to ≥200 μM 3-BP lactate dehydrogenase leakage was increased (p < 0.001). Reactive oxygen species production was increased in the cell cultures after a 1-h treatment at concentrations ≥100 μmol/l (p < 0.01), and caspase 3 activity was increased after a 20-h incubation with 150 μM and 200 μM 3-BP (p < 0.001). This toxic effect of 3-BP was also proved using primary mouse hepatocytes. In isolated mitochondria, 3-BP induced a dose- and time-dependent decrease of mitochondrial membrane potential during a 10-min incubation both with Complex I substrates glutamate + malate or Complex II substrate succinate, although this decrease was more pronounced with the latter. We also measured the effect of 3-BP on respiration of isolated mitochondria. ADP-activated respiration was inhibited by 20 μM 3-BP within 10 min. Similar effects were also found in permeabilized hepatocytes of both species.

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

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    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.

  17. Micropatterned co-culture of hepatocyte spheroids layered on non-parenchymal cells to understand heterotypic cellular interactions

    International Nuclear Information System (INIS)

    Otsuka, Hidenori; Sasaki, Kohei; Okimura, Saya; Nagamura, Masako; Nakasone, Yuichi

    2013-01-01

    Microfabrication and micropatterning techniques in tissue engineering offer great potential for creating and controlling cellular microenvironments including cell–matrix interactions, soluble stimuli and cell–cell interactions. Here, we present a novel approach to generate layered patterning of hepatocyte spheroids on micropatterned non-parenchymal feeder cells using microfabricated poly(ethylene glycol) (PEG) hydrogels. Micropatterned PEG-hydrogel-treated substrates with two-dimensional arrays of gelatin circular domains (ϕ = 100 μm) were prepared by photolithographic method. Only on the critical structure of PEG hydrogel with perfect protein rejection, hepatocytes were co-cultured with non-parenchymal cells to be led to enhanced hepatocyte functions. Then, we investigated the mechanism of the functional enhancement in co-culture with respect to the contributions of soluble factors and direct cell–cell interactions. In particular, to elucidate the influence of soluble factors on hepatocyte function, hepatocyte spheroids underlaid with fibroblasts (NIH/3T3 mouse fibroblasts) or endothelial cells (BAECs: bovine aortic endothelial cells) were compared with physically separated co-culture of hepatocyte monospheroids with NIH3T3 or BAEC using trans-well culture systems. Our results suggested that direct heterotypic cell-to-cell contact and soluble factors, both of these between hepatocytes and fibroblasts, significantly enhanced hepatocyte functions. In contrast, direct heterotypic cell-to-cell contact between hepatocytes and endothelial cells only contributed to enhance hepatocyte functions. This patterning technique can be a useful experimental tool for applications in basic science, drug screening and tissue engineering, as well as in the design of artificial liver devices. (paper)

  18. Radiation-induced PKC signaling system in cultured rat hepatocytes

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    Nakajima, Tetsuo; Yukawa, Osami

    1998-01-01

    Radiation effects on living organisms are mainly caused through reactive oxygen species (ROS) on living cells. It is known that ROS damages various membranes and the bio membranes play an important role in cellular signal transduction pathways. The effects of radiation on cellular signal transduction pathways in cultured rat hepatocytes have been studied

  19. Hepatocyte Hypoxia Inducible Factor-1 Mediates the Development of Liver Fibrosis in a Mouse Model of Nonalcoholic Fatty Liver Disease.

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    Omar A Mesarwi

    Full Text Available Obstructive sleep apnea (OSA is associated with the progression of non-alcoholic fatty liver disease (NAFLD to steatohepatitis and fibrosis. This progression correlates with the severity of OSA-associated hypoxia. In mice with diet induced obesity, hepatic steatosis leads to liver tissue hypoxia, which worsens with exposure to intermittent hypoxia. Emerging data has implicated hepatocyte cell signaling as an important factor in hepatic fibrogenesis. We hypothesized that hepatocyte specific knockout of the oxygen sensing α subunit of hypoxia inducible factor-1 (HIF-1, a master regulator of the global response to hypoxia, may be protective against the development of liver fibrosis.Wild-type mice and mice with hepatocyte-specific HIF-1α knockout (Hif1a-/-hep were fed a high trans-fat diet for six months, as a model of NAFLD. Hepatic fibrosis was evaluated by Sirius red stain and hydroxyproline assay. Liver enzymes, fasting insulin, and hepatic triglyceride content were also assessed. Hepatocytes were isolated from Hif1a-/-hep mice and wild-type controls and were exposed to sustained hypoxia (1% O2 or normoxia (16% O2 for 24 hours. The culture media was used to reconstitute type I collagen and the resulting matrices were examined for collagen cross-linking.Wild-type mice on a high trans-fat diet had 80% more hepatic collagen than Hif1a-/-hep mice (2.21 μg collagen/mg liver tissue, versus 1.23 μg collagen/mg liver tissue, p = 0.03, which was confirmed by Sirius red staining. Body weight, liver weight, mean hepatic triglyceride content, and fasting insulin were similar between groups. Culture media from wild-type mouse hepatocytes exposed to hypoxia allowed for avid collagen cross-linking, but very little cross-linking was seen when hepatocytes were exposed to normoxia, or when hepatocytes from Hif1a-/-hep mice were used in hypoxia or normoxia.Hepatocyte HIF-1 mediates an increase in liver fibrosis in a mouse model of NAFLD, perhaps due to liver

  20. In vitro culture of functionally active buffalo hepatocytes isolated by using a simplified manual perfusion method.

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    Santanu Panda

    Full Text Available In farm animals, there is no suitable cell line available to understand liver-specific functions. This has limited our understanding of liver function and metabolism in farm animals. Culturing and maintenance of functionally active hepatocytes is difficult, since they survive no more than few days. Establishing primary culture of hepatocytes can help in studying cellular metabolism, drug toxicity, hepatocyte specific gene function and regulation. Here we provide a simple in vitro method for isolation and short-term culture of functionally active buffalo hepatocytes.Buffalo hepatocytes were isolated from caudate lobes by using manual enzymatic perfusion and mechanical disruption of liver tissue. Hepatocyte yield was (5.3 ± 0.66×107 cells per gram of liver tissue with a viability of 82.3 ± 3.5%. Freshly isolated hepatocytes were spherical with well contrasted border. After 24 hours of seeding onto fibroblast feeder layer and different extracellular matrices like dry collagen, matrigel and sandwich collagen coated plates, hepatocytes formed confluent monolayer with frequent clusters. Cultured hepatocytes exhibited typical cuboidal and polygonal shape with restored cellular polarity. Cells expressed hepatocyte-specific marker genes or proteins like albumin, hepatocyte nuclear factor 4α, glucose-6-phosphatase, tyrosine aminotransferase, cytochromes, cytokeratin and α1-antitrypsin. Hepatocytes could be immunostained with anti-cytokeratins, anti-albumin and anti α1-antitrypsin antibodies. Abundant lipid droplets were detected in the cytosol of hepatocytes using oil red stain. In vitro cultured hepatocytes could be grown for five days and maintained for up to nine days on buffalo skin fibroblast feeder layer. Cultured hepatocytes were viable for functional studies.We developed a convenient and cost effective technique for hepatocytes isolation for short-term culture that exhibited morphological and functional characteristics of active hepatocytes

  1. 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.

  2. Cholesterol Enhances the Toxic Effect of Ethanol and Acetaldehyde in Primary Mouse Hepatocytes

    Directory of Open Access Journals (Sweden)

    Anayelly López-Islas

    2016-01-01

    Full Text Available Obesity and alcohol consumption are risk factors for hepatic steatosis, and both commonly coexist. Our objective was to evaluate the effect of ethanol and acetaldehyde on primary hepatocytes obtained from mice fed for two days with a high cholesterol (HC diet. HC hepatocytes increased lipid and cholesterol content. HC diet sensitized hepatocytes to the toxic effect of ethanol and acetaldehyde. Cyp2E1 content increased with HC diet, as well as in those treated with ethanol or acetaldehyde, while the activity of this enzyme determined in microsomes increased in the HC and in all ethanol treated hepatocytes, HC and CW. Oxidized proteins were increased in the HC cultures treated or not with the toxins. Transmission electron microscopy showed endoplasmic reticulum (ER stress and megamitochondria in hepatocytes treated with ethanol as in HC and the ethanol HC treated hepatocytes. ER stress determined by PERK content was increased in ethanol treated hepatocytes from HC mice and CW. Nuclear translocation of ATF6 was observed in HC hepatocytes treated with ethanol, results that indicate that lipids overload and ethanol treatment favor ER stress. Oxidative stress, ER stress, and mitochondrial damage underlie potential mechanisms for increased damage in steatotic hepatocyte treated with ethanol.

  3. Isolation of Kupffer Cells and Hepatocytes from a Single Mouse Liver

    DEFF Research Database (Denmark)

    Aparicio-Vergara, Marcela; Tencerova, Michaela; Morgantini, Cecilia

    2017-01-01

    Liver perfusion is a common technique used to isolate parenchymal and non-parenchymal liver cells for in vitro experiments. This method allows hepatic cells to be separated based on their size and weight, by centrifugation using a density gradient. To date, other methods allow the isolation of only...... one viable hepatic cellular fraction from a single mouse; either parenchymal (hepatocytes) or non-parenchymal cells (i.e., Kupffer cells or hepatic stellate cells). Here, we describe a method to isolate both hepatocytes and Kupffer cells from a single mouse liver, thereby providing the unique...... advantage of studying different liver cell types that have been isolated from the same organism....

  4. [The polyploidization characteristics of the hepatocytes of the mouse-like hamster Calomyscus mystax].

    Science.gov (United States)

    Anatskaia, O V; Malikov, V G; Meĭer, M N; Kudriavtsev, B N

    1995-01-01

    A cytophotometric measurement of DNA content in hepatocytes of maturing mouse-like hamsters was made. Cells belonging to ordinary mammalian ploidy classes 2c, 2c x 2, 4c, and 4c x 2 made about 90% of the hepatocyte population. The share of binucleated cells wa high (about 80%), the majority of these cells being 2c X 2 hepatocytes. Binucleated cells with tetraploid and diploid nuclei occur in almost every animal. An average hepatocyte ploidy level in mouse-like hamster is 4.6c. The main peculiarity of parenchymal liver cell populations is that up 5% of hepatocytes contain 3--11 nuclei of different ploidy classes. Multinucleated cells increase in number from 1.5% to 4% within the period from one year (the age of maturation) to two years. Later on their percentage does not change. It is found that in binucleated and multinucleated hepatocytes DNA synthesis can proceed asynchronously. Asynchrony in DNA synthesis elevates as the number of nuclei increases. Among the 2c x 2 and 2c x 3 cells an uneven distribution of 3H-thymidine label can occur, respectively, in 5 and in 50% cases, whereas all the cells with more than 3 nuclei display an uneven an uneven 3H-thymidin label distribution. The formation of multinucleated cells is supposed to be associated with asynchrony in DNA-synthesis in binucleated cells and with the restitution of mitosis.

  5. Inducibility of carbamoylphosphate synthetase (ammonia) in cultures of embryonic hepatocytes: ontogenesis of the responsiveness to hormones

    NARCIS (Netherlands)

    Lamers, W. H.; Zonneveld, D.; Charles, R.

    1984-01-01

    Glucocorticosteroids and cyclic AMP induce carbamoylphosphate synthetase (ammonia) (CPS) in rat hepatocytes. Using an enzyme immunoassay applied to hepatocyte cultures fixed in situ, it has been demonstrated that the capacity of hepatocytes to synthesize CPS in the presence of both hormones is

  6. Biotransformation of hydralazine (HDZ) in monolayer cultures of rabbit hepatocytes

    International Nuclear Information System (INIS)

    McQueen, C.A.; Rosado, R.R.

    1990-01-01

    Adverse reactions to HDZ have been associated with the acetylator polymorphism; slow acetylators are more likely to develop HDZ-induced lupus erythematosus. In studying the role of this polymorphism in susceptibility to HDZ toxicity, the biotransformation of HDZ was investigated in rabbit hepatocytes. New Zealand white rabbits, like humans, are classified as rapid or slow acetylators. Heptocytes were isolated from rapid acetylator rabbits by collagenase perfusion. Monolayer cultures were initiated and exposed to 14 C-HDZ. Since HDZ is unstable at neutral pH, parallel incubations were done in the absence of cells. Metabolites in the media were determined by reverse phase HPLC. Phthalazine (P), phthalazinone (PZ), triazoloph-thalazine (TP), methyl TP (MTP) and 3-hydroxy MTP were identified. In the absence of cells, more TP was formed than MTP, probably resulting from reaction of HDZ with components in the medium. In the presence of cells, there was a three-fold increase in MTP, while the amount of TP was relatively constant. Only trace amounts of P, PZ 3-hydroxy MTP were detected. These data indicate that monolayer cultures of rapid acetylator rabbit hepatocytes were capable of metabolizing HDZ with acetylation playing a major role. These studies are being extended to cells from slow acetylator rabbits

  7. 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

  8. Long-term culture and expansion of primary human hepatocytes

    NARCIS (Netherlands)

    Levy, G.; Bomze, D.; Heinz, S.; Ramachandran, S.D.; Noerenberg, A.; Cohen, M.; Shibolet, O.; Sklan, E.; Braspenning, J.C.; Nahmias, Y.

    2015-01-01

    Hepatocytes have a critical role in metabolism, but their study is limited by the inability to expand primary hepatocytes in vitro while maintaining proliferative capacity and metabolic function. Here we describe the oncostatin M (OSM)-dependent expansion of primary human hepatocytes by low

  9. Retroviral-mediated gene transfer and expression of human phenylalanine hydroxylase in primary mouse hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Peng, H.; Armentano, D.; Mackenzie-Graham, L.; Shen, R.F.; Darlington, G.; Ledley, F.D.; Woo, S.L.C. (Baylor College of Medicine, Houston, TX (USA))

    1988-11-01

    Genetic therapy for phenylketonuria (severe phenylalanine hydroxylase deficiency) may require introduction of a normal phenylalanine hydroxylase gene into hepatic cells of patients. The authors report development of a recombinant retrovirus based on the N2 vector for gene transfer and expression of human phenylalanine hydroxylase cDNA in primary mouse hepatocytes. This construct contains an internal promoter of the human {alpha}{sub 1}-antitrypsin gene driving transcription of the phenylalanine hydroxylase cDNA. Primary mouse hepatocytes were isolated from newborn mice, infected with the recombinant virus, and selected for expression of the neomycin-resistance gene. Hepatocytes transformed with the recombinant virus contained high levels of human phenylalanine hydroxylase mRNA transcripts originating from the retroviral and internal promoters. These results demonstrate that the transcriptional regulatory elements of the {alpha}{sub 1} antitrypsin gene retain their tissue-specific function in the recombinant provirus and establish a method for efficient transfer and high-level expression of human phenylalanine hydroxylase in primary hepatocytes.

  10. Retroviral-mediated gene transfer and expression of human phenylalanine hydroxylase in primary mouse hepatocytes

    International Nuclear Information System (INIS)

    Peng, H.; Armentano, D.; Mackenzie-Graham, L.; Shen, R.F.; Darlington, G.; Ledley, F.D.; Woo, S.L.C.

    1988-01-01

    Genetic therapy for phenylketonuria (severe phenylalanine hydroxylase deficiency) may require introduction of a normal phenylalanine hydroxylase gene into hepatic cells of patients. The authors report development of a recombinant retrovirus based on the N2 vector for gene transfer and expression of human phenylalanine hydroxylase cDNA in primary mouse hepatocytes. This construct contains an internal promoter of the human α 1 -antitrypsin gene driving transcription of the phenylalanine hydroxylase cDNA. Primary mouse hepatocytes were isolated from newborn mice, infected with the recombinant virus, and selected for expression of the neomycin-resistance gene. Hepatocytes transformed with the recombinant virus contained high levels of human phenylalanine hydroxylase mRNA transcripts originating from the retroviral and internal promoters. These results demonstrate that the transcriptional regulatory elements of the α 1 antitrypsin gene retain their tissue-specific function in the recombinant provirus and establish a method for efficient transfer and high-level expression of human phenylalanine hydroxylase in primary hepatocytes

  11. Efficient derivation of functional hepatocytes from mouse induced pluripotent stem cells by a combination of cytokines and sodium butyrate

    Institute of Scientific and Technical Information of China (English)

    ZHANG Qi; YANG Yang; ZHANG Jian; WANG Guo-ying; LIU Wei; QIU Dong-bo; HEI Zi-qing; YING Qi-long; CHEN Gui-hua

    2011-01-01

    Background Hepatocyte transplantation has been proposed as an alternative to whole-organ transplantation to support many forms of hepatic insufficiency.Unfortunately,the lack of donor livers makes it difficult to obtain enough viable human hepatocytes for hepatocyte-based therapies.Therefore,it is urgent to find new ways to provide ample hepatocytes.Induced pluripotent stem (iPS) cells,a breakthrough in stem cell research,may terminate these hinders for cell transplantation.For the promise of iPS cells to be realized in liver diseases,it is necessary to determine if and how efficient they can be differentiated into functional hepatocytes.Methods In this study,we directly compared the hepatic-differentiation capacity of mouse iPS cells and embryonic stem (ES) cells with three different induction approaches:conditions via embryonic body (EB) formation plus cytokines,conditions by combination of dimethyl sulfoxide and sodium butyrate and chemically defined,serum free monolayer conditions.Among these three induction conditions,more homogenous populations can be promoted under chemically defined,serum free conditions.The cells generated under these conditions exhibited hepatic functions in vitro,including glycogen storage,indocynine green (ICG) uptake and release as well as urea secretion.Although efficient hepatocytes differentiation from mouse iPS cells were observed,mouse iPS cells showed relatively lower hepatic induction efficiency compared with mouse ES cells.Results Mouse iPS cells would be efficiently differentiated into functional hepatocytes in vitro,which may be helpful in facilitating the development of hepatocytes for transplantation and for research on drug discovery.Conclusion We demonstrate that mouse iPS cells retain full potential for fetal liver development and describe procedures that facilitates the efficient generation of highly differentiated human hepatocyte-like cells from iPS cells in vitro.

  12. Induced Mitogenic Activity in AML-12 Mouse Hepatocytes Exposed to Low-dose Ultra-Wideband Electromagnetic Radiation

    Directory of Open Access Journals (Sweden)

    P. B. Tchounwou

    2005-04-01

    Full Text Available Ultra–wideband (UWB technology has increased with the use of various civilian and military applications. In the present study, we hypothesized that low-dose UWB electromagnetic radiation (UWBR could elicit a mitogenic effect in AML-12 mouse hepatocytes, in vitro. To test this hypothesis, we exposed AML-12 mouse hepatocytes, to UWBR in a specially constructed gigahertz transverse electromagnetic mode (GTEM cell. Cells were exposed to UWBR for 2 h at a temperature of 23°C, a pulse width of 10 ns, a repetition rate of 1 kHz, and field strength of 5-20 kV/m. UWB pulses were triggered by an external pulse generator for UWBR exposure but were not triggered for the sham exposure. We performed an MTT Assay to assess cell viability for UWBR-treated and sham-exposed hepatocytes. Data from viability studies indicated a time-related increase in hepatocytes at time intervals from 8-24 h post exposure. UWBR exerted a statistically significant (p < 0.05 dose-dependent response in cell viability in both serum-treated and serum free medium (SFM -treated hepatocytes. Western blot analysis of hepatocyte lysates demonstrated that cyclin A protein was induced in hepatocytes, suggesting that increased MTT activity after UWBR exposure was due to cell proliferation. This study indicates that UWBR has a mitogenic effect on AML-12 mouse hepatocytes and implicates a possible role for UWBR in hepatocarcinoma.

  13. Assessing Concordance of Drug-Induced Transcriptional Response in Rodent Liver and Cultured Hepatocytes.

    Directory of Open Access Journals (Sweden)

    Jeffrey J Sutherland

    2016-03-01

    Full Text Available The effect of drugs, disease and other perturbations on mRNA levels are studied using gene expression microarrays or RNA-seq, with the goal of understanding molecular effects arising from the perturbation. Previous comparisons of reproducibility across laboratories have been limited in scale and focused on a single model. The use of model systems, such as cultured primary cells or cancer cell lines, assumes that mechanistic insights derived from the models would have been observed via in vivo studies. We examined the concordance of compound-induced transcriptional changes using data from several sources: rat liver and rat primary hepatocytes (RPH from Drug Matrix (DM and open TG-GATEs (TG, human primary hepatocytes (HPH from TG, and mouse liver/HepG2 results from the Gene Expression Omnibus (GEO repository. Gene expression changes for treatments were normalized to controls and analyzed with three methods: 1 gene level for 9071 high expression genes in rat liver, 2 gene set analysis (GSA using canonical pathways and gene ontology sets, 3 weighted gene co-expression network analysis (WGCNA. Co-expression networks performed better than genes or GSA when comparing treatment effects within rat liver and rat vs. mouse liver. Genes and modules performed similarly at Connectivity Map-style analyses, where success at identifying similar treatments among a collection of reference profiles is the goal. Comparisons between rat liver and RPH, and those between RPH, HPH and HepG2 cells reveal lower concordance for all methods. We observe that the baseline state of untreated cultured cells relative to untreated rat liver shows striking similarity with toxicant-exposed cells in vivo, indicating that gross systems level perturbation in the underlying networks in culture may contribute to the low concordance.

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

    International Nuclear Information System (INIS)

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

    1986-01-01

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

  15. Gel entrapment culture of rat hepatocytes for investigation of tetracycline-induced toxicity

    International Nuclear Information System (INIS)

    Shen Chong; Meng Qin; Schmelzer, Eva; Bader, Augustinus

    2009-01-01

    This paper aimed to explore three-dimensionally cultured hepatocytes for testing drug-induced nonalcoholic steatohepatitis. Gel entrapped rat hepatocytes were applied for investigation of the tetracycline-induced steatohepatitis, while hepatocyte monolayer was set as a control. The toxic responses of hepatocytes were systematically evaluated by measuring cell viability, liver-specific function, lipid accumulation, oxidative stress, adenosine triphosphate content and mitochondrial membrane potential. The results suggested that gel entrapped hepatocytes showed cell death after 96 h of tetracycline treatment at 25 μM which is equivalent to toxic serum concentration in rats, while hepatocyte monolayer showed cell death at a high dose of 200 μM. The concentration-dependent accumulation of lipid as well as mitochondrial damage were regarded as two early events for tetracycline hepatotoxicity in gel entrapment culture due to their detectability ahead of subsequent increase of oxidative stress and a final cell death. Furthermore, the potent protection of fenofibrate and fructose-1,6-diphosphate were evidenced in only gel entrapment culture with higher expressions on the genes related to β-oxidation than hepatocyte monolayer, suggesting the mediation of lipid metabolism and mitochondrial damage in tetracycline toxicity. Overall, gel entrapped hepatocytes in three-dimension reflected more of the tetracycline toxicity in vivo than hepatocyte monolayer and thus was suggested as a more relevant system for evaluating steatogenic drugs.

  16. Repression of multiple CYP2D genes in mouse primary hepatocytes with a single siRNA construct.

    Science.gov (United States)

    Elraghy, Omaima; Baldwin, William S

    2015-01-01

    The Cyp2d subfamily is the second most abun-dant subfamily of hepatic drug-metabolizing CYPs. In mice, there are nine Cyp2d members that are believed to have redundant catalytic activity. We are testing and optimizing the ability of one short interfering RNA (siRNA) construct to knockdown the expression of multiple mouse Cyp2ds in primary hepatocytes. Expression of Cyp2d10, Cyp2d11, Cyp2d22, and Cyp2d26 was observed in the primary male mouse hepatocytes. Cyp2d9, which is male-specific and growth hormone-dependent, was not expressed in male primary hepatocytes, potentially because of its dependence on pulsatile growth hormone release from the anterior pituitary. Several different siRNAs at different concentrations and with different reagents were used to knockdown Cyp2d expression. siRNA constructs designed to repress only one construct often mildly repressed several Cyp2d isoforms. A construct designed to knockdown every Cyp2d isoform provided the best results, especially when incubated with transfection reagents designed specifically for primary cell culture. Interestingly, a construct designed to knockdown all Cyp2d isoforms, except Cyp2d10, caused a 2.5× increase in Cyp2d10 expression, presumably because of a compensatory response. However, while RNA expression is repressed 24 h after siRNA treatment, associated changes in Cyp2d-mediated metabolism are tenuous. Overall, this study provides data on the expression of murine Cyp2ds in primary cell lines, valuable information on designing siRNAs for silencing multiple murine CYPs, and potential pros and cons of using siRNA as a tool for repressing Cyp2d and estimating Cyp2d's role in murine xenobiotic metabolism.

  17. 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

  18. UDP-glucuronosyltransferase-dependent bioactivation of clofibric acid to a DNA-damaging intermediate in mouse hepatocytes.

    Science.gov (United States)

    Ghaoui, Roula; Sallustio, Benedetta C; Burcham, Philip C; Fontaine, Frank R

    2003-05-06

    Glucuronidation of a number of carboxyl-containing drugs generates reactive acyl glucuronide metabolites. These electrophilic species alkylate cell proteins and may be implicated in the pathogenesis of a number of toxic syndromes seen in patients receiving the parent aglycones. Whether acyl glucuronides also attack nuclear DNA is unknown, although the acyl glucuronide formed from clofibric acid was recently found to decrease the transfection efficiency of phage DNA and generate strand breaks in plasmid DNA in vitro. To determine if such a DNA damage occurs within a cellular environment, the comet assay (i.e. single-cell gel electrophoresis) was used to detect DNA lesions in the nuclear genome of isolated mouse hepatocytes cultured with clofibric acid. Overnight exposure to 50 microM and higher concentrations of clofibric acid produced concentration-dependent increases in the comet areas of hepatocyte nuclei, with 1 mM clofibrate producing a 3.6-fold elevation over controls. These effects closely coincided with culture medium concentrations of the glucuronide metabolite formed from clofibric acid, 1-O-beta-clofibryl glucuronide. Consistent with a role for glucuronidation in the DNA damage observed, the glucuronidation inhibitor borneol diminished glucuronide formation from 100 microM clofibrate by 98% and returned comet areas to baseline levels. Collectively, these results suggest that the acyl glucuronide formed from clofibric acid is capable of migrating from its site of formation within the endoplasmic reticulum to generate strand nicks in nuclear DNA.

  19. Transcriptional and metabolic flux profiling of triadimefon effects on cultured hepatocytes

    International Nuclear Information System (INIS)

    Iyer, Vidya V.; Ovacik, Meric A.; Androulakis, Ioannis P.; Roth, Charles M.; Ierapetritou, Marianthi G.

    2010-01-01

    Conazoles are a class of azole fungicides used to prevent fungal growth in agriculture, for treatment of fungal infections, and are found to be tumorigenic in rats and/or mice. In this study, cultured primary rat hepatocytes were treated to two different concentrations (0.3 and 0.15 mM) of triadimefon, which is a tumorigenic conazole in rat and mouse liver, on a temporal basis with daily media change. Following treatment, cells were harvested for microarray data ranging from 6 to 72 h. Supernatant was collected daily for three days, and the concentrations of various metabolites in the media and supernatant were quantified. Gene expression changes were most significant following exposure to 0.3 mM triadimefon and were characterized mainly by metabolic pathways related to carbohydrate, lipid and amino acid metabolism. Correspondingly, metabolic network flexibility analysis demonstrated a switch from fatty acid synthesis to fatty acid oxidation in cells exposed to triadimefon. It is likely that fatty acid oxidation is active in order to supply energy required for triadimefon detoxification. In 0.15 mM triadimefon treatment, the hepatocytes are able to detoxify the relatively low concentration of triadimefon with less pronounced changes in hepatic metabolism.

  20. Aging changes of macromolecular synthesis in the mitochondria of mouse hepatocytes as revealed by microscopic radioautography

    Energy Technology Data Exchange (ETDEWEB)

    Nagata, Tetsuji [Shinshu University, Matsumoto (Japan). Dept. of Anatomy and Cell Biology

    2007-07-01

    This mini-review reports aging changes of macromolecular synthesis in the mitochondria of mouse hepatocytes. We have observed the macromolecular synthesis, such as DNA, RNA and proteins, in the mitochondria of various mammalian cells by means of electron microscopic radioautography technique developed in our laboratory. The number of mitochondria per cell, number of labeled mitochondria per cell with 3H-thymidine, 3H-uridine and 3H-leucine, precursors for DNA, RNA and proteins, respectively, were counted and the labeling indices at various ages, from fetal to postnatal early days and several months to 1 and 2 years in senescence, were calculated, which showed variations due to aging. (author)

  1. Cultures of preimplantation mouse embryos

    International Nuclear Information System (INIS)

    Streffer, C.; Molls, M.

    1987-01-01

    In the preimplantation mouse embryos the chromosomal damage develops through several postradiation cell cycles and mitoses. New chromosome aberrations are seen during the second and third postradiation mitoses. Also, more micronuclei appear during later postradiation interphases. This is in agreement with the assumption that unrepaired chromosomal radiation damage develops during the cell generation cycle to such a form (i.e. double-strand breaks in DNA) that chromosomal breaks occur. This proposition is strengthened by the observation that radiation-induced damage is more rapidly expressed after neutron exposure (first or second postradiation mitosis) than after exposure to X rays at the one- or two-cell stage. The preimplantation mouse embryo culture is an inviting system for additional studies at the molecular level, especially now that within the last few years more sensitive methods have been developed for study of DNA and protein structure, regulation, and synthesis. The results from these studies of cultures of preimplantation mouse embryos present a favorable case for the study of complex biological systems under very defined conditions in vitro for extrapolation to effects in vivo

  2. Modulation of hepatocyte growth factor gene expression by estrogen in mouse ovary.

    Science.gov (United States)

    Liu, Y; Lin, L; Zarnegar, R

    1994-09-01

    Hepatocyte growth factor (HGF) is expressed in a variety of tissues and cell types under normal conditions and in response to various stimuli such as tissue injury. In the present study, we demonstrate that the transcription of the HGF gene is stimulated by estrogen in mouse ovary. A single injection of 17 beta-estradiol results in a dramatic and transient elevation of the levels of mouse HGF mRNA. Sequence analysis has found that two putative estrogen responsive elements (ERE) reside at -872 in the 5'-flanking region and at +511 in the first intron, respectively, of the mouse HGF gene. To test whether these ERE elements are responsible for estrogen induction of HGF gene expression, chimeric plasmids containing variable regions of the 5'-flanking sequence of HGF gene and the coding region for chloramphenicol acetyltransferase (CAT) gene were transiently transfected into both human endometrial carcinoma RL 95-2 cells and mouse fibroblast NIH 3T3 cells to assess hormone responsiveness. Transfection results indicate that the ERE elements of the mouse HGF gene can confer estrogen action to either homologous or heterologous promoters. Nuclear protein extracts either from RL95-2 cells transfected with the estrogen receptor expression vector or from mouse liver bound in vitro to ERE elements specifically, as shown by band shift assay. Therefore, our results demonstrate that the HGF gene is transcriptionally regulated by estrogen in mouse ovary; and such regulation is mediated via a direct interaction of the estrogen receptor complex with cis-acting ERE elements identified in the mouse HGF gene.

  3. The effect of hepatocyte growth factor on mouse oocyte in vitro maturation and subsequent fertilization and embryo development

    Directory of Open Access Journals (Sweden)

    Mohammad H. Bahadori

    2011-05-01

    Full Text Available Background: Oocyte invitro maturation is an enormously promising technology for the treatment of infertility, yet its clinical application remains limited owing to poor success rates. Therefore, this study was devised to evaluate the effect of hepatocyte growth factor (HGF on in vitro maturation of immature mouse oocytes and resulting embryos development. Materials and Method: Cumulus – oocyte complex and germinal vesicle were obtained from eighteen 6-8 weeks-old female NMRI mice 46-48 hours after administration of an injection of 5 IU PMSG (Pregnant Mares’ Serum Gonadotrophin. Oocytes were culture in TCM199 (Tissue culture medium-199 supplemented with dosages of 0, 10, 20, 50 and 100 ng/ml of HGF. After 24 hours, metaphase ІІ oocytes were co-incubated with sperms for 4-6 hours in T6 medium. Following isolation of two pronucleus embryos, cleavage of embryos was assessed in the same medium till blastocyst stage. The number of oocytes and embryos was recorded under an invert microscope and the rate of oocyte maturation, fertilization and embryos cleavage until blastocyst stage compared using of student χ2 test. Results: In all compared groups, oocytes growth and embryos development rate in the 20 ng/ml of HGF treatment group was significantly higher (p<0.05 than the control group (p<0.05.Conclusion: 20 ng/ml of HGF improved the nuclear maturation and embryo development up to blastocyst stage during culture condition

  4. Integrative cross-omics analysis in primary mouse hepatocytes unravels mechanisms of cyclosporin A-induced hepatotoxicity

    NARCIS (Netherlands)

    Hof, W.F.P.M.; Summeren, van A.; Lommen, A.; Coonen, M.L.J.; Brauers, K.; Herwijnen, van M.; Wodzig, W.K.W.H.; Kleinjans, J.C.S.

    2014-01-01

    The liver is responsible for drug metabolism and drug-induced hepatotoxicity is the most frequent reason for drug withdrawal, indicating that better pre-clinical toxicity tests are needed. In order to bypass animal models for toxicity screening, we exposed primary mouse hepatocytes for exploring the

  5. Acute Pre-/Post-Treatment with 8th Day SOD-Like Supreme (a Free Radical Scavenging Health Product Protects against Oxidant-Induced Injury in Cultured Cardiomyocytes and Hepatocytes In Vitro as Well as in Mouse Myocardium and Liver In Vivo

    Directory of Open Access Journals (Sweden)

    Pou Kuan Leong

    2017-04-01

    Full Text Available 8th Day superoxide dismutase (SOD-Like Supreme (SOD-Like Supreme, a free radical scavenging health product is an antioxidant-enriched fermentation preparation with free radical scavenging properties. In the present study, the cellular/tissue protective actions of SOD-Like Supreme against menadione toxicity in cultured H9c2 cardiomyocytes and in AML12 hepatocytes as well as oxidant-induced injury in the mouse myocardium and liver were investigated. SOD-Like Supreme was found to possess potent free radical scavenging activity in vitro as assessed by an oxygen radical absorbance capacity assay. Incubation with SOD-Like Supreme (0.5–3% (v/v was shown to protect against menadione-induced toxicity in H9c2 and AML12 cells, as evidenced by increases in cell viability. The ability of SOD-Like Supreme to protect against menadione cytotoxicity was associated with an elevation in the cellular reduced glutathione (GSH/oxidized glutathione (GSSG ratio in menadione-challenged cells. Consistent with the cell-based studies, pre-/post-treatment with SOD-Like Supreme (0.69 and 2.06 mL/kg, three intermittent doses per day for two consecutive days was found to protect against isoproterenol-induced myocardial injury and carbon tetrachloride hepatotoxicity in mice. The cardio/hepatoprotection afforded by SOD-Like Supreme was also paralleled by increases in myocardial/hepatic mitochondrial GSH/GSSG ratios in the SOD-Like Supreme-treated/oxidant-challenged mice. In conclusion, incubation/treatment with SOD-Like Supreme was found to protect against oxidant-induced injury in vitro and in vivo, presumably by virtue of its free radical scavenging activity.

  6. Uptake and disposition of mirex in hepatocytes and subcellular fractions in CD1 mouse liver

    International Nuclear Information System (INIS)

    Charles, A.K.; Rosenbaum, D.P.; Ashok, L.; Abraham, R.

    1985-01-01

    In vivo uptake and disposition of [ 14 C]mirex by CD1 mouse liver subcellular fractions and cells of different nuclear ploidy were examined following single or multiple doses of mirex injected intraperitoneally. Significant amounts of mirex were rapidly taken up by liver (21-29%), suggesting that liver is one of the primary sites of accumulation of the chemical. Among subcellular fractions, mirex was predominantly distributed in mitochondria and microsomes in the irreversibly bound form (about 20%), although its levels fluctuated considerably with time. Mirex was completely dissociated with trichloroacetic acid treatment from both nuclear and plasma membrane fractions, although the total uptake by these fractions was markedly high. The time course of uptake and concentration-dependent disposition of mirex revealed that polyploid hepatocytes selectively accumulated higher amounts of the chemical (two to three times) compared to diploid hepatocytes. The increased affinity of polyploid cells to mirex may indicate a greater susceptibility of this cell type to the chemical insult and also may suggest a possible early involvement of polyploids in the tumorigenic process in rodent livers

  7. Molecular dynamics study of lipid bilayers modeling the plasma membranes of mouse hepatocytes and hepatomas.

    Science.gov (United States)

    Andoh, Yoshimichi; Aoki, Noriyuki; Okazaki, Susumu

    2016-02-28

    Molecular dynamics (MD) calculations of lipid bilayers modeling the plasma membranes of normal mouse hepatocytes and hepatomas in water have been performed under physiological isothermal-isobaric conditions (310.15 K and 1 atm). The changes in the membrane properties induced by hepatic canceration were investigated and were compared with previous MD calculations included in our previous study of the changes in membrane properties induced by murine thymic canceration. The calculated model membranes for normal hepatocytes and hepatomas comprised 23 and 24 kinds of lipids, respectively. These included phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, lysophospholipids, and cholesterol. We referred to previously published experimental values for the mole fraction of the lipids adopted in the present calculations. The calculated structural and dynamic properties of the membranes such as lateral structure, order parameters, lateral self-diffusion constants, and rotational correlation times all showed that hepatic canceration causes plasma membranes to become more ordered laterally and less fluid. Interestingly, this finding contrasts with the less ordered structure and increased fluidity of plasma membranes induced by thymic canceration observed in our previous MD study.

  8. Comparative metabolism of honokiol in mouse, rat, dog, monkey, and human hepatocytes.

    Science.gov (United States)

    Jeong, Hyeon-Uk; Kim, Ju-Hyun; Kong, Tae Yeon; Choi, Won Gu; Lee, Hye Suk

    2016-04-01

    Honokiol has antitumor, antioxidative, anti-inflammatory, and antithrombotic effects. Here we aimed to identify the metabolic profile of honokiol in mouse, rat, dog, monkey, and human hepatocytes and to characterize the enzymes responsible for the glucuronidation and sulfation of honokiol. Honokiol had a high hepatic extraction ratio in all five species, indicating that it was extensively metabolized. A total of 32 metabolites, including 17 common and 15 different metabolites, produced via glucuronidation, sulfation, and oxidation of honokiol allyl groups were tentatively identified using liquid chromatography-high resolution quadrupole Orbitrap mass spectrometry. Glucuronidation of honokiol to M8 (honokiol-4-glucuronide) and M9 (honokiol-2'-glucuronide) was the predominant metabolic pathway in hepatocytes of all five species; however, interspecies differences between 4- and 2'-glucuronidation of honokiol were observed. UGT1A1, 1A8, 1A9, 2B15, and 2B17 played major roles in M8 formation, whereas UGT1A7 and 1A9 played major roles in M9 formation. Human cDNA-expressed SULT1C4 played a major role in M10 formation (honokiol-2'-sulfate), whereas SULT1A1*1, 1A1*2, and 1A2 played major roles in M11 formation (honokiol-4-sulfate). In conclusion, honokiol metabolism showed interspecies differences.

  9. Evaluation and optimization of hepatocyte culture media factors by design of experiments (DoE) methodology.

    Science.gov (United States)

    Dong, Jia; Mandenius, Carl-Fredrik; Lübberstedt, Marc; Urbaniak, Thomas; Nüssler, Andreas K N; Knobeloch, Daniel; Gerlach, Jörg C; Zeilinger, Katrin

    2008-07-01

    Optimization of cell culture media based on statistical experimental design methodology is a widely used approach for improving cultivation conditions. We applied this methodology to refine the composition of an established culture medium for growth of a human hepatoma cell line, C3A. A selection of growth factors and nutrient supplements were systematically screened according to standard design of experiments (DoE) procedures. The results of the screening indicated that the medium additives hepatocyte growth factor, oncostatin M, and fibroblast growth factor 4 significantly influenced the metabolic activities of the C3A cell line. Surface response methodology revealed that the optimum levels for these factors were 30 ng/ml for hepatocyte growth factor and 35 ng/ml for oncostatin M. Additional experiments on primary human hepatocyte cultures showed high variance in metabolic activities between cells from different individuals, making determination of optimal levels of factors more difficult. Still, it was possible to conclude that hepatocyte growth factor, epidermal growth factor, and oncostatin M had decisive effects on the metabolic functions of primary human hepatocytes.

  10. 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 ...

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

    International Nuclear Information System (INIS)

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

    1986-01-01

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

  12. Recovery of important physiological functions in 3D culture of immortal hepatocytes

    DEFF Research Database (Denmark)

    Wrzesinski, Krzysztof; Fey, S. J.

    2011-01-01

    to grow human liver cells in ‘3 dimensional’ cultures so that they behave very similar to the liver in our bodies. By growing the immortal hepatocytes in specially designed bioreactors they form small pieces of ‘pseudotissue’ which exhibit several of the functions seen in the adult liver. We have grown...

  13. Strong synergistic induction of CYP1A1 expression by andrographolide plus typical CYP1A inducers in mouse hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Jaruchotikamol, Atika [Department of Toxicology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194 (Japan); Jarukamjorn, Kanokwan [Department of Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen 40002 (Thailand); Sirisangtrakul, Wanna [Department of Toxicology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194 (Japan); Department of Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen 40002 (Thailand); Sakuma, Tsutomu; Kawasaki, Yuki [Department of Toxicology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194 (Japan); Nemoto, Nobuo [Department of Toxicology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194 (Japan)

    2007-10-15

    The effects of andrographolide, the major diterpenoid constituent of Andrographis paniculata, on the expression of cytochrome P450 superfamily 1 members, including CYP1A1, CYP1A2, and CYP1B1, as well as on aryl hydrocarbon receptor (AhR) expression in primary cultures of mouse hepatocytes were investigated in comparison with the effects of typical CYP1A inducers, including benz[a]anthracene, {beta}-naphthoflavone, and 2,3,7,8-tetrachlorodibenzo-p-dioxin. Andrographolide significantly induced the expression of CYP1A1 and CYP1A2 mRNAs in a concentration-dependent manner, as did the typical CYP1A inducers, but did not induce that of CYP1B1 or AhR. Interestingly, andrographolide plus the typical CYP1A inducers synergistically induced CYP1A1 expression, and the synergism was blocked by an AhR antagonist, resveratrol. The CYP1A1 enzyme activity showed a similar pattern of induction. This is the first report that shows that andrographolide has a potency to induce CYP1A1 enzyme and indicates that andrographolide could be a very useful compound for investigating the regulatory mechanism of the CYP1A1 induction pathway. In addition, our findings suggest preparing advice for rational administration of A. paniculata, according to its ability to induce CYP1A1 expression.

  14. Strong synergistic induction of CYP1A1 expression by andrographolide plus typical CYP1A inducers in mouse hepatocytes

    International Nuclear Information System (INIS)

    Jaruchotikamol, Atika; Jarukamjorn, Kanokwan; Sirisangtrakul, Wanna; Sakuma, Tsutomu; Kawasaki, Yuki; Nemoto, Nobuo

    2007-01-01

    The effects of andrographolide, the major diterpenoid constituent of Andrographis paniculata, on the expression of cytochrome P450 superfamily 1 members, including CYP1A1, CYP1A2, and CYP1B1, as well as on aryl hydrocarbon receptor (AhR) expression in primary cultures of mouse hepatocytes were investigated in comparison with the effects of typical CYP1A inducers, including benz[a]anthracene, β-naphthoflavone, and 2,3,7,8-tetrachlorodibenzo-p-dioxin. Andrographolide significantly induced the expression of CYP1A1 and CYP1A2 mRNAs in a concentration-dependent manner, as did the typical CYP1A inducers, but did not induce that of CYP1B1 or AhR. Interestingly, andrographolide plus the typical CYP1A inducers synergistically induced CYP1A1 expression, and the synergism was blocked by an AhR antagonist, resveratrol. The CYP1A1 enzyme activity showed a similar pattern of induction. This is the first report that shows that andrographolide has a potency to induce CYP1A1 enzyme and indicates that andrographolide could be a very useful compound for investigating the regulatory mechanism of the CYP1A1 induction pathway. In addition, our findings suggest preparing advice for rational administration of A. paniculata, according to its ability to induce CYP1A1 expression

  15. Usage of adenovirus expressing thymidine kinase mediated hepatocellular damage for enabling mouse liver repopulation with allogenic or xenogenic hepatocytes.

    Directory of Open Access Journals (Sweden)

    Daniel Moreno

    Full Text Available It has been shown that the liver of immunodeficient mice can be efficiently repopulated with human hepatocytes when subjected to chronic hepatocellular damage. Mice with such chimeric livers represent useful reagents for medical and clinical studies. However all previously reported models of humanized livers are difficult to implement as they involve cross-breeding of immunodeficient mice with mice exhibiting genetic alterations causing sustained hepatic injury. In this paper we attempted to create chimeric livers by inducing persistent hepatocellular damage in immunodeficient Rag2(-/- γc(-/- mice using an adenovirus encoding herpes virus thymidine kinase (AdTk and two consecutive doses of ganciclovir (GCV. We found that this treatment resulted in hepatocellular damage persisting for at least 10 weeks and enabled efficient engraftment and proliferation within the liver of either human or allogenic hepatocytes. Interestingly, while the nodules generated from the transplanted mouse hepatocytes were well vascularized, the human hepatocytes experienced progressive depolarization and exhibited reduced numbers of murine endothelial cells inside the nodules. In conclusion, AdTk/GCV-induced liver damage licenses the liver of immunodeficient mice for allogenic and xenogenic hepatocyte repopulation. This approach represents a simple alternative strategy for chimeric liver generation using immunodeficient mice without additional genetic manipulation of the germ line.

  16. 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

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

    International Nuclear Information System (INIS)

    Henkens, Tom; Papeleu, Peggy; Elaut, Greetje; Vinken, Mathieu; Rogiers, Vera; Vanhaecke, Tamara

    2007-01-01

    Histone deacetylase inhibitors (HDI) have been shown to increase differentiation-related gene expression in several tumor-derived cell lines by hyperacetylating core histones. Effects of HDI on primary cultured cells, however, have hardly been investigated. In the present study, the ability of trichostatin A (TSA), a prototype hydroxamate HDI, to counteract the loss of liver-specific functions in primary rat hepatocyte cultures has been investigated. Upon exposure to TSA, it was found that the cell viability of the cultured hepatocytes and their albumin secretion as a function of culture time were increased. TSA-treated hepatocytes also better maintained cytochrome P450 (CYP)-mediated phase I biotransformation capacity, whereas the activity of phase II glutathione S-transferases (GST) was not affected. Western blot and qRT-PCR analysis of CYP1A1, CYP2B1 and CYP3A11 protein and mRNA levels, respectively, further revealed that TSA acts at the transcriptional level. In addition, protein expression levels of the liver-enriched transcription factors (LETFs) hepatic nuclear factor 4 alpha (HNF4α) and CCAAT/enhancer binding protein alpha (C/EBPα) were accordingly increased by TSA throughout culture time. In conclusion, these findings indicate that TSA plays a major role in the preservation of the differentiated hepatic phenotype in culture. It is suggested that the effects of TSA on CYP gene expression are mediated via controlling the expression of LETFs

  18. Evaluation of Medicinal Plant Hepatotoxicity in Co-cultures of Hepatocytes and Monocytes

    Directory of Open Access Journals (Sweden)

    Bashar Saad

    2006-01-01

    Full Text Available Non-parenchymal cells might play an important role in the modulation of xenobiotic metabolism in liver and its pharmacological and toxicological consequences. Therefore, the role of cell-to-cell interactions in herbal induced liver toxicity was investigated in monocultures of cells from the human hepatocyte cell line (HepG2 and in co-cultures of cells from the HepG2 cell line and cells from the human monocyte cell line (THP1. Cells were treated with various concentrations (1–500 µg ml−1 of extracts of Pistacia palaestina, Juglans regia and Quercus ithaburensis for 24 h. Extracts from Cleome droserifolia, a known toxic plant, were taken as positive control. In the co-culture system, toxic effects were observed after exposure to extracts of Pistacia palestina and C. droserifolia. These two extracts significantly reduced by cell viability as measured the MTT test and the LDH assay. Whereas in hepatocyte cultures, only extracts of C. droserifolia were found to affect the cell viability. The production levels of albumin from hepatocytes were not affected by treatment with plant extracts in both culture systems. It seems that the observed reduction in cell viability after exposure to extracts of P. palestina in co-cultures but not in monocultures is a result of monocyte-derived factors. The use of liver cell co-cultures is therefore a useful approach to investigate the influence of intercellular communication on xenobiotic metabolism in liver.

  19. Cytotoxic mechanisms of hydrosulfide anion and cyanide anion in primary rat hepatocyte cultures

    International Nuclear Information System (INIS)

    Thompson, Rodney W.; Valentine, Holly L.; Valentine, William M.

    2003-01-01

    Hydrogen sulfide and hydrogen cyanide are known to compromise mitochondrial respiration through inhibition of cytochrome c oxidase and this is generally considered to be their primary mechanism of toxicity. Experimental studies and the efficiency of current treatment protocols suggest that H 2 S may exert adverse physiological effects through additional mechanisms. To evaluate the role of alternative mechanisms in H 2 S toxicity, the relative contributions of electron transport inhibition, uncoupling of mitochondrial respiration, and opening of the mitochondrial permeability transition pore (MPTP) to hydrosulfide and cyanide anion cytotoxicity in primary hepatocyte cultures were examined. Supplementation of hepatocytes with the glycolytic substrate, fructose, rescued hepatocytes from cyanide anion induced toxicity, whereas fructose supplementation increased hydrosulfide anion toxicity suggesting that hydrosulfide anion may compromise glycolysis in hepatocytes. Although inhibitors of the MPTP opening were protective for hydrosulfide anion, they had no effect on cyanide anion toxicity, consistent with an involvement of the permeability transition pore in hydrosulfide anion toxicity but not cyanide anion toxicity. Exposure of isolated rat liver mitochondria to hydrosulfide did not result in large amplitude swelling suggesting that if H 2 S induces the permeability transition it does so indirectly through a mechanism requiring other cellular components. Hydrosulfide anion did not appear to be an uncoupler of mitochondrial respiration in hepatocytes based upon the inability of oligomycin and fructose to protect hepatocytes from hydrosulfide anion toxicity. These findings support mechanisms additional to inhibition of cytochrome c oxidase in hydrogen sulfide toxicity. Further investigations are required to assess the role of the permeability transition in H 2 S toxicity, determine whether similar affects occur in other cell types or in vivo and evaluate whether this may

  20. 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.

  1. Metabolism of methyl-branched iodo palmitic acids in cultured hepatocytes

    International Nuclear Information System (INIS)

    Thomas, G.; Pepin, D.; Loriette, C.; Chambaz, J.; Bereziat, G.; Vidal, M.; Apparu, M.; Coornaert, S.

    1989-01-01

    The metabolic fate of methyl-branched iodo fatty acids was studied in primary culture of rat hepatocytes. We compared 16-iodo-2-R,S-methyl palmitic acid (2-Me), which can be β oxidized, with 16-iodo-3-R,S-methyl palmitic acid (3-Me) which can be β oxidized only after an initial α oxydation and with 16-iodo-2,2-dimethyl palmitic acid (2,2-Me 2 ) and 16-iodo-3,3-dimethyl palmitic acid (3,3-Me 2 ) which cannot be β oxidized at all. The normal fate of natural fatty acids was given by comparative experiments with [1- 14 C] palmitic acid. Monomethyl-branched iodo fatty acids were taken up in the same range as palmitic acid but more than dimethyl-branched iodo fatty acids. After a 15-h incubation, acido-soluble products (ASP) accounted for 75% of the radioactivity taken up as 16-iodo-2-methyl palmitic acid, 50% as other methyl-branched iodo fatty acids and only 30% as palmitic acid. Cultured hepatocytes, labelled for 3 h with the various fatty acids and reincubated for 12 h without fatty acid, secreted large amounts of free dimethyl-branched iodo fatty acids as compared to the monomethyl ones and palmitic acid. Only hepatocytes prelabelled with 16-[ 125 I]iodo-2,2-dimethyl palmitic acid exhibited an appreciable secretion of labeled triglycerides, but at a lower rate than with [1- 14 C] palmitic acid. Conversely, the 16-iodo-monomethyl palmitic acids remained chiefly in hepatocyte triglycerides. Minute amounts of 16-iodo-methyl-branched palmitic acids were found in hepatocyte or secreted phospholipids as compared with palmitic acid. (orig.)

  2. Utilization of supplemental methionine sources by primary cultures of chick hepatocytes

    International Nuclear Information System (INIS)

    Dibner, J.J.

    1983-01-01

    Utilization of 2-hydroxy-4-(methylthio) butanoic acid (HMB) as a substrate for protein synthesis was studied by using primary cultures of chick liver cells. Cultures were prepared by enzymatic dissociation of livers from week old Hubbard broiler chicks and were maintained for 4 days under nonproliferative conditions. Hepatocyte differentiation was verified by using dexamethasone induction of tyrosine aminotransferase activity. Conversion of [14C]HMB to L-methionine was shown by chromatographic analysis of hepatocyte protein hydrolysate and incorporation into protein was proven by cycloheximide inhibition of synthesis. When incorporation of HMB was compared to that of DL-methionine (DLM) equimolar quantities of the two sources were found in liver cell protein. These results support, at a cellular level, the conclusion that HMB and DLM are biochemically equivalent sources of methionine for protein synthesis

  3. Interleukin 6 regulates metallothionein gene expression and zinc metabolism in hepatocyte monolayer cultures

    International Nuclear Information System (INIS)

    Schroeder, J.J.; Cousins, R.J.

    1990-01-01

    Attention has focused on the cytokine interleukin 6 (IL-6) as a major mediator of acute-phase protein synthesis in hepatocytes in response to infection and tissue injury. The authors have evaluated the effects of IL-6 and IL-1α as well as extracellular zinc and glucocorticoid hormone on metal-lothionein gene expression and cellular zinc accumulation in rat hepatocyte monolayer cultures. Further, they have evaluated the teleological basis for cytokine mediation by examining cyto-protection from CCl 4 -induced damage. Incubation of hepatocytes with IL-6 led to concentration-dependent and time-dependent increases in metallothionein-1 and -2 mRNA and metallothionein protein. The level of each was increased within 3 hr after the addition of IL-6 at 10 ng/ml. Maximal increases the metallothionein mRNA and metallothionein protein were achieved after 12 hr and 36 hr, respectively. Concomitant with the up-regulation of metallothionein gene expression, IL-6 also increased cellular zinc. Responses to IL-6 required the synthetic glucocorticoid hormone dexamethasone and were optimized by increased extracellular zinc. Thus, IL-6 is a major cytokine mediator of metallothionein gene expression and zinc metabolism in hepatocytes and provides cytoprotection from CCl 4 -induced hepatotoxicity via a mode consistent with dependence upon increased cellular metallothionein synthesis and zinc accumulation

  4. Carbon Tetrachloride Increases Intracellular Calcium in Rat Liver and Hepatocyte Cultures

    Science.gov (United States)

    1986-05-12

    tible to destruction by CC14 ( Head ~ al., 1981). Thus, the activation of CC14 to a toxic moiety clearly depends upon metabolism by one or more... embryologic development (Wyllie, 1986). In contrast, Toyo-oka et al. (1985) could not establish that phospholipase& or proteases were involved in ischemic...D. M. Bissell, and u. A Meyer. (1977) Drug Metabolism in Adult Rat Hepatocyte& in Primary Monolayer Culture. Gastroenterology 72:1232-1239. Head , B

  5. Hanging Drop, A Best Three-Dimensional (3D) Culture Method for Primary Buffalo and Sheep Hepatocytes.

    Science.gov (United States)

    Shri, Meena; Agrawal, Himanshu; Rani, Payal; Singh, Dheer; Onteru, Suneel Kumar

    2017-04-26

    Livestock, having close resemblance to humans, could be a better source of primary hepatocytes than rodents. Herein, we successfully developed three-dimensional (3D) culturing system for primary sheep and buffalo hepatocytes. The 3D-structures of sheep hepatocytes were formed on the fifth-day and maintained until the tenth-day on polyHEMA-coated plates and in hanging drops with William's E media (HDW). Between the cultured and fresh cells, we observed a similar expression of GAPDH, HNF4α, ALB, CYP1A1, CK8 and CK18. Interestingly, a statistically significant increase was noted in the TAT, CPS, AFP, AAT, GSP and PCNA expression. In buffalo hepatocytes culture, 3D-like structures were formed on the third-day and maintained until the sixth-day on polyHEMA and HDW. The expression of HNF4α, GSP, CPS, AFP, AAT, PCNA and CK18 was similar between cultured and fresh cells. Further, a statistically significant increase in the TAT and CK8 expression, and a decrease in the GAPDH, CYP1A1 and ALB expression were noted. Among the culture systems, HDW maintained the liver transcript markers more or less similar to the fresh hepatocytes of the sheep and buffalo for ten and six days, respectively. Taken together, hanging drop is an efficient method for 3D culturing of primary sheep and buffalo hepatocytes.

  6. 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

  7. Effect of hepatocyte growth factor on radiation response of HeLa, V79, CHO and primary cultured parenchymal hepatocyte in vitro

    International Nuclear Information System (INIS)

    Yamazaki, Hideya; Inoue, Takehiro; Nose, Takayuki; Murayama, Shigeyuki; Teshima, Teruki; Ozeki, Syuji; Koizumi, Masahiko; Inoue, Toshihiko.

    1996-01-01

    Hepatocyte growth factor (HGF) is a multipotent cytokine enhancing regeneration of injured organs as liver, kidney and lung after injury. HGF enhances proliferation of various type of cells, inhibits proliferation of carcinoma cells, enhances motility of epithelial cells. We examined three cell lines (CHO, HeLa, V79) and primary cultured normal rat parenchymal hepatocytes to determine the effect of HGF on radiation response. HGF diminished survival of CHO and V79 cells determined by colony formation assay, whereas no significant change of survival was found in HeLa cells. No synergistic changes of survival were found when these three cell lines were irradiated with the addition of HGF. Thus, HGF did not enhance the radiation effect. We also analyzed the impact of irradiation with HGF on primary cultured normal rat parenchymal hepatocytes. At first, the release of glutamic-oxaloacetic amino-transaminase (GOT) in the supernatant was estimated. Irradiation (40 Gy) with or without HGF did not change GOT release in acute phase by 4 days after irradiation compared with the unirradiated control. Second, the DNA synthesis of rat parenchymal hepatocytes was analyzed using radioactive iodine-labeled deoxyuridine incorporation. HGF counteracted the suppression of DNA synthesis induced by irradiation. Thus, HGF may act as a mitogen even for irradiation-damaged normal cells. (author)

  8. Synergistic toxicity of ethanol and MDMA towards primary cultured rat hepatocytes

    International Nuclear Information System (INIS)

    Pontes, Helena; Sousa, Carla; Silva, Renata; Fernandes, Eduarda; Carmo, Helena; Remiao, Fernando; Carvalho, Felix; Bastos, Maria Lourdes

    2008-01-01

    Ethanol is frequently consumed along with 3,4-methylenedioxymethamphetamine (MDMA; ecstasy). Since both compounds are hepatotoxic and are metabolized in the liver, an increased deleterious interaction resulting from the concomitant use of these two drugs seems plausible. Another important feature of MDMA-induced toxicity is hyperthermia, an effect known to be potentiated after continuous exposure to ethanol. Considering the potential deleterious interaction, the aim of the present study was to evaluate the hepatotoxic effects of ethanol and MDMA mixtures to primary cultured rat hepatocytes and to elucidate the mechanism(s) underlying this interaction. For this purpose, the toxicity induced by MDMA to primary cultured rat hepatocytes in absence or in presence of ethanol was evaluated, under normothermic (36.5 deg. C) and hyperthermic (40.5 deg. C) conditions. While MDMA and ethanol, by themselves, had discrete effects on the analysed parameters, which were slightly aggravated under hyperthermia, the simultaneous incubation of MDMA and ethanol for 24 h, resulted in high cell death ratios accompanied by a significant disturbance of cellular redox status and decreased energy levels. Evaluation of apoptotic/necrotic features provided clear evidences that the cell death occurs preferentially through a necrotic pathway. All the evaluated parameters were dramatically aggravated when cells were incubated under hyperthermia. In conclusion, co-exposure of hepatocytes to ethanol and MDMA definitely results in a synergism of the hepatotoxic effects, through a disruption of the cellular redox status and enhanced cell death by a necrotic pathway in a temperature-dependent extent

  9. Preparation of Degradable Biological Carrier With LCC and its Application in Culture of Hepatocytes

    Science.gov (United States)

    Zhao, H. K.; Chen, X. K.; Wu, H. F.; Li, J. L.; Xie, Y. M.

    2018-05-01

    The purpose of this article is to extract lignin-carbohydrate complexes (LCC) with poplar as raw material, which was used to prepare bio-carrier by freeze-drying method. The chemical properties and morphological of LCC porous biological carriers were analyzed by GPC, FT-IR, scanning electron microscopy (SEM) and optical microscopy. The FT-IR spectrum results indicated that LCC which are composed of lignin and polysaccharide, with a typical LCC structure. Galactose have a specific ability to recognize liver cells owing to the presence of receptors on hepatocytes. Cell counting results showed that the cells increases fastest while the proliferation rate of the liver cell in LCC is obviously higher than that of control group. These results indicated that poplar LCC is very biocompatible, in which it might be a great potential biological carrier material for human hepatocyte culture.

  10. Impaired mitochondrial functions contribute to 3-bromopyruvate toxicity in primary rat and mouse hepatocytes

    Czech Academy of Sciences Publication Activity Database

    Sobotka, O.; Endlicher, R.; Drahota, Zdeněk; Kučera, O.; Rychtrmoc, D.; Raad, M.; Hakeem, K.; Červinková, Z.

    2016-01-01

    Roč. 48, č. 4 (2016), s. 363-373 ISSN 0145-479X Institutional support: RVO:67985823 Keywords : 3-bromopyruvate * toxicity * liver * hepatocyte * mitochondria Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.576, year: 2016

  11. 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

  12. Proteome analysis of a hepatocyte-specific BIRC5 (survivin)-knockout mouse model during liver regeneration.

    Science.gov (United States)

    Bracht, Thilo; Hagemann, Sascha; Loscha, Marius; Megger, Dominik A; Padden, Juliet; Eisenacher, Martin; Kuhlmann, Katja; Meyer, Helmut E; Baba, Hideo A; Sitek, Barbara

    2014-06-06

    The Baculoviral IAP repeat-containing protein 5 (BIRC5), also known as inhibitor of apoptosis protein survivin, is a member of the chromosomal passenger complex and a key player in mitosis. To investigate the function of BIRC5 in liver regeneration, we analyzed a hepatocyte-specific BIRC5-knockout mouse model using a quantitative label-free proteomics approach. Here, we present the analyses of the proteome changes in hepatocyte-specific BIRC5-knockout mice compared to wildtype mice, as well as proteome changes during liver regeneration induced by partial hepatectomy in wildtype mice and mice lacking hepatic BIRC5, respectively. The BIRC5-knockout mice showed an extensive overexpression of proteins related to cellular maintenance, organization and protein synthesis. Key regulators of cell growth, transcription and translation MTOR and STAT1/STAT2 were found to be overexpressed. During liver regeneration proteome changes representing a response to the mitotic stimulus were detected in wildtype mice. Mainly proteins corresponding to proliferation, cell cycle and cytokinesis were up-regulated. The hepatocyte-specific BIRC5-knockout mice showed impaired liver regeneration, which had severe consequences on the proteome level. However, several proteins with function in mitosis were found to be up-regulated upon the proliferative stimulus. Our results show that the E3 ubiquitin-protein ligase UHRF1 is strongly up-regulated during liver regeneration independently of BIRC5.

  13. Molecular mechanism of catalase activity change under sodium dodecyl sulfate-induced oxidative stress in the mouse primary hepatocytes.

    Science.gov (United States)

    Wang, Jing; Wang, Jiaxi; Xu, Chi; Liu, Rutao; Chen, Yadong

    2016-04-15

    Sodium dodecyl sulfate (SDS) contributes to adverse effects of organisms probably because of its ability to induce oxidative stress via changing the activity of antioxidant enzyme catalase (CAT). But the underlying molecular mechanisms still remain unclear. This study characterized the harmful effects of SDS-induced oxidative stress on the mouse primary hepatocytes as well as the structure and function of CAT molecule and investigated the underlying molecular mechanism. After 12h SDS (0.1μM to 0.2mM) exposure, no significant change was observed in CAT activity of the hepatocytes. After 0.5 and 0.8mM SDS exposure, the state of oxidative stress stimulated CAT production in the hepatocytes. The inhibition of CAT activity induced by directly interacting with SDS was unable to catch the synthesis of CAT and therefore resulted in the increased activity and elevated ROS level. Further molecular experiments showed that SDS prefers to bind to the interface with no direct effect on the active site and the structure of heme groups of CAT molecule. When the sites in the interface is saturated, SDS interacts with VAL 73, HIS 74, ASN 147 and PHE 152, the key residues of the enzyme activity, and leads to the decrease of CAT activity. Copyright © 2015 Elsevier B.V. All rights reserved.

  14. Therapeutic efficacy of human hepatocyte transplantation in a SCID/uPA mouse model with inducible liver disease.

    Directory of Open Access Journals (Sweden)

    Donna N Douglas

    2010-02-01

    Full Text Available Severe Combined Immune Deficient (SCID/Urokinase-type Plasminogen Activator (uPA mice undergo liver failure and are useful hosts for the propagation of transplanted human hepatocytes (HH which must compete with recipient-derived hepatocytes for replacement of the diseased liver parenchyma. While partial replacement by HH has proven useful for studies with Hepatitis C virus, complete replacement of SCID/uPA mouse liver by HH has never been achieved and limits the broader application of these mice for other areas of biomedical research. The herpes simplex virus type-1 thymidine kinase (HSVtk/ganciclovir (GCV system is a powerful tool for cell-specific ablation in transgenic animals. The aim of this study was to selectively eliminate murine-derived parenchymal liver cells from humanized SCID/uPA mouse liver in order to achieve mice with completely humanized liver parenchyma. Thus, we reproduced the HSVtk (vTK/GCV system of hepatic failure in SCID/uPA mice.In vitro experiments demonstrated efficient killing of vTK expressing hepatoma cells after GCV treatment. For in vivo experiments, expression of vTK was targeted to the livers of FVB/N and SCID/uPA mice. Hepatic sensitivity to GCV was first established in FVB/N mice since these mice do not undergo liver failure inherent to SCID/uPA mice. Hepatic vTK expression was found to be an integral component of GCV-induced pathologic and biochemical alterations and caused death due to liver dysfunction in vTK transgenic FVB/N and non-transplanted SCID/uPA mice. In SCID/uPA mice with humanized liver, vTK/GCV caused death despite extensive replacement of the mouse liver parenchyma with HH (ranging from 32-87%. Surprisingly, vTK/GCV-dependent apoptosis and mitochondrial aberrations were also localized to bystander vTK-negative HH.Extensive replacement of mouse liver parenchyma by HH does not provide a secure therapeutic advantage against vTK/GCV-induced cytotoxicity targeted to residual mouse hepatocytes

  15. 3D cultured immortalized human hepatocytes useful to develop drugs for blood-borne HCV

    International Nuclear Information System (INIS)

    Aly, Hussein Hassan; Shimotohno, Kunitada; Hijikata, Makoto

    2009-01-01

    Due to the high polymorphism of natural hepatitis C virus (HCV) variants, existing recombinant HCV replication models have failed to be effective in developing effective anti-HCV agents. In the current study, we describe an in vitro system that supports the infection and replication of natural HCV from patient blood using an immortalized primary human hepatocyte cell line cultured in a three-dimensional (3D) culture system. Comparison of the gene expression profile of cells cultured in the 3D system to those cultured in the existing 2D system demonstrated an up-regulation of several genes activated by peroxisome proliferator-activated receptor alpha (PPARα) signaling. Furthermore, using PPARα agonists and antagonists, we also analyzed the effect of PPARα signaling on the modulation of HCV replication using this system. The 3D in vitro system described in this study provides significant insight into the search for novel anti-HCV strategies that are specific to various strains of HCV.

  16. Mouse cell culture - Methods and protocols

    Directory of Open Access Journals (Sweden)

    CarloAlberto Redi

    2010-12-01

    Full Text Available The mouse is, out of any doubt, the experimental animal par excellence for many many colleagues within the scientific community, notably for those working in mammalian biology (in a broad sense, from basic genetic to modeling human diseases, starting at least from 1664 Robert Hooke experiments on air’s propertyn. Not surprising then that mouse cell cultures is a well established field of research itself and that there are several handbooks devoted to this discipline. Here, Andrew Ward and David Tosh provide a necessary update of the protocols currently needed. In fact, nearly half of the book is devoted to stem cells culture protocols, mainly embryonic, from a list of several organs (kidney, lung, oesophagus and intestine, pancreas and liver to mention some........

  17. Feasibility of direct oxygenation of primary-cultured rat hepatocytes using polyethylene glycol-decorated liposome-encapsulated hemoglobin (LEH).

    Science.gov (United States)

    Naruto, Hirosuke; Huang, Hongyun; Nishikawa, Masaki; Kojima, Nobuhiko; Mizuno, Atsushi; Ohta, Katsuji; Sakai, Yasuyuki

    2007-10-01

    We tested the short-term efficacy of liposome-encapsulated hemoglobin (LEH) in cultured rat hepatocytes. Supplementation with LEH (20% of the hemoglobin concentration of blood) did not lower albumin production in static culture, and completely reversed the cell death and deterioration in albumin production caused by an oxygen shortage in 2D flat-plate perfusion bioreactors.

  18. Effects of Kisspeptin-10 on Lipid Metabolism in Cultured Chicken Hepatocytes

    Directory of Open Access Journals (Sweden)

    J. Wu

    2012-09-01

    Full Text Available Our previous studies showed that kisspeptin-10 (Kp-10 injected in vivo can markedly increase lipid anabolism in liver of quails. In order to investigate the direct effect of Kp-10 on lipid metabolism of hepatocytes in birds, cells were separated from embryos livers and cultured in vitro with 0, 100 and 1,000 nM Kp-10, respectively. The results showed that after 24 h treatment, cells viability was not affected by 100 nM Kp-10, but showed a mild decrease with 1,000 nM Kp-10 compared to the control cells. Based on the results of the cell viability, 100 nM dosage of Kp-10 was selected for the further study and analysis. Compared with control cells, total cholesterol (Tch contents in 100 nM treated cells were increased by 51.23%, but did not reach statistical significance, while the level of triglyceride (TG, high density of lipoprotein-cholesterol (HDL-C and low density of lipoprotein-cholesterol (LDL-C were significantly increased. Real-time PCR results showed that ApoVLDL-II mRNA expression had a tendency to increase, genes including sterol regulatory element-binding protein-1 (SREBP-1, acetyl coenzyme A carboxylase α (ACCα, carnitine palmitoyltransferase 1 (CPT1, 3-hydroxyl-3-methylglutaryl-coenzyme A reductases (HMGCR and stearyl coenzyme A dehydrogenase-1 (SCD1 mRNA in hepatocytes were significantly down-regulated by 100 nM Kp-10. However, contrary to its gene expression, SREBP-1 protein expression was significantly up-regulated by 100 nM Kp-10. Some of the significant correlations in mRNA expression were found between genes encoding hepatic factors or enzymes involved in lipid metabolism in liver of birds. These results indicate that Kp-10 stimulates lipid synthesis directly in primary cultured hepatocytes of chickens.

  19. TGFbeta induces apoptosis and EMT in primary mouse hepatocytes independently of p53, p21Cip1 or Rb status

    International Nuclear Information System (INIS)

    Sheahan, Sharon; Bellamy, Christopher O; Harland, Stephen N; Harrison, David J; Prost, Sandrine

    2008-01-01

    TGFβ has pleiotropic effects that range from regulation of proliferation and apoptosis to morphological changes and epithelial-mesenchymal transition (EMT). Some evidence suggests that these effects may be interconnected. We have recently reported that P53, P21 Cip1 and pRB, three critical regulators of the G1/S transition are variably involved in TGFβ-induced cell cycle arrest in hepatocytes. As these proteins are also involved in the regulation of apoptosis in many circumstances, we investigated their contribution to other relevant TGFβ-induced effects, namely apoptosis and EMT, and examined how the various processes were interrelated. Primary mouse hepatocytes deficient in p53, p21 and/or Rb, singly or in combination were treated with TGFβ for 24 to 96 hours. Apoptosis was quantified according to morphology and by immunostaining for cleaved-capsase 3. Epithelial and mesenchymal marker expression was studied using immunocytochemistry and real time PCR. We found that TGFβ similarly induced morphological changes regardless of genotype and independently of proliferation index or sensitivity to inhibition of proliferation by TGFβ. Morphological changes were accompanied by decrease in E-cadherin and increased Snail expression but the mesenchymal markers (N-cadherin, SMAα and Vimentin) studied remained unchanged. TGFβ induced high levels of apoptosis in p53-/-, Rb-/-, p21 cip1 -/- and control hepatocytes although with slight differences in kinetics. This was unrelated to proliferation or changes in morphology and loss of cell-cell adhesion. However, hepatocytes deficient in both p53 and p21 cip1 were less sensitive to TGFβ-induced apoptosis. Although p53, p21 Cip1 and pRb are well known regulators of both proliferation and apoptosis in response to a multitude of stresses, we conclude that they are critical for TGFβ-driven inhibition of hepatocytes proliferation, but only slightly modulate TGFβ-induced apoptosis. This effect may depend on other parameters

  20. Inhibition of bile salt transport by drugs associated with liver injury in primary hepatocytes from human, monkey, dog, rat, and mouse.

    Science.gov (United States)

    Zhang, Jie; He, Kan; Cai, Lining; Chen, Yu-Chuan; Yang, Yifan; Shi, Qin; Woolf, Thomas F; Ge, Weigong; Guo, Lei; Borlak, Jürgen; Tong, Weida

    2016-08-05

    Interference of bile salt transport is one of the underlying mechanisms for drug-induced liver injury (DILI). We developed a novel bile salt transport activity assay involving in situ biosynthesis of bile salts from their precursors in primary human, monkey, dog, rat, and mouse hepatocytes in suspension as well as LC-MS/MS determination of extracellular bile salts transported out of hepatocytes. Glycine- and taurine-conjugated bile acids were rapidly formed in hepatocytes and effectively transported into the extracellular medium. The bile salt formation and transport activities were time‒ and bile-acid-concentration‒dependent in primary human hepatocytes. The transport activity was inhibited by the bile salt export pump (BSEP) inhibitors ketoconazole, saquinavir, cyclosporine, and troglitazone. The assay was used to test 86 drugs for their potential to inhibit bile salt transport activity in human hepatocytes, which included 35 drugs associated with severe DILI (sDILI) and 51 with non-severe DILI (non-sDILI). Approximately 60% of the sDILI drugs showed potent inhibition (with IC50 values monkey, dog, rat and mouse hepatocytes. Species differences in potency were observed with mouse being less sensitive than other species to inhibition of bile salt transport. In summary, a novel assay has been developed using hepatocytes in suspension from human and animal species that can be used to assess the potential for drugs and/or drug-derived metabolites to inhibit bile salt transport and/or formation activity. Drugs causing sDILI, except those by immune-mediated mechanism, are highly associated with potent inhibition of bile salt transport. Published by Elsevier Ireland Ltd.

  1. Coactivator PGC-1α regulates the fasting inducible xenobiotic-metabolizing enzyme CYP2A5 in mouse primary hepatocytes

    International Nuclear Information System (INIS)

    Arpiainen, Satu; Jaervenpaeae, Sanna-Mari; Manninen, Aki; Viitala, Pirkko; Lang, Matti A.; Pelkonen, Olavi; Hakkola, Jukka

    2008-01-01

    The nutritional state of organisms and energy balance related diseases such as diabetes regulate the metabolism of xenobiotics such as drugs, toxins and carcinogens. However, the mechanisms behind this regulation are mostly unknown. The xenobiotic-metabolizing cytochrome P450 (CYP) 2A5 enzyme has been shown to be induced by fasting and by glucagon and cyclic AMP (cAMP), which mediate numerous fasting responses. Peroxisome proliferator-activated receptor γ coactivator (PGC)-1α triggers many of the important hepatic fasting effects in response to elevated cAMP levels. In the present study, we were able to show that cAMP causes a coordinated induction of PGC-1α and CYP2A5 mRNAs in murine primary hepatocytes. Furthermore, the elevation of the PGC-1α expression level by adenovirus mediated gene transfer increased CYP2A5 transcription. Co-transfection of Cyp2a5 5' promoter constructs with the PGC-1α expression vector demonstrated that PGC-1α is able to activate Cyp2a5 transcription through the hepatocyte nuclear factor (HNF)-4α response element in the proximal promoter of the Cyp2a5 gene. Chromatin immunoprecipitation assays showed that PGC-1α binds, together with HNF-4α, to the same region at the Cyp2a5 proximal promoter. In conclusion, PGC-1α mediates the expression of CYP2A5 induced by cAMP in mouse hepatocytes through coactivation of transcription factor HNF-4α. This strongly suggests that PGC-1α is the major factor mediating the fasting response of CYP2A5

  2. Isoferritins in rat Kupffer cells, hepatocytes, and extrahepatic macrophages. Biosynthesis in cell suspensions and cultures in response to iron

    International Nuclear Information System (INIS)

    Doolittle, R.L.; Richter, G.W.

    1981-01-01

    Cultures of Kupffer cells and of hepatocytes, prepared from single rat livers, synthesized ferritin protein equally efficiently. In culture but not in suspension, both sorts of cells responded significantly to stimulation with iron by increased ferritin synthesis. As determined by isoelectric focusing, the isoferritin profiles of newly synthesized 14 -labeled Kupffer cell and hepatocyte ferritin were identical, each having three bands. However, unlabeled ferritin, extracted from nonparenchymal liver cells (mainly Kupffer and endothelial cells) of iron-loaded rats, contained an acidic isoferritin that was not present in hepatocyte ferritin. Investigation of ferritin synthesis in cultured peritoneal and alveolar macrophages yielded similar results. The isofocusing profile of newly synthesized peritoneal macrophage ferritin was indistinguishable from the profile of fresh Kupffer cell or hepatocyte ferritin. Thus, the three isoferritins common to Kupffer cells, hepatocytes, and extrahepatic macrophages are neither cell- nor tissue-specific. However, modifications on intracellular storage may affect the isofocusing properties. The findings, although consistent with the LnH24-n subunit model of ferritin protein, indicate identical restrictive genomic control of the H:L ratios in these sorts of cells. Further, they make it probable that Kupffer cell ferritin iron, originating by endogenous synthesis, is the principal source of Kupffer cell hemosiderin iron

  3. Dissociated cultures of newborn mouse brain

    International Nuclear Information System (INIS)

    Wiesmann, U.N.; Hofmann, K.; Burkhart, T.; Herschkowitz, N.

    1975-01-01

    The metabolism of 35 SO 4 -sulfated lipids and mucopolysaccharides was studied in dissociated brain cell cultures from newborn albino mouse brains. The cultures were maintained under an atmosphere of 40% O 2 and 5% CO 2 in apparent good health up to 30 days. Early morphological examination of the dissociated cells demonstrated an initial partial reaggregation of the cells, which later settled and became confluent bilayered cultures. Cell proliferation measured by DNA and protein determination, morphological differentiation and biochemical differentiation took place in the dissociated brain cell cultures analogous in some respects to the in vivo situation. A timed increase in the synthesis of a myelin precursor, cerebroside 35 SO 4 , was observed after 6 to 8 days in culture (DIC). A peak of cerebroside sulfate was evident at 17 DIC. No stable sulfatide was observed at any time. Protein-bound macromolecular 35 SO 4 -MPS was synthetized and secreted from the cells into the culture medium. Maximal synthesis and secretion occurred at 8 DIC. This culture system proves to be a useful model for studying some aspects of differentiation of brain cells under external conditions. (author)

  4. Effect of elevated total CoA levels on metabolic pathways in cultured hepatocytes

    International Nuclear Information System (INIS)

    Steffen, C.A.; Smith, C.M.

    1987-01-01

    Livers from fasted rats have 30% higher total CoA levels than fed rats. To determine whether this increase of total CoA influences metabolism, the rates of gluconeogenesis, fatty acid oxidation and ketogenesis were measured in hepatocytes with cyanamide (CYM) or pantothenate (PA) deficient medium used to vary total CoA levels independently of hormonal status. Primary cultures of rat hepatocytes were incubated 14 hrs with Bt 2 cAMP, dexamethasone + theophylline in PA deficient medium or with CYM (500 μM) + PA, rinsed and preincubated 0.5 hr to remove the CYM. Hepatocytes treated with CYM had total CoA levels 10-24% higher than PA deficient cells and lower rates of glucose production from lactate + pyruvate (L/P) or from alanine (0.23 +/- 0.05 and 0.089 +/- 0.02 μm/mg protein, respectively in CYM treated cells compared to 0.33 +/- 0.06 and 0.130 +/- 0.006 in PA deficient cells). This decrease was not due to CYM per se, as the direct addition of CYM stimulated glucose production from L/P. CYM treated cells with 15-40% higher total CoA and 30% higher fatty acyl-CoA levels had the same rates of [ 14 C]-palmitate oxidation as PA deficient cells. However, rates of ketogenesis were lower in CYM treated cells (163 +/- 11 nm/mg compared to 217 +/- 14 nm/mg protein). These results suggest that physiological alterations of hepatic total CoA levels are not necessary for fasting rates of gluconeogenesis, fatty acid oxidation and ketogenesis

  5. Antioxidant and cytoprotective properties of D-tagatose in cultured murine hepatocytes.

    Science.gov (United States)

    Paterna, J C; Boess, F; Stäubli, A; Boelsterli, U A

    1998-01-01

    D-Tagatose is a zero-energy producing ketohexose that is a powerful cytoprotective agent against chemically induced cell injury. To further explore the underlying mechanisms of cytoprotection, we investigated the effects of D-tagatose on both the generation of superoxide anion radicals and the consequences of oxidative stress driven by prooxidant compounds in intact cells. Primary cultures of hepatocytes derived from male C57BL/6 mice were exposed to the redox cycling drug nitrofurantoin (NFT). Lethal cell injury induced by 300 microM NFT was completely prevented by high concentrations (20 mM) of D-tagatose, whereas equimolar concentrations of glucose, mannitol, or xylose were ineffective. The extent of NFT-induced intracellular superoxide anion radical formation was not altered by D-tagatose, indicating that the ketohexose did not inhibit the reductive bioactivation of NFT. However, the NFT-induced decline of the intracellular GSH content was largely prevented by D-tagatose. The sugar also afforded complete protection against NFT toxicity in hepatocytes that had been chemically depleted of GSH. Furthermore, the ketohexose fully protected from increases in both membrane lipid peroxidation and protein carbonyl formation. In addition, D-tagatose completely prevented oxidative cell injury inflicted by toxic iron overload with ferric nitrilotriacetate (100 microM). In contrast, D-tagatose did not protect against lethal cell injury induced by tert-butyl hydroperoxide, a prooxidant which acts by hydroxyl radical-independent mechanisms and which is partitioned in the lipid bilayer. These results indicate that D-tagatose, which is a weak iron chelator, can antagonize the iron-dependent toxic consequences of intracellular oxidative stress in hepatocytes. The antioxidant properties of D-tagatose may result from sequestering the redox-active iron, thereby protecting more critical targets from the damaging potential of hydroxyl radical.

  6. 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)

  7. Arsenite Effects on Mitochondrial Bioenergetics in Human and Mouse Primary Hepatocytes Follow a Nonlinear Dose Response

    Directory of Open Access Journals (Sweden)

    Hemantkumar Chavan

    2017-01-01

    Full Text Available Arsenite is a known carcinogen and its exposure has been implicated in a variety of noncarcinogenic health concerns. Increased oxidative stress is thought to be the primary cause of arsenite toxicity and the toxic effect is thought to be linear with detrimental effects reported at all concentrations of arsenite. But the paradigm of linear dose response in arsenite toxicity is shifting. In the present study we demonstrate that arsenite effects on mitochondrial respiration in primary hepatocytes follow a nonlinear dose response. In vitro exposure of primary hepatocytes to an environmentally relevant, moderate level of arsenite results in increased oxidant production that appears to arise from changes in the expression and activity of respiratory Complex I of the mitochondrial proton circuit. In primary hepatocytes the excess oxidant production appears to elicit adaptive responses that promote resistance to oxidative stress and a propensity to increased proliferation. Taken together, these results suggest a nonlinear dose-response characteristic of arsenite with low-dose arsenite promoting adaptive responses in a process known as mitohormesis, with transient increase in ROS levels acting as transducers of arsenite-induced mitohormesis.

  8. A novel method to immobilize collagen on polypropylene film as substrate for hepatocyte culture

    International Nuclear Information System (INIS)

    Zhang Yong; Wang Wenjie; Feng Qingling; Cui Fuzhai; Xu Yingxin

    2006-01-01

    To improve the biocompatibility of polypropylene membrane with hepatocytes, film was firstly allowed to generate peroxide groups by ammonium peroxydisulfate (APS) thermal decomposition and was then carboxylated by free radical grafting polymerization using acrylic acid as the monomer and ferrous(II) ions as redox initiator. Collagen was then covalently immobilized through amide bonds between the residue amine groups and carboxylic ones in the presence of water-soluble carbodiimide. The film was characterized by Fourier transformed infrared spectroscopy in attenuated total reflection mode (ATR-FTIR), X-photoelectron spectroscopy (XPS) and confocal laser scanning microscopy (CLSM), etc. The experimental results show that there exist hydroperoxide groups after APS aqueous thermal decomposition, which are subsequently converted into the polymeric free radicals initiating the vinyl monomers to subject grafting polymerization. The maximum collagen immobilization content is about 10.5 μg/cm 2 by ninhydrin assay. Hepatocytes cultured on collagen immobilized film show stable phenotype and normal liver-specific functions more than 20 days as observed by scanning electronic microscopy (SEM) and biochemical parameters determination

  9. Expression profiling of interindividual variability following xenobiotic exposures in primary human hepatocyte cultures

    International Nuclear Information System (INIS)

    Goyak, Katy M.O.; Johnson, Mary C.; Strom, Stephen C.; Omiecinski, Curtis J.

    2008-01-01

    To examine the magnitude of human variability across the entire transcriptome after chemical challenge, we profiled gene expression responses to three different prototypic chemical inducers in primary human hepatocyte cultures from ten independent donors. Correlation between basal expression in any two hepatocyte donors ranged from r 2 values of 0.967 to 0.857, and chemical treatment tended to negatively impact correlation between donors. Including anticipated target genes, 10,812, 8373, and 7847 genes were changed in at least one donor by Aroclor 1254 (A1254), di(2-ethylhexyl) phthalate (DEHP), and phenobarbital (PB), respectively. A subset of these gene targets (n = 41) were altered with a high level of reproducibility in at least 9 donors, gene responses that correlated well with literature-reported mechanism of action. Filtering responses to the level of gene subsets clarified the biological impact associated with the respective chemical effectors, in lieu of substantial interindividual variation among donor responses. In these respects, the use of hierarchical clustering methods successfully grouped seven of the ten donors into chemical-specific rather than donor-specific clusters. However, at the whole-genome level, the magnitude of conserved gene expression changes among donors was surprisingly small, with fewer than 50% of the gene responses altered by a single chemical conserved in more than one donor. The use of higher level descriptors, such as those defined by the PANTHER classification system, may enable more consistent categorization of gene expression changes across individuals, as increased reproducibility was identified using this method

  10. A novel method to immobilize collagen on polypropylene film as substrate for hepatocyte culture

    Energy Technology Data Exchange (ETDEWEB)

    Zhang Yong [Biomaterials Laboratory, Department of Material Science and Engineering, Tsinghua University, Beijing 100084 (China); Wang Wenjie [Biomaterials Laboratory, Department of Material Science and Engineering, Tsinghua University, Beijing 100084 (China); Feng Qingling [Biomaterials Laboratory, Department of Material Science and Engineering, Tsinghua University, Beijing 100084 (China)]. E-mail: biomater@mail.tsinghua.edu.cn; Cui Fuzhai [Biomaterials Laboratory, Department of Material Science and Engineering, Tsinghua University, Beijing 100084 (China); Xu Yingxin [Institute of General Surgery, Chinese PLA General Hospital, Beijing 100853 (China)

    2006-05-15

    To improve the biocompatibility of polypropylene membrane with hepatocytes, film was firstly allowed to generate peroxide groups by ammonium peroxydisulfate (APS) thermal decomposition and was then carboxylated by free radical grafting polymerization using acrylic acid as the monomer and ferrous(II) ions as redox initiator. Collagen was then covalently immobilized through amide bonds between the residue amine groups and carboxylic ones in the presence of water-soluble carbodiimide. The film was characterized by Fourier transformed infrared spectroscopy in attenuated total reflection mode (ATR-FTIR), X-photoelectron spectroscopy (XPS) and confocal laser scanning microscopy (CLSM), etc. The experimental results show that there exist hydroperoxide groups after APS aqueous thermal decomposition, which are subsequently converted into the polymeric free radicals initiating the vinyl monomers to subject grafting polymerization. The maximum collagen immobilization content is about 10.5 {mu}g/cm{sup 2} by ninhydrin assay. Hepatocytes cultured on collagen immobilized film show stable phenotype and normal liver-specific functions more than 20 days as observed by scanning electronic microscopy (SEM) and biochemical parameters determination.

  11. The regulation of cytoskeletal and liver-specific gene expression during liver regeneration and primary hepatocyte culture

    International Nuclear Information System (INIS)

    Robinson, G.S.

    1989-01-01

    The focus of this dissertation is to determine what role(s) the extracellular matrix and expression of certain cytoskeletal genes play in the regulation of hepatocyte growth and the maintenance of a differential state. The expression of several cytoskeletal and liver-specific genes was examined during liver regeneration and in hepatocyte cultures maintained in a hormonally-defined, serum-free medium and plated on two different matrices: rat tail collagen and the EHS matrix. During liver regeneration and in hepatocytes cultured on rat tail collagen, there was a dramatic increase in tubulin mRNA levels coincident with but not linked to DNA synthesis. The message levels for other cytoskeletal genes similarly increased, while a decrease was observed in the mRNA levels of the liver-specific genes, serum albumin and alpha 1 inhibitor III. Hepatocytes cultured on the EHS matrix resulted in the maintenance of low levels of cytoskeletal gene expression and high levels of liver-specific gene expression, similar to that observed in the normal liver. Results from subcellar fractionation and two-dimensional gel electrophoresis of 35 S-labelled proteins paralleled the results seen at the mRNA level. Preliminary work suggests that microtubule organization may play a role in the expression of the liver-specific genes which encode secreted proteins. These studies, which compare hepatocytes cultured on collagen or the EHS matrix gel, reveal that both cell-cell and cell-matrix interactions play a major role in the maintenance of the differential phenotype in hepatocytes

  12. An inducible transgenic mouse model for immune mediated hepatitis showing clearance of antigen expressing hepatocytes by CD8+ T cells.

    Directory of Open Access Journals (Sweden)

    Marcin Cebula

    Full Text Available The liver has the ability to prime immune responses against neo antigens provided upon infections. However, T cell immunity in liver is uniquely modulated by the complex tolerogenic property of this organ that has to also cope with foreign agents such as endotoxins or food antigens. In this respect, the nature of intrahepatic T cell responses remains to be fully characterized. To gain deeper insight into the mechanisms that regulate the CD8+ T cell responses in the liver, we established a novel OVA_X_CreER(T2 mouse model. Upon tamoxifen administration OVA antigen expression is observed in a fraction of hepatocytes, resulting in a mosaic expression pattern. To elucidate the cross-talk of CD8+ T cells with antigen-expressing hepatocytes, we adoptively transferred K(b/OVA257-264-specific OT-I T cells to OVA_X_CreER(T2 mice or generated triple transgenic OVA_X CreER(T2_X_OT-I mice. OT-I T cells become activated in OVA_X_CreER(T2 mice and induce an acute and transient hepatitis accompanied by liver damage. In OVA_X_CreER(T2_X_OT-I mice, OVA induction triggers an OT-I T cell mediated, fulminant hepatitis resulting in 50% mortality. Surviving mice manifest a long lasting hepatitis, and recover after 9 weeks. In these experimental settings, recovery from hepatitis correlates with a complete loss of OVA expression indicating efficient clearance of the antigen-expressing hepatocytes. Moreover, a relapse of hepatitis can be induced upon re-induction of cured OVA_X_CreER(T2_X_OT-I mice indicating absence of tolerogenic mechanisms. This pathogen-free, conditional mouse model has the advantage of tamoxifen inducible tissue specific antigen expression that reflects the heterogeneity of viral antigen expression and enables the study of intrahepatic immune responses to both de novo and persistent antigen. It allows following the course of intrahepatic immune responses: initiation, the acute phase and antigen clearance.

  13. Bile canaliculi formation and biliary transport in 3D sandwich-cultured hepatocytes in dependence of the extracellular matrix composition.

    Science.gov (United States)

    Deharde, Daniela; Schneider, Christin; Hiller, Thomas; Fischer, Nicolas; Kegel, Victoria; Lübberstedt, Marc; Freyer, Nora; Hengstler, Jan G; Andersson, Tommy B; Seehofer, Daniel; Pratschke, Johann; Zeilinger, Katrin; Damm, Georg

    2016-10-01

    Primary human hepatocytes (PHH) are still considered as gold standard for investigation of in vitro metabolism and hepatotoxicity in pharmaceutical research. It has been shown that the three-dimensional (3D) cultivation of PHH in a sandwich configuration between two layers of extracellular matrix (ECM) enables the hepatocytes to adhere three dimensionally leading to formation of in vivo like cell-cell contacts and cell-matrix interactions. The aim of the present study was to investigate the influence of different ECM compositions on morphology, cellular arrangement and bile canaliculi formation as well as bile excretion processes in PHH sandwich cultures systematically. Freshly isolated PHH were cultured for 6 days between two ECM layers made of collagen and/or Matrigel in four different combinations. The cultures were investigated by phase contrast microscopy and immunofluorescence analysis with respect to cell-cell connections, repolarization as well as bile canaliculi formation. The influence of the ECM composition on cell activity and viability was measured using the XTT assay and a fluorescent dead or alive assay. Finally, the bile canalicular transport was analyzed by live cell imaging to monitor the secretion and accumulation of the fluorescent substance CDF in bile canaliculi. Using collagen and Matrigel in different compositions in sandwich cultures of hepatocytes, we observed differences in morphology, cellular arrangement and cell activity of PHH in dependence of the ECM composition. Sandwich-cultured hepatocytes with an underlay of collagen seem to represent the best in vivo tissue architecture in terms of formation of trabecular cell arrangement. Cultures overlaid with collagen were characterized by the formation of abundant bile canaliculi, while the bile canaliculi network in hepatocytes cultured on a layer of Matrigel and overlaid with collagen showed the most branched and stable canalicular network. All cultures showed a time-dependent leakage of

  14. DIFFERENCES IN PROPIONATE-INDUCED INHIBITION OF CHOLESTEROL AND TRIACYLGLYCEROL SYNTHESIS BETWEEN HUMAN AND RAT HEPATOCYTES IN PRIMARY CULTURE

    NARCIS (Netherlands)

    LIN, YG; VONK, RJ; SLOOFF, MJH; KUIPERS, F; SMIT, MJ

    Propionate is a short-chain fatty acid formed in the colon and supposedly involved in the cholesterol-lowering effect of soluble fibre. To explore the underlying mechanism(s) of this fibre action, we have used human hepatocytes in primary culture to study the effects of propionate on hepatic lipid

  15. Suppression of sterol 27-hydroxylase mRNA and transcriptional activity by bile acids in cultured rat hepatocytes

    NARCIS (Netherlands)

    Twisk, J.; Wit, E.C.M. de; Princen, H.M.G.

    1995-01-01

    In previous work we have demonstrated suppression of cholesterol 7α-hydroxylase by bile acids at the level of mRNA and transcription, resulting in a similar decline in bile acid synthesis in cultured rat hepatocytes. In view of the substantial contribution of the 'alternative' or '27-hydroxylase'

  16. Automated applications of sandwich-cultured hepatocytes in the evaluation of hepatic drug transport.

    Science.gov (United States)

    Perry, Cassandra H; Smith, William R; St Claire, Robert L; Brouwer, Kenneth R

    2011-04-01

    Predictions of the absorption, distribution, metabolism, excretion, and toxicity of compounds in pharmaceutical development are essential aspects of the drug discovery process. B-CLEAR is an in vitro system that uses sandwich-cultured hepatocytes to evaluate and predict in vivo hepatobiliary disposition (hepatic uptake, biliary excretion, and biliary clearance), transporter-based hepatic drug-drug interactions, and potential drug-induced hepatotoxicity. Automation of predictive technologies is an advantageous and preferred format in drug discovery. In this study, manual and automated studies are investigated and equivalence is demonstrated. In addition, automated applications using model probe substrates and inhibitors to assess the cholestatic potential of drugs and evaluate hepatic drug transport are examined. The successful automation of this technology provides a more reproducible and less labor-intensive approach, reducing potential operator error in complex studies and facilitating technology transfer.

  17. 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

  18. Dysplastic hepatocytes develop nuclear inclusions in a mouse model of viral hepatitis.

    Directory of Open Access Journals (Sweden)

    Priyanka Thakur

    Full Text Available Viral hepatitis resulting in chronic liver disease is an important clinical challenge and insight into the cellular processes that drive pathogenesis will be critical in order to develop new diagnostic and therapeutic options. Nuclear inclusions in viral and non-viral hepatitis are well documented and have diagnostic significance in some disease contexts. However, the origins and functional consequences of these nuclear inclusions remain elusive. To date the clinical observation of nuclear inclusions in viral and non-viral hepatitis has not been explored at depth in murine models of liver disease. Herein, we report that in a transgenic model of hepatitis B surface antigen mediated hepatitis, murine hepatocytes exhibit nuclear inclusions. Cells bearing nuclear inclusions were more likely to express markers of cell proliferation. We also established a correlation between these inclusions and oxidative stress. N-acetyl cysteine treatment effectively reduced oxidative stress levels, relieved endoplasmic reticulum (ER stress, and the number of nuclear inclusions we observed in the transgenic mice. Our results suggest that the presence of nuclear inclusions in hepatocytes correlates with oxidative stress and cellular proliferation in a model of antigen mediated hepatitis.

  19. Flux balance analysis predicts Warburg-like effects of mouse hepatocyte deficient in miR-122a.

    Directory of Open Access Journals (Sweden)

    Hua-Qing Wu

    2017-07-01

    Full Text Available The liver is a vital organ involving in various major metabolic functions in human body. MicroRNA-122 (miR-122 plays an important role in the regulation of liver metabolism, but its intrinsic physiological functions require further clarification. This study integrated the genome-scale metabolic model of hepatocytes and mouse experimental data with germline deletion of Mir122a (Mir122a-/- to infer Warburg-like effects. Elevated expression of MiR-122a target genes in Mir122a-/-mice, especially those encoding for metabolic enzymes, was applied to analyze the flux distributions of the genome-scale metabolic model in normal and deficient states. By definition of the similarity ratio, we compared the flux fold change of the genome-scale metabolic model computational results and metabolomic profiling data measured through a liquid-chromatography with mass spectrometer, respectively, for hepatocytes of 2-month-old mice in normal and deficient states. The Ddc gene demonstrated the highest similarity ratio of 95% to the biological hypothesis of the Warburg effect, and similarity of 75% to the experimental observation. We also used 2, 6, and 11 months of mir-122 knockout mice liver cell to examined the expression pattern of DDC in the knockout mice livers to show upregulated profiles of DDC from the data. Furthermore, through a bioinformatics (LINCS program prediction, BTK inhibitors and withaferin A could downregulate DDC expression, suggesting that such drugs could potentially alter the early events of metabolomics of liver cancer cells.

  20. 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

  1. 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

  2. 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

  3. Differential gene expression analysis of in vitro duck hepatitis B virus infected primary duck hepatocyte cultures

    Directory of Open Access Journals (Sweden)

    Issac Aneesh

    2011-07-01

    Full Text Available Abstract Background The human hepatitis B virus (HBV, a member of the hepadna viridae, causes acute or chronic hepatitis B, and hepatocellular carcinoma (HCC. The duck hepatitis B virus (DHBV infection, a dependable and reproducible model for hepadna viral studies, does not result in HCC unlike chronic HBV infection. Information on differential gene expression in DHBV infection might help to compare corresponding changes during HBV infection, and to delineate the reasons for this difference. Findings A subtractive hybridization cDNA library screening of in vitro DHBV infected, cultured primary duck hepatocytes (PDH identified cDNAs of 42 up-regulated and 36 down-regulated genes coding for proteins associated with signal transduction, cellular respiration, transcription, translation, ubiquitin/proteasome pathway, apoptosis, and membrane and cytoskeletal organization. Those coding for both novel as well as previously reported proteins in HBV/DHBV infection were present in the library. An inverse modulation of the cDNAs of ten proteins, reported to play role in human HCC, such as that of Y-box binding protein1, Platelet-activating factor acetylhydrolase isoform 1B, ribosomal protein L35a, Ferritin, α-enolase, Acid α-glucosidase and Caspase 3, copper-zinc superoxide dismutase (CuZnSOD, Filamin and Pyruvate dehydrogenase, was also observed in this in vitro study. Conclusions The present study identified cDNAs of a number of genes that are differentially modulated in in vitro DHBV infection of primary duck hepatocytes. Further correlation of this differential gene expression in in vivo infection models would be valuable to understand the little known aspects of the hepadnavirus biology.

  4. Kinetics of zinc uptake and exchange by primary cultures of rat hepatocytes

    International Nuclear Information System (INIS)

    Pattison, S.E.; Cousins, R.J.

    1986-01-01

    The kinetics of 65 Zn 2+ uptake and exchange by hepatocytes in primary culture have been examined in detail to provide a basis for analyzing hormonal regulation of hepatic zinc metabolism. 65 Zn 2+ uptake was found to be a biphasic process. The slow phase represents an exchange between Zn 2+ in the medium and preexisting, intracellular zinc pools. This exchange rate was saturable with a medium zinc concentration of 9.5 microM eliciting one-half the maximum exchange rate and a maximum exchange rate of 9.9 pmol Zn 2+ . min-1 . mg protein-1 in the presence of bovine serum albumin. In the absence of albumin, a secondary, nonsaturable uptake rate was observed. The slow phase was relatively selective, and of the divalent transition metal ions tested, only Cd 2+ and Mn 2+ caused inhibition. The rate of exchange suggests total hepatocyte zinc has a turnover rate of approximately 30 h. The fast phase of 65 Zn 2+ reflects net Zn 2+ accumulation into a labile pool. The initial rates for this process were too fast to be measured accurately, but steady-state measurements allowed determination of the labile pool size. The pool dimensions saturated in the presence [Kapp = 28.6 microM; pool capacity = 0.44 nmol Zn 2+ /mg protein] and absence [Kapp = 11.8 microM; pool capacity = 0.34 nmol Zn 2+ /mg protein] of bovine serum albumin. Kinetics and equilibria of Zn 2+ uptake into the labile pool suggest that the latter acts as a source of Zn 2+ for the slow-exchange phase. Dexamethasone stimulated slow Zn 2+ exchange and also increased the labile pool size. The data suggest physiological factors alter hepatic zinc metabolism by influencing both intracellular Zn 2+ pools

  5. Stimulation of albumin gene transcription by insulin in primary cultures of rat hepatocytes

    International Nuclear Information System (INIS)

    Lloyd, C.E.; Kalinyak, J.E.; Hutson, S.M.; Jefferson, L.S.

    1987-01-01

    The first goal of the work reported here was to prepare single-stranded DNA sequences for use in studies on the regulation of albumin gene expression. A double-stranded rat albumin cDNA clone was subcloned into the bacteriophage vector M13mp7. Single-stranded recombinant clones were screened for albumin sequences containing either the mRNA strand or the complementary strand. Two clones were selected that contained the 1200 nucleotide long 3' end of the albumin sequence. DNA from the clone containing the mRNA strand was used as a template for DNA polymerase I to prepare a radiolabeled, single-stranded cDNA to albumin mRNA. This radiolabeled cDNA probe was used to quantitate the relative abundance of albumin mRNA in samples of total cellular RNA. DNA from the clone containing the complementary strand was used to measure relative rates of albumin gene transcription in isolated nuclei. The second goal was to use the single-stranded DNA probes to investigate the mechanism of the insulin-mediated stimulation of albumin synthesis in primary cultures of rat hepatocytes. Addition of insulin to hepatocytes maintained in a chemically defined, serum-free medium for 40 h in the absence of any hormones resulted in a specific 1.5- to 2.5-fold stimulation of albumin gene transcription that was maximal at 3 h and was maintained above control values for at least 24 h. The rate of albumin gene transcription in nuclei isolated from livers of diabetic rats was reduced to 50% of the value recorded in control nuclei. Taken together, these findings demonstrate that insulin regulates synthesis of albumin at the level of gene transcription

  6. Property of hepatitis B virus replication in Tupaia belangeri hepatocytes

    International Nuclear Information System (INIS)

    Sanada, Takahiro; Tsukiyama-Kohara, Kyoko; Yamamoto, Naoki; Ezzikouri, Sayeh; Benjelloun, Soumaya; Murakami, Shuko; Tanaka, Yasuhito; Tateno, Chise; Kohara, Michinori

    2016-01-01

    The northern treeshrew (Tupaia belangeri) has been reported to be an effective candidate for animal infection model with hepatitis B virus (HBV). The objective of our study was to analyze the growth characteristics of HBV in tupaia hepatocytes and the host response to HBV infection. We established primary tupaia hepatocytes (3–6-week old tupaia) and infected them with HBV genotypes A, B and C, and all the genotypes proliferated as well as those in human primary hepatocytes (>10"5 copies/ml in culture supernatant). We next generated a chimeric mouse with tupaia liver by transplantation of tupaia primary hepatocytes to urokinase-type plasminogen activator cDNA (cDNA-uPA)/severe combined immunodeficient (SCID) mice and the replacement ratio with tupaia hepatocytes was found to be more than 95%. Infection of chimeric mice with HBV (genotypes B, C, and D) resulted in HBV-DNA level of 10"4-10"6 copies/ml after 8 weeks of infection, which were almost similar to that in humanized chimeric mouse. In contrast, serum HBV level in adult tupaia (1-year-old tupaia) was quite low (<10"3 copies/ml). Understanding the differences in the response to HBV infection in primary tupaia hepatocytes, chimeric mouse, and adult tupaia will contribute to elucidating the mechanism of persistent HBV infection and viral eradication. Thus, T. belangeri was found to be efficient for studying the host response to HBV infection, thereby providing novel insight into the pathogenesis of HBV. - Highlights: • Primary hepatocytes were established from tupaia that is a novel HBV infection model. • Tupaia primary hepatocytes were susceptible for HBV infection. • The immunodeficient chimeric mice with tupaia hepatocytes were established. • The chimeric mice with tupaia hepatocytes were susceptible for HBV infection.

  7. Property of hepatitis B virus replication in Tupaia belangeri hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Sanada, Takahiro [Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, 2-1-6, Kamikitazawa, Setagaya-ku, Tokyo 156-8506 (Japan); Tsukiyama-Kohara, Kyoko, E-mail: kkohara@vet.kagoshima-u.ac.jp [Transboundary Animal Diseases Centre, Joint Faculty of Veterinary Medicine, Kagoshima University, 1-21-24, Korimoto, Kagoshima-city, Kagoshima 890-0065 (Japan); Laboratory of Animal Hygiene, Joint Faculty of Veterinary Medicine, Kagoshima University, 1-21-24, Korimoto, Kagoshima, Kagoshima 890-0065 (Japan); Yamamoto, Naoki [Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, 2-1-6, Kamikitazawa, Setagaya-ku, Tokyo 156-8506 (Japan); Ezzikouri, Sayeh; Benjelloun, Soumaya [Viral Hepatitis Laboratory, Virology Unit, Institut Pasteur du Maroc, 1, Louis Pasteur, Casablanca 20360 (Morocco); Murakami, Shuko; Tanaka, Yasuhito [Department of Virology and Liver Unit, Nagoya City University Graduate School of Medical Sciences, Kawasumi 1, Mizuho-ku, Nagoya, Aichi 467-8601 (Japan); Tateno, Chise [PhoenixBio Co. Ltd., 3-4-1, Kagamiyama, Higashi-Hiroshima, Hiroshima 739-0046 (Japan); Kohara, Michinori, E-mail: kohara-mc@igakuken.or.jp [Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, 2-1-6, Kamikitazawa, Setagaya-ku, Tokyo 156-8506 (Japan)

    2016-01-08

    The northern treeshrew (Tupaia belangeri) has been reported to be an effective candidate for animal infection model with hepatitis B virus (HBV). The objective of our study was to analyze the growth characteristics of HBV in tupaia hepatocytes and the host response to HBV infection. We established primary tupaia hepatocytes (3–6-week old tupaia) and infected them with HBV genotypes A, B and C, and all the genotypes proliferated as well as those in human primary hepatocytes (>10{sup 5} copies/ml in culture supernatant). We next generated a chimeric mouse with tupaia liver by transplantation of tupaia primary hepatocytes to urokinase-type plasminogen activator cDNA (cDNA-uPA)/severe combined immunodeficient (SCID) mice and the replacement ratio with tupaia hepatocytes was found to be more than 95%. Infection of chimeric mice with HBV (genotypes B, C, and D) resulted in HBV-DNA level of 10{sup 4}-10{sup 6} copies/ml after 8 weeks of infection, which were almost similar to that in humanized chimeric mouse. In contrast, serum HBV level in adult tupaia (1-year-old tupaia) was quite low (<10{sup 3} copies/ml). Understanding the differences in the response to HBV infection in primary tupaia hepatocytes, chimeric mouse, and adult tupaia will contribute to elucidating the mechanism of persistent HBV infection and viral eradication. Thus, T. belangeri was found to be efficient for studying the host response to HBV infection, thereby providing novel insight into the pathogenesis of HBV. - Highlights: • Primary hepatocytes were established from tupaia that is a novel HBV infection model. • Tupaia primary hepatocytes were susceptible for HBV infection. • The immunodeficient chimeric mice with tupaia hepatocytes were established. • The chimeric mice with tupaia hepatocytes were susceptible for HBV infection.

  8. Acylcarnitine Profiles in Acetaminophen Toxicity in the Mouse: Comparison to Toxicity, Metabolism and Hepatocyte Regeneration

    Directory of Open Access Journals (Sweden)

    Jack Hinson

    2013-08-01

    Full Text Available High doses of acetaminophen (APAP result in hepatotoxicity that involves metabolic activation of the parent compound, covalent binding of the reactive intermediate N-acetyl-p-benzoquinone imine (NAPQI to liver proteins, and depletion of hepatic glutathione. Impaired fatty acid β-oxidation has been implicated in previous studies of APAP-induced hepatotoxicity. To better understand relationships between toxicity and fatty acid β-oxidation in the liver in APAP toxicity, metabolomic assays for long chain acylcarnitines were examined in relationship to established markers of liver toxicity, oxidative metabolism, and liver regeneration in a time course study in mice. Male B6C3F1 mice were treated with APAP (200 mg/kg IP or saline and sacrificed at 1, 2, 4, 8, 24 or 48 h after APAP. At 1 h, hepatic glutathione was depleted and APAP protein adducts were markedly increased. Alanine aminotransferase (ALT levels were elevated at 4 and 8 h, while proliferating cell nuclear antigen (PCNA expression, indicative of hepatocyte regeneration, was apparent at 24 h and 48 h. Elevations of palmitoyl, oleoyl and myristoyl carnitine were apparent by 2–4 h, concurrent with the onset of Oil Red O staining in liver sections. By 8 h, acylcarnitine levels were below baseline levels and remained low at 24 and 48 h. A partial least squares (PLS model suggested a direct association of acylcarnitine accumulation in serum to APAP protein adduct and hepatic glutathione levels in mice. Overall, the kinetics of serum acylcarnitines in APAP toxicity in mice followed a biphasic pattern involving early elevation after the metabolism phases of toxicity and later depletion of acylcarnitines.

  9. The biotransformation of tetrahydroaminoacridine (THA) in cultured hepatocytes as the cause for relative cytotoxicity in 3 species

    International Nuclear Information System (INIS)

    Smolarek, T.A.; Higgins, C.V.; Amacher, D.E.

    1990-01-01

    THA, a centrally acting anticholinesterase, shows promise for the treatment of Alzheimer's disease. However, its use has been associated with suspected human hepatotoxicity through an unknown mechanism. In this study, the cytotoxicity and biotransformation of THA was studied in rat, canine, and monkey primary hepatocyte cultures. Cytotoxicity was indicated by the release of ALT, AST, or LDH into culture medium over 24 hours. THA biotransformation was studied by exposing cells to 100 nM [ 3 H]-THA and then analyzing culture medium for labelled moieties by reversed-phase HPLC after 1,2, and 24 hrs in culture. THA was toxic to rat and canine cells at 200 μg/ml and to monkey cells at 100 μg/ml. About 98% of the THA was transformed by canine and rat cells to 3 metabolites in 2 hrs, but in monkey cell cultures, only 55% of THA was transformed to 2 metabolites in 2 hrs and 95% to 3 metabolites in 24 hrs. Quantitative differences were also noted in metabolite profiles between monkey and rat or canine cell cultures. The predominant metabolite at 2 hours, tentatively identified as 1-OH-THA, was greatly diminished or absent in canine and rat but not monkey cell cultures after 18 hours. Thus, unchanged THA or this 1-OH-THA metabolite may be responsible for the greater cytotoxicity in the monkey hepatocyte cultures

  10. Total hepatocellular disposition profiling of rosuvastatin and pitavastatin in sandwich-cultured human hepatocytes.

    Science.gov (United States)

    Kanda, Katsuhiro; Takahashi, Ryosuke; Yoshikado, Takashi; Sugiyama, Yuichi

    2018-04-09

    This study describes the total disposition profiling of rosuvastatin (RSV) and pitavastatin (PTV) using a single systematic procedure called D-PREX (Disposition Profile Exploration) in sandwich-cultured human hepatocytes (SCHH). The biliary excretion fractions of both statins were clearly observed, which were significantly decreased dependent on the concentration of Ko143, an inhibitor for breast cancer resistance protein (BCRP). Ko143 also decreased the basolateral efflux fraction of RSV, whereas that of PTV was not significantly affected. To understand these phenomena, effects of Ko143 on biliary excretion (BCRP and multidrug resistance-associated protein (MRP) 2) and basolateral efflux (MRP3 and MRP4) transporters were examined using transporter-expressing membrane vesicles. BCRP, MRP3 and MRP4-mediated transport of RSV was observed, and Ko143 inhibited these transporters except MRP3. BCRP and MRP4 also mediated the transport of PTV, but the Ko143-mediated inhibition was only clear for BCRP. These results might explain the Ko143-mediated complete and partial inhibition of the biliary excretion and the basolateral efflux of RSV, respectively, in SCHH. In conclusion, D-PREX with sequential sampling of supernatants prior to cell lysis enables the evaluation of total drug disposition profiles resulting from complex interplays of intracellular pathways, which would provide high-throughput evaluation of drug disposition during drug discovery. Copyright © 2018 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.

  11. Progesterone dose-dependently modulates hepatocyte growth factor production in 3T3-L1 mouse preadipocytes.

    Science.gov (United States)

    Ito, Tomoki; Yamaji, Daisuke; Kamikawa, Akihiro; Abd Eldaim, Mabrouk Attia; Okamatsu-Ogura, Yuko; Terao, Akira; Saito, Masayuki; Kimura, Kazuhiro

    2017-08-30

    It is well documented that estrogen is predominant inducer of hepatocyte growth factor (HGF) in a variety of cell types. However, the effect of progesterone (P) remains to be elusive. Thus, in the present study, we examined the effect of P and combined effect of P and 17β-estradiol (E2) on HGF expression and production in 3T3-L1 fibroblastic preadipocytes and mature adipocytes, as a model of stromal cells. Northern blot analysis showed that hgf mRNA expressed in preadipocytes was notably higher than that of mature adipocytes, and increased by treatment of preadipocytes with E2 or 10 nM P, but not with 1,000 nM P. The E2-induced hgf mRNA expression was enhanced by 10 nM P, but suppressed by 1,000 nM P. Western blot analysis revealed that biological active forms of HGF protein was found in the preadipocyte culture medium, while the lesser amount of HGF precursor protein was detected in the mature adipocyte culture medium. The amounts of HGF were changed dependently on the hgf mRNA expression levels. These results indicate that HGF production is intricately regulated by E2 and P at the transcriptional levels in 3T3-L1 cells, and may explain the changes in the HGF production during the mammary gland development, especially decrease in HGF expression during pregnancy when P concentration is high.

  12. 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.

  13. Improved Survival and Initiation of Differentiation of Human Induced Pluripotent Stem Cells to Hepatocyte-Like Cells upon Culture in William's E Medium followed by Hepatocyte Differentiation Inducer Treatment.

    Directory of Open Access Journals (Sweden)

    Minoru Tomizawa

    Full Text Available Hepatocyte differentiation inducer (HDI lacks both glucose and arginine, but is supplemented with galactose and ornithine, and is added together with other reagents such as apoptosis inhibitor and oncostatin M. Although human induced pluripotent stem (iPS cells initiate hepatocyte differentiation, most die within 7 days. In this study, we investigated both HDI and conventional media for their potential to improve cell survival.201B7 iPS cells were cultured in conventional media. This consisted of three cycles of 5-day culture in William's E (WE medium, followed by a 2-day culture in HDI.Expression levels of α-feto protein (AFP were higher in cells cultured in WE and in Dulbecco's Modified Eagle's Medium/Nutrient F-12 Ham (DF12. 201B7 cells expressed the highest AFP and albumin (ALB when cultured in HDI for 2 days following 7-day culture in WE. After three cycles of 5-day culture in WE followed by 2 days in HDI, 201B7 cells expressed AFP and ALB 54 ± 2.3 (average ± standard deviation and 73 ± 15.1 times higher, respectively, than those cultured in ReproFF (feeder-free condition.201B7 cells survived culture in WE for 7 days followed HDI for 2 days. After three cycles of culture under these conditions, hepatocyte differentiation was enhanced, as evidenced by increased AFP and ALB expression.

  14. Localization of Alkaline Phosphatase and Cathepsin D during Cell Restoration after Colchicine Treatment in Primary Cultures of Fetal Rat Hepatocytes

    International Nuclear Information System (INIS)

    Chida, Kohsuke; Taguchi, Meiko

    2011-01-01

    Localization of alkaline phosphatase (ALP) and cathepsin D (CAPD) in primary cultures of fetal rat hepatocytes was examined using double immunofluorescent staining in order to investigate the relationship between lysosome movement and the fate of ALP during cell restoration after microtubule disruption by colchicine. At 3 hr and 24 hr after colchicine treatment, numerous coarse dots containing ALP were observed throughout the cytoplasm, and some of these showed colocalization with CAPD. At 48 hr and 72 hr after colchicine treatment, although most of the dots containing ALP in the cytoplasm disappeared, dots containing CAPD remained. The present results suggest that the denatured ALP proteins remaining in the cytoplasm of hepatocytes during cell restoration after colchicine treatment are digested by lysosomes

  15. Simultaneous demonstration of acid phosphatase and glucose-6-phosphate dehydrogenase in mouse hepatocytes. A novel electron-microscopic dual staining enzyme-cytochemistry

    Directory of Open Access Journals (Sweden)

    S Matsubara

    2010-01-01

    Full Text Available Acid phosphatase (ACPase and glucose-6-phosphate dehydrogenase (G6PD play important roles in cell biology/disease pathophysiology in various organs including the liver. The purpose of the present report is to introduce a new enzymecytochemical method to simultaneously demonstrate the subcellular localization of ACPase and G6PD within the same hepatocyte in the mouse liver. The ultrastructural localization of ACPase and G6PD were demonstrated, with concomitant use of the cerium method and the copper-ferrocyanide method, respectively. ACPase labelings were localized in the lysosomes, and G6PD labelings were visible in the cytoplasm and on the cytosolic side of the endoplasmic reticulum of the hepatocyte. This novel double staining procedure may be a useful histochemical tool for the study of liver functions in both physiological and pathological conditions.

  16. 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

  17. Differentiation of monkey embryonic stem cells to hepatocytes by feeder-free dispersion culture and expression analyses of cytochrome p450 enzymes responsible for drug metabolism.

    Science.gov (United States)

    Maruyama, Junya; Matsunaga, Tamihide; Yamaori, Satoshi; Sakamoto, Sakae; Kamada, Noboru; Nakamura, Katsunori; Kikuchi, Shinji; Ohmori, Shigeru

    2013-01-01

    We reported previously that monkey embryonic stem cells (ESCs) were differentiated into hepatocytes by formation of embryoid bodies (EBs). However, this EB formation method is not always efficient for assays using a large number of samples simultaneously. A dispersion culture system, one of the differentiation methods without EB formation, is able to more efficiently provide a large number of feeder-free undifferentiated cells. A previous study demonstrated the effectiveness of the Rho-associated kinase inhibitor Y-27632 for feeder-free dispersion culture and induction of differentiation of monkey ESCs into neural cells. In the present study, the induction of differentiation of cynomolgus monkey ESCs (cmESCs) into hepatocytes was performed by the dispersion culture method, and the expression and drug inducibility of cytochrome P450 (CYP) enzymes in these hepatocytes were examined. The cmESCs were successfully differentiated into hepatocytes under feeder-free dispersion culture conditions supplemented with Y-27632. The hepatocytes differentiated from cmESCs expressed the mRNAs for three hepatocyte marker genes (α-fetoprotein, albumin, CYP7A1) and several CYP enzymes, as measured by real-time polymerase chain reaction. In particular, the basal expression of cmCYP3A4 (3A8) in these hepatocytes was detected at mRNA and enzyme activity (testosterone 6β-hydroxylation) levels. Furthermore, the expression and activity of cmCYP3A4 (3A8) were significantly upregulated by rifampicin. These results indicated the effectiveness of Y-27632 supplementation for feeder-free dispersed culture and induction of differentiation into hepatocytes, and the expression of functional CYP enzyme(s) in cmESC-derived hepatic cells.

  18. A transgenic rat hepatocyte - Kupffer cell co-culture model for evaluation of direct and macrophage-related effect of poly(amidoamine) dendrimers.

    Science.gov (United States)

    Jemnitz, Katalin; Bátai-Konczos, Attila; Szabó, Mónika; Ioja, Enikő; Kolacsek, Orsolya; Orbán, Tamás I; Török, György; Homolya, László; Kovács, Eszter; Jablonkai, István; Veres, Zsuzsa

    2017-02-01

    Increasing number of papers demonstrate that Kupffer cells (KCs) play a role in the development of drug induced liver injury (DILI). Furthermore, elevated intracellular Ca 2+ level of hepatocytes is considered as a common marker of DILI. Here we applied an in vitro model based on hepatocyte mono- and hepatocyte/KC co-cultures (H/KC) isolated from transgenic rats stably expressing the GCaMP2 fluorescent Ca 2+ sensor protein to investigate the effects of polycationic (G5), polyanionic (G4.5) and polyethylene-glycol coated neutral (G5 Peg) dendrimers known to accumulate in the liver, primarily in KCs. Following dendrimer exposure, hepatocyte homeostasis was measured by MTT cytotoxicity assay and by Ca 2+ imaging, while hepatocyte functions were studied by CYP2B1/2 inducibility, and bilirubin and taurocholate transport. G5 was significantly more cytotoxic than G4.5 for hepatocytes and induced Ca 2+ oscillation and sustained Ca 2+ signals at 1μM and10 μM, respectively both in hepatocytes and KCs. Dendrimer-induced Ca 2+ signals in hepatocytes were attenuated by macrophages. Activation of KCs by lipopolysaccharide and G5 decreased the inducibility of CYP2B1/2, which was restored by depleting the KCs with gadolinium-chloride and pentoxyphylline, suggesting a role of macrophages in the hindrance of CYP2B1/2 induction by G5 and lipopolysaccharide. In the H/KC, but not in the hepatocyte mono-culture, G5 reduced the canalicular efflux of bilirubin and stimulated the uptake and canalicular efflux of taurocholate. In conclusion, H/KC provides a good model for the prediction of hepatotoxic potential of drugs, especially of nanomaterials known to be trapped by macrophages, activation of which presumably contributes to DILI. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Metabolism of the synthetic cannabinoid 5F-PY-PICA by human and rat hepatocytes and identification of biliary analytical targets by directional efflux in sandwich-cultured rat hepatocytes using UHPLC-HR-MS/MS

    DEFF Research Database (Denmark)

    Mardal, Marie; Annaert, Pieter; Noble, Carolina

    2018-01-01

    Analytical strategies for detecting drugs in biological samples rely on information on metabolism and elimination. 5F-PY-PICA belongs to the group of synthetic cannabinoids that are known to undergo excretion into the bile. The aims of this study were the in vitro identification of metabolites of 5......F-PY-PICA and to determine which analytical targets are excreted into the bile and urine. Metabolites identified after incubation of 5F-PY-PICA with pooled human liver microsomes (pHLM), pooled human hepatocytes (pHH), or suspended and sandwich-cultured rat hepatocytes (SCRH). Rat hepatocytes were......-PY-PICA, M4, and M22 are proposed as analytical targets for bile analysis in forensic screening protocols, whereas M6 should be one of the main urinary targets for 5F-PY-PICA analysis....

  20. Hydrodynamic Delivery of Cre Protein to Lineage-Mark or Time-Stamp Mouse Hepatocytes In situ

    Science.gov (United States)

    Sonsteng, Katherine M.; Prigge, Justin R.; Talago, Emily A.; June, Ronald K.; Schmidt, Edward E.

    2014-01-01

    Cre-responsive fluorescent marker alleles are powerful tools for cell lineage tracing in mice; however their utility is limited by regulation of Cre activity. When targeting hepatocytes, hydrodynamic delivery of a Cre-expression plasmid can convert Cre-responsive alleles without inducing the intracellular or systemic antiviral responses often associated with viral-derived Cre-expression vectors. In this method, rapid high-volume intravenous inoculation induces hepatocyte-targeted uptake of extracellular molecules. Here we tested whether hydrodynamic delivery of Cre protein or Cre fused to the HIV-TAT cell-penetrating peptide could convert Cre-responsive reporters in hepatocytes of mice. Hydrodynamic delivery of 2 nmol of either Cre or TAT-Cre protein converted the reporter allele in 5 to 20% of hepatocytes. Neither protein gave detectable Cre activity in endothelia, non-liver organs, or non-hepatocyte cells in liver. Using mice homozygous for a Cre-responsive marker that directs red- (Cre-naïve) or green- (Cre-converted) fluorescent proteins to the nucleus, we assessed sub-saturation Cre-activity. One month after hydrodynamic inoculation with Cre protein, 58% of hepatocyte nuclei that were green were also red, indicating that less than half of the hepatocytes that had obtained enough Cre to convert one marker allele to green were able to convert all alleles. For comparison, one month after hydrodynamic delivery of a Cre-expression plasmid with a weak promoter, only 26% of the green nuclei were also red. Our results show that hydrodynamic delivery of Cre protein allows rapid allelic conversion in hepatocytes, but Cre-activity is sub-saturating so many cells will not convert multiple Cre-responsive alleles. PMID:24626158

  1. In vitro culture of mouse embryos amniotic fluid ID human

    African Journals Online (AJOL)

    1989-07-15

    Jul 15, 1989 ... Because human amniotic fluid is a physiological, balanced ultrafiltrate, it has been considered as an inexpensive alternative culture medium in. IVF. A study of the development of mouse embryos in human amniotic fluid was undertaken to assess the suitability of this as an optional culture medium in human ...

  2. The effects of gender, age, ethnicity, and liver cirrhosis on cytochrome P450 enzyme activity in human liver microsomes and inducibility in cultured human hepatocytes

    International Nuclear Information System (INIS)

    Parkinson, Andrew; Mudra, Daniel R.; Johnson, Cory; Dwyer, Anne; Carroll, Kathleen M.

    2004-01-01

    We have measured cytochrome P450 (CYP) activity in nearly 150 samples of human liver microsomes and 64 samples of cryopreserved human hepatocytes, and we have performed induction studies in over 90 preparations of cultured human hepatocytes. We have analyzed these data to examine whether the expression of CYP enzyme activity in liver microsomes and isolated hepatocytes or the inducibility of CYP enzymes in cultured hepatocytes is influenced by the gender, age, or ethnicity of the donor (the latter being limited to Caucasians, African Americans, and Hispanics due to a paucity of livers from Asian donors). In human liver microsomes, there were no statistically significant differences (P > 0.05) in CYP activity as a function of age, gender, or ethnicity with one exception. 7-Ethoxyresorufin O-dealkylase (CYP1A2) activity was greater in males than females, which is consistent with clinical observation. Liver microsomal testosterone 6β-hydroxylase (CYP3A4) activity was slightly greater in females than males, but the difference was not significant. However, in cryopreserved human hepatocytes, the gender difference in CYP3A4 activity (females = twice males) did reach statistical significance, which supports the clinical observation that females metabolize certain CYP3A4 substrates faster than do males. Compared with those from Caucasians and African Americans, liver microsomes from Hispanics had about twice the average activity of CYP2A6, CYP2B6, and CYP2C8 and half the activity of CYP1A2, although this apparent ethnic difference may be a consequence of the relatively low number of Hispanic donors. Primary cultures of hepatocytes were treated with β-naphthoflavone, an inducer of CYP1A2, phenobarbital or rifampin, both of which induce CYP2B6, CYP2C9, CYP2C19, and CYP3A4, albeit it to different extents. Induction of these CYP enzymes in freshly cultured hepatocytes did not appear to be influenced by the gender or age of the donor. Furthermore, CYP3A4 induction in

  3. Transcriptome of extracellular vesicles released by hepatocytes.

    Directory of Open Access Journals (Sweden)

    Felix Royo

    Full Text Available The discovery that the cells communicate through emission of vesicles has opened new opportunities for better understanding of physiological and pathological mechanisms. This discovery also provides a novel source for non-invasive disease biomarker research. Our group has previously reported that hepatocytes release extracellular vesicles with protein content reflecting the cell-type of origin. Here, we show that the extracellular vesicles released by hepatocytes also carry RNA. We report the messenger RNA composition of extracellular vesicles released in two non-tumoral hepatic models: primary culture of rat hepatocytes and a progenitor cell line obtained from a mouse foetal liver. We describe different subpopulations of extracellular vesicles with different densities and protein and RNA content. We also show that the RNA cargo of extracellular vesicles released by primary hepatocytes can be transferred to rat liver stellate-like cells and promote their activation. Finally, we provide in vitro and in vivo evidence that liver-damaging drugs galactosamine, acetaminophen, and diclofenac modify the RNA content of these vesicles. To summarize, we show that the extracellular vesicles secreted by hepatocytes contain various RNAs. These vesicles, likely to be involved in the activation of stellate cells, might become a new source for non-invasive identification of the liver toxicity markers.

  4. Oncostatin M induces upregulation of claudin-2 in rodent hepatocytes coinciding with changes in morphology and function of tight junctions

    International Nuclear Information System (INIS)

    Imamura, Masafumi; Kojima, Takashi; Lan, Mengdong; Son, Seiichi; Murata, Masaki; Osanai, Makoto; Chiba, Hideki; Hirata, Koichi; Sawada, Norimasa

    2007-01-01

    In rodent livers, integral tight junction (TJ) proteins claudin-1, -2, -3, -5 and -14 are detected and play crucial roles in the barrier to keep bile in bile canaculi away from the blood circulation. Claudin-2 shows a lobular gradient increasing from periportal to pericentral hepatocytes, whereas claudin-1 and -3 are expressed in the whole liver lobule. Although claudin-2 expression induces cation-selective channels in tight junctions of epithelial cells, the physiological functions and regulation of claudin-2 in hepatocytes remain unclear. Oncostatin M (OSM) is a multifunctional cytokine implicated in the differentiation of hepatocytes that induces formation of E-cadherin-based adherens junctions in fetal hepatocytes. In this study, we examined whether OSM could induce expression and function of claudin-2 in rodent hepatocytes, immortalized mouse and primary cultured proliferative rat hepatocytes. In the immortalized mouse and primary cultured proliferative rat hepatocytes, treatment with OSM markedly increased mRNA and protein of claudin-2 together with formation of developed networks of TJ strands. The increase of claudin-2 enhanced the paracellular barrier function which depended on molecular size. The increase of claudin-2 expression induced by OSM in rodent hepatocytes was regulated through distinct signaling pathways including PKC. These results suggest that expression of claudin-2 in rodent hepatocytes may play a specific role as controlling the size of paracellular permeability in the barrier to keep bile in bile canaculi

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

    Science.gov (United States)

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

  6. Bile acids exert negative feedback control on bile acid synthesis in cultured pig hepatocytes by suppression of cholesterol 7α-hydroxylase activity

    NARCIS (Netherlands)

    Kwekkeboom, J.; Princen, H.M.G.; Voorthuizen, E.M. van; Kempen, H.J.M.

    1990-01-01

    Feedback regulation of bile acid synthesis by its end products was studied in cultured hepatocytes of young weaned pigs. We previously showed that conversion of exogenous [14C] cholesterol into bile acids was suppressed by addition of bile acids to the culture medium. In the present study, the

  7. Effect of Non-Esterified Fatty Acids on Fatty Acid Metabolism-Related Genes in Calf Hepatocytes Cultured in Vitro

    Directory of Open Access Journals (Sweden)

    Peng Li

    2013-11-01

    Full Text Available Background: NEFA plays numerous roles in the metabolism of glucose, lipids, and proteins. A number of experimental studies have shown that NEFA may have an important role in fatty acid metabolism in the liver, especially in dairy cows that experience negative energy balance (NEB during early lactation. Methods: In this study, using fluorescent quantitative RT-PCR, ELISA, and primary hepatocytes cultured in vitro, we examined the effect of NEFA (0, 0.2, 0.4, 0.8, 1.6, and 3.2 mmol/L on fatty acid metabolism by monitoring the mRNA and protein expression of the following key enzymes: long chain acyl-CoA synthetase (ACSL, carnitine palmitoyltransferase IA (CPT IA, long chain acyl-CoA dehydrogenase (ACADL, and acetyl-CoA carboxylase (ACC. Results: The mRNA and protein expression levels of ACSL and ACADL markedly increased as the concentration of NEFA in the media was increased. The mRNA and protein expression levels of CPT IA were enhanced significantly when the NEFA concentrations increased from 0 to 1.6 mmol/L and decreased significantly when the NEFA concentrations increased from 1.6 to 3.2 mmol/L. The mRNA and protein expression of ACC decreased gradually with increasing concentrations of NEFA. Conclusion: These findings indicate that increased NEFA significantly promote the activation and β-oxidation of fatty acids, but very high NEFA concentrations may inhibit the translocation of fatty acids into mitochondria of hepatocytes. This may explain the development of ketosis or liver lipidosis in dairy cows. CPT IA might be the key control enzyme of the fatty acid oxidation process in hepatocytes.

  8. IL-6 modulates hepatocyte proliferation via induction of HGF/p21cip1: Regulation by SOCS3

    International Nuclear Information System (INIS)

    Sun Rui; Jaruga, Barbara; Kulkarni, Shailin; Sun Haoyu; Gao Bin

    2005-01-01

    The precise role of IL-6 in liver regeneration and hepatocyte proliferation is controversial and the role of SOCS3 in liver regeneration remains unknown. Here we show that in vitro treatment with IL-6 inhibited primary mouse hepatocyte proliferation. IL-6 induced p21 cip1 protein expression in primary mouse hepatocytes. Disruption of the p21 cip1 gene abolished the inhibitory effect of IL-6 on cell proliferation. Co-culture with nonparenchymal liver cells diminished IL-6 inhibition of hepatocyte proliferation, which was likely due to IL-6 stimulation of nonparenchymal cells to produce HGF. Finally, IL-6 induced higher levels of p21 cip1 protein expression and a slightly stronger inhibition of cell proliferation in SOCS3 +/- mouse hepatocytes compared to wild-type hepatocytes, while liver regeneration was enhanced and prolonged in SOCS3 +/- mice. Our findings suggest that IL-6 directly inhibits hepatocyte proliferation via a p21 cip1 -dependent mechanism and indirectly enhances hepatocyte proliferation via stimulating nonparenchymal cells to produce HGF. SOCS3 negatively regulates liver regeneration

  9. Effects of Mangifera indica L. aqueous extract (Vimang) on primary culture of rat hepatocytes.

    Science.gov (United States)

    Rodeiro, I; Donato, M T; Jiménez, N; Garrido, G; Delgado, R; Gómez-Lechón, M J

    2007-12-01

    Vimang is an aqueous extract from stem bark of Mangifera indica L. (Mango) with pharmacological properties. It is a mixture of polyphenols (as main components), terpenoids, steroids, fatty acids and microelements. In the present work we studied the cytotoxic effects of Vimang on rat hepatocytes, possible interactions of the extract with drug-metabolizing enzymes and its effects on GSH levels and lipid peroxidation. No cytotoxic effects were observed after 24 h exposure to Vimang of up to 1000 microg/mL, while a moderate cytotoxicity was observed after 48 and 72 h of exposure at higher concentrations (500 and 1000 microg/mL). The effect of the extract (50-400 microg/mL) on several P450 isozymes was evaluated. Exposure of hepatocytes to Vimang at concentrations of up to 100 microg/mL produced a significant reduction (60%) in 7-methoxyresorufin-O-demethylase (MROD; CYP1A2) activity, an increase (50%) in 7-penthoxyresorufin-O-depentylase (PROD; CYP2B1) activity, while no significant effect was observed with other isozymes. To our knowledge, this is the first report regarding the modulation of the activity of the P450 system by an extract of Mangifera indica L. The antioxidant properties of Vimang were also evaluated in t-butyl-hydroperoxide-treated hepatocytes. A 36-h pre-treatment of cells with Vimang (25-200 microg/mL) strongly inhibited the decrease of GSH levels and lipid peroxidation induced by t-butyl-hydroperoxide dose- and time-dependently.

  10. Dimethyl sulfoxide is a potent modulator of estrogen receptor isoforms and xenoestrogen biomarker responses in primary culture of salmon hepatocytes

    International Nuclear Information System (INIS)

    Mortensen, Anne S.; Arukwe, Augustine

    2006-01-01

    Dimethyl sulfoxide (DMSO) has been frequently used as carrier solvent in toxicological experiments where the most compelling DMSO attributes are its exceptionally low toxicity and environmental impact. We were inspired by recent and consistent observations that ethanol and DMSO modulate endocrine-disruptor biomarker responses in both in vitro and in vivo studies in our laboratory, to take a critical evaluation of these effects. Quantitative (real-time) polymerase chain reaction (PCR) method with specific primer pairs was used in this study to measure DMSO-induced time-dependent modulation of estrogen receptor (ER) isoforms, vitellogenin (Vtg) and zona radiata-protein (Zr-protein) gene expression patterns in primary culture of salmon hepatocytes. In addition, immunochemical analysis, using indirect enzyme linked immunosorbent assay (ELISA) with monoclonal (Vtg) and polyclonal (Zr-proteins) antibodies was used to detect and measure Vtg and Zr-proteins secreted in culture media. Salmon hepatocytes were isolated by a two-step collagenase perfusion method and exposed to 0.1% or 10 μL/L of DMSO after 48 h pre-culture. Cells were harvested at 12, 24, 48 and 72 h after exposure and analysed for ERα, ERβ, Vtg and Zr-protein gene expression using real-time PCR method. Media samples were collected at similar time-intervals for protein analysis. Our data show that DMSO-induced significant increase in ERα, ERβ, Vtg and Zr-protein genes in a time-dependent manner. Indirect ELISA analysis showed a time-specific effect of DMSO. The use of DMSO as carrier solvent in fish endocrine disruption studies should be re-evaluated. We recommend more investigation, using other endocrine-disruptor biomarkers in order to validate the suitability of common carrier solvents used in toxicology with the aim of setting new maximum allowable concentrations. In particular, given the high sensitivity of genomic approaches in toxicology, these results may have serious consequences for the

  11. Hepatic esterase activity is increased in hepatocyte-like cells derived from human embryonic stem cells using a 3D culture system.

    Science.gov (United States)

    Choi, Young-Jun; Kim, Hyemin; Kim, Ji-Woo; Yoon, Seokjoo; Park, Han-Jin

    2018-05-01

    The aim of the study is to generate a spherical three-dimensional (3D) aggregate of hepatocyte-like cells (HLCs) differentiated from human embryonic stem cells and to investigate the effect of the 3D environment on hepatic maturation and drug metabolism. Quantitative real-time PCR analysis indicated that gene expression of mature hepatocyte markers, drug-metabolizing enzymes, and hepatic transporters was significantly higher in HLCs cultured in the 3D system than in those cultured in a two-dimensional system (p formation, were increased in HLCs cultured in the 3D system. In particular, 3D spheroidal culture increased expression of CES1 and BCHE, which encode hepatic esterases (p 3D spheroidal culture enhances the maturation and drug metabolism of stem cell-derived HLCs, and this may help to optimize hepatic differentiation protocols for hepatotoxicity testing.

  12. Variations in DNA synthesis and mitotic indices in hepatocytes and sinusoid litoral cells of adult intact male mouse along a circadian time span.

    Science.gov (United States)

    Surur, J M; Moreno, F R; Badrán, A F; Llanos, J M

    1985-01-01

    Variations of DNA synthesis (DNAS) and mitotic indices along a circadian time span are described in the hepatocyte and sinusoid litoral cell populations of adult intact male mouse liver. Standardized (light from 0600 to 1800) mice were killed in groups of six to nine animals, every 2-4 hr along a circadian time span. Hepatocytes show significant peaks in the synthesis of DNA and the mitotic activity at 0200 and 1400, respectively. These results correspond to those previously described by us in young immature liver, regenerating liver and hepatomas. The phase differences between these peaks and the differences between their absolute values are discussed. Also considered are the practical consequences of our findings for experimental design. The curve of DNA synthesis of sinusoid litoral cells show a peak at 0200. The mitotic index show a bimodal waveform with peaks at 0800 and 2000. The existence of four different cell populations composing the so called sinusoid litoral cells and also the migration into and out of the liver of some macrophages considered as litoral (Kupffer) cells in our counts, makes interpretation of the curves somewhat complicated and deserves further analysis.

  13. Culture of Mouse Neural Stem Cell Precursors

    OpenAIRE

    Currle, D. Spencer; Hu, Jia Sheng; Kolski-Andreaco, Aaron; Monuki, Edwin S.

    2007-01-01

    Primary neural stem cell cultures are useful for studying the mechanisms underlying central nervous system development. Stem cell research will increase our understanding of the nervous system and may allow us to develop treatments for currently incurable brain diseases and injuries. In addition, stem cells should be used for stem cell research aimed at the detailed study of mechanisms of neural differentiation and transdifferentiation and the genetic and environmental signals that direct the...

  14. 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

  15. A novel rabbit anti-hepatocyte growth factor monoclonal neutralizing antibody inhibits tumor growth in prostate cancer cells and mouse xenografts

    International Nuclear Information System (INIS)

    Yu, Yanlan; Chen, Yicheng; Ding, Guoqing; Wang, Mingchao; Wu, Haiyang; Xu, Liwei; Rui, Xuefang; Zhang, Zhigen

    2015-01-01

    The hepatocyte growth factor and its receptor c-Met are correlated with castration-resistance in prostate cancer. Although HGF has been considered as an attractive target for therapeutic antibodies, the lack of cross-reactivity of monoclonal antibodies with human/mouse HGFs is a major obstacle in preclinical developments. We generated a panel of anti-HGF RabMAbs either blocking HGF/c-Met interaction or inhibiting c-Met phosphorylation. We selected one RabMAb with mouse cross-reactivity and demonstrated that it blocked HGF-stimulated downstream activation in PC-3 and DU145 cells. Anti-HGF RabMAb inhibited not only the growth of PC-3 cells but also HGF-dependent proliferation in HUVECs. We further demonstrated the efficacy and potency of the anti-HGF RabMAb in tumor xenograft mice models. Through these in vitro and in vivo experiments, we explored a novel therapeutic antibody for advanced prostate cancer. - Highlights: • HGF is an attractive target for castration-refractory prostate cancer. • We generated and characterized a panel of anti-HGF rabbit monoclonal antibodies. • More than half of these anti-HGF RabMAbs was cross-reactive with mouse HGF. • Anti-HGF RabMAb blocks HGF-stimulated phosphorylation and cell growth in vitro. • Anti-HGF RabMAb inhibits tumor growth and angiogenesis in xenograft mice

  16. A novel rabbit anti-hepatocyte growth factor monoclonal neutralizing antibody inhibits tumor growth in prostate cancer cells and mouse xenografts

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Yanlan; Chen, Yicheng; Ding, Guoqing; Wang, Mingchao; Wu, Haiyang; Xu, Liwei; Rui, Xuefang; Zhang, Zhigen, E-mail: srrshurology@163.com

    2015-08-14

    The hepatocyte growth factor and its receptor c-Met are correlated with castration-resistance in prostate cancer. Although HGF has been considered as an attractive target for therapeutic antibodies, the lack of cross-reactivity of monoclonal antibodies with human/mouse HGFs is a major obstacle in preclinical developments. We generated a panel of anti-HGF RabMAbs either blocking HGF/c-Met interaction or inhibiting c-Met phosphorylation. We selected one RabMAb with mouse cross-reactivity and demonstrated that it blocked HGF-stimulated downstream activation in PC-3 and DU145 cells. Anti-HGF RabMAb inhibited not only the growth of PC-3 cells but also HGF-dependent proliferation in HUVECs. We further demonstrated the efficacy and potency of the anti-HGF RabMAb in tumor xenograft mice models. Through these in vitro and in vivo experiments, we explored a novel therapeutic antibody for advanced prostate cancer. - Highlights: • HGF is an attractive target for castration-refractory prostate cancer. • We generated and characterized a panel of anti-HGF rabbit monoclonal antibodies. • More than half of these anti-HGF RabMAbs was cross-reactive with mouse HGF. • Anti-HGF RabMAb blocks HGF-stimulated phosphorylation and cell growth in vitro. • Anti-HGF RabMAb inhibits tumor growth and angiogenesis in xenograft mice.

  17. Comparison of the effects of the synthetic pyrethroid Metofluthrin and phenobarbital on CYP2B form induction and replicative DNA synthesis in cultured rat and human hepatocytes

    International Nuclear Information System (INIS)

    Hirose, Yukihiro; Nagahori, Hirohisa; Yamada, Tomoya; Deguchi, Yoshihito; Tomigahara, Yoshitaka; Nishioka, Kazuhiko; Uwagawa, Satoshi; Kawamura, Satoshi; Isobe, Naohiko; Lake, Brian G.; Okuno, Yasuyoshi

    2009-01-01

    High doses of Metofluthrin (MTF) have been shown to produce liver tumours in rats by a mode of action (MOA) involving activation of the constitutive androstane receptor leading to liver hypertrophy, induction of cytochrome P450 (CYP) forms and increased cell proliferation. The aim of this study was to compare the effects of MTF with those of the known rodent liver tumour promoter phenobarbital (PB) on the induction CYP2B forms and replicative DNA synthesis in cultured rat and human hepatocytes. Treatment with 50 μM MTF and 50 μM PB for 72 h increased CYP2B1 mRNA levels in male Wistar rat hepatocytes and CYP2B6 mRNA levels in human hepatocytes. Replicative DNA synthesis was determined by incorporation of 5-bromo-2'-deoxyuridine over the last 24 h of a 48 h treatment period. Treatment with 10-1000 μM MTF and 100-500 μM PB resulted in significant increases in replicative DNA synthesis in rat hepatocytes. While replicative DNA synthesis was increased in human hepatocytes treated with 5-50 ng/ml epidermal growth factor or 5-100 ng/ml hepatocyte growth factor, treatment with MTF and PB had no effect. These results demonstrate that while both MTF and PB induce CYP2B forms in both species, MTF and PB only induced replicative DNA synthesis in rat and not in human hepatocytes. These results provide further evidence that the MOA for MTF-induced rat liver tumour formation is similar to that of PB and some other non-genotoxic CYP2B form inducers and that the key event of increased cell proliferation would not occur in human liver

  18. Comparison of the effects of the synthetic pyrethroid Metofluthrin and phenobarbital on CYP2B form induction and replicative DNA synthesis in cultured rat and human hepatocytes.

    Science.gov (United States)

    Hirose, Yukihiro; Nagahori, Hirohisa; Yamada, Tomoya; Deguchi, Yoshihito; Tomigahara, Yoshitaka; Nishioka, Kazuhiko; Uwagawa, Satoshi; Kawamura, Satoshi; Isobe, Naohiko; Lake, Brian G; Okuno, Yasuyoshi

    2009-04-05

    High doses of Metofluthrin (MTF) have been shown to produce liver tumours in rats by a mode of action (MOA) involving activation of the constitutive androstane receptor leading to liver hypertrophy, induction of cytochrome P450 (CYP) forms and increased cell proliferation. The aim of this study was to compare the effects of MTF with those of the known rodent liver tumour promoter phenobarbital (PB) on the induction CYP2B forms and replicative DNA synthesis in cultured rat and human hepatocytes. Treatment with 50 microM MTF and 50 microM PB for 72 h increased CYP2B1 mRNA levels in male Wistar rat hepatocytes and CYP2B6 mRNA levels in human hepatocytes. Replicative DNA synthesis was determined by incorporation of 5-bromo-2'-deoxyuridine over the last 24 h of a 48 h treatment period. Treatment with 10-1000 microM MTF and 100-500 microM PB resulted in significant increases in replicative DNA synthesis in rat hepatocytes. While replicative DNA synthesis was increased in human hepatocytes treated with 5-50 ng/ml epidermal growth factor or 5-100 ng/ml hepatocyte growth factor, treatment with MTF and PB had no effect. These results demonstrate that while both MTF and PB induce CYP2B forms in both species, MTF and PB only induced replicative DNA synthesis in rat and not in human hepatocytes. These results provide further evidence that the MOA for MTF-induced rat liver tumour formation is similar to that of PB and some other non-genotoxic CYP2B form inducers and that the key event of increased cell proliferation would not occur in human liver.

  19. SREBP-1c Gene Silencing can Decrease Lipid Deposits in Bovine Hepatocytes Cultured in Vitro

    Directory of Open Access Journals (Sweden)

    Qinghua Deng

    2014-05-01

    Full Text Available Background: Fatty liver is a major metabolic disorder that occurs during early lactation in high-producing dairy cows. Sterol regulatory element-binding protein-1c (SREBP-1c is an important transcription factor that regulates lipid synthesis by regulating the expression of lipid metabolism genes. Methods: In this study, we reduced the expression of SREBP-1c by adenovirus-mediated SREBP-1c with a low expression vector (AD-GFP-SREBP-1c to study the effects of SREBP-1c on lipid deposits in bovine hepatocytes. The expression levels and enzyme activities of SERBP-1c and its target genes were determined by real-time PCR, western blot, and ELISA. Results: These results showed that Ad-GFP-SREBP-1c could inhibit SREBP-1c expression. The expression of the lipid synthesis enzyme acetyl-CoA carboxylase (ACC was down-regulated. The expression levels of the lipid oxidation enzymes long-chain fatty acyl-COA synthetase (ACSL-1, carnitine palmitoyltransferase І (CPT-І, carnitine palmitoyltransferase II (CPT- II, and β-hydroxyacyl-CoA-DH (HADH were significantly elevated. Furthermore, the expression levels of factors involved in the assembly and transport of very low-density lipoproteins (VLDLs, such as apolipoprotein B100 (ApoB, apolipoprotein E (ApoE, and microsomal triglyceride transfer protein (MTTP were decreased comparison with the negative control and the blank control groups, but the low-density lipoprotein receptor (LDLR was elevated. The concentrations of TG (triglyceride and VLDL were also reduced. Conclusion: These data suggest that low SREBP-1c expression can decrease lipid synthesis, increase lipid oxidation, and decrease the TG and VLDL content in bovine hepatocytes.

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

    International Nuclear Information System (INIS)

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

    2009-01-01

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

  1. Protective effect of curcumin and its analog on γ-radiation induced DNA damage and lipid peroxidation in cultured human lymphocytes and isolated rat hepatocytes in vitro

    International Nuclear Information System (INIS)

    Menon, Venugopal P.

    2007-01-01

    Ionizing radiation is known to induce oxidative stress through generation of reactive oxygen species (ROS) resulting in an imbalance of the pro-oxidant and antioxidant status in the cells, which is suggested to culminate in cell death. The present work was aimed to evaluate the radioprotective effect of curcumin and its analog on γ-radiation induced toxicity in cultured human lymphocytes and rat hepatocytes. Hepatocytes were isolated from the liver of rats by collagenase perfusion. The cellular changes were estimated using lipid peroxidative indices like thiobarbituric acid reactive substances (TBARS), the antioxidants superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and reduced glutathione (GSH). The DNA damage was analyzed by comet assay, cytokinesis blocked micro nucleus assay, dicentric aberrations and translocation frequency. Cell cycle distribution and measurement of the percentage of apoptotic cells were performed by flow cytometry analysis. To investigate whether the dietary agents like curcumin and its analog have a role on cell cycle regulation, we analyzed the changes in cell cycle profiles by using fluorescence activated cell sorter. The increase in the severity of DNA damage was observed with the increase dose (1, 2 and 4 Gy) of γ-radiation in cultured lymphocytes and hepatocytes. TBARS were increased significantly, whereas the levels of GSH and antioxidant enzymes were significantly decreased in γ-irradiated hepatocytes and lymphocytes. On pretreatment with curcumin and its analog (1, 5 and 10 μg/ml) showed a significant decrease in the levels of TBARS and DNA damage. The antioxidant enzymes were increased significantly along with the levels of GSH. The maximum protection of hepatocytes and lymphocytes was observed at 10 μg/ml curcumin and 5 μg/ml curcumin analog pretreatment. Thus, pretreatment with curcumin and its analog helps in protecting the normal hepatocytes and lymphocytes against γ-radiation induced cellular

  2. The partial pressure of oxygen affects biomarkers of oxidative stress in cultured rainbow trout (Oncorhynchus mykiss) hepatocytes.

    Science.gov (United States)

    Finne, E F; Olsvik, P A; Berntssen, M H G; Hylland, K; Tollefsen, K E

    2008-09-01

    Oxidative stress, the imbalance between production of reactive oxygen species and the cellular detoxification of these reactive compounds, is believed to be involved in the pathology of various diseases. Several biomarkers for oxidative stress have been proposed to serve as tools in toxicological and ecotoxicological research. Not only may exposure to various pro-oxidants create conditions of cellular oxidative stress, but hyperoxic conditions may also increase the production of reactive oxygen species. The objective of the current study was to determine the extent to which differences in oxygen partial pressure would affect biomarkers of oxidative stress in a primary culture of hepatocytes from rainbow trout (Oncorhynchus mykiss). Membrane integrity, metabolic activity, levels of total and oxidized glutathione (tGSH/GSSG) was determined, as well as mRNA expression levels of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), glutathione reductase (GSSG-R), gamma-glutamyl-cystein synthetase (GCS) and thioredoxin (TRX). The results show that different biomarkers of oxidative stress are affected when the cell culture is exposed to atmospheric oxygen, and that changes such as increased GSSG content and induction of GSSG-R and GSH-Px can be reduced by culturing the cells under lower oxygen tension. Oxygen tension may thus influence results of in vitro based cell research and is particularly important when assessing parameters in the antioxidant defence system. Further research is needed to establish the magnitude of this effect in different cellular systems.

  3. Commercially available media for flushing extracorporeal bioartificial liver systems prior to connection to the patient's circulation: an in vitro comparative study in two and three dimensional porcine hepatocyte cultures

    NARCIS (Netherlands)

    Flendrig, L. M.; Sommeijer, D.; Ladiges, N. C.; te Velde, A. A.; Maas, M. A.; Jörning, G. G.; Daalhuisen, J.; Chamuleau, R. A.

    1998-01-01

    Extracorporeal bioartificial liver (BAL) systems based on hepatocytes need to be flushed before clinical application, as hepatocyte culture media are not approved for medical use. Commercially available 0.9% NaCl solution and hemofiltration solution (both supplemented with 10% human albumin) were

  4. Property of hepatitis B virus replication in Tupaia belangeri hepatocytes.

    Science.gov (United States)

    Sanada, Takahiro; Tsukiyama-Kohara, Kyoko; Yamamoto, Naoki; Ezzikouri, Sayeh; Benjelloun, Soumaya; Murakami, Shuko; Tanaka, Yasuhito; Tateno, Chise; Kohara, Michinori

    2016-01-08

    The northern treeshrew (Tupaia belangeri) has been reported to be an effective candidate for animal infection model with hepatitis B virus (HBV). The objective of our study was to analyze the growth characteristics of HBV in tupaia hepatocytes and the host response to HBV infection. We established primary tupaia hepatocytes (3-6-week old tupaia) and infected them with HBV genotypes A, B and C, and all the genotypes proliferated as well as those in human primary hepatocytes (>10(5) copies/ml in culture supernatant). We next generated a chimeric mouse with tupaia liver by transplantation of tupaia primary hepatocytes to urokinase-type plasminogen activator cDNA (cDNA-uPA)/severe combined immunodeficient (SCID) mice and the replacement ratio with tupaia hepatocytes was found to be more than 95%. Infection of chimeric mice with HBV (genotypes B, C, and D) resulted in HBV-DNA level of 10(4)-10(6) copies/ml after 8 weeks of infection, which were almost similar to that in humanized chimeric mouse. In contrast, serum HBV level in adult tupaia (1-year-old tupaia) was quite low (<10(3) copies/ml). Understanding the differences in the response to HBV infection in primary tupaia hepatocytes, chimeric mouse, and adult tupaia will contribute to elucidating the mechanism of persistent HBV infection and viral eradication. Thus, T. belangeri was found to be efficient for studying the host response to HBV infection, thereby providing novel insight into the pathogenesis of HBV. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  5. Predictivity of dog co-culture model, primary human hepatocytes and HepG2 cells for the detection of hepatotoxic drugs in humans

    International Nuclear Information System (INIS)

    Atienzar, Franck A.; Novik, Eric I.; Gerets, Helga H.; Parekh, Amit; Delatour, Claude; Cardenas, Alvaro; MacDonald, James; Yarmush, Martin L.; Dhalluin, Stéphane

    2014-01-01

    Drug Induced Liver Injury (DILI) is a major cause of attrition during early and late stage drug development. Consequently, there is a need to develop better in vitro primary hepatocyte models from different species for predicting hepatotoxicity in both animals and humans early in drug development. Dog is often chosen as the non-rodent species for toxicology studies. Unfortunately, dog in vitro models allowing long term cultures are not available. The objective of the present manuscript is to describe the development of a co-culture dog model for predicting hepatotoxic drugs in humans and to compare the predictivity of the canine model along with primary human hepatocytes and HepG2 cells. After rigorous optimization, the dog co-culture model displayed metabolic capacities that were maintained up to 2 weeks which indicates that such model could be also used for long term metabolism studies. Most of the human hepatotoxic drugs were detected with a sensitivity of approximately 80% (n = 40) for the three cellular models. Nevertheless, the specificity was low approximately 40% for the HepG2 cells and hepatocytes compared to 72.7% for the canine model (n = 11). Furthermore, the dog co-culture model showed a higher superiority for the classification of 5 pairs of close structural analogs with different DILI concerns in comparison to both human cellular models. Finally, the reproducibility of the canine system was also satisfactory with a coefficient of correlation of 75.2% (n = 14). Overall, the present manuscript indicates that the dog co-culture model may represent a relevant tool to perform chronic hepatotoxicity and metabolism studies. - Highlights: • Importance of species differences in drug development. • Relevance of dog co-culture model for metabolism and toxicology studies. • Hepatotoxicity: higher predictivity of dog co-culture vs HepG2 and human hepatocytes

  6. Predictivity of dog co-culture model, primary human hepatocytes and HepG2 cells for the detection of hepatotoxic drugs in humans

    Energy Technology Data Exchange (ETDEWEB)

    Atienzar, Franck A., E-mail: franck.atienzar@ucb.com [UCB Pharma SA, Non-Clinical Development, Chemin du Foriest, 1420 Braine-l' Alleud (Belgium); Novik, Eric I. [H mu rel Corporation, 675 U.S. Highway 1, North Brunswick, NJ 08902 (United States); Gerets, Helga H. [UCB Pharma SA, Non-Clinical Development, Chemin du Foriest, 1420 Braine-l' Alleud (Belgium); Parekh, Amit [H mu rel Corporation, 675 U.S. Highway 1, North Brunswick, NJ 08902 (United States); Delatour, Claude; Cardenas, Alvaro [UCB Pharma SA, Non-Clinical Development, Chemin du Foriest, 1420 Braine-l' Alleud (Belgium); MacDonald, James [Chrysalis Pharma Consulting, LLC, 385 Route 24, Suite 1G, Chester, NJ 07930 (United States); Yarmush, Martin L. [Department of Biomedical Engineering, Rutgers University, Piscataway, NJ 08854 (United States); Dhalluin, Stéphane [UCB Pharma SA, Non-Clinical Development, Chemin du Foriest, 1420 Braine-l' Alleud (Belgium)

    2014-02-15

    Drug Induced Liver Injury (DILI) is a major cause of attrition during early and late stage drug development. Consequently, there is a need to develop better in vitro primary hepatocyte models from different species for predicting hepatotoxicity in both animals and humans early in drug development. Dog is often chosen as the non-rodent species for toxicology studies. Unfortunately, dog in vitro models allowing long term cultures are not available. The objective of the present manuscript is to describe the development of a co-culture dog model for predicting hepatotoxic drugs in humans and to compare the predictivity of the canine model along with primary human hepatocytes and HepG2 cells. After rigorous optimization, the dog co-culture model displayed metabolic capacities that were maintained up to 2 weeks which indicates that such model could be also used for long term metabolism studies. Most of the human hepatotoxic drugs were detected with a sensitivity of approximately 80% (n = 40) for the three cellular models. Nevertheless, the specificity was low approximately 40% for the HepG2 cells and hepatocytes compared to 72.7% for the canine model (n = 11). Furthermore, the dog co-culture model showed a higher superiority for the classification of 5 pairs of close structural analogs with different DILI concerns in comparison to both human cellular models. Finally, the reproducibility of the canine system was also satisfactory with a coefficient of correlation of 75.2% (n = 14). Overall, the present manuscript indicates that the dog co-culture model may represent a relevant tool to perform chronic hepatotoxicity and metabolism studies. - Highlights: • Importance of species differences in drug development. • Relevance of dog co-culture model for metabolism and toxicology studies. • Hepatotoxicity: higher predictivity of dog co-culture vs HepG2 and human hepatocytes.

  7. Transplantation of Porcine Hepatocytes Cultured with Polylactic Acid-O-Carboxymethylated Chitosan Nanoparticles Promotes Liver Regeneration in Acute Liver Failure Rats

    Directory of Open Access Journals (Sweden)

    Zhong Chen

    2011-01-01

    Full Text Available In this study, free porcine hepatocytes suspension (Group A, porcine hepatocytes embedded in collagen gel (Group B, porcine hepatocytes cultured with PLA-O-CMC nanoparticles and embedded in collagen gel (Group C, and PLA-O-CMC nanoparticles alone (Group D were transplanted into peritoneal cavity of ALF rats, respectively. The result showed that plasma HGF levels were elevated post-transplantation with a peak at 12 hr. The rats in Group C showed highest plasma HGF levels at 2, 6, 12, 24 and 36 hr post-transplantation and lowest HGF level at 48 hr. Plasma VEGF levels were elevated at 48 hr post-transplantation with a peak at 72 hr. The rats in Group C showed highest plasma HGF levels at 48, 72, and 96 hr post-transplantation. The liver functions in Group C were recovered most rapidly. Compared with Group B, Group C had significant high liver Kiel 67 antigen labeling index (Ki-67 LI at day 1 post-HTx (P<.05. Ki-67 LI in groups B and C was higher than that in groups A and D at days 5 and 7 post-HTx. In conclusion, intraperitoneal transplantation of porcine hepatocytes cultured with PLA-O-CMC nanoparticles and embedded in collagen gel can promote significantly liver regeneration in ALF rats.

  8. The use of cultured hepatocytes to investigate the metabolism of drugs and mechanisms of drug hepatotoxicity.

    Science.gov (United States)

    Gómez-Lechón, M J; Ponsoda, X; Bort, R; Castell, J V

    2001-01-01

    Hepatotoxins can be classified as intrinsic when they exert their effects on all individuals in a dose-dependent manner, and as idiosyncratic when their effects are the consequence of an abnormal metabolism of the drug by susceptible individuals (metabolic idiosyncrasy) or of an immune-mediated injury to hepatocytes (allergic hepatitis). Some xenobiotics are electrophilic, and others are biotransformed by the liver into highly reactive metabolites that are usually more toxic than the parent compound. This activation process is the key to many hepatotoxic phenomena. Mitochondria are a frequent target of hepatotoxic drugs, and the alteration of their function has immediate effects on the energy balance of cells (depletion of ATP). Lipid peroxidation, oxidative stress, alteration of Ca(2+) homeostasis, and covalent binding to cell macromolecules are the molecular mechanisms that are frequently involved in the toxicity of xenobiotics. Against these potential hazards, cells have their own defence mechanisms (for example, glutathione, DNA repair, suicide inactivation). Ultimately, toxicity is the balance between bioactivation and detoxification, which determines whether a reactive metabolite elicits a toxic effect. The ultimate goal of in vitro experiments is to generate the type of scientific information needed to identify compounds that are potentially toxic to man. For this purpose, both the design of the experiments and the interpretation of the results are critical.

  9. Effect of remifentanil on mitochondrial oxygen consumption of cultured human hepatocytes.

    Directory of Open Access Journals (Sweden)

    Siamak Djafarzadeh

    remifentanil increases cellular respiration of human hepatocytes and prevents TNF-α-induced mitochondrial dysfunction. The results were not explained by uncoupling of mitochondrial respiration.

  10. Cytoprotective effect of tocopherols in hepatocytes cultured with polyunsaturated fatty acids

    DEFF Research Database (Denmark)

    Mikkelsen, L.; Hansen, Harald S.; Grunnet, N.

    1994-01-01

    When highly unsaturated fatty acids are added to cell cultures, it can become important to include antioxidants in the culture medium to prevent cytotoxic peroxidation. To find an optimal antioxidant for this purpose, the effect of 50 µM a-tocopherol, ¿-tocopherol, a-tocopheryl acetate, a...... of thiobarbituric acid reactive substances in the cultures was also measured. a-Tocopheryl acid succinate was found to be the most effective cytoprotective compound, followed by N,N'-diphenyl-1,4-phenylenediamine, a- tocopherol, ¿-tocopherol and a-tocopheryl acetate, and a-tocopheryl phosphate was without effect....

  11. Induction of peroxisomal beta-oxidation by a microbial catabolite of cholic acid in rat liver and cultured rat hepatocytes.

    Science.gov (United States)

    Nishimaki-Mogami, T; Takahashi, A; Toyoda, K; Hayashi, Y

    1993-01-01

    The capability of (4R)-4-(2,3,4,6,6a beta,7,8,9,9a alpha,9b beta-decahydro-6a beta-methyl-3-oxo-1H-cyclopental[f]quinolin-7 beta-yl)valeric acid (DCQVA), a catabolite of cholic acid produced by enterobacteria, to induce peroxisome proliferation in vivo and in vitro was studied. Rats given 0.3% DCQVA in the diet for 2 weeks showed marked increases in peroxisomal beta-oxidation, mitochondrial 2,4-dienoyl-CoA reductase and microsomal laurate omega-oxidation activities in the liver compared with control rats given the diet without DCQVA. Cultured rat hepatocytes treated with DCQVA for 72 h also exhibited greatly enhanced beta-oxidation activity. The increased activity was concentration-dependent and the effective concentrations were comparable with those of clofibric acid that produced the same degree of induction in the assay. The results demonstrate that DCQVA is a potent peroxisome proliferator that occurs naturally in rat intestine. PMID:8216219

  12. Sandwich-Cultured Hepatocytes for Mechanistic Understanding of Hepatic Disposition of Parent Drugs and Metabolites by Transporter-Enzyme Interplay.

    Science.gov (United States)

    Matsunaga, Norikazu; Fukuchi, Yukina; Imawaka, Haruo; Tamai, Ikumi

    2018-05-01

    Functional interplay between transporters and drug-metabolizing enzymes is currently one of the hottest topics in the field of drug metabolism and pharmacokinetics. Uptake transporter-enzyme interplay is important to determine intrinsic hepatic clearance based on the extended clearance concept. Enzyme and efflux transporter interplay, which includes both sinusoidal (basolateral) and canalicular efflux transporters, determines the fate of metabolites formed in the liver. As sandwich-cultured hepatocytes (SCHs) maintain metabolic activities and form a canalicular network, the whole interplay between uptake and efflux transporters and drug-metabolizing enzymes can be investigated simultaneously. In this article, we review the utility and applicability of SCHs for mechanistic understanding of hepatic disposition of both parent drugs and metabolites. In addition, the utility of SCHs for mimicking species-specific disposition of parent drugs and metabolites in vivo is described. We also review application of SCHs for clinically relevant prediction of drug-drug interactions caused by drugs and metabolites. The usefulness of mathematical modeling of hepatic disposition of parent drugs and metabolites in SCHs is described to allow a quantitative understanding of an event in vitro and to develop a more advanced model to predict in vivo disposition. Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.

  13. Antioxidative and apoptotic properties of polyphenolic extracts from edible part of artichoke (Cynara scolymus L.) on cultured rat hepatocytes and on human hepatoma cells.

    Science.gov (United States)

    Miccadei, Stefania; Di Venere, Donato; Cardinali, Angela; Romano, Ferdinando; Durazzo, Alessandra; Foddai, Maria Stella; Fraioli, Rocco; Mobarhan, Sohrab; Maiani, Giuseppe

    2008-01-01

    Cultured rat hepatocytes and human hepatoma HepG2 cells were used to evaluate the hepatoprotective properties of polyphenolic extracts from the edible part of artichoke (AE). The hepatocytes were exposed to H2O2generated in situ by glucose oxidase and were treated with either AE, or pure chlorogenic acid (ChA) or with the well known antioxidant, N, N'-diphenyl-p-phenilenediamine (DPPD). Addition of glucose oxidase to the culture medium caused depletion of intracellular glutathione (GSH) content, accumulation of malondialdehyde (MDA) in the cultures, as a lipid peroxidation indicator, and cell death. These results demonstrated that AE protected cells from the oxidative stress caused by glucose oxidase, comparable to DPPD. Furthermore, AE, as well as ChA, prevented the loss of total GSH and the accumulation of MDA. Treatment of HepG2 cells for 24 h with AE reduced cell viability in a dose-dependent manner, however, ChA had no prominent effects on the cell death rate. Similarly, AE rather than ChA induced apoptosis, measured by flow cytometric analysis of annexin and by activation of caspase-3, in HepG2 cells. Our findings indicate that AE had a marked antioxidative potential that protects hepatocytes from an oxidative stress. Furthermore, AE reduced cell viability and had an apoptotic activity on a human liver cancer cell line.

  14. Disrupting Hepatocyte Cyp51 from Cholesterol Synthesis Leads to Progressive Liver Injury in the Developing Mouse and Decreases RORC Signalling

    Science.gov (United States)

    Urlep, Žiga; Lorbek, Gregor; Perše, Martina; Jeruc, Jera; Juvan, Peter; Matz-Soja, Madlen; Gebhardt, Rolf; Björkhem, Ingemar; Hall, Jason A.; Bonneau, Richard; Littman, Dan R.; Rozman, Damjana

    2017-01-01

    Development of mice with hepatocyte knockout of lanosterol 14α-demethylase (HCyp51-/-) from cholesterol synthesis is characterized by the progressive onset of liver injury with ductular reaction and fibrosis. These changes begin during puberty and are generally more aggravated in the knockout females. However, a subgroup of (pre)pubertal knockout mice (runts) exhibits a pronounced male prevalent liver dysfunction characterized by downregulated amino acid metabolism and elevated Casp12. RORC transcriptional activity is diminished in livers of all runt mice, in correlation with the depletion of potential RORC ligands subsequent to CYP51 disruption. Further evidence for this comes from the global analysis that identified a crucial overlap between hepatic Cyp51-/- and Rorc-/- expression profiles. Additionally, the reduction in RORA and RORC transcriptional activity was greater in adult HCyp51-/- females than males, which correlates well with their downregulated amino and fatty acid metabolism. Overall, we identify a global and sex-dependent transcriptional de-regulation due to the block in cholesterol synthesis during development of the Cyp51 knockout mice and provide in vivo evidence that sterol intermediates downstream of lanosterol may regulate the hepatic RORC activity.

  15. Model Based Targeting of IL-6-Induced Inflammatory Responses in Cultured Primary Hepatocytes to Improve Application of the JAK Inhibitor Ruxolitinib

    Directory of Open Access Journals (Sweden)

    Svantje Sobotta

    2017-10-01

    Full Text Available IL-6 is a central mediator of the immediate induction of hepatic acute phase proteins (APP in the liver during infection and after injury, but increased IL-6 activity has been associated with multiple pathological conditions. In hepatocytes, IL-6 activates JAK1-STAT3 signaling that induces the negative feedback regulator SOCS3 and expression of APPs. While different inhibitors of IL-6-induced JAK1-STAT3-signaling have been developed, understanding their precise impact on signaling dynamics requires a systems biology approach. Here we present a mathematical model of IL-6-induced JAK1-STAT3 signaling that quantitatively links physiological IL-6 concentrations to the dynamics of IL-6-induced signal transduction and expression of target genes in hepatocytes. The mathematical model consists of coupled ordinary differential equations (ODE and the model parameters were estimated by a maximum likelihood approach, whereas identifiability of the dynamic model parameters was ensured by the Profile Likelihood. Using model simulations coupled with experimental validation we could optimize the long-term impact of the JAK-inhibitor Ruxolitinib, a therapeutic compound that is quickly metabolized. Model-predicted doses and timing of treatments helps to improve the reduction of inflammatory APP gene expression in primary mouse hepatocytes close to levels observed during regenerative conditions. The concept of improved efficacy of the inhibitor through multiple treatments at optimized time intervals was confirmed in primary human hepatocytes. Thus, combining quantitative data generation with mathematical modeling suggests that repetitive treatment with Ruxolitinib is required to effectively target excessive inflammatory responses without exceeding doses recommended by the clinical guidelines.

  16. Toyocamycin attenuates free fatty acid-induced hepatic steatosis and apoptosis in cultured hepatocytes and ameliorates nonalcoholic fatty liver disease in mice.

    Science.gov (United States)

    Takahara, Ikuko; Akazawa, Yuko; Tabuchi, Maiko; Matsuda, Katsuya; Miyaaki, Hisamitsu; Kido, Youko; Kanda, Yasuko; Taura, Naota; Ohnita, Ken; Takeshima, Fuminao; Sakai, Yusuke; Eguchi, Susumu; Nakashima, Masahiro; Nakao, Kazuhiko

    2017-01-01

    A high serum level of saturated free fatty acids (FFAs) is associated with the development of nonalcoholic fatty liver disease (NAFLD). X-box binding protein-1 (XBP-1) is activated by FFA treatment upon splicing. XBP-1 is a transcription factor induced by the endoplasmic reticulum (ER) stress sensor endoribonuclease inositol-requiring enzyme 1 alpha (IRE1α). However, the role of XBP-1 in NAFLD remains relatively unexplored. Toyocamycin was recently reported to attenuate the activation of XBP-1, possibly by inducing a conformational change in IRE1α. In this study, we examined the effect of toyocamycin on hepatocyte lipoapoptosis and steatosis. We also explored the effects of toyocamycin in a mouse model of NAFLD. Huh-7 cells and isolated rat primary hepatocytes were treated with palmitic acid (PA), which is a saturated FFA, in the presence or absence of toyocamycin. In addition, male C57BL/6J mice were fed a diet rich in saturated fat, fructose, and cholesterol (FFC) for 4 months, after which the effect of toyocamycin was assessed. Toyocamycin attenuated FFA-induced steatosis. It also significantly reduced PA-induced hepatocyte lipoapoptosis. In addition, toyocamycin reduced the expression of cytosine-cytosine-adenosine-adenosine-thymidine enhancer-binding protein homologous protein (CHOP), which is a key player in ER stress-mediated apoptosis, as well as its downstream cell death modulator, death receptor 5. In the in vivo study, toyocamycin ameliorated the liver injury caused by FFC-induced NAFLD. It also reduced hepatic steatosis and the expression of lipogenic genes. The data we obtained suggest that toyocamycin attenuates hepatocyte lipogenesis and ameliorates NAFLD in vivo and may therefore be beneficial in the treatment of NAFLD in humans.

  17. Model Based Targeting of IL-6-Induced Inflammatory Responses in Cultured Primary Hepatocytes to Improve Application of the JAK Inhibitor Ruxolitinib.

    Science.gov (United States)

    Sobotta, Svantje; Raue, Andreas; Huang, Xiaoyun; Vanlier, Joep; Jünger, Anja; Bohl, Sebastian; Albrecht, Ute; Hahnel, Maximilian J; Wolf, Stephanie; Mueller, Nikola S; D'Alessandro, Lorenza A; Mueller-Bohl, Stephanie; Boehm, Martin E; Lucarelli, Philippe; Bonefas, Sandra; Damm, Georg; Seehofer, Daniel; Lehmann, Wolf D; Rose-John, Stefan; van der Hoeven, Frank; Gretz, Norbert; Theis, Fabian J; Ehlting, Christian; Bode, Johannes G; Timmer, Jens; Schilling, Marcel; Klingmüller, Ursula

    2017-01-01

    IL-6 is a central mediator of the immediate induction of hepatic acute phase proteins (APP) in the liver during infection and after injury, but increased IL-6 activity has been associated with multiple pathological conditions. In hepatocytes, IL-6 activates JAK1-STAT3 signaling that induces the negative feedback regulator SOCS3 and expression of APPs. While different inhibitors of IL-6-induced JAK1-STAT3-signaling have been developed, understanding their precise impact on signaling dynamics requires a systems biology approach. Here we present a mathematical model of IL-6-induced JAK1-STAT3 signaling that quantitatively links physiological IL-6 concentrations to the dynamics of IL-6-induced signal transduction and expression of target genes in hepatocytes. The mathematical model consists of coupled ordinary differential equations (ODE) and the model parameters were estimated by a maximum likelihood approach, whereas identifiability of the dynamic model parameters was ensured by the Profile Likelihood. Using model simulations coupled with experimental validation we could optimize the long-term impact of the JAK-inhibitor Ruxolitinib, a therapeutic compound that is quickly metabolized. Model-predicted doses and timing of treatments helps to improve the reduction of inflammatory APP gene expression in primary mouse hepatocytes close to levels observed during regenerative conditions. The concept of improved efficacy of the inhibitor through multiple treatments at optimized time intervals was confirmed in primary human hepatocytes. Thus, combining quantitative data generation with mathematical modeling suggests that repetitive treatment with Ruxolitinib is required to effectively target excessive inflammatory responses without exceeding doses recommended by the clinical guidelines.

  18. Glutathione deficiency induced by cystine and/or methionine deprivation does not affect thyroid hormone deiodination in cultured rat hepatocytes and monkey hepatocarcinoma cells

    International Nuclear Information System (INIS)

    Sato, K.; Robbins, J.

    1981-01-01

    To elucidate the recently advanced hypothesis that glutathione [L-gamma-glutamyl-L-cysteinyl glycine (GSH)] regulates deiodinating enzyme activities, accounting for the decreased conversion of T4 to T3 in the liver of fetal and starved animals, we investigated thyroid hormone metabolism in GSH-depleted neoplastic and normal hepatocytes. In monkey hepatocarcinoma cells, intracellular total GSH decreased below 10% of the control value (approximately 25 micrograms/mg protein) when cells were grown for 44 h in medium deficient in cystine and methionine or in cystine alone. The latter finding indicated that transsulfuration from methionine to cysteine was defective in these neoplastic cells. In primary cultured adult rat hepatocytes, on the other hand, the transsulfuration pathway was intact, and total GSH decreased below 10% of control (approximately 20 micrograms/mg protein) only in cells grown in cystine- and methionine-deficient medium. In both cell types, the oxidized GSH fraction remained constant (2-5% of total). Incubation with 125I-labeled T4 and T3, followed by chromatography, was used to evaluate 5-deiodination in hepatocarcinoma cells and both 5- and 5'-deiodination in normal hepatocytes. Deiodination was not decreased by GSH deficiency in either case, but was actually increased in hepatocarcinoma cells. This resulted from an increase in the Vmax of 5-deiodinase related to growth arrest. Diamide at 2 mM reversibly inhibited both 5'- and 5'-deiodination in rat hepatocytes, accompanied by decreased total GSH as well as increased GSH disulfide (27% of total). The data suggest that GSH is so abundant in the liver that hepatocytes can tolerate a greater than 90% decrease in intracellular concentration without any change in thyroid hormone deiodination and indicate that altered thyroid hormone metabolism in the fetus and in starvation cannot be accounted for by a decreased hepatic GSH concentration

  19. Hepatocyte nuclear factor 4 alpha is a key factor related to depression and physiological homeostasis in the mouse brain.

    Directory of Open Access Journals (Sweden)

    Kyosuke Yamanishi

    Full Text Available Major depressive disorder (MDD is a common psychiatric disorder that involves marked disabilities in global functioning, anorexia, and severe medical comorbidities. MDD is associated with not only psychological and sociocultural problems, but also pervasive physical dysfunctions such as metabolic, neurobiological and immunological abnormalities. Nevertheless, the mechanisms underlying the interactions between these factors have yet to be determined in detail. The aim of the present study was to identify the molecular mechanisms responsible for the interactions between MDD and dysregulation of physiological homeostasis, including immunological function as well as lipid metabolism, coagulation, and hormonal activity in the brain. We generated depression-like behavior in mice using chronic mild stress (CMS as a model of depression. We compared the gene expression profiles in the prefrontal cortex (PFC of CMS and control mice using microarrays. We subsequently categorized genes using two web-based bioinformatics applications: Ingenuity Pathway Analysis and The Database for Annotation, Visualization, and Integrated Discovery. We then confirmed significant group-differences by analyzing mRNA and protein expression levels not only in the PFC, but also in the thalamus and hippocampus. These web tools revealed that hepatocyte nuclear factor 4 alpha (Hnf4a may exert direct effects on various genes specifically associated with amine synthesis, such as genes involved in serotonin metabolism and related immunological functions. Moreover, these genes may influence lipid metabolism, coagulation, and hormonal activity. We also confirmed the significant effects of Hnf4a on both mRNA and protein expression levels in the brain. These results suggest that Hnf4a may have a critical influence on physiological homeostasis under depressive states, and may be associated with the mechanisms responsible for the interactions between MDD and the dysregulation of

  20. Human hepatocytes support the hypertrophic but not the hyperplastic response to the murine nongenotoxic hepatocarcinogen sodium phenobarbital in an in vivo study using a chimeric mouse with humanized liver.

    Science.gov (United States)

    Yamada, Tomoya; Okuda, Yu; Kushida, Masahiko; Sumida, Kayo; Takeuchi, Hayato; Nagahori, Hirohisa; Fukuda, Takako; Lake, Brian G; Cohen, Samuel M; Kawamura, Satoshi

    2014-11-01

    High doses of sodium phenobarbital (NaPB), a constitutive androstane receptor (CAR) activator, have been shown to produce hepatocellular tumors in rodents by a mitogenic mode of action (MOA) involving CAR activation. The effect of 1-week dietary treatment with NaPB on liver weight and histopathology, hepatic CYP2B enzyme activity and CYP2B/3A mRNA expression, replicative DNA synthesis and selected genes related to cell proliferation, and functional transcriptomic and metabolomic analyses was studied in male CD-1 mice, Wistar Hannover (WH) rats, and chimeric mice with human hepatocytes. The treatment of chimeric mice with 1000-1500-ppm NaPB resulted in plasma levels around 3-5-fold higher than those observed in human subjects given therapeutic doses of NaPB. NaPB produced dose-dependent increases in hepatic CYP2B activity and CYP2B/3A mRNA levels in all animal models. Integrated functional metabolomic and transcriptomic analyses demonstrated that the responses to NaPB in the human liver were clearly different from those in rodents. Although NaPB produced a dose-dependent increase in hepatocyte replicative DNA synthesis in CD-1 mice and WH rats, no increase in replicative DNA synthesis was observed in human hepatocyte-originated areas of chimeric mice. In addition, treatment with NaPB had no effect on Ki-67, PCNA, GADD45β, and MDM2 mRNA expression in chimeric mice, whereas significant increases were observed in CD-1 mice and/or WH rats. However, increases in hepatocyte replicative DNA synthesis were observed in chimeric mice both in vivo and in vitro after treatment epidermal growth factor. Thus, although NaPB could activate CAR in both rodent and human hepatocytes, NaPB did not increase replicative DNA synthesis in human hepatocytes of chimeric mice, whereas it was mitogenic to rat and mouse hepatocytes. As human hepatocytes are refractory to the mitogenic effects of NaPB, the MOA for NaPB-induced rodent liver tumor formation is thus not relevant for humans. © The

  1. Identification of a mouse synaptic glycoprotein gene in cultured neurons.

    Science.gov (United States)

    Yu, Albert Cheung-Hoi; Sun, Chun Xiao; Li, Qiang; Liu, Hua Dong; Wang, Chen Ran; Zhao, Guo Ping; Jin, Meilei; Lau, Lok Ting; Fung, Yin-Wan Wendy; Liu, Shuang

    2005-10-01

    Neuronal differentiation and aging are known to involve many genes, which may also be differentially expressed during these developmental processes. From primary cultured cerebral cortical neurons, we have previously identified various differentially expressed gene transcripts from cultured cortical neurons using the technique of arbitrarily primed PCR (RAP-PCR). Among these transcripts, clone 0-2 was found to have high homology to rat and human synaptic glycoprotein. By in silico analysis using an EST database and the FACTURA software, the full-length sequence of 0-2 was assembled and the clone was named as mouse synaptic glycoprotein homolog 2 (mSC2). DNA sequencing revealed transcript size of mSC2 being smaller than the human and rat homologs. RT-PCR indicated that mSC2 was expressed differentially at various culture days. The mSC2 gene was located in various tissues with higher expression in brain, lung, and liver. Functions of mSC2 in neurons and other tissues remain elusive and will require more investigation.

  2. The effect of laurel leaf extract against toxicity induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin in cultured rat hepatocytes.

    Science.gov (United States)

    Turkez, Hasan; Geyikoglu, Fatime

    2011-12-01

    2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a very toxic environmental pollutant that raises great public concern about its impact on human health. Recent studies indicate that laurel leaf extract exhibits antioxidant properties that can counter the toxic effects of certain compounds in the liver. The aim of this study was to assess how effective LE is against the toxicity of TCDD in a primary culture of rat hepatocytes. The extract (50 mg L(-1), 100 mg L(-1), and 200 mg L(-1)) was added to cultures alone or with TCDD (1.61 mg L(-1) and 3.22 mg L(-1)) for 48 hours. Cell viability was measured using the [3-(4,5-dimethyl-thiazol-2-yl) 2,5-diphenyltetrazolium bromide] (MTT) assay and the lactate dehydrogenase (LDH) cytotoxicity assay, while oxidative damage was assessed by measuring total antioxidant capacity (TAC) and total oxidative stress (TOS). DNA damage was also analysed using the micronucleus (MN) assay of the cultured hepatocytes. TCDD alone lowered, and laurel extract had no effect on cell viability. TCDD also increased TOS and significantly decreased TAC. It significantly increased the frequency of micronucleated hepatocytes in a dose-dependent manner. In cultures exposed to LE alone, TOS did not change and TAC significantly increased in a dose-dependent manner. Added to TCDD, laurel countered its toxic effects and showed protective effects against TCDD-mediated DNA damage. This points to the therapeutic potential of laurel against TCDD toxicity in the liver.

  3. Effects of dexamethasone and insulin on the synthesis of triacylglycerols and phosphatidylcholine and the secretion of very-low-density lipoproteins and lysophosphatidylcholine by monolayer cultures of rat hepatocytes.

    OpenAIRE

    Mangiapane, E H; Brindley, D N

    1986-01-01

    Rat hepatocytes in monolayer culture were preincubated for 19 h with 1 microM-dexamethasone, and the incubation was continued for a further 23 h with [14C]oleate, [3H]glycerol and 1 microM-dexamethasone. Dexamethasone increased the secretion of triacylglycerol into the medium in particles that had the properties of very-low-density lipoproteins. The increased secretion was matched by a decrease in the triacylglycerol and phosphatidylcholine that remained in the hepatocytes. Preincubating the ...

  4. Combined Stimulation with the Tumor Necrosis Factor α and the Epidermal Growth Factor Promotes the Proliferation of Hepatocytes in Rat Liver Cultured Slices

    Directory of Open Access Journals (Sweden)

    Francis Finot

    2012-01-01

    Full Text Available The culture liver slices are mainly used to investigate drug metabolism and xenobiotic-mediated liver injuries while apoptosis and proliferation remain unexplored in this culture model. Here, we show a transient increase in LDH release and caspase activities indicating an ischemic injury during the slicing procedure. Then, caspase activities decrease and remain low in cultured slices demonstrating a low level of apoptosis. The slicing procedure is also associated with the G0/G1 transition of hepatocytes demonstrated by the activation of stress and proliferation signalling pathways including the ERK1/2 and JNK1/2/3 MAPKinases and the transient upregulation of c-fos. The cells further progress up to mid-G1 phase as indicated by the sequential induction of c-myc and p53 mRNA levels after the slicing procedure and at 24 h of culture, respectively. The stimulation by epidermal growth factor induces the ERK1/2 phosphorylation but fails to activate expression of late G1 and S phase markers such as cyclin D1 and Cdk1 indicating that hepatocytes are arrested in mid-G1 phase of the cell cycle. However, we found that combined stimulation by the proinflammatory cytokine tumor necrosis factor α and the epidermal growth factor promotes the commitment to DNA replication as observed in vivo during the liver regeneration.

  5. Radioactive marking of proteins in cultured mouse embryos

    International Nuclear Information System (INIS)

    Nowak, J.

    1984-01-01

    The purpose of this work was to build an in vitro test system, with which on the one hand postimplantation embryos of the mouse could be cultured without morphological of physiological damage and on the other hand their protein could be as highly marked as possible. With this radioactively marked proteins were to be won, which are optimally suited for a high separation by two-dimensional electrophoresis. In addition incubation and preparation methods were found for the ages of day 10, 11 and 12 of the embryonic development. With the use of 3 H-marked amino acids in the culture medium it was determined that embryos without embryonic membranes, so-called N-embryos, built in more radioactivity into their proteins than the embryos with embryonic membranes, the so-called DAO-embryos or the DO-embryos. On the contrary, the embryos with intact blood circulation (DO-embryos) showed an even distribution of radioactive marker in their bodies. Since an even distribution of the marker in the embryo is a necessary prerequisite for a representative presentation of the proteins by 2DE, the DO-preparation was considered the best suited method. In order to increase the amount of radioactivity incorporated into the proteins of the DO-embryos, the concentration of the used isotope or the incubation length could be increased. A combination of both proved to be the best method. A 14 C-marked amino acid mixture of 20 μCi/corresponds to 20 μl instead of the usual 150 μCi 3 H-marked amino acids in a culture medium proved to be equally suitable. Notable changes which would have indicated a damaging affect of the used radioactivity or the in vitro culturing were not observed. The achieved methodical conditions were used for the presentation of the embryo proteins by two-dimensional electrophoresis and fluorography. (orig./MG) [de

  6. Diffusion chamber culture of mouse bone marrow cells, (1)

    International Nuclear Information System (INIS)

    Sigeta, Chiharu; Tanaka, Kimio; Kawakami, Masahito; Takahashi, Hiroshi; Ohkita, Takeshi

    1980-01-01

    Mouse bone marrow cells were cultured in diffusion chambers (DC) implanted in the peritoneal cavity of host mice. Host mice were subjected to (1) irradiation ( 60 Co 800 rad) and/or (2) phenylhydrazine induced anemia and then receiving irradiation ( 60 Co 600 rad). After culture periods of 3-7 days, the total number of cells in DC was increased. A marked increase in DC is due to the proliferation of granulocyte series. When host mice were subjected to anemia and irradiation, the start of cell proliferation in DC was delay about two days. On the whole, anemia and irradiation host reduced a little cell growth in DC. The number of immature granulocytes grown in DC in irradiated hosts or anemia and irradiated hosts increased and reached a plateu at day 5. During the plateu period, the proportions between immature and mature granulocytes in DC were kept constantly. The number of macrophages showed a two-phase increasing. Erythroid cells and lymphocytes rapidly disappeared from the chambers during 3 days. The number of erythroid cells was not significantly influenced even in anemia and irradiation hosts. (author)

  7. Insulin suppresses the AMPK signaling pathway to regulate lipid metabolism in primary cultured hepatocytes of dairy cows.

    Science.gov (United States)

    Li, Xinwei; Li, Yu; Ding, Hongyan; Dong, Jihong; Zhang, Renhe; Huang, Dan; Lei, Lin; Wang, Zhe; Liu, Guowen; Li, Xiaobing

    2018-05-01

    Dairy cows with type II ketosis display hepatic fat accumulation and hyperinsulinemia, but the underlying mechanism is not completely clear. This study aimed to clarify the regulation of lipid metabolism by insulin in cow hepatocytes. In vitro, cow hepatocytes were treated with 0, 1, 10, or 100 nm insulin in the presence or absence of AICAR (an AMP-activated protein kinase alpha (AMPKα) activator). The results showed that insulin decreased AMPKα phosphorylation. This inactivation of AMPKα increased the gene and protein expression levels of carbohydrate responsive element-binding protein (ChREBP) and sterol regulatory element-binding protein-1c (SREBP-1c), which downregulated the expression of lipogenic genes, thereby decreasing lipid biosynthesis. Furthermore, AMPKα inactivation decreased the gene and protein expression levels of peroxisome proliferator-activated receptor-α (PPARα), which upregulated the expression of lipid oxidation genes, thereby increasing lipid oxidation. In addition, insulin decreased the very low density lipoprotein (VLDL) assembly. Consequently, triglyceride content was significantly increased in insulin treated hepatocytes. Activation of AMPKα induced by AICAR could reverse the effect of insulin on PPARα, SREBP-1c, and ChREBP, thereby decreasing triglyceride content. These results indicate that insulin inhibits the AMPKα signaling pathway to increase lipid synthesis and decrease lipid oxidation and VLDL assembly in cow hepatocytes, thereby inducing TG accumulation. This mechanism could partly explain the causal relationship between hepatic fat accumulation and hyperinsulinemia in dairy cows with type II ketosis.

  8. Modulation of Xenobiotic Metabolizing Enzyme and Transporter Gene Expression in Primary Cultures of Human Hepatocytes by ToxCast Chemicals

    Science.gov (United States)

    ToxCast chemicals were assessed for induction or suppression of xenobiotic metabolizing enzyme and transporter gene expression using primary human hepatocytes. The mRNA levels of 14 target and 2 control genes were measured: ABCB1, ABCB11, ABCG2, SLCO1B1, CYP1A1, CYP1A2, CYP2B6, C...

  9. Sex-specific biotransformation and detoxification after xenobiotic exposure of primary cultured hepatocytes of European flounder (Platichthys flesus L.)

    NARCIS (Netherlands)

    Winzer, Katja; van Noorden, Cornelis J. F.; Köhler, Angela

    2002-01-01

    Sex-specific effects of sublethal concentrations of known effective pro-oxidants such as 100, 200 and 400 muM benzo[a]pyrene (B[a]p), 50 M nitrofurantoin (NF) and 100 muM hydrogen peroxide (H2O2) on biotransformation pathways were studied in isolated hepatocytes of immature female and male European

  10. Obeticholic acid, a selective farnesoid X receptor agonist, regulates bile acid homeostasis in sandwich-cultured human hepatocytes.

    Science.gov (United States)

    Zhang, Yuanyuan; Jackson, Jonathan P; St Claire, Robert L; Freeman, Kimberly; Brouwer, Kenneth R; Edwards, Jeffrey E

    2017-08-01

    Farnesoid X receptor (FXR) is a master regulator of bile acid homeostasis through transcriptional regulation of genes involved in bile acid synthesis and cellular membrane transport. Impairment of bile acid efflux due to cholangiopathies results in chronic cholestasis leading to abnormal elevation of intrahepatic and systemic bile acid levels. Obeticholic acid (OCA) is a potent and selective FXR agonist that is 100-fold more potent than the endogenous ligand chenodeoxycholic acid (CDCA). The effects of OCA on genes involved in bile acid homeostasis were investigated using sandwich-cultured human hepatocytes. Gene expression was determined by measuring mRNA levels. OCA dose-dependently increased fibroblast growth factor-19 (FGF-19) and small heterodimer partner (SHP) which, in turn, suppress mRNA levels of cholesterol 7-alpha-hydroxylase (CYP7A1), the rate-limiting enzyme for de novo synthesis of bile acids. Consistent with CYP7A1 suppression, total bile acid content was decreased by OCA (1 μmol/L) to 42.7 ± 20.5% relative to control. In addition to suppressing de novo bile acids synthesis, OCA significantly increased the mRNA levels of transporters involved in bile acid homeostasis. The bile salt excretory pump (BSEP), a canalicular efflux transporter, increased by 6.4 ± 0.8-fold, and the basolateral efflux heterodimer transporters, organic solute transporter α (OST α ) and OST β increased by 6.4 ± 0.2-fold and 42.9 ± 7.9-fold, respectively. The upregulation of BSEP and OST α and OST β, by OCA reduced the intracellular concentrations of d 8 -TCA, a model bile acid, to 39.6 ± 8.9% relative to control. These data demonstrate that OCA does suppress bile acid synthesis and reduce hepatocellular bile acid levels, supporting the use of OCA to treat bile acid-induced toxicity observed in cholestatic diseases. © 2017 Intercept Pharmaceuticals. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd, British Pharmacological Society and

  11. Glutathione depletion by valproic acid in sandwich-cultured rat hepatocytes: Role of biotransformation and temporal relationship with onset of toxicity

    International Nuclear Information System (INIS)

    Kiang, Tony K.L.; Teng Xiaowei; Surendradoss, Jayakumar; Karagiozov, Stoyan; Abbott, Frank S.; Chang, Thomas K.H.

    2011-01-01

    The present study was conducted in sandwich-cultured rat hepatocytes to investigate the chemical basis of glutathione (GSH) depletion by valproic acid (VPA) and evaluate the role of GSH depletion in VPA toxicity. Among the synthetic metabolites of VPA investigated, 4-ene-VPA and (E)-2,4-diene-VPA decreased cellular levels of total GSH, but only (E)-2,4-diene-VPA was more effective and more potent than the parent drug. The in situ generated, cytochrome P450-dependent 4-ene-VPA did not contribute to GSH depletion by VPA, as suggested by the experiment with a cytochrome P450 inhibitor, 1-aminobenzotriazole, to decrease the formation of this metabolite. In support of a role for metabolites, alpha-F-VPA and octanoic acid, which do not undergo biotransformation to form a 2,4-diene metabolite, CoA ester, or glucuronide, did not deplete GSH. A time course experiment showed that GSH depletion did not occur prior to the increase in 2',7'-dichlorofluorescein (a marker of oxidative stress), the decrease in [2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium] (WST-1) product formation (a marker of cell viability), or the increase in lactate dehydrogenase (LDH) release (a marker of necrosis) in VPA-treated hepatocytes. In conclusion, the cytochrome P450-mediated 4-ene-VPA pathway does not play a role in the in situ depletion of GSH by VPA, and GSH depletion is not an initiating event in VPA toxicity in sandwich-cultured rat hepatocytes.

  12. Proliferation of cultured mouse choroid plexus epithelial cells.

    Directory of Open Access Journals (Sweden)

    Basam Z Barkho

    Full Text Available The choroid plexus (ChP epithelium is a multifunctional tissue found in the ventricles of the brain. The major function of the ChP epithelium is to produce cerebrospinal fluid (CSF that bathes and nourishes the central nervous system (CNS. In addition to the CSF, ChP epithelial cells (CPECs produce and secrete numerous neurotrophic factors that support brain homeostasis, such as adult hippocampal neurogenesis. Accordingly, damage and dysfunction to CPECs are thought to accelerate and intensify multiple disease phenotypes, and CPEC regeneration would represent a potential therapeutic approach for these diseases. However, previous reports suggest that CPECs rarely divide, although this has not been extensively studied in response to extrinsic factors. Utilizing a cell-cycle reporter mouse line and live cell imaging, we identified scratch injury and the growth factors insulin-like growth factor 1 (IGF-1 and epidermal growth factor (EGF as extrinsic cues that promote increased CPEC expansion in vitro. Furthermore, we found that IGF-1 and EGF treatment enhances scratch injury-induced proliferation. Finally, we established whole tissue explant cultures and observed that IGF-1 and EGF promote CPEC division within the intact ChP epithelium. We conclude that although CPECs normally have a slow turnover rate, they expand in response to external stimuli such as injury and/or growth factors, which provides a potential avenue for enhancing ChP function after brain injury or neurodegeneration.

  13. [Isolation, purification and primary culture of adult mouse cardiac fibroblasts].

    Science.gov (United States)

    Li, Rujun; Gong, Kaizheng; Zhang, Zhengang

    2017-01-01

    Objective To establish a method for primary culture of adult mouse cardiac fibroblasts. Methods Myocardial tissues from adult mice were digested with 1 g/L trypsin and 0.8 g/L collagenase IV by oscillating water bath for a short time repeatedly. Cardiac fibroblasts and myocardial cells were isolated with differential adhesion method. Immunofluorescence staining was used to assess the purity of cardiac fibroblasts. The cell morphology was observed under an inverted phase contrast microscope. The proliferation of cardiac fibroblasts was analyzed by growth curve and CCK-8 assay. The Smad2/3 phosphorylation induced by TGF-β1 was detected by Western blotting. Results After 90 minutes of differential adhesion, adherent fibroblasts formed spherical cell mass and after 3 days, cells were spindle-shaped and proliferated rapidly. Cells were confluent after 5 days and the growth curve presented nearly "S" shape. The positive expression rate of vimentin was 95%. CCK-8 assay showed that the optimal cell proliferating activity was found from day 3 to day 5. The level of phosphorylated Smad2/3 obviously increased at the second passage induced by TGF-β1. Conclusion This method is economical and stable to isolate cardiac fibroblasts with high activity and high purity from adult mice.

  14. Chromosome preparations from microplate cultures of man, dog, rat, and mouse

    NARCIS (Netherlands)

    de Jong, B; Anders, GJPA; van der Meer, Ingrid H

    1976-01-01

    A simple method for making chromosomal preparations from 105 leukocytes from man, dog, mouse, and rat and from 0.01 ml total human and dog blood is developed. The leukocytes and the peripheral blood are cultured in Cooke microtiter plates in a culture volume of 0.1 ml. The culture medium is R.P.M.I.

  15. Hepatocyte heterogeneity in the metabolism of carbohydrates.

    Science.gov (United States)

    Jungermann, K; Thurman, R G

    1992-01-01

    Periportal and perivenous hepatocytes possess different amounts and activities of the rate-generating enzymes of carbohydrate and oxidative energy metabolism and thus different metabolic capacities. This is the basis of the model of metabolic zonation, according to which periportal cells catalyze predominantly the oxidative catabolism of fatty and amino acids as well as glucose release and glycogen formation via gluconeogenesis, and perivenous cells carry out preferentially glucose uptake for glycogen synthesis and glycolysis coupled to liponeogenesis. The input of humoral and nervous signals into the periportal and perivenous zones is different; gradients of oxygen, substrates and products, hormones and mediators and nerve densities exist which are important not only for the short-term regulation of carbohydrate metabolism but also for the long-term regulation of zonal gene expression. The specialization of periportal and perivenous hepatocytes in carbohydrate metabolism has been well characterized. In vivo evidence is provided by the complex metabolic situation termed the 'glucose paradox' and by zonal flux differences calculated on the basis of the distribution of enzymes and metabolites. In vitro evidence is given by the different flux rates determined with classical invasive techniques, e.g. in periportal-like and perivenous-like hepatocytes in cell culture, in periportal- and perivenous-enriched hepatocyte populations and in perfused livers during orthograde and retrograde flow, as well as with noninvasive techniques using miniature oxygen electrodes, e.g. in livers perfused in either direction. Differences of opinion in the interpretation of studies with invasive and noninvasive techniques by the authors are discussed. The declining gradient in oxygen concentrations, the decreasing glucagon/insulin ratio and the different innervation could be important factors in the zonal expression of the genes of carbohydrate-metabolizing enzymes. While it is clear that

  16. Comparison of the biological features between human fetal hepatocyte and immortalized L-02 hepatocyte in vitro

    International Nuclear Information System (INIS)

    Kong Weiwei; Teng Gaojun

    2004-01-01

    Objective: To evaluate the feasibilities of the potential donors in liver cell transplantation using the human fetal hepatocytes and immortalized L-02 hepatocytes by comparing their biological features. Methods: Human fetal hepatocytes were isolated from aborted fetal livers (gestational ages from 14 w to 24 w) by an improved two-stage perfusion method and cultured in a conditioned medium without any growth factors. α-fetal protein (AFP) and albumin (ALB) were detected by radioimmunoassay (RIA) and cytokeratin-19 (CK-19 ) was identified by cellular immunochemistry study. Immortalized L-02 hepatocytes were cultured in the same condition and the characteristic proteins were detected by the same methods. Results: The viability of human fetal hepatocytes was approximately 95% using the perfusion method, and the maximum survival time of the cultured hepatocytes was 3 weeks. The expression of AFP, ALB, and CK19 was detected at the same time, especially during Day 3 to Day 7 in the culture. By comparison, the proliferation ability of L-02 hepatocyte was greater, although with a lower level of ALB secretion. The expression of AFP and CK19 was not detected. Furthermore, during the long culture, L-02 hepatocytes may undergo a morphologic change and fail to express ALB. Conclusion: Human fetal hepatocyte may be a practical donor for hepatocyte transplantation with its high-level protein expression and potential bi-differentiation ability. In view of the absent expression of ALB and the morphologic change in culture, although with better proliferation, L-02 hepatocyte seems not useful for hepatocyte transplantation

  17. High-Affinity Low-Capacity and Low-Affinity High-Capacity N-Acetyl-2-Aminofluorene (AAF) Macromolecular Binding Sites Are Revealed During the Growth Cycle of Adult Rat Hepatocytes in Primary Culture.

    Science.gov (United States)

    Koch, Katherine S; Moran, Tom; Shier, W Thomas; Leffert, Hyam L

    2018-05-01

    Long-term cultures of primary adult rat hepatocytes were used to study the effects of N-acetyl-2-aminofluorene (AAF) on hepatocyte proliferation during the growth cycle; on the initiation of hepatocyte DNA synthesis in quiescent cultures; and, on hepatocyte DNA replication following the initiation of DNA synthesis. Scatchard analyses were used to identify the pharmacologic properties of radiolabeled AAF metabolite binding to hepatocyte macromolecules. Two classes of growth cycle-dependent AAF metabolite binding sites-a high-affinity low-capacity site (designated Site I) and a low-affinity high-capacity site (designated Site II)-associated with two spatially distinct classes of macromolecular targets, were revealed. Based upon radiolabeled AAF metabolite binding to purified hepatocyte genomic DNA or to DNA, RNA, proteins, and lipids from isolated nuclei, Site IDAY 4 targets (KD[APPARENT] ≈ 2-4×10-6 M and BMAX[APPARENT] ≈ 6 pmol/106 cells/24 h) were consistent with genomic DNA; and with AAF metabolized by a nuclear cytochrome P450. Based upon radiolabeled AAF binding to total cellular lysates, Site IIDAY 4 targets (KD[APPARENT] ≈ 1.5×10-3 M and BMAX[APPARENT] ≈ 350 pmol/106 cells/24 h) were consistent with cytoplasmic proteins; and with AAF metabolized by cytoplasmic cytochrome P450s. DNA synthesis was not inhibited by concentrations of AAF that saturated DNA binding in the neighborhood of the Site I KD. Instead, hepatocyte DNA synthesis inhibition required higher concentrations of AAF approaching the Site II KD. These observations raise the possibility that carcinogenic DNA adducts derived from AAF metabolites form below concentrations of AAF that inhibit replicative and repair DNA synthesis.

  18. A biomimetic porous hydrogel of gelatin and glycosaminoglycans cross-linked with transglutaminase and its application in the culture of hepatocytes

    International Nuclear Information System (INIS)

    De Colli, M; Massimi, M; Barbetta, A; Di Rosario, B L; Nardecchia, S; Dentini, M; Conti Devirgiliis, L

    2012-01-01

    The development of blended gelatin and glycosaminoglycan (GAG) scaffolds can potentially be used in many soft tissue engineering applications since these scaffolds mimic the structure and biological function of native extracellular matrix (ECM). In this study, we were able to obtain a gelatin–GAG scaffold by using a concentrated emulsion templating technique known as high internal phase emulsion (HIPE), in which a prevailing in volume organic phase is dispersed in the form of discrete droplets inside an aqueous solution of three biopolymers represented by gelatin, hyaluronic acid (HA) and chondroitin sulfate (CS) in the presence of a suitable surfactant. In order to preserve the bioactive potential of the biopolymers employed, the cross-linking procedure involved the use of transglutaminase (MTGase) that catalyzes the formation of covalent N-ε-(γ-glutamyl) lysine amide bonds. Since neither HA nor CS possess the necessary primary amino groups toward which MTGase is active, they were functionalized with the dipeptide glycine-lysine (GK). In this way the introduction of foreign cross-linking bridging units with an unpredictable biocompatibility was avoided. These enzymatic cross-linked gelatin–GAG scaffolds were tested in the culture of primary rat and C3A hepatocytes. Results underlined the good performance of this novel support in maintaining and promoting hepatocyte functions in vitro. (paper)

  19. Effects of carcinogen treatment on rat liver DNA synthesis in vivo and on nascent DNA synthesis and elongation in cultured hepatocytes

    International Nuclear Information System (INIS)

    Zurlo, J.; Mignano, J.E.; Eustice, D.C.; Poirier, M.C.; Yager, J.D.

    1986-01-01

    One objective of this study was to determine the effects of N-hydroxy-2-acetylaminofluorene (N-OH-AAF) treatment on DNA synthesis in regenerating rat liver. It was found that N-OH-AAF caused a dose-dependent inhibition of [ 3 H]thymidine incorporation into liver DNA. This inhibition was followed by a gradual, but incomplete recovery. The second objective of the study was to determine the effects of DNA damage on the size distribution and elongation of nascent hepatocyte DNA. Hepatocytes, which have been shown to demonstrate a pattern of inhibition and subsequent recovery of DNA synthesis following UV irradiation similar to that seen in vivo upon treatment with N-OH-AAF were cultured. The size distribution of nascent DNA after UV irradiation was determined by pH step gradient alkaline elution analysis. The results show that UV irradiation caused a dose-dependent decrease in the size distribution of nascent DNA suggesting an inhibition of elongation. The results show that resumption of DNA synthesis and nascent strand elongation occur on damaged templates. These observations along with previous studies support the idea that DNA damage leading to inhibition of DNA synthesis may induce SOS-type processes which if mutagenic may play a role in the initiation of carcinogenesis. (Auth.)

  20. Copper induces hepatocyte injury due to the endoplasmic reticulum stress in cultured cells and patients with Wilson disease

    International Nuclear Information System (INIS)

    Oe, Shinji; Miyagawa, Koichiro; Honma, Yuichi; Harada, Masaru

    2016-01-01

    Copper is an essential trace element, however, excess copper is harmful to human health. Excess copper-derived oxidants contribute to the progression of Wilson disease, and oxidative stress induces accumulation of abnormal proteins. It is known that the endoplasmic reticulum (ER) plays an important role in proper protein folding, and that accumulation of misfolded proteins disturbs ER homeostasis resulting in ER stress. However, copper-induced ER homeostasis disturbance has not been fully clarified. We treated human hepatoma cell line (Huh7) and immortalized-human hepatocyte cell line (OUMS29) with copper and chemical chaperones, including 4-phenylbutyrate and ursodeoxycholic acid. We examined copper-induced oxidative stress, ER stress and apoptosis by immunofluorescence microscopy and immunoblot analyses. Furthermore, we examined the effects of copper on carcinogenesis. Excess copper induced not only oxidative stress but also ER stress. Furthermore, excess copper induced DNA damage and reduced cell proliferation. Chemical chaperones reduced this copper-induced hepatotoxicity. Excess copper induced hepatotoxicity via ER stress. We also confirmed the abnormality of ultra-structure of the ER of hepatocytes in patients with Wilson disease. These findings show that ER stress plays a pivotal role in Wilson disease, and suggests that chemical chaperones may have beneficial effects in the treatment of Wilson disease.

  1. Copper induces hepatocyte injury due to the endoplasmic reticulum stress in cultured cells and patients with Wilson disease

    Energy Technology Data Exchange (ETDEWEB)

    Oe, Shinji, E-mail: ooes@med.uoeh-u.ac.jp; Miyagawa, Koichiro, E-mail: koichiro@med.uoeh-u.ac.jp; Honma, Yuichi, E-mail: y-homma@med.uoeh-u.ac.jp; Harada, Masaru, E-mail: msrharada@med.uoeh-u.ac.jp

    2016-09-10

    Copper is an essential trace element, however, excess copper is harmful to human health. Excess copper-derived oxidants contribute to the progression of Wilson disease, and oxidative stress induces accumulation of abnormal proteins. It is known that the endoplasmic reticulum (ER) plays an important role in proper protein folding, and that accumulation of misfolded proteins disturbs ER homeostasis resulting in ER stress. However, copper-induced ER homeostasis disturbance has not been fully clarified. We treated human hepatoma cell line (Huh7) and immortalized-human hepatocyte cell line (OUMS29) with copper and chemical chaperones, including 4-phenylbutyrate and ursodeoxycholic acid. We examined copper-induced oxidative stress, ER stress and apoptosis by immunofluorescence microscopy and immunoblot analyses. Furthermore, we examined the effects of copper on carcinogenesis. Excess copper induced not only oxidative stress but also ER stress. Furthermore, excess copper induced DNA damage and reduced cell proliferation. Chemical chaperones reduced this copper-induced hepatotoxicity. Excess copper induced hepatotoxicity via ER stress. We also confirmed the abnormality of ultra-structure of the ER of hepatocytes in patients with Wilson disease. These findings show that ER stress plays a pivotal role in Wilson disease, and suggests that chemical chaperones may have beneficial effects in the treatment of Wilson disease.

  2. Gene disruption of Plasmodium falciparum p52 results in attenuation of malaria liver stage development in cultured primary human hepatocytes.

    Directory of Open Access Journals (Sweden)

    Ben C L van Schaijk

    Full Text Available Difficulties with inducing sterile and long lasting protective immunity against malaria with subunit vaccines has renewed interest in vaccinations with attenuated Plasmodium parasites. Immunizations with sporozoites that are attenuated by radiation (RAS can induce strong protective immunity both in humans and rodent models of malaria. Recently, in rodent parasites it has been shown that through the deletion of a single gene, sporozoites can also become attenuated in liver stage development and, importantly, immunization with these sporozoites results in immune responses identical to RAS. The promise of vaccination using these genetically attenuated sporozoites (GAS depends on translating the results in rodent malaria models to human malaria. In this study, we perform the first essential step in this transition by disrupting, p52, in P. falciparum an ortholog of the rodent parasite gene, p36p, which we had previously shown can confer long lasting protective immunity in mice. These P. falciparum P52 deficient sporozoites demonstrate gliding motility, cell traversal and an invasion rate into primary human hepatocytes in vitro that is comparable to wild type sporozoites. However, inside the host hepatocyte development is arrested very soon after invasion. This study reveals, for the first time, that disrupting the equivalent gene in both P. falciparum and rodent malaria Plasmodium species generates parasites that become similarly arrested during liver stage development and these results pave the way for further development of GAS for human use.

  3. Assessment of the role of in situ generated (E)-2,4-diene-valproic acid in the toxicity of valproic acid and (E)-2-ene-valproic acid in sandwich-cultured rat hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Surendradoss, Jayakumar; Chang, Thomas K.H.; Abbott, Frank S., E-mail: frank.abbott@ubc.ca

    2012-11-01

    Valproic acid (VPA) undergoes cytochrome P450-mediated desaturation to form 4-ene-VPA, which subsequently yields (E)-2,4-diene-VPA by β-oxidation. Another biotransformation pathway involves β-oxidation of VPA to form (E)-2-ene-VPA, which also generates (E)-2,4-diene-VPA by cytochrome P450-mediated desaturation. Although the synthetic form of (E)-2,4-diene-VPA is more hepatotoxic than VPA as shown in various experimental models, there is no conclusive evidence to implicate the in situ generated (E)-2,4-diene-VPA in VPA hepatotoxicity. The present study investigated the effects of modulating the in situ formation of (E)-2,4-diene-VPA on markers of oxidative stress (formation of 2′,7′-dichlorofluorescein; DCF), steatosis (accumulation of BODIPY 558/568 C{sub 12}), necrosis (release of lactate dehydrogenase; LDH), and on cellular total glutathione (GSH) levels in sandwich-cultured rat hepatocytes treated with VPA or (E)-2-ene-VPA. Treatment with either of these chemicals alone increased each of the toxicity endpoints. In VPA-treated hepatocytes, (E)-2,4-diene-VPA was detected only at trace levels, even after phenobarbital (PB) pretreatment and there was no effect on the toxicity of VPA. Furthermore, pretreatment with a cytochrome P450 enzyme inhibitor, 1-aminobenzotriazole (1-ABT), did not influence the extent of VPA toxicity in both PB-pretreated and vehicle-pretreated hepatocytes. However, in (E)-2-ene-VPA-treated hepatocytes, PB pretreatment greatly enhanced the levels of (E)-2,4-diene-VPA and this was accompanied by a further enhancement of the effects of (E)-2-ene-VPA on DCF formation, BODIPY accumulation, LDH release, and GSH depletion. Pretreatment with 1-ABT reduced the concentrations of (E)-2,4-diene-VPA and the extent of (E)-2-ene-VPA toxicity; however, this occurred in PB-pretreated hepatocytes, but not in control hepatocytes. In conclusion, in situ generated (E)-2,4-diene-VPA is not responsible for the hepatocyte toxicity of VPA, whereas it

  4. Isolation, culture and adenoviral transduction of parietal cells from mouse gastric mucosa

    International Nuclear Information System (INIS)

    Gliddon, Briony L; Nguyen, Nhung V; Gunn, Priscilla A; Gleeson, Paul A; Driel, Ian R van

    2008-01-01

    Here we describe a method for the isolation of intact gastric glands from mice and primary culture and transfection of mouse gastric epithelial cells. Collagenase digestion of PBS-perfused mouse stomachs released large intact gastric glands that were plated on a basement membrane matrix. The heterogeneous gland cell cultures typically contain ∼60% parietal cells. Isolated mouse parietal cells remain viable in culture for up to 5 days and react strongly with an antibody specific to the gastric H + /K + ATPase. Isolated intact mouse gastric glands and primary cultures of mouse parietal cells respond to the secretagogue, histamine. Typical morphological changes from a resting to an acid-secreting active parietal cell were observed. In resting cultures of mouse parietal cells, the H + /K + ATPase displayed a cytoplasmic punctate staining pattern consistent with tubulovesicle element structures. Following histamine stimulation, an expansion of internal apical vacuole structures was observed together with a pronounced redistribution of the H + /K + ATPase from the cytoplasm to the apical vacuoles. A reproducible procedure to express genes of interest exogenously in these cultures of mouse parietal cells was also established. This method combines recombinant adenoviral transduction with magnetic field-assisted transfection resulting in ∼30% transduced parietal cells. Adenoviral-transduced parietal cells maintain their ability to undergo agonist-induced activation. This protocol will be useful for the isolation, culture and expression of genes in parietal cells from genetically modified mice and as such will be an invaluable tool for studying the complex exocytic and endocytic trafficking events of the H + /K + ATPase which underpin the regulation of acid secretion

  5. ART culture conditions change the probability of mouse embryo gestation through defined cellular and molecular responses

    NARCIS (Netherlands)

    Schwarzer, Caroline; Esteves, Telma Cristina; Arau´zo-Bravo, Marcos J.; le Gac, Severine; Nordhoff, Verena; Schlatt, Stefan; Boiani, Michele

    2012-01-01

    Do different human ART culture protocols prepare embryos differently for post-implantation development? ... Our data promote awareness that human ART culture media affect embryo development. Effects reported here in the mouse may apply also in human, because no ART medium presently available on the

  6. A Cell Culture Platform to Maintain Long-term Phenotype of Primary Human Hepatocytes and Endothelial Cells.

    Science.gov (United States)

    Ware, Brenton R; Durham, Mitchell J; Monckton, Chase P; Khetani, Salman R

    2018-03-01

    Modeling interactions between primary human hepatocytes (PHHs) and primary human liver sinusoidal endothelial cells (LSECs) in vitro can help elucidate human-specific mechanisms underlying liver physiology/disease and drug responses; however, existing hepatocyte/endothelial coculture models are suboptimal because of their use of rodent cells, cancerous cell lines, and/or nonliver endothelial cells. Hence, we sought to develop a platform that could maintain the long-term phenotype of PHHs and primary human LSECs. Primary human LSECs or human umbilical vein endothelial cells as the nonliver control were cocultivated with micropatterned PHH colonies (to control homotypic interactions) followed by an assessment of PHH morphology and functions (albumin and urea secretion, and cytochrome P-450 2A6 and 3A4 enzyme activities) over 3 weeks. Endothelial phenotype was assessed via gene expression patterns and scanning electron microscopy to visualize fenestrations. Hepatic responses in PHH/endothelial cocultures were benchmarked against responses in previously developed PHH/3T3-J2 fibroblast cocultures. Finally, PHH/fibroblast/endothelial cell tricultures were created and characterized as described previously. LSECs, but not human umbilical vein endothelial cells, induced PHH albumin secretion for ∼11 days; however, neither endothelial cell type could maintain PHH morphology and functions to the same magnitude/longevity as the fibroblasts. In contrast, both PHHs and endothelial cells displayed stable phenotype for 3 weeks in PHH/fibroblast/endothelial cell tricultures; furthermore, layered tricultures in which PHHs and endothelial cells were separated by a protein gel to mimic the space of Disse displayed similar functional levels as the coplanar tricultures. PHH/fibroblast/endothelial tricultures constitute a robust platform to elucidate reciprocal interactions between PHHs and endothelial cells in physiology, disease, and after drug exposure.

  7. Evaluation of an in vitro toxicogenetic mouse model for hepatotoxicity

    International Nuclear Information System (INIS)

    Martinez, Stephanie M.; Bradford, Blair U.; Soldatow, Valerie Y.; Kosyk, Oksana; Sandot, Amelia; Witek, Rafal; Kaiser, Robert; Stewart, Todd; Amaral, Kirsten; Freeman, Kimberly; Black, Chris; LeCluyse, Edward L.; Ferguson, Stephen S.; Rusyn, Ivan

    2010-01-01

    Numerous studies support the fact that a genetically diverse mouse population may be useful as an animal model to understand and predict toxicity in humans. We hypothesized that cultures of hepatocytes obtained from a large panel of inbred mouse strains can produce data indicative of inter-individual differences in in vivo responses to hepato-toxicants. In order to test this hypothesis and establish whether in vitro studies using cultured hepatocytes from genetically distinct mouse strains are feasible, we aimed to determine whether viable cells may be isolated from different mouse inbred strains, evaluate the reproducibility of cell yield, viability and functionality over subsequent isolations, and assess the utility of the model for toxicity screening. Hepatocytes were isolated from 15 strains of mice (A/J, B6C3F1, BALB/cJ, C3H/HeJ, C57BL/6J, CAST/EiJ, DBA/2J, FVB/NJ, BALB/cByJ, AKR/J, MRL/MpJ, NOD/LtJ, NZW/LacJ, PWD/PhJ and WSB/EiJ males) and cultured for up to 7 days in traditional 2-dimensional culture. Cells from B6C3F1, C57BL/6J, and NOD/LtJ strains were treated with acetaminophen, WY-14,643 or rifampin and concentration-response effects on viability and function were established. Our data suggest that high yield and viability can be achieved across a panel of strains. Cell function and expression of key liver-specific genes of hepatocytes isolated from different strains and cultured under standardized conditions are comparable. Strain-specific responses to toxicant exposure have been observed in cultured hepatocytes and these experiments open new opportunities for further developments of in vitro models of hepatotoxicity in a genetically diverse population.

  8. The Effects of Betaine on the Nuclear Fractal Dimension, Chromatin Texture, and Proliferative Activity in Hepatocytes in Mouse Model of Nonalcoholic Fatty Liver Disease.

    Science.gov (United States)

    Vesković, Milena; Labudović-Borović, Milica; Zaletel, Ivan; Rakočević, Jelena; Mladenović, Dušan; Jorgačević, Bojan; Vučević, Danijela; Radosavljević, Tatjana

    2018-04-01

    The effects of betaine on hepatocytes chromatin architecture changes were examined by using fractal and gray-level co-occurrence matrix (GLCM) analysis in methionine/choline-deficient (MCD) diet-induced, nonalcoholic fatty liver disease (NAFLD). Male C57BL/6 mice were divided into groups: (1) Control: standard diet; (2) BET: standard diet and betaine supplementation through drinking water (solution 1.5%); (3) MCD group: MCD diet for 6 weeks; (4) MCD+BET: fed with MCD diet + betaine for 6 weeks. Liver tissue was collected for histopathology, immunohistochemistry, and determination of fractal dimension and GLCM parameters. MCD diet induced diffuse micro- and macrovesicular steatosis accompanied with increased Ki67-positive hepatocyte nuclei. Steatosis and Ki67 immunopositivity were less prominent in the MCD+BET group compared with the MCD group. Angular second moment (ASM) and inverse difference moment (IDM) (textural homogeneity markers) were significantly increased in the MCD+BET group versus the MCD group (pMCD and the control group was evident. Heterogeneity parameters, contrast, and correlation were significantly increased in the MCD group versus the control (pMCD group (pMCD diet-induced NAFLD by reducing fat accumulation and inhibiting hepatocyte proliferation. Betaine supplementation increased nuclear homogeneity and chromatin complexity with reduction of entropy, contrast, and correlation.

  9. Mangifera indica L. extract and mangiferin modulate cytochrome P450 and UDP-glucuronosyltransferase enzymes in primary cultures of human hepatocytes.

    Science.gov (United States)

    Rodeiro, Idania; José Gómez-Lechón, M; Perez, Gabriela; Hernandez, Ivones; Herrera, José Alfredo; Delgado, Rene; Castell, José V; Teresa Donato, M

    2013-05-01

    The aqueous stem bark extract of Mangifera indica L. (MSBE) has been reported to have antioxidant, anti-inflammatory and analgesic properties. In previous studies, we showed that MSBE and mangiferin, its main component, lower the activity of some cytochrome P-450 (P450) enzymes in rat hepatocytes and human liver microsomes. In the present study, the effects of MSBE and mangiferin on several P450 enzymes and UDP-glucuronosyltransferases (UGTs) in human-cultured hepatocytes have been examined. After hepatocytes underwent a 48-h treatment with sub-cytotoxic concentrations of the products (50-250 µg/mL), a concentration-dependent decrease of the activity of the five P450 enzymes measured (CYP1A2, 2A6, 2C9, 2D6 and 3A4) was observed. For all the activities, a reduction of at least 50% at the highest concentration (250 µg/mL) was observed. In addition, UGT activities diminished. MSBE considerably reduced UGT1A9 activity (about 60% at 250 µg/mL) and lesser effects on the other UGTs. In contrast, 250 µg/mL mangiferin had greater effects on UGT1A1 and 2B7 than on UGT1A9 (about 55% vs. 35% reduction, respectively). Quantification of specific mRNAs revealed reduced CYP3A4 and 3A5 mRNAs content, and an increase in CYP1A1, CYP1A2, UGT1A1 and UGT1A9 mRNAs. No remarkable effects on the CYP2A6, 2B6, 2C9, 2C19, 2D6 and 2E1 levels were observed. Our results suggest that the activity and/or expression of major P450 and UGT enzymes is modulated by MSBE and that potential herb-drugs interactions could arise after a combined intake of this extract with conventional medicines. Therefore, the potential safety risks of this natural product derived by altering the ADMET properties of co-administered drugs should be examined. Copyright © 2012 John Wiley & Sons, Ltd.

  10. Establishment of mouse neuron and microglial cell co-cultured models and its action mechanism.

    Science.gov (United States)

    Zhang, Bo; Yang, Yunfeng; Tang, Jun; Tao, Yihao; Jiang, Bing; Chen, Zhi; Feng, Hua; Yang, Liming; Zhu, Gang

    2017-06-27

    The objective of this study is to establish a co-culture model of mouse neurons and microglial cells, and to analyze the mechanism of action of oxygen glucose deprivation (OGD) and transient oxygen glucose deprivation (tOGD) preconditioning cell models. Mouse primary neurons and BV2 microglial cells were successfully cultured, and the OGD and tOGD models were also established. In the co-culture of mouse primary neurons and microglial cells, the cell number of tOGD mouse neurons and microglial cells was larger than the OGD cell number, observed by a microscope. CCK-8 assay result showed that at 1h after treatment, the OD value in the control group is lower compared to all the other three groups (P control group compared to other three groups (P neurons cells were cultured. In the meantime mouse BV2 microglia cells were cultured. Two types of cells were co-cultured, and OGD and tOGD cell models were established. There were four groups in the experiment: control group (OGD), treatment group (tOGD+OGD), placebo group (tOGD+OGD+saline) and minocycline intervention group (tOGD+OGD+minocycline). CCK-8 kit was used to detect cell viability and flow cytometry was used to detect apoptosis. In this study, mouse primary neurons and microglial cells were co-cultured. The OGD and tOGD models were established successfully. tOGD was able to effectively protect neurons and microglial cells from damage, and inhibit the apoptosis caused by oxygen glucose deprivation.

  11. Isolation and culture of adult mouse vestibular nucleus neurons

    Science.gov (United States)

    Him, Aydın; Altuntaş, Serap; Öztürk, Gürkan; Erdoğan, Ender; Cengiz, Nureddin

    2017-12-19

    Background/aim: Isolated cell cultures are widely used to study neuronal properties due to their advantages. Although embryonic animals are preferred for culturing, their morphological or electrophysiological properties may not reflect adult neurons, which may be important in neurodegenerative diseases. This paper aims to develop a method for preparing isolated cell cultures of medial vestibular nucleus (MVN) from adult mice and describe its morphological and electrophysiological properties.Materials and methods: Vestibular nucleus neurons were mechanically and enzymatically isolated and cultured using a defined medium with known growth factors. Cell survival was measured with propidium iodide, and electrophysiological properties were investigated with current-clamp recording.Results: Vestibular neurons grew neurites in cultures, gaining adult-like morphological properties, and stayed viable for 3 days in culture. Adding bovine calf serum, nerve growth factor, or insulin-like growth factor into the culture medium enhanced neuronal viability. Current-clamp recording of the cultured neurons revealed tonic and phasic-type neurons with similar input resistance, resting membrane potential, action potential amplitude, and duration. Conclusion: Vestibular neurons from adult mice can be cultured, and regenerate axons in a medium containing appropriate growth factors. Culturing adult vestibular neurons provides a new method to study age-related pathologies of the vestibular system.

  12. Effects of dexamethasone and insulin on the synthesis of triacylglycerols and phosphatidylcholine and the secretion of very-low-density lipoproteins and lysophosphatidylcholine by monolayer cultures of rat hepatocytes.

    Science.gov (United States)

    Mangiapane, E H; Brindley, D N

    1986-01-01

    Rat hepatocytes in monolayer culture were preincubated for 19 h with 1 microM-dexamethasone, and the incubation was continued for a further 23 h with [14C]oleate, [3H]glycerol and 1 microM-dexamethasone. Dexamethasone increased the secretion of triacylglycerol into the medium in particles that had the properties of very-low-density lipoproteins. The increased secretion was matched by a decrease in the triacylglycerol and phosphatidylcholine that remained in the hepatocytes. Preincubating the hepatocytes for the total 42 h period with 36 nM-insulin decreased the amount of triacylglycerol in the medium and in the cells after the final incubation for 23 h with radioactive substrates. However, insulin had no significant effect on the triacylglycerol content of the cell and medium when it was present only in the final 23 h incubation. Insulin antagonized the effects of dexamethasone in stimulating the secretion of triacylglycerol from the hepatocytes, especially when it was present throughout the total 42 h period. The labelling of lysophosphatidylcholine in the medium when hepatocytes were incubated with [14C]oleate and [3H]glycerol was greater than that of phosphatidylcholine. The appearance of this lipid in the medium, unlike that of triacylglycerol and phosphatidylcholine, was not stimulated by dexamethasone, or inhibited by colchicine. However, the presence of lysophosphatidylcholine in the medium was decreased when the hepatocytes were incubated with both dexamethasone and insulin. These findings are discussed in relation to the control of the synthesis of glycerolipids and the secretion of very-low-density lipoproteins and lysophosphatidylcholine by the liver, particularly in relation to the interactions of glucocorticoids and insulin. PMID:3513755

  13. IN VITRO CULTURE OF FROZEN AND THAWED MOUSE OVA

    OpenAIRE

    KANAGAWA, Hiroshi

    1980-01-01

    Seventy mouse ova at the 4--8-cell stages were collected at room temperature from mice treated with pregnant mare's serum gonadotrophin and human chorionic gonadotrophin. Seventy ova were divided into 5 groups, and each group was placed in a 0.5 ml plastic straw with 0.2 ml of Brinster's medium. Then, the straws were immersed into an ice bath (0℃) for 5 minutes. Next, an equal volume of 2 M dimethyl sulfoxide was added to the sample straw. The medium with ova was then seeded at -6℃ and cooled...

  14. Inhibition of Drp1 protects against senecionine-induced mitochondria-mediated apoptosis in primary hepatocytes and in mice

    Directory of Open Access Journals (Sweden)

    Xiao Yang

    2017-08-01

    Full Text Available Pyrrolizidine alkaloids (PAs are a group of compounds found in various plants and some of them are widely consumed in the world as herbal medicines and food supplements. PAs are potent hepatotoxins that cause irreversible liver injury in animals and humans. However, the mechanisms by which PAs induce liver injury are not clear. In the present study, we determined the hepatotoxicity and molecular mechanisms of senecionine, one of the most common toxic PAs, in primary cultured mouse and human hepatocytes as well as in mice. We found that senecionine administration increased serum alanine aminotransferase levels in mice. H&E and TUNEL staining of liver tissues revealed increased hemorrhage and hepatocyte apoptosis in liver zone 2 areas. Mechanistically, senecionine induced loss of mitochondrial membrane potential, release of mitochondrial cytochrome c as well as mitochondrial JNK translocation and activation prior to the increased DNA fragmentation and caspase-3 activation in primary cultured mouse and human hepatocytes. SP600125, a specific JNK inhibitor, and ZVAD-fmk, a general caspase inhibitor, alleviated senecionine-induced apoptosis in primary hepatocytes. Interestingly, senecionine also caused marked mitochondria fragmentation in hepatocytes. Pharmacological inhibition of dynamin-related protein1 (Drp1, a protein that is critical to regulate mitochondrial fission, blocked senecionine-induced mitochondrial fragmentation and mitochondrial release of cytochrome c and apoptosis. More importantly, hepatocyte-specific Drp1 knockout mice were resistant to senecionine-induced liver injury due to decreased mitochondrial damage and apoptosis. In conclusion, our results uncovered a novel mechanism of Drp1-mediated mitochondrial fragmentation in senecionine-induced liver injury. Targeting Drp1-mediated mitochondrial fragmentation and apoptosis may be a potential avenue to prevent and treat hepatotoxicity induced by PAs. Keywords: Senecionine, Drp1

  15. 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

  16. Technique of the 'in vitro' fertilization and the culture of mouse embryos at preimplantation

    International Nuclear Information System (INIS)

    Kikuchi, Olivia Kimiko; Yamada, Takeshi

    1993-03-01

    The mammal embryo is an intensive cellular proliferating system, very radiosensitive and therefore adequate to the study of the biological effects of ionizing radiation. The technique of the in vitro fertilization and the culture of mouse embryos at preimplantation period, modified by Yamada et al (1982) to improve the efficiency of more than 95% of blastocyst formation is described. (author)

  17. Generation of Hepatocyte-like Cells from Human Induced Pluripotent Stem (iPS) Cells By Co-culturing Embryoid Body Cells with Liver Non-parenchymal Cell Line TWNT-1

    International Nuclear Information System (INIS)

    Javed, M. S.; Yaqoob, N.; Iwamuro, M.; Kobayashi, N.; Fujiwara, T.

    2014-01-01

    Objective: To generate a homogeneous population of patient-specific hepatocyte-like cells (HLCs) from human iPS cells those show the morphologic and phenotypic properties of primary human hepatocytes. Study Design: An experimental study. Place and Duration of Study: Department of Surgery, Okayama University, Graduate School of Medicine, Japan, from April to December 2011. Methodology: Human iPS cells were generated and maintained on ES qualified matrigel coated plates supplemented with mTeSR medium or alternatively on mitotically inactivated MEF feeder layer in DMEM/F12 medium containing 20% KOSR, 4ng/ml bFGF-2, 1 x 10-4 M 2-mercaptoethanol, 1 mmol/L NEAA, 2mM L-glutamine and 1% penicillin-streptomycin. iPS cells were differentiated to HLCs by sequential culture using a four step differentiation protocol: (I) Generation of embryoid bodies (EBs) in suspension culture; (II) Induction of definitive endoderm (DE) from 2 days old EBs by growth in human activin-A (100 ng/ml) and basic fibroblasts growth factor (bFGF2) (100 ng/ml) on matrigel coated plates; (III) Induction of hepatic progenitors by co-culture with non-parenchymal human hepatic stellate cell line (TWNT-1); and (IV) Maturation by culture in dexamethasone. Characterization was performed by RT-PCR and functional assays. Results: The generated HLCs showed microscopically morphological phenotype of human hepatocytes, expressed liver specific genes (ASGPR, Albumin, AFP, Sox17, Fox A2), secreted human liver-specific proteins such as albumin, synthesized urea and metabolized ammonia. Conclusion: Functional HLCs were generated from human iPS cells, which could be used for autologus hepatocyte transplantation for liver failure and as in vitro model for determining the metabolic and toxicological properties of drug compounds. (author)

  18. Hepatobiliary Clearance Prediction: Species Scaling From Monkey, Dog, and Rat, and In Vitro-In Vivo Extrapolation of Sandwich-Cultured Human Hepatocytes Using 17 Drugs.

    Science.gov (United States)

    Kimoto, Emi; Bi, Yi-An; Kosa, Rachel E; Tremaine, Larry M; Varma, Manthena V S

    2017-09-01

    Hepatobiliary elimination can be a major clearance pathway dictating the pharmacokinetics of drugs. Here, we first compared the dose eliminated in bile in preclinical species (monkey, dog, and rat) with that in human and further evaluated single-species scaling (SSS) to predict human hepatobiliary clearance. Six compounds dosed in bile duct-cannulated (BDC) monkeys showed biliary excretion comparable to human; and the SSS of hepatobiliary clearance with plasma fraction unbound correction yielded reasonable predictions (within 3-fold). Although dog SSS also showed reasonable predictions, rat overpredicted hepatobiliary clearance for 13 of 24 compounds. Second, we evaluated the translatability of in vitro sandwich-cultured human hepatocytes (SCHHs) to predict human hepatobiliary clearance for 17 drugs. For drugs with no significant active uptake in SCHH studies (i.e., with or without rifamycin SV), measured intrinsic biliary clearance was directly scalable with good predictability (absolute average fold error [AAFE] = 1.6). Drugs showing significant active uptake in SCHH, however, showed improved predictability when scaled based on extended clearance term (AAFE = 2.0), which incorporated sinusoidal uptake along with a global scaling factor for active uptake and the canalicular efflux clearance. In conclusion, SCHH is a useful tool to predict human hepatobiliary clearance, whereas BDC monkey model may provide further confidence in the prospective predictions. Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

  19. Metabolism of androstenone, 17β-estradiol and dihydrotestosterone in primary cultured pig hepatocytes and the role of 3β-hydroxysteroid dehydrogenase in this process.

    Directory of Open Access Journals (Sweden)

    Gang Chen

    Full Text Available Steroids metabolism plays an important role in mammals and contributes to quality of pig meat. The main objective of this study was to identify metabolites of androstenone, 17β-estradiol and dihydrotestosterone using primary cultured pig hepatocytes as a model system. The role of 3β-hydroxysteroid dehydrogenase (3βHSD in regulation of steroid metabolism was also validated using trilostane, a specific 3βHSD inhibitor. Steroid glucuronide conjugated metabolites were detected by liquid chromatography time of flight mass spectrometry (LC-TOF-MS. 3βHSD enzyme was essential for metabolism of androstenone to 5α-androst-16-en-3β-ol, which then formed androstenone glucuronide conjugate. Metabolism of 17β-estradiol was accompanied by formation of glucuronide-conjugated estrone and glucuronide-conjugated estradiol. The ratio of the two metabolites was around 5:1. 3βHSD enzyme was not involved in 17β-estradiol metabolism. 5α-Dihydrotestosterone-17β-glucuronide was identified as a dihydrotestosterone metabolite, and this metabolism was related to 3βHSD enzyme activity as demonstrated by inhibition study.

  20. Studies on the mechanism of fibrate-inhibited expression of plasminogen activator inhibitor-1 in cultured hepatocytes from cynomolgus monkey

    NARCIS (Netherlands)

    Arts, J.; Kooistra, T.

    1997-01-01

    Fibrates are widely used drugs in hyperlipidemic disorders. In addition to lowering serum triglyceride levels, fibrates have also been shown to reduce elevated plasma plasminogen activator inhibitor-1 (PAI-1) levels in vivo. We demonstrate that fibrates suppress PAI-1 synthesis in cultured

  1. ART culture conditions change the probability of mouse embryo gestation through defined cellular and molecular responses.

    Science.gov (United States)

    Schwarzer, Caroline; Esteves, Telma Cristina; Araúzo-Bravo, Marcos J; Le Gac, Séverine; Nordhoff, Verena; Schlatt, Stefan; Boiani, Michele

    2012-09-01

    Do different human ART culture protocols prepare embryos differently for post-implantation development? The type of ART culture protocol results in distinct cellular and molecular phenotypes in vitro at the blastocyst stage as well as subsequently during in vivo development. It has been reported that ART culture medium affects human development as measured by gestation rates and birthweights. However, due to individual variation across ART patients, it is not possible as yet to pinpoint a cause-effect relationship between choice of culture medium and developmental outcome. In a prospective study, 13 human ART culture protocols were compared two at a time against in vivo and in vitro controls. Superovulated mouse oocytes were fertilized in vivo using outbred and inbred mating schemes. Zygotes were cultured in medium or in the oviduct and scored for developmental parameters 96 h later. Blastocysts were either analyzed or transferred into fosters to measure implantation rates and fetal development. In total, 5735 fertilized mouse oocytes, 1732 blastocysts, 605 fetuses and 178 newborns were examined during the course of the study (December 2010-December 2011). Mice of the B6C3F1, C57Bl/6 and CD1 strains were used as oocyte donors, sperm donors and recipients for embryo transfer, respectively. In vivo fertilized B6C3F1 oocytes were allowed to cleave in 13 human ART culture protocols compared with mouse oviduct and optimized mouse medium (KSOM(aa)). Cell lineage composition of resultant blastocysts was analyzed by immunostaining and confocal microscopy (trophectoderm, Cdx2; primitive ectoderm, Nanog; primitive endoderm, Sox17), global gene expression by microarray analysis, and rates of development to midgestation and to term. Mouse zygotes show profound variation in blastocyst (49.9-91.9%) and fetal (15.7-62.0%) development rates across the 13 ART culture protocols tested (R(2)= 0.337). Two opposite protocols, human tubal fluid/multiblast (high fetal rate) and ISM1/ISM2

  2. Mouse embryonic stem cell culture for generation of three-dimensional retinal and cortical tissues.

    Science.gov (United States)

    Eiraku, Mototsugu; Sasai, Yoshiki

    2011-12-15

    Generation of compound tissues with complex structures is a major challenge in cell biology. In this article, we describe a protocol for mouse embryonic stem cell (ESC) culture for in vitro generation of three-dimensional retinal tissue, comparing it with the culture protocol for cortical tissue generation. Dissociated ESCs are reaggregated in a 96-well plate with reduced cell-plate adhesion and cultured as floating aggregates. Retinal epithelium is efficiently generated when ESC aggregates are cultured in serum-free medium containing extracellular matrix proteins, spontaneously forming hemispherical vesicles and then progressively transforming into a shape reminiscent of the embryonic optic cup in 9-10 d. In long-term culture, the ESC-derived optic cup generates a fully stratified retinal tissue consisting of all major neural retinal components. In contrast, the cortical differentiation culture can be started without exogenous extracellular matrix proteins, and it generates stratified cortical epithelia consisting of four distinct layers in 13 d.

  3. Bioconversion of α-linolenic acid into n-3 long-chain polyunsaturated fatty acid in hepatocytes and ad hoc cell culture optimisation.

    Directory of Open Access Journals (Sweden)

    Ramez Alhazzaa

    Full Text Available This study aimed to establish optimal conditions for a cell culture system that would allow the measurement of 18:3n-3 (ALA bioconversion into n-3 long-chain polyunsaturated fatty acid (n-3 LC-PUFA, and to determine the overall pathway kinetics. Using rat hepatocytes (FaO as model cells, it was established that a maximum 20:5n-3 (EPA production from 50 µM ALA initial concentration was achieved after 3 days of incubation. Next, it was established that a gradual increase in the ALA concentration from 0 up to 125 µM lead to a proportional increase in EPA, without concomitant increase in further elongated or desaturated products, such as 22:5n-3 (DPA and 22:6n-3 (DHA in 3 day incubations. Of interest, ALA bioconversion products were observed in the culture medium. Therefore, in vitro experiments disregarding the medium fatty acid content are underestimating the metabolism efficiency. The novel application of the fatty acid mass balance (FAMB method on cell culture system (cells with medium enabled quantifying the apparent enzymatic activities for the biosynthesis of n-3 LC-PUFA. The activity of the key enzymes was estimated and showed that, under these conditions, 50% (Km of the theoretical maximal (V max = 3654 µmol.g(-1 of cell protein.hour(-1 Fads2 activity on ALA can be achieved with 81 µM initial ALA. Interestingly, the apparent activity of Elovl2 (20:5n-3 elongation was the slowest amongst other biosynthesis steps. Therefore, the possible improvement of Elovl2 activity is suggested toward a more efficient DHA production from ALA. The present study proposed and described an ad hoc optimised cell culture conditions and methodology towards achieving a reliable experimental platform, using FAMB, to assist in studying the efficiency of ALA bioconversion into n-3 LC-PUFA in vitro. The FAMB proved to be a powerful and inexpensive method to generate a detailed description of the kinetics of n-3 LC-PUFA biosynthesis enzymes activities in vitro.

  4. Genomic analysis reveals a potential role for cell cycle perturbation in HCV-mediated apoptosis of cultured hepatocytes.

    Directory of Open Access Journals (Sweden)

    Kathie-Anne Walters

    2009-01-01

    Full Text Available The mechanisms of liver injury associated with chronic HCV infection, as well as the individual roles of both viral and host factors, are not clearly defined. However, it is becoming increasingly clear that direct cytopathic effects, in addition to immune-mediated processes, play an important role in liver injury. Gene expression profiling during multiple time-points of acute HCV infection of cultured Huh-7.5 cells was performed to gain insight into the cellular mechanism of HCV-associated cytopathic effect. Maximal induction of cell-death-related genes and appearance of activated caspase-3 in HCV-infected cells coincided with peak viral replication, suggesting a link between viral load and apoptosis. Gene ontology analysis revealed that many of the cell-death genes function to induce apoptosis in response to cell cycle arrest. Labeling of dividing cells in culture followed by flow cytometry also demonstrated the presence of significantly fewer cells in S-phase in HCV-infected relative to mock cultures, suggesting HCV infection is associated with delayed cell cycle progression. Regulation of numerous genes involved in anti-oxidative stress response and TGF-beta1 signaling suggest these as possible causes of delayed cell cycle progression. Significantly, a subset of cell-death genes regulated during in vitro HCV infection was similarly regulated specifically in liver tissue from a cohort of HCV-infected liver transplant patients with rapidly progressive fibrosis. Collectively, these data suggest that HCV mediates direct cytopathic effects through deregulation of the cell cycle and that this process may contribute to liver disease progression. This in vitro system could be utilized to further define the cellular mechanism of this perturbation.

  5. Effect of Fibroblast Co-culture on In Vitro Maturation and Fertilization of Mouse Preantral Follicles

    Directory of Open Access Journals (Sweden)

    Mahmoud Heidari

    2011-01-01

    Full Text Available Background: The aim of this study was to evaluate fibroblast co-culture on in vitro maturation andfertilization of prepubertal mouse preantral follicles.Materials and Methods: The ovaries of 12-14 day old mice were dissected and 120-150 μmintact preantral follicles with one or two layers of granulosa cells, and round oocytes were culturedindividually in α-minimal essential medium (α-MEM supplemented with 5% fetal bovine serum(FBS, 100 mIU/ml recombinant follicle stimulating hormone, 1% insulin, transferrin, seleniummix, 100 μg/ml penicillin and 50 μg/ml streptomycin as base medium for 12 days. A total number of226 follicules were cultured under two conditions: i base medium as control group (n=113; ii basemedium co-cultured with mouse embryonic fibroblast (MEF (n=113. Follicular diameters, alone,in addition to other factors were analyzed by student’s t-test and chi-square test, respectively.Results: The co-culture group showed significant differences (p<0.05 in growth rate (days 4, 6 and8 of the culture period and survival rate. However, there was no significant difference in antrumformation, ovulation rate and embryonic development of released oocytes. There were significantdifferences (p<0.05 in the estradiol and progesterone secretion at all days between the co-cultureand control groups.Conclusion: Fibroblast co-culture increased survival rate and steroid production of preantralfollicles by promoting granulosa cell proliferation.

  6. Hydrocortisone Diffusion Through Synthetic Membrane, Mouse Skin, and Epiderm™ Cultured Skin.

    Science.gov (United States)

    Christensen, John Mark; Chuong, Monica Chang; Le, Hang; Pham, Loan; Bendas, Ehab

    2011-03-01

    OBJECTIVES: The penetration of hydrocortisone (HC) from six topical over-the-counter products along with one prescription cream through cultured normal human-derived epidermal keratinocytes (Epiderm™), mouse skin and synthetic nylon membrane was performed as well as the effect hydrating the skin by pre-washing was explored using the Upright Franz Cell. METHOD AND RESULTS: Permeation of HC through EpiDerm™, mouse skin and synthetic membrane was highest with the topical HC gel formulation with prewash treatment of the membranes among seven products evaluated, 198 ± 32 µg/cm(2), 746.32 ± 12.43 µg/cm(2), and 1882 ± 395.18 µg/cm(2), respectively. Pre-washing to hydrate the skin enhanced HC penetration through EpiDerm™ and mouse skin. The 24-hour HC released from topical gel with prewash treatment was 198.495 ± 32 µg/cm(2) and 746.32 ± 12.43 µg/cm(2) while without prewash, the 24-h HC released from topical gel was 67.2 ± 7.41 µg/cm(2) and 653.43 ± 85.62 µg/cm(2) though EpiDerm™ and mouse skin, respectively. HC penetration through synthetic membrane was ten times greater than through mouse skin and EpiDerm™. Generally, the shape, pattern, and rank order of HC diffusion from each commercial product was similar through each membrane.

  7. Billion-scale production of hepatocyte-like cells from human induced pluripotent stem cells.

    Science.gov (United States)

    Yamashita, Tomoki; Takayama, Kazuo; Sakurai, Fuminori; Mizuguchi, Hiroyuki

    2018-02-19

    Human induced pluripotent stem (iPS) cell-derived hepatocyte-like cells are expected to be utilized in drug screening and regenerative medicine. However, hepatocyte-like cells have not been fully used in such applications because it is difficult to produce such cells on a large scale. In this study, we tried to establish a method to mass produce hepatocyte-like cells using a three-dimensional (3D) cell culture bioreactor called the Rotary Cell Culture System (RCCS). RCCS enabled us to obtain homogenous hepatocyte-like cells on a billion scale (>10 9  cells). The gene expression levels of some hepatocyte markers (alpha-1 antitrypsin, cytochrome (CYP) 1A2, CYP2D6, and hepatocyte nuclear factor 4alpha) were higher in 3D-cultured hepatocyte-like cells than in 2D-cultured hepatocyte-like cells. This result suggests that RCCS could provide more suitable conditions for hepatocyte maturation than the conventional 2D cell culture conditions. In addition, more than 90% of hepatocyte-like cells were positive for albumin and could uptake low-density lipoprotein in the culture medium. We succeeded in the large-scale production of homogenous and functional hepatocyte-like cells from human iPS cells. This technology will be useful in drug screening and regenerative medicine, which require enormous numbers of hepatocyte-like cells. Copyright © 2018 Elsevier Inc. All rights reserved.

  8. Antioxidant and Preventive Effects of Extract from Nymphaea candida Flower on In Vitro Immunological Liver Injury of Rat Primary Hepatocyte Cultures

    Directory of Open Access Journals (Sweden)

    Jun Zhao

    2011-01-01

    Full Text Available Nymphaea candida is traditional Uighur medicine that is commonly used to treat head pains, cough, hepatitis and hypertension in Xinjiang of China. In this article, the extract of N. candida was measured for antioxidant activity, using 1,1-diphenyl-2-picrylhydrazyl (DPPH radicals scavenging assay and reducing power determination, and compared with those of the positive controls of butylated hydroxytoluene (BHT and gallic acid (GA. The active extract was further purified by liquid-liquid partition to afford four fractions, of which the ethyl acetate-soluble (EA fraction (NCE exhibited the strongest antioxidant capacity with IC50 value of 12.6 g/mL for DPPH. Thirteen phenolic compounds were isolated from this fraction, and they all showed significant antioxidant activities in DPPH model system. Furthermore, NCE showed potent antioxidant capacity with IC50 value of 59.32 g/mL, 24.48 g/mL and 86.85 g/mL, for O2−, ·OH and H2O2 radicals, respectively. Moreover, NCE on BCG plus LPS-induced immunological liver injury was evaluated using primary cultured rat hepatocytes. NCE produced significant hepatoprotective effects as evidenced by decreased supernatant enzyme activities (AST—aspartate transaminase, P <  .01; ALT—alanine transferase, P <  .01 and nitric oxide (NO, P <  .01 production. These results revealed the in vitro antioxidant and hepatoprotective activities of NCE against immunological liver injury. Further investigations are necessary to verify these activities in vivo.

  9. Endogenous production of fibronectin is required for self-renewal of cultured mouse embryonic stem cells

    OpenAIRE

    Hunt, Geoffrey C.; Singh, Purva; Schwarzbauer, Jean E.

    2012-01-01

    Pluripotent cells are attached to the extracellular matrix (ECM) as they make cell fate decisions within the stem cell niche. Here we show that the ubiquitous ECM protein fibronectin is required for self-renewal decisions by cultured mouse embryonic stem (mES) cells. Undifferentiated mES cells produce fibronectin and assemble a fibrillar matrix. Increasing the level of substrate fibronectin increased cell spreading and integrin receptor signaling through focal adhesion kinase, while concomita...

  10. Organotypic hippocampal slice culture from the adult mouse brain: a versatile tool for translational neuropsychopharmacology.

    Science.gov (United States)

    Kim, Hyunjeong; Kim, Eosu; Park, Minsun; Lee, Eun; Namkoong, Kee

    2013-03-05

    One of the most significant barriers towards translational neuropsychiatry would be an unavailability of living brain tissues. Although organotypic brain tissue culture could be a useful alternative enabling observation of temporal changes induced by various drugs in living brain tissues, a proper method to establish a stable organotypic brain slice culture system using adult (rather than neonatal) hippocampus has been still elusive. In this study, we evaluated our simple method using the serum-free culture medium for successful adult organotypic hippocampal slice culture. Several tens of hippocampal slices from a single adult mouse (3-5 months old) were cultured in serum-free versus serum-containing conventional culture medium for 30 days and underwent various experiments to validate the effects of the existence of serum in the culture medium. Neither the excessive regression of neuronal viability nor metabolic deficiency was observed in the serum-free medium culture in contrast to the serum-containing medium culture. Despite such viability, newly generated immature neurons were scarcely detected in the serum-free culture, suggesting that the original neurons in the brain slice persist rather than being replaced by neurogenesis. Key structural features of in vivo neural tissue constituting astrocytes, neural processes, and pre- and post-synapses were also well preserved in the serum-free culture. In conclusion, using the serum-free culture medium, the adult hippocampal slice culture system will serve as a promising ex vivo tool for various fields of neuroscience, especially for studies on aging-related neuropsychiatric disorders or for high throughput screening of potential agents working against such disorders. Copyright © 2012 Elsevier Inc. All rights reserved.

  11. Rex3 (reduced in expression 3) as a new tumor marker in mouse hepatocarcinogenesis

    International Nuclear Information System (INIS)

    Braeuning, Albert; Jaworski, Maike; Schwarz, Michael; Koehle, Christoph

    2006-01-01

    In a previous microarray expression analysis, Rex3, a gene formerly not linked to tumor formation, was found to be highly overexpressed in both Ctnnb1-(β-Catenin) and Ha-ras-mutated mouse liver tumors. Subsequent analyses by in situ hybridization and real-time PCR confirmed a general liver tumor-specific overexpression of the gene (up to 400-fold). To investigate the role of Rex3 in liver tumors, hepatoma cells were transfected with FLAG- and Myc-tagged Rex3 expression vectors. Rex3 was shown to be exclusively localized to the cytoplasm, as determined by fluorescence microscopy and Western blotting. However, forced overexpression of Rex3 did not significantly affect proliferation or stress-induced apoptosis of transfected mouse hepatoma cells. Rex3 mRNA was determined in primary hepatocytes in culture by real-time PCR. In primary mouse hepatocytes, expression of Rex3 increased while cells dedifferentiated in culture. This effect was abolished when hepatocytes were maintained in a differentiated state. Furthermore, expression of Rex3 decreased in mouse liver with age of mice and the expression profile was highly correlated to that of the tumor markers α-fetoprotein and H19. The findings suggest a role of Rex3 as a marker for hepatocyte differentiation/dedifferentiation processes and tumor formation

  12. The antibiotic tiamulin is a potent inducer and inhibitor of cytochrome P4503A via the formation of a stable metabolic intermediate complex. Studies in primary hepatocyte cultures and liver microsomes of the pig.

    Science.gov (United States)

    Witkamp, R F; Nijmeijer, S M; Monshouwer, M; Van Miert, A S

    1995-05-01

    Tiamulin is a semisynthetic antibiotic frequently used in agricultural animals. The drug has been shown to produce clinically important--often lethal--interactions with other compounds that are simultaneously administered. To explain this, it has been suggested that tiamulin selectively inhibits oxidative drug metabolism via the formation of a cytochrome P450 metabolic intermediate complex. The aim of the present study was to provide further support for this hypothesis. When hepatic microsomes and cultured primary pig hepatocytes were incubated with tiamulin, a maximum in the absorbance spectrum at 455 nm was observed, which disappeared after adding KFe(CN)6. When hepatocytes were incubated with tiamulin for 72 hr, cytochrome P450 content and cytochrome P4503A apoprotein levels were increased. Tiamulin strongly inhibited and concentration dependently inhibited the hydroxylation rate of testosterone at the 6 beta-position in both microsomes and hepatocytes, and the microsomal N-demethylation rate of ethylmorphine. Other testosterone hydroxylations were inhibited to a lesser extent or not affected. The relative inhibition of the hydroxylation of testosterone at the 6 beta-position was more pronounced in microsomes from rifampicin- and triacetyloleandomycin-treated pigs. The results indicate that cytochrome P450 complex formation can at least partly explain the interactions observed with tiamulin. Tiamulin seems to be a strong, probably selective, inhibitor of the cytochrome P4503A subfamily and an interesting tool for further research.

  13. Effects of cortisol on the primary response of mouse spleen cell cultures to heterologous erythrocytes

    International Nuclear Information System (INIS)

    Dracott, B.N.

    1974-01-01

    Cell viability and the production of direct PFC were studied in mouse spleen cell cultures after cortisol treatment in vivo or in vitro at various times relative to primary stimulation with SRBC in vitro. Cortisol treatment in vivo reduced spleen cell numbers by 88 percent after 48 hr, but cultures of the remaining cells produced as many PFC in vitro as did cultures of equal numbers of normal spleen cells. In normal spleen cell cultures incubated with cortisol for 4 hr prior to the addition of antigen, peak responses of PFC/culture and PFC/10 6 cells occurred 24 hr later than in controls and averaged, respectively, 27 and 141 percent of control values. Minimum viable cell numbers were observed in cortisol-treated cultures after 3 days; thereafter cell numbers gradually increased. These results were not significantly altered when cultures were treated simultaneously with cortisol and antigen. The response was not suppressed if the addition of antigen preceded that of cortisol by more than 4 hr. Suppression was also considerably reduced if fetal calf serum was used when preparing cells for culture

  14. Inhibition of hepatocyte gap junctional intercellular communication by tumor promoters

    International Nuclear Information System (INIS)

    Ruch, R.J.

    1988-01-01

    The mechanisms by which tumor promoters enhance neoplasia are poorly understood. One effect common to most tumor promoters is their ability to inhibit the cell-to-cell exchange of small molecules and ions through gap junctions, i.e., gap junctional intercellular communication (IC). IC maybe necessary for normal growth control and the loss of IC may predispose cells to enhanced growth. In the present studies, the effects of liver tumor promoters and other agents on IC between rodent hepatocytes in primary culture has been studied. IC was detected between hepatocytes: (1) autoradiographically following the passage and incorporation of [5- 3 H]uridine nucleotides from pre-labeled donor hepatocytes to non-labeled, adjacent recipient hepatocytes and (2) by fluorescence microscopy after microinjection of fluorescent Lucifer Yellow CH dye into hepatocytes and visualizing dye spread into adjacent hepatocytes

  15. Effective PCR-based detection of Naegleria fowleri from cultured sample and PAM-developed mouse.

    Science.gov (United States)

    Kang, Heekyoung; Seong, Gi-Sang; Sohn, Hae-Jin; Kim, Jong-Hyun; Lee, Sang-Eun; Park, Mi Yeoun; Lee, Won-Ja; Shin, Ho-Joon

    2015-10-01

    Increasing numbers of Primary Amoebic Meningoencephalitis (PAM) cases due to Naegleria fowleri are becoming a serious issue in subtropical and tropical countries as a Neglected Tropical Disease (NTD). To establish a rapid and effective diagnostic tool, a PCR-based detection technique was developed based on previous PCR methods. Four kinds of primer pairs, Nfa1, Nae3, Nf-ITS, and Naegl, were employed in the cultured amoebic trophozoites and a mouse with PAM experimentally developed by N. fowleri inoculation (PAM-mouse). For the extraction of genomic DNA from N. fowleri trophozoites (1×10(6)), simple boiling with 10μl of PBS (pH 7.4) at 100°C for 30min was found to be the most rapid and efficient procedure, allowing amplification of 2.5×10(2) trophozoites using the Nfa-1 primer. The primers Nfa1 and Nae3 amplified only N. fowleri DNA, whereas the ITS primer detected N. fowleri and N. gruberi DNA. Using the PAM-mouse brain tissue, the Nfa1 primer was able to amplify the N. fowleri DNA 4 days post infection with 1ng/μl of genomic DNA being detectable. Using the PAM-mouse CSF, amplification of the N. fowleri DNA with the Nae3 primer was possible 5 days post infection showing a better performance than the Nfa1 primer at day 6. Copyright © 2015 Elsevier GmbH. All rights reserved.

  16. Differentiation of cartilaginous anlage in entire embryonic mouse limbs cultured in a rotating bioreactor.

    Science.gov (United States)

    Duke, P.; Oakley, C.; Montufar-Solis, D.

    The embryonic mammalian limb is sensitive both in vivo and in vitro to changes in gravitational force. Hypergravity of centrifugation and microgravity of space decreased size of elements due to precocious or delayed chondrogenesis respectively. In recapitulating spaceflight experiments, premetatarsals were cultured in suspension in a low stress, low sheer rotating bioreactor, and found to be shorter than those cultured in standard culture dishes, and cartilage development was delayed. This study only measured length of the metatarsals, and did not account for possible changes in width and/or in form of the skeletal elements. Shorter cartilage elements in limbbuds cultured in the bioreactor may be due to the ability of the system to reproduce a more in vivo 3D shape than traditional organ cultures. Tissues subjected to traditional organ cultures become flattened by their own weight, attachment to the filter, and restrictions imposed by nutrient diffusion. The purpose of the current experiment was to determine if entire limb buds could be successfully cultured in the bioreactor, and to compare the effects on 3D shape with that of culturing in a culture dish system. Fore and hind limbs from E11-E13 ICR mouse embryos were placed either in the bioreactor, in Trowell culture, or fixed as controls. Limbbuds were cultured for six days, fixed, and processed either as whole mounts or embedded for histology. Qualitative analysis revealed that the Trowell culture specimens were flattened, while bioreactor culture specimens had a more in vivo-like 3D limb shape. Sections of limbbuds from both types of cultures had excellent cartilage differentiation, with apparently more cell maturation, and hypertrophy in the specimens cultured in the bioreactor. Morphometric quantitation of the cartilaginous elements for comparisons of the two culture systems was complicated due to some limb buds fusing together during culture. This problem was especially noticeable in the younger limbs, and

  17. Differentiation of cartilaginous anlagen in entire embryonic mouse limbs cultured in a rotating bioreactor

    Science.gov (United States)

    Montufar-Solis, D.; Oakley, C. R.; Jefferson, Y.; Duke, P. J.

    2003-10-01

    Mechanisms involved in development of the embryonic limb have remained the same throughout eons of genetic and environmental evolution under Earth gravity (lg). During the spaceflight era it has been of interest to explore the ancient theory that form of the skeleton develops in response to gravity, and that changes in gravitational forces can change the developmental pattern of the limb. This has been shown in vivo and in vitro, allowing the hypergravity of centrifugation and microgravity of space to be used as tools to increase our knowledge of limb development. In recapitulations of spaceflight experiments, premetatarsals were cultured in suspension in a bioreactor, and found to be shorter and less differentiated than those cultured in standard culture dishes. This study only measured length of the metatarsals, and did not account for possible changes due to the skeletal elements having a more in vivo 3D shape while in suspension vs. flattened tissues compressed by their own weight. A culture system with an outcome closer to in vivo and that supports growth of younger limb buds than traditional systems will allow studies of early Hox gene expression, and contribute to the understanding of very early stages of development. The purpose of the current experiment was to determine if entire limb buds could be cultured in the bioreactor, and to compare the growth and differentiation with that of culturing in a culture dish system. Fore and hind limbs from E11-E13 ICR mouse embryos were cultured for six days, either in the bioreactor or in center-well organ culture dishes, fixed, and embedded for histology. E13 specimens grown in culture dishes were flat, while bioreactor culture specimens had a more in vivo-like 3D limb shape. Sections showed excellent cartilage differentiation in both culture systems, with more cell maturation, and hypertrophy in the specimens cultured in the bioreactor. Younger limb buds fused together during culture, so an additional set of El 1

  18. Establishment of c-myc-immortalized Kupffer cell line from a C57BL/6 mouse strain

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    Hiroshi Kitani

    2014-01-01

    Full Text Available We recently demonstrated in several mammalian species, a novel procedure to obtain liver-macrophages (Kupffer cells in sufficient numbers and purity using a mixed primary culture of hepatocytes. In this study, we applied this method to the C57BL/6 mouse liver and established an immortalized Kupffer cell line from this mouse strain. The hepatocytes from the C57BL/6 adult mouse liver were isolated by a two-step collagenase perfusion method and cultured in T25 culture flasks. Similar to our previous studies, the mouse hepatocytes progressively changed their morphology into a fibroblastic appearance after a few days of culture. After 7–10 days of culture, Kupffer-like cells, which were contaminants in the hepatocyte fraction at the start of the culture, actively proliferated on the mixed fibroblastic cell sheet. At this stage, a retroviral vector containing the human c-myc oncogene and neomycin resistance gene was introduced into the mixed culture. Gentle shaking of the culture flask, followed by the transfer and brief incubation of the culture supernatant, resulted in a quick and selective adhesion of Kupffer cells to a plastic dish surface. After selection with G418 and cloning by limiting dilutions, a clonal cell line (KUP5 was established. KUP5 cells displayed typical macrophage morphology and were stably passaged at 4–5 days intervals for more than 5 months, with a population doubling time of 19 h. KUP5 cells are immunocytochemically positive for mouse macrophage markers, such as Mac-1, F4/80. KUP5 cells exhibited substantial phagocytosis of polystyrene microbeads and the release of inflammatory cytokines upon lipopolysaccharide stimulation. Taken together, KUP5 cells provide a useful means to study the function of Kupffer cells in vitro.

  19. In vitro mouse spermatogenesis with an organ culture method in chemically defined medium.

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    Hiroyuki Sanjo

    Full Text Available We previously reported the successful induction and completion of mouse spermatogenesis by culturing neonatal testis tissues. The culture medium consisted of α-minimum essential medium (α-MEM, supplemented with Knockout serum replacement (KSR or AlbuMAX, neither of which were defined chemically. In this study, we formulated a chemically defined medium (CDM that can induce mouse spermatogenesis under organ culture conditions. It was found that bovine serum albumin (BSA purified through three different procedures had different effects on spermatogenesis. We also confirmed that retinoic acid (RA played crucial roles in the onset of spermatogonial differentiation and meiotic initiation. The added lipids exhibited weak promoting effects on spermatogenesis. Lastly, luteinizing hormone (LH, follicle stimulating hormone (FSH, triiodothyronine (T3, and testosterone (T combined together promoted spermatogenesis until round spermatid production. The CDM, however, was not able to produce elongated spermatids. It was also unable to induce spermatogenesis from the very early neonatal period, before 2 days postpartum, leaving certain factors necessary for spermatogenic induction in mice unidentified. Nonetheless, the present study provided important basic information on testis organ culture and spermatogenesis in vitro.

  20. Temporal kinetics and concentration-response relationships for induction of CYP1A, CYP2B, and CYP3A in primary cultures of beagle dog hepatocytes.

    Science.gov (United States)

    Graham, Richard A; Tyler, Lindsey O; Krol, Wojciech L; Silver, Ivin S; Webster, Lindsey O; Clark, Philip; Chen, Liangfu; Banks, Troy; LeCluyse, Edward L

    2006-01-01

    Compared to other species, little information is available on the xenobiotic-induced regulation of cytochrome P450 enzymes in the beagle dog. Dogs are widely used in the pharmaceutical industry for many study types, including those that will impact decisions on compound progression. The purpose of this study was (1) to determine the temporal kinetics of drug-induced changes in canine CYP1A, CYP2B, and CYP3A mRNA and enzymatic activity, and (2) to characterize concentration-response relationships for CYP1A2, CYP2B11, and CYP3A12 using primary cultures of canine hepatocytes treated with beta-naphthoflavone (BNF), phenobarbital (PB), and rifampin (RIF), respectively. CYP1A1 and CYP1A2 mRNA exhibited maximal expression (12,700-fold and 206-fold, respectively) after 36 h of treatment with BNF. PB treatment, but not RIF treatment, caused maximal induction of CYP2B11 mRNA (149-fold) after 48 h of treatment. CYP3A12 and CYP3A26 mRNA levels were increased maximally after 72 h of treatment with PB and RIF (CYP3A12, 35-fold and 18-fold, and CYP3A26, 72-fold and 22-fold with PB and RIF treatment, respectively). Concentration-response relationships for BNF induced 7-ethoxyresorufin O-dealkylation (EROD) (EC(50) = 7.8 +/- 4.2 microM), PB induced 7-benzyloxyresorufin O-dealkylation (BROD) (EC(50) = 123 +/- 30 microM), and PB and RIF induced testosterone 6beta-hydroxylation (EC(50) = 132 +/- 28 microM and 0.98 +/- 0.16 microM) resembled the relationship for human CYP induction compared to that of rodent. Interestingly, RIF had no effect on CYP2B11 expression, which represents a species difference overlooked in previous investigations. Overall, the induction of dog CYP1A, CYP2B, and CYP3A exhibits characteristics that are intermediate to those of rodent and human. (c) 2006 Wiley Periodicals, Inc.

  1. 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

  2. N-Acetyl-2-Aminofluorene (AAF) Processing in Adult Rat Hepatocytes in Primary Culture Occurs by High-Affinity Low-Velocity and Low-Affinity High-Velocity AAF Metabolite-Forming Systems.

    Science.gov (United States)

    Koch, Katherine S; Moran, Tom; Shier, W Thomas; Leffert, Hyam L

    2018-05-01

    N-acetyl-2-aminofluorene (AAF) is a procarcinogen used widely in physiological investigations of chemical hepatocarcinogenesis. Its metabolic pathways have been described extensively, yet little is known about its biochemical processing, growth cycle expression, and pharmacological properties inside living hepatocytes-the principal cellular targets of this hepatocarcinogen. In this report, primary monolayer adult rat hepatocyte cultures and high specific-activity [ring G-3 H]-N-acetyl-2-aminofluorene were used to extend previous observations of metabolic activation of AAF by highly differentiated, proliferation-competent hepatocytes in long-term cultures. AAF metabolism proceeded by zero-order kinetics. Hepatocytes processed significant amounts of procarcinogen (≈12 μg AAF/106 cells/day). Five ring-hydroxylated and one deacetylated species of AAF were secreted into the culture media. Extracellular metabolite levels varied during the growth cycle (days 0-13), but their rank quantitative order was time invariant: 5-OH-AAF > 7-OH-AAF > 3-OH-AAF > N-OH-AAF > aminofluorene (AF) > 1-OH-AAF. Lineweaver-Burk analyses revealed two principal classes of metabolism: System I (high-affinity and low-velocity), Km[APPARENT] = 1.64 × 10-7  M and VMAX[APPARENT] = 0.1 nmol/106 cells/day and System II (low-affinity and high-velocity), Km[APPARENT] = 3.25 × 10-5  M and VMAX[APPARENT] = 1000 nmol/106 cells/day. A third system of metabolism of AAF to AF, with Km[APPARENT] and VMAX[APPARENT] constants of 9.6 × 10-5  M and 4.7 nmol/106 cells/day, was also observed. Evidence provided in this report and its companion paper suggests selective roles and intracellular locations for System I- and System II-mediated AAF metabolite formation during hepatocarcinogenesis, although some of the molecules and mechanisms responsible for multi-system processing remain to be fully defined.

  3. Cultured embryonic non-innervated mouse muzzle is capable of generating a whisker pattern.

    Science.gov (United States)

    Andrés, F L; Van Der Loos, H

    1983-01-01

    The whisker pattern on the muzzle of the mouse is mapped in the contralateral parietal neocortex, each whisker follicle projecting to its own multineuronal unit ('barrel'). To determine the role, if any, of the peripheral innervation in the establishment of the vibrissal array, we cultured non-innervated prospective whiskerpads from 9- and 10-day-old embryos, mostly on chorioallantoic membrane. The results show that skin, alone, is capable of generating the whisker pattern, thus adducing a strong argument for the hypothesis that the central brain maps have their origin in the periphery. Copyright © 1983. Published by Elsevier Ltd.

  4. Lipofection of cultured mouse muscle cells: a direct comparison of Lipofectamine and DOSPER.

    Science.gov (United States)

    Dodds, E; Dunckley, M G; Naujoks, K; Michaelis, U; Dickson, G

    1998-04-01

    Cationic lipid-DNA complexes (lipoplexes) have been widely used as gene transfer vectors which avoid the adverse immunogenicity and potential for viraemia of viral vectors. With the long-term aim of gene transfer into skeletal muscle in vivo, we describe a direct in vitro comparison of two commercially available cationic lipid formulations, Lipofectamine and DOSPER. Optimisation of transfection was performed in the C2C12 mouse muscle cell line, before further studies in primary mouse myoblasts and C2C12 myotubes. Reporter gene constructs expressing either E. coli beta-galactosidase or green fluorescent protein (GFP) were used in order to evaluate transfection efficiency by histochemical staining or FACS analysis, respectively. Both lipid formulations were able to promote efficient, reproducible gene transfer in C2C12 cells, and to transfect primary mouse myoblast cultures successfully. However, DOSPER exhibited the important advantage of being able to transfect cells in the presence of serum of both bovine and murine origin. This feature allowed increased cell survival during in vitro transfections, and may be advantageous for direct in vivo gene transfer efficacy.

  5. Isolation of an 18,000-dalton hypusine-containing protein from cultured mouse neuroblastoma cells

    International Nuclear Information System (INIS)

    Dou, Q.P.; Chen, K.Y.

    1987-01-01

    An 18,000-dalton protein can be metabolically labeled by [ 3 H]putrescine or spermidine in mammalian cells. The labeling is due to a post-translational conversion of a lysine residue to hypusine residue. Previous studies indicated that the labeling is growth-dependent and is greatly diminished in mouse neuroblastoma cells after differentiation. To further study the physiological functions of this protein in the differentiation of mouse neuroblastoma cells, they have developed a simple procedure to purify this protein from cultured NB-15 mouse neuroblastoma cells. The 4-steps procedure included a Cibacron-Blue column, an omega-diaminooctyl-agarose column, a Sephadex G-50 column, and a Mono Q column. The procedure resulted in a 500-fold purification and the preparation appeared to be homogenous as judged by SDS-PAGE. Peptide map analysis using V-8 protease digestion method indicated that the 18,000-dalton hypusine-containing protein from NB-15 cells was identical to eukaryotic initiation factor 4D isolated from rabbit reticulocytes. This purification scheme also enabled them to detect a very faintly labeled protein in NB-15 cells. This weakly labeled protein had an apparent molecular weight of 22,000-dalton and pI of 5.0

  6. Establishment of an immortalized mouse dermal papilla cell strain with optimized culture strategy

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    Haiying Guo

    2018-01-01

    Full Text Available Dermal papilla (DP plays important roles in hair follicle regeneration. Long-term culture of mouse DP cells can provide enough cells for research and application of DP cells. We optimized the culture strategy for DP cells from three dimensions: stepwise dissection, collagen I coating, and optimized culture medium. Based on the optimized culture strategy, we immortalized primary DP cells with SV40 large T antigen, and established several immortalized DP cell strains. By comparing molecular expression and morphologic characteristics with primary DP cells, we found one cell strain named iDP6 was similar with primary DP cells. Further identifications illustrate that iDP6 expresses FGF7 and α-SMA, and has activity of alkaline phosphatase. During the process of characterization of immortalized DP cell strains, we also found that cells in DP were heterogeneous. We successfully optimized culture strategy for DP cells, and established an immortalized DP cell strain suitable for research and application of DP cells.

  7. A preliminary study for constructing a bioartificial liver device with induced pluripotent stem cell-derived hepatocytes

    Directory of Open Access Journals (Sweden)

    Iwamuro Masaya

    2012-12-01

    Full Text Available Abstract Background Bioartificial liver systems, designed to support patients with liver failure, are composed of bioreactors and functional hepatocytes. Immunological rejection of the embedded hepatocytes by the host immune system is a serious concern that crucially degrades the performance of the device. Induced pluripotent stem (iPS cells are considered a desirable source for bioartificial liver systems, because patient-derived iPS cells are free from immunological rejection. The purpose of this paper was to test the feasibility of a bioartificial liver system with iPS cell-derived hepatocyte-like cells. Methods Mouse iPS cells were differentiated into hepatocyte-like cells by a multi-step differentiation protocol via embryoid bodies and definitive endoderm. Differentiation of iPS cells was evaluated by morphology, PCR assay, and functional assays. iPS cell-derived hepatocyte-like cells were cultured in a bioreactor module with a pore size of 0.2 μm for 7 days. The amount of albumin secreted into the circulating medium was analyzed by ELISA. Additionally, after a 7-day culture in a bioreactor module, cells were observed by a scanning electron microscope. Results At the final stage of the differentiation program, iPS cells changed their morphology to a polygonal shape with two nucleoli and enriched cytoplasmic granules. Transmission electron microscope analysis revealed their polygonal shape, glycogen deposition in the cytoplasm, microvilli on their surfaces, and a duct-like arrangement. PCR analysis showed increased expression of albumin mRNA over the course of the differentiation program. Albumin and urea production was also observed. iPS-Heps culture in bioreactor modules showed the accumulation of albumin in the medium for up to 7 days. Scanning electron microscopy revealed the attachment of cell clusters to the hollow fibers of the module. These results indicated that iPS cells were differentiated into hepatocyte-like cells after culture

  8. 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.

  9. 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.

  10. Inhibition of chondroitin sulfate glycosaminoglycans incorporation affected odontoblast differentiation in cultured embryonic mouse molars.

    Science.gov (United States)

    Liu, Lipei; Chen, Weiting; Li, Lefeng; Xu, Fangfang; Jiang, Beizhan

    2017-12-01

    Chondroitin sulfate proteoglycan (CSPG) is an important component of extracellular matrix (ECM), it is composed of a core protein and one or more chondroitin sulfate glycosaminoglycan side chains (CS-GAGs). To investigate the roles of its CS-GAGs in dentinogenesis, the mouse mandibular first molar tooth germs at early bell stage were cultivated with or without β-xyloside. As expected, the CS-GAGs were inhibited on their incorporation to CSPGs by β-xyloside, accompanied by the change of morphology of the cultured tooth germs. The histological results and the transmission electron microscopy (TEM) investigation indicated that β-xyloside exhibited obvious inhibiting effects on odontoblasts differentiation compared with the control group. Meanwhile the results of immunohistochemistry, in situ hybridization and quantitative RT-PCR for type I collagen, dentin matrix acidic phosphoprotein 1 and dentin sialophosphoprotein, the products of differentiated odontoblasts, further proved that odontoblasts differentiation was inhibited. Collagen fibers detected in TEM decreased and arranged in disorder as well. Thus we conclude that the inhibition of CS-GAGs incorporation to CSPGs can affect odontoblast differentiation in cultured embryonic mouse molars.

  11. A refined characterisation of the NeoHepatocyte phenotype necessitates a reappraisal of the transdifferentiation hypothesis.

    Science.gov (United States)

    Riquelme, Paloma; Wundt, Judith; Hutchinson, James A; Brulport, Marc; Jun, Yu; Sotnikova, Anna; Girreser, Ulrich; Braun, Felix; Gövert, Felix; Soria, Bernat; Nüssler, Andreas; Clement, Bernd; Hengstler, Jan G; Fändrich, Fred

    2009-03-01

    Under certain culture conditions human peripheral blood monocytes may be induced to express phenotypic markers of non-haematopoietic lineages, including hepatocyte-defining traits. One such example, the NeoHepatocyte, was previously shown to express a broad panel of hepatocyte-like marker antigens and metabolic activities, both in vitro and following engraftment in the liver of immunodeficient mice. In this report, a refined description of NeoHepatocytes, with regard to their expression of xenobiotic-metabolising enzymes, morphology, hepatocyte marker expression and cell surface phenotype, is presented in comparison with human macrophages in defined states of activation. Contrary to prior assertions, it would seem more likely that NeoHepatocytes express particular hepatocyte-defining genes during a normal programme of macrophage differentiation rather than undergoing a process of transdifferentiation to become hepatocyte-like cells.

  12. Direct induction of hepatocyte-like cells from immortalized human bone marrow mesenchymal stem cells by overexpression of HNF4α

    International Nuclear Information System (INIS)

    Hu, Xiaojun; Xie, Peiyi; Li, Weiqiang; Li, Zhengran; Shan, Hong

    2016-01-01

    Hepatocytes from human bone marrow-derived mesenchymal stem cells (hBM-MSCs) are expected to be a useful source for cell transplantation. However, relatively low efficiency and repeatability of hepatic differentiation of human BM-MSCs remains an obstacle for clinical translation. Hepatocyte nuclear factor 4 alpha (HNF4α), a critical transcription factor, plays an essential role in the entire process of liver development. In this study, immortalized hBM-MSCs, UE7T-13 cells were transduced with a lentiviral vector containing HNF4α. The typical fibroblast-like morphology of the MSCs changed, and polygonal, epithelioid cells grew out after HNF4α transduction. In hepatocyte culture medium, HNF4α-transduced MSCs (E7-hHNF4α cells) strongly expressed the albumin (ALB), CYP2B6, alpha-1 antitrypsin (AAT), and FOXA2 mRNA and exhibited morphology markedly similar to that of mature hepatocytes. The E7-hHNF4α cells showed hepatic functions such as Indocyanine green (ICG) uptake and release, glycogen storage, urea production and ALB secretion. Approximately 28% of E7-hHNF4α cells expressed both ALB and AAT. Furthermore, these E7-hHNF4α cells via superior mesenteric vein (SMV) injection expressed human ALB in mouse chronic injured liver. In conclusion, this study represents a novel strategy by directly inducing hepatocyte-like cells from MSCs. - Highlights: • We overexpressed HNF4α in immortalized BM-MSCs by lentiviral transduction. • HNF4α-transduced MSCs transdifferentiated into hepatocytes with mature hepatic metabolic functions. • Our study represents a novel strategy by direct induction of hepatocyte-like cells from MSCs.

  13. Hepatocyte polyploidization and its association with pathophysiological processes.

    Science.gov (United States)

    Wang, Min-Jun; Chen, Fei; Lau, Joseph T Y; Hu, Yi-Ping

    2017-05-18

    A characteristic cellular feature of the mammalian liver is the progressive polyploidization of the hepatocytes, where individual cells acquire more than two sets of chromosomes. Polyploidization results from cytokinesis failure that takes place progressively during the course of postnatal development. The proportion of polyploidy also increases with the aging process or with cellular stress such as surgical resection, toxic stimulation, metabolic overload, or oxidative damage, to involve as much as 90% of the hepatocytes in mice and 40% in humans. Hepatocyte polyploidization is generally considered an indicator of terminal differentiation and cellular senescence, and related to the dysfunction of insulin and p53/p21 signaling pathways. Interestingly, the high prevalence of hepatocyte polyploidization in the aged mouse liver can be reversed when the senescent hepatocytes are serially transplanted into young mouse livers. Here we review the current knowledge on the mechanism of hepatocytes polyploidization during postnatal growth, aging, and liver diseases. The biologic significance of polyploidization in senescent reversal, within the context of new ways to think of liver aging and liver diseases is considered.

  14. SphK1 mediates hepatic inflammation in a mouse model of NASH induced by high saturated fat feeding and initiates proinflammatory signaling in hepatocytes.

    Science.gov (United States)

    Geng, Tuoyu; Sutter, Alton; Harland, Michael D; Law, Brittany A; Ross, Jessica S; Lewin, David; Palanisamy, Arun; Russo, Sarah B; Chavin, Kenneth D; Cowart, L Ashley

    2015-12-01

    Steatohepatitis occurs in up to 20% of patients with fatty liver disease and leads to its primary disease outcomes, including fibrosis, cirrhosis, and increased risk of hepatocellular carcinoma. Mechanisms that mediate this inflammation are of major interest. We previously showed that overload of saturated fatty acids, such as that which occurs with metabolic syndrome, induced sphingosine kinase 1 (SphK1), an enzyme that generates sphingosine-1-phosphate (S1P). While data suggest beneficial roles for S1P in some contexts, we hypothesized that it may promote hepatic inflammation in the context of obesity. Consistent with this, we observed 2-fold elevation of this enzyme in livers from humans with nonalcoholic fatty liver disease and also in mice with high saturated fat feeding, which recapitulated the human disease. Mice exhibited activation of NFκB, elevated cytokine production, and immune cell infiltration. Importantly, SphK1-null mice were protected from these outcomes. Studies in cultured cells demonstrated saturated fatty acid induction of SphK1 message, protein, and activity, and also a requirement of the enzyme for NFκB signaling and increased mRNA encoding TNFα and MCP1. Moreover, saturated fat-induced NFκB signaling and elevation of TNFα and MCP1 mRNA in HepG2 cells was blocked by targeted knockdown of S1P receptor 1, supporting a role for this lipid signaling pathway in inflammation in nonalcoholic fatty liver disease.

  15. mRNA transfection of mouse and human neural stem cell cultures.

    Directory of Open Access Journals (Sweden)

    Samuel McLenachan

    Full Text Available The use of synthetic mRNA as an alternative gene delivery vector to traditional DNA-based constructs provides an effective method for inducing transient gene expression in cell cultures without genetic modification. Delivery of mRNA has been proposed as a safer alternative to viral vectors in the induction of pluripotent cells for regenerative therapies. Although mRNA transfection of fibroblasts, dendritic and embryonic stem cells has been described, mRNA delivery to neurosphere cultures has not been previously reported. Here we sought to establish an efficient method for delivering mRNA to primary neurosphere cultures. Neurospheres derived from the subventricular zone of adult mice or from human embryonic stem cells were transfected with EGFP mRNA by lipofection and electroporation. Transfection efficiency and expression levels were monitored by flow cytometry. Cell survival following transfection was examined using live cell counting and the MTT assay. Both lipofection and electroporation provided high efficiency transfection of neurospheres. In comparison with lipofection, electroporation resulted in increased transfection efficiencies, but lower expression per cell and shorter durations of expression. Additional rounds of lipofection renewed EGFP expression in neurospheres, suggesting this method may be suitable for reprogramming applications. In summary, we have developed a protocol for achieving high efficiency transfection rates in mouse and human neurosphere cell culture that can be applied for future studies of gene function studies in neural stem cells, such as defining efficient differentiation protocols for glial and neuronal linages.

  16. mRNA Transfection of Mouse and Human Neural Stem Cell Cultures

    Science.gov (United States)

    McLenachan, Samuel; Zhang, Dan; Palomo, Ana Belén Alvarez; Edel, Michael J.; Chen, Fred K.

    2013-01-01

    The use of synthetic mRNA as an alternative gene delivery vector to traditional DNA-based constructs provides an effective method for inducing transient gene expression in cell cultures without genetic modification. Delivery of mRNA has been proposed as a safer alternative to viral vectors in the induction of pluripotent cells for regenerative therapies. Although mRNA transfection of fibroblasts, dendritic and embryonic stem cells has been described, mRNA delivery to neurosphere cultures has not been previously reported. Here we sought to establish an efficient method for delivering mRNA to primary neurosphere cultures. Neurospheres derived from the subventricular zone of adult mice or from human embryonic stem cells were transfected with EGFP mRNA by lipofection and electroporation. Transfection efficiency and expression levels were monitored by flow cytometry. Cell survival following transfection was examined using live cell counting and the MTT assay. Both lipofection and electroporation provided high efficiency transfection of neurospheres. In comparison with lipofection, electroporation resulted in increased transfection efficiencies, but lower expression per cell and shorter durations of expression. Additional rounds of lipofection renewed EGFP expression in neurospheres, suggesting this method may be suitable for reprogramming applications. In summary, we have developed a protocol for achieving high efficiency transfection rates in mouse and human neurosphere cell culture that can be applied for future studies of gene function studies in neural stem cells, such as defining efficient differentiation protocols for glial and neuronal linages. PMID:24386231

  17. mRNA transfection of mouse and human neural stem cell cultures.

    Science.gov (United States)

    McLenachan, Samuel; Zhang, Dan; Palomo, Ana Belén Alvarez; Edel, Michael J; Chen, Fred K

    2013-01-01

    The use of synthetic mRNA as an alternative gene delivery vector to traditional DNA-based constructs provides an effective method for inducing transient gene expression in cell cultures without genetic modification. Delivery of mRNA has been proposed as a safer alternative to viral vectors in the induction of pluripotent cells for regenerative therapies. Although mRNA transfection of fibroblasts, dendritic and embryonic stem cells has been described, mRNA delivery to neurosphere cultures has not been previously reported. Here we sought to establish an efficient method for delivering mRNA to primary neurosphere cultures. Neurospheres derived from the subventricular zone of adult mice or from human embryonic stem cells were transfected with EGFP mRNA by lipofection and electroporation. Transfection efficiency and expression levels were monitored by flow cytometry. Cell survival following transfection was examined using live cell counting and the MTT assay. Both lipofection and electroporation provided high efficiency transfection of neurospheres. In comparison with lipofection, electroporation resulted in increased transfection efficiencies, but lower expression per cell and shorter durations of expression. Additional rounds of lipofection renewed EGFP expression in neurospheres, suggesting this method may be suitable for reprogramming applications. In summary, we have developed a protocol for achieving high efficiency transfection rates in mouse and human neurosphere cell culture that can be applied for future studies of gene function studies in neural stem cells, such as defining efficient differentiation protocols for glial and neuronal linages.

  18. Induction of different activated phenotypes of mouse peritoneal macrophages grown in different tissue culture media.

    Science.gov (United States)

    Kawakami, Tomoya; Koike, Atsushi; Amano, Fumio

    2017-08-01

    The role of activated macrophages in the host defense against pathogens or tumor cells has been investigated extensively. Many researchers have been using various culture media in in vitro experiments using macrophages. We previously reported that J774.1/JA-4 macrophage-like cells showed great differences in their activated macrophage phenotypes, such as production of reactive oxygen, nitric oxide (NO) or cytokines depending on the culture medium used, either F-12 (Ham's F-12 nutrient mixture) or Dulbecco modified Eagle's medium (DMEM). To examine whether a difference in the culture medium would influence the functions of primary macrophages, we used BALB/c mouse peritoneal macrophages in this study. Among the activated macrophage phenotypes, the expression of inducible NO synthase in LPS- and/or IFN-γ-treated peritoneal macrophages showed the most remarkable differences between F-12 and DMEM; i.e., NO production by LPS- and/or IFN-γ-treated cells was far lower in DMEM than in F-12. Similar results were obtained with C57BL mouse peritoneal macrophages. Besides, dilution of F-12 medium with saline resulted in a slight decrease in NO production, whereas that of DMEM with saline resulted in a significant increase, suggesting the possibility that DMEM contained some inhibitory factor(s) for NO production. However, such a difference in NO production was not observed when macrophage-like cell lines were examined. These results suggest that phenotypes of primary macrophages could be changed significantly with respect to host inflammatory responses by the surrounding environment including nutritional factors and that these altered macrophage phenotypes might influence the biological host defense.

  19. Shenfu injection attenuates neurotoxicity of bupivacaine in cultured mouse spinal cord neurons

    Institute of Scientific and Technical Information of China (English)

    XIONG Li-ze; WANG Qiang; LIU Mu-yun; PENG Ye; LI Qing-bo; LU Zhi-hong; LEI Chong

    2007-01-01

    Background Our previous in vivo study in the rat demonstrates that Shenfu injection, a clinically used extract preparation from Chinese herbs, attenuates neural and cardiac toxicity induced by intravenous infusion of bupivacaine, a local anesthetic. This study was designed to investigate whether bupivacaine could induce a toxic effect in primary cultured mouse spinal cord neuron and if so, whether the Shenfu injection had a similar neuroprotective effect in the cell model. Methods The spinal cords from 11- to 14-day-old fetal mice were minced and incubated. Cytarabine was added into the medium to inhibit the proliferation of non-neuronal cells. The immunocytochemical staining of β-tubulin was used to determine the identity of cultured cells. The cultured neurons were randomly assigned into three sets treated with various doses of bupivacaine, Shenfu and bupivacaine+Shenfu, for 48 hours respectively. Cell viability in each group was analyzed by methyl thiazoleterazolium (MTT) assay. Results The viability of the cultured neurons treated with bupivacaine at concentrations of 0.01%, 0.02%, 0.04% and 0.08% was decreased in a dose-dependent manner. Although the Shenfu injection at concentrations ranging from 1/50 to 1/12.5 (V/V) had no significant influence on the viability of cultured neurons (P<0.05 vs control), the injection significantly increased the cellular viability of cultured neurons pretreated with 0.03% bupivacaine (P<0.05). Conclusion Although Shenfu injection itself has no effect on spinal neurons, it was able to reduce the bupivacaine induced neurotoxicity in vitro.

  20. The potential of induced pluripotent stem cell derived hepatocytes.

    Science.gov (United States)

    Hannoun, Zara; Steichen, Clara; Dianat, Noushin; Weber, Anne; Dubart-Kupperschmitt, Anne

    2016-07-01

    Orthotopic liver transplantation remains the only curative treatment for liver disease. However, the number of patients who die while on the waiting list (15%) has increased in recent years as a result of severe organ shortages; furthermore the incidence of liver disease is increasing worldwide. Clinical trials involving hepatocyte transplantation have provided encouraging results. However, transplanted cell function appears to often decline after several months, necessitating liver transplantation. The precise aetiology of the loss of cell function is not clear, but poor engraftment and immune-mediated loss appear to be important factors. Also, primary human hepatocytes (PHH) are not readily available, de-differentiate, and die rapidly in culture. Hepatocytes are available from other sources, such as tumour-derived human hepatocyte cell lines and immortalised human hepatocyte cell lines or porcine hepatocytes. However, all these cells suffer from various limitations such as reduced or differences in functions or risk of zoonotic infections. Due to their significant potential, one possible inexhaustible source of hepatocytes is through the directed differentiation of human induced pluripotent stem cells (hiPSCs). This review will discuss the potential applications and existing limitations of hiPSC-derived hepatocytes in regenerative medicine, drug screening, in vitro disease modelling and bioartificial livers. Copyright © 2016 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

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

    2014-01-01

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

  2. Reconstruction of mouse testicular cellular microenvironments in long-term seminiferous tubule culture.

    Directory of Open Access Journals (Sweden)

    Juho-Antti Mäkelä

    Full Text Available Research on spermatogonia is hampered by complex architecture of the seminiferous tubule, poor viability of testicular tissue ex vivo and lack of physiologically relevant long-term culture systems. Therefore there is a need for an in vitro model that would enable long term survival and propagation of spermatogonia. We aimed at the most simplified approach to enable all different cell types within the seminiferous tubules to contribute to the creation of a niche for spermatogonia. In the present study we describe the establishment of a co-culture of mouse testicular cells that is based on proliferative and migratory activity of seminiferous tubule cells and does not involve separation, purification or differential plating of individual cell populations. The co-culture is composed of the constituents of testicular stem cell niche: Sertoli cells [identified by expression of Wilm's tumour antigen 1 (WT1 and secretion of glial cell line-derived neurotrophic factor, GDNF], peritubular myoid cells (expressing alpha smooth muscle actin, αSMA and spermatogonia [expressing MAGE-B4, PLZF (promyelocytic leukaemia zinc finger, LIN28, Gpr125 (G protein-coupled receptor 125, CD9, c-Kit and Nanog], and can be maintained for at least five weeks. GDNF was found in the medium at a sufficient concentration to support proliferating spermatogonial stem cells (SSCs that were able to start spermatogenic differentiation after transplantation to an experimentally sterile recipient testis. Gdnf mRNA levels were elevated by follicle-stimulating hormone (FSH which shows that the Sertoli cells in the co-culture respond to physiological stimuli. After approximately 2-4 weeks of culture a spontaneous formation of cord-like structures was monitored. These structures can be more than 10 mm in length and branch. They are formed by peritubular myoid cells, Sertoli cells, fibroblasts and spermatogonia as assessed by gene expression profiling. In conclusion, we have managed to

  3. Susceptibility of mouse minute virus to inactivation by heat in two cell culture media types.

    Science.gov (United States)

    Schleh, Marc; Romanowski, Peter; Bhebe, Prince; Zhang, Li; Chinniah, Shivanthi; Lawrence, Bill; Bashiri, Houman; Gaduh, Asri; Rajurs, Viveka; Rasmussen, Brian; Chuck, Alice; Dehghani, Houman

    2009-01-01

    Viral contaminations of biopharmaceutical manufacturing cell culture facilities are a significant threat and one for which having a risk mitigation strategy is highly desirable. High temperature, short time (HTST) mammalian cell media treatment may potentially safeguard manufacturing facilities from such contaminations. HTST is thought to inactivate virions by denaturing proteins of the viral capsid, and there is evidence that HTST provides ample virucidal efficacy against nonenveloped or naked viruses such as mouse minute virus (MMV), a parvovirus. The aim of the studies presented herein was to further delineate the susceptibility of MMV, known to have contaminated mammalian cell manufacturing facilities, to heat by exposing virus-spiked cell culture media to a broad range of temperatures and for various times of exposure. The results of these studies show that HTST is capable of inactivating MMV by three orders of magnitude or more. Thus, we believe that HTST is a useful technology for the purposes of providing a barrier to adventitious contamination of mammalian cell culture processes in the biopharmaceutical industry. 2009 American Institute of Chemical Engineers

  4. Flow cytometric measurement of the metabolism of benzo [a] pyrene by mouse liver cells in culture

    International Nuclear Information System (INIS)

    Bartholomew, J.C.; Wade, C.G.; Dougherty, K.

    1984-01-01

    The metabolism of benzo[a]pyrene in individual cells was monitored by flow cytometry. The measurements are based on the alterations that occur in the fluorescence emission spectrum of benzo[a]pyrene when it is converted to various metabolities. Using present instrumentation the technique could easily detect 1 x 10/sup 6/ molecules per cells of benzo [a]pyrene and 1 x 10/sup 7/ molecules per cell of the diol epoxide. The analysis of C3H IOT 1/2 mouse fibroblasts growing in culture indicated that there was heterogeneity in the conversion of the parent compound into diol epoxide derivative suggesting that some variation in sensitivity to transformation by benzo[a]pyrene may be due to differences in cellular metabolism

  5. Radiation effects on cultured mouse embryos in relation to cell division cycle

    International Nuclear Information System (INIS)

    Domon, M.

    1982-01-01

    The authors have worked with mouse embryos in vitro asking first, what are the suitable parameters to define the radiation sensitivity of embryos, and second what is a major factor determining it. The LD 50 was adopted as a parameter of the radiation sensitivity of a population in a mouse embryo system in culture. The fertilized ova were collected into Whitten's medium at various times during the pronuclear and 2-cell stages of development. They were irradiated in chambers with X-rays at doses of 0 to 800 rads. After the embryos were cultured, a set of the lethal fractions for various X-ray doses were obtained. Regarding the radiation sensitivity variation of the embryos, the LD 50 varied from 100 to 200 rads during the pronuclear stage and from 100 to 600 rads during the 2-cell stage. The embryos during the pronuclear stage were most radioresistant at early G 2 phase, followed by an increase in the sensitivity. The embryos during the 2-cell stage were also most radioresistant at early G 2 phase and were more sensitive when they got close to either the first or the second cleavage division. Furthermore, it seems that the factor 6 of the large variation was due to the extremely long G 2 period, 14 hrs for the 2-cell embryos. That is, the pooled 2-cell embryos were in a relative sense well synchronized with G 2 phase. In contrast, the synchrony was poor during the pronuclear stage, which led to less variation of the LD 50 for the pronuclear embryos. It is concluded that during the early cleavage stages of mice, radiosensitivity is mainly governed by the content of cells of various cell cycle ages in the embryo. (Namekawa, K.)

  6. Immortalized human hepatocytes as a tool for the study of hepatocytic (de-)differentiation

    NARCIS (Netherlands)

    Schippers, IJ; Moshage, H; Roelofsen, H; Muller, M; Heymans, HSA; Ruiters, M; Kuipers, F

    Primary human hepatocytes were immortalized by stable transfection with a recombinant plasmid containing the early region of simian virus (SV) 40. The cells were cultured in serum-free, hormonally defined medium during the immortalization procedure. Foci of dividing cells were seen after 3 months.

  7. The survival of cultured mouse cerebellar granule cells is not dependent on elevated potassium-ion concentration

    DEFF Research Database (Denmark)

    Mogensen, Helle Smidt; Hack, N; Balázs, R

    1994-01-01

    The effects of K(+)-induced membrane depolarization were studied on the survival and biochemical parameters in mouse and rat cerebellar granule cells grown in micro-well cultures. Cell numbers were determined by estimating DNA content using the Hoechst 33258 fluorochrome binding assay. DNA from d...

  8. Minute changes to the culture environment of mouse pre-implantation embryos affect the health of the conceptus

    Directory of Open Access Journals (Sweden)

    George Koustas

    2016-07-01

    Conclusions: Exposing mouse pre-implantation embryos to ambient air at 37.0 °C, even for brief periods for routine micromanipulations is detrimental to normal embryonic development. Our results highlight the importance of how small alterations in the culture environment can have major consequences for the health of the embryo.

  9. EXPOSURE TO A P13KINASE INHIBITOR PRODUCED DYSMORPHOGENESIS IN NEURULATION-STAGED MOUSE EMBRYOS IN CULTURE

    Science.gov (United States)

    The haloacetic acids (HAA) are a family of chemicals that are drinking water disinfection byproducts. We previously reported that bromo- and chloro-acetic acids alter embryonic development when mouse conceptuses are directly exposed to these xenobiotics in whole embryo culture. C...

  10. A fast and simple dot-immunobinding assay for quantifiction of mouse immunoglobulins in hybridoma culture supernatants

    Czech Academy of Sciences Publication Activity Database

    Sulimenko, Tetyana; Dráber, Pavel

    2004-01-01

    Roč. 289, - (2004), s. 89-95 ISSN 0022-1759 R&D Projects: GA AV ČR IBS5052301; GA MŠk LN00A026; GA MŠk 1P04OE158 Keywords : dot-immunobinding assay * hybridoma culture superntatants * mouse immunoglobulins Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.464, year: 2004

  11. In vitro culture of pre-implanted mouse embryos. A model system for studying combined effects

    International Nuclear Information System (INIS)

    Streffer, C.; Beuningen, D. van; Molls, M.; Pon, A.; Schulz, S.; Zamboglou, N.

    1978-01-01

    Studies on combined effects, e.g. interaction between chemical toxicants and ionizing radiation, are difficult to perform, as they are dependent on many factors (substance concentration, radiation dose, sequence of treatments, etc.). In order to obtain data from such studies it is necessary to establish a comparatively simple experimental model system. We have established such a model system by studying combined effects on pre-implanted mouse embryos cultured in vitro. This system has the following advantages: (1) The embryos can be cultivated for several days in vitro; (2) Their physiological intactness can be tested; and (3) Cell proliferation, cell killing and chromosomal damage can be investigated comparatively easily. The embryos are isolated at the 2-cell stage and incubated in a culture medium in vitro. The development of the embryos is followed under the microscope until the development of blastocysts or the hatching of blastocysts is observed. These blastocysts can be transplanted to fostered mice and the development of normal animals determined. The proliferation kinetics can be studied easily, and the methods are described. A method has also been developed to measure the DNA content of individual cells by microscope fluorometry. After treatment of the embryos with ionizing radiation or drugs the release of micronuclei has been observed from the cell nuclei, which is an expression for chromosomal damage. Substances or radionuclides can be added to the culture medium or external irradiation can be performed during the culture period. Also the combined effects of radiation and heating can be studied. The effects of X-rays and tritiated compounds have also been investigated. The combined effects of radiation with antibiotics such as actinomycin D, and environmental toxicants such as lead, have been determined. The system described has been useful to evaluate cytological, teratogenic and cytogenetic effects

  12. Estrogenic and anti-estrogenic influences in cultured brown trout hepatocytes: Focus on the expression of some estrogen and peroxisomal related genes and linked phenotypic anchors.

    Science.gov (United States)

    Madureira, Tânia Vieira; Malhão, Fernanda; Pinheiro, Ivone; Lopes, Célia; Ferreira, Nádia; Urbatzka, Ralph; Castro, L Filipe C; Rocha, Eduardo

    2015-12-01

    Estrogens, estrogenic mimics and anti-estrogenic compounds are known to target estrogen receptors (ER) that can modulate other nuclear receptor signaling pathways, such as those controlled by the peroxisome proliferator-activated receptor (PPAR), and alter organelle (inc. peroxisome) morphodynamics. By using primary isolated brown trout (Salmo trutta f. fario) hepatocytes after 72 and 96h of exposure we evaluated some effects in selected molecular targets and in peroxisomal morphological features caused by: (1) an ER agonist (ethinylestradiol-EE2) at 1, 10 and 50μM; (2) an ER antagonist (ICI 182,780) at 10 and 50μM; and (3) mixtures of both (Mix I-10μM EE2 and 50μM ICI; Mix II-1μM EE2 and 10μM ICI and Mix III-1μM EE2 and 50μM ICI). The mRNA levels of the estrogenic targets (ERα, ERβ-1 and vitellogenin A-VtgA) and the peroxisome structure/function related genes (catalase, urate oxidase-Uox, 17β-hydroxysteroid dehydrogenase 4-17β-HSD4, peroxin 11α-Pex11α and PPARα) were analyzed by real-time polymerase chain reaction (RT-PCR). Stereology combined with catalase immunofluorescence revealed a significant reduction in peroxisome volume densities at 50μM of EE2 exposure. Concomitantly, at the same concentration, electron microscopy showed smaller peroxisome profiles, exacerbated proliferation of rough endoplasmic reticulum, and a generalized cytoplasmic vacuolization of hepatocytes. Catalase and Uox mRNA levels decreased in all estrogenic stimuli conditions. VtgA and ERα mRNA increased after all EE2 treatments, while ERβ-1 had an inverse pattern. The EE2 action was reversed by ICI 182,780 in a concentration-dependent manner, for VtgA, ERα and Uox. Overall, our data show the great value of primary brown trout hepatocytes to study the effects of estrogenic/anti-estrogenic inputs in peroxisome kinetics and in ER and PPARα signaling, backing the still open hypothesis of crosstalk interactions between these pathways and calling for more mechanistic

  13. Antifatigue Activity of Liquid Cultured Tricholoma matsutake Mycelium Partially via Regulation of Antioxidant Pathway in Mouse

    Directory of Open Access Journals (Sweden)

    Quan Li

    2015-01-01

    Full Text Available Tricholoma matsutake has been popular as food and biopharmaceutical materials in Asian countries for its various pharmacological activities. The present study aims to analyze the antifatigue effects on enhancing exercise performance of Tricholoma matsutake fruit body (ABM and liquid cultured mycelia (TM in mouse model. Two-week Tricholoma matsutake treatment significantly enhances the exercise performance in weight-loaded swimming, rotating rod, and forced running test. In TM- and ABM-treated mice, some factors were observed at 60 min after swimming compared with nontreated mice, such as the increased levels of adenosine triphosphate (ATP, antioxidative enzymes, and glycogen and the reduced levels of malondialdehyde and reactive oxygen species in muscle, liver, and/or serum. Further data obtained from western blot show that CM and ABM have strongly enhanced the activation of 5′-AMP-activated protein kinase (AMPK, and the expressions of peroxisome proliferator have activated receptor γ coactivator-1α (PGC-1α and phosphofructokinase-1 (PFK-1 in liver. Our data suggest that both Tricholoma matsutake fruit body and liquid cultured mycelia possess antifatigue effects related to AMPK-linked antioxidative pathway. The information uncovered in our study may serve as a valuable resource for further identification and provide experimental evidence for clinical trials of Tricholoma matsutake as an effective agent against fatigue related diseases.

  14. Differential radiosensitivity of mouse embryonic neurons and glia in cell culture

    International Nuclear Information System (INIS)

    Dambergs, R.; Kidson, C.

    1977-01-01

    The responses of neurons and glial cells to ultraviolet and γ-radiation were studied in cell cultures of embryonic mouse brains. A decrease in the ratio of glia to neurons occurred after both forms of irradiation. [ 3 H]thymidine labelling followed by autoradiography revealed that all glia were capable of replication, whereas 70 percent of neurons were non-replicating under the conditions of the study. Ultraviolet radiation caused a decrease in the proportion of replicating neurons but did not affect the proportion of replicating glia, whereas γ-radiation caused a decrease in DNA replication in both cell types. Levels of ultraviolet radiation-induced unscheduled DNA synthesis were lower in neurons than in glia. It is concluded that sensitivity to both ionizing and ultraviolet radiation of neurons and glial cells in embryonic brain cultures is determined primarily by the capacity for and state of DNA replication. Neurons which have already reached the stage of terminal differentiation are more resistant than replicating neurons of glial cells

  15. Neuroprotective effect of the endogenous neural peptide apelin in cultured mouse cortical neurons

    International Nuclear Information System (INIS)

    Zeng, Xiang Jun; Yu, Shan Ping; Zhang, Like; Wei, Ling

    2010-01-01

    The adipocytokine apelin and its G protein-coupled APJ receptor were initially isolated from a bovine stomach and have been detected in the brain and cardiovascular system. Recent studies suggest that apelin can protect cardiomyocytes from ischemic injury. Here, we investigated the effect of apelin on apoptosis in mouse primary cultures of cortical neurons. Exposure of the cortical cultures to a serum-free medium for 24 h induced nuclear fragmentation and apoptotic death; apelin-13 (1.0-5.0 nM) markedly prevented the neuronal apoptosis. Apelin neuroprotective effects were mediated by multiple mechanisms. Apelin-13 reduced serum deprivation (SD)-induced ROS generation, mitochondria depolarization, cytochrome c release and activation of caspase-3. Apelin-13 prevented SD-induced changes in phosphorylation status of Akt and ERK1/2. In addition, apelin-13 attenuated NMDA-induced intracellular Ca 2+ accumulation. These results indicate that apelin is an endogenous neuroprotective adipocytokine that may block apoptosis and excitotoxic death via cellular and molecular mechanisms. It is suggested that apelins may be further explored as a potential neuroprotective reagent for ischemia-induced brain damage.

  16. Estrogenic and anti-estrogenic influences in cultured brown trout hepatocytes: Focus on the expression of some estrogen and peroxisomal related genes and linked phenotypic anchors

    International Nuclear Information System (INIS)

    Madureira, Tânia Vieira; Malhão, Fernanda; Pinheiro, Ivone; Lopes, Célia; Ferreira, Nádia; Urbatzka, Ralph; Castro, L. Filipe C.; Rocha, Eduardo

    2015-01-01

    Highlights: • Evidence of crosstalk between estrogens and peroxisomal pathways in brown trout. • VtgA and ERα mRNA levels increased after 1, 10 and 50 μM of ethinylestradiol (EE2). • ERβ-1, catalase and urate oxidase mRNA levels decreased after estrogenic stimuli. • Estrogenic effects in VtgA, ERα and Uox mRNA levels were reverted by ICI 182,780. • Immunofluorescence/electron microscopy shows smaller peroxisomes after 50 μM of EE2. - Abstract: Estrogens, estrogenic mimics and anti-estrogenic compounds are known to target estrogen receptors (ER) that can modulate other nuclear receptor signaling pathways, such as those controlled by the peroxisome proliferator-activated receptor (PPAR), and alter organelle (inc. peroxisome) morphodynamics. By using primary isolated brown trout (Salmo trutta f. fario) hepatocytes after 72 and 96 h of exposure we evaluated some effects in selected molecular targets and in peroxisomal morphological features caused by: (1) an ER agonist (ethinylestradiol—EE2) at 1, 10 and 50 μM; (2) an ER antagonist (ICI 182,780) at 10 and 50 μM; and (3) mixtures of both (Mix I—10 μM EE2 and 50 μM ICI; Mix II—1 μM EE2 and 10 μM ICI and Mix III—1 μM EE2 and 50 μM ICI). The mRNA levels of the estrogenic targets (ERα, ERβ-1 and vitellogenin A—VtgA) and the peroxisome structure/function related genes (catalase, urate oxidase—Uox, 17β-hydroxysteroid dehydrogenase 4—17β-HSD4, peroxin 11α—Pex11α and PPARα) were analyzed by real-time polymerase chain reaction (RT-PCR). Stereology combined with catalase immunofluorescence revealed a significant reduction in peroxisome volume densities at 50 μM of EE2 exposure. Concomitantly, at the same concentration, electron microscopy showed smaller peroxisome profiles, exacerbated proliferation of rough endoplasmic reticulum, and a generalized cytoplasmic vacuolization of hepatocytes. Catalase and Uox mRNA levels decreased in all estrogenic stimuli conditions. VtgA and ERα m

  17. Estrogenic and anti-estrogenic influences in cultured brown trout hepatocytes: Focus on the expression of some estrogen and peroxisomal related genes and linked phenotypic anchors

    Energy Technology Data Exchange (ETDEWEB)

    Madureira, Tânia Vieira, E-mail: tvmadureira@icbas.up.pt [Interdisciplinary Centre of Marine and Environmental Research (CIIMAR/CIMAR), U.Porto—University of Porto, Rua dos Bragas 289, P 4050-123 Porto (Portugal); Institute of Biomedical Sciences Abel Salazar, U.Porto (ICBAS)—University of Porto, Laboratory of Histology and Embryology, Department of Microscopy, Rua Jorge Viterbo Ferreira 228, P 4050-313 Porto (Portugal); Malhão, Fernanda; Pinheiro, Ivone; Lopes, Célia; Ferreira, Nádia [Interdisciplinary Centre of Marine and Environmental Research (CIIMAR/CIMAR), U.Porto—University of Porto, Rua dos Bragas 289, P 4050-123 Porto (Portugal); Institute of Biomedical Sciences Abel Salazar, U.Porto (ICBAS)—University of Porto, Laboratory of Histology and Embryology, Department of Microscopy, Rua Jorge Viterbo Ferreira 228, P 4050-313 Porto (Portugal); Urbatzka, Ralph [Interdisciplinary Centre of Marine and Environmental Research (CIIMAR/CIMAR), U.Porto—University of Porto, Rua dos Bragas 289, P 4050-123 Porto (Portugal); Castro, L. Filipe C. [Interdisciplinary Centre of Marine and Environmental Research (CIIMAR/CIMAR), U.Porto—University of Porto, Rua dos Bragas 289, P 4050-123 Porto (Portugal); Faculty of Sciences (FCUP), U.Porto—University of Porto, Department of Biology, Rua do Campo Alegre, P 4169-007 Porto (Portugal); Rocha, Eduardo [Interdisciplinary Centre of Marine and Environmental Research (CIIMAR/CIMAR), U.Porto—University of Porto, Rua dos Bragas 289, P 4050-123 Porto (Portugal); Institute of Biomedical Sciences Abel Salazar, U.Porto (ICBAS)—University of Porto, Laboratory of Histology and Embryology, Department of Microscopy, Rua Jorge Viterbo Ferreira 228, P 4050-313 Porto (Portugal)

    2015-12-15

    Highlights: • Evidence of crosstalk between estrogens and peroxisomal pathways in brown trout. • VtgA and ERα mRNA levels increased after 1, 10 and 50 μM of ethinylestradiol (EE2). • ERβ-1, catalase and urate oxidase mRNA levels decreased after estrogenic stimuli. • Estrogenic effects in VtgA, ERα and Uox mRNA levels were reverted by ICI 182,780. • Immunofluorescence/electron microscopy shows smaller peroxisomes after 50 μM of EE2. - Abstract: Estrogens, estrogenic mimics and anti-estrogenic compounds are known to target estrogen receptors (ER) that can modulate other nuclear receptor signaling pathways, such as those controlled by the peroxisome proliferator-activated receptor (PPAR), and alter organelle (inc. peroxisome) morphodynamics. By using primary isolated brown trout (Salmo trutta f. fario) hepatocytes after 72 and 96 h of exposure we evaluated some effects in selected molecular targets and in peroxisomal morphological features caused by: (1) an ER agonist (ethinylestradiol—EE2) at 1, 10 and 50 μM; (2) an ER antagonist (ICI 182,780) at 10 and 50 μM; and (3) mixtures of both (Mix I—10 μM EE2 and 50 μM ICI; Mix II—1 μM EE2 and 10 μM ICI and Mix III—1 μM EE2 and 50 μM ICI). The mRNA levels of the estrogenic targets (ERα, ERβ-1 and vitellogenin A—VtgA) and the peroxisome structure/function related genes (catalase, urate oxidase—Uox, 17β-hydroxysteroid dehydrogenase 4—17β-HSD4, peroxin 11α—Pex11α and PPARα) were analyzed by real-time polymerase chain reaction (RT-PCR). Stereology combined with catalase immunofluorescence revealed a significant reduction in peroxisome volume densities at 50 μM of EE2 exposure. Concomitantly, at the same concentration, electron microscopy showed smaller peroxisome profiles, exacerbated proliferation of rough endoplasmic reticulum, and a generalized cytoplasmic vacuolization of hepatocytes. Catalase and Uox mRNA levels decreased in all estrogenic stimuli conditions. VtgA and ERα m

  18. Low-protein diet induces, whereas high-protein diet reduces hepatic FGF21 production in mice, but glucose and not amino acids up-regulate FGF21 in cultured hepatocytes.

    Science.gov (United States)

    Chalvon-Demersay, Tristan; Even, Patrick C; Tomé, Daniel; Chaumontet, Catherine; Piedcoq, Julien; Gaudichon, Claire; Azzout-Marniche, Dalila

    2016-10-01

    Fibroblast growth factor 21 (FGF21) is a polypeptide secreted by the liver and involved in several metabolic processes such as thermogenesis and lipid oxidation. The nutritional mechanisms controlling FGF21 production are poorly understood. This study aimed to investigate how dietary carbohydrates and proteins impact FGF21 production and how in turn, FGF21 is involved in the metabolic adaptation to changes in the carbohydrate and protein contents of the diet. For that purpose, we fed 25 male C57BL/6 mice diets composed of different protein and carbohydrate contents (normal-protein and carbohydrate diet (N=9, NPNC), low-protein high-carbohydrate diet (N=8, LPHC), high-protein low-carbohydrate diet (N=8, HPLC) for 3 weeks. We measured liver Fgf21 gene expression, synthesis and secretion as well as different parameters related to energy and glucose metabolism. We also investigated the direct role of amino acids and glucose in the control of Fgf21 gene expression in hepatocyte primary cultures (n=6). In vivo, FGF21 responds acutely to LPHC intake whereas under an HPLC diet, plasma FGF21 circulating levels are low in the fasted and refed states. In hepatocytes, Fgf21 expression was controlled by glucose but not amino acids. Both diets increased the thermic effect of feeding (TEF) and ketogenesis was increased in fasted HPLC mice. The results presented suggest that dietary glucose, rather than amino acids, directly controls FGF21 secretion, and that FGF21 may be involved in the increased TEF response to LPHC. The effects of the HPLC diet on ketogenesis and TEF are probably controlled by other metabolic pathways. Copyright © 2016 Elsevier Inc. All rights reserved.

  19. Source-Related Effects of Wastewater on Transcription Factor (AhR, CAR and PXR-Mediated Induction of Gene Expression in Cultured Rat Hepatocytes and Their Association with the Prevalence of Antimicrobial-Resistant Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Keerthi S Guruge

    Full Text Available Extracts of wastewater collected from 4 sewage treatment plants (STPs receiving effluents from different sources in South India were investigated for their levels of transcription factor-mediated gene induction in primary cultured rat hepatocytes. In addition, the relation between gene induction levels and the prevalence of antimicrobial-resistant Escherichia coli (E. coli in wastewater was examined. STP-3, which treats only hospital wastewater, exhibited significantly greater induction potency of all 6 drug metabolizing cytochrome P450 (CYP genes examined, CYP1A1, 1A2, 1B1, 2B15, 3A1, and 3A2, whereas the wastewater at STP-1, which exclusively receives domestic sewage, showed significantly diminished levels of induction of 3 CYP genes when compared to the levels of CYP induction at STP-2, which receives mixed wastewater. Samples collected during the monsoon season showed a significantly altered gene induction capacity compared to that of samples from the pre-monsoon period. The data suggest that the toxicity of wastewater in STPs was not significantly diminished during the treatment process. The chemical-gene interaction data predicted that a vast number of chemicals present in the wastewater would stimulate the genes studied in the rat hepatocytes. The multivariable logistic regression analysis demonstrated that the prevalence of isolates resistant to cefotaxime, imipenem and streptomycin was significantly correlated with the levels of induction of at least three CYP-isozymes in STP wastewater. In addition, the resistance of isolates in treatment plants was not altered by the treatment steps, whereas the sampling season did have an impact on the resistance to specific antimicrobials. The identification of receptor-mediated gene regulation capacities offers important data not limited to the (synergistic physiological role of chemicals in biological systems but may provide new insight into the link between the effects of known/unknown drugs and

  20. Controlled cell morphology and liver-specific function of engineered primary hepatocytes by fibroblast layer cell densities.

    Science.gov (United States)

    Sakai, Yusuke; Koike, Makiko; Kawahara, Daisuke; Hasegawa, Hideko; Murai, Tomomi; Yamanouchi, Kosho; Soyama, Akihiko; Hidaka, Masaaki; Takatsuki, Mitsuhisa; Fujita, Fumihiko; Kuroki, Tamotsu; Eguchi, Susumu

    2018-03-05

    Engineered primary hepatocytes, including co-cultured hepatocyte sheets, are an attractive to basic scientific and clinical researchers because they maintain liver-specific functions, have reconstructed cell polarity, and have high transplantation efficiency. However, co-culture conditions regarding engineered primary hepatocytes were suboptimal in promoting these advantages. Here we report that the hepatocyte morphology and liver-specific function levels are controlled by the normal human diploid fibroblast (TIG-118 cell) layer cell density. Primary rat hepatocytes were plated onto TIG-118 cells, previously plated 3 days before at 1.04, 5.21, and 26.1×10 3  cells/cm 2 . Hepatocytes plated onto lower TIG-118 cell densities expanded better during the early culture period. The hepatocytes gathered as colonies and only exhibited small adhesion areas because of the pushing force from proliferating TIG-118 cells. The smaller areas of each hepatocyte result in the development of bile canaliculi. The highest density of TIG-118 cells downregulated albumin synthesis activity of hepatocytes. The hepatocytes may have undergone apoptosis associated with high TGF-β1 concentration and necrosis due to a lack of oxygen. These occurrences were supported by apoptotic chromatin condensation and high expression of both proteins HIF-1a and HIF-1b. Three types of engineered hepatocyte/fibroblast sheets comprising different TIG-118 cell densities were harvested after 4 days of hepatocyte culture and showed a complete cell sheet format without any holes. Hepatocyte morphology and liver-specific function levels are controlled by TIG-118 cell density, which helps to design better engineered hepatocytes for future applications such as in vitro cell-based assays and transplantable hepatocyte tissues. Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  1. A novel cell culture model for studying differentiation and apoptosis in the mouse mammary gland

    International Nuclear Information System (INIS)

    Gordon, Katrina E; Binas, Bert; Chapman, Rachel S; Kurian, Kathreena M; Clarkson, Richard W E; John Clark, A; Birgitte Lane, E; Watson, Christine J

    2000-01-01

    This paper describes the derivation and characterization of a novel, conditionally immortal mammary epithelial cell line named KIM-2. These cells were derived from mid-pregnant mammary glands of a mouse harbouring one to two copies of a transgene comprised of the ovine β-lactoglobulin milk protein gene promoter, driving expression of a temperature-sensitive variant of simian virus-40 (SV40) large T antigen (T-Ag). KIM-2 cells have a characteristic luminal epithelial cell morphology and a stable, nontransformed phenotype at the semipermissive temperature of 37°C. In contrast, at the permissive temperature of 33°C the cells have an elongated spindle-like morphology and become transformed after prolonged culture. Differentiation of KIM-2 cells at 37°C, in response to lactogenic hormones, results in the formation of polarized dome-like structures with tight junctions. This is accompanied by expression of the milk protein genes that encode β-casein and whey acidic protein (WAP), and activation of the prolactin signalling molecule, signal transducer and activator of transcription (STAT)5. Fully differentiated KIM-2 cultures at 37°C become dependent on lactogenic hormones for survival and undergo extensive apoptosis upon hormone withdrawal, as indicated by nuclear morphology and flow cytometric analysis. KIM-2 cells can be genetically modified by stable transfection and clonal lines isolated that retain the characteristics of untransfected cells. KIM-2 cells are a valuable addition, therefore, to currently available lines of mammary epithelial cells. Their capacity for extensive differentiation in the absence of exogenously added basement membrane, and ability to undergo apoptosis in response to physiological signals will provide an invaluable model system for the study of signal transduction pathways and transcriptional regulatory mechanisms that control differentiation and involution in the mammary gland

  2. Endogenous production of fibronectin is required for self-renewal of cultured mouse embryonic stem cells.

    Science.gov (United States)

    Hunt, Geoffrey C; Singh, Purva; Schwarzbauer, Jean E

    2012-09-10

    Pluripotent cells are attached to the extracellular matrix (ECM) as they make cell fate decisions within the stem cell niche. Here we show that the ubiquitous ECM protein fibronectin is required for self-renewal decisions by cultured mouse embryonic stem (mES) cells. Undifferentiated mES cells produce fibronectin and assemble a fibrillar matrix. Increasing the level of substrate fibronectin increased cell spreading and integrin receptor signaling through focal adhesion kinase, while concomitantly inducing the loss of Nanog and Oct4 self-renewal markers. Conversely, reducing fibronectin production by mES cells growing on a feeder-free gelatin substrate caused loss of cell adhesion, decreased integrin signaling, and decreased expression of self-renewal markers. These effects were reversed by providing the cells with exogenous fibronectin, thereby restoring adhesion to the gelatin substrate. Interestingly, mES cells do not adhere directly to the gelatin substrate, but rather adhere indirectly through gelatin-bound fibronectin, which facilitates self-renewal via its effects on cell adhesion. These results provide new insights into the mechanism of regulation of self-renewal by growth on a gelatin-coated surface. The effects of increasing or decreasing fibronectin levels show that self-renewal depends on an intermediate level of cell-fibronectin interactions. By providing cell adhesive signals that can act with other self-renewal factors to maintain mES cell pluripotency, fibronectin is therefore a necessary component of the self-renewal signaling pathway in culture. Copyright © 2012 Elsevier Inc. All rights reserved.

  3. Effects of 4-aminopyridine on organelle movement in cultured mouse dorsal root ganglion neurites.

    Science.gov (United States)

    Hiruma, Hiromi; Kawakami, Tadashi

    2010-03-01

    Aminopyridines, widely used as a K(+) channel blocker, are membrane-permeable weak bases and have the ability to form vacuoles in the cytoplasm. The vacuoles originate from acidic organelles such as lysosomes. Here, we investigated the effects of 4-aminopyridine (4-AP) on organelle movement in neurites of cultured mouse dorsal root ganglion (DRG) neurons by using video-enhanced microscopy. Some experiments were carried out using fluorescent dyes for lysosomes and mitochondria and confocal microscopy. Treatment of DRG neurons with 4 mM 4-AP caused Brownian movement of some lysosomes within 5 min. The Brownian movement gradually became rapid and vacuoles were formed around individual lysosomes 10-20 min after the start of treatment. Axonal transport of organelles was inhibited by 4-AP. Lysosomes showing Brownian movement were not transported in longitudinal direction of the neurite and the transport of mitochondria was interrupted by vacuoles. The 4-AP-induced Brownian movement of lysosomes with vacuole formation and inhibition of axonal transport were prevented by the simultaneous treatment with vacuolar H(+) ATPase inhibitor bafilomycin A1 or in Cl(-)-free SO(4)(2-) medium. These results indicate that changes in organelle movement by 4-AP are related to vacuole formation and the vacuolar H(+) ATPase and Cl(-) are required for the effects of 4-AP.

  4. Chromosome alterations in the X-ray-induced transformants of cultured mouse cell line

    International Nuclear Information System (INIS)

    Kodama, Seiji; Komatsu, Kenshi; Okumura, Yutaka; Sasaki, M.S.

    1989-01-01

    Mouse m5S cells were subjected to soft X-ray irradiation. Twenty-four transformants were separated as indicators of focus formation. Two clones, cl.4103 and cl.6310, were chosen for the analysis of chromosome alterations in transformants. A parent strain, m5S/1M, served as the control. Anchorage independence (AG) was not detected in the control strain, irrespective of culture conditions and population doubling number (PDN). In the case of transformants, the frequency of AG was increased with increasing PDN for cl.4103, and was constant for cl.6310, irrespective of PDN. Karyotype of m5S/1M was 42, X, -Y, +der (6) t(6;13), t(8;8), +8, +15. In addition, -13, der(10) and -der(6)t(6;13), der(5), +mar occurred as karyotype alterations for cl.4103 and cl. 6310, respectively. The present experiment indicated that chromosome alterations secondary to primary alterations occur in a high frequency in the transforming process of X-ray irradiated cells, and that the secondary chromosome alterations result in selective proliferation of transformed clones. (Namekawa, K)

  5. Microdrop preparation factors influence culture-media osmolality, which can impair mouse embryo preimplantation development.

    Science.gov (United States)

    Swain, J E; Cabrera, L; Xu, X; Smith, G D

    2012-02-01

    Because media osmolality can impact embryo development, the effect of conditions during microdrop preparation on osmolality was examined. Various sizes of microdrops were prepared under different laboratory conditions. Drops were pipetted directly onto a dish and covered by oil (standard method) or pipetted on the dish, overlaid with oil before removing the underlying media and replaced with fresh media (wash-drop method). Drops were made at 23°C or on a heated stage (37°C) and with or without airflow. Osmolality was assessed at 5 min and 24h. The biological impact of osmolality change was demonstrated by culturing 1-cell mouse embryos in media with varying osmolality. Reduced drop volume, increased temperature and standard method were associated with a significant increase in osmolality at both 5 min and 24h (P-values media with elevated osmolality (>310mOsm/kg; P<0.05). Procedures in the IVF laboratory can alter osmolality and impact embryo development. Copyright © 2011 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

  6. Neurogenic and neurotrophic effects of BDNF peptides in mouse hippocampal primary neuronal cell cultures.

    Directory of Open Access Journals (Sweden)

    Maria del Carmen Cardenas-Aguayo

    Full Text Available The level of brain-derived neurotrophic factor (BDNF, a member of the neurotrophin family, is down regulated in Alzheimer's disease (AD, Parkinson's disease (PD, depression, stress, and anxiety; conversely the level of this neurotrophin is increased in autism spectrum disorders. Thus, modulating the level of BDNF can be a potential therapeutic approach for nervous system pathologies. In the present study, we designed five different tetra peptides (peptides B-1 to B-5 corresponding to different active regions of BDNF. These tetra peptides were found to be non-toxic, and they induced the expression of neuronal markers in mouse embryonic day 18 (E18 primary hippocampal neuronal cultures. Additionally, peptide B-5 induced the expression of BDNF and its receptor, TrkB, suggesting a positive feedback mechanism. The BDNF peptides induced only a moderate activation (phosphorylation at Tyr 706 of the TrkB receptor, which could be blocked by the Trk's inhibitor, K252a. Peptide B-3, when combined with BDNF, potentiated the survival effect of this neurotrophin on H(2O(2-treated E18 hippocampal cells. Peptides B-3 and B-5 were found to work as partial agonists and as partial antagonists competing with BDNF to activate the TrkB receptor in a dose-dependent manner. Taken together, these results suggest that the described BDNF tetra peptides are neurotrophic, can modulate BDNF signaling in a partial agonist/antagonist way, and offer a novel therapeutic approach to neural pathologies where BDNF levels are dysregulated.

  7. Development of a new mouse palate organ culture system and effect of X-irradiation on palatogenesis

    International Nuclear Information System (INIS)

    Hiranuma, Hiroko; Jikko, Akitoshi; Maeda, Takashi; Furukawa, Souhei

    1999-01-01

    On the basis of an already established suspension system of organ culture for mouse palate explants, we developed a new culture system, which has several advantages over the previous methods. We used a 48-well culture plate in which the explants can be cultured individually, and only 300 μl of medium is needed for each well. In order to optimize the culture results, we systematically studied the influence of main ''culture conditions'' such as tilt degree of the culturing palate, rotation speed, and addition of ascorbic acid to the medium. This system allows culturing of palates from day 13.5 of gestation to day 16.5 under serum-free conditions using a chemically defined medium, which resulted in 78% of the palates growing fused. Utilizing this culture system, the direct effect of X-irradiation on palataogesis was analyzed. A 4 Gy dose of X-irradiation was administrated at the beginning of culture period. The incidence of palatal fusion was not significantly different from that of the non-irradiated group. (author)

  8. In vitro culture of individual mouse preimplantation embryos: the role of embryo density, microwells, oxygen, timing and conditioned media.

    Science.gov (United States)

    Kelley, Rebecca L; Gardner, David K

    2017-05-01

    Single embryo culture is suboptimal compared with group culture, but necessary for embryo monitoring, and culture systems should be improved for single embryos. Pronucleate mouse embryos were used to assess the effect of culture conditions on single embryo development. Single culture either before or after compaction reduced cell numbers (112.2 ± 3.1; 110.2 ± 3.5) compared with group culture throughout (127.0 ± 3.4; P media volume from 20 µl to 2 µl increased blastocyst cell numbers in single embryos cultured in 5% oxygen (84.4 ± 3.2 versus 97.8 ± 2.8; P Culture in microwell plates for the EmbryoScope and Primo Vision time-lapse systems changed cleavage timings and increased inner cell mass cell number (24.1 ± 1.0; 23.4 ± 1.2) compared with a 2 µl microdrop (18.4 ± 1.0; P media to single embryos increased hatching rate and blastocyst cell number (91.5 ± 4.7 versus 113.1 ± 4.4; P culture before or after compaction is therefore detrimental; oxygen, media volume and microwells influence single embryo development; and embryo-conditioned media may substitute for group culture. Copyright © 2017 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

  9. Transcription factors ETF, E2F, and SP-1 are involved in cytokine-independent proliferation of murine hepatocytes.

    Science.gov (United States)

    Zellmer, Sebastian; Schmidt-Heck, Wolfgang; Godoy, Patricio; Weng, Honglei; Meyer, Christoph; Lehmann, Thomas; Sparna, Titus; Schormann, Wiebke; Hammad, Seddik; Kreutz, Clemens; Timmer, Jens; von Weizsäcker, Fritz; Thürmann, Petra A; Merfort, Irmgard; Guthke, Reinhard; Dooley, Steven; Hengstler, Jan G; Gebhardt, Rolf

    2010-12-01

    The cellular basis of liver regeneration has been intensely investigated for many years. However, the mechanisms initiating hepatocyte "plasticity" and priming for proliferation are not yet fully clear. We investigated alterations in gene expression patterns during the first 72 hours of C57BL/6N mouse hepatocyte culture on collagen monolayers (CM), which display a high basal frequency of proliferation in the absence of cytokines. Although many metabolic genes were down-regulated, genes related to mitogen-activated protein kinase (MAPK) signaling and cell cycle were up-regulated. The latter genes showed an overrepresentation of transcription factor binding sites (TFBS) for ETF (TEA domain family member 2), E2F1 (E2F transcription factor 1), and SP-1 (Sp1 transcription factor) (P ETF, E2F1, and SP-1 and displayed increased expression of E2F1. Cultivation of murine hepatocytes on CM primes cells for proliferation through cytokine-independent activation of MAPK signaling. The transcription factors ETF, E2F1, and SP-1 seem to play a pronounced role in mediating proliferation-dependent differential gene expression. Similar events, but on a shorter time-scale, occur very early after liver damage in vivo. Copyright © 2010 American Association for the Study of Liver Diseases.

  10. Culture conditions have an impact on the maturation of traceable, transplantable mouse embryonic stem cell-derived otic progenitor cells.

    Science.gov (United States)

    Abboud, Nesrine; Fontbonne, Arnaud; Watabe, Isabelle; Tonetto, Alain; Brezun, Jean Michel; Feron, François; Zine, Azel

    2017-09-01

    The generation of replacement inner ear hair cells (HCs) remains a challenge and stem cell therapy holds the potential for developing therapeutic solutions to hearing and balance disorders. Recent developments have made significant strides in producing mouse otic progenitors using cell culture techniques to initiate HC differentiation. However, no consensus has been reached as to efficiency and therefore current methods remain unsatisfactory. In order to address these issues, we compare the generation of otic and HC progenitors from embryonic stem (ES) cells in two cell culture systems: suspension vs. adherent conditions. In the present study, an ES cell line derived from an Atoh1-green fluorescent protein (GFP) transgenic mouse was used to track the generation of otic progenitors, initial HCs and to compare these two differentiation systems. We used a two-step short-term differentiation method involving an induction period of 5 days during which ES cells were cultured in the presence of Wnt/transforming growth factor TGF-β inhibitors and insulin-like growth factor IGF-1 to suppress mesoderm and reinforce presumptive ectoderm and otic lineages. The generated embryoid bodies were then differentiated in medium containing basic fibroblast growth factor (bFGF) for an additional 5 days using either suspension or adherent culture methods. Upon completion of differentiation, quantitative polymerase chain reaction analysis and immunostaining monitored the expression of otic/HC progenitor lineage markers. The results indicate that cells differentiated in suspension cultures produced cells expressing otic progenitor/HC markers at a higher efficiency compared with the production of these cell types within adherent cultures. Furthermore, we demonstrated that a fraction of these cells can incorporate into ototoxin-injured mouse postnatal cochlea explants and express MYO7A after transplantation. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons

  11. Primary culture of glial cells from mouse sympathetic cervical ganglion: a valuable tool for studying glial cell biology.

    Science.gov (United States)

    de Almeida-Leite, Camila Megale; Arantes, Rosa Maria Esteves

    2010-12-15

    Central nervous system glial cells as astrocytes and microglia have been investigated in vitro and many intracellular pathways have been clarified upon various stimuli. Peripheral glial cells, however, are not as deeply investigated in vitro despite its importance role in inflammatory and neurodegenerative diseases. Based on our previous experience of culturing neuronal cells, our objective was to standardize and morphologically characterize a primary culture of mouse superior cervical ganglion glial cells in order to obtain a useful tool to study peripheral glial cell biology. Superior cervical ganglia from neonatal C57BL6 mice were enzymatically and mechanically dissociated and cells were plated on diluted Matrigel coated wells in a final concentration of 10,000cells/well. Five to 8 days post plating, glial cell cultures were fixed for morphological and immunocytochemical characterization. Glial cells showed a flat and irregular shape, two or three long cytoplasm processes, and round, oval or long shaped nuclei, with regular outline. Cell proliferation and mitosis were detected both qualitative and quantitatively. Glial cells were able to maintain their phenotype in our culture model including immunoreactivity against glial cell marker GFAP. This is the first description of immunocytochemical characterization of mouse sympathetic cervical ganglion glial cells in primary culture. This work discusses the uses and limitations of our model as a tool to study many aspects of peripheral glial cell biology. Copyright © 2010 Elsevier B.V. All rights reserved.

  12. Culture medium, gas atmosphere and MAPK inhibition affect regulation of RNA-binding protein targets during mouse preimplantation development.

    Science.gov (United States)

    Calder, Michele D; Watson, Patricia H; Watson, Andrew J

    2011-11-01

    During oogenesis, mammalian oocytes accumulate maternal mRNAs that support the embryo until embryonic genome activation. RNA-binding proteins (RBP) may regulate the stability and turnover of maternal and embryonic mRNAs. We hypothesised that varying embryo culture conditions, such as culture medium, oxygen tension and MAPK inhibition, affects regulation of RBPs and their targets during preimplantation development. STAU1, ELAVL1, KHSRP and ZFP36 proteins and mRNAs were detected throughout mouse preimplantation development, whereas Elavl2 mRNA decreased after the two-cell stage. Potential target mRNAs of RBP regulation, Gclc, Slc2a1 and Slc7a1 were detected during mouse preimplantation development. Gclc mRNA was significantly elevated in embryos cultured in Whitten's medium compared with embryos cultured in KSOMaa, and Gclc mRNA was elevated under high-oxygen conditions. Inhibition of the p38 MAPK pathway reduced Slc7a1 mRNA expression while inhibition of ERK increased Slc2a1 mRNA expression. The half-lives of the potential RBP mRNA targets are not regulated in parallel; Slc2a1 mRNA displayed the longest half-life. Our results indicate that mRNAs and proteins encoding five RBPs are present during preimplantation development and more importantly, demonstrate that expression of RBP target mRNAs are regulated by culture medium, gas atmosphere and MAPK pathways.

  13. Evaluation of hollow fiber culture for large-scale production of mouse embryonic stem cell-derived hematopoietic stem cells.

    Science.gov (United States)

    Nakano, Yu; Iwanaga, Shinya; Mizumoto, Hiroshi; Kajiwara, Toshihisa

    2018-03-03

    Hematopoietic stem cells (HSCs) have the ability to differentiate into all types of blood cells and can be transplanted to treat blood disorders. However, it is difficult to obtain HSCs in large quantities because of the shortage of donors. Recent efforts have focused on acquiring HSCs by differentiation of pluripotent stem cells. As a conventional differentiation method of pluripotent stem cells, the formation of embryoid bodies (EBs) is often employed. However, the size of EBs is limited by depletion of oxygen and nutrients, which prevents them from being efficient for the production of HSCs. In this study, we developed a large-scale hematopoietic differentiation approach for mouse embryonic stem (ES) cells by applying a hollow fiber (HF)/organoid culture method. Cylindrical organoids, which had the potential for further spontaneous differentiation, were established inside of hollow fibers. Using this method, we improved the proliferation rate of mouse ES cells to produce an increased HSC population and achieved around a 40-fold higher production volume of HSCs in HF culture than in conventional EB culture. Therefore, the HF/organoid culture method may be a new mass culture method to acquire pluripotent stem cell-derived HSCs.

  14. Investigation of testosterone, androstenone, and estradiol metabolism in HepG2 cells and primary culture pig hepatocytes and their effects on 17βHSD7 gene expression.

    Directory of Open Access Journals (Sweden)

    Gang Chen

    Full Text Available Steroid metabolism is important in various species. The accumulation of androgen metabolite, androstenone, in pig adipose tissue is negatively associated with pork flavor, odour and makes the meat unfit for human consumption. The 17β-hydroxysteroid dehydrogenase type 7 (17βHSD7 expressed abundantly in porcine liver, and it was previously suggested to be associated with androstenone levels. Understanding the enzymes and metabolic pathways responsible for androstenone as well as other steroids metabolism is important for improving the meat quality. At the same time, metabolism of steroids is known to be species- and tissue-specific. Therefore it is important to investigate between-species variations in the hepatic steroid metabolism and to elucidate the role of 17βHSD7 in this process. Here we used an effective methodological approach, liquid chromatography coupled with mass spectrometry, to investigate species-specific metabolism of androstenone, testosterone and beta-estradiol in HepG2 cell line, and pig cultured hepatocytes. Species- and concentration-depended effect of steroids on 17βHSD7 gene expression was also investigated. It was demonstrated that the investigated steroids can regulate the 17βHSD7 gene expression in HepG2 and primary cultured porcine hepatocytes in a concentration-dependent and species-dependent pattern. Investigation of steroid metabolites demonstrated that androstenone formed a 3'-hydroxy compound 3β-hydroxy-5α-androst-16-ene. Testosterone was metabolized to 4-androstene-3,17-dione. Estrone was found as the metabolite for β-estradiol. Inhibition study with 17βHSD inhibitor apigenin showed that apigenin didn't affect androstenone metabolism. Apigenin at high concentration (50 µM tends to inhibit testosterone metabolism but this inhibition effect was negligible. Beta-estradiol metabolism was notably inhibited with apigenin at high concentration. The study also established that the level of testosterone and

  15. E2f1 mediates high glucose-induced neuronal death in cultured mouse retinal explants.

    Science.gov (United States)

    Wang, Yujiao; Zhou, Yi; Xiao, Lirong; Zheng, Shijie; Yan, Naihong; Chen, Danian

    2017-10-02

    Diabetic retinopathy (DR) is the most common complication of diabetes and remains one of the major causes of blindness in the world; infants born to diabetic mothers have higher risk of developing retinopathy of prematurity (ROP). While hyperglycemia is a major risk factor, the molecular and cellular mechanisms underlying DR and diabetic ROP are poorly understood. To explore the consequences of retinal cells under high glucose, we cultured wild type or E2f1 -/- mouse retinal explants from postnatal day 8 with normal glucose, high osmotic or high glucose media. Explants were also incubated with cobalt chloride (CoCl 2 ) to mimic the hypoxic condition. We showed that, at 7 days post exposure to high glucose, retinal explants displayed elevated cell death, ectopic cell division and intact retinal vascular plexus. Cell death mainly occurred in excitatory neurons, such as ganglion and bipolar cells, which were also ectopically dividing. Many Müller glial cells reentered the cell cycle; some had irregular morphology or migrated to other layers. High glucose inhibited the hyperoxia-induced blood vessel regression of retinal explants. Moreover, inactivation of E2f1 rescued high glucose-induced ectopic division and cell death of retinal neurons, but not ectopic cell division of Müller glial cells and vascular phenotypes. This suggests that high glucose has direct but distinct effects on retinal neurons, glial cells and blood vessels, and that E2f1 mediates its effects on retinal neurons. These findings shed new light onto mechanisms of DR and the fetal retinal abnormalities associated with maternal diabetes, and suggest possible new therapeutic strategies.

  16. Dose-response relationship of cadmium or radiation-induced embryotoxicity in mouse whole embryo culture

    International Nuclear Information System (INIS)

    Nakashima, Kiyohito; Kawamata, Akitoshi; Matsuoka, Masato; Wakisaka, Takashi; Fujiki, Yoshishige

    1988-01-01

    Mouse embryos of B6C3F 1 strain were exposed in vitro to 1.2 to 2.2 μM cadmium chloride (Cd) or to 100 to 320 R x-rays, and the effects of the exposure on development were examined after 39 h of culture. Development of embryos was assessed from lethality, formation of the neural tube defect, diameter and protein of yolk sac, crown-rump and head lengths, embryonic protein, and number of somites. Incidence of the neural tube defect increased from 3.4 to 100% by 1.2 to 2.0 μM Cd, while embryo deaths increased from 13.8 to 93.3% by 2.0 to 2.2 μM Cd. Embryonic protein was significantly reduced at the teratogenic range, but the number of somites was only affected by 1.6 to 2.0 μM Cd. X-irradiation at 100 to 320 R induced the neural tube defect in 2.9 to 72.7% of the embryos. An embryolethal effect was observed only at the 320 R dose. Crown-rump and head lengths and embryonic protein were significantly affected at the teratogenic range, but the diameter and protein of yolk sac and number of somites were hardly affected. Cadmium- or radiation-induced response data of both teratogenicity and endpoints indicating inhibition of embryonic development were acceptably fitted to a linear log-probit regression. These regressions suggest that as an estimation of interference in development of embryos, embryonic protein and head length are sensitive endpoints while the number of somites is an insensitive criterion. (author)

  17. Effect of culture medium volume and embryo density on early mouse embryonic development: tracking the development of the individual embryo.

    Science.gov (United States)

    Dai, Shan-Jun; Xu, Chang-Long; Wang, Jeffrey; Sun, Ying-Pu; Chian, Ri-Cheng

    2012-07-01

    To determine the optimal volume or density of embryos for the well-of-the-well (WOW) system in order to track the development of individual embryos and to determine whether the WOW system can reverse the negative impact of culturing embryos singly. (1) Mouse embryos (groups of nine at the 2-cell stage) were cultured in 6.25 μl, 12.50 μl, 25.00 μl and 50.00 μl of droplets of culture medium under paraffin oil; (2) Groups of three, six, nine and twelve embryos at the 2-cell stage were cultured in 50 μl of droplet of culture medium under paraffin oil; (3) Groups of nine embryos at the 2-cell stage were cultured in 50 μl of droplet under paraffin oil with or without nine micro-wells made on the bottom of the Petri dish into each of which were placed one of the nine embryos (WOW system). Also single 2-cell stage embryos was cultured individually in 5.5 μl of droplet of culture medium under paraffin oil with or without a single micro-well made on the bottom of the Petri dish (WOW system for single culture). At the end of culture, the percentages of blastocyst development, hatching and hatched blastocysts were compared in each group. The blastocysts were fixed for differential staining. The blastocyst development was significantly higher (P WOW system. The blastocyst development was not improved when single embryo cultured individually in a micro-well was compared to single embryo cultured individually without micro-well. The total cell numbers of blastocysts were significantly higher in group embryo culture than single embryo culture regardless of whether the WOW system was used. In addition, the total cell numbers of blastocysts were significantly higher (P WOW system than without. Group embryo culture is superior to single embryo culture for blastocyst development. The WOW system with 50 μl of droplet of culture medium can be used to track the individual development of embryo cultured in groups while preserving good embryonic development. The reduced

  18. Cytochrome P4501A induction in avian hepatocyte cultures exposed to polychlorinated biphenyls: Comparisons with AHR1-mediated reporter gene activity and in ovo toxicity

    Energy Technology Data Exchange (ETDEWEB)

    Manning, Gillian E., E-mail: gmann017@uottawa.ca [Centre for Advanced Research in Environmental Genomics, Department of Biology, University of Ottawa, Ottawa, ON, Canada K1N 6N5 (Canada); Environment Canada, National Wildlife Research Centre, Ottawa, ON, Canada K1A 0H3 (Canada); Mundy, Lukas J., E-mail: lukas.mundy@ec.gc.ca [Centre for Advanced Research in Environmental Genomics, Department of Biology, University of Ottawa, Ottawa, ON, Canada K1N 6N5 (Canada); Environment Canada, National Wildlife Research Centre, Ottawa, ON, Canada K1A 0H3 (Canada); Crump, Doug, E-mail: doug.crump@ec.gc.ca [Environment Canada, National Wildlife Research Centre, Ottawa, ON, Canada K1A 0H3 (Canada); Jones, Stephanie P., E-mail: stephanie.jones@ec.gc.ca [Environment Canada, National Wildlife Research Centre, Ottawa, ON, Canada K1A 0H3 (Canada); Chiu, Suzanne, E-mail: suzanne.chiu@ec.gc.ca [Environment Canada, National Wildlife Research Centre, Ottawa, ON, Canada K1A 0H3 (Canada); Klein, Jeff, E-mail: jeffery@well-labs.com [Wellington Laboratories Inc., Research Division, Guelph, ON, Canada N1G 3chsM5 (Canada); Konstantinov, Alex, E-mail: alex@well-labs.com [Wellington Laboratories Inc., Research Division, Guelph, ON, Canada N1G 3chsM5 (Canada); Potter, Dave, E-mail: dpotter@well-labs.com [Wellington Laboratories Inc., Research Division, Guelph, ON, Canada N1G 3chsM5 (Canada); Kennedy, Sean W., E-mail: sean.kennedy@ec.gc.ca [Centre for Advanced Research in Environmental Genomics, Department of Biology, University of Ottawa, Ottawa, ON, Canada K1N 6N5 (Canada); Environment Canada, National Wildlife Research Centre, Ottawa, ON, Canada K1A 0H3 (Canada)

    2013-01-01

    Avian-specific toxic equivalency factors (TEFs) were developed by the World Health Organization to simplify environmental risk assessments of dioxin-like compounds (DLCs), but TEFs do not account for differences in the toxic and biochemical potencies of DLCs among species of birds. Such variability may be due to differences in species sensitivity to individual DLCs. The sensitivity of avian species to DLCs was recently associated with the identity of amino acids 324 and 380 in the aryl hydrocarbon receptor 1 (AHR1) ligand binding domain. A luciferase reporter gene (LRG) assay, measuring AHR1-mediated induction of a cytochrome P450 1A5 (CYP1A5) reporter gene, in combination with a species' AHR1 ligand binding domain sequence, were also shown to predict avian species sensitivity to polychlorinated biphenyls (PCBs) and PCB relative potency in a given species. The goals of the present study were to (1) characterize the concentration-dependent effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin and PCBs 126, 77, 105 and 118 on induction of ethoxyresorufin O-deethylase (EROD) activity and CYP1A4/5 mRNA in chicken, ring-necked pheasant and Japanese quail embryo hepatocytes and (2) compare these in vitro results to those previously generated by the LRG assay and in ovo toxicity studies. EROD activity and CYP1A4/5 mRNA expression data support and complement the findings of the LRG assay. CYP1A enzyme activity and mRNA expression were significantly correlated both with luciferase activity and in ovo toxicity induced by PCBs. Relative potency values were generally similar between the LRG and EROD assays and indicate that the relative potency of some PCBs may differ among species. -- Highlights: ► The chicken isn't the most sensitive species to CYP1A induction by PCB 105 and 118. ► The relative potency of PCBs differs between avian species. ► EROD activity was correlated with luciferase activity from the LRG assay. ► EROD activity was a better predictor of

  19. Role of the nuclear xenobiotic receptors CAR and PXR in induction of cytochromes P450 by non-dioxinlike polychlorinated biphenyls in cultured rat hepatocytes

    International Nuclear Information System (INIS)

    Gährs, Maike; Roos, Robert; Andersson, Patrik L.; Schrenk, Dieter

    2013-01-01

    Polychlorinated biphenyls (PCBs) are among the most ubiquitously detectable ‘persistent organic pollutants’. In contrast to ‘dioxinlike’ (DL) PCBs, less is known about the molecular mode of action of the larger group of the ‘non-dioxinlike’ (NDL) PCBs. Owing to the life-long exposure of the human population, a carcinogenic, i.e., tumor-promoting potency of NDL-PCBs has to be considered in human risk assessment. A major problem in risk assessment of NDL-PCBs is dioxin-like impurities that can occur in commercially available NDL-PCB standards. In the present study, we analyzed the induction of CYP2B1 and CYP3A1 in primary rat hepatocytes using a number of highly purified NDL-PCBs with various degrees of chlorination and substitution patterns. Induction of these enzymes is mediated by the nuclear xenobiotic receptors CAR (Constitutive androstane receptor) and PXR (Pregnane X receptor). For CYP2B1 induction, concentration–response analysis revealed a very narrow window of EC 50 estimates, being in the range of 1–4 μM for PCBs 28 and 52, and between 0.4 and 1 μM for PCBs 101, 138, 153 and 180. CYP3A1 induction was less sensitive to NDL-PCBs, the most pronounced induction being achieved at 100 μM with the higher chlorinated congeners. Using okadaic acid and small interfering RNAs targeting CAR and PXR, we could demonstrate that CAR plays a major role and PXR a minor role in NDL-PCB-driven induction of CYPs, both effects showing no stringent structure–activity relationship. As the only obvious relevant determinant, the degree of chlorination was found to be positively correlated with the inducing potency of the congeners. - Highlights: • We analyzed six highly purified NDL-PCBs for CYP2B1 and CYP3A1 expression. • CAR plays a major, PXR a minor role in NDL-PCB-driven induction of CYPs. • The degree of chlorination seems to be the major parameter for the inducing potency. • There exists a competition between CAR and PXR. • Activated PXR may

  20. Cytochrome P4501A induction in avian hepatocyte cultures exposed to polychlorinated biphenyls: Comparisons with AHR1-mediated reporter gene activity and in ovo toxicity

    International Nuclear Information System (INIS)

    Manning, Gillian E.; Mundy, Lukas J.; Crump, Doug; Jones, Stephanie P.; Chiu, Suzanne; Klein, Jeff; Konstantinov, Alex; Potter, Dave; Kennedy, Sean W.

    2013-01-01

    Avian-specific toxic equivalency factors (TEFs) were developed by the World Health Organization to simplify environmental risk assessments of dioxin-like compounds (DLCs), but TEFs do not account for differences in the toxic and biochemical potencies of DLCs among species of birds. Such variability may be due to differences in species sensitivity to individual DLCs. The sensitivity of avian species to DLCs was recently associated with the identity of amino acids 324 and 380 in the aryl hydrocarbon receptor 1 (AHR1) ligand binding domain. A luciferase reporter gene (LRG) assay, measuring AHR1-mediated induction of a cytochrome P450 1A5 (CYP1A5) reporter gene, in combination with a species' AHR1 ligand binding domain sequence, were also shown to predict avian species sensitivity to polychlorinated biphenyls (PCBs) and PCB relative potency in a given species. The goals of the present study were to (1) characterize the concentration-dependent effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin and PCBs 126, 77, 105 and 118 on induction of ethoxyresorufin O-deethylase (EROD) activity and CYP1A4/5 mRNA in chicken, ring-necked pheasant and Japanese quail embryo hepatocytes and (2) compare these in vitro results to those previously generated by the LRG assay and in ovo toxicity studies. EROD activity and CYP1A4/5 mRNA expression data support and complement the findings of the LRG assay. CYP1A enzyme activity and mRNA expression were significantly correlated both with luciferase activity and in ovo toxicity induced by PCBs. Relative potency values were generally similar between the LRG and EROD assays and indicate that the relative potency of some PCBs may differ among species. -- Highlights: ► The chicken isn't the most sensitive species to CYP1A induction by PCB 105 and 118. ► The relative potency of PCBs differs between avian species. ► EROD activity was correlated with luciferase activity from the LRG assay. ► EROD activity was a better predictor of toxicity than CYP

  1. Role of the nuclear xenobiotic receptors CAR and PXR in induction of cytochromes P450 by non-dioxinlike polychlorinated biphenyls in cultured rat hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Gährs, Maike; Roos, Robert [University of Kaiserslautern, Food Chemistry and Toxicology, Erwin-Schroedinger-Str. 52, D-67663 Kaiserslautern (Germany); Andersson, Patrik L. [Umeå University, Department of Chemistry, Linnaeus väg 6, SE-901 87 Umeå (Sweden); Schrenk, Dieter, E-mail: schrenk@rhrk.uni-kl.de [University of Kaiserslautern, Food Chemistry and Toxicology, Erwin-Schroedinger-Str. 52, D-67663 Kaiserslautern (Germany)

    2013-10-01

    Polychlorinated biphenyls (PCBs) are among the most ubiquitously detectable ‘persistent organic pollutants’. In contrast to ‘dioxinlike’ (DL) PCBs, less is known about the molecular mode of action of the larger group of the ‘non-dioxinlike’ (NDL) PCBs. Owing to the life-long exposure of the human population, a carcinogenic, i.e., tumor-promoting potency of NDL-PCBs has to be considered in human risk assessment. A major problem in risk assessment of NDL-PCBs is dioxin-like impurities that can occur in commercially available NDL-PCB standards. In the present study, we analyzed the induction of CYP2B1 and CYP3A1 in primary rat hepatocytes using a number of highly purified NDL-PCBs with various degrees of chlorination and substitution patterns. Induction of these enzymes is mediated by the nuclear xenobiotic receptors CAR (Constitutive androstane receptor) and PXR (Pregnane X receptor). For CYP2B1 induction, concentration–response analysis revealed a very narrow window of EC{sub 50} estimates, being in the range of 1–4 μM for PCBs 28 and 52, and between 0.4 and 1 μM for PCBs 101, 138, 153 and 180. CYP3A1 induction was less sensitive to NDL-PCBs, the most pronounced induction being achieved at 100 μM with the higher chlorinated congeners. Using okadaic acid and small interfering RNAs targeting CAR and PXR, we could demonstrate that CAR plays a major role and PXR a minor role in NDL-PCB-driven induction of CYPs, both effects showing no stringent structure–activity relationship. As the only obvious relevant determinant, the degree of chlorination was found to be positively correlated with the inducing potency of the congeners. - Highlights: • We analyzed six highly purified NDL-PCBs for CYP2B1 and CYP3A1 expression. • CAR plays a major, PXR a minor role in NDL-PCB-driven induction of CYPs. • The degree of chlorination seems to be the major parameter for the inducing potency. • There exists a competition between CAR and PXR. • Activated PXR

  2. Generation of Otic Sensory Neurons from Mouse Embryonic Stem Cells in 3D Culture

    Directory of Open Access Journals (Sweden)

    Michael Perny

    2017-12-01

    Full Text Available The peripheral hearing process taking place in the cochlea mainly depends on two distinct sensory cell types: the mechanosensitive hair cells and the spiral ganglion neurons (SGNs. The first respond to the mechanical stimulation exerted by sound pressure waves on their hair bundles by releasing neurotransmitters and thereby activating the latter. Loss of these sensorineural cells is associated with permanent hearing loss. Stem cell-based approaches aiming at cell replacement or in vitro drug testing to identify potential ototoxic, otoprotective, or regenerative compounds have lately gained attention as putative therapeutic strategies for hearing loss. Nevertheless, they rely on efficient and reliable protocols for the in vitro generation of cochlear sensory cells for their implementation. To this end, we have developed a differentiation protocol based on organoid culture systems, which mimics the most important steps of in vivo otic development, robustly guiding mouse embryonic stem cells (mESCs toward otic sensory neurons (OSNs. The stepwise differentiation of mESCs toward ectoderm was initiated using a quick aggregation method in presence of Matrigel in serum-free conditions. Non-neural ectoderm was induced via activation of bone morphogenetic protein (BMP signaling and concomitant inhibition of transforming growth factor beta (TGFβ signaling to prevent mesendoderm induction. Preplacodal and otic placode ectoderm was further induced by inhibition of BMP signaling and addition of fibroblast growth factor 2 (FGF2. Delamination and differentiation of SGNs was initiated by plating of the organoids on a 2D Matrigel-coated substrate. Supplementation with brain-derived neurotrophic factor (BDNF and neurotrophin-3 (NT-3 was used for further maturation until 15 days of in vitro differentiation. A large population of neurons with a clear bipolar morphology and functional excitability was derived from these cultures. Immunostaining and gene expression

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

    Science.gov (United States)

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

    2013-01-01

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

  4. The influence of serum substituents on serum-free Vero cell conditioned culture media manufactured from Dulbecco's modified Eagle medium in mouse embryo culture.

    Science.gov (United States)

    Lee, Jong-Seon; Kim, Ju-Hwan; Seo, Young-Seok; Yang, Jung-Bo; Kim, Yong-Il; Kim, Hye-Jin; Lee, Ki-Hwan

    2013-09-01

    This study was conducted to examine the influences of supplementation of the serum substituents and available period of serum-free Vero cell conditioned media (SF-VCM) manufactured from Dulbecco's modified Eagle medium cultured with Vero cells for in vitro development of mouse preimplantation embryos. A total of 1,099 two-cell embryos collected from imprinting control region mice were cultured in SF-VCM with 10% and 20% human follicular fluid (hFF), serum substitute supplement (SSS), and serum protein substitute (SPS). Development of embryos was observed every 24 hours. Results between different groups were analyzed by chi-square test, and considered statistically significant when P-value was less than 0.05. The rates of embryonic development cultured in SF-VCM supplemented with serum substituents were significantly higher compare with serum-free group (P media up to 4 weeks did not affect on embryonic development.

  5. Mouse pancreas tissue slice culture facilitates long-term studies of exocrine and endocrine cell physiology in situ.

    Science.gov (United States)

    Marciniak, Anja; Selck, Claudia; Friedrich, Betty; Speier, Stephan

    2013-01-01

    Studies on pancreatic cell physiology rely on the investigation of exocrine and endocrine cells in vitro. Particularly, in the case of the exocrine tissue these studies have suffered from a reduced functional viability of acinar cells in culture. As a result not only investigations on dispersed acinar cells and isolated acini were limited in their potential, but also prolonged studies on pancreatic exocrine and endocrine cells in an intact pancreatic tissue environment were unfeasible. To overcome these limitations, we aimed to establish a pancreas tissue slice culture platform to allow long-term studies on exocrine and endocrine cells in the intact pancreatic environment. Mouse pancreas tissue slice morphology was assessed to determine optimal long-term culture settings for intact pancreatic tissue. Utilizing optimized culture conditions, cell specificity and function of exocrine acinar cells and endocrine beta cells were characterized over a culture period of 7 days. We found pancreas tissue slices cultured under optimized conditions to have intact tissue specific morphology for the entire culture period. Amylase positive intact acini were present at all time points of culture and acinar cells displayed a typical strong cell polarity. Amylase release from pancreas tissue slices decreased during culture, but maintained the characteristic bell-shaped dose-response curve to increasing caerulein concentrations and a ca. 4-fold maximal over basal release. Additionally, endocrine beta cell viability and function was well preserved until the end of the observation period. Our results show that the tissue slice culture platform provides unprecedented maintenance of pancreatic tissue specific morphology and function over a culture period for at least 4 days and in part even up to 1 week. This analytical advancement now allows mid -to long-term studies on the cell biology of pancreatic disorder pathogenesis and therapy in an intact surrounding in situ.

  6. Mouse pancreas tissue slice culture facilitates long-term studies of exocrine and endocrine cell physiology in situ.

    Directory of Open Access Journals (Sweden)

    Anja Marciniak

    Full Text Available Studies on pancreatic cell physiology rely on the investigation of exocrine and endocrine cells in vitro. Particularly, in the case of the exocrine tissue these studies have suffered from a reduced functional viability of acinar cells in culture. As a result not only investigations on dispersed acinar cells and isolated acini were limited in their potential, but also prolonged studies on pancreatic exocrine and endocrine cells in an intact pancreatic tissue environment were unfeasible. To overcome these limitations, we aimed to establish a pancreas tissue slice culture platform to allow long-term studies on exocrine and endocrine cells in the intact pancreatic environment. Mouse pancreas tissue slice morphology was assessed to determine optimal long-term culture settings for intact pancreatic tissue. Utilizing optimized culture conditions, cell specificity and function of exocrine acinar cells and endocrine beta cells were characterized over a culture period of 7 days. We found pancreas tissue slices cultured under optimized conditions to have intact tissue specific morphology for the entire culture period. Amylase positive intact acini were present at all time points of culture and acinar cells displayed a typical strong cell polarity. Amylase release from pancreas tissue slices decreased during culture, but maintained the characteristic bell-shaped dose-response curve to increasing caerulein concentrations and a ca. 4-fold maximal over basal release. Additionally, endocrine beta cell viability and function was well preserved until the end of the observation period. Our results show that the tissue slice culture platform provides unprecedented maintenance of pancreatic tissue specific morphology and function over a culture period for at least 4 days and in part even up to 1 week. This analytical advancement now allows mid -to long-term studies on the cell biology of pancreatic disorder pathogenesis and therapy in an intact surrounding in situ.

  7. Neuroglobin overexpression inhibits oxygen-glucose deprivation-induced mitochondrial permeability transition pore opening in primary cultured mouse cortical neurons.

    Science.gov (United States)

    Yu, Zhanyang; Liu, Ning; Li, Yadan; Xu, Jianfeng; Wang, Xiaoying

    2013-08-01

    Neuroglobin (Ngb) is an endogenous neuroprotective molecule against hypoxic/ischemic brain injury, but the underlying mechanisms remain largely undefined. Our recent study revealed that Ngb can bind to voltage-dependent anion channel (VDAC), a regulator of mitochondria permeability transition (MPT). In this study we examined the role of Ngb in MPT pore (mPTP) opening following oxygen-glucose deprivation (OGD) in primary cultured mouse cortical neurons. Co-immunoprecipitation (Co-IP) and immunocytochemistry showed that the binding between Ngb and VDAC was increased after OGD compared to normoxia, indicating the OGD-enhanced Ngb-VDAC interaction. Ngb overexpression protected primary mouse cortical neurons from OGD-induced neuronal death, to an extent comparable to mPTP opening inhibitor, cyclosporine A (CsA) pretreatment. We further measured the role of Ngb in OGD-induced mPTP opening using Ngb overexpression and knockdown approaches in primary cultured neurons, and recombinant Ngb exposure to isolated mitochondria. Same as CsA pretreatment, Ngb overexpression significantly reduced OGD-induced mPTP opening markers including mitochondria swelling, mitochondrial NAD(+) release, and cytochrome c (Cyt c) release in primary cultured neurons. Recombinant Ngb incubation significantly reduced OGD-induced NAD(+) release and Cyt c release from isolated mitochondria. In contrast, Ngb knockdown significantly increased OGD-induced neuron death, and increased OGD-induced mitochondrial NAD(+) release and Cyt c release as well, and these outcomes could be rescued by CsA pretreatment. In summary, our results demonstrated that Ngb overexpression can inhibit OGD-induced mPTP opening in primary cultured mouse cortical neurons, which may be one of the molecular mechanisms of Ngb's neuroprotection. Copyright © 2013 Elsevier Inc. All rights reserved.

  8. Identification and characterization of a 29-kilodalton protein from Mycobacterium tuberculosis culture filtrate recognized by mouse memory effector cells

    DEFF Research Database (Denmark)

    Rosenkrands, I; Rasmussen, P.B.; Carnio, M

    1998-01-01

    Culture filtrate proteins from Mycobacterium tuberculosis induce protective immunity in various animal models of tuberculosis. Two molecular mass regions (6 to 10 kDa and 24 to 36 kDa) of short-term culture filtrate are preferentially recognized by Th1 cells in animal models as well as by patients...... the antigen 85 complex was selected. The 29-kDa antigen (CFP29) was purified from M. tuberculosis short-term culture filtrate by thiophilic adsorption chromatography, anion-exchange chromatography, and gel filtration, In its native form, CFP29 forms a polymer with a high molecular mass. CFP29 was mapped......, and they both elicited the release of high levels of gamma interferon from mouse memory effector cells isolated during the recall of protective immunity to tuberculosis. Interspecies analysis by immunoblotting and PCR demonstrated that CFP29 is widely distributed in mycobacterial species....

  9. Direct effect of gonadal and contraceptive steroids on insulin release from mouse pancreatic islets in organ culture

    DEFF Research Database (Denmark)

    Nielsen, Jens Høiriis

    1984-01-01

    Sex steroids are supposed to contribute to the normal glucose homeostasis and to the altered glucose and insulin metabolism in pregnancy and during contraception. In the present study isolated mouse pancreatic islets were maintained in tissue culture medium RPMI 1640 supplemented with 0.5% newborn...... calf serum and 100 ng/ml of one of the following steroids: oestradiol, progesterone, testosterone, megestrol acetate, medroxyprogesterone, chlormadinone acetate, norethynodrel, norethindrone acetate, and ethynyloestradiol. Release of insulin to the culture medium was measured during a 2 week culture...... in the presence of oestradiol, progesterone, or testosterone were subjected to 30 min stimulation with 5.5, 11, 22 mmol/l glucose, only the progesterone-treated islets released more insulin in response to glucose than the control islets. It is concluded that progesterone and its derivatives have a direct effect...

  10. Eight-Shaped Hatching Increases the Risk of Inner Cell Mass Splitting in Extended Mouse Embryo Culture.

    Directory of Open Access Journals (Sweden)

    Zheng Yan

    Full Text Available Increased risk of monozygotic twinning (MZT has been shown to be associated with assisted reproduction techniques, particularly blastocyst culture. Interestingly, inner cell mass (ICM splitting in human '8'-shaped hatching blastocysts that resulted in MZT was reported. However, the underlying cause of MZT is not known. In this study, we investigated in a mouse model whether in vitro culture leads to ICM splitting and its association with hatching types. Blastocyst hatching was observed in: (i in vivo developed blastocysts and (ii-iii in vitro cultured blastocysts following in vivo or in vitro fertilization. We found that '8'-shaped hatching occurred with significantly higher frequency in the two groups of in vitro cultured blastocysts than in the group of in vivo developed blastocysts (24.4% and 20.4% versus 0.8%, respectively; n = 805, P < 0.01. Moreover, Oct4 immunofluorescence staining was performed to identify the ICM in the hatching and hatched blastocysts. Scattered and split distribution of ICM cells was observed around the small zona opening of '8'-shaped hatching blastocysts. This occurred at a high frequency in the in vitro cultured groups. Furthermore, we found more double OCT4-positive masses, suggestive of increased ICM splitting in '8'-shaped hatching and hatched blastocysts than in 'U'-shaped hatching and hatched blastocysts (12.5% versus 1.9%, respectively; n = 838, P < 0.01. Therefore, our results demonstrate that extended in vitro culture can cause high frequencies of '8'-shaped hatching, and '8'-shaped hatching that may disturb ICM herniation leading to increased risk of ICM splitting in mouse blastocysts. These results may provide insights into the increased risk of human MZT after in vitro fertilization and blastocyst transfer.

  11. Integrin-linked kinase is involved in matrix-induced hepatocyte differentiation

    International Nuclear Information System (INIS)

    Gkretsi, Vasiliki; Bowen, William C.; Yang, Yu; Wu, Chuanyue; Michalopoulos, George K.

    2007-01-01

    Hepatocytes have restricted proliferative capacity in culture and when cultured without matrix, lose the hepatocyte-specific gene expression and characteristic cellular micro-architecture. Overlay of matrix-preparations on de-differentiated hepatocytes restores differentiation. Integrin-linked kinase (ILK) is a cell-matrix-adhesion protein crucial in fundamental processes such as differentiation and survival. In this study, we investigated the role of ILK, and its binding partners PINCH, α-parvin, and Mig-2 in matrix-induced hepatocyte differentiation. We report here that ILK is present in the liver and localizes at cell-matrix adhesions of cultured hepatocytes. We also show that ILK, PINCH, α-parvin, and Mig-2 expression level is dramatically reduced in the re-differentiated hepatocytes. Interestingly, hepatocytes lacking ILK undergo matrix-induced differentiation but their differentiation is incomplete, as judged by monitoring cell morphology and production of albumin. Our results show that ILK and cell-matrix adhesion proteins play an important role in the process of matrix-induced hepatocyte differentiation

  12. Differentiation of mouse embryonic stem cells into cardiomyocytes via the hanging-drop and mass culture methods.

    Science.gov (United States)

    Fuegemann, Christopher J; Samraj, Ajoy K; Walsh, Stuart; Fleischmann, Bernd K; Jovinge, Stefan; Breitbach, Martin

    2010-12-01

    Herein, we describe two protocols for the in vitro differentiation of mouse embryonic stem cells (mESCs) into cardiomyocytes. mESCs are pluripotent and can be differentiated into cells of all three germ layers, including cardiomyocytes. The methods described here facilitate the differentiation of mESCs into the different cardiac subtypes (atrial-, ventricular-, nodal-like cells). The duration of cell culture determines whether preferentially early- or late-developmental stage cardiomyocytes can be obtained preferentially. This approach allows the investigation of cardiomyocyte development and differentiation in vitro, and also allows for the enrichment and isolation of physiologically intact cardiomyocytes for transplantation purposes. © 2010 by John Wiley & Sons, Inc.

  13. A Functional Assay for Putative Mouse and Human Definitive Endoderm using Chick Whole-Embryo Cultures

    DEFF Research Database (Denmark)

    Johannesson, Martina; Semb, Tor Henrik; Serup, Palle

    2012-01-01

    . Thus, the purpose of this study is to describe a method whereby the in vivo functionality of DE derived from ESCs can be assessed. Methods: By directed differentiation, putative DE was derived from human and mouse ESCs. This putative DE was subsequently transplanted into the endoderm of chick embryos...... to determine any occurrence of integration. Putative DE was analyzed by gene and protein expression prior to transplantation and 48 h post transplantation. Results: Putative DE, derived from mouse and human ESCs, was successfully integrated within the chick endoderm. Endoderm-specific genes were expressed...... result show that putative DE integrates with the chick endoderm and participate in the development of the chicken gut, indicating the generation of functional DE from ESCs. This functional assay can be used to assess the generation of functional DE derived from both human and mouse ESCs and provides...

  14. Characterization of primary and secondary cultures of astrocytes prepared from mouse cerebral cortex

    DEFF Research Database (Denmark)

    Skytt, Dorte Marie; Madsen, Karsten Kirkegaard; Pajecka, Kamilla

    2010-01-01

    Astrocyte cultures were prepared from cerebral cortex of new-born and 7-day-old mice and additionally, the cultures from new-born animals were passaged as secondary cultures. The cultures were characterized by immunostaining for the astrocyte markers glutamine synthetase (GS), glial fibrillary ac...... cerebral cortex of 7-day-old mice have metabolic and functional properties indistinguishable from those of classical astrocyte cultures prepared from neocortex of new-born animals. This provides flexibility with regard to preparation and use of these cultures for a variety of purposes....

  15. Role of macrophages in the immune response to hepatocytes

    International Nuclear Information System (INIS)

    Bumgardner, G.L.; Chen, S.; Almond, S.P.; Ascher, N.L.; Payne, W.D.; Matas, A.J.

    1990-01-01

    The purpose of this study was to determine the role of host macrophages in the development of allospecific cytolytic T cells (allo-CTLs) in response to purified allogeneic MHC Class I+, Class II- hepatocytes in vivo in hepatocyte sponge matrix allografts (HC-SMA). Depletion of antigen-presenting cells (APCs) from responder splenocytes in mixed lymphocyte hepatocyte culture (MLHC) inhibits the development of allo-CTLs in response to purified hepatocytes. First the ability of sponge macrophages to function as accessory cells in indirect presentation of hepatocyte Class I antigen was tested in MLHC. We found that addition of irradiated sponge cells (a source of sponge macrophages) restored the development of allo-CTLs in MLHC depleted of responder APCs. Therefore, radioresistant sponge macrophages can function as accessory cells in MLHC. We next employed silica as an immunotherapy targeted against host macrophages and assessed the effect on development of allo-CTLs in HC-SMA. We found that local (intrasponge) silica treatment completely inhibited the development of allo-CTLs in HC-SMA. Combined local and systemic silica treatment resulted in inhibition of allocytotoxicity comparable to local silica treatment alone in the doses tested. We conclude that host macrophages which infiltrate HC-SMA can function as accessory cells in vitro in MLHC and that both infiltrating host macrophages and lymphocytes participate in the development of an alloimmune response to purified hepatocytes in vivo. This interaction may involve indirect antigen presentation of hepatocyte Class I antigen by macrophages to host lymphocytes which accumulate in HC-SMA

  16. Radiotoxicity and incorporation of methyl-tritiated-thymidine on preimplantation mouse embryo. In vitro fertilization and culture

    International Nuclear Information System (INIS)

    Kikuchi, O.K.; Ohyama, H.; Yamada, T.

    1993-04-01

    In the present work different concentrations of methyl- 3 H-thymidine was added to the culture medium micro drops containing the mouse zygotes at pro nuclear stage and the embryos were cultured in vitro at 37 0 C in a humidified atmosphere with 5% of CO 2 for four days up to the expanded blastocyst stage, the established end point to calculate the L D 50 lethal dose. The blastocyst formation rate decreased with increasing concentration of tritium in medium and a value of 2.4 X 10 3 Bq/ml for L D 50 was obtained. The 3 H-Td R incorporation by the embryo during the preimplantation period was low at the beginning and increased quickly during the morula and the blastocyst development. (author)

  17. Functional crosstalk in culture between macrophages and trigeminal sensory neurons of a mouse genetic model of migraine

    Directory of Open Access Journals (Sweden)

    Franceschini Alessia

    2012-11-01

    Full Text Available Abstract Background Enhanced activity of trigeminal ganglion neurons is thought to underlie neuronal sensitization facilitating the onset of chronic pain attacks, including migraine. Recurrent headache attacks might establish a chronic neuroinflammatory ganglion profile contributing to the hypersensitive phenotype. Since it is difficult to study this process in vivo, we investigated functional crosstalk between macrophages and sensory neurons in primary cultures from trigeminal sensory ganglia of wild-type (WT or knock-in (KI mice expressing the Cacna1a gene mutation (R192Q found in familial hemiplegic migraine-type 1. After studying the number and morphology of resident macrophages in culture, the consequences of adding host macrophages on macrophage phagocytosis and membrane currents mediated by pain-transducing P2X3 receptors on sensory neurons were examined. Results KI ganglion cultures constitutively contained a larger number of active macrophages, although no difference in P2X3 receptor expression was found. Co-culturing WT or KI ganglia with host macrophages (active as much as resident cells strongly stimulated single cell phagocytosis. The same protocol had no effect on P2X3 receptor expression in WT or KI co-cultures, but it largely enhanced WT neuron currents that grew to the high amplitude constitutively seen for KI neurons. No further potentiation of KI neuronal currents was observed. Conclusions Trigeminal ganglion cultures from a genetic mouse model of migraine showed basal macrophage activation together with enhanced neuronal currents mediated by P2X3 receptors. This phenotype could be replicated in WT cultures by adding host macrophages, indicating an important functional crosstalk between macrophages and sensory neurons.

  18. Functional crosstalk in culture between macrophages and trigeminal sensory neurons of a mouse genetic model of migraine.

    Science.gov (United States)

    Franceschini, Alessia; Nair, Asha; Bele, Tanja; van den Maagdenberg, Arn Mjm; Nistri, Andrea; Fabbretti, Elsa

    2012-11-21

    Enhanced activity of trigeminal ganglion neurons is thought to underlie neuronal sensitization facilitating the onset of chronic pain attacks, including migraine. Recurrent headache attacks might establish a chronic neuroinflammatory ganglion profile contributing to the hypersensitive phenotype. Since it is difficult to study this process in vivo, we investigated functional crosstalk between macrophages and sensory neurons in primary cultures from trigeminal sensory ganglia of wild-type (WT) or knock-in (KI) mice expressing the Cacna1a gene mutation (R192Q) found in familial hemiplegic migraine-type 1. After studying the number and morphology of resident macrophages in culture, the consequences of adding host macrophages on macrophage phagocytosis and membrane currents mediated by pain-transducing P2X3 receptors on sensory neurons were examined. KI ganglion cultures constitutively contained a larger number of active macrophages, although no difference in P2X3 receptor expression was found. Co-culturing WT or KI ganglia with host macrophages (active as much as resident cells) strongly stimulated single cell phagocytosis. The same protocol had no effect on P2X3 receptor expression in WT or KI co-cultures, but it largely enhanced WT neuron currents that grew to the high amplitude constitutively seen for KI neurons. No further potentiation of KI neuronal currents was observed. Trigeminal ganglion cultures from a genetic mouse model of migraine showed basal macrophage activation together with enhanced neuronal currents mediated by P2X3 receptors. This phenotype could be replicated in WT cultures by adding host macrophages, indicating an important functional crosstalk between macrophages and sensory neurons.

  19. Identification of a population of cells with hematopoietic stem cell properties in mouse aorta-gonad-mesonephros cultures

    International Nuclear Information System (INIS)

    Nobuhisa, Ikuo; Ohtsu, Naoki; Okada, Seiji; Nakagata, Naomi; Taga, Tetsuya

    2007-01-01

    The aorta-gonad-mesonephros (AGM) region is a primary source of definitive hematopoietic cells in the midgestation mouse embryo. In cultures of dispersed AGM regions, adherent cells containing endothelial cells are observed first, and then non-adherent hematopoietic cells are produced. Here we report on the characterization of hematopoietic cells that emerge in the AGM culture. Based on the expression profiles of CD45 and c-Kit, we defined three cell populations: CD45 low c-Kit + cells that had the ability to form hematopoietic cell colonies in methylcellulose media and in co-cultures with stromal cells; CD45 low c-Kit - cells that showed a granulocyte morphology; CD45 high c-Kit low/- that exhibited a macrophage morphology. In co-cultures of OP9 stromal cells and freshly prepared AGM cultures, CD45 low c-Kit + cells from the AGM culture had the abilities to reproduce CD45 low c-Kit + cells and differentiate into CD45 low c-Kit - and CD45 high c-Kit low/- cells, whereas CD45 low c-Kit - and CD45 high c-Kit low/- did not produce CD45 low c-Kit + cells. Furthermore, CD45 low c-Kit + cells displayed a long-term repopulating activity in adult hematopoietic tissue when transplanted into the liver of irradiated newborn mice. These results indicate that CD45 low c-Kit + cells from the AGM culture have the potential to reconstitute multi-lineage hematopoietic cells

  20. Investigation of the interaction of radiation and cardiotoxic anticancer agents using a fetal mouse heart organ culture system

    International Nuclear Information System (INIS)

    Kimler, B.F.; Rethorst, R.D.; Cox, G.G.

    1985-01-01

    The fetal mouse heart organ culture was utilized in an attempt to predict the cardiotoxic effects of combinations of radiation, Adriamycin (ADR), and Dihydroxyanthraquinone (DHAQ), antineoplastic agents which have been shown to produce clinical cardiomyopathy. Seventeen-day fetal hearts were removed and placed in a culture system of micro-titer plates. A single heart was placed in each well on a piece of aluminum mesh to keep the heart above the culture medium but bathed by capillary action. The plates were then placed in a 100% oxygen environment at 37 0 C. Treatments were performed on day 1 after culture: radiation doses (Cs-137) of 10, 20, or 40 Gy; drug treatment with 10, 30, or 100 μg/ml of ADR; 5, 20, or 50 μg/ml of DHAQ; and combinations and sequences of drug and radiation. Hearts were checked every day for functional activity as evidenced by a continuous heart beat. Untreated hearts beat rhythmically for up to 9 days; treated hearts stopped beating earlier. Using an endpoint of functional retention time, dose response curves were obtained for all individual agents and for combinations of agents. This system may help to predict the cardiotoxic effects that result from the use of these drugs and radiation. It may also aid in the development of new anthracycline chemotherapeutic agents that lack cardiotoxicity

  1. Human hepatocyte depletion in the presence of HIV-1 infection in dual reconstituted humanized mice

    Science.gov (United States)

    Wang, Weimin; Cheng, Yan; Makarov, Edward; Ganesan, Murali; Gebhart, Catherine L.; Gorantla, Santhi; Osna, Natalia

    2018-01-01

    ABSTRACT Human immunodeficiency virus type 1 (HIV-1) infection impairs liver function, and liver diseases have become a leading cause of morbidity in infected patients. The immunopathology of liver damage caused by HIV-1 remains unclear. We used chimeric mice dually reconstituted with a human immune system and hepatocytes to address the relevance of the model to pathobiology questions related to human hepatocyte survival in the presence of systemic infection. TK-NOG males were transplanted with mismatched human hematopoietic stem/progenitor cells and hepatocytes, human albumin concentration and the presence of human immune cells in blood were monitored for hepatocytes and immune reconstitution, and mice were infected with HIV-1. HIV-1-infected animals showed a decline in human albumin concentration with a significant reduction in percentage of human hepatocytes compared to uninfected mice. The decrease in human albumin levels correlated with a decline in CD4+ cells in the liver and with an increase in HIV-1 viral load. HIV-1 infection elicited proinflammatory response in the immunological milieu of the liver in HIV-infected mice compared to uninfected animals, as determined by upregulation of IL23, CXCL10 and multiple toll-like receptor expression. The inflammatory reaction associated with HIV-1 infection in vivo could contribute to the depletion and dysfunction of hepatocytes. The dual reconstituted TK-NOG mouse model is a feasible platform to investigate hepatocyte-related HIV-1 immunopathogenesis. This article has an associated First Person interview with the first author of the paper. PMID:29361613

  2. Human hepatocyte depletion in the presence of HIV-1 infection in dual reconstituted humanized mice

    Directory of Open Access Journals (Sweden)

    Raghubendra Singh Dagur

    2018-02-01

    Full Text Available Human immunodeficiency virus type 1 (HIV-1 infection impairs liver function, and liver diseases have become a leading cause of morbidity in infected patients. The immunopathology of liver damage caused by HIV-1 remains unclear. We used chimeric mice dually reconstituted with a human immune system and hepatocytes to address the relevance of the model to pathobiology questions related to human hepatocyte survival in the presence of systemic infection. TK-NOG males were transplanted with mismatched human hematopoietic stem/progenitor cells and hepatocytes, human albumin concentration and the presence of human immune cells in blood were monitored for hepatocytes and immune reconstitution, and mice were infected with HIV-1. HIV-1-infected animals showed a decline in human albumin concentration with a significant reduction in percentage of human hepatocytes compared to uninfected mice. The decrease in human albumin levels correlated with a decline in CD4+ cells in the liver and with an increase in HIV-1 viral load. HIV-1 infection elicited proinflammatory response in the immunological milieu of the liver in HIV-infected mice compared to uninfected animals, as determined by upregulation of IL23, CXCL10 and multiple toll-like receptor expression. The inflammatory reaction associated with HIV-1 infection in vivo could contribute to the depletion and dysfunction of hepatocytes. The dual reconstituted TK-NOG mouse model is a feasible platform to investigate hepatocyte-related HIV-1 immunopathogenesis. This article has an associated First Person interview with the first author of the paper.

  3. DNA repair ability of cultured cells derived from mouse embryos in comparison with human cells

    International Nuclear Information System (INIS)

    Yaki, T.

    1982-01-01

    DNA repair in mouse cells derived from embryos of 3 inbred strains were investigated in comparison with that in human cells. The levels of unscheduled DNA synthesis after UV irradiation appeared to change at different passages, but capacities of host-cell reactivation of UV-irradiated herpes simplex virus were always reduced to the same levels as those in xeroderma pigmentosum cells. This implied that mouse cells are reduced in excision-repair capacities and that the apparently high levels of unscheduled DNA synthesis at certain passages are not quantitatively related to high levels of cell survival. Essentially no differences in DNA repair were noted among 3 strains - BALB/c, C3H/He and C57BL/10. (orig.)

  4. Transdifferentiated rat pancreatic progenitor cells (AR42J-B13/H) respond to phenobarbital in a rat hepatocyte-specific manner.

    Science.gov (United States)

    Osborne, M; Haltalli, M; Currie, R; Wright, J; Gooderham, N J

    2016-07-01

    Phenobarbital (PB) is known to produce species-specific effects in the rat and mouse, being carcinogenic in certain mouse strains, but only in rats if treated after a DNA damaging event. PB treatment in the rat and mouse also produces disparate effects on cell signalling and miRNA expression profiles. These responses are induced by short term and prolonged PB exposure, respectively, with the latter treatments being difficult to examine mechanistically in primary hepatocytes due to rapid loss of the original hepatic phenotype and limited sustainability in culture. Here we explore the rat hepatocyte-like B13/H cell line as a model for hepatic response to PB exposure in both short-term and longer duration treatments. We demonstrate that PB with Egf treatment in the B13/H cells resulted in a significant increase in Erk activation, as determined by the ratio of phospho-Erk to total Erk, compared to Egf alone. We also show that an extended treatment with PB in the B13/H cells produces a miRNA response similar to that seen in the rat in vivo, via the time-dependent induction of miR-182/96. Additionally, we confirm that B13/H cells respond to Car activators in a typical rat-specific manner. These data suggest that the B13/H cells produce temporal responses to PB that are comparable to those reported in short-term primary rat hepatocyte cultures and in the longer term are similar to those in the rat in vivo. Finally, we also show that Car-associated miR-122 expression is decreased by PB treatment in B13/H cells, a PB-induced response that is common to the rat, mouse and human. We conclude that the B13/H cell system produces a qualitative response comparable to the rat, which is different to the response in the mouse, and that this model could be a useful tool for exploring the functional consequences of PB-sensitive miRNA changes and resistance to PB-mediated tumours in the rat. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  5. No evidence for protective erythropoietin alpha signalling in rat hepatocytes

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    Frede Stilla

    2009-04-01

    Full Text Available Abstract Background Recombinant human erythropoietin alpha (rHu-EPO has been reported to protect the liver of rats and mice from ischemia-reperfusion injury. However, direct protective effects of rHu-EPO on hepatocytes and the responsible signalling pathways have not yet been described. The aim of the present work was to study the protective effect of rHu-EPO on warm hypoxia-reoxygenation and cold-induced injury to hepatocytes and the rHu-EPO-dependent signalling involved. Methods Loss of viability of isolated rat hepatocytes subjected to hypoxia/reoxygenation or incubated at 4°C followed by rewarming was determined from released lactate dehydrogenase activity in the absence and presence of rHu-EPO (0.2–100 U/ml. Apoptotic nuclear morphology was assessed by fluorescence microscopy using the nuclear fluorophores H33342 and propidium iodide. Erythropoietin receptor (EPOR, EPO and Bcl-2 mRNAs were quantified by real time PCR. Activation of JAK-2, STAT-3 and STAT-5 in hepatocytes and rat livers perfused in situ was assessed by Western blotting. Results In contrast to previous in vivo studies on ischemia-reperfusion injury to the liver, rHu-EPO was without any protective effect on hypoxic injury, hypoxia-reoxygenation injury and cold-induced apoptosis to isolated cultured rat hepatocytes. EPOR mRNA was identified in these cells but specific detection of the EPO receptor protein was not possible due to the lack of antibody specificity. Both, in the cultured rat hepatocytes (10 U/ml for 15 minutes and in the rat liver perfused in situ with rHu-EPO (8.9 U/ml for 15 minutes no evidence for EPO-dependent signalling was found as indicated by missing effects of rHu-EPO on phosphorylation of JAK-2, STAT-3 and STAT-5 and on the induction of Bcl-2 mRNA. Conclusion Together, these results indicate the absence of any protective EPO signalling in rat hepatocytes. This implies that the protection provided by rHu-EPO in vivo against ischemia-reperfusion and

  6. Magnetic cell labeling of primary and stem cell-derived pig hepatocytes for MRI-based cell tracking of hepatocyte transplantation.

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    Dwayne R Roach

    Full Text Available Pig hepatocytes are an important investigational tool for optimizing hepatocyte transplantation schemes in both allogeneic and xenogeneic transplant scenarios. MRI can be used to serially monitor the transplanted cells, but only if the hepatocytes can be labeled with a magnetic particle. In this work, we describe culture conditions for magnetic cell labeling of cells from two different pig hepatocyte cell sources; primary pig hepatocytes (ppHEP and stem cell-derived hepatocytes (PICM-19FF. The magnetic particle is a micron-sized iron oxide particle (MPIO that has been extensively studied for magnetic cell labeling for MRI-based cell tracking. ppHEP could endocytose MPIO with labeling percentages as high as 70%, achieving iron content as high as ~55 pg/cell, with >75% viability. PICM-19FF had labeling >97%, achieving iron content ~38 pg/cell, with viability >99%. Extensive morphological and functional assays indicated that magnetic cell labeling was benign to the cells. The results encourage the use of MRI-based cell tracking for the development and clinical use of hepatocyte transplantation methodologies. Further, these results generally highlight the importance of functional cell assays in the evaluation of contrast agent biocompatibility.

  7. Effects of Treatment with Bone Morphogenetic Protein 4 and Co-culture on Expression of Piwil2 Gene in Mouse Differentiated Embryonic Stem Cells

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    Mehdi Forouzandeh-Moghadam

    2009-01-01

    Full Text Available Background: Specific growth factors and feeder layers seem to have important roles in in vitroembryonic stem cells (ESCs differentiation. In this study,the effects of bone morphogenetic protein4 (BMP4 and mouse embryonic fibroblasts (MEFs co-culture system on germ cell differentiationfrom mouse ESCs were studied.Materials and Methods: Cell suspension was prepared from one-day-old embryoid body (EBand cultured for four days in DMEM medium containing 20% fetal bovine serum (FBS in thefollowing groups: simple culture (SC, simple culture with BMP4 (SCB, co-culture (CO-C andco-culture with BMP4 (CO-CB. Expression of piwi-like homolog 2 (Piwil2, the germ cell-specificgene, was evaluated in the different study groups by using quantitative real time polymerase chainreaction (RT-PCR. Testis was used as a positive control.Results: The maximum and minimum Piwil2 expression was observed in SC and SCB groups,respectively. A significant difference was observed in Piwil2 expression between SCB and otherstudy groups (p<0.05.Conclusion: The findings of this study showed that neither the addition of BMP4 in culture mediumnor the use of MEFs as a feeder layer have a positive effect on late germ cell induction from mouseESCs.

  8. Apoptosis induced by glufosinate ammonium in the neuroepithelium of developing mouse embryos in culture.

    Science.gov (United States)

    Watanabe, T

    1997-01-24

    Glufosinate ammonium structurally resembles glutamate and blocks glutamine synthetase. Glufosinate was recently found to be dysmorphogenic in mammals in vitro. The present study examined the cell death induced specifically by glufosinate in the neuroepithelium of mouse embryos. Electron micrograph revealed characteristic chromatin condensation and segregation, extracellular apoptotic bodies, and cell fragments phagocytosed in macrophages in the neuroepithelium of the brain vesicle and neural tube. Moreover neuroepithelial cells undergoing DNA fragmentation were histochemically identified. DNA gel electrophoresis of the neuroepithelial layer revealed a DNA ladder. These observations demonstrate that glufosinate specifically induced apoptosis in the neuroepithelium of embryos.

  9. Optimization of the isolation and cultivation of Cyprinus carpio primary hepatocytes.

    Science.gov (United States)

    Yanhong, Fan; Chenghua, He; Guofang, Liu; Haibin, Zhang

    2008-10-01

    The aquatic environment is affected by numerous chemical contaminants. There is an increasing need to identify these chemicals and to evaluate their potential toxicity towards aquatic life. In this research we optimized techniques for primary cell culture of Cyprinus carpio hepatocytes as one adjunct model for ecotoxicological evaluation of the potential hazards of xenobiotics in the aquatic environment. In this study, Cyprinus carpio hepatocytes were isolated by mechanical separation, two-step collagenase perfusion, and pancreatin digestion. The hepatocytes or parenchymal cells could be separated from cell debris and from non-parenchymal cells by low-speed centrifugation (Percoll gradient centrifugation). The harvested hepatocytes were suspended in DMEM, M199 (cultured in 5% CO(2)), or L-15 (cultured without 5% CO(2)) medium then cultured at 17, 27, or 37 degrees C. Cell yield was counted by use of a hemocytometer, and the viability of the cells was assessed by use of the Trypan blue exclusion test. Results from these studies showed that the best method of isolation was pancreatin digestion (the cell yield was 2.7 x 10(8) per g (liver weight) and the viability was 98.4%) and the best medium was M199 (cultured in 5% CO(2)) or L-15 (cultured without 5% CO(2)). The optimum culture temperature was 27 degrees C. The primary hepatocytes culture of Cyprimus carpio grew well and satisfied requirements for most toxicological experiments in this condition.

  10. Correlation of initiating potency of skin carcinogens with potency to induce resistance to terminal differentiation in cultured mouse keratinocytes

    International Nuclear Information System (INIS)

    Kilkenny, A.E.; Morgan, D.; Spangler, E.F.; Yuspa, S.H.

    1985-01-01

    The induction by chemical carcinogens of resistance to terminal differentiation in cultured mouse keratinocytes has been proposed to represent a cellular change associated with the initiation phase of skin carcinogenesis. Previous results with this culture model indicated that the number of differentiation-resistant foci was correlated with the dose and known potency for several chemical carcinogens. Assay conditions were optimized to provide quantitative results for screening a variety of carcinogens for their potency as inducers of foci resistant to terminal differentiation. Eight skin initiators of varying potency and from different chemical classes and ultraviolet light were studied for their activity to induce this alteration in cultured epidermal cells from newborn BALB/c mice. There was an excellent positive correlation for the potency of these agents as initiators in vivo and as inducers of altered differentiation in vitro. The induction of resistant foci was independent of the relative cytotoxic effects of each agent except where cytotoxicity was extensive and reduced the number of foci. The results support the hypothesis that initiation of carcinogenesis in skin results in an alteration in the program of epidermal cell differentiation. The results also suggest that the assay is useful for identifying relative potency classes (strong, moderate, weak) of initiating agents

  11. Cell death induced on cell cultures and nude mouse skin by non-thermal, nanosecond-pulsed generated plasma.

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    Arnaud Duval

    Full Text Available Non-thermal plasmas are gaseous mixtures of molecules, radicals, and excited species with a small proportion of ions and energetic electrons. Non-thermal plasmas can be generated with any high electro-magnetic field. We studied here the pathological effects, and in particular cell death, induced by nanosecond-pulsed high voltage generated plasmas homogeneously applied on cell cultures and nude mouse skin. In vitro, Jurkat cells and HMEC exhibited apoptosis and necrosis, in dose-dependent manner. In vivo, on nude mouse skin, cell death occurred for doses above 113 J/cm(2 for the epidermis, 281 J/cm(2 for the dermis, and 394 J/cm(2 for the hypodermis. Using electron microscopy, we characterized apoptosis for low doses and necrosis for high doses. We demonstrated that these effects were not related to thermal, photonic or pH variations, and were due to the production of free radicals. The ability of cold plasmas to generate apoptosis on cells in suspension and, without any sensitizer, on precise skin areas, opens new fields of application in dermatology for extracorporeal blood cell treatment and the eradication of superficial skin lesions.

  12. Mouse preimplantation embryo responses to culture medium osmolarity include increased expression of CCM2 and p38 MAPK activation

    Directory of Open Access Journals (Sweden)

    Watson Andrew J

    2007-01-01

    Full Text Available Abstract Background Mechanisms that confer an ability to respond positively to environmental osmolarity are fundamental to ensuring embryo survival during the preimplantation period. Activation of p38 mitogen-activated protein kinase (MAPK occurs following exposure to hyperosmotic treatment. Recently, a novel scaffolding protein called Osmosensing Scaffold for MEKK3 (OSM was linked to p38 MAPK activation in response to sorbitol-induced hypertonicity. The human ortholog of OSM is cerebral cavernous malformation 2 (CCM2. The present study was conducted to investigate whether CCM2 is expressed during mouse preimplantation development and to determine whether this scaffolding protein is associated with p38 MAPK activation following exposure of preimplantation embryos to hyperosmotic environments. Results Our results indicate that Ccm2 along with upstream p38 MAPK pathway constituents (Map3k3, Map2k3, Map2k6, and Map2k4 are expressed throughout mouse preimplantation development. CCM2, MAP3K3 and the phosphorylated forms of MAP2K3/MAP2K6 and MAP2K4 were also detected throughout preimplantation development. Embryo culture in hyperosmotic media increased p38 MAPK activity in conjunction with elevated CCM2 levels. Conclusion These results define the expression of upstream activators of p38 MAPK during preimplantation development and indicate that embryo responses to hyperosmotic environments include elevation of CCM2 and activation of p38 MAPK.

  13. Nonylphenol-mediated CYP induction is PXR-dependent: The use of humanized mice and human hepatocytes suggests that hPXR is less sensitive than mouse PXR to nonylphenol treatment

    International Nuclear Information System (INIS)

    Mota, Linda C.; Barfield, Christina; Hernandez, Juan P.; Baldwin, William S.

    2011-01-01

    Nonylphenol (NP), a by-product of alkylphenol ethoxylates, is a pervasive surfactant that activates the xenosensing nuclear receptor, the pregnane X-receptor (PXR) in transactivation assays in vitro. We are interested in determining if NP activates PXR in vivo, determining if hPXR and mPXR act similarly, and investigating the role of PXR in protecting individuals from NP. Wild-type (WT), PXR-null, and humanized PXR (hPXR) mice were treated with NP at 0, 50 or 75 mg/kg/day for one week, and cytochrome P450 (CYP) induction, liver histopathology, and serum NP concentrations were examined. WT mice treated with NP showed induction of Cyp2b, and male-specific induction of Cyp2c and Cyp3a. CYPs were not induced in PXR-null mice, demonstrating that PXR is necessary for NP-mediated CYP induction. CAR-mediated CYP induction was not observed in the PXR-null mice despite previous data demonstrating that NP is also a CAR activator. hPXR mice only showed moderate Cyp induction, suggesting that hPXR is not as sensitive to NP as mPXR in vivo. NP-mediated Cyp3a induction from three human hepatocyte donors was not significant, confirming that hPXR is not very sensitive to NP-mediated CYP induction. Lastly, mice with PXR (mPXR and hPXR) showed lower NP serum concentrations than PXR-null mice treated with NP suggesting that PXR plays a role in decreasing liver toxicity by basally regulating phase I-III detoxification enzymes that promote the metabolism and elimination of NP. In summary, PXR is required for NP-mediated CYP-induction, mPXR mediates greater CYP induction than hPXR in vivo, and the presence of PXR, especially mPXR, is associated with altered histopathology and increased clearance of NP.

  14. Flow cytometric measurement of the metabolism of benzo[a]pyrene by mouse liver cells in culture

    International Nuclear Information System (INIS)

    Bartholomew, J.C.; Wade, C.G.; Dougherty, K.K.

    1984-01-01

    The metabolism of benzo[a]pyrene in individual cells was monitored by flow cytometry. The measurements are based on the alterations that occur in the fluorescence emission spectrum of benzo[a]pyrene when it is converted to various metabolites. Using present instrumentation the technique could easily detect 1x10 6 molecules per cells of benzo[a]pyrene and 1x10 7 molecules per cell of the diol epoxide. The analysis of C3H IOT 1/2 mouse fibroblasts growing in culture indicated that there was heterogeneity in the conversion of the parent compound into diol epoxide derivatives suggesting that some variation in sensitivity to transformation by benzo[a]pyrene may be due to differences in cellular metabolism. The technique allows sensitive detection of metabolites in viable cells, and provides a new approach to the study of factors that influence both metabolism and transformation. (orig.)

  15. Protective effects of cultured and fermented ginseng extracts against scopolamine-induced memory loss in a mouse model.

    Science.gov (United States)

    Han, Song-Hee; Kim, Sung-June; Yun, Young Won; Nam, Sang Yoon; Lee, Hu-Jang; Lee, Beom-Jun

    2018-03-01

    This study was performed to investigate the effect of a concentrate of fermented wild ginseng root culture (HLJG0701) on memory improvement in the scopolamine (SPL)-induced memory-deficient mouse model. Eight-week-old male ICR mice were used to evaluate the protective effect of HLJG0701 against the SPL-induced memory loss animal model. The Morris water maze test, which measures hippocampus-dependent learning ability, and the Y-maze test, a short-term memory assessment test, were performed and related markers were analyzed. HLJG0701-treated groups displayed significantly reduced acetylcholinesterase activity and increased acetylcholine level compared with the SPL-administered group (SPL-G) ( P memory loss by inhibiting acetylcholinesterase activity and preventing acetylcholine deficiency.

  16. Acute response of mouse kidney clonogens to fractionated irradiation in situ and then assayed in primary culture

    International Nuclear Information System (INIS)

    Yeemin Jen; Hendry, J.H.

    1991-01-01

    The radiosensitivity of mouse kidney cells after in situ single-dose, 2, 8, and 16 fraction X-irradiations was measured in primary culture using a clonogenic assay. The assay was made 12 h after single doses or 12 h after the last dose of the multifraction regimens. When analysed using the linear-quadratic model, as predicted the individual α components for all the different fractionation schedules were not significantly different, and the changes in the β values were consistent with those expected on the basis of the reciprocal fraction numbers. When all four data sets were integrated to derive a common α/β ratio, the result was 4.4±1.3 (1SE) Gy, or 2.8±0.9 Gy (a better fit) if the single-dose data set was excluded. These values fall into the range reported for kidney using assays of tissue function at long times after irradiation. (author)

  17. Effect of combined treatments of pepleomycin with x-ray in cultured cells of a mouse squamous cell carcinoma

    International Nuclear Information System (INIS)

    Yokota, Masahiko; Wakamatsu, Yoshiko; Sairenji, Eiko; Ohara, Hirosi.

    1986-01-01

    Cultured SQ-1979 cells of C3H mouse squamous cell carcinomas in logarithmic growth phase were treated with either pepleomycin (PEP) or bleomycin (BLM), alone or in combination with X-rays. The lethal effect of PEP on the SQ cells was greater than that of BLM. The survival curves of the cells were biphasic at concentrations examined (5 to 60 μg/ml) of PEP. The survival depended on the concentration and treatment time up to 3 hr. The cells treated with BLM showed biphasic survival curves also. Regarding combined effects of either PEP or BLM and X-rays, there was no significant difference in the survival curves between the group with X-rays and the group with X-rays and drug. When a drug was given 4 hr after exposure to X-rays rather than before that, synergistic action of the drug and X-rays was attained. This was marked for PEP. (Namekawa, K.)

  18. mRNA transfection of mouse and human neural stem cell cultures

    OpenAIRE

    McLenachan, Samuel; Zhang, D.; Palomo, A.B.; Edel, Michael John; Chen, F.K.

    2013-01-01

    The use of synthetic mRNA as an alternative gene delivery vector to traditional DNA-based constructs provides an effective method for inducing transient gene expression in cell cultures without genetic modification. Delivery of mRNA has been proposed as a safer alternative to viral vectors in the induction of pluripotent cells for regenerative therapies. Although mRNA transfection of fibroblasts, dendritic and embryonic stem cells has been described, mRNA delivery to neurosphere cultures has ...

  19. 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

  20. Acidosis-induced downregulation of hepatocyte mitochondrial aquaporin-8 and ureagenesis from ammonia.

    Science.gov (United States)

    Molinas, Sara M; Soria, Leandro R; Marrone, Julieta; Danielli, Mauro; Trumper, Laura; Marinelli, Raúl A

    2015-08-01

    It has been proposed that, during metabolic acidosis, the liver downregulates mitochondrial ammonia detoxification via ureagenesis, a bicarbonate-consuming process. Since we previously demonstrated that hepatocyte mitochondrial aquaporin-8 channels (mtAQP8) facilitate the uptake of ammonia and its metabolism into urea, we studied whether mtAQP8 is involved in the liver adaptive response to acidosis. Primary cultured rat hepatocytes were adapted to acidosis by exposing them to culture medium at pH 7.0 for 40 h. Control cells were exposed to pH 7.4. Hepatocytes exposed to acid medium showed a decrease in mtAQP8 protein expression (-30%, p ammonia was assessed by incubating the cells with (15)N-labeled ammonia and measuring (15)N-labeled urea synthesis by nuclear magnetic resonance. Reduced ureagenesis was found in acidified hepatocytes (-31%, p ammonia in response to acidosis.

  1. Proliferation of mouse endometrial stromal cells in culture is highly sensitive to lysophosphatidic acid signaling

    International Nuclear Information System (INIS)

    Aikawa, Shizu; Kano, Kuniyuki; Inoue, Asuka; Aoki, Junken

    2017-01-01

    Endometrial stromal cells (ESCs) proliferate rapidly both in vivo and in vitro. Here we show that proliferation of ESCs in vitro is strongly dependent on lysophosphatidic acid (LPA) signaling. LPA is produced by autotaxin (ATX) and induces various kinds of cellular processes including migration, proliferation and inhibition of cell death possibly through six G protein-coupled receptors (LPA 1-6 ). We found that ESCs proliferated rapidly in vitro in an autocrine manner and that the proliferation was prominently suppressed by either an ATX inhibitor (ONO-8430506) or an LPA 1/3 antagonist (Ki16425). Among the cells lines tested, mouse ESCs were the most sensitive to these inhibitors. Proliferation of ESCs isolated from either LPA 1 - or LPA 3 -deficient mice was comparable to proliferation of ESCs isolated from control mice. An LPA receptor antagonist (AM095), which was revealed to be a dual LPA 1 /LPA 3 antagonist, also suppressed the proliferation of ESCs. The present results show that LPA signaling has a critical role in the proliferation of ESCs, and that this role is possibly mediated redundantly by LPA 1 and LPA 3 . - Highlights: • Uterine endometrial stromal cells (ESCs) proliferate rapidly both in vivo and in vitro. • ESCs proliferated in vitro in an autocrine fashion. • Proliferation of mouse ESCs was prominently suppressed by inhibitors of lysophosphatidic acid (LPA) signaling. • LPA receptors, LPA 1 and LPA 3 , had redundant role in supporting the proliferation of ESCs.

  2. TGFbeta Induces Binucleation/Polyploidization in Hepatocytes through a Src-Dependent Cytokinesis Failure.

    Directory of Open Access Journals (Sweden)

    Marco De Santis Puzzonia

    Full Text Available In all mammals, the adult liver shows binucleated as well as mononucleated polyploid hepatocytes. The hepatic polyploidization starts after birth with an extensive hepatocyte binucleation and generates hepatocytes of several ploidy classes. While the functional significance of hepatocyte polyploidy is becoming clearer, how it is triggered and maintained needs to be clarified. Aim of this study was to identify a major inducer of hepatocyte binucleation/polyploidization and the cellular and molecular mechanisms involved. We found that, among several cytokines analyzed, known to be involved in early liver development and/or mass control, TGFbeta1 was capable to induce, together with the expected morphological changes, binucleation in hepatocytes in culture. Most importantly, the pharmacological inhibition of TGFbeta signaling in healthy mice during weaning, when the physiological binucleation occurs, induced a significant decrease of hepatocyte binucleation rate, without affecting cell proliferation and hepatic index. The TGFbeta-induced hepatocyte binucleation resulted from a cytokinesis failure, as assessed by video microscopy, and is associated with a delocalization of the cytokinesis regulator RhoA-GTPase from the mid-body of dividing cells. The use of specific chemical inhibitors demonstrated that the observed events are Src-dependent. Finally, the restoration of a fully epithelial phenotype by TGFbeta withdrawal gave rise to a cell progeny capable to maintain the polyploid state. In conclusion, we identified TGFbeta as a major inducer of hepatocyte binucleation both in vitro and in vivo, thus ascribing a novel role to this pleiotropic cytokine. The production of binucleated/tetraploid hepatocytes is due to a cytokinesis failure controlled by the molecular axis TGFbeta/Src/RhoA.

  3. TGFbeta Induces Binucleation/Polyploidization in Hepatocytes through a Src-Dependent Cytokinesis Failure.

    Science.gov (United States)

    De Santis Puzzonia, Marco; Cozzolino, Angela Maria; Grassi, Germana; Bisceglia, Francesca; Strippoli, Raffaele; Guarguaglini, Giulia; Citarella, Franca; Sacchetti, Benedetto; Tripodi, Marco; Marchetti, Alessandra; Amicone, Laura

    2016-01-01

    In all mammals, the adult liver shows binucleated as well as mononucleated polyploid hepatocytes. The hepatic polyploidization starts after birth with an extensive hepatocyte binucleation and generates hepatocytes of several ploidy classes. While the functional significance of hepatocyte polyploidy is becoming clearer, how it is triggered and maintained needs to be clarified. Aim of this study was to identify a major inducer of hepatocyte binucleation/polyploidization and the cellular and molecular mechanisms involved. We found that, among several cytokines analyzed, known to be involved in early liver development and/or mass control, TGFbeta1 was capable to induce, together with the expected morphological changes, binucleation in hepatocytes in culture. Most importantly, the pharmacological inhibition of TGFbeta signaling in healthy mice during weaning, when the physiological binucleation occurs, induced a significant decrease of hepatocyte binucleation rate, without affecting cell proliferation and hepatic index. The TGFbeta-induced hepatocyte binucleation resulted from a cytokinesis failure, as assessed by video microscopy, and is associated with a delocalization of the cytokinesis regulator RhoA-GTPase from the mid-body of dividing cells. The use of specific chemical inhibitors demonstrated that the observed events are Src-dependent. Finally, the restoration of a fully epithelial phenotype by TGFbeta withdrawal gave rise to a cell progeny capable to maintain the polyploid state. In conclusion, we identified TGFbeta as a major inducer of hepatocyte binucleation both in vitro and in vivo, thus ascribing a novel role to this pleiotropic cytokine. The production of binucleated/tetraploid hepatocytes is due to a cytokinesis failure controlled by the molecular axis TGFbeta/Src/RhoA.

  4. Thymic epithelial cells. I. Expression of strong suppressive (veto) activity in mouse thymic epithelial cell cultures

    DEFF Research Database (Denmark)

    Claesson, Mogens Helweg; Ropke, C

    1990-01-01

    We show that thymic epithelial cells grown under serum-free conditions in a chemically defined culture medium can act as veto cells in vitro. The veto activity of thymic epithelial cells results in inactivation of specific alloreactive cytotoxic T-cell precursors at the clonal level. It is conclu......We show that thymic epithelial cells grown under serum-free conditions in a chemically defined culture medium can act as veto cells in vitro. The veto activity of thymic epithelial cells results in inactivation of specific alloreactive cytotoxic T-cell precursors at the clonal level...

  5. Optimization of the isolation and cultivation of Cyprinus carpio primary hepatocytes

    OpenAIRE

    Yanhong, Fan; Chenghua, He; Guofang, Liu; Haibin, Zhang

    2008-01-01

    The aquatic environment is affected by numerous chemical contaminants. There is an increasing need to identify these chemicals and to evaluate their potential toxicity towards aquatic life. In this research we optimized techniques for primary cell culture of Cyprinus carpio hepatocytes as one adjunct model for ecotoxicological evaluation of the potential hazards of xenobiotics in the aquatic environment. In this study, Cyprinus carpio hepatocytes were isolated by mechanical separation, two-st...

  6. Organ culture of C57BL/6 mouse arteries with LPS as an in vitro model of vascular inflammation

    DEFF Research Database (Denmark)

    Outzen, Emilie Middelbo; Mehryar, Rahila; Boonen, Harrie C.M.

    Background: Vascular inflammation is believed to be involved in the pathogenesis of various cardiovascular diseases, the study of which often involves use of the mouse strain C57BL/6. In vivo studies can, however, be difficult to control and interpret. Aim of the study: To set up and characterise...... an in vitro model for studying vascular inflammation and function in cultured arteries from C57BL/6 mice. Methods: Segments of abdominal aorta and mesenteric arteries (MA) were incubated for 24 hours at 37̊C and 95% O2/5% CO2 in DMEM ± 100 ng/mL LPS. Aorta segments were frozen for molecular studies...... was achieved at a normalisation factor of 0.9 (0.91 ± 0.06, mean ± SEM, n = 9) as observed (0.85 ± 0.06, mean ± SEM, n = 3) and previously described in rat MA (Mulvany and Halpern, 1977). Furthermore, preliminary findings show that organ culture with 100 ng/mL LPS decreases endothelium-dependent dilation of C...

  7. Stage-specific synthesis and fucosylation of plasma membrane proteins by mouse pachytene spermatocytes and round spermatids in culture

    International Nuclear Information System (INIS)

    Gerton, G.L.; Millette, C.F.

    1986-01-01

    Little is known about the ability of mammalian spermatogenic cells to synthesize plasma membrane components in the presence or absence of Sertoli cells. In this study, purified populations (greater than 90%) of pachytene spermatocytes or round spermatids were isolated by unit gravity sedimentation and cultured for 20-24 h in the presence of [ 35 S]methionine or [ 3 H] fucose. Cell viabilities remained over 90% during the course of these experiments. Plasma membranes were purified from these cells and analyzed by two-dimensional gel electrophoresis. Qualitatively, the same plasma membrane proteins were synthesized by both cell types with the exception of the major Concanavalin A-binding glycoprotein, p151; the synthesis of p151 is greatly diminished or inhibited after meiosis. [3H]Fucose was incorporated into at least 6 common glycoproteins of both cells. Eight components fucosylated with molecular weights from 35,000 to 120,000 were specific to pachytene spermatocyte membranes. One fast-migrating fucosylated component may represent an uncharacterized lipid whose synthesis is terminated after meiosis. Round spermatids specifically fucosylated two components with molecular weights of 45,000 and 80,000. These results demonstrate the viability of germ cells of the male mouse in short-term culture and show that they are capable of synthesizing and fucosylating plasma membrane components in the absence of Sertoli cells

  8. Identification of Hedgehog signaling outcomes in mouse testis development using a hanging drop-culture system.

    Science.gov (United States)

    Szczepny, Anette; Hogarth, Cathryn A; Young, Julia; Loveland, Kate L

    2009-02-01

    The Hedgehog (Hh) signaling pathway affects fetal testis growth. Recently, we described the dynamic cellular production of Hh signaling pathway components in juvenile and adult rodent testes. The Hh signaling is understood to regulate cord formation in the fetal testis, but minimal knowledge exists regarding how Hh signaling impacts the postnatal testis. To investigate this, we employed hanging drop cultures, which are used routinely in embryoid body formation. This approach has the advantage of using small media volume, and we examined its suitability for short-term culture of both murine embryonic gonads and adult testis tubules. The effects of cyclopamine, a specific Hh signaling inhibitor, were examined following culture of Embryonic Day 11.5 urogenital ridges (as control) and adult seminiferous tubule fragments for 24-48 h using histological, cell proliferation, and gene expression analyses. Cultured embryonic testes displayed generally normal cord structure, anti-Müllerian hormone (Amh) expression, and cell proliferation; known Hh target gene expression (Gli1, osteopontin, official symbol Spp1, and Amh) was altered in response to cyclopamine. Cultured adult tubules exhibited some loss of seminiferous epithelium organization over 48 h. Spermatogonia continued to proliferate, however, and no significant loss of viability was noted overall. Addition of cyclopamine significantly affected levels of Gli1, Igfbp6, Ccnd2 (cyclin D2), Ccnb1 (cyclin B1), Spp1, Kit, and Amh mRNAs; these genes have been shown previously to be expressed in Sertoli and germ cells. These novel results identify Hh target genes in the testis and demonstrate this signaling pathway likely affects cell survival and differentiation in the context of normal adult testis.

  9. YAP Inhibition Restores Hepatocyte Differentiation in Advanced HCC, Leading to Tumor Regression

    Directory of Open Access Journals (Sweden)

    Julien Fitamant

    2015-03-01

    Full Text Available Defective Hippo/YAP signaling in the liver results in tissue overgrowth and development of hepatocellular carcinoma (HCC. Here, we uncover mechanisms of YAP-mediated hepatocyte reprogramming and HCC pathogenesis. YAP functions as a rheostat in maintaining metabolic specialization, differentiation, and quiescence within the hepatocyte compartment. Increased or decreased YAP activity reprograms subsets of hepatocytes to different fates associated with deregulation of the HNF4A, CTNNB1, and E2F transcriptional programs that control hepatocyte quiescence and differentiation. Importantly, treatment with small interfering RNA-lipid nanoparticles (siRNA-LNPs targeting YAP restores hepatocyte differentiation and causes pronounced tumor regression in a genetically engineered mouse HCC model. Furthermore, YAP targets are enriched in an aggressive human HCC subtype characterized by a proliferative signature and absence of CTNNB1 mutations. Thus, our work reveals Hippo signaling as a key regulator of the positional identity of hepatocytes, supports targeting of YAP using siRNA-LNPs as a paradigm of differentiation-based therapy, and identifies an HCC subtype that is potentially responsive to this approach.

  10. DISTINCT FUNCTIONS OF JNK AND C-JUN IN OXIDANT-INDUCED HEPATOCYTE DEATH

    Science.gov (United States)

    Amir, Muhammad; Liu, Kun; Zhao, Enpeng; Czaja, Mark J.

    2013-01-01

    Overactivation of c-Jun N-terminal kinase (JNK)/c-Jun signaling is a central mechanism of hepatocyte injury and death including that from oxidative stress. However, the functions of JNK and c-Jun are still unclear, and this pathway also inhibits hepatocyte death. Previous studies of menadione-induced oxidant stress demonstrated that toxicity resulted from sustained JNK/c-Jun activation as death was blocked by the c-Jun dominant negative TAM67. To further delineate the function of JNK/c-Jun signaling in hepatocyte injury from oxidant stress, the effects of direct JNK inhibition on menadione-induced death were examined. In contrast to the inhibitory effect of TAM67, pharmacological JNK inhibition by SP600125 sensitized the rat hepatocyte cell line RALA255-10G to death from menadione. SP600125 similarly sensitized mouse primary hepatocytes to menadione toxicity. Death from SP600125/menadione was c-Jun dependent as it was blocked by TAM67, but independent of c-Jun phosphorylation. Death occurred by apoptosis and necrosis and activation of the mitochondrial death pathway. Short hairpin RNA knockdowns of total JNK or JNK2 sensitized to death from menadione, whereas a jnk1 knockdown was protective. Jnk2 null mouse primary hepatocytes were also sensitized to menadione death. JNK inhibition magnified decreases in cellular ATP content and β-oxidation induced by menadione. This effect mediated cell death as chemical inhibition of β-oxidation also sensitized cells to death from menadione, and supplementation with the β-oxidation substrate oleate blocked death. Components of the JNK/c-Jun signaling pathway have opposing functions in hepatocyte oxidant stress with JNK2 mediating resistance to cell death and c-Jun promoting death. PMID:22644775

  11. 2,3,7,8-Tetrachlorodibenzo-p-dioxin specifically reduces mRNA for the mineralization-related dentin sialophosphoprotein in cultured mouse embryonic molar teeth

    International Nuclear Information System (INIS)

    Kiukkonen, Anu; Sahlberg, Carin; Lukinmaa, Pirjo-Liisa; Alaluusua, Satu; Peltonen, Eija; Partanen, Anna-Maija

    2006-01-01

    Previous studies show that the most toxic dioxin congener, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), interferes with mineralization of the dental matrices in developing mouse and rat teeth. Culture of mouse embryonic molar teeth with TCDD leads to the failure of enamel to be deposited and dentin to undergo mineralization. Lactationally exposed rats show defectively matured enamel and retardation of dentin mineralization. To see if the impaired mineralization is associated with changes in the expression of dentin sialophosphoprotein (Dspp), Bono1 and/or matrix metalloproteinase-20 (MMP-20), thought to be involved in mineralization of the dental hard tissues, we cultured mouse (NMRI) E18 mandibular molars for 3, 5 or 7 days and exposed them to 1 μM TCDD after 2 days of culture. As detected by in situ hybridization of tissue sections, localization and intensity of Bono1 and MMP-20 expression showed no definite difference between the control and exposed tooth explants, suggesting that TCDD does not affect their expression. On the contrary, TCDD reduced or prevented the expression of Dspp in secretory odontoblasts and decreased it in presecretory ameloblasts. The results suggest that the retardation of dentin mineralization by TCDD in mouse molar teeth involves specific interference with Dspp expression

  12. LDLR expression and localization are altered in mouse and human cell culture models of Alzheimer's disease.

    Directory of Open Access Journals (Sweden)

    Jose F Abisambra

    Full Text Available BACKGROUND: Alzheimer's disease (AD is a chronic neurodegenerative disorder and the most common form of dementia. The major molecular risk factor for late-onset AD is expression of the epsilon-4 allele of apolipoprotein E (apoE, the major cholesterol transporter in the brain. The low-density lipoprotein receptor (LDLR has the highest affinity for apoE and plays an important role in brain cholesterol metabolism. METHODOLOGY/PRINCIPAL FINDINGS: Using RT-PCR and western blotting techniques we found that over-expression of APP caused increases in both LDLR mRNA and protein levels in APP transfected H4 neuroglioma cells compared to H4 controls. Furthermore, immunohistochemical experiments showed aberrant localization of LDLR in H4-APP neuroglioma cells, Abeta-treated primary neurons, and in the PSAPP transgenic mouse model of AD. Finally, immunofluorescent staining of LDLR and of gamma- and alpha-tubulin showed a change in LDLR localization preferentially away from the plasma membrane that was paralleled by and likely the result of a disruption of the microtubule-organizing center and associated microtubule network. CONCLUSIONS/SIGNIFICANCE: These data suggest that increased APP expression and Abeta exposure alters microtubule function, leading to reduced transport of LDLR to the plasma membrane. Consequent deleterious effects on apoE uptake and function will have implications for AD pathogenesis and/or progression.

  13. Krp1 (Sarcosin) promotes lateral fusion of myofibril assembly intermediates in cultured mouse cardiomyocytes

    International Nuclear Information System (INIS)

    Greenberg, Cynthia C.; Connelly, Patricia S.; Daniels, Mathew P.; Horowits, Robert

    2008-01-01

    Krp1, also called sarcosin, is a cardiac and skeletal muscle kelch repeat protein hypothesized to promote the assembly of myofibrils, the contractile organelles of striated muscles, through interaction with N-RAP and actin. To elucidate its role, endogenous Krp1 was studied in primary embryonic mouse cardiomyocytes. While immunofluorescence showed punctate Krp1 distribution throughout the cell, detergent extraction revealed a significant pool of Krp1 associated with cytoskeletal elements. Reduction of Krp1 expression with siRNA resulted in specific inhibition of myofibril accumulation with no effect on cell spreading. Immunostaining analysis and electron microscopy revealed that cardiomyocytes lacking Krp1 contained sarcomeric proteins with longitudinal periodicities similar to mature myofibrils, but fibrils remained thin and separated. These thin myofibrils were degraded by a scission mechanism distinct from the myofibril disassembly pathway observed during cell division in the developing heart. The data are consistent with a model in which Krp1 promotes lateral fusion of adjacent thin fibrils into mature, wide myofibrils and contribute insight into mechanisms of myofibrillogenesis and disassembly

  14. Developmental and dysmorphogenic effects of glufosinate ammonium on mouse embryos in culture.

    Science.gov (United States)

    Watanabe, T; Iwase, T

    1996-01-01

    The effects of glufosinate ammonium on embryonic development in mice were examined using whole embryo and micromass cultures of midbrain and limb bud cells. In day 8 embryos cultured for 48 hr, glufosinate caused significant overall embryonic growth retardation and increased embryolethality to 37.5% at 10 micrograms/ml (5.0 x 10(-5) M). All embryos in the treated groups exhibited specific morphological defects including hypoplasia of the prosencephalon (forebrain) (100%) and visceral arches (100%). In day 10 embryos cultured for 24 hr, glufosinate significantly reduced the crown-rump length and the number of somite pairs, and produced a high incidence of morphological defects (84.6%) at 10 micrograms/ml. These embryos were characterized by blister in the lateral head (100%), hypoplasia of prosencephalon (57.1%), and cleft lips (42.9%) at 20 micrograms/ml (10.0 x 10(-5) M). Histological examination of the treated embryos showed numerous cell death (pyknotic debris) present throughout the neuroepithelium in the brain vesicle and neural tube, but did not involve the underlying mesenchyme. In micromass culture, glufosinate inhibited the differentiation of midbrain cells in day 12 embryos with 50% inhibition occurring at 0.55 microgram/ml (2.8 x 10(-6) M). The ratios of 50% inhibition concentration for cell proliferation to cell differentiation in limb bud cells were 0.76 and 1.52 in day 11 and 12 embryos, respectively. These findings indicate that glufosinate ammonium is embryotoxic in vitro. In addition to causing growth retardation, glufosinate specifically affected the neuroepithelium of the brain vesicle and neural tube, leading to neuroepithelial cell death.

  15. Chondroregulatory action of prolactin on proliferation and differentiation of mouse chondrogenic ATDC5 cells in 3-dimensional micromass cultures

    International Nuclear Information System (INIS)

    Seriwatanachai, Dutmanee; Krishnamra, Nateetip; Charoenphandhu, Narattaphol

    2012-01-01

    Highlights: ► Mouse chondrogenic ATDC5 cells expressed PRL receptor mRNAs and proteins. ► Low PRL concentration (10 ng/mL) increased chondrocyte viability and differentiation. ► Higher PRL concentrations (⩾100 ng/mL) decreased viability and increased apoptosis. -- Abstract: A recent investigation in lactating rats has provided evidence that the lactogenic hormone prolactin (PRL) increases endochondral bone growth and bone elongation, presumably by accelerating apoptosis of hypertrophic chondrocytes in the growth plate and/or subsequent chondrogenic matrix mineralization. Herein, we demonstrated the direct chondroregulatory action of PRL on proliferation, differentiation and apoptosis of chondrocytes in 3-dimensional micromass culture of mouse chondrogenic ATDC5 cell line. The results showed that ATDC5 cells expressed PRL receptor (PRLR) transcripts, and responded typically to PRL by downregulating PRLR expression. Exposure to a low PRL concentration of 10 ng/mL, comparable to the normal levels in male and non-pregnant female rats, increased chondrocyte viability, differentiation, proteoglycan accumulation, and mRNA expression of several chondrogenic differentiation markers, such as Sox9, ALP and Hspg2. In contrast, high PRL concentrations of ⩾100 ng/mL, comparable to the levels in pregnancy or lactation, decreased chondrocyte viability by inducing apoptosis, with no effect on chondrogenic marker expression. It could be concluded that chondrocytes directly but differentially responded to non-pregnant and pregnant/lactating levels of PRL, thus suggesting the stimulatory effect of PRL on chondrogenesis in young growing individuals, and supporting the hypothesis of hypertrophic chondrocyte apoptosis in the growth plate of lactating rats.

  16. Chondroregulatory action of prolactin on proliferation and differentiation of mouse chondrogenic ATDC5 cells in 3-dimensional micromass cultures

    Energy Technology Data Exchange (ETDEWEB)

    Seriwatanachai, Dutmanee [Center of Calcium and Bone Research (COCAB), Faculty of Science, Mahidol University, Bangkok (Thailand); Krishnamra, Nateetip [Center of Calcium and Bone Research (COCAB), Faculty of Science, Mahidol University, Bangkok (Thailand); Department of Physiology, Faculty of Science, Mahidol University, Bangkok (Thailand); Charoenphandhu, Narattaphol, E-mail: naratt@narattsys.com [Center of Calcium and Bone Research (COCAB), Faculty of Science, Mahidol University, Bangkok (Thailand); Department of Physiology, Faculty of Science, Mahidol University, Bangkok (Thailand)

    2012-03-30

    Highlights: Black-Right-Pointing-Pointer Mouse chondrogenic ATDC5 cells expressed PRL receptor mRNAs and proteins. Black-Right-Pointing-Pointer Low PRL concentration (10 ng/mL) increased chondrocyte viability and differentiation. Black-Right-Pointing-Pointer Higher PRL concentrations ( Greater-Than-Or-Slanted-Equal-To 100 ng/mL) decreased viability and increased apoptosis. -- Abstract: A recent investigation in lactating rats has provided evidence that the lactogenic hormone prolactin (PRL) increases endochondral bone growth and bone elongation, presumably by accelerating apoptosis of hypertrophic chondrocytes in the growth plate and/or subsequent chondrogenic matrix mineralization. Herein, we demonstrated the direct chondroregulatory action of PRL on proliferation, differentiation and apoptosis of chondrocytes in 3-dimensional micromass culture of mouse chondrogenic ATDC5 cell line. The results showed that ATDC5 cells expressed PRL receptor (PRLR) transcripts, and responded typically to PRL by downregulating PRLR expression. Exposure to a low PRL concentration of 10 ng/mL, comparable to the normal levels in male and non-pregnant female rats, increased chondrocyte viability, differentiation, proteoglycan accumulation, and mRNA expression of several chondrogenic differentiation markers, such as Sox9, ALP and Hspg2. In contrast, high PRL concentrations of Greater-Than-Or-Slanted-Equal-To 100 ng/mL, comparable to the levels in pregnancy or lactation, decreased chondrocyte viability by inducing apoptosis, with no effect on chondrogenic marker expression. It could be concluded that chondrocytes directly but differentially responded to non-pregnant and pregnant/lactating levels of PRL, thus suggesting the stimulatory effect of PRL on chondrogenesis in young growing individuals, and supporting the hypothesis of hypertrophic chondrocyte apoptosis in the growth plate of lactating rats.

  17. Hypoxia-inducible factor-dependent production of profibrotic mediators by hypoxic hepatocytes.

    Science.gov (United States)

    Copple, Bryan L; Bustamante, Juan J; Welch, Timothy P; Kim, Nam Deuk; Moon, Jeon-Ok

    2009-08-01

    During the development of liver fibrosis, mediators are produced that stimulate cells in the liver to differentiate into myofibroblasts and to produce collagen. Recent studies demonstrated that the transcription factor, hypoxia-inducible factor-1alpha (HIF-1alpha), is critical for upregulation of profibrotic mediators, such as platelet-derived growth factor-A (PDGF-A), PDGF-B and plasminogen activator inhibitor-1 (PAI-1) in the liver, during the development of fibrosis. What remains unknown is the cell type-specific regulation of these genes by HIF-1alpha in liver cell types. Accordingly, the hypothesis was tested that HIF-1alpha is activated in hypoxic hepatocytes and regulates the production of profibrotic mediators by these cells. In this study, hepatocytes were isolated from the livers of control and HIF-1alpha- or HIF-1beta-deficient mice and exposed to hypoxia. Exposure of primary mouse hepatocytes to 1% oxygen stimulated nuclear accumulation of HIF-1alpha and upregulated PAI-1, vascular endothelial cell growth factor and the vasoactive peptides adrenomedullin-1 (ADM-1) and ADM-2. In contrast, the levels of PDGF-A and PDGF-B mRNAs were unaffected in these cells by hypoxia. Exposure of HIF-1alpha-deficient hepatocytes to 1% oxygen only partially prevented upregulation of these genes, suggesting that other hypoxia-regulated transcription factors, such as HIF-2alpha, may also regulate these genes. In support of this, HIF-2alpha was activated in hypoxic hepatocytes, and exposure of HIF-1beta-deficient hepatocytes to 1% oxygen completely prevented upregulation of PAI-1, vascular endothelial cell growth factor and ADM-1, suggesting that HIF-2alpha may also contribute to upregulation of these genes in hypoxic hepatocytes. Collectively, our results suggest that HIFs may be important regulators of profibrotic and vasoactive mediators by hypoxic hepatocytes.

  18. Role of CYP2B in Phenobarbital-Induced Hepatocyte Proliferation in Mice.

    Science.gov (United States)

    Li, Lei; Bao, Xiaochen; Zhang, Qing-Yu; Negishi, Masahiko; Ding, Xinxin

    2017-08-01

    Phenobarbital (PB) promotes liver tumorigenesis in rodents, in part through activation of the constitutive androstane receptor (CAR) and the consequent changes in hepatic gene expression and increases in hepatocyte proliferation. A typical effect of CAR activation by PB is a marked induction of Cyp2b10 expression in the liver; the latter has been suspected to be vital for PB-induced hepatocellular proliferation. This hypothesis was tested here by using a Cyp2a(4/5)bgs -null (null) mouse model in which all Cyp2b genes are deleted. Adult male and female wild-type (WT) and null mice were treated intraperitoneally with PB at 50 mg/kg once daily for 5 successive days and tested on day 6. The liver-to-body weight ratio, an indicator of liver hypertrophy, was increased by 47% in male WT mice, but by only 22% in male Cyp2a(4/5)bgs -null mice, by the PB treatment. The fractions of bromodeoxyuridine-positive hepatocyte nuclei, assessed as a measure of the rate of hepatocyte proliferation, were also significantly lower in PB-treated male null mice compared with PB-treated male WT mice. However, whereas few proliferating hepatocytes were detected in saline-treated mice, many proliferating hepatocytes were still detected in PB-treated male null mice. In contrast, female WT mice were much less sensitive than male WT mice to PB-induced hepatocyte proliferation, and PB-treated female WT and PB-treated female null mice did not show significant difference in rates of hepatocyte proliferation. These results indicate that CYP2B induction plays a significant, but partial, role in PB-induced hepatocyte proliferation in male mice. U.S. Government work not protected by U.S. copyright.

  19. Arsenic compromises conducting airway epithelial barrier properties in primary mouse and immortalized human cell cultures.

    Directory of Open Access Journals (Sweden)

    Cara L Sherwood

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

  20. Milligram quantities of homogeneous recombinant full-length mouse Munc18c from Escherichia coli cultures.

    Directory of Open Access Journals (Sweden)

    Asma Rehman

    Full Text Available Vesicle fusion is an indispensable cellular process required for eukaryotic cargo delivery. The Sec/Munc18 protein Munc18c is essential for insulin-regulated trafficking of glucose transporter4 (GLUT4 vesicles to the cell surface in muscle and adipose tissue. Previously, our biophysical and structural studies have used Munc18c expressed in SF9 insect cells. However to maximize efficiency, minimize cost and negate any possible effects of post-translational modifications of Munc18c, we investigated the use of Escherichia coli as an expression host for Munc18c. We were encouraged by previous reports describing Munc18c production in E. coli cultures for use in in vitro fusion assay, pulldown assays and immunoprecipitations. Our approach differs from the previously reported method in that it uses a codon-optimized gene, lower temperature expression and autoinduction media. Three N-terminal His-tagged constructs were engineered, two with a tobacco etch virus (TEV or thrombin protease cleavage site to enable removal of the fusion tag. The optimized protocol generated 1-2 mg of purified Munc18c per L of culture at much reduced cost compared to Munc18c generated using insect cell culture. The purified recombinant Munc18c protein expressed in bacteria was monodisperse, monomeric, and functional. In summary, we developed methods that decrease the cost and time required to generate functional Munc18c compared with previous insect cell protocols, and which generates sufficient purified protein for structural and biophysical studies.

  1. Peptide-enhanced mRNA transfection in cultured mouse cardiac fibroblasts and direct reprogramming towards cardiomyocyte-like cells

    Directory of Open Access Journals (Sweden)

    Lee K

    2015-03-01

    Full Text Available Kunwoo Lee,1,2 Pengzhi Yu,3 Nithya Lingampalli,1 Hyun Jin Kim,1 Richard Tang,1 Niren Murthy1,2 1Department of Bioengineering, University of California, Berkeley, CA, USA; 2UC Berkeley and UCSF Joint Graduate Program in Bioengineering, Berkeley/San Francisco, CA, USA; 3Gladstone Institute of Cardiovascular Disease, San Francisco, CA, USA Abstract: The treatment of myocardial infarction is a major challenge in medicine due to the inability of heart tissue to regenerate. Direct reprogramming of endogenous cardiac fibroblasts into functional cardiomyocytes via the delivery of transcription factor mRNAs has the potential to regenerate cardiac tissue and to treat heart failure. Even though mRNA delivery to cardiac fibroblasts has the therapeutic potential, mRNA transfection in cardiac fibroblasts has been challenging. Herein, we develop an efficient mRNA transfection in cultured mouse cardiac fibroblasts via a polyarginine-fused heart-targeting peptide and lipofectamine complex, termed C-Lipo and demonstrate the partial direct reprogramming of cardiac fibroblasts towards cardiomyocyte cells. C-Lipo enabled the mRNA-induced direct cardiac reprogramming due to its efficient transfection with low toxicity, which allowed for multiple transfections of Gata4, Mef2c, and Tbx5 (GMT mRNAs for a period of 2 weeks. The induced cardiomyocyte-like cells had α-MHC promoter-driven GFP expression and striated cardiac muscle structure from a-actinin immunohistochemistry. GMT mRNA transfection of cultured mouse cardiac fibroblasts via C-Lipo significantly increased expression of the cardiomyocyte marker genes, Actc1, Actn2, Gja1, Hand2, and Tnnt2, after 2 weeks of transfection. Moreover, this study provides the first direct evidence that the stoichiometry of the GMT reprogramming factors influence the expression of cardiomyocyte marker genes. Our results demonstrate that mRNA delivery is a potential approach for cardiomyocyte generation. Keywords: direct cardiac

  2. Differentiating Mouse Embryonic Stem Cells into Embryoid Bodies by Hanging-Drop Cultures.

    Science.gov (United States)

    Behringer, Richard; Gertsenstein, Marina; Nagy, Kristina Vintersten; Nagy, Andras

    2016-12-01

    Embryonic stem (ES) cells can develop into many types of differentiated tissues if they are placed into a differentiating environment. This can occur in vivo when the ES cells are injected into or aggregated with an embryo, or in vitro if their culture conditions are modified to induce differentiation. There are an increasing number of differentiating culture conditions that can bias the differentiation of ES cells into desired cell types. Determining the mechanisms that control ES cell differentiation into therapeutically important cell types is a quickly growing area of research. Knowledge gained from these studies may eventually lead to the use of stem cells to repair specific damaged tissues. Many times ES cell differentiation proceeds through an intermediate stage called the embryoid body (EB). EBs are round structures composed of ES cells that have undergone some of the initial stages of differentiation. EBs can then be manipulated further to generate more specific cell types. This protocol describes a method to differentiate ES cells into EBs. It produces EBs of comparable size. This aspect is important because the differentiation processes taking place inside an EB are influenced by its size. © 2016 Cold Spring Harbor Laboratory Press.

  3. In vitro fertilization and embryo culture strongly impact the placental transcriptome in the mouse model.

    Directory of Open Access Journals (Sweden)

    Patricia Fauque

    Full Text Available BACKGROUND: Assisted Reproductive Technologies (ART are increasingly used in humans; however, their impact is now questioned. At blastocyst stage, the trophectoderm is directly in contact with an artificial medium environment, which can impact placental development. This study was designed to carry out an in-depth analysis of the placental transcriptome after ART in mice. METHODOLOGY/PRINCIPAL FINDINGS: Blastocysts were transferred either (1 after in vivo fertilization and development (control group or (2 after in vitro fertilization and embryo culture. Placentas were then analyzed at E10.5. Six percent of transcripts were altered at the two-fold threshold in placentas of manipulated embryos, 2/3 of transcripts being down-regulated. Strikingly, the X-chromosome harbors 11% of altered genes, 2/3 being induced. Imprinted genes were modified similarly to the X. Promoter composition analysis indicates that FOXA transcription factors may be involved in the transcriptional deregulations. CONCLUSIONS: For the first time, our study shows that in vitro fertilization associated with embryo culture strongly modify the placental expression profile, long after embryo manipulations, meaning that the stress of artificial environment is memorized after implantation. Expression of X and imprinted genes is also greatly modulated probably to adapt to adverse conditions. Our results highlight the importance of studying human placentas from ART.

  4. L-cysteine supplementation upregulates glutathione (GSH) and vitamin D binding protein (VDBP) in hepatocytes cultured in high glucose and in vivo in liver, and increases blood levels of GSH, VDBP, and 25-hydroxy-vitamin D in Zucker diabetic fatty rats.

    Science.gov (United States)

    Jain, Sushil K; Kanikarla-Marie, Preeti; Warden, Cassandra; Micinski, David

    2016-05-01

    Vitamin D binding protein (VDBP) status has an effect on and can potentially improve the status of 25(OH) vitamin D and increase the metabolic actions of 25(OH) vitamin D under physiological and pathological conditions. Diabetes is associated with lower levels of glutathione (GSH) and 25(OH) vitamin D. This study examined the hypothesis that upregulation of GSH will also upregulate blood levels of VDBP and 25(OH) vitamin D in type 2 diabetic rats. L-cysteine (LC) supplementation was used to upregulate GSH status in a FL83B hepatocyte cell culture model and in vivo using Zucker diabetic fatty (ZDF) rats. Results show that LC supplementation upregulates both protein and mRNA expression of VDBP and vitamin D receptor (VDR) and GSH status in hepatocytes exposed to high glucose, and that GSH deficiency, induced by glutamate cysteine ligase knockdown, resulted in the downregulation of GSH, VDBP, and VDR and an increase in oxidative stress levels in hepatocytes. In vivo, LC supplementation increased GSH and protein and mRNA expression of VDBP and vitamin D 25-hydroxylase (CYP2R1) in the liver, and simultaneously resulted in elevated blood levels of LC and GSH, as well as increases in VDBP and 25(OH) vitamin D levels, and decreased inflammatory biomarkers in ZDF rats compared with those in placebo-supplemented ZDF rats consuming a similar diet. LC supplementation may provide a novel approach by which to raise blood levels of VDBP and 25(OH) vitamin D in type 2 diabetes. © 2016 The Authors. Molecular Nutrition & Food Research Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Functional properties of hepatocytes in vitro are correlated with cell polarity maintenance.

    Science.gov (United States)

    Zeigerer, Anja; Wuttke, Anne; Marsico, Giovanni; Seifert, Sarah; Kalaidzidis, Yannis; Zerial, Marino

    2017-01-01

    Exploring the cell biology of hepatocytes in vitro could be a powerful strategy to dissect the molecular mechanisms underlying the structure and function of the liver in vivo. However, this approach relies on appropriate in vitro cell culture systems that can recapitulate the cell biological and metabolic features of the hepatocytes in the liver whilst being accessible to experimental manipulations. Here, we adapted protocols for high-resolution fluorescence microscopy and quantitative image analysis to compare two primary hepatocyte culture systems, monolayer and collagen sandwich, with respect to the distribution of two distinct populations of early endosomes (APPL1 and EEA1-positive), endocytic capacity, metabolic and signaling activities. In addition to the re-acquisition of hepatocellular polarity, primary hepatocytes grown in collagen sandwich but not in monolayer culture recapitulated the apico-basal distribution of EEA1 endosomes observed in liver tissue. We found that such distribution correlated with the organization of the actin cytoskeleton in vitro and, surprisingly, was dependent on the nutritional state in vivo. Hepatocytes in collagen sandwich also exhibited faster kinetics of low-density lipoprotein (LDL) and epidermal growth factor (EGF) internalization, showed improved insulin sensitivity and preserved their ability for glucose production, compared to hepatocytes in monolayer cultures. Although no in vitro culture system can reproduce the exquisite structural features of liver tissue, our data nevertheless highlight the ability of the collagen sandwich system to recapitulate key structural and functional properties of the hepatocytes in the liver and, therefore, support the usage of this system to study aspects of hepatocellular biology in vitro. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  6. Augmenter of liver regeneration (ALR) protects human hepatocytes against apoptosis

    International Nuclear Information System (INIS)

    Ilowski, Maren; Kleespies, Axel; Toni, Enrico N. de; Donabauer, Barbara; Jauch, Karl-Walter; Hengstler, Jan G.; Thasler, Wolfgang E.

    2011-01-01

    Research highlights: → ALR decreases cytochrome c release from mitochondria. → ALR protects hepatocytes against apoptosis induction by ethanol, TRAIL, anti-Apo, TGF-β and actinomycin D. → ALR exerts a liver-specific anti-apoptotic effect. → A possible medical usage of ALR regarding protection of liver cells during apoptosis inducing therapies. -- Abstract: Augmenter of liver regeneration (ALR) is known to support liver regeneration and to stimulate proliferation of hepatocytes. However, it is not known if ALR exerts anti-apoptotic effects in human hepatocytes and whether this protective effect is cell type specific. This is relevant, because compounds that protect the liver against apoptosis without undesired effects, such as protection of metastatic tumour cells, would be appreciated in several clinical settings. Primary human hepatocytes (phH) and organotypic cancer cell lines were exposed to different concentrations of apoptosis inducers (ethanol, TRAIL, anti-Apo, TGF-β, actinomycin D) and cultured with or without recombinant human ALR (rhALR). Apoptosis was evaluated by the release of cytochrome c from mitochondria and by FACS with propidium iodide (PI) staining. ALR significantly decreased apoptosis induced by ethanol, TRAIL, anti-Apo, TGF-β and actinomycin D. Further, the anti-apoptotic effect of ALR was observed in primary human hepatocytes and in HepG2 cells but not in bronchial (BC1), colonic (SW480), gastric (GC1) and pancreatic (L3.6PL) cell lines. Therefore, the hepatotrophic growth factor ALR acts in a liver specific manner with regards to both its mitogenic and its anti-apoptotic effect. Unlike the growth factors HGF and EGF, rhALR acts in a liver specific manner. Therefore, ALR is a promising candidate for further evaluation as a possible hepatoprotective factor in clinical settings.

  7. Augmenter of liver regeneration (ALR) protects human hepatocytes against apoptosis

    Energy Technology Data Exchange (ETDEWEB)

    Ilowski, Maren [Liver Regeneration Group, Department of Surgery, Grosshadern Hospital, Ludwig Maximilians University, Munich (Germany); Kleespies, Axel [Department of Surgery, Grosshadern Hospital, Ludwig Maximilians University, Munich (Germany); Toni, Enrico N. de [Department of Medicine II, Grosshadern Hospital, Ludwig Maximilians University, Munich (Germany); Donabauer, Barbara [Liver Regeneration Group, Department of Surgery, Grosshadern Hospital, Ludwig Maximilians University, Munich (Germany); Jauch, Karl-Walter [Department of Surgery, Grosshadern Hospital, Ludwig Maximilians University, Munich (Germany); Hengstler, Jan G. [Leibniz Research Centre for Working Environment and Human Factors, Technical University, Dortmund (Germany); Thasler, Wolfgang E., E-mail: wolfgang.thasler@med.uni-muenchen.de [Liver Regeneration Group, Department of Surgery, Grosshadern Hospital, Ludwig Maximilians University, Munich (Germany); Department of Surgery, Grosshadern Hospital, Ludwig Maximilians University, Munich (Germany)

    2011-01-07

    Research highlights: {yields} ALR decreases cytochrome c release from mitochondria. {yields} ALR protects hepatocytes against apoptosis induction by ethanol, TRAIL, anti-Apo, TGF-{beta} and actinomycin D. {yields} ALR exerts a liver-specific anti-apoptotic effect. {yields} A possible medical usage of ALR regarding protection of liver cells during apoptosis inducing therapies. -- Abstract: Augmenter of liver regeneration (ALR) is known to support liver regeneration and to stimulate proliferation of hepatocytes. However, it is not known if ALR exerts anti-apoptotic effects in human hepatocytes and whether this protective effect is cell type specific. This is relevant, because compounds that protect the liver against apoptosis without undesired effects, such as protection of metastatic tumour cells, would be appreciated in several clinical settings. Primary human hepatocytes (phH) and organotypic cancer cell lines were exposed to different concentrations of apoptosis inducers (ethanol, TRAIL, anti-Apo, TGF-{beta}, actinomycin D) and cultured with or without recombinant human ALR (rhALR). Apoptosis was evaluated by the release of cytochrome c from mitochondria and by FACS with propidium iodide (PI) staining. ALR significantly decreased apoptosis induced by ethanol, TRAIL, anti-Apo, TGF-{beta} and actinomycin D. Further, the anti-apoptotic effect of ALR was observed in primary human hepatocytes and in HepG2 cells but not in bronchial (BC1), colonic (SW480), gastric (GC1) and pancreatic (L3.6PL) cell lines. Therefore, the hepatotrophic growth factor ALR acts in a liver specific manner with regards to both its mitogenic and its anti-apoptotic effect. Unlike the growth factors HGF and EGF, rhALR acts in a liver specific manner. Therefore, ALR is a promising candidate for further evaluation as a possible hepatoprotective factor in clinical settings.

  8. Nicotine-induced Disturbances of Meiotic Maturation in Cultured Mouse Oocytes: Alterations of Spindle Integrity and Chromosome Alignment

    Directory of Open Access Journals (Sweden)

    Zenzes Maria

    2004-09-01

    Full Text Available Abstract We investigated whether nicotine exposure in vitro of mouse oocytes affects spindle and chromosome function during meiotic maturation (M-I and M-II. Oocytes in germinal vesicle (GV stage were cultured in nicotine for 8 h or for 16 h, to assess effects in M-I and in metaphase II (M-II. The latter culture setting used the three protocols: 8 h nicotine then 8 h medium (8N + 8M; 16 h nicotine (16N; 8 h medium then 8 h nicotine (8M + 8N. Non-toxic concentrations of nicotine at 1.0, 2.5, 5.0 and 10.0 mmol/L were used. Spindle-chromosome configurations were analyzed with wide-field optical sectioning microscopy. In 8 h cultures, nicotine exposure resulted in dose-related increased proportions of M-I oocytes with defective spindle-chromosome configurations. A dose-related delayed entry into anaphase I was also detected. In 16 h cultures, nicotine exposure for the first 8 h (8N + 8M, or for 16 h (16N, resulted in dose- and time-related increased proportions of oocytes arrested in M-I (10 mmol/L; 8 h: 53.2%, controls 9.6%; 16 h: 87.6%, controls 8.5%. Defects in M-I spindles and chromosomes caused M-I arrest leading to dose-related decreased proportions of oocytes that reached metaphase-II (10 mmol/L 8 h: 46.8%, controls 90.4%;16 h: 12.4%, controls 91.5%. A delayed anaphase-I affected the normal timing of M-II, leading to abnormal oocytes with dispersed chromosomes, or with double spindles and no polar body. Nicotine exposure during the second 8 h (8M + 8N resulted in dose-related, increased proportions of M-II oocytes with defective spindles and chromosomes (10 mmol/L: 42.9%, controls 2.0%. Nicotine has no adverse effects on GV break down, but induces spindle and chromosome defects compromising oocyte meiotic maturation and development.

  9. Human spermatogonial stem cells display limited proliferation in vitro under mouse spermatogonial stem cell culture conditions.

    Science.gov (United States)

    Medrano, Jose V; Rombaut, Charlotte; Simon, Carlos; Pellicer, Antonio; Goossens, Ellen

    2016-11-01

    To study the ability of human spermatogonial stem cells (hSSCs) to proliferate in vitro under mouse spermatogonial stem cell (mSSC) culture conditions. Experimental basic science study. Reproductive biology laboratory. Cryopreserved testicular tissue with normal spermatogenesis obtained from three donors subjected to orchiectomy due to a prostate cancer treatment. Testicular cells used to create in vitro cell cultures corresponding to the following groups: [1] unsorted human testicular cells, [2] differentially plated human testicular cells, and [3] cells enriched with major histocompatibility complex class 1 (HLA - )/epithelial cell surface antigen (EPCAM + ) in coculture with inactivated testicular feeders from the same patient. Analyses and characterization including immunocytochemistry and quantitative reverse-transcription polymerase chain reaction for somatic and germ cell markers, testosterone and inhibin B quantification, and TUNEL assay. Putative hSSCs appeared in singlets, doublets, or small groups of up to four cells in vitro only when testicular cells were cultured in StemPro-34 medium supplemented with glial cell line-derived neurotrophic factor (GDNF), leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF). Fluorescence-activated cell sorting with HLA - /EPCAM + resulted in an enrichment of 27% VASA + /UTF1 + hSSCs, compared to 13% in unsorted controls. Coculture of sorted cells with inactivated testicular feeders gave rise to an average density of 112 hSSCs/cm 2 after 2 weeks in vitro compared with unsorted cells (61 hSSCs/cm 2 ) and differentially plated cells (49 hSSCS/cm 2 ). However, putative hSSCs rarely stained positive for the proliferation marker Ki67, and their presence was reduced to the point of almost disappearing after 4 weeks in vitro. We found that hSSCs show limited proliferation in vitro under mSSC culture conditions. Coculture of HLA - /EPCAM + sorted cells with testicular

  10. Hepatocyte produced matrix metalloproteinases are regulated by CD147 in liver fibrogenesis.

    Science.gov (United States)

    Calabro, Sarah R; Maczurek, Annette E; Morgan, Alison J; Tu, Thomas; Wen, Victoria W; Yee, Christine; Mridha, Auvro; Lee, Maggie; d'Avigdor, William; Locarnini, Stephen A; McCaughan, Geoffrey W; Warner, Fiona J; McLennan, Susan V; Shackel, Nicholas A

    2014-01-01

    The classical paradigm of liver injury asserts that hepatic stellate cells (HSC) produce, remodel and turnover the abnormal extracellular matrix (ECM) of fibrosis via matrix metalloproteinases (MMPs). In extrahepatic tissues MMP production is regulated by a number of mechanisms including expression of the glycoprotein CD147. Previously, we have shown that CD147 is expressed on hepatocytes but not within the fibrotic septa in cirrhosis [1]. Therefore, we investigated if hepatocytes produce MMPs, regulated by CD147, which are capable of remodelling fibrotic ECM independent of the HSC. Non-diseased, fibrotic and cirrhotic livers were examined for MMP activity and markers of fibrosis in humans and mice. CD147 expression and MMP activity were co-localised by in-situ zymography. The role of CD147 was studied in-vitro with siRNA to CD147 in hepatocytes and in-vivo in mice with CCl4 induced liver injury using ãCD147 antibody intervention. In liver fibrosis in both human and mouse tissue MMP expression and activity (MMP-2, -9, -13 and -14) increased with progressive injury and localised to hepatocytes. Additionally, as expected, MMPs were abundantly expressed by activated HSC. Further, with progressive fibrosis there was expression of CD147, which localised to hepatocytes but not to HSC. Functionally significant in-vitro regulation of hepatocyte MMP production by CD147 was demonstrated using siRNA to CD147 that decreased hepatocyte MMP-2 and -9 expression/activity. Further, in-vivo α-CD147 antibody intervention decreased liver MMP-2, -9, -13, -14, TGF-β and α-SMA expression in CCl4 treated mice compared to controls. We have shown that hepatocytes produce active MMPs and that the glycoprotein CD147 regulates hepatocyte MMP expression. Targeting CD147 regulates hepatocyte MMP production both in-vitro and in-vivo, with the net result being reduced fibrotic matrix turnover in-vivo. Therefore, CD147 regulation of hepatocyte MMP is a novel pathway that could be targeted by

  11. Hepatocyte produced matrix metalloproteinases are regulated by CD147 in liver fibrogenesis.

    Directory of Open Access Journals (Sweden)

    Sarah R Calabro

    Full Text Available The classical paradigm of liver injury asserts that hepatic stellate cells (HSC produce, remodel and turnover the abnormal extracellular matrix (ECM of fibrosis via matrix metalloproteinases (MMPs. In extrahepatic tissues MMP production is regulated by a number of mechanisms including expression of the glycoprotein CD147. Previously, we have shown that CD147 is expressed on hepatocytes but not within the fibrotic septa in cirrhosis [1]. Therefore, we investigated if hepatocytes produce MMPs, regulated by CD147, which are capable of remodelling fibrotic ECM independent of the HSC.Non-diseased, fibrotic and cirrhotic livers were examined for MMP activity and markers of fibrosis in humans and mice. CD147 expression and MMP activity were co-localised by in-situ zymography. The role of CD147 was studied in-vitro with siRNA to CD147 in hepatocytes and in-vivo in mice with CCl4 induced liver injury using ãCD147 antibody intervention.In liver fibrosis in both human and mouse tissue MMP expression and activity (MMP-2, -9, -13 and -14 increased with progressive injury and localised to hepatocytes. Additionally, as expected, MMPs were abundantly expressed by activated HSC. Further, with progressive fibrosis there was expression of CD147, which localised to hepatocytes but not to HSC. Functionally significant in-vitro regulation of hepatocyte MMP production by CD147 was demonstrated using siRNA to CD147 that decreased hepatocyte MMP-2 and -9 expression/activity. Further, in-vivo α-CD147 antibody intervention decreased liver MMP-2, -9, -13, -14, TGF-β and α-SMA expression in CCl4 treated mice compared to controls.We have shown that hepatocytes produce active MMPs and that the glycoprotein CD147 regulates hepatocyte MMP expression. Targeting CD147 regulates hepatocyte MMP production both in-vitro and in-vivo, with the net result being reduced fibrotic matrix turnover in-vivo. Therefore, CD147 regulation of hepatocyte MMP is a novel pathway that could be

  12. Immunoprecipitation assay of alpha-fetoprotein synthesis by cultured mouse hepatoma cells treated with estrogens and glucocorticoids.

    Science.gov (United States)

    Rosebrock, J A; Parker, C L; Kute, T E

    1981-01-01

    This investigation was to study the biosynthesis of 3H-labeled alpha-fetoprotein (AFP) by cultured mouse hepatoma (HEPA-2) cells. Both the function and regulation of this oncodevelopmental gene are unknown. However, evidence indicates that mechanisms controlling the expression of AFP involve aspects of both normal embryonic development and neoplastic transformation. the secretion of AFP was analyzed during different phases of the growth cycle to provide information on AFP production using standard culture conditions. The highest rate of secretion occurred during the stationary phase, followed by the late logarithmic and early logarithmic phases of growth, respectively. The production of AFP was then determined following the addition of glucocorticoids and estrogens in an attempt to understand hormonal factors that may be involved. Studies utilizing estradiol-17 beta indicated that the secretion of AFP did not appear to be sensitive to this steroid even though sucrose density gradient analysis of HEPA-2 cytosol, for estrogenic receptors, revealed competitive binding moieties on the 8S and 4S regions of the gradient. In contrast, the secretion of the total complement of proteins, including AFP, was significantly stimulated by the glucocorticoids, dexamethasone and corticosterone. Analysis of HEPA-2 cytosol for glucocorticoid receptors revealed binding components in the 7S and 3-4S regions of the gradient. The 3H-dexamethasone binding appeared to be stereospecific since nonlabeled dexamethasone, but not nonlabeled estradiol-17 beta, effectively displaced the bound radioactivity. The glucocorticoid-binding component in HEPA-2 therefore displayed characteristics reported for glucocorticoid receptors in normal liver and other hepatomas.

  13. Tumorigenic risk of human induced pluripotent stem cell explants cultured on mouse SNL76/7 feeder cells

    Energy Technology Data Exchange (ETDEWEB)

    Kamada, Mizuna; Mitsui, Youji, E-mail: y-mitsui8310@hb.tp1.jp; Kumazaki, Tsutomu; Kawahara, Yuta; Matsuo, Taira; Takahashi, Tomoko, E-mail: t-takahashi@kph.bunri-u.ac.jp

    2014-10-24

    Highlights: • hiPS cell explants formed malignant tumors when SNL76/7 feeder cells were used. • Multi type tumors developed by interaction of SNL76/7 feeder cells with hiPS cells. • Tumorigenic risk occurs by co-culture of hiPS cells with SNL76/7 feeder cells. - Abstract: The potential for tumor formation from transplanted human induced pluripotent stem cell (hiPSC) derivatives represents a high risk in their application to regenerative medicine. We examined the genetic origin and characteristics of tumors, that were formed when 13 hiPSC lines, established by ourselves, and 201B7 hiPSC from Kyoto University were transplanted into severe combined immune-deficient (SCID) mice. Though teratomas formed in 58% of mice, five angiosarcomas, one malignant solitary fibrous tumor and one undifferentiated pleomorphic sarcoma formed in the remaining mice. Three malignant cell lines were established from the tumors, which were derived from mitomycin C (MMC)-treated SNL76/7 (MMC-SNL) feeder cells, as tumor development from fusion cells between MMC-SNL and hiPSCs was negative by genetic analysis. While parent SNL76/7 cells produced malignant tumors, neither MMC-SNL nor MMC-treated mouse embryo fibroblast (MEF) produced malignant tumors. When MMC-SNL feeder cells were co-cultured with hiPSCs, growing cell lines were generated, that expressed genes similar to the parent SNL76/7 cells. Thus, hiPSCs grown on MMC-SNL feeder cells have a high risk of generating feeder-derived malignant tumors. The possible mechanism(s) of growth restoration and the formation of multiple tumor types are discussed with respect of the interactions between MMC-SNL and hiPSC.

  14. Heme oxygenase attenuates angiotensin II-mediated superoxide production in cultured mouse thick ascending loop of Henle cells.

    Science.gov (United States)

    Kelsen, Silvia; Patel, Bijal J; Parker, Lawson B; Vera, Trinity; Rimoldi, John M; Gadepalli, Rama S V; Drummond, Heather A; Stec, David E

    2008-10-01

    Heme oxygenase (HO)-1 induction can attenuate the development of angiotensin II (ANG II)-dependent hypertension. However, the mechanism by which HO-1 lowers blood pressure is not clear. The goal of this study was to test the hypothesis that induction of HO-1 can reduce the ANG II-mediated increase in superoxide production in cultured thick ascending loop of Henle (TALH) cells. Studies were performed on an immortalized cell line of mouse TALH (mTALH) cells. HO-1 was induced in cultured mTALH cells by treatment with cobalt protoporphyrin (CoPP, 10 microM) or hemin (50 microM) or by transfection with a plasmid containing the human HO-1 isoform. Treatment of mTALH cells with 10(-9) M ANG II increased dihydroethidium (DHE) fluorescence (an index of superoxide levels) from 35.5+/-5 to 136+/-18 relative fluorescence units (RFU)/microm2. Induction of HO-1 via CoPP, hemin, or overexpression of the human HO-1 isoform significantly reduced ANG II-induced DHE fluorescence to 64+/-5, 64+/-8, and 41+/-4 RFU/microm2, respectively. To determine which metabolite of HO-1 is responsible for reducing ANG II-mediated increases in superoxide production in mTALH cells, cells were preincubated with bilirubin or carbon monoxide (CO)-releasing molecule (CORM)-A1 (each at 100 microM) before exposure to ANG II. DHE fluorescence averaged 80+/-7 RFU/microm2 after incubation with ANG II and was significantly decreased to 55+/-7 and 53+/-4 RFU/microm2 after pretreatment with bilirubin and CORM-A1. These results demonstrate that induction of HO-1 in mTALH cells reduces the levels of ANG II-mediated superoxide production through the production of both bilirubin and CO.

  15. Transdifferentiation of mouse adipose-derived stromal cells into acinar cells of the submandibular gland using a co-culture system

    International Nuclear Information System (INIS)

    Lee, Jingu; Park, Sangkyu; Roh, Sangho

    2015-01-01

    A loss of salivary gland function often occurs after radiation therapy in head and neck tumors, though secretion of saliva by the salivary glands is essential for the health and maintenance of the oral environment. Transplantation of salivary acinar cells (ACs), in part, may overcome the side effects of therapy. Here we directly differentiated mouse adipose-derived stromal cells (ADSCs) into ACs using a co-culture system. Multipotent ADSCs can be easily collected from stromal vascular fractions of adipose tissues. The isolated ADSCs showed positive expression of markers such as integrin beta-1 (CD29), cell surface glycoprotein (CD44), endoglin (CD105), and Nanog. The cells were able to differentiate into adipocytes, osteoblasts, and neural-like cells after 14 days in culture. ADSCs at passage 2 were co-cultured with mouse ACs in AC culture medium using the double-chamber (co-culture system) to avoid mixing the cell types. The ADSCs in this co-culture system expressed markers of ACs, such as α-amylases and aquaporin5, in both mRNA and protein. ADSCs cultured in AC-conditioned medium also expressed AC markers. Cellular proliferation and senescence analyses demonstrated that cells in the co-culture group showed lower senescence and a higher proliferation rate than the AC-conditioned medium group at Days 14 and 21. The results above imply direct conversion of ADSCs into ACs under the co-culture system; therefore, ADSCs may be a stem cell source for the therapy for salivary gland damage. - Highlights: • ADSCs could transdifferentiate into acinar cells (ACs) using ACs co-culture (CCA). • Transdifferentiated ADSCs expressed ACs markers such as α-amylase and aquaporin5. • High proliferation and low senescence were presented in CCA at Day 14. • Transdifferentiation of ADSCs into ACs using CCA may be an appropriate method for cell-based therapy

  16. Transdifferentiation of mouse adipose-derived stromal cells into acinar cells of the submandibular gland using a co-culture system

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Jingu; Park, Sangkyu; Roh, Sangho, E-mail: sangho@snu.ac.kr

    2015-05-15

    A loss of salivary gland function often occurs after radiation therapy in head and neck tumors, though secretion of saliva by the salivary glands is essential for the health and maintenance of the oral environment. Transplantation of salivary acinar cells (ACs), in part, may overcome the side effects of therapy. Here we directly differentiated mouse adipose-derived stromal cells (ADSCs) into ACs using a co-culture system. Multipotent ADSCs can be easily collected from stromal vascular fractions of adipose tissues. The isolated ADSCs showed positive expression of markers such as integrin beta-1 (CD29), cell surface glycoprotein (CD44), endoglin (CD105), and Nanog. The cells were able to differentiate into adipocytes, osteoblasts, and neural-like cells after 14 days in culture. ADSCs at passage 2 were co-cultured with mouse ACs in AC culture medium using the double-chamber (co-culture system) to avoid mixing the cell types. The ADSCs in this co-culture system expressed markers of ACs, such as α-amylases and aquaporin5, in both mRNA and protein. ADSCs cultured in AC-conditioned medium also expressed AC markers. Cellular proliferation and senescence analyses demonstrated that cells in the co-culture group showed lower senescence and a higher proliferation rate than the AC-conditioned medium group at Days 14 and 21. The results above imply direct conversion of ADSCs into ACs under the co-culture system; therefore, ADSCs may be a stem cell source for the therapy for salivary gland damage. - Highlights: • ADSCs could transdifferentiate into acinar cells (ACs) using ACs co-culture (CCA). • Transdifferentiated ADSCs expressed ACs markers such as α-amylase and aquaporin5. • High proliferation and low senescence were presented in CCA at Day 14. • Transdifferentiation of ADSCs into ACs using CCA may be an appropriate method for cell-based therapy.

  17. Caloric restriction mimetic 2-deoxyglucose maintains cytoarchitecture and reduces tau phosphorylation in primary culture of mouse hippocampal pyramidal neurons.

    Science.gov (United States)

    Bele, M S; Gajare, K A; Deshmukh, A A

    2015-06-01

    Typical form of neurons is crucially important for their functions. This is maintained by microtubules and associated proteins like tau. Hyperphosphorylation of tau is a major concern in neurodegenerative diseases. Glycogen synthase kinase3β (GSK3β) and cyclin-dependent protein kinase 5 (Cdk5) are the enzymes that govern tau phosphorylation. Currently, efforts are being made to target GSK3β and Cdk5 as possible therapeutic avenues to control tau phosphorylation and treat neurodegenerative diseases related to taupathies. In a number of studies, caloric restriction mimetic 2-deoxyglucose (C6H12O5) was found to be beneficial in improving the brain functions. However, no reports are available on the effect of 2-deoxyglucose 2-DG on tau phosphorylation. In the present study, hippocampal pyramidal neurons from E17 mouse embryos were isolated and cultured on poly-L-lysine-coated coverslips. Neurons from the experimental group were treated with 10 mM 2-deoxyglucose. The treatment of 2-DG resulted in healthier neuronal morphology in terms of significantly lower number of cytoplasmic vacuoles, little or no membrane blebbings, maintained axon hillock and intact neurites. There were decreased immunofluorescence signals for GSK3β, pTau at Ser262, Cdk5 and pTau at Ser235 suggesting decreased tau phosphorylation, which was further confirmed by Western blotting. The results indicate the beneficial effects of 2-DG in controlling the tau phosphorylation and maintaining the healthy neuronal cytoarchitecture.

  18. Combined data preprocessing and multivariate statistical analysis characterizes fed-batch culture of mouse hybridoma cells for rational medium design.

    Science.gov (United States)

    Selvarasu, Suresh; Kim, Do Yun; Karimi, Iftekhar A; Lee, Dong-Yup

    2010-10-01

    We present an integrated framework for characterizing fed-batch cultures of mouse hybridoma cells producing monoclonal antibody (mAb). This framework systematically combines data preprocessing, elemental balancing and statistical analysis technique. Initially, specific rates of cell growth, glucose/amino acid consumptions and mAb/metabolite productions were calculated via curve fitting using logistic equations, with subsequent elemental balancing of the preprocessed data indicating the presence of experimental measurement errors. Multivariate statistical analysis was then employed to understand physiological characteristics of the cellular system. The results from principal component analysis (PCA) revealed three major clusters of amino acids with similar trends in their consumption profiles: (i) arginine, threonine and serine, (ii) glycine, tyrosine, phenylalanine, methionine, histidine and asparagine, and (iii) lysine, valine and isoleucine. Further analysis using partial least square (PLS) regression identified key amino acids which were positively or negatively correlated with the cell growth, mAb production and the generation of lactate and ammonia. Based on these results, the optimal concentrations of key amino acids in the feed medium can be inferred, potentially leading to an increase in cell viability and productivity, as well as a decrease in toxic waste production. The study demonstrated how the current methodological framework using multivariate statistical analysis techniques can serve as a potential tool for deriving rational medium design strategies. Copyright © 2010 Elsevier B.V. All rights reserved.

  19. The metabolic role of isoleucine in detoxification of ammonia in cultured mouse neurons and astrocytes.

    Science.gov (United States)

    Johansen, Maja L; Bak, Lasse K; Schousboe, Arne; Iversen, Peter; Sørensen, Michael; Keiding, Susanne; Vilstrup, Hendrik; Gjedde, Albert; Ott, Peter; Waagepetersen, Helle S

    2007-06-01

    Cerebral hyperammonemia is a hallmark of hepatic encephalopathy, a debilitating condition arising secondary to liver disease. Pyruvate oxidation including tricarboxylic acid (TCA) cycle metabolism has been suggested to be inhibited by hyperammonemia at the pyruvate and alpha-ketoglutarate dehydrogenase steps. Catabolism of the branched-chain amino acid isoleucine provides both acetyl-CoA and succinyl-CoA, thus by-passing both the pyruvate dehydrogenase and the alpha-ketoglutarate dehydrogenase steps. Potentially, this will enable the TCA cycle to work in the face of ammonium-induced inhibition. In addition, this will provide the alpha-ketoglutarate carbon skeleton for glutamate and glutamine synthesis by glutamate dehydrogenase and glutamine synthetase (astrocytes only), respectively, both reactions fixing ammonium. Cultured cerebellar neurons (primarily glutamatergic) or astrocytes were incubated in the presence of either [U-13C]glucose (2.5 mM) and isoleucine (1 mM) or [U-13C]isoleucine and glucose. Cell cultures were treated with an acute ammonium chloride load of 2 (astrocytes) or 5 mM (neurons and astrocytes) and incorporation of 13C-label into glutamate, aspartate, glutamine and alanine was determined employing mass spectrometry. Labeling from [U-13C]glucose in glutamate and aspartate increased as a result of ammonium-treatment in both neurons and astrocytes, suggesting that the TCA cycle was not inhibited. Labeling in alanine increased in neurons but not in astrocytes, indicating elevated glycolysis in neurons. For both neurons and astrocytes, labeling from [U-13C]isoleucine entered glutamate and aspartate albeit to a lower extent than from [U-13C]glucose. Labeling in glutamate and aspartate from [U-13C]isoleucine was decreased by ammonium treatment in neurons but not in astrocytes, the former probably reflecting increased metabolism of unlabeled glucose. In astrocytes, ammonia treatment resulted in glutamine production and release to the medium, partially

  20. Propofol pretreatment attenuates LPS-induced granulocyte-macrophage colony-stimulating factor production in cultured hepatocytes by suppressing MAPK/ERK activity and NF-κB translocation

    International Nuclear Information System (INIS)

    Jawan, Bruno; Kao, Y.-H.; Goto, Shigeru; Pan, M.-C.; Lin, Y.-C.; Hsu, L.-W.; Nakano, Toshiaki; Lai, C.-Y.; Sun, C.-K.; Cheng, Y.-F.; Tai, M.-H.

    2008-01-01

    Propofol (PPF), a widely used intravenous anesthetic for induction and maintenance of anesthesia during surgeries, was found to possess suppressive effect on host immunity. This study aimed at investigating whether PPF plays a modulatory role in the lipopolysaccharide (LPS)-induced inflammatory cytokine expression in a cell line of rat hepatocytes. Morphological observation and viability assay showed that PPF exhibits no cytotoxicity at concentrations up to 300 μM after 48 h incubation. Pretreatment with 100 μM PPF for 24 h prior to LPS stimulation was performed to investigate the modulatory effect on LPS-induced inflammatory gene production. The results of semi-quantitative RT-PCR demonstrated that PPF pretreatment significantly suppressed the LPS-induced toll-like receptor (TLR)-4, CD14, tumor necrosis factor (TNF)-α, and granulocyte-macrophage colony-stimulating factor (GM-CSF) gene expression. Western blotting analysis showed that PPF pretreatment potentiated the LPS-induced TLR-4 downregulation. Flow cytometrical analysis revealed that PPF pretreatment showed no modulatory effect on the LPS-upregulated CD14 expression on hepatocytes. In addition, PPF pretreatment attenuated the phosphorylation of mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) and IκBα, as well as the nuclear translocation of NF-κB primed by LPS. Moreover, addition of PD98059, a MAPK kinase inhibitor, significantly suppressed the LPS-induced NF-κB nuclear translocation and GM-CSF production, suggesting that the PPF-attenuated GM-CSF production in hepatocytes may be attributed to its suppressive effect on MAPK/ERK signaling pathway. In conclusion, PPF as an anesthetic may clinically benefit those patients who are vulnerable to sepsis by alleviating sepsis-related inflammatory response in livers

  1. Generation of hyaline cartilaginous tissue from mouse adult dermal fibroblast culture by defined factors

    Science.gov (United States)

    Hiramatsu, Kunihiko; Sasagawa, Satoru; Outani, Hidetatsu; Nakagawa, Kanako; Yoshikawa, Hideki; Tsumaki, Noriyuki

    2011-01-01

    Repair of cartilage injury with hyaline cartilage continues to be a challenging clinical problem. Because of the limited number of chondrocytes in vivo, coupled with in vitro de-differentiation of chondrocytes into fibrochondrocytes, which secrete type I collagen and have an altered matrix architecture and mechanical function, there is a need for a novel cell source that produces hyaline cartilage. The generation of induced pluripotent stem (iPS) cells has provided a tool for reprogramming dermal fibroblasts to an undifferentiated state by ectopic expression of reprogramming factors. Here, we show that retroviral expression of two reprogramming factors (c-Myc and Klf4) and one chondrogenic factor (SOX9) induces polygonal chondrogenic cells directly from adult dermal fibroblast cultures. Induced cells expressed marker genes for chondrocytes but not fibroblasts, i.e., the promoters of type I collagen genes were extensively methylated. Although some induced cell lines formed tumors when subcutaneously injected into nude mice, other induced cell lines generated stable homogenous hyaline cartilage–like tissue. Further, the doxycycline-inducible induction system demonstrated that induced cells are able to respond to chondrogenic medium by expressing endogenous Sox9 and maintain chondrogenic potential after substantial reduction of transgene expression. Thus, this approach could lead to the preparation of hyaline cartilage directly from skin, without generating iPS cells. PMID:21293062

  2. Tumor Necrosis Factor-α-Induced Ototoxicity in Mouse Cochlear Organotypic Culture.

    Directory of Open Access Journals (Sweden)

    Qian Wu

    Full Text Available Tumor necrosis factor (TNF-α is a cytokine involved in acute inflammatory phase reactions, and is the primary upstream mediator in the cochlear inflammatory response. Treatment of the organ of Corti with TNF-α can induce hair cell damage. However, the resulting morphological changes have not been systematically examined. In the present study, cochlear organotypic cultures from neonatal mice were treated with various concentrations and durations of TNF-α to induce inflammatory responses. Confocal microscopy was used to evaluate the condition of hair cells and supporting cells following immunohistochemical staining. In addition, the ultrastructure of the stereocilia bundle, hair cells, and supporting cells were examined by scanning and transmission electron microscopy. TNF-α treatment resulted in a fusion and loss of stereocilia bundles in hair cells, swelling of mitochondria, and vacuolation and degranulation of the endoplasmic reticulum. Disruption of tight junctions between hair cells and supporting cells was also observed at high concentrations. Hair cell loss was preceded by apoptosis of Deiters' and pillar cells. Taken together, these findings detail the morphological changes in the organ of Corti after TNF-α treatment, and provide an in vitro model of inflammatory-induced ototoxicity.

  3. Prolongation of liver-specific function for primary hepatocytes maintenance in 3D printed architectures.

    Science.gov (United States)

    Kim, Yohan; Kang, Kyojin; Yoon, Sangtae; Kim, Ji Sook; Park, Su A; Kim, Wan Doo; Lee, Seung Bum; Ryu, Ki-Young; Jeong, Jaemin; Choi, Dongho

    2018-01-23

    Isolated primary hepatocytes from the liver are very similar to in vivo native liver hepatocytes, but they have the disadvantage of a limited lifespan in 2D culture. Although a sandwich culture and 3D organoids with mesenchymal stem cells (MSCs) as an attractive assistant cell source to extend lifespan can be used, it cannot fully reproduce the in vivo architecture. Moreover, long-term 3D culture leads to cell death because of hypoxic stress. Therefore, to overcome the drawback of 2D and 3D organoids, we try to use a 3D printing technique using alginate hydrogels with primary hepatocytes and MSCs. The viability of isolated hepatocytes was more than 90%, and the cells remained alive for 7 days without morphological changes in the 3D hepatic architecture with MSCs. Compared to a 2D system, the expression level of functional hepatic genes and proteins was higher for up to 7 days in the 3D hepatic architecture. These results suggest that both the 3D bio-printing technique and paracrine molecules secreted by MSCs supported long-term culture of hepatocytes without morphological changes. Thus, this technique allows for widespread expansion of cells while forming multicellular aggregates, may be applied to drug screening and could be an efficient method for developing an artificial liver.

  4. Palmitoylethanolamide Blunts Amyloid-β42-Induced Astrocyte Activation and Improves Neuronal Survival in Primary Mouse Cortical Astrocyte-Neuron Co-Cultures.

    Science.gov (United States)

    Beggiato, Sarah; Borelli, Andrea Celeste; Ferraro, Luca; Tanganelli, Sergio; Antonelli, Tiziana; Tomasini, Maria Cristina

    2018-01-01

    Based on the pivotal role of astrocytes in brain homeostasis and the strong metabolic cooperation existing between neurons and astrocytes, it has been suggested that astrocytic dysfunctions might cause and/or contribute to neuroinflammation and neurodegenerative processes. Therapeutic approaches aimed at both neuroprotection and neuroinflammation reduction may prove particularly effective in slowing the progression of these diseases. The endogenous lipid mediator palmitoylethanolamide (PEA) displayed neuroprotective and anti(neuro)inflammatory properties, and demonstrated interesting potential as a novel treatment for Alzheimer's disease. We firstly evaluated whether astrocytes could participate in regulating the Aβ42-induced neuronal damage, by using primary mouse astrocytes cell cultures and mixed astrocytes-neurons cultures. Furthermore, the possible protective effects of PEA against Aβ42-induced neuronal toxicity have also been investigated by evaluating neuronal viability, apoptosis, and morphometric parameters. The presence of astrocytes pre-exposed to Aβ42 (0.5μM; 24 h) induced a reduction of neuronal viability in primary mouse astrocytes-neurons co-cultures. Furthermore, under these experimental conditions, an increase in the number of neuronal apoptotic nuclei and a decrease in the number of MAP-2 positive neurons were observed. Finally, astrocytic Aβ42 pre-exposure induced an increase in the number of neurite aggregations/100μm as compared to control (i.e., untreated) astrocytes-neurons co-cultures. These effects were not observed in neurons cultured in the presence of astrocytes pre-exposed to PEA (0.1μM), applied 1 h before and maintained during Aβ42 treatment. Astrocytes contribute to Aβ42-induced neurotoxicity and PEA, by blunting Aβ42-induced astrocyte activation, improved neuronal survival in mouse astrocyte-neuron co-cultures.

  5. Interleukin-1 inhibition facilitates recovery from liver injury and promotes regeneration of hepatocytes in alcoholic hepatitis in mice.

    Science.gov (United States)

    Iracheta-Vellve, Arvin; Petrasek, Jan; Gyogyosi, Benedek; Bala, Shashi; Csak, Timea; Kodys, Karen; Szabo, Gyongyi

    2017-07-01

    Inflammation and impaired hepatocyte regeneration contribute to liver failure in alcoholic hepatitis (AH). Interleukin (IL)-1 is a key inflammatory cytokine in the pathobiology of AH. The role of IL-1 in liver regeneration in the recovery phase of alcohol-induced liver injury is unknown. In this study, we tested IL-1 receptor antagonist to block IL-1 signalling in a mouse model of acute-on-chronic liver injury on liver inflammation and hepatocyte regeneration in AH. We observed that inhibition of IL-1 signalling decreased liver inflammation and neutrophil infiltration, and resulted in enhanced regeneration of hepatocytes and increased rate of recovery from liver injury in AH. Our novel findings suggest that IL-1 drives sustained liver inflammation and impaired hepatocyte regeneration even after cessation of ethanol exposure. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  6. Non-viral ex vivo hepatic gene transfer by in situ lipofection of liver and intraperitoneal transplantation of hepatocytes.

    Science.gov (United States)

    Rangarajan, P N; Vatsala, P G; Ashok, M S; Srinivas, V K; Habibullah, C M; Padmanaban, G

    1997-04-29

    Perfusion of liver with plasmid DNA-lipofectin complexes via the portal vein results in efficient accumulation of the vector in hepatocytes. Such hepatocytes, when administered intraperitoneally into a hepatectomized rat, repopulate the liver and express the transgene efficiently. This procedure obviates the need for large-scale hepatocyte culture for ex vivo gene transfer. Further, intraperitoneal transplantation is a simple and cost-effective strategy of introducing genetically modified hepatocytes into liver. Thus, in situ lipofection of liver and intraperitoneal transfer of hepatocytes can be developed into a novel method of non-viral ex vivo gene transfer technique that has applications in the treatment of metabolic disorders of liver and hepatic gene therapy.

  7. Towards a defined ECM and small molecule based monolayer culture system for the expansion of mouse and human intestinal stem cells.

    Science.gov (United States)

    Tong, Zhixiang; Martyn, Keir; Yang, Andy; Yin, Xiaolei; Mead, Benjamin E; Joshi, Nitin; Sherman, Nicholas E; Langer, Robert S; Karp, Jeffrey M

    2018-02-01

    Current ISC culture systems face significant challenges such as animal-derived or undefined matrix compositions, batch-to-batch variability (e.g. Matrigel-based organoid culture), and complexity of assaying cell aggregates such as organoids which renders the research and clinical translation of ISCs challenging. Here, through screening for suitable ECM components, we report a defined, collagen based monolayer culture system that supports the growth of mouse and human intestinal epithelial cells (IECs) enriched for an Lgr5 + population comparable or higher to the levels found in a standard Matrigel-based organoid culture. The system, referred to as the Bolstering Lgr5 Transformational (BLT) Sandwich culture, comprises a collagen IV-coated porous substrate and a collagen I gel overlay which sandwich an IEC monolayer in between. The distinct collagen cues synergistically regulate IEC attachment, proliferation, and Lgr5 expression through maximizing the engagement of distinct cell surface adhesion receptors (i.e. integrin α2β1, integrin β4) and cell polarity. Further, we apply our BLT Sandwich system to identify that the addition of a bone morphogenetic protein (BMP) receptor inhibitor (LDN-193189) improves the expansion of Lgr5-GFP + cells from mouse small intestinal crypts by nearly 2.5-fold. Notably, the BLT Sandwich culture is capable of expanding human-derived IECs with higher LGR5 mRNA levels than conventional Matrigel culture, providing superior expansion of human LGR5 + ISCs. Considering the key roles Lgr5 + ISCs play in intestinal epithelial homeostasis and regeneration, we envision that our BLT Sandwich culture system holds great potential for understanding and manipulating ISC biology in vitro (e.g. for modeling ISC-mediated gut diseases) or for expanding a large number of ISCs for clinical utility (e.g. for stem cell therapy). Copyright © 2017 Elsevier Ltd. All rights reserved.

  8. Deletion of hepatocyte nuclear factor-1-beta in an infant with prune belly syndrome.

    Science.gov (United States)

    Haeri, Sina; Devers, Patricia L; Kaiser-Rogers, Kathleen A; Moylan, Vincent J; Torchia, Beth S; Horton, Amanda L; Wolfe, Honor M; Aylsworth, Arthur S

    2010-08-01

    Prune belly syndrome is a rare congenital disorder characterized by deficiency of abdominal wall muscles, cryptorchidism, and urinary tract anomalies. We have had the opportunity to study a baby with prune belly syndrome associated with an apparently de novo 1.3-megabase interstitial 17q12 microdeletion that includes the hepatocyte nuclear factor-1-beta gene at 17q12. One previous patient, an adult, has been reported with prune belly syndrome and a hepatocyte nuclear factor-1-beta microdeletion. Hepatocyte nuclear factor-1-beta is a widely expressed transcription factor that regulates tissue-specific gene expression and is expressed in numerous tissues including mesonephric duct derivatives, the renal tubule of the metanephros, and the developing prostate of the mouse. Mutations in hepatocyte nuclear factor-1-beta cause the "renal cysts and diabetes syndrome," isolated renal cystic dysplasia, and a variety of other malformations. Based on its expression pattern and the observation of two affected cases, we propose that haploinsufficiency of hepatocyte nuclear factor-1-beta may be causally related to the production of the prune belly syndrome phenotype through a mechanism of prostatic and ureteral hypoplasia that results in severe obstructive uropathy with urinary tract and abdominal distension. Copyright Thieme Medical Publishers.

  9. Glutamic Acid as Enhancer of Protein Synthesis Kinetics in Hepatocytes from Old Rats.

    Science.gov (United States)

    Brodsky, V Y; Malchenko, L A; Butorina, N N; Lazarev Konchenko, D S; Zvezdina, N D; Dubovaya, T K

    2017-08-01

    Dense cultures of hepatocytes from old rats (~2 years old, body weight 530-610 g) are different from similar cultures of hepatocytes from young rats by the low amplitude of protein synthesis rhythm. Addition of glutamic acid (0.2, 0.4, or 0.6 mg/ml) into the culture medium with hepatocytes of old rats resulted in increase in the oscillation amplitudes of the protein synthesis rhythm to the level of young rats. A similar action of glutamic acid on the protein synthesis kinetics was observed in vivo after feeding old rats with glutamic acid. Inhibition of metabotropic receptors of glutamic acid with α-methyl-4-carboxyphenylglycine (0.01 mg/ml) abolished the effect of glutamic acid. The amplitude of oscillation of the protein synthesis rhythm in a cell population characterizes synchronization of individual oscillations caused by direct cell-cell communications. Hence, glutamic acid, acting as a receptor-dependent transmitter, enhanced direct cell-cell communications of hepatocytes that were decreased with aging. As differentiated from other known membrane signaling factors (gangliosides, norepinephrine, serotonin, dopamine), glutamic acid can penetrate into the brain and thus influence the communications and protein synthesis kinetics that are disturbed with aging not only in hepatocytes, but also in neurons.

  10. Factor VIIa binding and internalization in hepatocytes

    DEFF Research Database (Denmark)

    Hjortoe, G; Sorensen, B B; Petersen, L C

    2005-01-01

    The liver is believed to be the primary clearance organ for coagulation proteases, including factor VIIa (FVIIa). However, at present, clearance mechanisms for FVIIa in liver are unknown. To obtain information on the FVIIa clearance mechanism, we investigated the binding and internalization...... no effect. HEPG2 cells internalized FVIIa with a rate of 10 fmol 10(-5) cells h(-1). In contrast to HEPG2 cells, FVIIa binding to primary rat hepatocytes was completely independent of TF, and excess unlabeled FVIIa partly reduced the binding of 125I-FVIIa to rat hepatocytes. Further, compared with HEPG2...... cells, three- to fourfold more FVIIa bound to rat primary hepatocytes, and the bound FVIIa was internalized at a faster rate. Similar FVIIa binding and internalization profiles were observed in primary human hepatocytes. Plasma inhibitors had no effect on FVIIa binding and internalization in hepatocytes...

  11. Hepatocytes Determine the Hypoxic Microenvironment and Radiosensitivity of Colorectal Cancer Cells Through Production of Nitric Oxide That Targets Mitochondrial Respiration

    Energy Technology Data Exchange (ETDEWEB)

    Jiang, Heng; Verovski, Valeri N.; Leonard, Wim; Law, Ka Lun; Vermeersch, Marieke; Storme, Guy; Van den Berge, Dirk; Gevaert, Thierry; Sermeus, Alexandra [Department of Radiotherapy, Universitair Ziekenhuis Brussel, Vrije Universiteit Brussel, Brussels (Belgium); De Ridder, Mark, E-mail: mark.deridder@uzbrussel.be [Department of Radiotherapy, Universitair Ziekenhuis Brussel, Vrije Universiteit Brussel, Brussels (Belgium)

    2013-03-01

    Purpose: To determine whether host hepatocytes may reverse hypoxic radioresistance through nitric oxide (NO)-induced oxygen sparing, in a model relevant to colorectal cancer (CRC) liver metastases. Methods and Materials: Hepatocytes and a panel of CRC cells were incubated in a tissue-mimetic coculture system with diffusion-limited oxygenation, and oxygen levels were monitored by an oxygen-sensing fluorescence probe. To activate endogenous NO production, cocultures were exposed to a cytokine mixture, and the expression of inducible nitric oxide synthase was analyzed by reverse transcription–polymerase chain reaction, Western blotting, and NO/nitrite production. The mitochondrial targets of NO were examined by enzymatic activity. To assess hypoxic radioresponse, cocultures were irradiated and reseeded for colonies. Results: Resting hepatocytes consumed 10-40 times more oxygen than mouse CT26 and human DLD-1, HT29, HCT116, and SW480 CRC cells, and thus seemed to be the major effectors of hypoxic conditioning. As a result, hepatocytes caused uniform radioprotection of tumor cells at a 1:1 ratio. Conversely, NO-producing hepatocytes radiosensitized all CRC cell lines more than 1.5-fold, similar to the effect of selective mitochondrial inhibitors. The radiosensitizing effect was associated with a respiratory self-arrest of hepatocytes at the level of aconitase and complex II, which resulted in profound reoxygenation of tumor cells through oxygen sparing. Nitric oxide–producing hepatocytes were at least 10 times more active than NO-producing macrophages to reverse hypoxia-induced radioresistance. Conclusions: Hepatocytes were the major determinants of the hypoxic microenvironment and radioresponse of CRC cells in our model of metabolic hypoxia. We provide evidence that reoxygenation and radiosensitization of hypoxic CRC cells can be achieved through oxygen sparing induced by endogenous NO production in host hepatocytes.

  12. Hepatocytes Determine the Hypoxic Microenvironment and Radiosensitivity of Colorectal Cancer Cells Through Production of Nitric Oxide That Targets Mitochondrial Respiration

    International Nuclear Information System (INIS)

    Jiang, Heng; Verovski, Valeri N.; Leonard, Wim; Law, Ka Lun; Vermeersch, Marieke; Storme, Guy; Van den Berge, Dirk; Gevaert, Thierry; Sermeus, Alexandra; De Ridder, Mark

    2013-01-01

    Purpose: To determine whether host hepatocytes may reverse hypoxic radioresistance through nitric oxide (NO)-induced oxygen sparing, in a model relevant to colorectal cancer (CRC) liver metastases. Methods and Materials: Hepatocytes and a panel of CRC cells were incubated in a tissue-mimetic coculture system with diffusion-limited oxygenation, and oxygen levels were monitored by an oxygen-sensing fluorescence probe. To activate endogenous NO production, cocultures were exposed to a cytokine mixture, and the expression of inducible nitric oxide synthase was analyzed by reverse transcription–polymerase chain reaction, Western blotting, and NO/nitrite production. The mitochondrial targets of NO were examined by enzymatic activity. To assess hypoxic radioresponse, cocultures were irradiated and reseeded for colonies. Results: Resting hepatocytes consumed 10-40 times more oxygen than mouse CT26 and human DLD-1, HT29, HCT116, and SW480 CRC cells, and thus seemed to be the major effectors of hypoxic conditioning. As a result, hepatocytes caused uniform radioprotection of tumor cells at a 1:1 ratio. Conversely, NO-producing hepatocytes radiosensitized all CRC cell lines more than 1.5-fold, similar to the effect of selective mitochondrial inhibitors. The radiosensitizing effect was associated with a respiratory self-arrest of hepatocytes at the level of aconitase and complex II, which resulted in profound reoxygenation of tumor cells through oxygen sparing. Nitric oxide–producing hepatocytes were at least 10 times more active than NO-producing macrophages to reverse hypoxia-induced radioresistance. Conclusions: Hepatocytes were the major determinants of the hypoxic microenvironment and radioresponse of CRC cells in our model of metabolic hypoxia. We provide evidence that reoxygenation and radiosensitization of hypoxic CRC cells can be achieved through oxygen sparing induced by endogenous NO production in host hepatocytes

  13. Characterization of genetically engineered mouse hepatoma cells with inducible liver functions by overexpression of liver-enriched transcription factors.

    Science.gov (United States)

    Yamamoto, Hideaki; Tonello, Jane Marie; Sambuichi, Takanori; Kawabe, Yoshinori; Ito, Akira; Kamihira, Masamichi

    2018-01-01

    New cell sources for the research and therapy of organ failure could significantly alleviate the shortage of donor livers that are available to patients who suffer from liver disease. Liver carcinoma derived cells, or hepatoma cells, are the ideal cells for developing bioartificial liver systems. Such cancerous liver cells are easy to prepare in large quantities and can be maintained over long periods under standard culture conditions, unlike primary hepatocytes. However, hepatoma cells possess only a fraction of the functions of primary hepatocytes. In a previous study, by transducing cells with liver-enriched transcription factors that could be inducibly overexpressed-hepatocyte nuclear factor (HNF)1α, HNF1β, HNF3β [FOXA2], HNF4α, HNF6, CCAAT/enhancer binding protein (C/EBP)α, C/EBPβ and C/EBPγ-we created mouse hepatoma cells with high liver-specific gene expression called the Hepa/8F5 cell line. In the present study, we performed functional and genetic analyses to characterize the Hepa/8F5 cell line. Further, in three-dimensional cultures, the function of these cells improved significantly compared to parental cells. Ultimately, these cells might become a new resource that can be used in basic and applied hepatic research. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  14. Optimization of the isolation and cultivation of Cyprinus carpio primary hepatocytes

    Science.gov (United States)

    Yanhong, Fan; Chenghua, He; Guofang, Liu

    2008-01-01

    The aquatic environment is affected by numerous chemical contaminants. There is an increasing need to identify these chemicals and to evaluate their potential toxicity towards aquatic life. In this research we optimized techniques for primary cell culture of Cyprinus carpio hepatocytes as one adjunct model for ecotoxicological evaluation of the potential hazards of xenobiotics in the aquatic environment. In this study, Cyprinus carpio hepatocytes were isolated by mechanical separation, two-step collagenase perfusion, and pancreatin digestion. The hepatocytes or parenchymal cells could be separated from cell debris and from non-parenchymal cells by low-speed centrifugation (Percoll gradient centrifugation). The harvested hepatocytes were suspended in DMEM, M199 (cultured in 5% CO2), or L-15 (cultured without 5% CO2) medium then cultured at 17, 27, or 37 °C. Cell yield was counted by use of a hemocytometer, and the viability of the cells was assessed by use of the Trypan blue exclusion test. Results from these studies showed that the best method of isolation was pancreatin digestion (the cell yield was 2.7 × 108 per g (liver weight) and the viability was 98.4%) and the best medium was M199 (cultured in 5% CO2) or L-15 (cultured without 5% CO2). The optimum culture temperature was 27 °C. The primary hepatocytes culture of Cyprimus carpio grew well and satisfied requirements for most toxicological experiments in this condition. PMID:19002769

  15. Adropin induction of lipoprotein lipase expression in tilapia hepatocytes.

    Science.gov (United States)

    Lian, Anji; Wu, Keqiang; Liu, Tianqiang; Jiang, Nan; Jiang, Quan

    2016-01-01

    The peptide hormone adropin plays a role in energy homeostasis. However, biological actions of adropin in non-mammalian species are still lacking. Using tilapia as a model, we examined the role of adropin in lipoprotein lipase (LPL) regulation in hepatocytes. To this end, the structural identity of tilapia adropin was established by 5'/3'-rapid amplification of cDNA ends (RACE). The transcripts of tilapia adropin were ubiquitously expressed in various tissues with the highest levels in the liver and hypothalamus. The prolonged fasting could elevate tilapia hepatic adropin gene expression, whereas no effect of fasting was observed on hypothalamic adropin gene levels. In primary cultures of tilapia hepatocytes, synthetic adropin was effective in stimulating LPL release, cellular LPL content, and total LPL production. The increase in LPL production also occurred with parallel rises in LPL gene levels. In parallel experiments, adropin could elevate cAMP production and up-regulate protein kinase A (PKA) and PKC activities. Using a pharmacological approach, cAMP/PKA and PLC/inositol trisphosphate (IP3)/PKC cascades were shown to be involved in adropin-stimulated LPL gene expression. Parallel inhibition of p38MAPK and Erk1/2, however, were not effective in these regards. Our findings provide, for the first time, evidence that adropin could stimulate LPL gene expression via direct actions in tilapia hepatocytes through the activation of multiple signaling mechanisms. © 2016 Society for Endocrinology.

  16. Homocysteine inhibits hepatocyte proliferation via endoplasmic reticulum stress.

    Directory of Open Access Journals (Sweden)

    Xue Yu

    Full Text Available Homocysteine is an independent risk factor for coronary, cerebral, and peripheral vascular diseases. Recent studies have shown that levels of homocysteine are elevated in patients with impaired hepatic function, but the precise role of homocysteine in the development of hepatic dysfunction is unclear. In this study, we examined the effect of homocysteine on hepatocyte proliferation in vitro. Our results demonstrated that homocysteine inhibited hepatocyte proliferation by up-regulating protein levels of p53 as well as mRNA and protein levels of p21(Cip1 in primary cultured hepatocytes. Homocysteine induced cell growth arrest in p53-positive hepatocarcinoma cell line HepG2, but not in p53-null hepatocarcinoma cell line Hep3B. A p53 inhibitor pifithrin-α inhibited the expression of p21(Cip1 and attenuated homocysteine-induced cell growth arrest. Homocysteine induced TRB3 expression via endoplasmic reticulum stress pathway, resulting in Akt dephosphorylation. Knock-down of endogenous TRB3 significantly suppressed the inhibitory effect of homocysteine on cell proliferation and the phosphorylation of Akt. LiCl reversed homocysteine-mediated cell growth arrest by inhibiting TRB3-mediated Akt dephosphorylation. These results demonstrate that both TRB3 and p21(Cip1 are critical molecules in the homocysteine signaling cascade and provide a mechanistic explanation for impairment of liver regeneration in hyperhomocysteinemia.

  17. A fat option for the pig: Hepatocytic differentiated mesenchymal stem cells for translational research

    Energy Technology Data Exchange (ETDEWEB)

    Brückner, Sandra, E-mail: sandra.brueckner@medizin.uni-leipzig.de [University Hospital Leipzig, Department of Visceral, Transplantation, Thoracic and Vascular Surgery, Liebigstraße 21, Leipzig D-04103 (Germany); Tautenhahn, Hans-Michael, E-mail: hans-michael.tautenhahn@medizin.uni-leipzig.de [University Hospital Leipzig, Department of Visceral, Transplantation, Thoracic and Vascular Surgery, Liebigstraße 21, Leipzig D-04103 (Germany); TRM, Translational Centre for Regenerative Medicine, Philipp-Rosenthal-Str. 55, Leipzig D-04103 (Germany); Winkler, Sandra, E-mail: sandra.pelz@medizin.uni-leipzig.de [University Hospital Leipzig, Department of Visceral, Transplantation, Thoracic and Vascular Surgery, Liebigstraße 21, Leipzig D-04103 (Germany); Stock, Peggy, E-mail: peggy.stock@medizin.uni-leipzig.de [University Hospital Leipzig, Department of Visceral, Transplantation, Thoracic and Vascular Surgery, Liebigstraße 21, Leipzig D-04103 (Germany); Dollinger, Matthias, E-mail: matthias.dollinger@uniklinik-ulm.de [University Hospital Ulm, First Department of Medicine, Albert-Einstein-Allee 23, Ulm D-89081 (Germany); Christ, Bruno, E-mail: bruno.christ@medizin.uni-leipzig.de [University Hospital Leipzig, Department of Visceral, Transplantation, Thoracic and Vascular Surgery, Liebigstraße 21, Leipzig D-04103 (Germany); TRM, Translational Centre for Regenerative Medicine, Philipp-Rosenthal-Str. 55, Leipzig D-04103 (Germany)

    2014-02-15

    Study background: Extended liver resection is the only curative treatment option of liver cancer. Yet, the residual liver may not accomplish the high metabolic and regenerative capacity needed, which frequently leads to acute liver failure. Because of their anti-inflammatory and -apoptotic as well as pro-proliferative features, mesenchymal stem cells differentiated into hepatocyte-like cells might provide functional and regenerative compensation. Clinical translation of basic research requires pre-clinical approval in large animals. Therefore, we characterized porcine mesenchymal stem cells (MSC) from adipose tissue and bone marrow and their hepatocyte differentiation potential for future assessment of functional liver support after surgical intervention in the pig model. Methods: Mesenchymal surface antigens and multi-lineage differentiation potential of porcine MSC isolated by collagenase digestion either from bone marrow or adipose tissue (subcutaneous/visceral) were assessed by flow cytometry. Morphology and functional properties (urea-, glycogen synthesis and cytochrome P450 activity) were determined during culture under differentiation conditions and compared with primary porcine hepatocytes. Results: MSC from porcine adipose tissue and from bone marrow express the typical mesenchymal markers CD44, CD29, CD90 and CD105 but not haematopoietic markers. MSC from both sources displayed differentiation into the osteogenic as well as adipogenic lineage. After hepatocyte differentiation, expression of CD105 decreased significantly and cells adopted the typical polygonal morphology of hepatocytes. Glycogen storage was comparable in adipose tissue- and bone marrow-derived cells. Urea synthesis was about 35% lower in visceral than in subcutaneous adipose tissue-derived MSC. Cytochrome P450 activity increased significantly during differentiation and was twice as high in hepatocyte-like cells generated from bone marrow as from adipose tissue. Conclusion: The hepatocyte

  18. A fat option for the pig: Hepatocytic differentiated mesenchymal stem cells for translational research

    International Nuclear Information System (INIS)

    Brückner, Sandra; Tautenhahn, Hans-Michael; Winkler, Sandra; Stock, Peggy; Dollinger, Matthias; Christ, Bruno

    2014-01-01

    Study background: Extended liver resection is the only curative treatment option of liver cancer. Yet, the residual liver may not accomplish the high metabolic and regenerative capacity needed, which frequently leads to acute liver failure. Because of their anti-inflammatory and -apoptotic as well as pro-proliferative features, mesenchymal stem cells differentiated into hepatocyte-like cells might provide functional and regenerative compensation. Clinical translation of basic research requires pre-clinical approval in large animals. Therefore, we characterized porcine mesenchymal stem cells (MSC) from adipose tissue and bone marrow and their hepatocyte differentiation potential for future assessment of functional liver support after surgical intervention in the pig model. Methods: Mesenchymal surface antigens and multi-lineage differentiation potential of porcine MSC isolated by collagenase digestion either from bone marrow or adipose tissue (subcutaneous/visceral) were assessed by flow cytometry. Morphology and functional properties (urea-, glycogen synthesis and cytochrome P450 activity) were determined during culture under differentiation conditions and compared with primary porcine hepatocytes. Results: MSC from porcine adipose tissue and from bone marrow express the typical mesenchymal markers CD44, CD29, CD90 and CD105 but not haematopoietic markers. MSC from both sources displayed differentiation into the osteogenic as well as adipogenic lineage. After hepatocyte differentiation, expression of CD105 decreased significantly and cells adopted the typical polygonal morphology of hepatocytes. Glycogen storage was comparable in adipose tissue- and bone marrow-derived cells. Urea synthesis was about 35% lower in visceral than in subcutaneous adipose tissue-derived MSC. Cytochrome P450 activity increased significantly during differentiation and was twice as high in hepatocyte-like cells generated from bone marrow as from adipose tissue. Conclusion: The hepatocyte

  19. Force spectroscopy of hepatocytic extracellular matrix components

    Energy Technology Data Exchange (ETDEWEB)

    Yongsunthon, R., E-mail: YongsuntR@Corning.com [Corning Incorporated, SP-FR-01, R1S32D, Corning, NY 14831 (United States); Baker, W.A.; Bryhan, M.D.; Baker, D.E.; Chang, T.; Petzold, O.N.; Walczak, W.J.; Liu, J.; Faris, R.A.; Senaratne, W.; Seeley, L.A.; Youngman, R.E. [Corning Incorporated, SP-FR-01, R1S32D, Corning, NY 14831 (United States)

    2009-07-15

    We present atomic force microscopy and force spectroscopy data of live hepatocytes (HEPG2/C3A liver cell line) grown in Eagle's Minimum Essential Medium, a complex solution of salts and amino acids commonly used for cell culture. Contact-mode imaging and force spectroscopy of this system allowed correlation of cell morphology and extracellular matrix (ECM) properties with substrate properties. Force spectroscopy analysis of cellular 'footprints' indicated that the cells secrete large polymers (e.g., 3.5 {mu}m contour length and estimated MW 1000 kDa) onto their substrate surface. Although definitive identification of the polymers has not yet been achieved, fluorescent-labeled antibody staining has specified the presence of ECM proteins such as collagen and laminin in the cellular footprints. The stretched polymers appear to be much larger than single molecules of known ECM components, such as collagen and heparan sulfate proteoglycan, thus suggesting that the cells create larger entangled, macromolecular structures from smaller components. There is strong evidence which suggests that the composition of the ECM is greatly influenced by the hydrophobicity of the substrate surface, with preferential production and/or adsorption of larger macromolecules on hydrophobic surfaces.

  20. AR42J-B-13 cell: An expandable progenitor to generate an unlimited supply of functional hepatocytes

    International Nuclear Information System (INIS)

    Wallace, Karen; Fairhall, Emma A.; Charlton, Keith A.; Wright, Matthew C.

    2010-01-01

    Hepatocytes are the preparation of choice for Toxicological research in vitro. However, despite the fact that hepatocytes proliferate in vivo during liver regeneration, they are resistant to proliferation in vitro, do not tolerate sub-culture and tend to enter a de-differentiation program that results in a loss of hepatic function. These limitations have resulted in the search for expandable rodent and human cells capable of being directed to differentiate into functional hepatocytes. Research with stem cells suggests that it may be possible to provide the research community with hepatocytes in vitro although to date, significant challenges remain, notably generating a sufficiently pure population of hepatocytes with a quantitative functionality comparable with hepatocytes. This paper reviews work with the AR42J-B-13 (B-13) cell line. The B-13 cell was cloned from the rodent AR42J pancreatic cell line, express genes associated with pancreatic acinar cells and readily proliferates in simple culture media. When exposed to glucocorticoid, 75-85% of the cells trans-differentiate into hepatocyte-like (B-13/H) cells functioning at a level quantitatively similar to freshly isolated rat hepatocytes (with the remaining cells retaining the B-13 phenotype). Trans-differentiation of pancreatic acinar cells also appears to occur in vivo in rats treated with glucocorticoid; in mice with elevated circulating glucocorticoid and in humans treated for long periods with glucocorticoid. The B-13 response to glucocorticoid therefore appears to be related to a real pathophysiological response of a pancreatic cell to glucocorticoid. An understanding of how this process occurs and if it can be generated or engineered in human cells would result in a cell line with the ability to generate an unlimited supply of functional human hepatocytes in a cost effective manner.

  1. Neuroprotective effects of the AMPA antagonist PNQX in oxygen-glucose deprivation in mouse hippocampal slice cultures and global cerebral ischemia in gerbils

    DEFF Research Database (Denmark)

    Montero, Maria; Nielsen, Marianne; Rønn, Lars Christian B

    2007-01-01

    PNQX (9-methyl-amino-6-nitro-hexahydro-benzo(F)quinoxalinedione) is a selective AMPA antagonist with demonstrated neuroprotective effects in focal ischemia in rats. Here we report corresponding effects in mouse hippocampal slice cultures subjected to oxygen and glucose deprivation (OGD) and in tr......PNQX (9-methyl-amino-6-nitro-hexahydro-benzo(F)quinoxalinedione) is a selective AMPA antagonist with demonstrated neuroprotective effects in focal ischemia in rats. Here we report corresponding effects in mouse hippocampal slice cultures subjected to oxygen and glucose deprivation (OGD......) and in transient global cerebral ischemia in gerbils. For in vitro studies, hippocampal slice cultures derived from 7-day-old mice and grown for 14 days, were submersed in oxygen-glucose deprived medium for 30 min and exposed to PNQX for 24 h, starting together with OGD, immediately after OGD, or 2 h after OGD...... stained for the neurodegeneration marker Fluoro-Jade B and immunostained for the astroglial marker glial fibrillary acidic protein revealed a significant PNQX-induced decrease in neuronal cell death and astroglial activation. We conclude that, PNQX provided neuroprotection against both global cerebral...

  2. Specific binding of an immunoreactive and biologically active 125I-labeled substance P derivative to mouse mesencephalic cells in primary culture

    International Nuclear Information System (INIS)

    Beaujouan, J.C.; Torrens, Y.; Herbet, A.; Daguet, M.C.; Glowinski, J.; Prochiantz, A.

    1982-01-01

    Binding characteristics of 125 I-labeled Bolton-Hunter substance P ([ 125 I]BHSP), a radioactive analogue of substance P, were studied with mesencephalic primary cultures prepared from embryonic mouse brain. Nonspecific binding represented no more than 20% of the total binding observed on the cells. In contrast, significant specific binding--saturable, reversible, and temperature-dependent--was demonstrated. Scatchard analysis of concentration-dependent binding saturation indicates a single population of noninteracting sites with a high affinity (Kd . 169 pM). Substance P and different substance P analogues were tested for their competitive potencies with regard to [ 125 I]BHSP binding. BHSP itself, substance P, (Tyr8)-substance P, and (nor-Leu11)-substance P strongly inhibited the binding. Good inhibition was also obtained with physalaemin and eledoisin, two peptides structurally related to substance P. When substance P C-terminal fragments were tested for their ability to compete with [ 125 I]BHSP binding, a good relationship was found between competitive activity and peptide length. Regional distribution of [ 125 I]BHSP binding sites was found using primary cultures obtained from different regions of embryonic mouse brain. Mesencephalic, hypothalamic, and striatal cultures had the highest [ 125 I]BHSP binding capacities, whereas cortical, hippocampal, and cerebellar cells shared only little binding activity. Finally, when mesencephalic cells were grown under conditions impairing glial development, [ 125 I]BHSP binding was not affected, demonstrating that binding sites are located on neuronal cells

  3. Live cell imaging of cytosolic NADH/NAD+ ratio in hepatocytes and liver slices.

    Science.gov (United States)

    Masia, Ricard; McCarty, William J; Lahmann, Carolina; Luther, Jay; Chung, Raymond T; Yarmush, Martin L; Yellen, Gary

    2018-01-01

    Fatty liver disease (FLD), the most common chronic liver disease in the United States, may be caused by alcohol or the metabolic syndrome. Alcohol is oxidized in the cytosol of hepatocytes by alcohol dehydrogenase (ADH), which generates NADH and increases cytosolic NADH/NAD + ratio. The increased ratio may be important for development of FLD, but our ability to examine this question is hindered by methodological limitations. To address this, we used the genetically encoded fluorescent sensor Peredox to obtain dynamic, real-time measurements of cytosolic NADH/NAD + ratio in living hepatocytes. Peredox was expressed in dissociated rat hepatocytes and HepG2 cells by transfection, and in mouse liver slices by tail-vein injection of adeno-associated virus (AAV)-encoded sensor. Under control conditions, hepatocytes and liver slices exhibit a relatively low (oxidized) cytosolic NADH/NAD + ratio as reported by Peredox. The ratio responds rapidly and reversibly to substrates of lactate dehydrogenase (LDH) and sorbitol dehydrogenase (SDH). Ethanol causes a robust dose-dependent increase in cytosolic NADH/NAD + ratio, and this increase is mitigated by the presence of NAD + -generating substrates of LDH or SDH. In contrast to hepatocytes and slices, HepG2 cells exhibit a relatively high (reduced) ratio and show minimal responses to substrates of ADH and SDH. In slices, we show that comparable results are obtained with epifluorescence imaging and two-photon fluorescence lifetime imaging (2p-FLIM). Live cell imaging with Peredox is a promising new approach to investigate cytosolic NADH/NAD + ratio in hepatocytes. Imaging in liver slices is particularly attractive because it allows preservation of liver microanatomy and metabolic zonation of hepatocytes. NEW & NOTEWORTHY We describe and validate a new approach for measuring free cytosolic NADH/NAD + ratio in hepatocytes and liver slices: live cell imaging with the fluorescent biosensor Peredox. This approach yields dynamic, real

  4. Optimization of a serum-free culture medium for mouse embryonic stem cells using design of experiments (DoE) methodology.

    Science.gov (United States)

    Knöspel, Fanny; Schindler, Rudolf K; Lübberstedt, Marc; Petzolt, Stephanie; Gerlach, Jörg C; Zeilinger, Katrin

    2010-12-01

    The in vitro culture behaviour of embryonic stem cells (ESC) is strongly influenced by the culture conditions. Current culture media for expansion of ESC contain some undefined substances. Considering potential clinical translation work with such cells, the use of defined media is desirable. We have used Design of Experiments (DoE) methods to investigate the composition of a serum-free chemically defined culture medium for expansion of mouse embryonic stem cells (mESC). Factor screening analysis according to Plackett-Burman revealed that insulin and leukaemia inhibitory factor (LIF) had a significant positive influence on the proliferation activity of the cells, while zinc and L: -cysteine reduced the cell growth. Further analysis using minimum run resolution IV (MinRes IV) design indicates that following factor adjustment LIF becomes the main factor for the survival and proliferation of mESC. In conclusion, DoE screening assays are applicable to develop and to refine culture media for stem cells and could also be employed to optimize culture media for human embryonic stem cells (hESC).

  5. Generation of human hepatocytes by stem cell technology: definition of the hepatocyte.

    Science.gov (United States)

    Hengstler, Jan G; Brulport, Marc; Schormann, Wiebke; Bauer, Alexander; Hermes, Matthias; Nussler, Andreas K; Fandrich, Fred; Ruhnke, Maren; Ungefroren, Hendrik; Griffin, Louise; Bockamp, Ernesto; Oesch, Franz; von Mach, Marc-Alexander

    2005-06-01

    Since 1999, numerous articles have reported the generation of hepatocytes from different types of extrahepatic stem or precursor cells. This opens exciting new possibilities for pharmacology and toxicology, as well as for cell therapy. Hepatocyte marker expression, including albumin, cytokeratin 18, c-met, alpha-fetoprotein and cytochrome P450 3A4 and -2B6, has been observed after transplantation of different types of human stem cells into the liver of laboratory animals or in vitro after incubation with cytokines. These intriguing observations have prompted scientists to classify stem cell-derived cell populations as hepatocytes. However, this conclusion may be premature. It has been shown that factors of the liver microenvironment can induce expression of a limited number of hepatocyte marker genes in nonhepatic cell types. To conclude on the grounds of a limited number of markers that these cells are true hepatocytes is not indicated. In this case one should carefully evaluate crucial hepatocyte-defining enzymatic properties. The present article: i) reviews studies describing the fate of extrahepatic human stem and precursor cells in livers of laboratory animals, including the possibility of cell fusion; and ii) critically discusses the phenotype of stem cells after application of various differentiation protocols aimed at generating human hepatocytes. In addition, the necessary criteria needed for defining a true hepatocyte are suggested. Establishing the necessary properties for stem cell-derived hepatocytes is timely and reasonable, and thus avoids further misleading semantic confusion. Finally, it is essential to understand that the definition of a bona fide hepatocyte should not be limited to qualitative assays, such as reverse transcriptase polymerase chain reaction and immunohistochemistry, but has to include a quantitative analysis of enzymatic activities, which allows direct comparison with primary hepatocytes. Although the stem cell-derived-hepatocyte

  6. Enhancement of the predicted drug hepatotoxicity in gel entrapped hepatocytes within polysulfone-g-poly (ethylene glycol) modified hollow fiber

    International Nuclear Information System (INIS)

    Shen Chong; Zhang Guoliang; Meng Qin

    2010-01-01

    Collagen gel-based 3D cultures of hepatocytes have been proposed for evaluation of drug hepatotoxicity because of their more reliability than traditional monolayer culture. The collagen gel entrapment of hepatocytes in hollow fibers has been proven to well reflect the drug hepatotoxicity in vivo but was limited by adsorption of hydrophobic drugs onto hollow fibers. This study aimed to investigate the impact of hollow fibers on hepatocyte performance and drug hepatotoxicity. Polysulfone-g-poly (ethylene glycol) (PSf-g-PEG) hollow fiber was fabricated and applied for the first time to suppress the drug adsorption. Then, the impact of hollow fibers was evaluated by detecting the hepatotoxicity of eight selected drugs to gel entrapped hepatocytes within PSf and PSf-g-PEG hollow fibers, or without hollow fibers. The hepatocytes in PSf-g-PEG hollow fiber showed the highest sensitivity to drug hepatotoxicity, while those in PSf hollow fiber and cylindrical gel without hollow fiber underestimated the hepatotoxicity due to either drug adsorption or low hepatic functions. Therefore, the 3D culture of gel entrapped hepatocytes within PSf-g-PEG hollow fiber would be a promising tool for investigation of drug hepatotoxicity in vitro.

  7. Foxa1 reduces lipid accumulation in human hepatocytes and is down-regulated in nonalcoholic fatty liver.

    Directory of Open Access Journals (Sweden)

    Marta Moya

    Full Text Available Triglyceride accumulation in nonalcoholic fatty liver (NAFL results from unbalanced lipid metabolism which, in the liver, is controlled by several transcription factors. The Foxa subfamily of winged helix/forkhead box (Fox transcription factors comprises three members which play important roles in controlling both metabolism and homeostasis through the regulation of multiple target genes in the liver, pancreas and adipose tissue. In the mouse liver, Foxa2 is repressed by insulin and mediates fasting responses. Unlike Foxa2 however, the role of Foxa1 in the liver has not yet been investigated in detail. In this study, we evaluate the role of Foxa1 in two human liver cell models, primary cultured hepatocytes and HepG2 cells, by adenoviral infection. Moreover, human and rat livers were analyzed to determine Foxa1 regulation in NAFL. Results demonstrate that Foxa1 is a potent inhibitor of hepatic triglyceride synthesis, accumulation and secretion by repressing the expression of multiple target genes of these pathways (e.g., GPAM, DGAT2, MTP, APOB. Moreover, Foxa1 represses the fatty acid transporter protein FATP2 and lowers fatty acid uptake. Foxa1 also increases the breakdown of fatty acids by inducing peroxisomal fatty acid β-oxidation and ketone body synthesis. Finally, Foxa1 is able to largely up-regulate UCP1, thereby dissipating energy and consistently decreasing the mitochondria membrane potential. We also report that human and rat NAFL have a reduced Foxa1 expression, possibly through a protein kinase C-dependent pathway. We conclude that Foxa1 is an antisteatotic factor that coordinately tunes several lipid metabolic pathways to block triglyceride accumulation in hepatocytes. However, Foxa1 is down-regulated in human and rat NAFL and, therefore, increasing Foxa1 levels could protect from steatosis. Altogether, we suggest that Foxa1 could be a novel therapeutic target for NAFL disease and insulin resistance.

  8. Assessment of mitochondrial dysfunction-related, drug-induced hepatotoxicity in primary rat hepatocytes

    International Nuclear Information System (INIS)

    Liu, Cong; Sekine, Shuichi; Ito, Kousei

    2016-01-01

    Evidence that mitochondrial dysfunction plays a central role in drug-induced liver injury is rapidly accumulating. In contrast to physiological conditions, in which almost all adenosine triphosphate (ATP) in hepatocytes is generated in mitochondria via aerobic respiration, the high glucose content and limited oxygen supply of conventional culture systems force primary hepatocytes to generate most ATP via cytosolic glycolysis. Thus, such anaerobically poised cells are resistant to xenobiotics that impair mitochondrial function, and are not suitable to identify drugs with mitochondrial liabilities. In this study, primary rat hepatocytes were cultured in galactose-based medium, instead of the conventional glucose-based medium, and in hyperoxia to improve the reliance of energy generation on aerobic respiration. Activation of mitochondria was verified by diminished cellular lactate release and increased oxygen consumption. These conditions improved sensitivity to the mitochondrial complex I inhibitor rotenone. Since oxidative stress is also a general cause of mitochondrial impairment, cells were exposed to test compounds in the presence of transferrin to increase the generation of reactive oxygen species via increased uptake of iron. Finally, 14 compounds with reported mitochondrial liabilities were tested to validate this new drug-induced mitochondrial toxicity assay. Overall, the culture of primary rat hepatocytes in galactose, hyperoxia and transferrin is a useful model for the identification of mitochondrial dysfunction-related drug-induced hepatotoxicity. - Highlights: • Drug-induced mitochondrial toxicity was evaluated using primary rat hepatocytes. • Galactose and hyperoxia could activate OXPHOS in primary rat hepatocytes. • Cells with enhanced OXPHOS exhibit improved sensitivity to mitochondrial toxins. • Transferrin potentiate mitochondrial toxicity via increased ROS production.

  9. 3D-printed gelatin scaffolds of differing pore geometry modulate hepatocyte function and gene expression.

    Science.gov (United States)

    Lewis, Phillip L; Green, Richard M; Shah, Ramille N

    2018-03-15

    Three dimensional (3D) printing is highly amenable to the fabrication of tissue-engineered organs of a repetitive microstructure such as the liver. The creation of uniform and geometrically repetitive tissue scaffolds can also allow for the control over cellular aggregation and nutrient diffusion. However, the effect of differing geometries, while controlling for pore size, has yet to be investigated in the context of hepatocyte function. In this study, we show the ability to precisely control pore geometry of 3D-printed gelatin scaffolds. An undifferentiated hepatocyte cell line (HUH7) demonstrated high viability and proliferation when seeded on 3D-printed scaffolds of two different geometries. However, hepatocyte specific functions (albumin secretion, CYP activity, and bile transport) increases in more interconnected 3D-printed gelatin cultures compared to a less interconnected geometry and to 2D controls. Additionally, we also illustrate the disparity between gene expression and protein function in simple 2D culture modes, and that recreation of a physiologically mimetic 3D environment is necessary to induce both expression and function of cultured hepatocytes. Three dimensional (3D) printing provides tissue engineers the ability spatially pattern cells and materials in precise geometries, however the biological effects of scaffold geometry on soft tissues such as the liver have not been rigorously investigated. In this manuscript, we describe a method to 3D print gelatin into well-defined repetitive geometries that show clear differences in biological effects on seeded hepatocytes. We show that a relatively simple and widely used biomaterial, such as gelatin, can significantly modulate biological processes when fabricated into specific 3D geometries. Furthermore, this study expands upon past research into hepatocyte aggregation by demonstrating how it can be manipulated to enhance protein function, and how function and expression may not precisely correlate in

  10. Assessment of mitochondrial dysfunction-related, drug-induced hepatotoxicity in primary rat hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Cong; Sekine, Shuichi, E-mail: ssekine@faculty.chiba-u.jp; Ito, Kousei

    2016-07-01

    Evidence that mitochondrial dysfunction plays a central role in drug-induced liver injury is rapidly accumulating. In contrast to physiological conditions, in which almost all adenosine triphosphate (ATP) in hepatocytes is generated in mitochondria via aerobic respiration, the high glucose content and limited oxygen supply of conventional culture systems force primary hepatocytes to generate most ATP via cytosolic glycolysis. Thus, such anaerobically poised cells are resistant to xenobiotics that impair mitochondrial function, and are not suitable to identify drugs with mitochondrial liabilities. In this study, primary rat hepatocytes were cultured in galactose-based medium, instead of the conventional glucose-based medium, and in hyperoxia to improve the reliance of energy generation on aerobic respiration. Activation of mitochondria was verified by diminished cellular lactate release and increased oxygen consumption. These conditions improved sensitivity to the mitochondrial complex I inhibitor rotenone. Since oxidative stress is also a general cause of mitochondrial impairment, cells were exposed to test compounds in the presence of transferrin to increase the generation of reactive oxygen species via increased uptake of iron. Finally, 14 compounds with reported mitochondrial liabilities were tested to validate this new drug-induced mitochondrial toxicity assay. Overall, the culture of primary rat hepatocytes in galactose, hyperoxia and transferrin is a useful model for the identification of mitochondrial dysfunction-related drug-induced hepatotoxicity. - Highlights: • Drug-induced mitochondrial toxicity was evaluated using primary rat hepatocytes. • Galactose and hyperoxia could activate OXPHOS in primary rat hepatocytes. • Cells with enhanced OXPHOS exhibit improved sensitivity to mitochondrial toxins. • Transferrin potentiate mitochondrial toxicity via increased ROS production.

  11. Pretreatment with mixed-function oxidase inducers increases the sensitivity of the hepatocyte/DNA repair assay

    International Nuclear Information System (INIS)

    Shaddock, J.G.; Heflich, R.H.; McMillan, D.C.; Hinson, J.A.; Casciano, D.A.

    1989-01-01

    A recent National Toxicology Program evaluation indicates that the rat hepatocyte/DNA repair assay has a high false-negative rate and that it is insensitive to some genotoxic hepatocarcinogens as well as other species and organ-specific carcinogens. In this study, the authors examined whether the sensitivity of the hepatocyte/DNA repair assay might be increased through animal pretreatment with various hepatic mixed-function oxidase inducers, i.e., Aroclor 1254, phenobarbital, and 3,3',4,4'-tetrachloroazobenzene (TCAB). The effects on unscheduled DNA synthesis (UDS), a measured of DNA damage and repair, were studied in cultures exposed to known and/or potential carcinogens that had been evaluated as negative or questionable or that produced conflicting results with hepatocytes isolated from uninduced animals. 4,4'-Oxydianiline, 1-nitropy-rene, and TCAB produced concentration-dependent increases in UDS in hepatocytes from rats pretreated with Aroclor 1254. 4,4'-Oxydianiline and TCAB also induced a dose-dependent increase in DNA repair in hepatocytes from rats pretreated with phenobarbital, whereas 1-nitropyrene was negative. These data indicate that the limited sensitivity to chemical carcinogens displayed by the hepatocyte/DNA repair assay may be increased by using hepatocytes isolated from animals exposed to hepatic mixed-function oxidase inducers

  12. Pretreatment with mixed-function oxidase inducers increases the sensitivity of the hepatocyte/DNA repair assay

    Energy Technology Data Exchange (ETDEWEB)

    Shaddock, J.G.; Heflich, R.H.; McMillan, D.C.; Hinson, J.A.; Casciano, D.A. (National Center for Toxicological Research, Jefferson, AK (USA) Univ. of Arkansas for Medical Sciences, Little Rock (USA))

    1989-01-01

    A recent National Toxicology Program evaluation indicates that the rat hepatocyte/DNA repair assay has a high false-negative rate and that it is insensitive to some genotoxic hepatocarcinogens as well as other species and organ-specific carcinogens. In this study, the authors examined whether the sensitivity of the hepatocyte/DNA repair assay might be increased through animal pretreatment with various hepatic mixed-function oxidase inducers, i.e., Aroclor 1254, phenobarbital, and 3,3{prime},4,4{prime}-tetrachloroazobenzene (TCAB). The effects on unscheduled DNA synthesis (UDS), a measured of DNA damage and repair, were studied in cultures exposed to known and/or potential carcinogens that had been evaluated as negative or questionable or that produced conflicting results with hepatocytes isolated from uninduced animals. 4,4{prime}-Oxydianiline, 1-nitropy-rene, and TCAB produced concentration-dependent increases in UDS in hepatocytes from rats pretreated with Aroclor 1254. 4,4{prime}-Oxydianiline and TCAB also induced a dose-dependent increase in DNA repair in hepatocytes from rats pretreated with phenobarbital, whereas 1-nitropyrene was negative. These data indicate that the limited sensitivity to chemical carcinogens displayed by the hepatocyte/DNA repair assay may be increased by using hepatocytes isolated from animals exposed to hepatic mixed-function oxidase inducers.

  13. Development of an in vitro culture method for stepwise differentiation of mouse embryonic stem cells and induced pluripotent stem cells into mature osteoclasts.

    Science.gov (United States)

    Nishikawa, Keizo; Iwamoto, Yoriko; Ishii, Masaru

    2014-05-01

    The development of methods for differentiation of embryonic stem cells (ESCs) and induced pluripotent stem cell (iPSCs) into functional cells have helped to analyze the mechanism regulating cellular processes and to explore cell-based assays for drug discovery. Although several reports have demonstrated methods for differentiation of mouse ESCs into osteoclast-like cells, it remains unclear whether these methods are applicable for differentiation of iPSCs to osteoclasts. In this study, we developed a simple method for stepwise differentiation of mouse ESCs and iPSCs into bone-resorbing osteoclasts based upon a monoculture approach consisting of three steps. First, based on conventional hanging-drop methods, embryoid bodies (EBs) were produced from mouse ESCs or iPSCs. Second, EBs were cultured in medium supplemented with macrophage colony-stimulating factor (M-CSF), and differentiated to osteoclast precursors, which expressed CD11b. Finally, ESC- or iPSC-derived osteoclast precursors stimulated with receptor activator of nuclear factor-B ligand (RANKL) and M-CSF formed large multinucleated osteoclast-like cells that expressed tartrate-resistant acid phosphatase and were capable of bone resorption. Molecular analysis showed that the expression of osteoclast marker genes such as Nfatc1, Ctsk, and Acp5 are increased in a RANKL-dependent manner. Thus, our procedure is simple and easy and would be helpful for stem cell-based bone research.

  14. Endocrine disruption screening by protein and gene expression of vitellogenin in freshly isolated and cryopreserved rainbow trout hepatocytes.

    Science.gov (United States)

    Markell, Lauren K; Mingoia, Robert T; Peterson, Heather M; Yao, Jianhong; Waters, Stephanie M; Finn, James P; Nabb, Diane L; Han, Xing

    2014-08-18

    Xenobiotics may activate the estrogen receptor, resulting in alteration of normal endocrine functions in animals and humans. Consequently, this necessitates development of assay end points capable of identifying estrogenic xenobiotics. In the present study, we screened the potential estrogenicity of chemicals via their ability to induce vitellogenin (VTG) expression in cultured primary hepatocytes from male trout. A routine method for VTG detection measures the secretion of the protein by enzyme-linked immunosorbent assay (ELISA) in freshly isolated trout hepatocytes. However, this lengthy (6 days) culturing procedure requires that hepatocyte isolation is performed each time the assay is run. We optimized this methodology by investigating the utility of cryopreserved hepatocytes, shortening the incubation time, performing a quantitative real-time PCR (qPCR) method for VTG quantification, and verifying the model system with reference chemicals 17β-estradiol, estrone, diethylstilbestrol, hexestrol, genistein, and a negative control, corticosterone. To test the performance of both freshly isolated and cryopreserved hepatocytes, mRNA was collected from hepatocytes following 24 h treatment for VTG gene expression analysis, whereas cell culture media was collected for a VTG ELISA 96 h post-treatment. EC50 values were obtained for each reference chemical except for corticosterone, which exhibited no induction of VTG gene or protein level. Our results show linear concordance between ELISA and qPCR detection methods. Although there was approximately 50% reduction in VTG inducibility following cryopreservation, linear concordance of EC50 values was found between freshly isolated and cryopreserved hepatocytes, indicating that cryopreservation does not alter the functional assessment of estrogen receptor activation and therefore VTG expression. These studies demonstrate that qPCR is a sensitive and specific method for detecting VTG gene expression that can be used together

  15. Selective insulin resistance in hepatocyte senescence

    International Nuclear Information System (INIS)

    Aravinthan, Aloysious; Challis, Benjamin; Shannon, Nicholas; Hoare, Matthew; Heaney, Judith; Alexander, Graeme J.M.

    2015-01-01

    Insulin resistance has been described in association with chronic liver disease for decades. Hepatocyte senescence has been demonstrated in chronic liver disease and as many as 80% of hepatocytes show a senescent phenotype in advanced liver disease. The aim of this study was to understand the role of hepatocyte senescence in the development of insulin resistance. Senescence was induced in HepG2 cells via oxidative stress. The insulin metabolic pathway was studied in control and senescent cells following insulin stimulation. GLUT2 and GLUT4 expressions were studied in HepG2 cells and human liver tissue. Further, GLUT2 and GLUT4 expressions were studied in three independent chronic liver disease cohorts. Signalling impairment distal to Akt in phosphorylation of AS160 and FoxO1 was evident in senescent HepG2 cells. Persistent nuclear localisation of FoxO1 was demonstrated in senescent cells despite insulin stimulation. Increased GLUT4 and decreased GLUT2 expressions were evident in senescent cells, human cirrhotic liver tissue and publically available liver disease datasets. Changes in GLUT expressions were associated with a poor clinical prognosis. In conclusion, selective insulin resistance is evident in senescent HepG2 cells and changes in GLUT expressions can be used as surrogate markers of hepatocyte senescence. - Highlights: • Senescent hepatocytes demonstrate selective insulin resistance. • GLUT changes act as markers of hepatocyte senescence and have prognostic value. • Study offers insight into long noticed intimacy of cirrhosis and insulin resistance

  16. Selective insulin resistance in hepatocyte senescence

    Energy Technology Data Exchange (ETDEWEB)

    Aravinthan, Aloysious [Division of Gastroenterology and Hepatology, Department of Medicine, University of Cambridge, Cambridge (United Kingdom); Challis, Benjamin [Institute of Metabolic Sciences, University of Cambridge, Cambridge (United Kingdom); Shannon, Nicholas [Cancer Research UK Cambridge Institute, Cambridge (United Kingdom); Hoare, Matthew [Division of Gastroenterology and Hepatology, Department of Medicine, University of Cambridge, Cambridge (United Kingdom); Cancer Research UK Cambridge Institute, Cambridge (United Kingdom); Heaney, Judith [Division of Gastroenterology and Hepatology, Department of Medicine, University of Cambridge, Cambridge (United Kingdom); Foundation for Liver Research, Institute of Hepatology, London (United Kingdom); Alexander, Graeme J.M., E-mail: gja1000@doctors.org.uk [Division of Gastroenterology and Hepatology, Department of Medicine, University of Cambridge, Cambridge (United Kingdom)

    2015-02-01

    Insulin resistance has been described in association with chronic liver disease for decades. Hepatocyte senescence has been demonstrated in chronic liver disease and as many as 80% of hepatocytes show a senescent phenotype in advanced liver disease. The aim of this study was to understand the role of hepatocyte senescence in the development of insulin resistance. Senescence was induced in HepG2 cells via oxidative stress. The insulin metabolic pathway was studied in control and senescent cells following insulin stimulation. GLUT2 and GLUT4 expressions were studied in HepG2 cells and human liver tissue. Further, GLUT2 and GLUT4 expressions were studied in three independent chronic liver disease cohorts. Signalling impairment distal to Akt in phosphorylation of AS160 and FoxO1 was evident in senescent HepG2 cells. Persistent nuclear localisation of FoxO1 was demonstrated in senescent cells despite insulin stimulation. Increased GLUT4 and decreased GLUT2 expressions were evident in senescent cells, human cirrhotic liver tissue and publically available liver disease datasets. Changes in GLUT expressions were associated with a poor clinical prognosis. In conclusion, selective insulin resistance is evident in senescent HepG2 cells and changes in GLUT expressions can be used as surrogate markers of hepatocyte senescence. - Highlights: • Senescent hepatocytes demonstrate selective insulin resistance. • GLUT changes act as markers of hepatocyte senescence and have prognostic value. • Study offers insight into long noticed intimacy of cirrhosis and insulin resistance.

  17. Demonstration of neuron-glia transfer of precursors for GABA biosynthesis in a co-culture system of dissociated mouse cerebral cortex.

    Science.gov (United States)

    Leke, Renata; Bak, Lasse K; Schousboe, Arne; Waagepetersen, Helle S

    2008-12-01

    Co-cultures of neurons and astrocytes were prepared from dissociated embryonic mouse cerebral cortex and cultured for 7 days. To investigate if these cultures may serve as a functional model system to study neuron-glia interaction with regard to GABA biosynthesis, the cells were incubated either in media containing [U-(13)C]glutamine (0.1, 0.3 and 0.5 mM) or 1 mM acetate plus 2.5 mM glucose plus 1 mM lactate. In the latter case one of the 3 substrates was uniformly (13)C labeled. Cellular contents and (13)C labeling of glutamate, GABA, aspartate and glutamine were determined in the cells after an incubation period of 2.5 h. The GABA biosynthetic machinery exhibited the expected complexity with regard to metabolic compartmentation and involvement of TCA cycle activity as seen in other culture systems containing GABAergic neurons. Metabolism of acetate clearly demonstrated glial synthesis of glutamine and its transfer to the neuronal compartment. It is concluded that this co-culture system serves as a reliable model in which functional and pharmacological aspects of GABA biosynthesis can be investigated.

  18. Hepatocyte polarization is essential for the productive entry of the hepatitis B virus.

    Science.gov (United States)

    Schulze, Andreas; Mills, Kerry; Weiss, Thomas S; Urban, Stephan

    2012-02-01

    Human hepatitis B virus (HBV) is characterized by a high species specificity and a distinct liver tropism. Within the liver, HBV replication occurs in differentiated and polarized hepatocytes. Accordingly, the in vitro HBV infection of primary human hepatocytes (PHHs) and the human hepatoma cell line, HepaRG, is restricted to differentiated, hepatocyte-like cells. Though preparations of PHH contain up to 100% hepatic cells, cultures of differentiated HepaRG cells are a mixture of hepatocyte-like and biliary-like epithelial cells. We used PHH and HepaRG cells and compared the influence of virus inoculation dose, cell differentiation, and polarization on productive HBV infection. At multiplicities of genome equivalents (mge) >8,000, almost 100% of PHHs could be infected. In contrast, only a subset of HepaRG cells stained positive for HBcAg at comparable or even higher mge. Infection predominantly occurred at the edges of islands of hepatocyte-like HepaRG cells. This indicates a limited accessibility of the HBV receptor, possibly as a result of its polar sorting. Multidrug resistance protein 2 (MRP2), a marker selectively transported to the apical (i.e., canalicular) cell membrane, revealed two polarization phenotypes of HepaRG cells. HBV infection within the islands of hepatocyte-like HepaRG cells preferentially occurred in cells that resemble PHH, exhibiting canalicular structures. However, disruption of cell-cell junctions allowed the additional infection of cells that do not display a PHH-like polarization. HBV enters hepatocytes via the basolateral membrane. This model, at least partially, explains the difference of PHH and HepaRG cells in infection efficacy, provides insights into natural HBV infection, and establishes a basis for optimization of the HepaRG infection system. Copyright © 2011 American Association for the Study of Liver Diseases.

  19. LPS, but not Angiotensin ll, lnduces Direct Pro-lnflammatory Effects in Cultured Mouse Arteries and Human Endothelial and Vascular Smooth Muscle Cells

    DEFF Research Database (Denmark)

    Outzen, Emilie M; Zaki, Marina; Mehryar, Rahila

    2017-01-01

    resistance-sized arteries (MRA) supported by experiments in cultured human primary endothelial and vascular smooth muscle cells. Results showed that 24-hr organ culture of mouse MRA with 10 nM Ang II had, unlike 100 ng/mL LPS, no effects on IL-6 or MCP-1 secretion, VCAM1 mRNA expression or endothelial......]-Ang II had no concentration- or time-dependent effects on IL-6 and MCP-1 secretion in human umbilical vein endothelial cells (HUVEC) and human aortic smooth muscle cells (HASMC). AGTR1 or AGTR2 mRNA expression were undetectable in HUVEC, whereas HASMC expressed only AGTR1 mRNA. In summary, contrary...... rights reserved....

  20. Insulin: its binding to specific receptors and its stimulation of DNA synthesis and 2',3'-cyclic nucleotide phosphohydrolase in embryonic mouse brain cell cultures

    International Nuclear Information System (INIS)

    Shanker, G.; Pieringer, R.A.

    1986-01-01

    Previously, the authors demonstrated that ornithine decarboxylase was stimulated by insulin in cultures of embryonic mouse brain cells. In the present work, they have investigated the presence and specificity of insulin receptors in these cultures. A time study showed that maximum binding of 125 [I] labelled insulin was around 75 min. Other studies measured the influence of concentration and age on insulin binding. A displacement study using increasing concentrations of cold insulin, glucagon or growth hormone demonstrated that the specificity of the receptors for insulin was rather high. It was also found that insulin displayed a clear dose-dependent stimulation of thymidine incorporation into the brain cells. Insulin also stimulated the glial enzyme 2':3'-cyclic nucleotide phosphohydrolase (CNP-ase). The results suggest a dual role for insulin; it regulates both cell proliferation as well as differentiation

  1. Changes in sensitivity to radiation and to bleomycin occurring during the life history of monolayer cultures of a mouse tumour cell line

    International Nuclear Information System (INIS)

    Twentyman, P.R.; Bleehen, N.M.

    1975-01-01

    The response to X-radiation and to bleomycin has been measured at a number of times during the life of monolayer cultures of EMT6 mouse tumour cells. Little change in radiation sensitivity was seen at any time and no loss of the shoulder to the survival curve occurred. Cultures in early plateau phase (where a considerable amount of cell proliferation is balanced by cell loss) showed a reduced sensitivity to bleomycin when compared with cells in exponential growth. However, after a longer period in plateau phase, when proliferation had virtually ceased, the sensitivity became greater than that of exponential phase cells. These findings are discussed with reference to the conflicting results of other workers. (author)

  2. Dopamine release in organotypic cultures of foetal mouse mesencephalon: effects of depolarizing agents, pargyline, nomifensine, tetrodotoxin and calcium

    DEFF Research Database (Denmark)

    Larsen, Trine R; Rossen, Sine; Gramsbergen, Jan B

    2008-01-01

    Organotypic mesencephalic cultures provide an attractive in vitro alternative to study development of the nigrostriatal system and pathophysiological mechanisms related to Parkinson's disease. However, dopamine (DA) release mechanisms have been poorly characterized in such cultures. We report her...

  3. Kinetics of the membrane current mediated by serotonin 5-HT3 receptors in cultured mouse neuroblastoma cells.

    NARCIS (Netherlands)

    Neijt, H.C.; Plomp, J.J.; Vijverberg, H.P.M.

    1989-01-01

    1. Ionic currents mediated by serotonin 5-HT3 receptors were studied in the mouse neuroblastoma cell line N1E-115, using suction pipettes for intracellular perfusion and voltage clamp recording. The dependence of the kinetics of the membrane current on serotonin concentration was investigated. 2. At

  4. The distinct nature of hematopoietic stem cell subpopulations studied in long-term stroma-associated culture in the mouse

    NARCIS (Netherlands)

    J.P. van der Sluijs

    1993-01-01

    textabstractIn this thesis a number of experiments is described which give evidence for the notion of a distinct nature of CFU-S and cells with LTRA in the mouse. The distinct nature is illustrated by the difference in growth characteristics of CFU-S and LTRA cells on preestablished bone

  5. Choline or methionine reverses impaired secretion of VLDL by hepatocytes from choline-deficient rats

    International Nuclear Information System (INIS)

    Yao, Z.; Vance, D.E.

    1987-01-01

    Male rats fed a choline-deficient (CD) diet for three days accumulated triacylglycerol (TG) in the liver. Hepatocytes from these rats were cultured and maintained in a medium + choline. The rate of secretion of TG was reduced by 50% in the CD cells. Correspondingly, [ 3 H]oleate and [ 3 H]glycerol were incorporated at a 2-fold higher rate into TG secreted by choline-supplemented cells compared to CD cells. Isolation of lipoprotein fractions by ultracentrifugation showed that the reduced secretion of TG by CD hepatocytes was mainly due to an impaired secretion of very low density lipoprotein (VLDL). Incorporation of [ 3 H]leucine into secreted apoB/sub H/, apoB/sub L/ and apoE was markedly reduced in CD cells compared to choline-supplemented cells. Secretion of high density lipoprotein was not reduced in the CD hepatocytes. Normal secretion of VLDL was resumed upon addition of methionine to the CD cells

  6. Effect of long-term culture of mouse embryonic stem cells under low oxygen concentration as well as on glycosaminoglycan hyaluronan on cell proliferation and differentiation.

    Science.gov (United States)

    Ramírez, M Á; Pericuesta, E; Yáñez-Mó, M; Palasz, A; Gutiérrez-Adán, A

    2011-02-01

    Maintaining undifferentiated stem cells in defined conditions is of critical importance to improve their in vitro culture. We have evaluated the effects of culturing mouse stem (mES) cells under physiological oxygen concentration as well as by replacing fibroblast feeder layer (mEF) with gelatin or glycosaminoglycan hyaluronan (HA), on cell proliferation and differentiation. After 3 days culture or after long-term cell culture under different conditions, levels of apoptotic cell death were determined by cell cycle and TUNEL (TdT-mediated dUTP nick end labelling) assays and levels of cell proliferation by CFSE (5-(and-6)-carboxyfluorescein diacetate succinimidyl ester) labelling. We assessed spontaneous differentiation into cardiomyocytes and mRNA expression of pluripotency and differentiation biomarkers. After 3 days culture under hypoxic conditions, levels of proliferation and apoptosis of mES cells were higher, in correlation with increase in intracellular reactive oxygen species. However, when cells were continuously grown for 1 month under those conditions, the level of apoptosis was, in all cases, under 4%. Hypoxia reduced spontaneous differentiation of mES into cardiomyocytes. Long-term culture on HA was more effective in maintaining the pluripotent state of the mES cells when compared to that on gelatin. Level of terminal differentiation was highest on mEF, intermediate on HA and lowest on gelatin. Our data suggest that hypoxia is not necessary for maintaining pluripotency of mES cells and appeared to be detrimental during ES differentiation. Moreover, HA may offer a valuable alternative for long-term culture of mES cells in vitro. © 2010 Blackwell Publishing Ltd.

  7. Collagen metabolism and basement membrane formation in cultures of mouse mammary epithelial cells: Induction of assembly on fibrillar type I collagen substrata

    International Nuclear Information System (INIS)

    David, G.; van der Schueren, B.; van den Berghe, H.; Nusgens, B.; Van Cauwenberge, D.; Lapiere, C.

    1987-01-01

    Collagen metabolism was compared in cultures of mouse mammary epithelial cells maintained on plastic or fibrillar type I collagen gel substrata. The accumulation of dialysable and non-dialysable [ 3 H]hydroxyproline and the identification of the collagens produced suggest no difference between substrata in the allover rates of collagen synthesis and degradation. The proportion of the [ 3 H]collagen which accumulates in the monolayers of cultures on collagen, however, markedly exceeds that of cultures on plastic. Cultures on collagen deposit a sheet-like layer of extracellular matrix materials on the surface of the collagen fibers. Transformed cells on collagen produce and accumulate more [ 3 H]collage, yet are less effective in basement membrane formation than normal cells, indicting that the accumulation of collagen alone and the effect of interstitial collagen thereupon do not suffice. Thus, exogenous fibrillar collagen appears to enhance, but is not sufficient for proper assembly of collagenous basement membrane components near the basal epithelial cell surface

  8. In-vitro developmental potential of individual mouse blastomeres cultured with and without zona pellucida: future implications for human assisted reproduction.

    Science.gov (United States)

    Illmensee, K; Kaskar, K; Zavos, P M

    2006-08-01

    This study was designed to compare the developmental potential of individual blastomeres derived from 2-, 4-, 6- and 8-cell mouse embryos cultured with and without zona pellucida (ZP). In the first series, one, three, five and seven blastomeres were biopsied from 2-, 4-, 6- and 8-cell embryos respectively, and inserted individually into empty ZP recipients, leaving the remaining blastomere within its original ZP. In the second series, the same protocol was used except that the biopsied blastomeres were cultured without ZP and compared with the remaining blastomere within its original ZP. For the first series, individual blastomeres derived from 2-, 4-, 6- and 8-cell embryos cultured with ZP showed blastocyst development of 82.4, 68.6, 44.4 and 23.1% respectively, with corresponding hatching rates of 70.6, 60.0, 25.9 and 7.7%. For the second series, individual blastomeres cultured without ZP progressed with blastocyst development of 73.3, 64.5, 35.7 and 22.7% respectively. Blastocyst multiplication was achieved most efficiently when using individual blastomeres from 4- and 6-cell embryos. This is the first report on comparative in-vitro propagation of single blastomeres derived from various cleavage stages in a mammalian species. Blastomere cloning with its multiple applications may be envisaged for human assisted reproductive technologies.

  9. Hepatocyte specific expression of human cloned genes

    Energy Technology Data Exchange (ETDEWEB)

    Cortese, R

    1986-01-01

    A large number of proteins are specifically synthesized in the hepatocyte. Only the adult liver expresses the complete repertoire of functions which are required at various stages during development. There is therefore a complex series of regulatory mechanisms responsible for the maintenance of the differentiated state and for the developmental and physiological variations in the pattern of gene expression. Human hepatoma cell lines HepG2 and Hep3B display a pattern of gene expression similar to adult and fetal liver, respectively; in contrast, cultured fibroblasts or HeLa cells do not express most of the liver specific genes. They have used these cell lines for transfection experiments with cloned human liver specific genes. DNA segments coding for alpha1-antitrypsin and retinol binding protein (two proteins synthesized both in fetal and adult liver) are expressed in the hepatoma cell lines HepG2 and Hep3B, but not in HeLa cells or fibroblasts. A DNA segment coding for haptoglobin (a protein synthesized only after birth) is only expressed in the hepatoma cell line HepG2 but not in Hep3B nor in non hepatic cell lines. The information for tissue specific expression is located in the 5' flanking region of all three genes. In vivo competition experiments show that these DNA segments bind to a common, apparently limiting, transacting factor. Conventional techniques (Bal deletions, site directed mutagenesis, etc.) have been used to precisely identify the DNA sequences responsible for these effects. The emerging picture is complex: they have identified multiple, separate transcriptional signals, essential for maximal promoter activation and tissue specific expression. Some of these signals show a negative effect on transcription in fibroblast cell lines.

  10. The loss of imprinted DNA methylation in mouse blastocysts is inflicted to a similar extent by in vitro follicle culture and ovulation induction.

    Science.gov (United States)

    Saenz-de-Juano, M D; Billooye, K; Smitz, J; Anckaert, E

    2016-06-01

    Does in vitro follicle culture (IFC) have an effect on maintenance of imprinted DNA methylation in preimplantation mouse embryos? We report similar alterations in the methylation pattern of H19 imprinted maternally expressed transcript (H19), small nuclear ribonucleoprotein polypeptide N (Snrpn) and mesoderm specific transcript (Mest) imprinted genes in mouse blastocysts obtained after ovulation induction and IFC. Furthermore, we observed no differences in the gene expression of maternal effect proteins related with imprinting maintenance between superovulated in vivo grown or IFC oocytes. Assisted reproductive technology is associated with adverse post-natal outcomes such as increased risk of premature birth, altered birthweight, congenital anomalies and genomic imprinting syndromes in human and in animal models. Previous studies have shown that ovulation induction allowed normal imprinting establishment in mouse oocytes, but interfered with imprinting maintenance during preimplantation . Normal imprinting establishment was also observed in mouse oocytes derived from a standardized IFC from the early pre-antral follicle stage. The methylation profiles of differentially methylated regions (DMRs) of three key imprinted genes (H19, Snrpn and Mest) were compared at hatched blastocyst stage between embryos obtained from IFC or superovulated oocytes, each subjected to IVF and preimplantation in vitro culture (IVC); in non-manipulated in vivo produced late blastocyst (control) and in in vivo produced 2-cell embryos that were in vitro cultured until the hatched blastocyst stage (to assess the effect of IVC). Two different mice strains (Mus musculus C57BL/6J X CBA/Ca and Mus musculus B6 (CAST7)) were used to discriminate between maternal and paternal alleles of imprinted genes. Additionally, a limiting-dilution bisulfite-sequencing technique was carried out on individual embryos in order to avoid amplification bias. To assess whether IFC and ovulation induction

  11. The oligodendroglial precursor cell line Oli-neu represents a cell culture system to examine functional expression of the mouse gap junction gene connexin29 (Cx29

    Directory of Open Access Journals (Sweden)

    Goran Christoph Söhl

    2013-06-01

    Full Text Available The potential gap junction forming mouse connexin29 (Cx29 protein is concomitantly expressed with connexin32 (Cx32 in peripheral myelin forming Schwann cells and together with both Cx32 and connexin47 (Cx47 in oligodendrocytes of the CNS. To study the genomic structure and functional expression of Cx29, either primary cells or cell culture systems might be selected, from which the latter are easier to cultivate. Both structure and expression of Cx29 is still not fully understood. In the mouse sciatic nerve, brain and the oligodendroglial precursor cell line Oli-neu the Cx29 gene is processed in two transcript isoforms both harbouring a unique reading frame. In contrast to Cx32 and Cx47, only Cx29 protein is abundantly expressed in undifferentiated as well as differentiated Oli-neu cells but the absence of Etbr dye transfer after microinjection concealed the function of Cx29 mediated gap junction communication between those cells. Although HeLa cells stably transfected with Cx29 or Cx29-eGFP neither demonstrated any permeability for Lucifer yellow nor for neurobiotin, blocking of Etbr uptake from the media by gap junction blockers does suppose a role of Cx29 in hemi-channel function. Thus, we conclude that, due to its high abundance of Cx29 expression and its reproducible culture conditions, the oligodendroglial precursor cell line Oli-neu might constitute an appropriate cell culture system to study molecular mechanisms or putative extracellular stimuli to functionally open Cx29 channels or hemi-channels.

  12. Effectiveness of mesenchymal stems cells cultured by hanging drop vs. conventional culturing on the repair of hypoxic-ischemic-damaged mouse brains, measured by stemness gene expression

    Directory of Open Access Journals (Sweden)

    Lou Yongli

    2016-01-01

    Full Text Available In this study, we investigated the therapeutic effects of Human Mesenchymal Stem Cells (hMSCs cultured by hanging drop and conventional culturing methods on cerebellar repair in hypoxic-ischemic (HI brain injured mice. Real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR was used to analyze the expression levels of three stemness genes, Oct4, Sox2 and Nanog, and the migration related gene CXCR4. MSC prepared by hanging drop or conventional techniques were administered intranasally to nine day old mice, and analyzed by MRI at day 28. Results indicate that the MSCs, especially the hanging drop cultured MSCs, significantly improved the mice’s cerebellar damage repair. MSCs derived from the hanging drop culture were smaller than those from the conventional culture. The gene expression levels were significantly increased for the MSCs derived from the hanging drop culture. The mechanism might relate to the fact that the hanging drop cultured MSCs can be kept in an undifferentiated state, resulting in its higher expression level of migration receptor of CXCR4.

  13. Anti-osteoporotic activity of harpagide by regulation of bone formation in osteoblast cell culture and ovariectomy-induced bone loss mouse models.

    Science.gov (United States)

    Chung, Hwa-Jin; Kyung Kim, Won; Joo Park, Hyen; Cho, Lan; Kim, Me-Riong; Kim, Min Jeong; Shin, Joon-Shik; Ho Lee, Jin; Ha, In-Hyuk; Kook Lee, Sang

    2016-02-17

    Harpagide, an iridoid glucoside, is a constituent of the root of Harpagophytum procumbens var. sublobatum (Engl.) Stapf, Devil's claw which has been used in patients with osteoarthritis (OA). In the present study, we investigated the anti-osteoporotic potential of harpagide and its underlying mechanism of action in in vitro cell culture and in vivo bone loss animal models. Harpagide was obtained from the alkalic hydrolysis of harpagoside, a major constituent of H. procumbens var. sublobatum Analysis of biomarkers for bone formation in osteoblastic MC3T3-E1 cells and bone resorption in osteoclast cells derived from mouse bone marrow cells was performed to evaluate the mechanism of action. The protective activity of harpagide against bone loss was also evaluated in ovariectomized (OVX) mouse model. Harpagide improved bone properties by stimulating the process of differentiation and maturation of osteoblast cells and suppressing the process of RANKL-induced differentiation of osteoclast cells. In OVX-induced bone loss mouse model, oral administration of harpagide significantly improved recovery of bone mineral density, trabecular bone volume, and trabecular number in the femur. Harpagide also prevented increase of trabecular separation and structure model index induced by OVX. Harpagide effectively inhibited the serum levels of biochemical markers of bone loss, including alkaline phosphatase, osteocalcin, C-terminal telopeptide, and tartrate-resistant acid phosphatase. Taken together, the present study demonstrates that harpagide has a potential for prevention of bone loss in OVX mice by regulating the stimulation of osteoblast differentiation and the suppression of osteoclast formation. Therefore, these findings suggest that harpagide might serve as a bioactive compound derived from H. procumbens var. sublobatum for improvement of age-dependent bone destruction disease. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  14. Apelin ameliorates TNF-α-induced reduction of glycogen synthesis in the hepatocytes through G protein-coupled receptor APJ.

    Directory of Open Access Journals (Sweden)

    Jiaojiao Chu

    Full Text Available Apelin, a novel adipokine, is the specific endogenous ligand of G protein-coupled receptor APJ. Consistent with its putative role as an adipokine, apelin has been linked to states of insulin resistance. However, the function of apelin in hepatic insulin resistance, a vital part of insulin resistance, and its underlying mechanisms still remains unclear. Here we define the impacts of apelin on TNF-α-induced reduction of glycogen synthesis in the hepatocytes. Our studies indicate that apelin reversed TNF-α-induced reduction of glycogen synthesis in HepG2 cells, mouse primary hepatocytes and liver tissues of C57BL/6J mice by improving JNK-IRS1-AKT-GSK pathway. Moreover, Western blot revealed that APJ, but not apelin, expressed in the hepatocytes and liver tissues of mice. We found that F13A, a competitive antagonist for G protein-coupled receptor APJ, suppressed the effects of apelin on TNF-α-induced reduction of glycogen synthesis in the hepatocytes, suggesting APJ is involved in the function of apelin. In conclusion, we show novel evidence suggesting that apelin ameliorates TNF-α-induced reduction of glycogen synthesis in the hepatocytes through G protein-coupled receptor APJ. Apelin appears as a beneficial adipokine with anti-insulin resistance properties, and thus as a promising therapeutic target in metabolic disorders.

  15. Hyperinsulinemia is associated with increased soluble insulin receptors release from hepatocytes

    Directory of Open Access Journals (Sweden)

    Marcia eHiriart

    2014-06-01

    Full Text Available It has been generally assumed that insulin circulates freely in blood. However it can also interact with plasma proteins. Insulin receptors are located in the membrane of target cells and consist of an alpha and beta subunits with a tyrosine kinase cytoplasmic domain. The ectodomain, called soluble insulin receptor (SIR has been found elevated in patients with diabetes mellitus. We explored if insulin binds to SIRs in circulation under physiological conditions and hypothesize that this SIR may be released by hepatocytes in response to high insulin concentrations. The presence of SIR in rat and human plasmas and the culture medium of hepatocytes was explored using Western blot analysis. A purification protocol was performed to isolated SIR using affinity, gel filtration and ion exchange chromatographies. A modified reverse hemolytic plaque assay was used to measure SIR release from cultured hepatocytes. Incubation with 1 nmol l-1 insulin induces the release of the insulin receptor ectodomains from normal rat hepatocytes. This effect can be partially prevented by blocking protease activity. Furthermore, plasma levels of SIR were higher in a model of metabolic syndrome, where rats are hyperinsulinemic. We also found increased SIR levels in hyperinsulinemic humans. SIR may be an important regulator of the amount of free insulin in circulation. In hyperinsulinemia the amount of this soluble receptor increases, this could lead to higher amounts of insulin bound to this receptor, rather than free insulin, which is the biologically active form of the hormone. This observation could enlighten the mechanisms of insulin resistance.

  16. Effectiveness of mesenchymal stems cells cultured by hanging drop vs. conventional culturing on the repair of hypoxic-ischemic-damaged mouse brains, measured by stemness gene expression

    OpenAIRE

    Lou Yongli; Guo Dewei; Zhang Hui; Song Laijun

    2016-01-01

    In this study, we investigated the therapeutic effects of Human Mesenchymal Stem Cells (hMSCs) cultured by hanging drop and conventional culturing methods on cerebellar repair in hypoxic-ischemic (HI) brain injured mice. Real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) was used to analyze the expression levels of three stemness genes, Oct4, Sox2 and Nanog, and the migration related gene CXCR4. MSC prepared by hanging drop or conventional techniques were adminis...

  17. Histamine Induces Alzheimer’s Disease-Like Blood Brain Barrier Breach and Local Cellular Responses in Mouse Brain Organotypic Cultures

    Directory of Open Access Journals (Sweden)

    Jonathan C. Sedeyn

    2015-01-01

    Full Text Available Among the top ten causes of death in the United States, Alzheimer’s disease (AD is the only one that cannot be cured, prevented, or even slowed down at present. Significant efforts have been exerted in generating model systems to delineate the mechanism as well as establishing platforms for drug screening. In this study, a promising candidate model utilizing primary mouse brain organotypic (MBO cultures is reported. For the first time, we have demonstrated that the MBO cultures exhibit increased blood brain barrier (BBB permeability as shown by IgG leakage into the brain parenchyma, astrocyte activation as evidenced by increased expression of glial fibrillary acidic protein (GFAP, and neuronal damage-response as suggested by increased vimentin-positive neurons occur upon histamine treatment. Identical responses—a breakdown of the BBB, astrocyte activation, and neuronal expression of vimentin—were then demonstrated in brains from AD patients compared to age-matched controls, consistent with other reports. Thus, the histamine-treated MBO culture system may provide a valuable tool in combating AD.

  18. Temperature shift and host cell contact up-regulate sporozoite expression of Plasmodium falciparum genes involved in hepatocyte infection.

    Directory of Open Access Journals (Sweden)

    Anthony Siau

    Full Text Available Plasmodium sporozoites are deposited in the skin by Anopheles mosquitoes. They then find their way to the liver, where they specifically invade hepatocytes in which they develop to yield merozoites infective to red blood cells. Relatively little is known of the molecular interactions during these initial obligatory phases of the infection. Recent data suggested that many of the inoculated sporozoites invade hepatocytes an hour or more after the infective bite. We hypothesised that this pre-invasive period in the mammalian host prepares sporozoites for successful hepatocyte infection. Therefore, the genes whose expression becomes modified prior to hepatocyte invasion would be those likely to code for proteins implicated in the subsequent events of invasion and development. We have used P. falciparum sporozoites and their natural host cells, primary human hepatocytes, in in vitro co-culture system as a model for the pre-invasive period. We first established that under co-culture conditions, sporozoites maintain infectivity for an hour or more, in contrast to a drastic loss in infectivity when hepatocytes were not included. Thus, a differential transcriptome of salivary gland sporozoites versus sporozoites co-cultured with hepatocytes was established using a pan-genomic P. falciparum microarray. The expression of 532 genes was found to have been up-regulated following co-culture. A fifth of these genes had no orthologues in the genomes of Plasmodium species used in rodent models of malaria. Quantitative RT-PCR analysis of a selection of 21 genes confirmed the reliability of the microarray data. Time-course analysis further indicated two patterns of up-regulation following sporozoite co-culture, one transient and the other sustained, suggesting roles in hepatocyte invasion and liver stage development, respectively. This was supported by functional studies of four hitherto uncharacterized proteins of which two were shown to be sporozoite surface

  19. Differentiation of human umbilical cord mesenchymal stromal cells into low immunogenic hepatocyte-like cells.

    Science.gov (United States)

    Zhao, Qinjun; Ren, Hongying; Li, Xiyuan; Chen, Zhong; Zhang, Xiangyu; Gong, Wei; Liu, Yongjun; Pang, Tianxiang; Han, Zhong Chao

    2009-01-01

    Mesenchymal stromal cells (MSC) isolated from several human tissues have been known to differentiate into the hepatic lineage in vitro, but the immunogenicity of the differentiated hepatocyte-like cells (DHC) has not been reported. Umbilical cord (UC) MSC are thought to be an attractive cell source for cell therapy because of their young age and low infection rate compared with adult tissue MSC. Hepatic differentiation of UC-MSC was induced with a 2-step protocol. The expressions of hepatic markers were detected by RT-PCR and immunofluorescence staining. Albumin production and urea secretion were measured by ELISA and colorimetric assay respectively. The immunosuppressive properties of DHC was detected by mixed lymphocyte culture. After incubation with specific growth factors, including hepatocyte growth factor (HGF) and basic fibroblast growth factor (bFGF), UC MSC exhibited a high hepatic differentiation ability in an adherent culture condition. The differentiated UC MSC showed hepatocyte-like morphology and expressed several liver-specific markers at gene and protein levels. Furthermore, the DHC exhibited hepatocyte-specific functions, including albumin secretion, low-density lipoprotein uptake and urea production. More importantly, DHC did not express major histocompatibility complex (MHC) II antigen and were not able to induce lymphocyte proliferation in mixed lymphocyte culture, as undifferentiated UC MSC did. Our results indicate that UC MSC are able to differentiate into functional hepatocyte-like cells that still retain their low immunogenicity in vitro. More importantly, DHC incorporated into the parenchyma of liver when transplanted into mice with CCl(4)-induced liver injury. Therefore, DHC may be an ideal source for cell therapy of liver diseases.

  20. Independent, parallel pathways to CXCL10 induction in HCV-infected hepatocytes.

    Science.gov (United States)

    Brownell, Jessica; Wagoner, Jessica; Lovelace, Erica S; Thirstrup, Derek; Mohar, Isaac; Smith, Wesley; Giugliano, Silvia; Li, Kui; Crispe, I Nicholas; Rosen, Hugo R; Polyak, Stephen J

    2013-10-01

    The pro-inflammatory chemokine CXCL10 is induced by HCV infection in vitro and in vivo, and is associated with outcome of IFN (interferon)-based therapy. We studied how hepatocyte sensing of early HCV infection via TLR3 (Toll-like receptor 3) and RIG-I (retinoic acid inducible gene I) led to expression of CXCL10. CXCL10, type I IFN, and type III IFN mRNAs and proteins were measured in PHH (primary human hepatocytes) and hepatocyte lines harboring functional or non-functional TLR3 and RIG-I pathways following HCV infection or exposure to receptor-specific stimuli. HuH7 human hepatoma cells expressing both TLR3 and RIG-I produced maximal CXCL10 during early HCV infection. Neutralization of type I and type III IFNs had no impact on virus-induced CXCL10 expression in TLR3+/RIG-I+ HuH7 cells, but reduced CXCL10 expression in PHH. PHH cultures were positive for monocyte, macrophage, and dendritic cell mRNAs. Immunodepletion of non-parenchymal cells (NPCs) eliminated marker expression in PHH cultures, which then showed no IFN requirement for CXCL10 induction during HCV infection. Immunofluorescence studies also revealed a positive correlation between intracellular HCV Core and CXCL10 protein expression (r(2) = 0.88, p ≤ 0.001). While CXCL10 induction in hepatocytes during the initial phase of HCV infection is independent of hepatocyte-derived type I and type III IFNs, NPC-derived IFNs contribute to CXCL10 induction during HCV infection in PHH cultures. Copyright © 2013 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

  1. Detoxification of ammonia in mouse cortical GABAergic cell cultures increases neuronal oxidative metabolism and reveals an emerging role for release of glucose-derived alanine

    DEFF Research Database (Denmark)

    Leke, Renata; Bak, Lasse Kristoffer; Anker, Malene

    2011-01-01

    Cerebral hyperammonemia is believed to play a pivotal role in the development of hepatic encephalopathy (HE), a debilitating condition arising due to acute or chronic liver disease. In the brain, ammonia is thought to be detoxified via the activity of glutamine synthetase, an astrocytic enzyme....... Moreover, it has been suggested that cerebral tricarboxylic acid (TCA) cycle metabolism is inhibited and glycolysis enhanced during hyperammonemia. The aim of this study was to characterize the ammonia-detoxifying mechanisms as well as the effects of ammonia on energy-generating metabolic pathways...... in a mouse neuronal-astrocytic co-culture model of the GABAergic system. We found that 5 mM ammonium chloride affected energy metabolism by increasing the neuronal TCA cycle activity and switching the astrocytic TCA cycle toward synthesis of substrate for glutamine synthesis. Furthermore, ammonia exposure...

  2. Modulator effects of interleukin-1beta and tumor necrosis factor-alpha on AMPA-induced excitotoxicity in mouse organotypic hippocampal slice cultures

    DEFF Research Database (Denmark)

    Bernardino, Liliana; Xapelli, Sara; Silva, Ana P

    2005-01-01

    The inflammatory cytokines interleukin-1beta and tumor necrosis factor-alpha (TNF-alpha) have been identified as mediators of several forms of neurodegeneration in the brain. However, they can produce either deleterious or beneficial effects on neuronal function. We investigated the effects...... of mouse recombinant TNF-alpha (10 ng/ml) enhanced excitotoxicity when the cultures were simultaneously exposed to AMPA and to this cytokine. Decreasing the concentration of TNF-alpha to 1 ng/ml resulted in neuroprotection against AMPA-induced neuronal death independently on the application protocol....... By using TNF-alpha receptor (TNFR) knock-out mice, we demonstrated that the potentiation of AMPA-induced toxicity by TNF-alpha involves TNF receptor-1, whereas the neuroprotective effect is mediated by TNF receptor-2. AMPA exposure was associated with activation and proliferation of microglia as assessed...

  3. Connexin 32 and connexin 43 are involved in lineage restriction of hepatic progenitor cells to hepatocytes

    Directory of Open Access Journals (Sweden)

    Haiyun Pei

    2017-11-01

    Full Text Available Abstract Background Bi-potential hepatic progenitor cells can give rise to both hepatocytes and cholangiocytes, which is the last phase and critical juncture in terms of sequentially hepatic lineage restriction from any kind of stem cells. If their differentiation can be controlled, it might access to functional hepatocytes to develop pharmaceutical and biotechnology industries as well as cell therapies for end-stage liver diseases. Methods In this study, we investigated the influence of Cx32 and Cx43 on hepatocyte differentiation of WB-F344 cells by in vitro gain and loss of function analyses. An inhibitor of Cx32 was also used to make further clarification. To reveal p38 MAPK pathway is closely related to Cxs, rats with 70% partial hepatectomy were injected intraperitoneally with a p38 inhibitor, SB203580. Besides, the effects of p38 MAPK pathway on differentiation of hepatoblasts isolated from fetal rat livers were evaluated by addition of SB203580 in culture medium. Results In vitro gain and loss of function analyses showed overexpression of Connexin 32 and knockdown of Connexin 43 promoted hepatocytes differentiation from hepatic progenitor cells. In addition, in vitro and ex vivo research revealed inhibition of p38 mitogen-activated protein kinase pathway can improve hepatocytes differentiation correlating with upregulation of Connexin 32 expression and downregulation of Connexin 43 expression. Conclusions Here we demonstrate that Connexins play crucial roles in facilitating differentiation of hepatic progenitors. Our work further implicates that regulators of Connexins and their related pathways might provide new insights to improve lineage restriction of stem cells to mature hepatocytes.

  4. Stimulation of MMP-11 (stromelysin-3) expression in mouse fibroblasts by cytokines, collagen and co-culture with human breast cancer cell lines

    International Nuclear Information System (INIS)

    Selvey, Saxon; Haupt, Larisa M; Thompson, Erik W; Matthaei, Klaus I; Irving, Michael G; Griffiths, Lyn R

    2004-01-01

    Matrix metalloproteinases (MMPs) are central to degradation of the extracellular matrix and basement membrane during both normal and carcinogenic tissue remodeling. MT1-MMP (MMP-14) and stromelysin-3 (MMP-11) are two members of the MMP family of proteolytic enzymes that have been specifically implicated in breast cancer progression. Expressed in stromal fibroblasts adjacent to epithelial tumour cells, the mechanism of MT1-MMP and MMP-11 induction remains unknown. To investigate possible mechanisms of induction, we examined the effects of a number of plausible regulatory agents and treatments that may physiologically influence MMP expression during tumour progression. Thus NIH3T3 and primary mouse embryonic fibroblasts (MEFs) were: a) treated with the cytokines IL-1β, IL-2, IL-6, IL-8 and TGF-β for 3, 6, 12, 24, and 48 hours; b) grown on collagens I, IV and V; c) treated with fibronectin, con-A and matrigel; and d) co-cultured with a range of HBC (human breast cancer) cell lines of varied invasive and metastatic potential. Competitive quantitative RT-PCR indicated that MMP-11 expression was stimulated to a level greater than 100%, by 48 hour treatments of IL-1β, IL-2, TGF-β, fibronectin and collagen V. No other substantial changes in expression of MMP-11 or MT1-MMP in either tested fibroblast culture, under any treatment conditions, were observed. We have demonstrated significant MMP-11 stimulation in mouse fibroblasts using cytokines, matrix constituents and HBC cell lines, and also some inhibition of MT1-MMP. Our data suggest that the regulation of these genes in the complex stromal-epithelial interactions that occur in human breast carcinoma, is influenced by several mechanisms

  5. Stimulation of MMP-11 (stromelysin-3 expression in mouse fibroblasts by cytokines, collagen and co-culture with human breast cancer cell lines

    Directory of Open Access Journals (Sweden)

    Matthaei Klaus I

    2004-07-01

    Full Text Available Abstract Background Matrix metalloproteinases (MMPs are central to degradation of the extracellular matrix and basement membrane during both normal and carcinogenic tissue remodeling. MT1-MMP (MMP-14 and stromelysin-3 (MMP-11 are two members of the MMP family of proteolytic enzymes that have been specifically implicated in breast cancer progression. Expressed in stromal fibroblasts adjacent to epithelial tumour cells, the mechanism of MT1-MMP and MMP-11 induction remains unknown. Methods To investigate possible mechanisms of induction, we examined the effects of a number of plausible regulatory agents and treatments that may physiologically influence MMP expression during tumour progression. Thus NIH3T3 and primary mouse embryonic fibroblasts (MEFs were: a treated with the cytokines IL-1β, IL-2, IL-6, IL-8 and TGF-β for 3, 6, 12, 24, and 48 hours; b grown on collagens I, IV and V; c treated with fibronectin, con-A and matrigel; and d co-cultured with a range of HBC (human breast cancer cell lines of varied invasive and metastatic potential. Results Competitive quantitative RT-PCR indicated that MMP-11 expression was stimulated to a level greater than 100%, by 48 hour treatments of IL-1β, IL-2, TGF-β, fibronectin and collagen V. No other substantial changes in expression of MMP-11 or MT1-MMP in either tested fibroblast culture, under any treatment conditions, were observed. Conclusion We have demonstrated significant MMP-11 stimulation in mouse fibroblasts using cytokines, matrix constituents and HBC cell lines, and also some inhibition of MT1-MMP. Our data suggest that the regulation of these genes in the complex stromal-epithelial interactions that occur in human breast carcinoma, is influenced by several mechanisms.

  6. Technique of the `in vitro` fertilization and the culture of mouse embryos at preimplantation; Tecnica de fertilizacao `in vitro` e cultura de embrioes de camundongo durante a pre-implantacao

    Energy Technology Data Exchange (ETDEWEB)

    Kikuchi, Olivia Kimiko [Instituto de Pesquisas Energeticas e Nucleares (IPEN), Sao Paulo, SP (Brazil); Yamada, Takeshi [National Inst. of Radiological Sciences, Chiba (Japan)

    1993-03-01

    The mammal embryo is an intensive cellular proliferating system, very radiosensitive and therefore adequate to the study of the biological effects of ionizing radiation. The technique of the in vitro fertilization and the culture of mouse embryos at preimplantation period, modified by Yamada et al (1982) to improve the efficiency of more than 95% of blastocyst formation is described. (author) 2 refs., 7 figs.

  7. Hepatocyte Growth Factor Reduces Free Cholesterol-Mediated Lipotoxicity in Primary Hepatocytes by Countering Oxidative Stress

    Directory of Open Access Journals (Sweden)

    Mayra Domínguez-Pérez

    2016-01-01

    Full Text Available Cholesterol overload in the liver has shown toxic effects by inducing the aggravation of nonalcoholic fatty liver disease to steatohepatitis and sensitizing to damage. Although the mechanism of damage is complex, it has been demonstrated that oxidative stress plays a prominent role in the process. In addition, we have proved that hepatocyte growth factor induces an antioxidant response in hepatic cells; in the present work we aimed to figure out the protective effect of this growth factor in hepatocytes overloaded with free cholesterol. Hepatocytes from mice fed with a high-cholesterol diet were treated or not with HGF, reactive oxygen species present in cholesterol overloaded hepatocytes significantly decreased, and this effect was particularly associated with the increase in glutathione and related enzymes, such as γ-gamma glutamyl cysteine synthetase, GSH peroxidase, and GSH-S-transferase. Our data clearly indicate that HGF displays an antioxidant response by inducing the glutathione-related protection system.

  8. Insulin and branched-chain amino acid depletion during mouse preimplantation embryo culture programmes body weight gain and raised blood pressure during early postnatal life.

    Science.gov (United States)

    Velazquez, Miguel A; Sheth, Bhavwanti; Smith, Stephanie J; Eckert, Judith J; Osmond, Clive; Fleming, Tom P

    2018-02-01

    Mouse maternal low protein diet exclusively during preimplantation development (Emb-LPD) is sufficient to programme altered growth and cardiovascular dysfunction in offspring. Here, we use an in vitro model comprising preimplantation culture in medium depleted in insulin and branched-chain amino acids (BCAA), two proposed embryo programming inductive factors from Emb-LPD studies, to examine the consequences for blastocyst organisation and, after embryo transfer (ET), postnatal disease origin. Two-cell embryos were cultured to blastocyst stage in defined KSOM medium supplemented with four combinations of insulin and BCAA concentrations. Control medium contained serum insulin and uterine luminal fluid amino acid concentrations (including BCAA) found in control mothers from the maternal diet model (N-insulin+N-bcaa). Experimental medium (three groups) contained 50% reduction in insulin and/or BCAA (L-insulin+N-bcaa, N-insulin+L-bcaa, and L-insulin+N-bcaa). Lineage-specific cell numbers of resultant blastocysts were not affected by treatment. Following ET, a combined depletion of insulin and BCAA during embryo culture induced a non sex-specific increase in birth weight and weight gain during early postnatal life. Furthermore, male offspring displayed relative hypertension and female offspring reduced heart/body weight, both characteristics of Emb-LPD offspring. Combined depletion of metabolites also resulted in a strong positive correlation between body weight and glucose metabolism that was absent in the control group. Our results support the notion that composition of preimplantation culture medium can programme development and associate with disease origin affecting postnatal growth and cardiovascular phenotypes and implicate two important nutritional mediators in the inductive mechanism. Our data also have implications for human assisted reproductive treatment (ART) practice. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  9. Culture media-based selection of endothelial cells, pericytes, and perivascular-resident macrophage-like melanocytes from the young mouse vestibular system.

    Science.gov (United States)

    Zhang, Jinhui; Chen, Songlin; Cai, Jing; Hou, Zhiqiang; Wang, Xiaohan; Kachelmeier, Allan; Shi, Xiaorui

    2017-03-01

    The vestibular blood-labyrinth barrier (BLB) is comprised of perivascular-resident macrophage-like melanocytes (PVM/Ms) and pericytes (PCs), in addition to endothelial cells (ECs) and basement membrane (BM), and bears strong resemblance to the cochlear BLB in the stria vascularis. Over the past few decades, in vitro cell-based models have been widely used in blood-brain barrier (BBB) and blood-retina barrier (BRB) research, and have proved to be powerful tools for studying cell-cell interactions in their respective organs. Study of both the vestibular and strial BLB has been limited by the unavailability of primary culture cells from these barriers. To better understand how barrier component cells interact in the vestibular system to control BLB function, we developed a novel culture medium-based method for