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Sample records for adult mouse hepatocytes

  1. 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.

  2. 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.

  3. RNA synthesis in primary cultures of adult rat hepatocytes

    International Nuclear Information System (INIS)

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

    1983-01-01

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

  4. Alternative Cell Sources to Adult Hepatocytes for Hepatic Cell Therapy.

    Science.gov (United States)

    Pareja, Eugenia; Gómez-Lechón, María José; Tolosa, Laia

    2017-01-01

    Adult hepatocyte transplantation is limited by scarce availability of suitable donor liver tissue for hepatocyte isolation. New cell-based therapies are being developed to supplement whole-organ liver transplantation, to reduce the waiting-list mortality rate, and to obtain more sustained and significant metabolic correction. Fetal livers and unsuitable neonatal livers for organ transplantation have been proposed as potential useful sources of hepatic cells for cell therapy. However, the major challenge is to use alternative cell sources for transplantation that can be derived from reproducible methods. Different types of stem cells with hepatic differentiation potential are eligible for generating large numbers of functional hepatocytes for liver cell therapy to treat degenerative disorders, inborn hepatic metabolic diseases, and organ failure. Clinical trials are designed to fully establish the safety profile of such therapies and to define target patient groups and standardized protocols.

  5. 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

  6. Phenotypic and functional analyses show stem cell-derived hepatocyte-like cells better mimic fetal rather than adult hepatocytes.

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    Baxter, Melissa; Withey, Sarah; Harrison, Sean; Segeritz, Charis-Patricia; Zhang, Fang; Atkinson-Dell, Rebecca; Rowe, Cliff; Gerrard, Dave T; Sison-Young, Rowena; Jenkins, Roz; Henry, Joanne; Berry, Andrew A; Mohamet, Lisa; Best, Marie; Fenwick, Stephen W; Malik, Hassan; Kitteringham, Neil R; Goldring, Chris E; Piper Hanley, Karen; Vallier, Ludovic; Hanley, Neil A

    2015-03-01

    Hepatocyte-like cells (HLCs), differentiated from pluripotent stem cells by the use of soluble factors, can model human liver function and toxicity. However, at present HLC maturity and whether any deficit represents a true fetal state or aberrant differentiation is unclear and compounded by comparison to potentially deteriorated adult hepatocytes. Therefore, we generated HLCs from multiple lineages, using two different protocols, for direct comparison with fresh fetal and adult hepatocytes. Protocols were developed for robust differentiation. Multiple transcript, protein and functional analyses compared HLCs to fresh human fetal and adult hepatocytes. HLCs were comparable to those of other laboratories by multiple parameters. Transcriptional changes during differentiation mimicked human embryogenesis and showed more similarity to pericentral than periportal hepatocytes. Unbiased proteomics demonstrated greater proximity to liver than 30 other human organs or tissues. However, by comparison to fresh material, HLC maturity was proven by transcript, protein and function to be fetal-like and short of the adult phenotype. The expression of 81% phase 1 enzymes in HLCs was significantly upregulated and half were statistically not different from fetal hepatocytes. HLCs secreted albumin and metabolized testosterone (CYP3A) and dextrorphan (CYP2D6) like fetal hepatocytes. In seven bespoke tests, devised by principal components analysis to distinguish fetal from adult hepatocytes, HLCs from two different source laboratories consistently demonstrated fetal characteristics. HLCs from different sources are broadly comparable with unbiased proteomic evidence for faithful differentiation down the liver lineage. This current phenotype mimics human fetal rather than adult hepatocytes. Copyright © 2014 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

  7. 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....

  8. [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.

  9. 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.

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

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    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.

  11. 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

  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. 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.

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

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    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.

  15. 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.

  16. 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.

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

    International Nuclear Information System (INIS)

    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

  18. 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.

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

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    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)

  20. 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

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

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    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.

  2. 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

  3. 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

  4. 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.

  5. 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.

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

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    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.

  7. 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

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

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    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.

  9. Hepatocyte nuclear factor 4A improves hepatic differentiation of immortalized adult human hepatocytes and improves liver function and survival.

    Science.gov (United States)

    Hang, Hua-Lian; Liu, Xin-Yu; Wang, Hai-Tian; Xu, Ning; Bian, Jian-Min; Zhang, Jian-Jun; Xia, Lei; Xia, Qiang

    2017-11-15

    Immortalized human hepatocytes (IHH) could provide an unlimited supply of hepatocytes, but insufficient differentiation and phenotypic instability restrict their clinical application. This study aimed to determine the role of hepatocyte nuclear factor 4A (HNF4A) in hepatic differentiation of IHH, and whether encapsulation of IHH overexpressing HNF4A could improve liver function and survival in rats with acute liver failure (ALF). Primary human hepatocytes were transduced with lentivirus-mediated catalytic subunit of human telomerase reverse transcriptase (hTERT) to establish IHH. Cells were analyzed for telomerase activity, proliferative capacity, hepatocyte markers, and tumorigenicity (c-myc) expression. Hepatocyte markers, hepatocellular functions, and morphology were studied in the HNF4A-overexpressing IHH. Hepatocyte markers and karyotype analysis were completed in the primary hepatocytes using shRNA knockdown of HNF4A. Nuclear translocation of β-catenin was assessed. Rat models of ALF were treated with encapsulated IHH or HNF4A-overexpressing IHH. A HNF4A-positive IHH line was established, which was non-tumorigenic and conserved properties of primary hepatocytes. HNF4A overexpression significantly enhanced mRNA levels of genes related to hepatic differentiation in IHH. Urea levels were increased by the overexpression of HNF4A, as measured 24h after ammonium chloride addition, similar to that of primary hepatocytes. Chromosomal abnormalities were observed in primary hepatocytes transfected with HNF4A shRNA. HNF4α overexpression could significantly promote β-catenin activation. Transplantation of HNF4A overexpressing IHH resulted in better liver function and survival of rats with ALF compared with IHH. HNF4A improved hepatic differentiation of IHH. Transplantation of HNF4A-overexpressing IHH could improve the liver function and survival in a rat model of ALF. Copyright © 2017 Elsevier Inc. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Casey J Guenthner

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

  11. 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

  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.

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    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. 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.

  16. 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

  17. 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.

  18. 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

  19. 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.

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

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    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.

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

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    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.

  2. 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.

  3. 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.

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

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    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.

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

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    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.

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

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    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

  7. 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.

  8. 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

  9. 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.

  10. 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.

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

    Science.gov (United States)

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

    1993-01-01

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

  12. 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.

  13. 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

  14. 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.

  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. ATM localization and gene expression in the adult mouse eye.

    Science.gov (United States)

    Leemput, Julia; Masson, Christel; Bigot, Karine; Errachid, Abdelmounaim; Dansault, Anouk; Provost, Alexandra; Gadin, Stéphanie; Aoufouchi, Said; Menasche, Maurice; Abitbol, Marc

    2009-01-01

    High levels of metabolism and oxygen consumption in most adult murine ocular compartments, combined with exposure to light and ultraviolet (UV) radiation, are major sources of oxidative stress, causing DNA damage in ocular cells. Of all mammalian body cells, photoreceptor cells consume the largest amount of oxygen and generate the highest levels of oxidative damage. The accumulation of such damage throughout life is a major factor of aging tissues. Several multiprotein complexes have recently been identified as the major sensors and mediators involved in the maintenance of DNA integrity. The activity of these complexes initially seemed to be restricted to dividing cells, given their ultimate role in major cell cycle checkpoints. However, it was later established that they are also active in post-mitotic cells. Recent findings demonstrate that the DNA damage response (DDR) is essential for the development, maintenance, and normal functioning of the adult central nervous system. One major molecular factor in the DDR is the protein, ataxia telangiectasia mutated (ATM). It is required for the rapid induction of cellular responses to DNA double-strand breaks. These cytotoxic DNA lesions may be caused by oxidative damage. To understand how ATM prevents oxidative stress and participates in the maintenance of genomic integrity and cell viability of the adult retina, we determined the ATM expression patterns and studied its localization in the adult mouse eye. Atm gene expression was analyzed by RT-PCR experiments and its localization by in situ hybridization on adult mouse ocular and cerebellar tissue sections. ATM protein expression was determined by western blot analysis of proteins homogenates extracted from several mouse tissues and its localization by immunohistochemistry experiments performed on adult mouse ocular and cerebellar tissue sections. In addition, subcellular localization was realized by confocal microscopy imaging of ocular tissue sections, with a special

  18. A Comprehensive Atlas of the Adult Mouse Penis

    Science.gov (United States)

    Phillips, Tiffany R.; Wright, David K.; Gradie, Paul E.; Johnston, Leigh A.; Pask, Andrew J.

    2016-01-01

    Mice are routinely used to study the development of the external genitalia and, in particular, the process of male urethral closure. This is because misplacement of the male penile urethra, or hypospadias, is amongst the most common birth defects reported in humans. While mice present a tractable model to study penile development, several structures differ between mice and humans, and there is a lack of consensus in the literature on their annotation and developmental origins. Defining the ontology of the mouse prepuce is especially important for the relevance and interpretation of mouse models of hypospadias to human conditions. We have developed a detailed annotation of the adult mouse penis that addresses these differences and enables an accurate comparison of murine and human hypospadias phenotypes. Through MRI data, gross morphology and section histology, we define the origin of the mouse external and internal prepuces, their relationship to the single human foreskin as well as provide a comprehensive view of the various structures of the mouse penis and their associated muscle attachments within the body. These data are combined to annotate structures in a novel 3D adult penis atlas that can be downloaded, viewed at any angle, and manipulated to examine the relationship of various structures. PMID:26112156

  19. New Insights on the Morphology of Adult Mouse Penis1

    Science.gov (United States)

    Rodriguez, Esequiel; Weiss, Dana A.; Yang, Jennifer H.; Menshenina, Julia; Ferretti, Max; Cunha, Tristan J.; Barcellos, Dale; Chan, Lok Yun; Risbridger, Gail; Cunha, Gerald R.; Baskin, Laurence S.

    2011-01-01

    ABSTRACT The adult mouse penis represents the end point of masculine sex differentiation of the embryonic genital tubercle and contains bone, cartilage, the urethra, erectile bodies, several types of epithelium, and many individual cell types arrayed into specific anatomical structures. Using contemporary high-resolution imaging techniques, we sought to provide new insights to the current description of adult mouse penile morphology to enable understanding of penile abnormalities, including hypospadias. Examination of serial transverse and longitudinal sections, scanning electron microscopy, and three-dimensional (3D) reconstruction provided a new appreciation of the individual structures in the adult mouse penis and their 3D interrelationships. In so doing, we discovered novel paired erectile bodies, the male urogenital mating protuberance (MUMP), and more accurately described the urethral meatus. These morphological observations were quantified by morphometric analysis and now provide accurate morphological end points of sex differentiation of mouse penis that will be the foundation of future studies to identify normal and abnormal penile development. PMID:21918128

  20. 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.

  1. 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

  2. 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

  3. [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.

  4. A developmentally plastic adult mouse kidney cell line spontaneously generates multiple adult kidney structures

    International Nuclear Information System (INIS)

    Webb, Carol F.; Ratliff, Michelle L.; Powell, Rebecca; Wirsig-Wiechmann, Celeste R.; Lakiza, Olga; Obara, Tomoko

    2015-01-01

    Despite exciting new possibilities for regenerative therapy posed by the ability to induce pluripotent stem cells, recapitulation of three-dimensional kidneys for repair or replacement has not been possible. ARID3a-deficient mouse tissues generated multipotent, developmentally plastic cells. Therefore, we assessed the adult mouse ARID3a−/− kidney cell line, KKPS5, which expresses renal progenitor surface markers as an alternative cell source for modeling kidney development. Remarkably, these cells spontaneously developed into multicellular nephron-like structures in vitro, and engrafted into immunocompromised medaka mesonephros, where they formed mouse nephron structures. These data implicate KKPS5 cells as a new model system for studying kidney development. - Highlights: • An ARID3a-deficient mouse kidney cell line expresses multiple progenitor markers. • This cell line spontaneously forms multiple nephron-like structures in vitro. • This cell line formed mouse kidney structures in immunocompromised medaka fish kidneys. • Our data identify a novel model system for studying kidney development

  5. A developmentally plastic adult mouse kidney cell line spontaneously generates multiple adult kidney structures

    Energy Technology Data Exchange (ETDEWEB)

    Webb, Carol F., E-mail: carol-webb@omrf.org [Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK (United States); Immunobiology and Cancer Research, Oklahoma Medical Research Foundation, Oklahoma City, OK (United States); Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, OK (United States); Ratliff, Michelle L., E-mail: michelle-ratliff@omrf.org [Immunobiology and Cancer Research, Oklahoma Medical Research Foundation, Oklahoma City, OK (United States); Powell, Rebecca, E-mail: rebeccapowell@gmail.com [Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK (United States); Wirsig-Wiechmann, Celeste R., E-mail: celeste-wirsig@ouhsc.edu [Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK (United States); Lakiza, Olga, E-mail: olga-lakiza@ouhsc.edu [Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK (United States); Obara, Tomoko, E-mail: tomoko-obara@ouhsc.edu [Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK (United States)

    2015-08-07

    Despite exciting new possibilities for regenerative therapy posed by the ability to induce pluripotent stem cells, recapitulation of three-dimensional kidneys for repair or replacement has not been possible. ARID3a-deficient mouse tissues generated multipotent, developmentally plastic cells. Therefore, we assessed the adult mouse ARID3a−/− kidney cell line, KKPS5, which expresses renal progenitor surface markers as an alternative cell source for modeling kidney development. Remarkably, these cells spontaneously developed into multicellular nephron-like structures in vitro, and engrafted into immunocompromised medaka mesonephros, where they formed mouse nephron structures. These data implicate KKPS5 cells as a new model system for studying kidney development. - Highlights: • An ARID3a-deficient mouse kidney cell line expresses multiple progenitor markers. • This cell line spontaneously forms multiple nephron-like structures in vitro. • This cell line formed mouse kidney structures in immunocompromised medaka fish kidneys. • Our data identify a novel model system for studying kidney development.

  6. 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.

  7. Beta-adrenergic control of phosphatidylcholine synthesis by transmethylation in hepatocytes from juvenile, adult and adrenalectomized rats.

    Science.gov (United States)

    Marin-Cao, D; Alvarez Chiva, V; Mato, J M

    1983-01-01

    Changes in isoprenaline-sensitive phospholipid methyltransferase were studied in hepatocytes isolated from juvenile, mature and adrenalectomized rats. Isoprenaline produced greater stimulation of cyclic AMP accumulation in juvenile and mature adrenalectomized rats than in mature animals. Similarly, isoprenaline stimulated phospholipid methyltransferase in juvenile and mature adrenalectomized rats but had no effect in mature animals. Isoprenaline-mediated activation of phospholipid methyltransferase in adrenalectomized rats was time- and dose-dependent. In hepatocytes isolated from adrenalectomized rats incubated with [Me-3H]methionine or [3H]-ethanolamine the addition of isoprenaline increased the amount of radioactivity incorporated into phosphatidylcholine. The activation by isoprenaline of phospholipid methyltransferase was abolished by the beta-blocker propranolol and by insulin. These results indicate that rat liver the occupation of functional beta-receptors causes a stimulation of phospholipid methylation. It is suggested that, as reported previously, cyclic AMP activates phospholipid methyltransferase. PMID:6320796

  8. Protein composition and synthesis in the adult mouse spinal cord

    International Nuclear Information System (INIS)

    Stodieck, L.S.; Luttges, M.W.

    1983-01-01

    Properties of spinal cord proteins were studied in adult mice subjected to unilateral crush or electrical stimulation of sciatic nerve. The protein composition of spinal tissue was determined using SDS-polyacrylamide gel electrophoresis coupled with subcellular fractionation. Comparisons of mouse spinal cord and brain revealed similarities in the types but differences in the concentrations of myelin associated proteins, nuclear histones and other proteins. Comparisons with sciatic nerve proteins demonstrated differences in types of proteins but similarities in the concentration of myelin proteins and nuclear histones. The short term (less than 2 hrs.) incorporation of radioactive amino acids into spinal cord proteins revealed heterogeneous rates of incorporation. Neither nerve crush six days prior to testing nor sciatic nerve stimulation had a significant effect on the protein composition or amino acid incorporation rates of spinal cord tissue. These observations suggest that known differences in spinal cord function following alterations in nerve input may be dependent upon different mechanisms than have been found in the brain

  9. Dissection of Hippocampal Dentate Gyrus from Adult Mouse

    Science.gov (United States)

    Hagihara, Hideo; Toyama, Keiko; Yamasaki, Nobuyuki; Miyakawa, Tsuyoshi

    2009-01-01

    The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its remarkable neuronal cell plasticity, and its involvement in epilepsy, neurodegenerative diseases, and psychiatric disorders. The hippocampus is composed of distinct regions; the dentate gyrus, which comprises mainly granule neurons, and Ammon's horn, which comprises mainly pyramidal neurons, and the two regions are connected by both anatomic and functional circuits. Many different mRNAs and proteins are selectively expressed in the dentate gyrus, and the dentate gyrus is a site of adult neurogenesis; that is, new neurons are continually generated in the adult dentate gyrus. To investigate mRNA and protein expression specific to the dentate gyrus, laser capture microdissection is often used. This method has some limitations, however, such as the need for special apparatuses and complicated handling procedures. In this video-recorded protocol, we demonstrate a dissection technique for removing the dentate gyrus from adult mouse under a stereomicroscope. Dentate gyrus samples prepared using this technique are suitable for any assay, including transcriptomic, proteomic, and cell biology analyses. We confirmed that the dissected tissue is dentate gyrus by conducting real-time PCR of dentate gyrus-specific genes, tryptophan 2,3-dioxygenase (TDO2) and desmoplakin (Dsp), and Ammon's horn enriched genes, Meis-related gene 1b (Mrg1b) and TYRO3 protein tyrosine kinase 3 (Tyro3). The mRNA expressions of TDO2 and Dsp in the dentate gyrus samples were detected at obviously higher levels, whereas Mrg1b and Tyro3 were lower levels, than those in the Ammon's horn samples. To demonstrate the advantage of this method, we performed DNA microarray analysis using samples of whole hippocampus and dentate gyrus. The mRNA expression of TDO2 and Dsp, which are expressed selectively in the dentate gyrus, in the whole hippocampus of alpha

  10. 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.

  11. 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.

  12. 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.

  13. 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.

    Science.gov (United States)

    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

  14. Cerebellar stem cells do not produce neurons and astrocytes in adult mouse

    International Nuclear Information System (INIS)

    Su, Xin; Guan, Wuqiang; Yu, Yong-Chun; Fu, Yinghui

    2014-01-01

    Highlights: • No new neurons and astrocytes are generated in adult mouse cerebellum. • Very few mash1 + or nestin + stem cells exist, and most of them are quiescent. • Cell proliferation rate is diversified among cerebellar regions and decreases over time. - Abstract: Although previous studies implied that cerebellar stem cells exist in some adult mammals, little is known about whether these stem cells can produce new neurons and astrocytes. In this study by bromodeoxyuridine (BrdU) intraperitoneal (i.p.) injection, we found that there are abundant BrdU + cells in adult mouse cerebellum, and their quantity and density decreases significantly over time. We also found cell proliferation rate is diversified in different cerebellar regions. Among these BrdU + cells, very few are mash1 + or nestin + stem cells, and the vast majority of cerebellar stem cells are quiescent. Data obtained by in vivo retrovirus injection indicate that stem cells do not produce neurons and astrocytes in adult mouse cerebellum. Instead, some cells labeled by retrovirus are Iba1 + microglia. These results indicate that very few stem cells exist in adult mouse cerebellum, and none of these stem cells contribute to neurogenesis and astrogenesis under physiological condition

  15. Doublecortin-like knockdown in the adult mouse brain : implications for neurogenesis, neuroplasticity and behaviour

    NARCIS (Netherlands)

    Saaltink, Dirk-Jan

    2014-01-01

    The results in this thesis showed for the first time doublecortin-like (DCL)-specific expression in the adult mouse brain. Besides the expected regions with the capacity to generate new neurons (hippocampus and olfactory forebrain), DCL expression was found in three novel brain areas namely

  16. MicroRNA expression in the adult mouse central nervous system

    DEFF Research Database (Denmark)

    Bak, Mads; Silahtaroglu, Asli; Møller, Morten

    2008-01-01

    distinct areas of the adult mouse central nervous system (CNS). Microarray profiling in combination with real-time RT-PCR and LNA (locked nucleic acid)-based in situ hybridization uncovered 44 miRNAs displaying more than threefold enrichment in the spinal cord, cerebellum, medulla oblongata, pons......RNA-related gene regulatory networks in the mammalian central nervous system. Udgivelsesdato: 2008-Mar...

  17. 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.

  18. Regulation by commensal bacteria of neurogenesis in the subventricular zone of adult mouse brain.

    Science.gov (United States)

    Sawada, Naoki; Kotani, Takenori; Konno, Tasuku; Setiawan, Jajar; Nishigaito, Yuka; Saito, Yasuyuki; Murata, Yoji; Nibu, Ken-Ichi; Matozaki, Takashi

    2018-04-15

    In the mouse olfactory bulb (OB), interneurons such as granule cells and periglomerular cells are continuously replaced by adult-born neurons, which are generated in the subventricular zone (SVZ) of the brain. We have now investigated the role of commensal bacteria in regulation of such neuronal cell turnover in the adult mouse brain. Administration of mixture of antibiotics to specific pathogen-free (SPF) mice markedly attenuated the incorporation of bromodeoxyuridine (BrdU) into the SVZ cells. The treatment with antibiotics also reduced newly generated BrdU-positive neurons in the mouse OB. In addition, the incorporation of BrdU into the SVZ cells of germ-free (GF) mice was markedly reduced compared to that apparent for SPF mice. In contrast, the reduced incorporation of BrdU into the SVZ cells of GF mice was recovered by their co-housing with SPF mice, suggesting that commensal bacteria promote the incorporation of BrdU into the SVZ cells. Finally, we found that administration of ampicillin markedly attenuated the incorporation of BrdU into the SVZ cells of SPF mice. Our results thus suggest that ampicillin-sensitive commensal bacteria regulate the neurogenesis in the SVZ of adult mouse brain. Copyright © 2018 Elsevier Inc. All rights reserved.

  19. 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.

  20. Transcriptome signature of the adult mouse choroid plexus

    Directory of Open Access Journals (Sweden)

    Marques Fernanda

    2011-01-01

    Full Text Available Abstract Background Although the gene expression profile of several tissues in humans and in rodent animal models has been explored, analysis of the complete choroid plexus (CP transcriptome is still lacking. A better characterization of the CP transcriptome can provide key insights into its functions as one of the barriers that separate the brain from the periphery and in the production of cerebrospinal fluid. Methods This work extends further what is known about the mouse CP transcriptome through a microarray analysis of CP tissue from normal mice under physiological conditions. Results We found that the genes most highly expressed are those implicated in energy metabolism (oxidative phosphorylation, glycolysis/gluconeogenesis and in ribosomal function, which is in agreement with the secretory nature of the CP. On the other hand, genes encoding for immune mediators are among those with lower expression in basal conditions. In addition, we found genes known to be relevant during brain development, and not previously identified to be expressed in the CP, including those encoding for various axonal guidance and angiogenesis molecules and for growth factors. Some of these are known to influence the neural stem cell niche in the subventricular zone, highlighting the involvement of the CP as a likely modulator of neurogenesis. Interestingly, our observations confirm that the CP transcriptome is unique, displaying low homology with that of other tissues. Of note, we describe here that the closest similarity is with the transcriptome of the endothelial cells of the blood-brain barrier. Conclusions Based on the data presented here, it will now be possible to further explore the function of particular proteins of the CP secretome in health and in disease.

  1. Epigenetic transgenerational inheritance of vinclozolin induced mouse adult onset disease and associated sperm epigenome biomarkers.

    Science.gov (United States)

    Guerrero-Bosagna, Carlos; Covert, Trevor R; Haque, Md M; Settles, Matthew; Nilsson, Eric E; Anway, Matthew D; Skinner, Michael K

    2012-12-01

    The endocrine disruptor vinclozolin has previously been shown to promote epigenetic transgenerational inheritance of adult onset disease in the rat. The current study was designed to investigate the transgenerational actions of vinclozolin on the mouse. Transient exposure of the F0 generation gestating female during gonadal sex determination promoted transgenerational adult onset disease in F3 generation male and female mice, including spermatogenic cell defects, testicular abnormalities, prostate abnormalities, kidney abnormalities and polycystic ovarian disease. Pathology analysis demonstrated 75% of the vinclozolin lineage animals developed disease with 34% having two or more different disease states. Interestingly, the vinclozolin induced transgenerational disease was observed in the outbred CD-1 strain, but not the inbred 129 mouse strain. Analysis of the F3 generation sperm epigenome identified differential DNA methylation regions that can potentially be utilized as epigenetic biomarkers for transgenerational exposure and disease. Copyright © 2012 Elsevier Inc. All rights reserved.

  2. MRI visualization of endogenous neural progenitor cell migration along the RMS in the adult mouse brain

    DEFF Research Database (Denmark)

    Vreys, Ruth; Vande Velde, Greetje; Krylychkina, Olga

    2010-01-01

    The adult rodent brain contains neural progenitor cells (NPCs), generated in the subventricular zone (SVZ), which migrate along the rostral migratory stream (RMS) towards the olfactory bulb (OB) where they differentiate into neurons. The aim of this study was to visualize endogenous NPC migration...... by a longitudinal MRI study and validated with histology. Here, we visualized endogenous NPC migration in the mouse brain by in vivo MRI and demonstrated accumulation of MPIO-labeled NPCs in the OB over time with ex vivo MRI. Furthermore, we investigated the influence of in situ injection of MPIOs on adult...

  3. Moderate activation of IKK2-NF-kB in unstressed adult mouse liver induces cytoprotective genes and lipogenesis without apparent signs of inflammation or fibrosis.

    Science.gov (United States)

    Lu, Hong; Lei, Xiaohong; Zhang, Qinghao

    2015-07-30

    The NF-kB signaling, regulated by IKK1-p52/RelB and IKK2-p65, is activated by various stresses to protect or damage the liver, in context-specific manners. Two previous studies of liver-specific expression of constitutive active IKK2 (IKK2ca) showed that strong activation of IKK2-NF-kB in mouse livers caused inflammation, insulin resistance, and/or fibrosis. The purpose of this study was to understand how moderate activation of IKK2-NF-kB in adult mouse livers alters hepatic gene expression and pathophysiology. We generated mice with adult hepatocyte-specific activation of Ikk2 (Liv-Ikk2ca) using Alb-cre mice and Ikk2ca Rosa26 knockin mice in which a moderate expression of Ikk2ca transgene was driven by the endogenous Rosa26 promoter. Surprisingly, compared to wild-type mice, adult male Liv-Ikk2ca mice had higher hepatic mRNA expression of Ikk2 and classical NF-kB targets (e.g. Lcn2 and A20), as well as IKK1, NIK, and RelB, but no changes in markers of inflammation or fibrosis. Blood levels of IL-6 and MCP-1 remained unchanged, and histology analysis showed a lack of injury or infiltration of inflammatory cells in livers of Liv-Ikk2ca mice. Moreover, Liv-Ikk2ca mice had lower mRNA expression of prooxidative enzymes Cyp2e1 and Cyp4a14, higher expression of antioxidative enzymes Sod2, Gpx1, and Nqo1, without changes in key enzymes for fatty acid oxidation, glucose utilization, or gluconeogenesis. In parallel, Liv-Ikk2ca mice and wild-type mice had similar levels of hepatic reduced glutathione, endogenous reactive oxygen species, and lipid peroxidation. Additionally, Liv-Ikk2ca mice had higher Cyp3a11 without down-regulation of most drug processing genes. Regarding nuclear proteins of NF-kB subunits, Liv-Ikk2ca mice had moderately higher p65 and p50 but much higher RelB. Results of ChIP-qPCR showed that the binding of p50 to multiple NF-kB-target genes was markedly increased in Liv-Ikk2ca mice. Additionally, Liv-Ikk2ca mice had moderate increase in triglycerides in

  4. Adult Mouse Liver Contains Two Distinct Populations of Cholangiocytes

    Directory of Open Access Journals (Sweden)

    Bin Li

    2017-08-01

    Full Text Available The biliary system plays an important role in several acquired and genetic disorders of the liver. We have previously shown that biliary duct epithelium contains cells giving rise to proliferative Lgr5+ organoids in vitro. However, it remained unknown whether all biliary cells or only a specific subset had this clonogenic activity. The cell surface protease ST14 was identified as a positive marker for the clonogenic subset of cholangiocytes and was used to separate clonogenic and non-clonogenic duct cells by fluorescence-activated cell sorting. Only ST14hi duct cells had the ability to generate organoids that could be serially passaged. The gene expression profiles of clonogenic and non-clonogenic duct cells were similar, but several hundred genes were differentially expressed. RNA fluorescence in situ hybridization showed that clonogenic duct cells are interspersed among regular biliary epithelium at a ∼1:3 ratio. We conclude that adult murine cholangiocytes can be subdivided into two populations differing in their proliferative capacity.

  5. The time point of β-catenin knockout in hepatocytes determines their response to xenobiotic activation of the constitutive androstane receptor

    International Nuclear Information System (INIS)

    Ganzenberg, Katrin; Singh, Yasmin; Braeuning, Albert

    2013-01-01

    The constitutive androstane receptor (CAR) controls the expression of drug-metabolizing enzymes and regulates hepatocyte proliferation. Studies with transgenic mice with an early postnatal conditional hepatocyte-specific knockout of the β-catenin gene Ctnnb1 revealed that β-catenin deficiency decreases the magnitude of induction of drug-metabolizing enzymes by CAR activators, abrogates zonal differences in the hepatocytes’ susceptibility to these compounds, and impacts on hepatocyte proliferation. These data, however, do not allow distinguishing between effects caused by β-catenin deficiency during postnatal liver development and acute effects of β-catenin deficiency in the adult animal at the time point of CAR activation. Therefore, CAR activation was now studied in a different mouse model allowing for the hepatocyte-specific knockout of β-catenin in adult mice. Treatment of these mice with 3 mg/kg body weight of the model CAR activator 1,4-bis-[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) confirmed previous findings related to the coordinate regulation of drug metabolism by β-catenin and CAR. More importantly, the present study clarified that the impact of β-catenin signaling on CAR-mediated enzyme induction in the liver is not merely due to developmental defects caused by a postnatal lack of β-catenin, but depends on the presence of β-catenin at the time point of xenobiotic treatment. The study also revealed interesting differences between the two mouse models: hepatic zonation of TCPOBOP-dependent induction of drug-metabolizing enzymes was restored in mice with late knockout of β-catenin, and the strong proliferative response of female mice was exclusively abolished when using animals with a late β-catenin knockout. This suggests a β-catenin-dependent postnatal priming of hepatocytes during postnatal liver development, later affecting the proliferative response of adult animals to CAR-activating xenobiotics

  6. An adult passive transfer mouse model to study desmoglein 3 signaling in pemphigus vulgaris.

    Science.gov (United States)

    Schulze, Katja; Galichet, Arnaud; Sayar, Beyza S; Scothern, Anthea; Howald, Denise; Zymann, Hillard; Siffert, Myriam; Zenhäusern, Denise; Bolli, Reinhard; Koch, Peter J; Garrod, David; Suter, Maja M; Müller, Eliane J

    2012-02-01

    Evidence has accumulated that changes in intracellular signaling downstream of desmoglein 3 (Dsg3) may have a significant role in epithelial blistering in the autoimmune disease pemphigus vulgaris (PV). Currently, most studies on PV involve passive transfer of pathogenic antibodies into neonatal mice that have not finalized epidermal morphogenesis, and do not permit analysis of mature hair follicles (HFs) and stem cell niches. To investigate Dsg3 antibody-induced signaling in the adult epidermis at defined stages of the HF cycle, we developed a model with passive transfer of AK23 (a mouse monoclonal pathogenic anti-Dsg3 antibody) into adult 8-week-old C57Bl/6J mice. Validated using histopathological and molecular methods, we found that this model faithfully recapitulates major features described in PV patients and PV models. Two hours after AK23 transfer, we observed widening of intercellular spaces between desmosomes and EGFR activation, followed by increased Myc expression and epidermal hyperproliferation, desmosomal Dsg3 depletion, and predominant blistering in HFs and oral mucosa. These data confirm that the adult passive transfer mouse model is ideally suited for detailed studies of Dsg3 antibody-mediated signaling in adult skin, providing the basis for investigations on novel keratinocyte-specific therapeutic strategies.

  7. Brain transcriptional stability upon prion protein-encoding gene invalidation in zygotic or adult mouse

    Directory of Open Access Journals (Sweden)

    Béringue Vincent

    2010-07-01

    Full Text Available Abstract Background The physiological function of the prion protein remains largely elusive while its key role in prion infection has been expansively documented. To potentially assess this conundrum, we performed a comparative transcriptomic analysis of the brain of wild-type mice with that of transgenic mice invalidated at this locus either at the zygotic or at the adult stages. Results Only subtle transcriptomic differences resulting from the Prnp knockout could be evidenced, beside Prnp itself, in the analyzed adult brains following microarray analysis of 24 109 mouse genes and QPCR assessment of some of the putatively marginally modulated loci. When performed at the adult stage, neuronal Prnp disruption appeared to sequentially induce a response to an oxidative stress and a remodeling of the nervous system. However, these events involved only a limited number of genes, expression levels of which were only slightly modified and not always confirmed by RT-qPCR. If not, the qPCR obtained data suggested even less pronounced differences. Conclusions These results suggest that the physiological function of PrP is redundant at the adult stage or important for only a small subset of the brain cell population under classical breeding conditions. Following its early reported embryonic developmental regulation, this lack of response could also imply that PrP has a more detrimental role during mouse embryogenesis and that potential transient compensatory mechanisms have to be searched for at the time this locus becomes transcriptionally activated.

  8. Sertoli cells maintain Leydig cell number and peritubular myoid cell activity in the adult mouse testis.

    Directory of Open Access Journals (Sweden)

    Diane Rebourcet

    Full Text Available The Sertoli cells are critical regulators of testis differentiation and development. In the adult, however, their known function is restricted largely to maintenance of spermatogenesis. To determine whether the Sertoli cells regulate other aspects of adult testis biology we have used a novel transgenic mouse model in which Amh-Cre induces expression of the receptor for Diphtheria toxin (iDTR specifically within Sertoli cells. This causes controlled, cell-specific and acute ablation of the Sertoli cell population in the adult animal following Diphtheria toxin injection. Results show that Sertoli cell ablation leads to rapid loss of all germ cell populations. In addition, adult Leydig cell numbers decline by 75% with the remaining cells concentrated around the rete and in the sub-capsular region. In the absence of Sertoli cells, peritubular myoid cell activity is reduced but the cells retain an ability to exclude immune cells from the seminiferous tubules. These data demonstrate that, in addition to support of spermatogenesis, Sertoli cells are required in the adult testis both for retention of the normal adult Leydig cell population and for support of normal peritubular myoid cell function. This has implications for our understanding of male reproductive disorders and wider androgen-related conditions affecting male health.

  9. 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.

  10. Identification and characterization of adult mouse meniscus stem/progenitor cells.

    Science.gov (United States)

    Gamer, Laura W; Shi, Rui Rui; Gendelman, Ashira; Mathewson, Dylan; Gamer, Jackson; Rosen, Vicki

    Meniscal damage is a common problem that accelerates the onset of knee osteoarthritis. Stem cell-based tissue engineering treatment approaches have shown promise in preserving meniscal tissue and restoring meniscal function. The purpose of our study was to identify meniscus-derived stem/progenitor cells (MSPCs) from mouse, a model system that allows for in vivo analysis of the mechanisms underlying meniscal injury and healing. MSPCs were isolated from murine menisci grown in explant culture and characterized for stem cell properties. Flow cytometry was used to detect the presence of surface antigens related to stem cells, and qRT-PCR was used to examine the gene expression profile of MSPCs. Major proteins associated with MSPCs were localized in the adult mouse knee using immunohistochemistry. Our data show that MSPCs have universal stem cell-like properties including clonogenicity and multi-potentiality. MSPCs expressed the mesenchymal stem cell markers CD44, Sca-1, CD90, and CD73 and when cultured had elevated levels of biglycan and collagen type I, important extracellular matrix components of adult meniscus. MSPC also expressed significant levels of Lox and Igf-1, genes associated with the embryonic meniscus. Localization studies showed staining for these same proteins in the superficial and outer zones of the adult mouse meniscus, regions thought to harbor endogenous repair cells. MSPCs represent a novel resident stem cell population in the murine meniscus. Analysis of MSPCs in mice will allow for a greater understanding of the cell biology of the meniscus, essential information for enhancing therapeutic strategies for treating knee joint injury and disease.

  11. BAG3 regulates contractility and Ca2+ homeostasis in adult mouse ventricular myocytes

    OpenAIRE

    Feldman, Arthur M.; Gordon, Jennifer; Wang, JuFang; Song, Jianliang; Zhang, Xue-Qian; Myers, Valerie D.; Tilley, Douglas G.; Gao, Erhe; Hoffman, Nicholas E.; Tomar, Dhanendra; Madesh, Muniswamy; Rabinowitz, Joseph; Koch, Walter J.; Su, Feifei; Khalili, Kamel

    2016-01-01

    Bcl2-associated athanogene 3 (BAG3) is a 575 amino acid anti-apoptotic protein that is constitutively expressed in the heart. BAG3 mutations, including mutations leading to loss of protein, are associated with familial cardiomyopathy. Furthermore, BAG3 levels have been found to be reduced in end-stage non-familial failing myocardium. In contrast to neonatal myocytes in which BAG3 is found in the cytoplasm and involved in protein quality control and apoptosis, in adult mouse left ventricular (...

  12. A novel type of self-beating cardiomyocytes in adult mouse ventricles

    International Nuclear Information System (INIS)

    Omatsu-Kanbe, Mariko; Matsuura, Hiroshi

    2009-01-01

    This study was designed to investigate the presence of resident heart cells that are distinct from terminally-differentiated cardiomyocytes. Adult mouse heart was coronary perfused with collagenase, and ventricles were excised and further digested. After spinning cardiomyocyte-containing fractions down, the supernatant fraction was collected and cultured without adding any chemicals. Two to five days after plating, some of rounded cells adhered to the culture dish, gradually changed their shape and then started self-beating. These self-beating cells did not appreciably proliferate but underwent a further morphological maturation process to form highly branched shapes with many projections. These cells were mostly multinucleated, well sarcomeric-organized and expressed cardiac marker proteins, defined as atypically-shaped cardiomyocytes (ACMs). Patch-clamp experiments revealed that ACMs exhibited spontaneous action potentials arising from the preceding slow diastolic depolarization. We thus found a novel type of resident heart cells in adult cardiac ventricles that spontaneously develop into self-beating cardiomyocytes.

  13. Cathepsin B-dependent motor neuron death after nerve injury in the adult mouse

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Li; Wu, Zhou; Baba, Masashi [Department of Aging Science and Pharmacology, Faculty of Dental Sciences, Kyushu University, Maidashi 3-1-1, Fukuoka 812-8582 (Japan); Peters, Christoph [Institute fuer Molekulare Medizin und Zellforshung, Albert-Ludwings-Universitaet Freiburg, D-79104 Freiburg (Germany); Uchiyama, Yasuo [Department of Cell Biology and Neuroscience, Juntendo University Graduate School of Medicine, Tokyo (Japan); Nakanishi, Hiroshi, E-mail: nakan@dent.kyushu-u.ac.jp [Department of Aging Science and Pharmacology, Faculty of Dental Sciences, Kyushu University, Maidashi 3-1-1, Fukuoka 812-8582 (Japan)

    2010-08-27

    Research highlights: {yields} Cathepsin B (CB), a lysosomal cysteine protease, is expressed in neuron and glia. {yields} CB increased in hypogrossal nucleus neurons after nerve injury in adult mice. {yields} CB-deficiency significantly increased the mean survival ratio of injured neurons. {yields} Thus, CB plays a critical role in axotomy-induced neuronal death in adult mice. -- Abstract: There are significant differences in the rate of neuronal death after peripheral nerve injury between species. The rate of neuronal death of motor neurons after nerve injury in the adult rats is very low, whereas that in adult mice is relatively high. However, the understanding of the mechanism underlying axotomy-induced motor neuron death in adult mice is limited. Cathepsin B (CB), a typical cysteine lysosomal protease, has been implicated in three major morphologically distinct pathways of cell death; apoptosis, necrosis and autophagic cell death. The possible involvement of CB in the neuronal death of hypogrossal nucleus (HGN) neurons after nerve injury in adult mice was thus examined. Quantitative analyses showed the mean survival ratio of HGN neurons in CB-deficient (CB-/-) adult mice after nerve injury was significantly greater than that in the wild-type mice. At the same time, proliferation of microglia in the injured side of the HGN of CB-/- adult mice was markedly reduced compared with that in the wild-type mice. On the injured side of the HGN in the wild-type adult mice, both pro- and mature forms of CB markedly increased in accordance with the increase in the membrane-bound form of LC3 (LC3-II), a marker protein of autophagy. Furthermore, the increase in CB preceded an increase in the expression of Noxa, a major executor for axotomy-induced motor neuron death in the adult mouse. Conversely, expression of neither Noxa or LC3-II was observed in the HGN of adult CB-/- mice after nerve injury. These observations strongly suggest that CB plays a critical role in axotomy

  14. Cathepsin B-dependent motor neuron death after nerve injury in the adult mouse

    International Nuclear Information System (INIS)

    Sun, Li; Wu, Zhou; Baba, Masashi; Peters, Christoph; Uchiyama, Yasuo; Nakanishi, Hiroshi

    2010-01-01

    Research highlights: → Cathepsin B (CB), a lysosomal cysteine protease, is expressed in neuron and glia. → CB increased in hypogrossal nucleus neurons after nerve injury in adult mice. → CB-deficiency significantly increased the mean survival ratio of injured neurons. → Thus, CB plays a critical role in axotomy-induced neuronal death in adult mice. -- Abstract: There are significant differences in the rate of neuronal death after peripheral nerve injury between species. The rate of neuronal death of motor neurons after nerve injury in the adult rats is very low, whereas that in adult mice is relatively high. However, the understanding of the mechanism underlying axotomy-induced motor neuron death in adult mice is limited. Cathepsin B (CB), a typical cysteine lysosomal protease, has been implicated in three major morphologically distinct pathways of cell death; apoptosis, necrosis and autophagic cell death. The possible involvement of CB in the neuronal death of hypogrossal nucleus (HGN) neurons after nerve injury in adult mice was thus examined. Quantitative analyses showed the mean survival ratio of HGN neurons in CB-deficient (CB-/-) adult mice after nerve injury was significantly greater than that in the wild-type mice. At the same time, proliferation of microglia in the injured side of the HGN of CB-/- adult mice was markedly reduced compared with that in the wild-type mice. On the injured side of the HGN in the wild-type adult mice, both pro- and mature forms of CB markedly increased in accordance with the increase in the membrane-bound form of LC3 (LC3-II), a marker protein of autophagy. Furthermore, the increase in CB preceded an increase in the expression of Noxa, a major executor for axotomy-induced motor neuron death in the adult mouse. Conversely, expression of neither Noxa or LC3-II was observed in the HGN of adult CB-/- mice after nerve injury. These observations strongly suggest that CB plays a critical role in axotomy-induced mortor neuron

  15. 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.

  16. Nogo-A is a reliable oligodendroglial marker in adult human and mouse CNS and in demyelinated lesions

    DEFF Research Database (Denmark)

    Kuhlmann, Tanja; Remington, Leah; Maruschak, Brigitte

    2007-01-01

    to be strongly expressed in mature oligodendrocytes in vivo. In the present investigation we analyzed the expression patterns of Nogo-A in adult mouse and human CNS as well as in demyelinating animal models and multiple sclerosis lesions. Nogo-A expression was compared with that of other frequently used...... oligodendroglial markers such as CC1, CNP, and in situ hybridization for proteolipid protein mRNA. Nogo-A strongly and reliably labeled oligodendrocytes in the adult CNS as well as in demyelinating lesions and thus represents a valuable tool for the identification of oligodendrocytes in human and mouse CNS tissue...

  17. A brain-specific gene cluster isolated from the region of the mouse obesity locus is expressed in the adult hypothalamus and during mouse development

    Energy Technology Data Exchange (ETDEWEB)

    Laig-Webster, M.; Lim, M.E.; Chehab, F.F. [Univ. of California, San Francisco, CA (United States)

    1994-09-01

    The molecular defect underlying an autosomal recessive form of genetic obesity in a classical mouse model C57 BL/6J-ob/ob has not yet been elucidated. Whereas metabolic and physiological disturbances such as diabetes and hypertension are associated with obesity, the site of expression and the nature of the primary lesion responsible for this cascade of events remains elusive. Our efforts aimed at the positional cloning of the ob gene by YAC contig mapping and gene identification have resulted in the cloning of a brain-specific gene cluster from the ob critical region. The expression of this gene cluster is remarkably complex owing to the multitude of brain-specific mRNA transcripts detected on Northern blots. cDNA cloning of these transcripts suggests that they are expressed from different genes as well as by alternate splicing mechanisms. Furthermore, the genomic organization of the cluster appears to consist of at least two identical promoters displaying CpG islands characteristic of housekeeping genes, yet clearly involving tissue-specific expression. Sense and anti-sense synthetic RNA probes were derived from a common DNA sequence on 3 cDNA clones and hybridized to 8-16 days mouse embryonic stages and mouse adult brain sections. Expression in development was noticeable as of the 11th day of gestation and confined to the central nervous system mainly in the telencephalon and spinal cord. Coronal and sagittal sections of the adult mouse brain showed expression only in 3 different regions of the brain stem. In situ hybridization to mouse hypothalamus sections revealed the presence of a localized and specialized group of cells expressing high levels of mRNA, suggesting that this gene cluster may also be involved in the regulation of hypothalamic activities. The hypothalamus has long been hypothesized as a primary candidate tissue for the expression of the obesity gene mainly because of its well-established role in the regulation of energy metabolism and food intake.

  18. The Thoc1 encoded ribonucleoprotein is required for myeloid progenitor cell homeostasis in the adult mouse.

    Science.gov (United States)

    Pitzonka, Laura; Ullas, Sumana; Chinnam, Meenalakshmi; Povinelli, Benjamin J; Fisher, Daniel T; Golding, Michelle; Appenheimer, Michelle M; Nemeth, Michael J; Evans, Sharon; Goodrich, David W

    2014-01-01

    Co-transcriptionally assembled ribonucleoprotein (RNP) complexes are critical for RNA processing and nuclear export. RNPs have been hypothesized to contribute to the regulation of coordinated gene expression, and defects in RNP biogenesis contribute to genome instability and disease. Despite the large number of RNPs and the importance of the molecular processes they mediate, the requirements for individual RNP complexes in mammalian development and tissue homeostasis are not well characterized. THO is an evolutionarily conserved, nuclear RNP complex that physically links nascent transcripts with the nuclear export apparatus. THO is essential for early mouse embryonic development, limiting characterization of the requirements for THO in adult tissues. To address this shortcoming, a mouse strain has been generated allowing inducible deletion of the Thoc1 gene which encodes an essential protein subunit of THO. Bone marrow reconstitution was used to generate mice in which Thoc1 deletion could be induced specifically in the hematopoietic system. We find that granulocyte macrophage progenitors have a cell autonomous requirement for Thoc1 to maintain cell growth and viability. Lymphoid lineages are not detectably affected by Thoc1 loss under the homeostatic conditions tested. Myeloid lineages may be more sensitive to Thoc1 loss due to their relatively high rate of proliferation and turnover.

  19. The Thoc1 encoded ribonucleoprotein is required for myeloid progenitor cell homeostasis in the adult mouse.

    Directory of Open Access Journals (Sweden)

    Laura Pitzonka

    Full Text Available Co-transcriptionally assembled ribonucleoprotein (RNP complexes are critical for RNA processing and nuclear export. RNPs have been hypothesized to contribute to the regulation of coordinated gene expression, and defects in RNP biogenesis contribute to genome instability and disease. Despite the large number of RNPs and the importance of the molecular processes they mediate, the requirements for individual RNP complexes in mammalian development and tissue homeostasis are not well characterized. THO is an evolutionarily conserved, nuclear RNP complex that physically links nascent transcripts with the nuclear export apparatus. THO is essential for early mouse embryonic development, limiting characterization of the requirements for THO in adult tissues. To address this shortcoming, a mouse strain has been generated allowing inducible deletion of the Thoc1 gene which encodes an essential protein subunit of THO. Bone marrow reconstitution was used to generate mice in which Thoc1 deletion could be induced specifically in the hematopoietic system. We find that granulocyte macrophage progenitors have a cell autonomous requirement for Thoc1 to maintain cell growth and viability. Lymphoid lineages are not detectably affected by Thoc1 loss under the homeostatic conditions tested. Myeloid lineages may be more sensitive to Thoc1 loss due to their relatively high rate of proliferation and turnover.

  20. Expression of the Norrie disease gene (Ndp) in developing and adult mouse eye, ear, and brain.

    Science.gov (United States)

    Ye, Xin; Smallwood, Philip; Nathans, Jeremy

    2011-01-01

    The Norrie disease gene (Ndp) codes for a secreted protein, Norrin, that activates canonical Wnt signaling by binding to its receptor, Frizzled-4. This signaling system is required for normal vascular development in the retina and for vascular survival in the cochlea. In mammals, the pattern of Ndp expression beyond the retina is poorly defined due to the low abundance of Norrin mRNA and protein. Here, we characterize Ndp expression during mouse development by studying a knock-in mouse that carries the coding sequence of human placental alkaline phosphatase (AP) inserted at the Ndp locus (Ndp(AP)). In the CNS, Ndp(AP) expression is apparent by E10.5 and is dynamic and complex. The anatomically delimited regions of Ndp(AP) expression observed prenatally in the CNS are replaced postnatally by widespread expression in astrocytes in the forebrain and midbrain, Bergman glia in the cerebellum, and Müller glia in the retina. In the developing and adult cochlea, Ndp(AP) expression is closely associated with two densely vascularized regions, the stria vascularis and a capillary plexus between the organ of Corti and the spiral ganglion. These observations suggest the possibility that Norrin may have developmental and/or homeostatic functions beyond the retina and cochlea. Copyright © 2010 Elsevier B.V. All rights reserved.

  1. Meis1 Is Required for Adult Mouse Erythropoiesis, Megakaryopoiesis and Hematopoietic Stem Cell Expansion.

    Directory of Open Access Journals (Sweden)

    Michelle Erin Miller

    Full Text Available Meis1 is recognized as an important transcriptional regulator in hematopoietic development and is strongly implicated in the pathogenesis of leukemia, both as a Hox transcription factor co-factor and independently. Despite the emerging recognition of Meis1's importance in the context of both normal and leukemic hematopoiesis, there is not yet a full understanding of Meis1's functions and the relevant pathways and genes mediating its functions. Recently, several conditional mouse models for Meis1 have been established. These models highlight a critical role for Meis1 in adult mouse hematopoietic stem cells (HSCs and implicate reactive oxygen species (ROS as a mediator of Meis1 function in this compartment. There are, however, several reported differences between these studies in terms of downstream progenitor populations impacted and effectors of function. In this study, we describe further characterization of a conditional knockout model based on mice carrying a loxP-flanked exon 8 of Meis1 which we crossed onto the inducible Cre localization/expression strains, B6;129-Gt(ROSA26Sor(tm1(Cre/ERTNat/J or B6.Cg-Tg(Mx1-Cre1Cgn/J. Findings obtained from these two inducible Meis1 knockout models confirm and extend previous reports of the essential role of Meis1 in adult HSC maintenance and expansion and provide new evidence that highlights key roles of Meis1 in both megakaryopoiesis and erythropoiesis. Gene expression analyses point to a number of candidate genes involved in Meis1's role in hematopoiesis. Our data additionally support recent evidence of a role of Meis1 in ROS regulation.

  2. Phenotyping of nNOS neurons in the postnatal and adult female mouse hypothalamus.

    Science.gov (United States)

    Chachlaki, Konstantina; Malone, Samuel A; Qualls-Creekmore, Emily; Hrabovszky, Erik; Münzberg, Heike; Giacobini, Paolo; Ango, Fabrice; Prevot, Vincent

    2017-10-15

    Neurons expressing nitric oxide (NO) synthase (nNOS) and thus capable of synthesizing NO play major roles in many aspects of brain function. While the heterogeneity of nNOS-expressing neurons has been studied in various brain regions, their phenotype in the hypothalamus remains largely unknown. Here we examined the distribution of cells expressing nNOS in the postnatal and adult female mouse hypothalamus using immunohistochemistry. In both adults and neonates, nNOS was largely restricted to regions of the hypothalamus involved in the control of bodily functions, such as energy balance and reproduction. Labeled cells were found in the paraventricular, ventromedial, and dorsomedial nuclei as well as in the lateral area of the hypothalamus. Intriguingly, nNOS was seen only after the second week of life in the arcuate nucleus of the hypothalamus (ARH). The most dense and heavily labeled population of cells was found in the organum vasculosum laminae terminalis (OV) and the median preoptic nucleus (MEPO), where most of the somata of the neuroendocrine neurons releasing GnRH and controlling reproduction are located. A great proportion of nNOS-immunoreactive neurons in the OV/MEPO and ARH were seen to express estrogen receptor (ER) α. Notably, almost all ERα-immunoreactive cells of the OV/MEPO also expressed nNOS. Moreover, the use of EYFP Vglut2 , EYFP Vgat , and GFP Gad67 transgenic mouse lines revealed that, like GnRH neurons, most hypothalamic nNOS neurons have a glutamatergic phenotype, except for nNOS neurons of the ARH, which are GABAergic. Altogether, these observations are consistent with the proposed role of nNOS neurons in physiological processes. © 2017 Wiley Periodicals, Inc.

  3. 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.

  4. Establishment of a tamoxifen-inducible Cre-driver mouse strain for widespread and temporal genetic modification in adult mice.

    Science.gov (United States)

    Ichise, Hirotake; Hori, Akiko; Shiozawa, Seiji; Kondo, Saki; Kanegae, Yumi; Saito, Izumu; Ichise, Taeko; Yoshida, Nobuaki

    2016-07-29

    Temporal genetic modification of mice using the ligand-inducible Cre/loxP system is an important technique that allows the bypass of embryonic lethal phenotypes and access to adult phenotypes. In this study, we generated a tamoxifen-inducible Cre-driver mouse strain for the purpose of widespread and temporal Cre recombination. The new line, named CM32, expresses the GFPneo-fusion gene in a wide variety of tissues before FLP recombination and tamoxifen-inducible Cre after FLP recombination. Using FLP-recombined CM32 mice (CM32Δ mice) and Cre reporter mouse lines, we evaluated the efficiency of Cre recombination with and without tamoxifen administration to adult mice, and found tamoxifen-dependent induction of Cre recombination in a variety of adult tissues. In addition, we demonstrated that conditional activation of an oncogene could be achieved in adults using CM32Δ mice. CM32Δ;T26 mice, which harbored a Cre recombination-driven, SV40 large T antigen-expressing transgene, were viable and fertile. No overt phenotype was found in the mice up to 3 months after birth. Although they displayed pineoblastomas (pinealoblastomas) and/or thymic enlargement due to background Cre recombination by 6 months after birth, they developed epidermal hyperplasia when administered tamoxifen. Collectively, our results suggest that the CM32Δ transgenic mouse line can be applied to the assessment of adult phenotypes in mice with loxP-flanked transgenes.

  5. Wnt3 and Gata4 regulate axon regeneration in adult mouse DRG neurons.

    Science.gov (United States)

    Duan, Run-Shan; Liu, Pei-Pei; Xi, Feng; Wang, Wei-Hua; Tang, Gang-Bin; Wang, Rui-Ying; Saijilafu; Liu, Chang-Mei

    2018-05-05

    Neurons in the adult central nervous system (CNS) have a poor intrinsic axon growth potential after injury, but the underlying mechanisms are largely unknown. Wingless-related mouse mammary tumor virus integration site (WNT) family members regulate neural stem cell proliferation, axon tract and forebrain development in the nervous system. Here we report that Wnt3 is an important modulator of axon regeneration. Downregulation or overexpression of Wnt3 in adult dorsal root ganglion (DRG) neurons enhances or inhibits their axon regeneration ability respectively in vitro and in vivo. Especially, we show that Wnt3 modulates axon regeneration by repressing mRNA translation of the important transcription factor Gata4 via binding to the three prime untranslated region (3'UTR). Downregulation of Gata4 could restore the phenotype exhibited by Wnt3 downregulation in DRG neurons. Taken together, these data indicate that Wnt3 is a key intrinsic regulator of axon growth ability of the nervous system. Copyright © 2018 Elsevier Inc. All rights reserved.

  6. Dynamic Remodeling of Pericytes In Vivo Maintains Capillary Coverage in the Adult Mouse Brain

    Directory of Open Access Journals (Sweden)

    Andrée-Anne Berthiaume

    2018-01-01

    Full Text Available Summary: Direct contact and communication between pericytes and endothelial cells is critical for maintenance of cerebrovascular stability and blood-brain barrier function. Capillary pericytes have thin processes that reach hundreds of micrometers along the capillary bed. The processes of adjacent pericytes come in close proximity but do not overlap, yielding a cellular chain with discrete territories occupied by individual pericytes. Little is known about whether this pericyte chain is structurally dynamic in the adult brain. Using in vivo two-photon imaging in adult mouse cortex, we show that while pericyte somata were immobile, the tips of their processes underwent extensions and/or retractions over days. The selective ablation of single pericytes provoked exuberant extension of processes from neighboring pericytes to contact uncovered regions of the endothelium. Uncovered capillary regions had normal barrier function but were dilated until pericyte contact was regained. Pericyte structural plasticity may be critical for cerebrovascular health and warrants detailed investigation. : Pericyte-endothelial contact is important for many aspects of cerebrovascular health. Berthiaume et al. use longitudinal two-photon imaging to show that the processes of brain capillary pericytes are structurally plastic in vivo. Their processes can grow hundreds of micrometers to ensure contact with exposed endothelium following ablation of a single pericyte. Keywords: capillary, pericyte, endothelium, blood-brain barrier, blood flow, plasticity, two-photon imaging, Alzheimer’s disease, dementia, stroke

  7. Cell proliferation, movement and differentiation during maintenance of the adult mouse adrenal cortex.

    Directory of Open Access Journals (Sweden)

    Su-Ping Chang

    Full Text Available Appropriate maintenance and regeneration of adult endocrine organs is important in both normal physiology and disease. We investigated cell proliferation, movement and differentiation in the adult mouse adrenal cortex, using different 5-bromo-2'-deoxyuridine (BrdU labelling regimens and immunostaining for phenotypic steroidogenic cell markers. Pulse-labelling showed that cell division was largely confined to the outer cortex, with most cells moving inwards towards the medulla at around 13-20 µm per day, though a distinct labelled cell population remained in the outer 10% of the cortex. Pulse-chase-labelling coupled with phenotypic immunostaining showed that, unlike cells in the inner cortex, most BrdU-positive outer cortical cells did not express steroidogenic markers, while co-staining for BrdU and Ki67 revealed that some outer cortical BrdU-positive cells were induced to proliferate following acute adrenocorticotropic hormone (ACTH treatment. Extended pulse-chase-labelling identified cells in the outer cortex which retained BrdU label for up to 18-23 weeks. Together, these observations are consistent with the location of both slow-cycling stem/progenitor and transiently amplifying cell populations in the outer cortex. Understanding the relationships between these distinct adrenocortical cell populations will be crucial to clarify mechanisms underpinning adrenocortical maintenance and long-term adaptation to pathophysiological states.

  8. Expression of a truncated receptor protein tyrosine phosphatase kappa in the brain of an adult transgenic mouse

    DEFF Research Database (Denmark)

    Shen, P; Canoll, P D; Sap, J

    1999-01-01

    that goal, we have used this mouse model to map the distribution of the truncated RPTP-kappa/beta-geo fusion protein in the adult mouse brain using beta-galactosidase as a marker enzyme. Visualization of the beta-galactosidase activity revealed a non-random pattern of expression, and identified cells......-6596]. Nevertheless, since the transgene's expression is driven by the endogenous RPTP-kappa promoter, distribution of the truncated RPTP-kappa/beta-geo fusion protein should reflect the regional and cellular expression of wild-type RPTP-kappa, and thus may identify sites where RPTP-kappa is important. Towards...

  9. Rhythmic ganglion cell activity in bleached and blind adult mouse retinas.

    Science.gov (United States)

    Menzler, Jacob; Channappa, Lakshmi; Zeck, Guenther

    2014-01-01

    In retinitis pigmentosa--a degenerative disease which often leads to incurable blindness--the loss of photoreceptors deprives the retina from a continuous excitatory input, the so-called dark current. In rodent models of this disease this deprivation leads to oscillatory electrical activity in the remaining circuitry, which is reflected in the rhythmic spiking of retinal ganglion cells (RGCs). It remained unclear, however, if the rhythmic RGC activity is attributed to circuit alterations occurring during photoreceptor degeneration or if rhythmic activity is an intrinsic property of healthy retinal circuitry which is masked by the photoreceptor's dark current. Here we tested these hypotheses by inducing and analysing oscillatory activity in adult healthy (C57/Bl6) and blind mouse retinas (rd10 and rd1). Rhythmic RGC activity in healthy retinas was detected upon partial photoreceptor bleaching using an extracellular high-density multi-transistor-array. The mean fundamental spiking frequency in bleached retinas was 4.3 Hz; close to the RGC rhythm detected in blind rd10 mouse retinas (6.5 Hz). Crosscorrelation analysis of neighbouring wild-type and rd10 RGCs (separation distance rhythmic RGC spiking in these retinas is driven by a network of presynaptic neurons. The inhibition of glutamatergic ganglion cell input or the inhibition of gap junctional coupling abolished the rhythmic pattern. In rd10 and rd1 retinas the presynaptic network leads to local field potentials, whereas in bleached retinas additional pharmacological disinhibition is required to achieve detectable field potentials. Our results demonstrate that photoreceptor bleaching unmasks oscillatory activity in healthy retinas which shares many features with the functional phenotype detected in rd10 retinas. The quantitative physiological differences advance the understanding of the degeneration process and may guide future rescue strategies.

  10. Adult plasticity in the subcortical auditory pathway of the maternal mouse.

    Directory of Open Access Journals (Sweden)

    Jason A Miranda

    Full Text Available Subcortical auditory nuclei were traditionally viewed as non-plastic in adulthood so that acoustic information could be stably conveyed to higher auditory areas. Studies in a variety of species, including humans, now suggest that prolonged acoustic training can drive long-lasting brainstem plasticity. The neurobiological mechanisms for such changes are not well understood in natural behavioral contexts due to a relative dearth of in vivo animal models in which to study this. Here, we demonstrate in a mouse model that a natural life experience with increased demands on the auditory system - motherhood - is associated with improved temporal processing in the subcortical auditory pathway. We measured the auditory brainstem response to test whether mothers and pup-naïve virgin mice differed in temporal responses to both broadband and tone stimuli, including ultrasonic frequencies found in mouse pup vocalizations. Mothers had shorter latencies for early ABR peaks, indicating plasticity in the auditory nerve and the cochlear nucleus. Shorter interpeak latency between waves IV and V also suggest plasticity in the inferior colliculus. Hormone manipulations revealed that these cannot be explained solely by estrogen levels experienced during pregnancy and parturition in mothers. In contrast, we found that pup-care experience, independent of pregnancy and parturition, contributes to shortening auditory brainstem response latencies. These results suggest that acoustic experience in the maternal context imparts plasticity on early auditory processing that lasts beyond pup weaning. In addition to establishing an animal model for exploring adult auditory brainstem plasticity in a neuroethological context, our results have broader implications for models of perceptual, behavioral and neural changes that arise during maternity, where subcortical sensorineural plasticity has not previously been considered.

  11. Adult plasticity in the subcortical auditory pathway of the maternal mouse.

    Science.gov (United States)

    Miranda, Jason A; Shepard, Kathryn N; McClintock, Shannon K; Liu, Robert C

    2014-01-01

    Subcortical auditory nuclei were traditionally viewed as non-plastic in adulthood so that acoustic information could be stably conveyed to higher auditory areas. Studies in a variety of species, including humans, now suggest that prolonged acoustic training can drive long-lasting brainstem plasticity. The neurobiological mechanisms for such changes are not well understood in natural behavioral contexts due to a relative dearth of in vivo animal models in which to study this. Here, we demonstrate in a mouse model that a natural life experience with increased demands on the auditory system - motherhood - is associated with improved temporal processing in the subcortical auditory pathway. We measured the auditory brainstem response to test whether mothers and pup-naïve virgin mice differed in temporal responses to both broadband and tone stimuli, including ultrasonic frequencies found in mouse pup vocalizations. Mothers had shorter latencies for early ABR peaks, indicating plasticity in the auditory nerve and the cochlear nucleus. Shorter interpeak latency between waves IV and V also suggest plasticity in the inferior colliculus. Hormone manipulations revealed that these cannot be explained solely by estrogen levels experienced during pregnancy and parturition in mothers. In contrast, we found that pup-care experience, independent of pregnancy and parturition, contributes to shortening auditory brainstem response latencies. These results suggest that acoustic experience in the maternal context imparts plasticity on early auditory processing that lasts beyond pup weaning. In addition to establishing an animal model for exploring adult auditory brainstem plasticity in a neuroethological context, our results have broader implications for models of perceptual, behavioral and neural changes that arise during maternity, where subcortical sensorineural plasticity has not previously been considered.

  12. Transplantation of adult mouse iPS cell-derived photoreceptor precursors restores retinal structure and function in degenerative mice.

    Directory of Open Access Journals (Sweden)

    Budd A Tucker

    2011-04-01

    Full Text Available This study was designed to determine whether adult mouse induced pluripotent stem cells (iPSCs, could be used to produce retinal precursors and subsequently photoreceptor cells for retinal transplantation to restore retinal function in degenerative hosts. iPSCs were generated using adult dsRed mouse dermal fibroblasts via retroviral induction of the transcription factors Oct4, Sox2, KLF4 and c-Myc. As with normal mouse ES cells, adult dsRed iPSCs expressed the pluripotency genes SSEA1, Oct4, Sox2, KLF4, c-Myc and Nanog. Following transplantation into the eye of immune-compromised retinal degenerative mice these cells proceeded to form teratomas containing tissue comprising all three germ layers. At 33 days post-differentiation a large proportion of the cells expressed the retinal progenitor cell marker Pax6 and went on to express the photoreceptor markers, CRX, recoverin, and rhodopsin. When tested using calcium imaging these cells were shown to exhibit characteristics of normal retinal physiology, responding to delivery of neurotransmitters. Following subretinal transplantation into degenerative hosts differentiated iPSCs took up residence in the retinal outer nuclear layer and gave rise to increased electro retinal function as determined by ERG and functional anatomy. As such, adult fibroblast-derived iPSCs provide a viable source for the production of retinal precursors to be used for transplantation and treatment of retinal degenerative disease.

  13. Doublecortin (DCX is not essential for survival and differentiation of newborn neurons in the adult mouse dentate gyrus

    Directory of Open Access Journals (Sweden)

    Jagroop eDhaliwal

    2016-01-01

    Full Text Available In the adult brain, expression of the microtubule-associated protein Doublecortin (DCX is associated with neural progenitor cells (NPCs that give rise to new neurons in the dentate gyrus. Many studies quantify the number of DCX-expressing cells as a proxy for the level of adult neurogenesis, yet no study has determined the effect of removing DCX from adult hippocampal NPCs. Here, we use a retroviral and inducible mouse transgenic approach to either knockdown or knockout DCX from adult NPCs in the dentate gyrus and examine how this affects cell survival and neuronal maturation. Our results demonstrate that shRNA-mediated knockdown of DCX or Cre-mediated recombination in floxed DCX mice does not alter hippocampal neurogenesis and does not change the neuronal fate of the NPCs. Together these findings show that the survival and maturation of adult-generated hippocampal neurons does not require DCX.

  14. Expression of a truncated receptor protein tyrosine phosphatase kappa in the brain of an adult transgenic mouse

    DEFF Research Database (Denmark)

    Shen, P; Canoll, P D; Sap, J

    1999-01-01

    processes such as axonal growth and target recognition, as has been demonstrated for certain Drosophila RPTPs. The brain distribution of RPTP-kappa-expressing cells has not been determined, however. In a gene-trap mouse model with a beta-gal+neo (beta-geo) insertion in the endogenous RPTP-kappa gene......-6596]. Nevertheless, since the transgene's expression is driven by the endogenous RPTP-kappa promoter, distribution of the truncated RPTP-kappa/beta-geo fusion protein should reflect the regional and cellular expression of wild-type RPTP-kappa, and thus may identify sites where RPTP-kappa is important. Towards...... that goal, we have used this mouse model to map the distribution of the truncated RPTP-kappa/beta-geo fusion protein in the adult mouse brain using beta-galactosidase as a marker enzyme. Visualization of the beta-galactosidase activity revealed a non-random pattern of expression, and identified cells...

  15. The impact of maternal separation on adult mouse behaviour and on the total neuron number in the mouse hippocampus

    DEFF Research Database (Denmark)

    Fabricius, K.; Wörtwein, Gitta; Pakkenberg, B.

    2008-01-01

    , the number of errors made by the MS24 mice compared to controls and in total distance moved. The mice were subsequently sacrificed and the total number of neurons estimated in the hippocampus using the optical fractionator. We found a significant loss of neurons in the dentate gyrus in MS mice compared...... to controls. Apparently a single maternal separation can impact the number of neurons in mouse hippocampus either by a decrease of neurogenesis or as an increase in neuron apoptosis. This study is the first to assess the result of maternal separation combining behaviour and stereology Udgivelsesdato: 2008/2...

  16. Adeno-associated virus-mediated gene delivery into the scala media of the normal and deafened adult mouse ear.

    Science.gov (United States)

    Kilpatrick, L A; Li, Q; Yang, J; Goddard, J C; Fekete, D M; Lang, H

    2011-06-01

    Murine models are ideal for studying cochlear gene transfer, as many hearing loss-related mutations have been discovered and mapped within the mouse genome. However, because of the small size and delicate nature, the membranous labyrinth of the mouse is a challenging target for the delivery of viral vectors. To minimize injection trauma, we developed a procedure for the controlled release of adeno-associated viruses (AAVs) into the scala media of adult mice. This procedure poses minimal risk of injury to structures of the cochlea and middle ear, and allows for near-complete preservation of low and middle frequency hearing. In this study, transduction efficiency and cellular specificity of AAV vectors (serotypes 1, 2, 5, 6 and 8) were investigated in normal and drug-deafened ears. Using the cytomegalovirus promoter to drive gene expression, a variety of cell types were transduced successfully, including sensory hair cells and supporting cells, as well as cells in the auditory nerve and spiral ligament. Among all five serotypes, inner hair cells were the most effectively transduced cochlear cell type. All five serotypes of AAV vectors transduced cells of the auditory nerve, though serotype 8 was the most efficient vector for transduction. Our findings indicate that efficient AAV inoculation (via the scala media) can be performed in adult mouse ears, with hearing preservation a realistic goal. The procedure we describe may also have applications for intra-endolymphatic drug delivery in many mouse models of human deafness.

  17. Quantitative expression profile of distinct functional regions in the adult mouse brain.

    Directory of Open Access Journals (Sweden)

    Takeya Kasukawa

    Full Text Available The adult mammalian brain is composed of distinct regions with specialized roles including regulation of circadian clocks, feeding, sleep/awake, and seasonal rhythms. To find quantitative differences of expression among such various brain regions, we conducted the BrainStars (B* project, in which we profiled the genome-wide expression of ∼50 small brain regions, including sensory centers, and centers for motion, time, memory, fear, and feeding. To avoid confounds from temporal differences in gene expression, we sampled each region every 4 hours for 24 hours, and pooled the samples for DNA-microarray assays. Therefore, we focused on spatial differences in gene expression. We used informatics to identify candidate genes with expression changes showing high or low expression in specific regions. We also identified candidate genes with stable expression across brain regions that can be used as new internal control genes, and ligand-receptor interactions of neurohormones and neurotransmitters. Through these analyses, we found 8,159 multi-state genes, 2,212 regional marker gene candidates for 44 small brain regions, 915 internal control gene candidates, and 23,864 inferred ligand-receptor interactions. We also found that these sets include well-known genes as well as novel candidate genes that might be related to specific functions in brain regions. We used our findings to develop an integrated database (http://brainstars.org/ for exploring genome-wide expression in the adult mouse brain, and have made this database openly accessible. These new resources will help accelerate the functional analysis of the mammalian brain and the elucidation of its regulatory network systems.

  18. Hepatocyte heterogeneity in the metabolism of carbohydrates.

    Science.gov (United States)

    Jungermann, K; Thurman, R G

    1992-01-01

    the hepatocytes sense the glucagon/insulin gradients via the respective hormone receptors, it is not known how they sense different oxygen tensions; the O2 sensor may be an oxygen-binding heme protein. The zonal separation of glucose release and uptake appears to be important for the liver to operate as a 'glucostat'. Thus, zonation of carbohydrate metabolism develops gradually during the first weeks of life, in part before and in part with weaning, when (in rat and mouse) the fat- and protein-rich but carbohydrate-poor nutrition via milk is replaced by carbohydrate-rich food. Similarly, zonation of carbohydrate metabolism adapts to longer lasting alterations in the need of a 'glucostat', such as starvation, diabetes, portocaval anastomoses or partial hepatectomy.

  19. Repolarization of hepatocytes in culture.

    Science.gov (United States)

    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.

  20. Visualizing form and function in organotypic slices of the adult mouse parotid gland.

    Science.gov (United States)

    Warner, Jennifer D; Peters, Christian G; Saunders, Rudel; Won, Jong Hak; Betzenhauser, Matthew J; Gunning, William T; Yule, David I; Giovannucci, David R

    2008-09-01

    An organotypic slice preparation of the adult mouse parotid salivary gland amenable to a variety of optical assessments of fluid and protein secretion dynamics is described. The semi-intact preparation rendered without the use of enzymatic treatment permitted live-cell imaging and multiphoton analysis of cellular and supracellular signals. Toward this end we demonstrated that the parotid slice is a significant addition to the repertoire of tools available to investigators to probe exocrine structure and function since there is currently no cell culture system that fully recapitulates parotid acinar cell biology. Importantly, we show that a subpopulation of the acinar cells of parotid slices can be maintained in short-term culture and retain their morphology and function for up to 2 days. This in vitro model system is a significant step forward compared with enzymatically dispersed acini that rapidly lose their morphological and functional characteristics over several hours, and it was shown to be long enough for the expression and trafficking of exogenous protein following adenoviral infection. This system is compatible with a variety of genetic and physiological approaches used to study secretory function.

  1. Olfactory discrimination training up-regulates and reorganizes expression of microRNAs in adult mouse hippocampus

    OpenAIRE

    Smalheiser, Neil R; Lugli, Giovanni; Lenon, Angela L; Davis, John M; Torvik, Vetle I; Larson, John

    2010-01-01

    Adult male mice (strain C57Bl/6J) were trained to execute nose-poke responses for water reinforcement; then they were randomly assigned to either of two groups: olfactory discrimination training (exposed to two odours with reward contingent upon correctly responding to one odour) or pseudo-training (exposed to two odours with reward not contingent upon response). These were run in yoked fashion and killed when the discrimination-trained mouse reached a learning criterion of 70% correct respon...

  2. 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

  3. 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

  4. PAX6 MiniPromoters drive restricted expression from rAAV in the adult mouse retina

    Directory of Open Access Journals (Sweden)

    Jack W Hickmott

    2016-01-01

    Full Text Available Current gene therapies predominantly use small, strong, and readily available ubiquitous promoters. However, as the field matures, the availability of small, cell-specific promoters would be greatly beneficial. Here we design seven small promoters from the human paired box 6 (PAX6 gene and test them in the adult mouse retina using recombinant adeno-associated virus. We chose the retina due to previous successes in gene therapy for blindness, and the PAX6 gene since it is: well studied; known to be driven by discrete regulatory regions; expressed in therapeutically interesting retinal cell types; and mutated in the vision-loss disorder aniridia, which is in need of improved therapy. At the PAX6 locus, 31 regulatory regions were bioinformatically predicted, and nine regulatory regions were constructed into seven MiniPromoters. Driving Emerald GFP, these MiniPromoters were packaged into recombinant adeno-associated virus, and injected intravitreally into postnatal day 14 mice. Four MiniPromoters drove consistent retinal expression in the adult mouse, driving expression in combinations of cell-types that endogenously express Pax6: ganglion, amacrine, horizontal, and Müller glia. Two PAX6-MiniPromoters drive expression in three of the four cell types that express PAX6 in the adult mouse retina. Combined, they capture all four cell types, making them potential tools for research, and PAX6-gene therapy for aniridia.

  5. PAX6 MiniPromoters drive restricted expression from rAAV in the adult mouse retina.

    Science.gov (United States)

    Hickmott, Jack W; Chen, Chih-Yu; Arenillas, David J; Korecki, Andrea J; Lam, Siu Ling; Molday, Laurie L; Bonaguro, Russell J; Zhou, Michelle; Chou, Alice Y; Mathelier, Anthony; Boye, Sanford L; Hauswirth, William W; Molday, Robert S; Wasserman, Wyeth W; Simpson, Elizabeth M

    2016-01-01

    Current gene therapies predominantly use small, strong, and readily available ubiquitous promoters. However, as the field matures, the availability of small, cell-specific promoters would be greatly beneficial. Here we design seven small promoters from the human paired box 6 (PAX6) gene and test them in the adult mouse retina using recombinant adeno-associated virus. We chose the retina due to previous successes in gene therapy for blindness, and the PAX6 gene since it is: well studied; known to be driven by discrete regulatory regions; expressed in therapeutically interesting retinal cell types; and mutated in the vision-loss disorder aniridia, which is in need of improved therapy. At the PAX6 locus, 31 regulatory regions were bioinformatically predicted, and nine regulatory regions were constructed into seven MiniPromoters. Driving Emerald GFP, these MiniPromoters were packaged into recombinant adeno-associated virus, and injected intravitreally into postnatal day 14 mice. Four MiniPromoters drove consistent retinal expression in the adult mouse, driving expression in combinations of cell-types that endogenously express Pax6: ganglion, amacrine, horizontal, and Müller glia. Two PAX6-MiniPromoters drive expression in three of the four cell types that express PAX6 in the adult mouse retina. Combined, they capture all four cell types, making them potential tools for research, and PAX6-gene therapy for aniridia.

  6. MYC gene delivery to adult mouse utricles stimulates proliferation of postmitotic supporting cells in vitro.

    Science.gov (United States)

    Burns, Joseph C; Yoo, James J; Atala, Anthony; Jackson, John D

    2012-01-01

    The inner ears of adult humans and other mammals possess a limited capacity for regenerating sensory hair cells, which can lead to permanent auditory and vestibular deficits. During development and regeneration, undifferentiated supporting cells within inner ear sensory epithelia can self-renew and give rise to new hair cells; however, these otic progenitors become depleted postnatally. Therefore, reprogramming differentiated supporting cells into otic progenitors is a potential strategy for restoring regenerative potential to the ear. Transient expression of the induced pluripotency transcription factors, Oct3/4, Klf4, Sox2, and c-Myc reprograms fibroblasts into neural progenitors under neural-promoting culture conditions, so as a first step, we explored whether ectopic expression of these factors can reverse supporting cell quiescence in whole organ cultures of adult mouse utricles. Co-infection of utricles with adenoviral vectors separately encoding Oct3/4, Klf4, Sox2, and the degradation-resistant T58A mutant of c-Myc (c-MycT58A) triggered significant levels of supporting cell S-phase entry as assessed by continuous BrdU labeling. Of the four factors, c-MycT58A alone was both necessary and sufficient for the proliferative response. The number of BrdU-labeled cells plateaued between 5-7 days after infection, and then decreased ~60% by 3 weeks, as many cycling cells appeared to enter apoptosis. Switching to differentiation-promoting culture medium at 5 days after ectopic expression of c-MycT58A temporarily attenuated the loss of BrdU-labeled cells and accompanied a very modest but significant expansion of the sensory epithelium. A small number of the proliferating cells in these cultures labeled for the hair cell marker, myosin VIIA, suggesting they had begun differentiating towards a hair cell fate. The results indicate that ectopic expression of c-MycT58A in combination with methods for promoting cell survival and differentiation may restore regenerative

  7. MYC gene delivery to adult mouse utricles stimulates proliferation of postmitotic supporting cells in vitro.

    Directory of Open Access Journals (Sweden)

    Joseph C Burns

    Full Text Available The inner ears of adult humans and other mammals possess a limited capacity for regenerating sensory hair cells, which can lead to permanent auditory and vestibular deficits. During development and regeneration, undifferentiated supporting cells within inner ear sensory epithelia can self-renew and give rise to new hair cells; however, these otic progenitors become depleted postnatally. Therefore, reprogramming differentiated supporting cells into otic progenitors is a potential strategy for restoring regenerative potential to the ear. Transient expression of the induced pluripotency transcription factors, Oct3/4, Klf4, Sox2, and c-Myc reprograms fibroblasts into neural progenitors under neural-promoting culture conditions, so as a first step, we explored whether ectopic expression of these factors can reverse supporting cell quiescence in whole organ cultures of adult mouse utricles. Co-infection of utricles with adenoviral vectors separately encoding Oct3/4, Klf4, Sox2, and the degradation-resistant T58A mutant of c-Myc (c-MycT58A triggered significant levels of supporting cell S-phase entry as assessed by continuous BrdU labeling. Of the four factors, c-MycT58A alone was both necessary and sufficient for the proliferative response. The number of BrdU-labeled cells plateaued between 5-7 days after infection, and then decreased ~60% by 3 weeks, as many cycling cells appeared to enter apoptosis. Switching to differentiation-promoting culture medium at 5 days after ectopic expression of c-MycT58A temporarily attenuated the loss of BrdU-labeled cells and accompanied a very modest but significant expansion of the sensory epithelium. A small number of the proliferating cells in these cultures labeled for the hair cell marker, myosin VIIA, suggesting they had begun differentiating towards a hair cell fate. The results indicate that ectopic expression of c-MycT58A in combination with methods for promoting cell survival and differentiation may restore

  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. Effect of cyanotoxins on the hypothalamic-pituitary-gonadal axis in male adult mouse.

    Science.gov (United States)

    Xiong, Xiaolu; Zhong, Anyuan; Xu, Huajun

    2014-01-01

    Microcystins LR (MC-LR) are hepatotoxic cyanotoxins that have been shown to induce reproductive toxicity, and Hypothalamic-Pituitary-Gonadal Axis (HPG) is responsible for the control of reproductive functions. However, few studies have been performed to evaluate the effects of MC-LR on HPG axis. This study aimed to investigate the MC-LR-induced toxicity in the reproductive system of mouse and focus on the HPG axis. Adult male C57BL/6 mice were exposed to various concentrations of MC-LR (0, 3.75, 7.50, 15.00 and 30.00 µg/kg body weight per day) for 1 to 14 days, and it was found that exposure to different concentrations of MC-LR significantly disturbed sperm production in the mice testes in a dose- and time-dependent manner. To elucidate the associated possible mechanisms, the serum levels of testosterone, follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were assessed. Meanwhile, PCR assays were employed to detect alterations in a series of genes involved in HPG axis, such as FSH, LH, gonadotropin-releasing hormone (GnRH) and their complement receptors. Furthermore, the effect of MC-LR on the viability and testosterone production of Leydig cells were tested in vitro. MC-LR significantly impaired the spermatogenesis of mice possibly through the direct or indirect inhibition of GnRH synthesis at the hypothalamic level, which resulted in reduction of serum levels of LH that lead to suppression of testosterone production in the testis of mice. MC-LR may be a GnRH toxin that would disrupt the reproductive system of mice.

  10. Effect of cyanotoxins on the hypothalamic-pituitary-gonadal axis in male adult mouse.

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    Xiaolu Xiong

    Full Text Available Microcystins LR (MC-LR are hepatotoxic cyanotoxins that have been shown to induce reproductive toxicity, and Hypothalamic-Pituitary-Gonadal Axis (HPG is responsible for the control of reproductive functions. However, few studies have been performed to evaluate the effects of MC-LR on HPG axis. This study aimed to investigate the MC-LR-induced toxicity in the reproductive system of mouse and focus on the HPG axis.Adult male C57BL/6 mice were exposed to various concentrations of MC-LR (0, 3.75, 7.50, 15.00 and 30.00 µg/kg body weight per day for 1 to 14 days, and it was found that exposure to different concentrations of MC-LR significantly disturbed sperm production in the mice testes in a dose- and time-dependent manner. To elucidate the associated possible mechanisms, the serum levels of testosterone, follicle-stimulating hormone (FSH and luteinizing hormone (LH were assessed. Meanwhile, PCR assays were employed to detect alterations in a series of genes involved in HPG axis, such as FSH, LH, gonadotropin-releasing hormone (GnRH and their complement receptors. Furthermore, the effect of MC-LR on the viability and testosterone production of Leydig cells were tested in vitro.MC-LR significantly impaired the spermatogenesis of mice possibly through the direct or indirect inhibition of GnRH synthesis at the hypothalamic level, which resulted in reduction of serum levels of LH that lead to suppression of testosterone production in the testis of mice.MC-LR may be a GnRH toxin that would disrupt the reproductive system of mice.

  11. Uptake of ingested bovine lactoferrin and its accumulation in adult mouse tissues.

    Science.gov (United States)

    Fischer, Romy; Debbabi, Hajer; Blais, Anne; Dubarry, Michel; Rautureau, Michèle; Boyaka, Prosper N; Tome, Daniel

    2007-10-01

    Lactoferrin is a glycoprotein with antimicrobial and immunoregulatory properties, which is found in milk, other external secretions, and in the secondary granules of neutrophils. The present study examined the time course of uptake and the pattern of tissue accumulation of bovine lactoferrin (bLf) following intragastric intubation of a single dose to adult naïve mice or to mice daily fed bLf for 4 weeks. Following ingestion, bLf was transferred from the intestine into peripheral blood in a form with intact molecular weight (80 kDa) and localized within 10 to 20 min after oral administration in the liver, kidneys, gall bladder, spleen, and brain of both groups of mice. Immunoreactive bLf could also be detected in the luminal contents of the stomach, small intestine and colon 1 h after intragastric intubation. Interestingly, serum and tissue accumulation of bLf was approximately 50% lower in mice chronically fed this protein than in those given only the single oral dose. Furthermore, significant levels of bLf-specific IgA and IgG antibodies as well as bLf-containing IgA- and IgG immune complexes were detected in mice chronically fed bLf but not in those fed only once. Taken together, these results indicate that bLf resists major proteolytic degradation in the intestinal lumen and is readily absorbed in an antigenic form in blood and various mouse tissues. Chronic ingestion of lactoferrin reduces its uptake, probably through mechanisms such as immune exclusion, which minimize potential harmful reactions to food products.

  12. BAG3 regulates contractility and Ca(2+) homeostasis in adult mouse ventricular myocytes.

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    Feldman, Arthur M; Gordon, Jennifer; Wang, JuFang; Song, Jianliang; Zhang, Xue-Qian; Myers, Valerie D; Tilley, Douglas G; Gao, Erhe; Hoffman, Nicholas E; Tomar, Dhanendra; Madesh, Muniswamy; Rabinowitz, Joseph; Koch, Walter J; Su, Feifei; Khalili, Kamel; Cheung, Joseph Y

    2016-03-01

    Bcl2-associated athanogene 3 (BAG3) is a 575 amino acid anti-apoptotic protein that is constitutively expressed in the heart. BAG3 mutations, including mutations leading to loss of protein, are associated with familial cardiomyopathy. Furthermore, BAG3 levels have been found to be reduced in end-stage non-familial failing myocardium. In contrast to neonatal myocytes in which BAG3 is found in the cytoplasm and involved in protein quality control and apoptosis, in adult mouse left ventricular (LV) myocytes BAG3 co-localized with Na(+)-K(+)-ATPase and L-type Ca(2+) channels in the sarcolemma and t-tubules. BAG3 co-immunoprecipitated with β1-adrenergic receptor, L-type Ca(2+) channels and phospholemman. To simulate decreased BAG3 protein levels observed in human heart failure, we targeted BAG3 by shRNA (shBAG3) in adult LV myocytes. Reducing BAG3 by 55% resulted in reduced contraction and [Ca(2+)]i transient amplitudes in LV myocytes stimulated with isoproterenol. L-type Ca(2+) current (ICa) and sarcoplasmic reticulum (SR) Ca(2+) content but not Na(+)/Ca(2+) exchange current (INaCa) or SR Ca(2+) uptake were reduced in isoproterenol-treated shBAG3 myocytes. Forskolin or dibutyryl cAMP restored ICa amplitude in shBAG3 myocytes to that observed in WT myocytes, consistent with BAG3 having effects upstream and at the level of the receptor. Resting membrane potential and action potential amplitude were unaffected but APD50 and APD90 were prolonged in shBAG3 myocytes. Protein levels of Ca(2+) entry molecules and other important excitation-contraction proteins were unchanged in myocytes with lower BAG3. Our findings that BAG3 is localized at the sarcolemma and t-tubules while modulating myocyte contraction and action potential duration through specific interaction with the β1-adrenergic receptor and L-type Ca(2+) channel provide novel insight into the role of BAG3 in cardiomyopathies and increased arrhythmia risks in heart failure. Copyright © 2016 Elsevier Ltd. All rights

  13. BAG3 regulates contractility and Ca2+ homeostasis in adult mouse ventricular myocytes

    Science.gov (United States)

    Feldman, Arthur M.; Gordon, Jennifer; Wang, JuFang; Song, Jianliang; Zhang, Xue-Qian; Myers, Valerie D.; Tilley, Douglas G.; Gao, Erhe; Hoffman, Nicholas E.; Tomar, Dhanendra; Madesh, Muniswamy; Rabinowitz, Joseph; Koch, Walter J.; Su, Feifei; Khalili, Kamel; Cheung, Joseph Y.

    2016-01-01

    Bcl2-associated athanogene 3 (BAG3) is a 575 amino acid anti-apoptotic protein that is constitutively expressed in the heart. BAG3 mutations, including mutations leading to loss of protein, are associated with familial cardiomyopathy. Furthermore, BAG3 levels have been found to be reduced in end-stage non-familial failing myocardium. In contrast to neonatal myocytes in which BAG3 is found in the cytoplasm and involved in protein quality control and apoptosis, in adult mouse left ventricular (LV) myocytes BAG3 co-localized with Na+-K+-ATPase and L-type Ca2+ channels in the sarcolemma and t-tubules. BAG3 co-immunoprecipitated with β1-adrenergic receptor, L-type Ca2+ channels and phospholemman. To simulate decreased BAG3 protein levels observed in human heart failure, we targeted BAG3 by shRNA (shBAG3) in adult LV myocytes. Reducing BAG3 by 55% resulted in reduced contraction and [Ca2+]i transient amplitudes in LV myocytes stimulated with isoproterenol. L-type Ca2+ current (ICa) and sarcoplasmic reticulum (SR) Ca2+ content but not Na+/Ca2+ exchange current (INaCa) or SR Ca2+ uptake were reduced in isoproterenol-treated shBAG3 myocytes. Forskolin or dibutyrl cAMP restored ICa amplitude in shBAG3 myocytes to that observed in WT myocytes, consistent with BAG3 having effects upstream and at the level of the receptor. Resting membrane potential and action potential amplitude were unaffected but APD50 and APD90 were prolonged in shBAG3 myocytes. Protein levels of Ca2+ entry molecules and other important excitation-contraction proteins were unchanged in myocytes with lower BAG3. Our findings that BAG3 is localized at the sarcolemma and t-tubules while modulating myocyte contraction and action potential duration through specific interaction with the β1-adrenergic receptor and L-type Ca2+ channel provide novel insight into the role of BAG3 in cardiomyopathies and increased arrhythmia risks in heart failure. PMID:26796036

  14. Oligodendrocyte- and Neuron-Specific Nogo-A Restrict Dendritic Branching and Spine Density in the Adult Mouse Motor Cortex.

    Science.gov (United States)

    Zemmar, Ajmal; Chen, Chia-Chien; Weinmann, Oliver; Kast, Brigitt; Vajda, Flora; Bozeman, James; Isaad, Noel; Zuo, Yi; Schwab, Martin E

    2018-06-01

    Nogo-A has been well described as a myelin-associated inhibitor of neurite outgrowth and functional neuroregeneration after central nervous system (CNS) injury. Recently, a new role of Nogo-A has been identified as a negative regulator of synaptic plasticity in the uninjured adult CNS. Nogo-A is present in neurons and oligodendrocytes. However, it is yet unclear which of these two pools regulate synaptic plasticity. To address this question we used newly generated mouse lines in which Nogo-A is specifically knocked out in (1) oligodendrocytes (oligoNogo-A KO) or (2) neurons (neuroNogo-A KO). We show that both oligodendrocyte- and neuron-specific Nogo-A KO mice have enhanced dendritic branching and spine densities in layer 2/3 cortical pyramidal neurons. These effects are compartmentalized: neuronal Nogo-A affects proximal dendrites whereas oligodendrocytic Nogo-A affects distal regions. Finally, we used two-photon laser scanning microscopy to measure the spine turnover rate of adult mouse motor cortex layer 5 cells and find that both Nogo-A KO mouse lines show enhanced spine remodeling after 4 days. Our results suggest relevant control functions of glial as well as neuronal Nogo-A for synaptic plasticity and open new possibilities for more selective and targeted plasticity enhancing strategies.

  15. 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.

  16. Disruption of Ah Receptor Signaling during Mouse Development Leads to Abnormal Cardiac Structure and Function in the Adult.

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    Vinicius S Carreira

    Full Text Available The Developmental Origins of Health and Disease (DOHaD Theory proposes that the environment encountered during fetal life and infancy permanently shapes tissue physiology and homeostasis such that damage resulting from maternal stress, poor nutrition or exposure to environmental agents may be at the heart of adult onset disease. Interference with endogenous developmental functions of the aryl hydrocarbon receptor (AHR, either by gene ablation or by exposure in utero to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, a potent AHR ligand, causes structural, molecular and functional cardiac abnormalities and altered heart physiology in mouse embryos. To test if embryonic effects progress into an adult phenotype, we investigated whether Ahr ablation or TCDD exposure in utero resulted in cardiac abnormalities in adult mice long after removal of the agent. Ten-months old adult Ahr-/- and in utero TCDD-exposed Ahr+/+ mice showed sexually dimorphic abnormal cardiovascular phenotypes characterized by echocardiographic findings of hypertrophy, ventricular dilation and increased heart weight, resting heart rate and systolic and mean blood pressure, and decreased exercise tolerance. Underlying these effects, genes in signaling networks related to cardiac hypertrophy and mitochondrial function were differentially expressed. Cardiac dysfunction in mouse embryos resulting from AHR signaling disruption seems to progress into abnormal cardiac structure and function that predispose adults to cardiac disease, but while embryonic dysfunction is equally robust in males and females, the adult abnormalities are more prevalent in females, with the highest severity in Ahr-/- females. The findings reported here underscore the conclusion that AHR signaling in the developing heart is one potential target of environmental factors associated with cardiovascular disease.

  17. Thalamocortical Projection Neuron and Interneuron Numbers in the Visual Thalamic Nuclei of the Adult C57BL/6 Mouse.

    Science.gov (United States)

    Evangelio, Marian; García-Amado, María; Clascá, Francisco

    2018-01-01

    A key parameter to constrain predictive, bottom-up circuit models of a given brain domain is the number and position of the neuronal populations involved. These include not only the neurons whose bodies reside within the domain, but also the neurons in distant regions that innervate the domain. The mouse visual cortex receives its main subcortical input from the dorsal lateral geniculate nucleus (dLGN) and the lateral posterior (LP) complex of the thalamus. The latter consists of three different nuclei: lateral posterior lateral (LPL), lateral posterior medial rostral (LPMR), and lateral posterior medial caudal (LPMC), each exhibiting specific patterns of connections with the various visual cortical areas. Here, we have determined the number of thalamocortical projection neurons and interneurons in the LP complex and dLGN of the adult C57BL/6 male mouse. We combined Nissl staining and histochemical and immunolabeling methods for consistently delineating nuclei borders, and applied unbiased stereological cell counting methods. Thalamic interneurons were identified using GABA immunolabeling. The C57BL/6 dLGN contains ∼21,200 neurons, while LP complex contains ∼31,000 total neurons. The dLGN and LP are the only nuclei of the mouse dorsal thalamus containing substantial numbers GABA-immunoreactive interneurons. These interneurons, however, are scarcer than previously estimated; they are 5.6% of dLGN neurons and just 1.9% of the LP neurons. It can be thus inferred that the dLGN contains ∼20,000 and the LP complex ∼30,400 thalamocortical projection neurons (∼12,000 in LPL, 15,200 in LPMR, and 4,200 in LPMC). The present dataset is relevant for constraining models of mouse visual thalamocortical circuits, as well as for quantitative comparisons between genetically modified mouse strains, or across species.

  18. C/EBPalpha and C/EBPbeta are required for Sebocyte differentiation and stratified squamous differentiation in adult mouse skin.

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    John S House

    Full Text Available C/EBPalpha and C/EBPbeta are bZIP transcription factors that are highly expressed in the interfollicular epidermis and sebaceous glands of skin and yet germ line deletion of either family member alone has only mild or no effect on keratinocyte biology and their role in sebocyte biology has never been examined. To address possible functional redundancies and reveal functional roles of C/EBPalpha and C/EBPbeta in postnatal skin, mouse models were developed in which either family member could be acutely ablated alone or together in the epidermis and sebaceous glands of adult mice. Acute removal of either C/EBPalpha or C/EBPbeta alone in adult mouse skin revealed modest to no discernable changes in epidermis or sebaceous glands. In contrast, co-ablation of C/EBPalpha and C/EBPbeta in postnatal epidermis resulted in disruption of stratified squamous differentiation characterized by hyperproliferation of basal and suprabasal keratinocytes and a defective basal to spinous keratinocyte transition involving an expanded basal compartment and a diminished and delayed spinous compartment. Acute co-ablation of C/EBPalpha and C/EBPbeta in sebaceous glands resulted in severe morphological defects, and sebocyte differentiation was blocked as determined by lack of sebum production and reduced expression of stearoyl-CoA desaturase (SCD3 and melanocortin 5 receptor (MC5R, two markers of terminal sebocyte differentiation. Specialized sebocytes of Meibomian glands and preputial glands were also affected. Our results indicate that in adult mouse skin, C/EBPalpha and C/EBPbeta are critically involved in regulating sebocyte differentiation and epidermal homeostasis involving the basal to spinous keratinocyte transition and basal cell cycle withdrawal.

  19. 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

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

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    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.

  1. CRMP5 regulates generation and survival of newborn neurons in olfactory and hippocampal neurogenic areas of the adult mouse brain.

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    Alexandra Veyrac

    Full Text Available The Collapsin Response Mediator Proteins (CRMPS are highly expressed in the developing brain, and in adult brain areas that retain neurogenesis, ie: the olfactory bulb (OB and the dentate gyrus (DG. During brain development, CRMPs are essentially involved in signaling of axon guidance and neurite outgrowth, but their functions in the adult brain remain largely unknown. CRMP5 has been initially identified as the target of auto-antibodies involved in paraneoplasic neurological diseases and further implicated in a neurite outgrowth inhibition mediated by tubulin binding. Interestingly, CRMP5 is also highly expressed in adult brain neurogenic areas where its functions have not yet been elucidated. Here we observed in both neurogenic areas of the adult mouse brain that CRMP5 was present in proliferating and post-mitotic neuroblasts, while they migrate and differentiate into mature neurons. In CRMP5(-/- mice, the lack of CRMP5 resulted in a significant increase of proliferation and neurogenesis, but also in an excess of apoptotic death of granule cells in the OB and DG. These findings provide the first evidence that CRMP5 is involved in the generation and survival of newly generated neurons in areas of the adult brain with a high level of activity-dependent neuronal plasticity.

  2. Neuron-Enriched Gene Expression Patterns are Regionally Anti-Correlated with Oligodendrocyte-Enriched Patterns in the Adult Mouse and Human Brain.

    Science.gov (United States)

    Tan, Powell Patrick Cheng; French, Leon; Pavlidis, Paul

    2013-01-01

    An important goal in neuroscience is to understand gene expression patterns in the brain. The recent availability of comprehensive and detailed expression atlases for mouse and human creates opportunities to discover global patterns and perform cross-species comparisons. Recently we reported that the major source of variation in gene transcript expression in the adult normal mouse brain can be parsimoniously explained as reflecting regional variation in glia to neuron ratios, and is correlated with degree of connectivity and location in the brain along the anterior-posterior axis. Here we extend this investigation to two gene expression assays of adult normal human brains that consisted of over 300 brain region samples, and perform comparative analyses of brain-wide expression patterns to the mouse. We performed principal components analysis (PCA) on the regional gene expression of the adult human brain to identify the expression pattern that has the largest variance. As in the mouse, we observed that the first principal component is composed of two anti-correlated patterns enriched in oligodendrocyte and neuron markers respectively. However, we also observed interesting discordant patterns between the two species. For example, a few mouse neuron markers show expression patterns that are more correlated with the human oligodendrocyte-enriched pattern and vice-versa. In conclusion, our work provides insights into human brain function and evolution by probing global relationships between regional cell type marker expression patterns in the human and mouse brain.

  3. Hippo Signaling Influences HNF4A and FOXA2 Enhancer Switching during Hepatocyte Differentiation

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    Olivia Alder

    2014-10-01

    Full Text Available Summary: Cell fate acquisition is heavily influenced by direct interactions between master regulators and tissue-specific enhancers. However, it remains unclear how lineage-specifying transcription factors, which are often expressed in both progenitor and mature cell populations, influence cell differentiation. Using in vivo mouse liver development as a model, we identified thousands of enhancers that are bound by the master regulators HNF4A and FOXA2 in a differentiation-dependent manner, subject to chromatin remodeling, and associated with differentially expressed target genes. Enhancers exclusively occupied in the embryo were found to be responsive to developmentally regulated TEAD2 and coactivator YAP1. Our data suggest that Hippo signaling may affect hepatocyte differentiation by influencing HNF4A and FOXA2 interactions with temporal enhancers. In summary, transcription factor-enhancer interactions are not only tissue specific but also differentiation dependent, which is an important consideration for researchers studying cancer biology or mammalian development and/or using transformed cell lines. : It is unclear how key transcription factors are critical for both lineage specification during embryonic development and maintenance of a differentiated, adult phenotype. By profiling the enhancer occupancy of the key transcription factors HNF4A and FOXA2 during mouse liver development, Alder et al. have found that YAP1 can influence enhancer interactions and target gene expression levels. Enhancer switching enables HNF4A and FOXA2 to fulfill distinct roles during organ development.

  4. PPARg mRNA in the adult mouse hypothalamus: distribution and regulation in response to dietary challenges

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    Yang eLiu

    2015-09-01

    Full Text Available Peroxisome proliferator-activated receptor gamma (PPARg is a ligand-activated transcription factor that was originally identified as a regulator of peroxisome proliferation and adipocyte differentiation. Emerging evidence suggests that functional PPARg signaling also occurs within the hypothalamus. However, the exact distribution and identities of PPARg-expressing hypothalamic cells remains under debate. The present study systematically mapped PPARg mRNA expression in the adult mouse brain using in situ hybridization histochemistry. PPARg mRNA was found to be expressed at high levels outside the hypothalamus including the neocortex, the olfactory bulb, the organ of the vasculosum of the lamina terminalis, and the subfornical organ. Within the hypothalamus, PPARg was present at moderate levels in the suprachiasmatic nucleus and the ependymal of the 3rd ventricle. In all examined feeding-related hypothalamic nuclei, PPARg was expressed at very low levels that were close to the limit of detection. Using qPCR techniques, we demonstrated that PPARg mRNA expression was upregulated in the suprachiasmatic nucleus in response to fasting. Double in situ hybridization further demonstrated that PPARg was primarily expressed in neurons. Collectively, our observations provide a comprehensive map of PPARg distribution and regulation in the intact adult mouse hypothalamus.

  5. A Novel Procedure for Rapid Imaging of Adult Mouse Brains with MicroCT Using Iodine-Based Contrast.

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    Ryan Anderson

    Full Text Available High-resolution Magnetic Resonance Imaging (MRI has been the primary modality for obtaining 3D cross-sectional anatomical information in animals for soft tissue, particularly brain. However, costs associated with MRI can be considerably high for large phenotypic screens for gross differences in the structure of the brain due to pathology and/or experimental manipulations. MicroCT (mCT, especially benchtop mCT, is becoming a common laboratory equipment with throughput rates equal or faster than any form of high-resolution MRI at lower costs. Here we explore adapting previously developed contrast based mCT to image adult mouse brains in-situ. We show that 2% weight per volume (w/v iodine-potassium iodide solution can be successfully used to image adult mouse brains within 48 hours post-mortem when a structural support matrix is used. We demonstrate that hydrogel can be effectively used as a perfusant which limits the tissue shrinkage due to iodine.

  6. Anthocyanins protect against LPS-induced oxidative stress-mediated neuroinflammation and neurodegeneration in the adult mouse cortex.

    Science.gov (United States)

    Khan, Muhammad Sohail; Ali, Tahir; Kim, Min Woo; Jo, Myeung Hoon; Jo, Min Gi; Badshah, Haroon; Kim, Myeong Ok

    2016-11-01

    Several studies provide evidence that reactive oxygen species (ROS) are key mediators of various neurological disorders. Anthocyanins are polyphenolic compounds and are well known for their anti-oxidant and neuroprotective effects. In this study, we investigated the neuroprotective effects of anthocyanins (extracted from black soybean) against lipopolysaccharide (LPS)-induced ROS-mediated neuroinflammation and neurodegeneration in the adult mouse cortex. Intraperitoneal injection of LPS (250 μg/kg) for 7 days triggers elevated ROS and oxidative stress, which induces neuroinflammation and neurodegeneration in the adult mouse cortex. Treatment with 24 mg/kg/day of anthocyanins for 14 days in LPS-injected mice (7 days before and 7 days co-treated with LPS) attenuated elevated ROS and oxidative stress compared to mice that received LPS-injection alone. The immunoblotting results showed that anthocyanins reduced the level of the oxidative stress kinase phospho-c-Jun N-terminal Kinase 1 (p-JNK). The immunoblotting and morphological results showed that anthocyanins treatment significantly reduced LPS-induced-ROS-mediated neuroinflammation through inhibition of various inflammatory mediators, such as IL-1β, TNF-α and the transcription factor NF- k B. Anthocyanins treatment also reduced activated astrocytes and microglia in the cortex of LPS-injected mice, as indicated by reductions in GFAP and Iba-1, respectively. Anthocyanins also prevent overexpression of various apoptotic markers, i.e., Bax, cytosolic cytochrome C, cleaved caspase-3 and PARP-1. Immunohistochemical fluoro-jade B (FJB) and Nissl staining indicated that anthocyanins prevent LPS-induced neurodegeneration in the mouse cortex. Our results suggest that dietary flavonoids, such as anthocyanins, have antioxidant and neuroprotective activities that could be beneficial to various neurological disorders. Copyright © 2016 Elsevier Ltd. All rights reserved.

  7. Maternal western diet primes non-alcoholic fatty liver disease in adult mouse offspring

    NARCIS (Netherlands)

    Pruis, M. G. M.; Lendvai, A.; Bloks, V. W.; Zwier, M. V.; Baller, J. F. W.; de Bruin, A.; Groen, A. K.; Plosch, T.

    AimMetabolic programming via components of the maternal diet during gestation may play a role in the development of different aspects of the metabolic syndrome. Using a mouse model, we aimed to characterize the role of maternal western-type diet in the development of non-alcoholic fatty liver

  8. The Satellite Cell in Male and Female, Developing and Adult Mouse Muscle: Distinct Stem Cells for Growth and Regeneration

    Science.gov (United States)

    Neal, Alice; Boldrin, Luisa; Morgan, Jennifer Elizabeth

    2012-01-01

    Satellite cells are myogenic cells found between the basal lamina and the sarcolemma of the muscle fibre. Satellite cells are the source of new myofibres; as such, satellite cell transplantation holds promise as a treatment for muscular dystrophies. We have investigated age and sex differences between mouse satellite cells in vitro and assessed the importance of these factors as mediators of donor cell engraftment in an in vivo model of satellite cell transplantation. We found that satellite cell numbers are increased in growing compared to adult and in male compared to female adult mice. We saw no difference in the expression of the myogenic regulatory factors between male and female mice, but distinct profiles were observed according to developmental stage. We show that, in contrast to adult mice, the majority of satellite cells from two week old mice are proliferating to facilitate myofibre growth; however a small proportion of these cells are quiescent and not contributing to this growth programme. Despite observed changes in satellite cell populations, there is no difference in engraftment efficiency either between satellite cells derived from adult or pre-weaned donor mice, male or female donor cells, or between male and female host muscle environments. We suggest there exist two distinct satellite cell populations: one for muscle growth and maintenance and one for muscle regeneration. PMID:22662253

  9. 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.

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

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    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.

  11. 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.

  12. Metabolism of six CYP probe substrates in fetal hepatocytes

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    Abdul Naveed Shaik

    2016-06-01

    Full Text Available Cytochrome P-450 (CYP are the most common drug metabolizing enzymes and are abundantly expressed in liver apart from kidney, lungs, intestine, brain etc. Their expression levels change with physiological conditions and disease states. The expression of these CYPs is less in human foetus and neonates compared to adults, which results in lower clearance of xenobiotics in infants and neonates compared to adults. Hepatocytes are the cells which are largely used to study these CYPs. We have isolated hepatocytes from aborted foetus to study the metabolism of six probe substrates: phenacetin, diclofenac, S-mephenytoin, dextromethorphan, nifedipine and testosterone. The results obtained show the expression of various CYPs (CYP1A2, CYP2C19, CYP2C9, CYP2D6, and CYP3A4 in human foetus and their involvement in metabolism of CYP probe substrates.

  13. Distribution and densitometry mapping of L1-CAM Immunoreactivity in the adult mouse brain – light microscopic observation

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    Yamasaki Hironobu

    2003-04-01

    Full Text Available Abstract Background The importance of L1 expression in the matured brain is suggested by physiological and behavioral studies showing that L1 is related to hippocampal plasticity and fear conditioning. The distribution of L1 in mouse brain might provide a basis for understanding its role in the brain. Results We examined the overall distribution of L1 in the adult mouse brain by immunohistochemistry using two polyclonal antibodies against different epitopes for L1. Immunoreactive L1 was widely but unevenly distributed from the olfactory bulb to the upper cervical cord. The accumulation of immunoreactive L1 was greatest in a non-neuronal element of the major fibre bundles, i.e. the lateral olfactory tract, olfactory and temporal limb of the anterior commissure, corpus callosum, stria terminalis, globus pallidus, fornix, mammillothalamic tract, solitary tract, and spinal tract of the trigeminal nerve. High to highest levels of non-neuronal and neuronal L1 were found in the grey matter; i.e. the piriform and entorhinal cortices, hypothalamus, reticular part of the substantia nigra, periaqueductal grey, trigeminal spinal nucleus etc. High to moderate density of neuronal L1 was found in the olfactory bulb, layer V of the cerebral cortex, amygdala, pontine grey, superior colliculi, cerebellar cortex, solitary tract nucleus etc. Only low to lowest levels of neuronal L1 were found in the hippocampus, grey matter in the caudate-putamen, thalamus, cerebellar nuclei etc. Conclusion L1 is widely and unevenly distributed in the matured mouse brain, where immunoreactivity was present not only in neuronal elements; axons, synapses and cell soma, but also in non-neuronal elements.

  14. Gene expression of drug metabolizing enzymes in adult and aged mouse liver: A modulation by immobilization stress

    International Nuclear Information System (INIS)

    Mikhailova, O.N.; Gulyaeva, L.F.; Filipenko, M.L.

    2005-01-01

    The role of stress in the regulation of enzymatic systems involved in the biotransformation of xenobiotics, as well as endogenous substrates in the liver was investigated using single immobilization stress as a model. Adult (3 months of age) and aged (26 months) C3H/a male mice were used. Cytochrome P450 1A1 and 1A2 (CYP1A1 and CYP1A2), glutathione S-transferase M1 (GSTM1), aryl hydrocarbon receptor (AHR), aryl hydrocarbon receptor nuclear translocator (ARNT) and catechol-O-methyltransferase (COMT) mRNA levels in the mouse liver were measured by a semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) method. Excluding CYP1A1, experiments revealed significant differences in the expression of these genes between adult- and aged-control animals. The influence of stress on the expression of genes studied was shown to be higher in adult mice than in aged ones. Our results clearly demonstrate the lack of response or even the attenuation of gene expression in aged animals that may play an important role in age-related pathologies and diseases

  15. The Phospholipase D2 Knock Out Mouse Has Ectopic Purkinje Cells and Suffers from Early Adult-Onset Anosmia.

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    Matthieu M Vermeren

    Full Text Available Phospholipase D2 (PLD2 is an enzyme that produces phosphatidic acid (PA, a lipid messenger molecule involved in a number of cellular events including, through its membrane curvature properties, endocytosis. The PLD2 knock out (PLD2KO mouse has been previously reported to be protected from insult in a model of Alzheimer's disease. We have further analysed a PLD2KO mouse using mass spectrophotometry of its lipids and found significant differences in PA species throughout its brain. We have examined the expression pattern of PLD2 which allowed us to define which region of the brain to analyse for defect, notably PLD2 was not detected in glial-rich regions. The expression pattern lead us to specifically examine the mitral cells of olfactory bulbs, the Cornus Amonis (CA regions of the hippocampus and the Purkinje cells of the cerebellum. We find that the change to longer PA species correlates with subtle architectural defect in the cerebellum, exemplified by ectopic Purkinje cells and an adult-onset deficit of olfaction. These observations draw parallels to defects in the reelin heterozygote as well as the effect of high fat diet on olfaction.

  16. Taurine in drinking water recovers learning and memory in the adult APP/PS1 mouse model of Alzheimer's disease

    Science.gov (United States)

    Kim, Hye Yun; Kim, Hyunjin V.; Yoon, Jin H.; Kang, Bo Ram; Cho, Soo Min; Lee, Sejin; Kim, Ji Yoon; Kim, Joo Won; Cho, Yakdol; Woo, Jiwan; Kim, YoungSoo

    2014-01-01

    Alzheimer's disease (AD) is a lethal progressive neurological disorder affecting the memory. Recently, US Food and Drug Administration mitigated the standard for drug approval, allowing symptomatic drugs that only improve cognitive deficits to be allowed to accelerate on to clinical trials. Our study focuses on taurine, an endogenous amino acid found in high concentrations in humans. It has demonstrated neuroprotective properties against many forms of dementia. In this study, we assessed cognitively enhancing property of taurine in transgenic mouse model of AD. We orally administered taurine via drinking water to adult APP/PS1 transgenic mouse model for 6 weeks. Taurine treatment rescued cognitive deficits in APP/PS1 mice up to the age-matching wild-type mice in Y-maze and passive avoidance tests without modifying the behaviours of cognitively normal mice. In the cortex of APP/PS1 mice, taurine slightly decreased insoluble fraction of Aβ. While the exact mechanism of taurine in AD has not yet been ascertained, our results suggest that taurine can aid cognitive impairment and may inhibit Aβ-related damages. PMID:25502280

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

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    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.

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

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    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

  19. The morphological changes of adult mouse testes after 60Co Gamma-radiation

    International Nuclear Information System (INIS)

    Koruji, M.; Movahedin, M.; Gourabi, H.; Jabbary Arfaee, A.

    2008-01-01

    Cytotoxic therapy can lead to prolonged azoospermia or even sterility. In the present study, we investigated the morphological changes of mouse testes after γ-Radiation. Methods: After anesthetizing of NMRI mice, testes and their surrounding tissues were irradiated using a cobalt therapy machine. Four experimental groups were irradiated with fractionated doses of: 1.5+8, 1.5+12 and 1.5+16 Gy (with an interval of 24 h) and single dose of 14 Gy. Non-irradiated mice were considered as control group. Testes were removed 4, 6 and 8 weeks following irradiation, weighed and processed for light microscopic study. Diameters of seminiferous tubules and their lumens, epithelium thickness, percentage of different types of tubules and number of spermatogenic cell were measured. Moreover, sperm count motility and viability rates were evaluated in epididymis. Results: Number of normal tubules, epithelium thickness, tubules diameter and lumen diameter were significantly reduced with high dose irradiation in comparison with control testes. The recovery was observed after 8 weeks. Epididymal sperm count, motility and viability rates were significantly decreased in the irradiated mice comparing non-irradiated ones. These parameters were increased after 8 weeks. Conclusion: According to the results, irradiation can cause temporary azoospermia in mouse and this effect is reversible after 8 weeks

  20. Quiescent Oct4+ Neural Stem Cells (NSCs) Repopulate Ablated Glial Fibrillary Acidic Protein+ NSCs in the Adult Mouse Brain.

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    Reeve, Rachel L; Yammine, Samantha Z; Morshead, Cindi M; van der Kooy, Derek

    2017-09-01

    Adult primitive neural stem cells (pNSCs) are a rare population of glial fibrillary acidic protein (GFAP) - Oct4 + cells in the mouse forebrain subependymal zone bordering the lateral ventricles that give rise to clonal neurospheres in leukemia inhibitory factor in vitro. pNSC neurospheres can be passaged to self-renew or give rise to GFAP + NSCs that form neurospheres in epidermal growth factor and fibroblast growth factor 2, which we collectively refer to as definitive NSCs (dNSCs). Label retention experiments using doxycycline-inducible histone-2B (H2B)-green fluorescent protein (GFP) mice and several chase periods of up to 1 year quantified the adult pNSC cell cycle time as 3-5 months. We hypothesized that while pNSCs are not very proliferative at baseline, they may exist as a reserve pool of NSCs in case of injury. To test this function of pNSCs, we obtained conditional Oct4 knockout mice, Oct4 fl/fl ;Sox1 Cre (Oct4 CKO ), which do not yield adult pNSC-derived neurospheres. When we ablated the progeny of pNSCs, namely all GFAP + dNSCs, in these Oct4 CKO mice, we found that dNSCs did not recover as they do in wild-type mice, suggesting that pNSCs are necessary for dNSC repopulation. Returning to the H2B-GFP mice, we observed that the cytosine β-d-arabinofuranoside ablation of proliferating cells including dNSCs-induced quiescent pNSCs to proliferate and significantly dilute their H2B-GFP label. In conclusion, we demonstrate that pNSCs are the most quiescent stem cells in the adult brain reported to date and that their lineage position upstream of GFAP + dNSCs allows them to repopulate a depleted neural lineage. Stem Cells 2017;35:2071-2082. © 2017 AlphaMed Press.

  1. Hepatocyte Growth Factor-c-MET Signaling Mediates the Development of Nonsensory Structures of the Mammalian Cochlea and Hearing.

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    Shibata, Shumei; Miwa, Toru; Wu, Hsiao-Huei; Levitt, Pat; Ohyama, Takahiro

    2016-08-03

    The stria vascularis is a nonsensory structure that is essential for auditory hair cell function by maintaining potassium concentration of the scala media. During mouse embryonic development, a subpopulation of neural crest cell-derived melanocytes migrates and incorporates into a subregion of the cochlear epithelium, forming the intermediate cell layer of the stria vascularis. The relation of this developmental process to stria vascularis function is currently unknown. In characterizing the molecular differentiation of developing peripheral auditory structures, we discovered that hepatocyte growth factor (Hgf) is expressed in the future stria vascularis of the cochlear epithelium. Its receptor tyrosine kinase, c-Met, is expressed in the cochlear epithelium and melanocyte-derived intermediate cells in the stria vascularis. Genetic dissection of HGF signaling via c-MET reveals that the incorporation of the melanocytes into the future stria vascularis of the cochlear duct requires c-MET signaling. In addition, inactivation of either the ligand or receptor developmentally resulted in a profound hearing loss at young adult stages. These results suggest a novel connection between HGF signaling and deafness via melanocyte deficiencies. We found the roles of hepatocyte growth factor (HGF) signaling in stria vascularis development for the first time and that lack of HGF signaling in the inner ear leads to profound hearing loss in the mouse. Our findings reveal a novel mechanism that may underlie human deafness DFNB39 and DFNB97. Our findings reveal an additional example of context-dependent c-MET signaling diversity, required here for proper cellular invasion developmentally that is essential for specific aspects of auditory-related organogenesis. Copyright © 2016 the authors 0270-6474/16/368200-10$15.00/0.

  2. Hepatocyte Growth Factor–c-MET Signaling Mediates the Development of Nonsensory Structures of the Mammalian Cochlea and Hearing

    Science.gov (United States)

    Shibata, Shumei; Miwa, Toru; Wu, Hsiao-Huei; Levitt, Pat

    2016-01-01

    The stria vascularis is a nonsensory structure that is essential for auditory hair cell function by maintaining potassium concentration of the scala media. During mouse embryonic development, a subpopulation of neural crest cell-derived melanocytes migrates and incorporates into a subregion of the cochlear epithelium, forming the intermediate cell layer of the stria vascularis. The relation of this developmental process to stria vascularis function is currently unknown. In characterizing the molecular differentiation of developing peripheral auditory structures, we discovered that hepatocyte growth factor (Hgf) is expressed in the future stria vascularis of the cochlear epithelium. Its receptor tyrosine kinase, c-Met, is expressed in the cochlear epithelium and melanocyte-derived intermediate cells in the stria vascularis. Genetic dissection of HGF signaling via c-MET reveals that the incorporation of the melanocytes into the future stria vascularis of the cochlear duct requires c-MET signaling. In addition, inactivation of either the ligand or receptor developmentally resulted in a profound hearing loss at young adult stages. These results suggest a novel connection between HGF signaling and deafness via melanocyte deficiencies. SIGNIFICANCE STATEMENT We found the roles of hepatocyte growth factor (HGF) signaling in stria vascularis development for the first time and that lack of HGF signaling in the inner ear leads to profound hearing loss in the mouse. Our findings reveal a novel mechanism that may underlie human deafness DFNB39 and DFNB97. Our findings reveal an additional example of context-dependent c-MET signaling diversity, required here for proper cellular invasion developmentally that is essential for specific aspects of auditory-related organogenesis. PMID:27488639

  3. 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.

  4. CRISPR/Cas9 Engineering of Adult Mouse Liver Demonstrates That the Dnajb1-Prkaca Gene Fusion Is Sufficient to Induce Tumors Resembling Fibrolamellar Hepatocellular Carcinoma.

    Science.gov (United States)

    Engelholm, Lars H; Riaz, Anjum; Serra, Denise; Dagnæs-Hansen, Frederik; Johansen, Jens V; Santoni-Rugiu, Eric; Hansen, Steen H; Niola, Francesco; Frödin, Morten

    2017-12-01

    Fibrolamellar hepatocellular carcinoma (FL-HCC) is a primary liver cancer that predominantly affects children and young adults with no underlying liver disease. A somatic, 400 Kb deletion on chromosome 19 that fuses part of the DnaJ heat shock protein family (Hsp40) member B1 gene (DNAJB1) to the protein kinase cAMP-activated catalytic subunit alpha gene (PRKACA) has been repeatedly identified in patients with FL-HCC. However, the DNAJB1-PRKACA gene fusion has not been shown to induce liver tumorigenesis. We used the CRISPR/Cas9 technique to delete in mice the syntenic region on chromosome 8 to create a Dnajb1-Prkaca fusion and monitored the mice for liver tumor development. We delivered CRISPR/Cas9 vectors designed to juxtapose exon 1 of Dnajb1 with exon 2 of Prkaca to create the Dnajb1-Prkaca gene fusion associated with FL-HCC, or control Cas9 vector, via hydrodynamic tail vein injection to livers of 8-week-old female FVB/N mice. These mice did not have any other engineered genetic alterations and were not exposed to liver toxins or carcinogens. Liver tissues were collected 14 months after delivery; genomic DNA was analyzed by PCR to detect the Dnajb1-Prkaca fusion, and tissues were characterized by histology, immunohistochemistry, RNA sequencing, and whole-exome sequencing. Livers from 12 of the 15 mice given the vectors to induce the Dnajb1-Prkaca gene fusion, but none of the 11 mice given the control vector, developed neoplasms. The tumors contained the Dnajb1-Prkaca gene fusion and had histologic and cytologic features of human FL-HCCs: large polygonal cells with granular, eosinophilic, and mitochondria-rich cytoplasm, prominent nucleoli, and markers of hepatocytes and cholangiocytes. In comparing expression levels of genes between the mouse tumor and non-tumor liver cells, we identified changes similar to those detected in human FL-HCC, which included genes that affect cell cycle and mitosis regulation. Genomic analysis of mouse neoplasms induced by

  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

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    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. Expression of tryptophan 2,3-dioxygenase in mature granule cells of the adult mouse dentate gyrus

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    Ohira, Koji

    2010-09-01

    Full Text Available Abstract New granule cells are continuously generated in the dentate gyrus of the adult hippocampus. During granule cell maturation, the mechanisms that differentiate new cells not only describe the degree of cell differentiation, but also crucially regulate the progression of cell differentiation. Here, we describe a gene, tryptophan 2,3-dioxygenase (TDO, whose expression distinguishes stem cells from more differentiated cells among the granule cells of the adult mouse dentate gyrus. The use of markers for proliferation, neural progenitors, and immature and mature granule cells indicated that TDO was expressed in mature cells and in some immature cells. In mice heterozygous for the alpha-isoform of calcium/calmodulin-dependent protein kinase II, in which dentate gyrus granule cells fail to mature normally, TDO immunoreactivity was substantially downregulated in the dentate gyrus granule cells. Moreover, a 5-bromo-2'-deoxyuridine labeling experiment revealed that new neurons began to express TDO between 2 and 4 wk after the neurons were generated, when the axons and dendrites of the granule cells developed and synaptogenesis occurred. These findings indicate that TDO might be required at a late-stage of granule cell development, such as during axonal and dendritic growth, synaptogenesis and its maturation.

  7. Expression of the Argonaute protein PiwiL2 and piRNAs in adult mouse mesenchymal stem cells

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    Wu, Qiuling; Ma, Qi; Shehadeh, Lina A.; Wilson, Amber; Xia, Linghui; Yu, Hong [Department of Molecular and Cellular Pharmacology, Vascular Biology Institute, University of Miami Miller School of Medicine, Miami, FL 33136 (United States); Webster, Keith A., E-mail: kwebster@med.miami.edu [Department of Molecular and Cellular Pharmacology, Vascular Biology Institute, University of Miami Miller School of Medicine, Miami, FL 33136 (United States)

    2010-06-11

    Piwi (P-element-induced wimpy testis) first discovered in Drosophila is a member of the Argonaute family of micro-RNA binding proteins with essential roles in germ-cell development. The murine homologue of PiwiL2, also known as Mili is selectively expressed in the testes, and mice bearing targeted mutations of the PiwiL2 gene are male-sterile. PiwiL2 proteins are thought to protect the germ line genome by suppressing retrotransposons, stabilizing heterochromatin structure, and regulating target genes during meiosis and mitosis. Here, we report that PiwiL2 and associated piRNAs (piRs) may play similar roles in adult mouse mesenchymal stem cells. We found that PiwiL2 is expressed in the cytoplasm of metaphase mesenchymal stem cells from the bone marrow of adult and aged mice. Knockdown of PiwiL2 with a specific siRNA enhanced cell proliferation, significantly increased the number of cells in G1/S and G2/M cell cycle phases and was associated with increased expression of cell cycle genes CCND1, CDK8, microtubule regulation genes, and decreased expression of tumor suppressors Cables 1, LATS, and Cxxc4. The results suggest broader roles for Piwi in genome surveillance beyond the germ line and a possible role in regulating the cell cycle of mesenchymal stem cells.

  8. Expression of the Argonaute protein PiwiL2 and piRNAs in adult mouse mesenchymal stem cells

    International Nuclear Information System (INIS)

    Wu, Qiuling; Ma, Qi; Shehadeh, Lina A.; Wilson, Amber; Xia, Linghui; Yu, Hong; Webster, Keith A.

    2010-01-01

    Piwi (P-element-induced wimpy testis) first discovered in Drosophila is a member of the Argonaute family of micro-RNA binding proteins with essential roles in germ-cell development. The murine homologue of PiwiL2, also known as Mili is selectively expressed in the testes, and mice bearing targeted mutations of the PiwiL2 gene are male-sterile. PiwiL2 proteins are thought to protect the germ line genome by suppressing retrotransposons, stabilizing heterochromatin structure, and regulating target genes during meiosis and mitosis. Here, we report that PiwiL2 and associated piRNAs (piRs) may play similar roles in adult mouse mesenchymal stem cells. We found that PiwiL2 is expressed in the cytoplasm of metaphase mesenchymal stem cells from the bone marrow of adult and aged mice. Knockdown of PiwiL2 with a specific siRNA enhanced cell proliferation, significantly increased the number of cells in G1/S and G2/M cell cycle phases and was associated with increased expression of cell cycle genes CCND1, CDK8, microtubule regulation genes, and decreased expression of tumor suppressors Cables 1, LATS, and Cxxc4. The results suggest broader roles for Piwi in genome surveillance beyond the germ line and a possible role in regulating the cell cycle of mesenchymal stem cells.

  9. Genistein exposure inhibits growth and alters steroidogenesis in adult mouse antral follicles

    Energy Technology Data Exchange (ETDEWEB)

    Patel, Shreya, E-mail: Shreya.patel214@gmail.com [Department of Comparative Biosciences, University of Illinois, 2001 S. Lincoln Ave, Urbana, IL 61802 (United States); Peretz, Jackye, E-mail: Jackye.peretz@gmail.com [Department of Comparative Biosciences, University of Illinois, 2001 S. Lincoln Ave, Urbana, IL 61802 (United States); Pan, Yuan-Xiang, E-mail: yxpan@illinois.edu [Department of Food Science and Human Nutrition, University of Illinois, 905 S. Goodwin, Urbana, IL 61801 (United States); Helferich, William G., E-mail: helferic@illinois.edu [Department of Food Science and Human Nutrition, University of Illinois, 905 S. Goodwin, Urbana, IL 61801 (United States); Flaws, Jodi A., E-mail: jflaws@illinois.edu [Department of Comparative Biosciences, University of Illinois, 2001 S. Lincoln Ave, Urbana, IL 61802 (United States)

    2016-02-15

    Genistein is a naturally occurring isoflavone phytoestrogen commonly found in plant products such as soybeans, lentils, and chickpeas. Genistein, like other phytoestrogens, has the potential to mimic, enhance, or impair the estradiol biosynthesis pathway, thereby potentially altering ovarian follicle growth. Previous studies have inconsistently indicated that genistein exposure may alter granulosa cell proliferation and hormone production, but no studies have examined the effects of genistein on intact antral follicles. Thus, this study was designed to test the hypothesis that genistein exposure inhibits follicle growth and steroidogenesis in intact antral follicles. To test this hypothesis, antral follicles isolated from CD-1 mice were cultured with vehicle (dimethyl sulfoxide; DMSO) or genistein (6.0 and 36 μM) for 18–96 h. Every 24 h, follicle diameters were measured to assess growth. At the end of each culture period, the media were pooled to measure hormone levels, and the cultured follicles were collected to measure expression of cell cycle regulators and steroidogenic enzymes. The results indicate that genistein (36 μM) inhibits growth of mouse antral follicles. Additionally, genistein (6.0 and 36 μM) increases progesterone, testosterone, and dehydroepiandrosterone (DHEA) levels, but decreases estrone and estradiol levels. The results also indicate that genistein alters the expression of steroidogenic enzymes at 24, 72 and 96 h, and the expression of cell cycle regulators at 18 h. These data indicate that genistein exposure inhibits antral follicle growth by inhibiting the cell cycle, alters sex steroid hormone levels, and dysregulates steroidogenic enzymes in cultured mouse antral follicles. - Highlights: • Genistein exposure inhibits antral follicle growth. • Genistein exposure alters expression of cell cycle regulators. • Genistein exposure alters sex steroid hormones. • Genistein exposure alters expression of steroidogenic enzymes.

  10. Genistein exposure inhibits growth and alters steroidogenesis in adult mouse antral follicles

    International Nuclear Information System (INIS)

    Patel, Shreya; Peretz, Jackye; Pan, Yuan-Xiang; Helferich, William G.; Flaws, Jodi A.

    2016-01-01

    Genistein is a naturally occurring isoflavone phytoestrogen commonly found in plant products such as soybeans, lentils, and chickpeas. Genistein, like other phytoestrogens, has the potential to mimic, enhance, or impair the estradiol biosynthesis pathway, thereby potentially altering ovarian follicle growth. Previous studies have inconsistently indicated that genistein exposure may alter granulosa cell proliferation and hormone production, but no studies have examined the effects of genistein on intact antral follicles. Thus, this study was designed to test the hypothesis that genistein exposure inhibits follicle growth and steroidogenesis in intact antral follicles. To test this hypothesis, antral follicles isolated from CD-1 mice were cultured with vehicle (dimethyl sulfoxide; DMSO) or genistein (6.0 and 36 μM) for 18–96 h. Every 24 h, follicle diameters were measured to assess growth. At the end of each culture period, the media were pooled to measure hormone levels, and the cultured follicles were collected to measure expression of cell cycle regulators and steroidogenic enzymes. The results indicate that genistein (36 μM) inhibits growth of mouse antral follicles. Additionally, genistein (6.0 and 36 μM) increases progesterone, testosterone, and dehydroepiandrosterone (DHEA) levels, but decreases estrone and estradiol levels. The results also indicate that genistein alters the expression of steroidogenic enzymes at 24, 72 and 96 h, and the expression of cell cycle regulators at 18 h. These data indicate that genistein exposure inhibits antral follicle growth by inhibiting the cell cycle, alters sex steroid hormone levels, and dysregulates steroidogenic enzymes in cultured mouse antral follicles. - Highlights: • Genistein exposure inhibits antral follicle growth. • Genistein exposure alters expression of cell cycle regulators. • Genistein exposure alters sex steroid hormones. • Genistein exposure alters expression of steroidogenic enzymes.

  11. 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)

  12. Molecular properties of adult mouse gastric and intestinal epithelial progenitors in their niches

    DEFF Research Database (Denmark)

    Giannakis, Marios; Stappenbeck, Thaddeus S; Mills, Jason C

    2006-01-01

    pathways. Wnt/beta-catenin, phosphoinositide-3/Akt kinase, insulin-like growth factor-1, vascular endothelial growth factor, integrin, and gamma-aminobutyric acid receptor signaling cascades, plus glycerolipid, fatty acid, and amino acid metabolic pathways are among those prominently represented in adult...

  13. Myogenin regulates exercise capacity and skeletal muscle metabolism in the adult mouse.

    Directory of Open Access Journals (Sweden)

    Jesse M Flynn

    2010-10-01

    Full Text Available Although skeletal muscle metabolism is a well-studied physiological process, little is known about how it is regulated at the transcriptional level. The myogenic transcription factor myogenin is required for skeletal muscle development during embryonic and fetal life, but myogenin's role in adult skeletal muscle is unclear. We sought to determine myogenin's function in adult muscle metabolism. A Myog conditional allele and Cre-ER transgene were used to delete Myog in adult mice. Mice were analyzed for exercise capacity by involuntary treadmill running. To assess oxidative and glycolytic metabolism, we performed indirect calorimetry, monitored blood glucose and lactate levels, and performed histochemical analyses on muscle fibers. Surprisingly, we found that Myog-deleted mice performed significantly better than controls in high- and low-intensity treadmill running. This enhanced exercise capacity was due to more efficient oxidative metabolism during low- and high-intensity exercise and more efficient glycolytic metabolism during high-intensity exercise. Furthermore, Myog-deleted mice had an enhanced response to long-term voluntary exercise training on running wheels. We identified several candidate genes whose expression was altered in exercise-stressed muscle of mice lacking myogenin. The results suggest that myogenin plays a critical role as a high-level transcriptional regulator to control the energy balance between aerobic and anaerobic metabolism in adult skeletal muscle.

  14. 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

  15. 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

  16. The familial dysautonomia disease gene IKBKAP is required in the developing and adult mouse central nervous system

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    Marta Chaverra

    2017-05-01

    Full Text Available Hereditary sensory and autonomic neuropathies (HSANs are a genetically and clinically diverse group of disorders defined by peripheral nervous system (PNS dysfunction. HSAN type III, known as familial dysautonomia (FD, results from a single base mutation in the gene IKBKAP that encodes a scaffolding unit (ELP1 for a multi-subunit complex known as Elongator. Since mutations in other Elongator subunits (ELP2 to ELP4 are associated with central nervous system (CNS disorders, the goal of this study was to investigate a potential requirement for Ikbkap in the CNS of mice. The sensory and autonomic pathophysiology of FD is fatal, with the majority of patients dying by age 40. While signs and pathology of FD have been noted in the CNS, the clinical and research focus has been on the sensory and autonomic dysfunction, and no genetic model studies have investigated the requirement for Ikbkap in the CNS. Here, we report, using a novel mouse line in which Ikbkap is deleted solely in the nervous system, that not only is Ikbkap widely expressed in the embryonic and adult CNS, but its deletion perturbs both the development of cortical neurons and their survival in adulthood. Primary cilia in embryonic cortical apical progenitors and motile cilia in adult ependymal cells are reduced in number and disorganized. Furthermore, we report that, in the adult CNS, both autonomic and non-autonomic neuronal populations require Ikbkap for survival, including spinal motor and cortical neurons. In addition, the mice developed kyphoscoliosis, an FD hallmark, indicating its neuropathic etiology. Ultimately, these perturbations manifest in a developmental and progressive neurodegenerative condition that includes impairments in learning and memory. Collectively, these data reveal an essential function for Ikbkap that extends beyond the peripheral nervous system to CNS development and function. With the identification of discrete CNS cell types and structures that depend on

  17. Transplant of Hepatocytes, Undifferentiated Mesenchymal Stem Cells, and In Vitro Hepatocyte-Differentiated Mesenchymal Stem Cells in a Chronic Liver Failure Experimental Model: A Comparative Study.

    Science.gov (United States)

    El Baz, Hanan; Demerdash, Zeinab; Kamel, Manal; Atta, Shimaa; Salah, Faten; Hassan, Salwa; Hammam, Olfat; Khalil, Heba; Meshaal, Safa; Raafat, Inas

    2018-02-01

    Liver transplant is the cornerstone line of treatment for chronic liver diseases; however, the long list of complications and obstacles stand against this operation. Searching for new modalities for treatment of chronic liver illness is a must. In the present research, we aimed to compare the effects of transplant of undifferentiated human mesenchymal stem cells, in vitro differentiated mesenchymal stem cells, and adult hepatocytes in an experimental model of chronic liver failure. Undifferentiated human cord blood mesenchymal stem cells were isolated, pro-pagated, and characterized by morphology, gene expression analysis, and flow cytometry of surface markers and in vitro differentiated into hepatocyte-like cells. Rat hepatocytes were isolated by double perfusion technique. An animal model of chronic liver failure was developed, and undifferentiated human cord blood mesenchymal stem cells, in vitro hepato-genically differentiated mesenchymal stem cells, or freshly isolated rat hepatocytes were transplanted into a CCL4 cirrhotic experimental model. Animals were killed 3 months after transplant, and liver functions and histopathology were assessed. Compared with the cirrhotic control group, the 3 cell-treated groups showed improved alanine aminotransferase, aspartate aminotransferase, albumin, and bilirubin levels, with best results shown in the hepatocyte-treated group. Histopathologic examination of the treated groups showed improved fibrosis, with best results obtained in the undifferentiated mesenchymal stem cell-treated group. Both adult hepatocytes and cord blood mesenchymal stem cells proved to be promising candidates for cell-based therapy in liver regeneration on an experimental level. Improved liver function was evident in the hepatocyte-treated group, and fibrosis control was more evident in the undifferentiated mesenchymal stem cell-treated group.

  18. Olfactory discrimination training up-regulates and reorganizes expression of microRNAs in adult mouse hippocampus.

    Science.gov (United States)

    Smalheiser, Neil R; Lugli, Giovanni; Lenon, Angela L; Davis, John M; Torvik, Vetle I; Larson, John

    2010-02-26

    Adult male mice (strain C57Bl/6J) were trained to execute nose-poke responses for water reinforcement; then they were randomly assigned to either of two groups: olfactory discrimination training (exposed to two odours with reward contingent upon correctly responding to one odour) or pseudo-training (exposed to two odours with reward not contingent upon response). These were run in yoked fashion and killed when the discrimination-trained mouse reached a learning criterion of 70% correct responses in 20 trials, occurring after three sessions (a total of approximately 40 min of training). The hippocampus was dissected bilaterally from each mouse (N = 7 in each group) and profiling of 585 miRNAs (microRNAs) was carried out using multiplex RT-PCR (reverse transcription-PCR) plates. A significant global up-regulation of miRNA expression was observed in the discrimination training versus pseudo-training comparison; when tested individually, 29 miRNAs achieved significance at P = 0.05. miR-10a showed a 2.7-fold increase with training, and is predicted to target several learning-related mRNAs including BDNF (brain-derived neurotrophic factor), CAMK2b (calcium/calmodulin-dependent protein kinase IIβ), CREB1 (cAMP-response-element-binding protein 1) and ELAVL2 [ELAV (embryonic lethal, abnormal vision, Drosophila)-like; Hu B]. Analysis of miRNA pairwise correlations revealed the existence of several miRNA co-expression modules that were specific to the training group. These in vivo results indicate that significant, dynamic and co-ordinated changes in miRNA expression accompany early stages of learning.

  19. Olfactory Discrimination Training Up-Regulates and Reorganizes Expression of MicroRNAs in Adult Mouse Hippocampus

    Directory of Open Access Journals (Sweden)

    Neil R Smalheiser

    2010-01-01

    Full Text Available Adult male mice (strain C57Bl/6J were trained to execute nose-poke responses for water reinforcement; then they were randomly assigned to either of two groups: Olfactory discrimination training (exposed to two odours with reward contingent upon correctly responding to one odour or pseudo-training (exposed to two odours with reward not contingent upon response. These were run in yoked fashion and killed when the discrimination-trained mouse reached a learning criterion of 70% correct responses in 20 trials, occurring after three sessions (a total of ~40 min of training. The hippocampus was dissected bilaterally from each mouse (N=7 in each group and profiling of 585 miRNAs (microRNAs was carried out using multiplex RT–PCR (reverse transcription–PCR plates. A significant global up-regulation of miRNA expression was observed in the discrimination training versus pseudo-training comparison; when tested individually, 29 miRNAs achieved significance at P=0.05. miR-10a showed a 2.7-fold increase with training, and is predicted to target several learning-related mRNAs including BDNF (brain-derived neurotrophic factor, CAMK2b (calcium/calmodulin-dependent protein kinase IIβ, CREB1 (cAMP-response-element-binding protein 1 and ELAVL2 [ELAV (embryonic lethal, abnormal vision, Drosophila-like; Hu B]. Analysis of miRNA pairwise correlations revealed the existence of several miRNA co-expression modules that were specific to the training group. These in vivo results indicate that significant, dynamic and co-ordinated changes in miRNA expression accompany early stages of learning.

  20. Transmitter release in the neuromuscular synapse of the protein kinase C theta-deficient adult mouse.

    Science.gov (United States)

    Besalduch, Núria; Santafé, Manel M; Garcia, Neus; Gonzalez, Carmen; Tomás, Marta; Tomás, Josep; Lanuza, Maria A

    2011-04-01

    We studied structural and functional features of the neuromuscular junction in adult mice (P30) genetically deficient in the protein kinase C (PKC) theta isoform. Confocal and electron microscopy shows that there are no differences in the general morphology of the endplates between PKC theta-deficient and wild-type (WT) mice. Specifically, there is no difference in the density of the synaptic vesicles. However, the myelin sheath is not as thick in the intramuscular nerve fibers of the PKC theta-deficient mice. We found a significant reduction in the size of evoked endplate potentials and in the frequency of spontaneous, asynchronous, miniature endplate potentials in the PKC theta-deficient neuromuscular preparations in comparison with the WT, but the mean amplitude of the spontaneous potentials is not different. These changes indicate that PKC theta has a presynaptic role in the function of adult neuromuscular synapses. Copyright © 2010 Wiley-Liss, Inc.

  1. Adult neurogenesis and specific replacement of interneuron subtypes in the mouse main olfactory bulb

    Directory of Open Access Journals (Sweden)

    LaRocca Greg

    2007-11-01

    Full Text Available Abstract Background New neurons are generated in the adult brain from stem cells found in the subventricular zone (SVZ. These cells proliferate in the SVZ, generating neuroblasts which then migrate to the main olfactory bulb (MOB, ending their migration in the glomerular layer (GLL and the granule cell layer (GCL of the MOB. Neuronal populations in these layers undergo turnover throughout life, but whether all neuronal subtypes found in these areas are replaced and when neurons begin to express subtype-specific markers is not known. Results Here we use BrdU injections and immunohistochemistry against (calretinin, calbindin, N-copein, tyrosine hydroxylase and GABA and show that adult-generated neurons express markers of all major subtypes of neurons in the GLL and GCL. Moreover, the fractions of new neurons that express subtype-specific markers at 40 and 75 days post BrdU injection are very similar to the fractions of all neurons expressing these markers. We also show that many neurons in the glomerular layer do not express NeuN, but are readily and specifically labeled by the fluorescent nissl stain Neurotrace. Conclusion The expression of neuronal subtype-specific markers by new neurons in the GLL and GCL changes rapidly during the period from 14–40 days after BrdU injection before reaching adult levels. This period may represent a critical window for cell fate specification similar to that observed for neuronal survival.

  2. Mitochondrial DNA deletion mutations in adult mouse cardiac side population cells

    International Nuclear Information System (INIS)

    Lushaj, Entela B.; Lozonschi, Lucian; Barnes, Maria; Anstadt, Emily; Kohmoto, Takushi

    2012-01-01

    We investigated the presence and potential role of mitochondrial DNA (mtDNA) deletion mutations in adult cardiac stem cells. Cardiac side population (SP) cells were isolated from 12-week-old mice. Standard polymerase chain reaction (PCR) was used to screen for the presence of mtDNA deletion mutations in (a) freshly isolated SP cells and (b) SP cells cultured to passage 10. When present, the abundance of mtDNA deletion mutation was analyzed in single cell colonies. The effect of different levels of deletion mutations on SP cell growth and differentiation was determined. MtDNA deletion mutations were found in both freshly isolated and cultured cells from 12-week-old mice. While there was no significant difference in the number of single cell colonies with mtDNA deletion mutations from any of the groups mentioned above, the abundance of mtDNA deletion mutations was significantly higher in the cultured cells, as determined by quantitative PCR. Within a single clonal cell population, the detectable mtDNA deletion mutations were the same in all cells and unique when compared to deletions of other colonies. We also found that cells harboring high levels of mtDNA deletion mutations (i.e. where deleted mtDNA comprised more than 60% of total mtDNA) had slower proliferation rates and decreased differentiation capacities. Screening cultured adult stem cells for mtDNA deletion mutations as a routine assessment will benefit the biomedical application of adult stem cells.

  3. Geminin Participates in Differentiation Decisions of Adult Neural Stem Cells Transplanted in the Hemiparkinsonian Mouse Brain.

    Science.gov (United States)

    Taouki, Ioanna; Tasiudi, Eve; Lalioti, Maria-Eleni; Kyrousi, Christina; Skavatsou, Eleni; Kaplani, Konstantina; Lygerou, Zoi; Kouvelas, Elias D; Mitsacos, Adamantia; Giompres, Panagiotis; Taraviras, Stavros

    2017-08-15

    Neural stem cells have been considered as a source of stem cells that can be used for cell replacement therapies in neurodegenerative diseases, as they can be isolated and expanded in vitro and can be used for autologous grafting. However, due to low percentages of survival and varying patterns of differentiation, strategies that will enhance the efficacy of transplantation are under scrutiny. In this article, we have examined whether alterations in Geminin's expression, a protein that coordinates the balance between self-renewal and differentiation, can improve the properties of stem cells transplanted in 6-OHDA hemiparkinsonian mouse model. Our results indicate that, in the absence of Geminin, grafted cells differentiating into dopaminergic neurons were decreased, while an increased number of oligodendrocytes were detected. The number of proliferating multipotent cells was not modified by the absence of Geminin. These findings encourage research related to the impact of Geminin on transplantations for neurodegenerative disorders, as an important molecule in influencing differentiation decisions of the cells composing the graft.

  4. 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.

  5. Characterizing newly repopulated microglia in the adult mouse: impacts on animal behavior, cell morphology, and neuroinflammation.

    Directory of Open Access Journals (Sweden)

    Monica R P Elmore

    Full Text Available Microglia are the primary immune cell in the brain and are postulated to play important roles outside of immunity. Administration of the dual colony-stimulating factor 1 receptor (CSF1R/c-Kit kinase inhibitor, PLX3397, to adult mice results in the elimination of ~99% of microglia, which remain eliminated for as long as treatment continues. Upon removal of the inhibitor, microglia rapidly repopulate the entire adult brain, stemming from a central nervous system (CNS resident progenitor cell. Using this method of microglial elimination and repopulation, the role of microglia in both healthy and diseased states can be explored. Here, we examine the responsiveness of newly repopulated microglia to an inflammatory stimulus, as well as determine the impact of these cells on behavior, cognition, and neuroinflammation. Two month-old wild-type mice were placed on either control or PLX3397 diet for 21 d to eliminate microglia. PLX3397 diet was then removed in a subset of animals to allow microglia to repopulate and behavioral testing conducted beginning at 14 d repopulation. Finally, inflammatory profiling of the microglia-repopulated brain in response to lipopolysaccharide (LPS; 0.25 mg/kg or phosphate buffered saline (PBS was determined 21 d after inhibitor removal using quantitative real time polymerase chain reaction (RT-PCR, as well as detailed analyses of microglial morphologies. We find mice with repopulated microglia to perform similarly to controls by measures of behavior, cognition, and motor function. Compared to control/resident microglia, repopulated microglia had larger cell bodies and less complex branching in their processes, which resolved over time after inhibitor removal. Inflammatory profiling revealed that the mRNA gene expression of repopulated microglia was similar to normal resident microglia and that these new cells appear functional and responsive to LPS. Overall, these data demonstrate that newly repopulated microglia function

  6. Decreasing maternal myostatin programs adult offspring bone strength in a mouse model of osteogenesis imperfecta.

    Science.gov (United States)

    Oestreich, Arin K; Kamp, William M; McCray, Marcus G; Carleton, Stephanie M; Karasseva, Natalia; Lenz, Kristin L; Jeong, Youngjae; Daghlas, Salah A; Yao, Xiaomei; Wang, Yong; Pfeiffer, Ferris M; Ellersieck, Mark R; Schulz, Laura C; Phillips, Charlotte L

    2016-11-22

    During fetal development, the uterine environment can have effects on offspring bone architecture and integrity that persist into adulthood; however, the biochemical and molecular mechanisms remain unknown. Myostatin is a negative regulator of muscle mass. Parental myostatin deficiency (Mstn tm1Sjl/+ ) increases muscle mass in wild-type offspring, suggesting an intrauterine programming effect. Here, we hypothesized that Mstn tm1Sjl/+ dams would also confer increased bone strength. In wild-type offspring, maternal myostatin deficiency altered fetal growth and calvarial collagen content of newborn mice and conferred a lasting impact on bone geometry and biomechanical integrity of offspring at 4 mo of age, the age of peak bone mass. Second, we sought to apply maternal myostatin deficiency to a mouse model with osteogenesis imperfecta (Col1a2 oim ), a heritable connective tissue disorder caused by abnormalities in the structure and/or synthesis of type I collagen. Femora of male Col1a2 oim/+ offspring from natural mating of Mstn tm1Sjl/+ dams to Col1a2 oim/+ sires had a 15% increase in torsional ultimate strength, a 29% increase in tensile strength, and a 24% increase in energy to failure compared with age, sex, and genotype-matched offspring from natural mating of Col1a2 oim/+ dams to Col1a2 oim/+ sires. Finally, increased bone biomechanical strength of Col1a2 oim/+ offspring that had been transferred into Mstn tm1Sjl/+ dams as blastocysts demonstrated that the effects of maternal myostatin deficiency were conferred by the postimplantation environment. Thus, targeting the gestational environment, and specifically prenatal myostatin pathways, provides a potential therapeutic window and an approach for treating osteogenesis imperfecta.

  7. Morphological analysis of activity-reduced adult-born neurons in the mouse olfactory bulb

    Directory of Open Access Journals (Sweden)

    Jeffrey E Dahlen

    2011-05-01

    Full Text Available Adult born neurons are added to the olfactory bulb (OB throughout life in rodents. While many factors have been identified as regulating the survival and integration of adult-born neurons (ABNs into existing circuitry, the understanding of how these factors affect ABN morphology and connectivity is limited. Here we compare how cell intrinsic (siRNA knock down of voltage gated sodium channels NaV1.1-1.3 and circuit level (naris occlusion reductions in activity affect ABN morphology during integration into the OB. We found that both manipulations reduce the number of dendritic spines (and thus likely the number of reciprocal synaptic connections formed with the surrounding circuitry and inhibited dendritic ramification of ABNs. Further, we identified regions of ABN apical dendrites where the largest and most significant decreases occur following siRNA knock down or naris occlusion. In siRNA knock down cells, reduction of spines is observed in proximal regions of the apical dendrite. This suggests that distal regions of the dendrite may remain active independent of NaV1.1-1.3 channel expression, perhaps facilitated by activation of T-type calcium channels and NMDA receptors. By contrast, circuit level reduction of activity by naris occlusion resulted in a global depression of spine number. Together, these results indicate that ABNs retain the ability to develop their typical overall morphological features regardless of experienced activity, and activity modulates the number and location of formed connections.

  8. Evidence of functional duplicity of Nestin expression in the adult mouse midbrain

    Directory of Open Access Journals (Sweden)

    Parisa Farzanehfar

    2017-03-01

    Full Text Available Whether or not neurogenesis occurs in the adult substantia nigra pars compacta (SNc is an important question relevant for developing better treatments for the motor symptoms of Parkinson's disease (PD. Although controversial, it is generally believed that dividing cells here remain undifferentiated or differentiate into glia, not neurons. However, there is a suggestion that Nestin-expressing neural precursor cells (NPCs in the adult SNc have a propensity to differentiate into neurons, which we sought to confirm in the present study. Adult (>8-weeks old transgenic NesCreERT2/GtROSA or NesCreERT2/R26eYFP mice were used to permanently label Nestin-expressing cells and their progeny with β-galactosidase (β-gal or enhanced yellow fluorescent protein (eYFP, respectively. Most β-gal+ or eYFP+ cells were found in the ependymal lining of the midbrain aqueduct (Aq and in the midline ventral to Aq. Smaller but significant numbers were in the periaqueductal gray (PAG, the ventral tegmental area (VTA, and in SNc. Low-level basal proliferation was evidenced by a modest increase in number of β-gal+ or eYFP+ cells over time, fewer β-gal+ or eYFP+ cells when mice were administered the anti-mitotic agent Cytarabine, and incorporation of the proliferation marker bromodeoxyuridine (BrdU in a very small number of β-gal+ cells. No evidence of migration was found, including no immunoreactivity against the migration markers doublecortin (DCX or polysialic acid neural cell adhesion molecule (PSA-NCAM, and no dispersal of β-gal+ or eYFP+ cells through the midbrain parenchyma over time. However, β-gal+ or eYFP+ cells did increase in size and express higher levels of mature neuronal genes over time, indicating growth and neuronal differentiation. In mice whose SNc dopamine neurons had been depleted with 6-hydroxy-dopamine, a model of PD, there were ~2-fold more β-gal+ cells in SNc specifically, although the proportion that were also NeuN+ was not affected

  9. Hericium erinaceus Extract Reduces Anxiety and Depressive Behaviors by Promoting Hippocampal Neurogenesis in the Adult Mouse Brain.

    Science.gov (United States)

    Ryu, Sun; Kim, Hyoun Geun; Kim, Joo Youn; Kim, Seong Yun; Cho, Kyung-Ok

    2018-02-01

    Versatile biological activities of Hericium erinaceus (HE) have been reported in many brain diseases. However, roles of HE in major psychiatric disorders such as depression and anxiety remain to be investigated. Therefore, we evaluated whether HE could reduce anxiety and depressive behaviors in the adult mouse and its underlying mechanisms. Male C57BL/6 mice were administered HE (20 or 60 mg/kg, p.o.) or saline once a day for 4 weeks. Open field and tail suspension tests were performed 30 min after the last administration of HE, followed by forced swim test 2 days later. We found that chronic administration of HE showed anxiolytic and antidepressant-like effects. To elucidate possible mechanisms, proliferative activity of the hippocampal progenitor cells was assessed by immunohistochemistry of proliferating cell nuclear antigen (PCNA) and Ki67. Moreover, to evaluate neuronal survival in the dentate gyrus, 5-bromo-2'-deoxyuridine (BrdU) (120 mg/kg, i.p.) was given at the first day of HE administration, followed by isolation of the brains 4 weeks later. HE (60 mg/kg) increased the number of PCNA- and Ki67-positive cells in the subgranular zone of the hippocampus, indicating increased proliferation of hippocampal progenitors. In addition, BrdU- and BrdU/NeuN-positive cells in the dentate gyrus were significantly increased when treated with HE (60 mg/kg) compared with the saline-treated group, demonstrating enhanced neurogenesis by HE treatment. Taken together, the results indicate that chronic HE administration can exert anxiolytic and antidepressant-like effects, possibly by enhancing adult hippocampal neurogenesis.

  10. Action of plasma and liver extract from adult mice on the mitotic activity of young mouse liver.

    Science.gov (United States)

    García, A L; Inda, A M; Echave Llanos, J M

    1991-06-01

    Inbred C3HS male mice, standardized for periodicity analysis were used. A hundred and seventy 25 +/- 2 days old mice were injected at 16:00 hs with saline, plasma or liver extract from 27 mice 90 days old. Controls were made at 08/16, 12/20, 16/24, 08/40, 12/44, 16/48, 08/64, 12/68 and 16/72 (time of day/time post-injection). The mitotic activity of the hepatocytes and litoral cells were determined. The injection of small doses of extract and plasma inhibits the mitotic activity of hepatocytes during the first and second following days. A compensatory wave appears in the third day. The extract inhibits the mitotic activity of litoral cells in the first day of control only, whereas the plasma inhibits this variable in the second and third day.

  11. Induced Neural Stem Cells Achieve Long-Term Survival and Functional Integration in the Adult Mouse Brain

    Directory of Open Access Journals (Sweden)

    Kathrin Hemmer

    2014-09-01

    Full Text Available Differentiated cells can be converted directly into multipotent neural stem cells (i.e., induced neural stem cells [iNSCs]. iNSCs offer an attractive alternative to induced pluripotent stem cell (iPSC technology with regard to regenerative therapies. Here, we show an in vivo long-term analysis of transplanted iNSCs in the adult mouse brain. iNSCs showed sound in vivo long-term survival rates without graft overgrowths. The cells displayed a neural multilineage potential with a clear bias toward astrocytes and a permanent downregulation of progenitor and cell-cycle markers, indicating that iNSCs are not predisposed to tumor formation. Furthermore, the formation of synaptic connections as well as neuronal and glial electrophysiological properties demonstrated that differentiated iNSCs migrated, functionally integrated, and interacted with the existing neuronal circuitry. We conclude that iNSC long-term transplantation is a safe procedure; moreover, it might represent an interesting tool for future personalized regenerative applications.

  12. EMMPRIN (basigin/CD147) expression is not correlated with MMP activity during adult mouse mammary gland development.

    Science.gov (United States)

    Szymanowska, Malgorzata; Hendry, Kay A K; Robinson, Claire; Kolb, Andreas F

    2009-01-01

    Extracellular matrix metalloproteinase inducer (EMMPRIN/basigin/CD147) is a cell surface protein, which has been associated with the induction of matrix metalloproteinase (MMP) genes during cancer metastasis. EMMPRIN plays a role in a variety of physiological processes as is evident by the diverse deficiencies detectable in EMMPRIN knockout mice. We have analysed the role of EMMPRIN in the induction of MMP genes during mammary gland differentiation and involution. Co-transfection studies showed that EMMPRIN has diverse effects on MMP promoter activity in different mammary and non-mammary cell lines. Expression of EMMPRIN mRNA is enhanced markedly by insulin in a mammary gland cell line but appears to have no direct effect on MMP gene expression in these cells. Microarray analysis and quantitative PCR show that EMMPRIN is expressed throughout mammary gland differentiation in the mouse. Its expression decreases during early pregnancy and briefly after induction of mammary gland involution by litter removal. Immunohistochemical analysis shows that EMMPRIN expression is limited to the stromal compartment during pregnancy, whereas it is strongly expressed in the epithelium during lactation. In summary the data argue against a causal role for EMMPRIN for the induction of MMP gene expression during adult mammary gland development. These data therefore support a physiological role for EMMPRIN other than MMP induction in mammary gland biology. 2008 Wiley-Liss, Inc.

  13. Induced neural stem cells achieve long-term survival and functional integration in the adult mouse brain.

    Science.gov (United States)

    Hemmer, Kathrin; Zhang, Mingyue; van Wüllen, Thea; Sakalem, Marna; Tapia, Natalia; Baumuratov, Aidos; Kaltschmidt, Christian; Kaltschmidt, Barbara; Schöler, Hans R; Zhang, Weiqi; Schwamborn, Jens C

    2014-09-09

    Differentiated cells can be converted directly into multipotent neural stem cells (i.e., induced neural stem cells [iNSCs]). iNSCs offer an attractive alternative to induced pluripotent stem cell (iPSC) technology with regard to regenerative therapies. Here, we show an in vivo long-term analysis of transplanted iNSCs in the adult mouse brain. iNSCs showed sound in vivo long-term survival rates without graft overgrowths. The cells displayed a neural multilineage potential with a clear bias toward astrocytes and a permanent downregulation of progenitor and cell-cycle markers, indicating that iNSCs are not predisposed to tumor formation. Furthermore, the formation of synaptic connections as well as neuronal and glial electrophysiological properties demonstrated that differentiated iNSCs migrated, functionally integrated, and interacted with the existing neuronal circuitry. We conclude that iNSC long-term transplantation is a safe procedure; moreover, it might represent an interesting tool for future personalized regenerative applications. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  14. Distribution of ELOVL4 in the Developing and Adult Mouse Brain

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    David M. Sherry

    2017-05-01

    Full Text Available ELOngation of Very Long chain fatty acids (ELOVL-4 is essential for the synthesis of very long chain-fatty acids (fatty acids with chain lengths ≥ 28 carbons. The functions of ELOVL4 and its very long-chain fatty acid products are poorly understood at present. However, mutations in ELOVL4 cause neurodevelopmental or neurodegenerative diseases that vary according to the mutation and inheritance pattern. Heterozygous inheritance of different ELOVL4 mutations causes Stargardt-like Macular Dystrophy or Spinocerebellar Ataxia type 34. Homozygous inheritance of ELOVL4 mutations causes more severe disease characterized by seizures, intellectual disability, ichthyosis, and premature death. To better understand ELOVL4 and very long chain fatty acid function in the brain, we examined ELOVL4 expression in the mouse brain between embryonic day 18 and postnatal day 60 by immunolabeling using ELOVL4 and other marker antibodies. ELOVL4 was widely expressed in a region- and cell type-specific manner, and was restricted to cell bodies, consistent with its known localization to endoplasmic reticulum. ELOVL4 labeling was most prominent in gray matter, although labeling also was present in some cells located in white matter. ELOVL4 was widely expressed in the developing brain by embryonic day 18 and was especially pronounced in regions underlying the lateral ventricles and other neurogenic regions. The basal ganglia in particular showed intense ELOVL4 labeling at this stage. In the postnatal brain, cerebral cortex, hippocampus, cerebellum, thalamus, hypothalamus, midbrain, pons, and medulla all showed prominent ELOVL4 labeling, although ELOVL4 distribution was not uniform across all cells or subnuclei within these regions. In contrast, the basal ganglia showed little ELOVL4 labeling in the postnatal brain. Double labeling studies showed that ELOVL4 was primarily expressed by neurons, although presumptive oligodendrocytes located in white matter tracts also showed

  15. PPARs Expression in Adult Mouse Neural Stem Cells: Modulation of PPARs during Astroglial Differentiaton of NSC

    Directory of Open Access Journals (Sweden)

    A. Cimini

    2007-01-01

    Full Text Available PPAR isotypes are involved in the regulation of cell proliferation, death, and differentiation, with different roles and mechanisms depending on the specific isotype and ligand and on the differentiated, undifferentiated, or transformed status of the cell. Differentiation stimuli are integrated by key transcription factors which regulate specific sets of specialized genes to allow proliferative cells to exit the cell cycle and acquire specialized functions. The main differentiation programs known to be controlled by PPARs both during development and in the adult are placental differentiation, adipogenesis, osteoblast differentiation, skin differentiation, and gut differentiation. PPARs may also be involved in the differentiation of macrophages, brain, and breast. However, their functions in this cell type and organs still awaits further elucidation. PPARs may be involved in cell proliferation and differentiation processes of neural stem cells (NSC. To this aim, in this work the expression of the three PPAR isotypes and RXRs in NSC has been investigated.

  16. 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...

  17. 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.

  18. 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

  19. 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

  20. Potassium Bromate-induced Changes in the Adult Mouse Cerebellum Are Ameliorated by Vanillin.

    Science.gov (United States)

    Ben Saad, Hajer; Driss, Dorra; Jaballi, Imen; Ghozzi, Hanen; Boudawara, Ons; Droguet, Michael; Magné, Christian; Nasri, Monsef; Zeghal, Khaled Mounir; Hakim, Ahmed; Ben Amara, Ibtissem

    2018-02-01

    The current study aimed to elucidate the effect of vanillin on behavioral changes, oxidative stress, and histopathological changes induced by potassium bromate (KBrO3), an environmental pollutant, in the cerebellum of adult mice. The animals were divided into four groups: group 1 served as a control, group 2 received KBrO3, group 3 received KBrO3 and vanillin, and group 4 received only vanillin. We then measured behavioral changes, oxidative stress, and molecular and histological changes in the cerebellum. We observed significant behavioral changes in KBrO3-exposed mice. When investigating redox homeostasis in the cerebellum, we found that mice treated with KBrO3 had increased lipid peroxidation and protein oxidation in the cerebellum. These effects were accompanied by decreased Na+-K+ and Mg2+ ATPase activity and antioxidant enzyme gene expression when compared to the control group. Additionally, there was a significant increase in cytokine gene expression in KBrO3-treated mice. Microscopy revealed that KBrO3 intoxication resulted in numerous degenerative changes in the cerebellum that were substantially ameliorated by vanillin supplementation. Co-administration of vanillin blocked the biochemical and molecular anomalies induced by KBrO3. Our results demonstrate that vanillin is a potential therapeutic agent for oxidative stress associated with neurodegenerative diseases. Copyright © 2018 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.

  1. An ex vivo spinal cord injury model to study ependymal cells in adult mouse tissue.

    Science.gov (United States)

    Fernandez-Zafra, Teresa; Codeluppi, Simone; Uhlén, Per

    2017-08-15

    Traumatic spinal cord injury is characterized by an initial cell loss that is followed by a concerted cellular response in an attempt to restore the damaged tissue. Nevertheless, little is known about the signaling mechanisms governing the cellular response to injury. Here, we have established an adult ex vivo system that exhibits multiple hallmarks of spinal cord injury and allows the study of complex processes that are difficult to address using animal models. We have characterized the ependymal cell response to injury in this model system and found that ependymal cells can become activated, proliferate, migrate out of the central canal lining and differentiate in a manner resembling the in vivo situation. Moreover, we show that these cells respond to external adenosine triphosphate and exhibit spontaneous Ca 2+ activity, processes that may play a significant role in the regulation of their response to spinal cord injury. This model provides an attractive tool to deepen our understanding of the ependymal cell response after spinal cord injury, which may contribute to the development of new treatment options for spinal cord injury. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. The electrocorticograms of the aged mouse x-irradiated at juvenile or young adult

    International Nuclear Information System (INIS)

    Minamisawa, Takeru; Sasaki, Shunsaku.

    1984-01-01

    The electrocorticograms (ECoGs) of the (C57BL/6 x C3H)F 1 mice irradiated at juvenile or young adult were studied when they attained the age of 24-26 months. One group of mice was irradiated 35 days post partum (35-DPP) and another 105 days (105-DPP). All the animals were irradiated with 300 R of X-rays to whole body. The ECoGs were recorded from the freely moving animals with the permanently implanted electrodes fixed over the visual cortical surface. The resulted ECoGs were divided into 3 patterns: wakefulness (W), slow wave sleep (SWS), and paradoxical sleep (PS). Six parameters of the 3 patterns were compared among the 2 irradiated groups and the non-irradiated control group. The mean SWS- and PS-cycle times, and mean SWS length were significantly longer in the 35-DPP group than in the control group. Changes in the ECoGs were less profound in the 105-DPP group than those in the 35-DPP group: only a significant change due to irradiation at 105-DPP was a decrease in the ratio of the total PS time to the total sleep time (TST = total SWS time + total PS time). There was no difference in the body weight and brain weight among the 2 irradiated groups and the control group. (author)

  3. Mash1-expressing cells could differentiate to type III cells in adult mouse taste buds.

    Science.gov (United States)

    Takagi, Hiroki; Seta, Yuji; Kataoka, Shinji; Nakatomi, Mitsushiro; Toyono, Takashi; Kawamoto, Tatsuo

    2018-03-10

    The gustatory cells in taste buds have been identified as paraneuronal; they possess characteristics of both neuronal and epithelial cells. Like neurons, they form synapses, store and release transmitters, and are capable of generating an action potential. Like epithelial cells, taste cells have a limited life span and are regularly replaced throughout life. However, little is known about the molecular mechanisms that regulate taste cell genesis and differentiation. In the present study, to begin to understand these mechanisms, we investigated the role of Mash1-positive cells in regulating adult taste bud cell differentiation through the loss of Mash1-positive cells using the Cre-loxP system. We found that the cells expressing type III cell markers-aromatic L-amino acid decarboxylase (AADC), carbonic anhydrase 4 (CA4), glutamate decarboxylase 67 (GAD67), neural cell adhesion molecule (NCAM), and synaptosomal-associated protein 25 (SNAP25)-were significantly reduced in the circumvallate taste buds after the administration of tamoxifen. However, gustducin and phospholipase C beta2 (PLC beta2)-markers of type II taste bud cells-were not significantly changed in the circumvallate taste buds after the administration of tamoxifen. These results suggest that Mash1-positive cells could be differentiated to type III cells, not type II cells in the taste buds.

  4. 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.

  5. Analysis of Adult Female Mouse (Mus musculus) Group Behavior on the International Space Station (ISS)

    Science.gov (United States)

    Solomides, P.; Moyer, E. L.; Talyansky, Y.; Choi, S.; Gong, C.; Globus, R. K.; Ronca, A. E.

    2016-01-01

    As interest in long duration effects of space habitation increases, understanding the behavior of model organisms living within the habitats engineered to fly them is vital for designing, validating, and interpreting future spaceflight studies. A handful of papers have previously reported behavior of mice and rats in the weightless environment of space. The Rodent Research Hardware and Operations Validation (Rodent Research-1; RR1) utilized the Rodent Habitat (RH) developed at NASA Ames Research Center to fly mice on the ISS (International Space Station). Ten adult (16-week-old) female C57BL/6 mice were launched on September 21st, 2014 in an unmanned Dragon Capsule, and spent 37 days in microgravity. Here we report group behavioral phenotypes of the RR1 Flight (FLT) and environment-matched Ground Control (GC) mice in the Rodent Habitat (RH) during this long-duration flight. Video was recorded for 33 days on the ISS, permitting daily assessments of overall health and well-being of the mice, and providing a valuable repository for detailed behavioral analysis. We previously reported that, as compared to GC mice, RR1 FLT mice exhibited the same range of behaviors, including eating, drinking, exploration, self- and allo-grooming, and social interactions at similar or greater levels of occurrence. Overall activity was greater in FLT as compared to GC mice, with spontaneous ambulatory behavior, including organized 'circling' or 'race-tracking' behavior that emerged within the first few days of flight following a common developmental sequence, and comprised the primary dark cycle activity persisting throughout the remainder of the experiment. Participation by individual mice increased dramatically over the course of the flight. Here we present a detailed analysis of 'race-tracking' behavior in which we quantified: (1) Complete lap rotations by individual mice; (2) Numbers of collisions between circling mice; (3) Lap directionality; and (4) Recruitment of mice into a group

  6. Expression of extracellular matrix components is disrupted in the immature and adult estrogen receptor β-null mouse ovary.

    Directory of Open Access Journals (Sweden)

    Alexandra Zalewski

    Full Text Available Within the ovary, Estrogen Receptor β (ERβ is localized to the granulosa cells of growing follicles. 17β-estradiol (E2 acting via ERβ augments the actions of follicle stimulating hormone in granulosa cells, leading to granulosa cell differentiation and formation of a preovulatory follicle. Adult ERβ-null females are subfertile and possess ovaries with reduced numbers of growing follicles and corpora lutea. Because the majority of E2 production by granulosa cells occurs once puberty is reached, a role for ERβ in the ovary prior to puberty has not been well examined. We now provide evidence that lack of ERβ disrupts gene expression as early as post-natal day (PND 13, and in particular, we identify a number of genes of the extracellular matrix (ECM that are significantly higher in ERβ-null follicles than in wildtype (WT follicles. Considerable changes occur to the ECM occur during normal folliculogenesis to allow for the dramatic growth, cellular differentiation, and reorganization of the follicle from the primary to preovulatory stage. Using quantitative PCR and immunofluorescence, we now show that several ECM genes are aberrantly overexpressed in ERβ-null follicles. We find that Collagen11a1, a protein highly expressed in cartilage, is significantly higher in ERβ-null follicles than WT follicles as early as PND 13, and this heightened expression continues through PND 23-29 into adulthood. Similarly, Nidogen 2, a highly conserved basement membrane glycoprotein, is elevated in ERβ-null follicles at PND 13 into adulthood, and is elevated specifically in the ERβ-null focimatrix, a basal lamina-like matrix located between granulosa cells. Focimatrix laminin and Collagen IV expression were also higher in ERβ-null ovaries than in WT ovaries at various ages. Our findings suggest two novel observations: a that ERβ regulates granulosa cell gene expression ovary prior to puberty, and b that ERβ regulates expression of ECM components in the

  7. Deep-brain magnetic stimulation promotes adult hippocampal neurogenesis and alleviates stress-related behaviors in mouse models for neuropsychiatric disorders

    Science.gov (United States)

    2014-01-01

    Background Repetitive Transcranial Magnetic Stimulation (rTMS)/ Deep-brain Magnetic Stimulation (DMS) is an effective therapy for various neuropsychiatric disorders including major depression disorder. The molecular and cellular mechanisms underlying the impacts of rTMS/DMS on the brain are not yet fully understood. Results Here we studied the effects of deep-brain magnetic stimulation to brain on the molecular and cellular level. We examined the adult hippocampal neurogenesis and hippocampal synaptic plasticity of rodent under stress conditions with deep-brain magnetic stimulation treatment. We found that DMS promotes adult hippocampal neurogenesis significantly and facilitates the development of adult new-born neurons. Remarkably, DMS exerts anti-depression effects in the learned helplessness mouse model and rescues hippocampal long-term plasticity impaired by restraint stress in rats. Moreover, DMS alleviates the stress response in a mouse model for Rett syndrome and prolongs the life span of these animals dramatically. Conclusions Deep-brain magnetic stimulation greatly facilitates adult hippocampal neurogenesis and maturation, also alleviates depression and stress-related responses in animal models. PMID:24512669

  8. 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

  9. Purification of oogonial stem cells from adult mouse and human ovaries: an assessment of the literature and a view toward the future.

    Science.gov (United States)

    Woods, Dori C; White, Yvonne A R; Tilly, Jonathan L

    2013-01-01

    Contemporary claims that mitotically active female germ line or oogonial stem cells (OSCs) exist and support oogenesis during postnatal life in mammals have been debated in the field of reproductive biology since March 2004, when a mouse study posed the first serious challenge to the dogma of a fixed pool of oocytes being endowed at birth in more than 50 years. Other studies have since been put forth that further question the validity of this dogma, including the isolation of OSCs from neonatal and adult mouse ovaries by 4 independent groups using multiple strategies. Two of these groups also reported that isolated mouse OSCs, once transplanted back into ovaries of adult female mice, differentiate into fully functional eggs that ovulate, fertilize, and produce healthy embryos and offspring. Arguably, one of the most significant advances in this emerging field was provided by a new research study published this year, which reported the successful isolation and functional characterization of OSCs from ovaries of reproductive age women. Two commentaries on this latest work, one cautiously supportive and one highly skeptical, were published soon afterward. This article evaluates the current literature regarding postnatal oogenesis in mammals and discusses important next steps for future work on OSC biology and function.

  10. Bone morphogenetic protein-2 is a negative regulator of hepatocyte proliferation downregulated in the regenerating liver

    NARCIS (Netherlands)

    Xu, Cui-Ping; Ji, Wen-Min; van den Brink, Gijs R.; Peppelenbosch, Maikel P.

    2006-01-01

    To characterize the expression and dynamic changes of bone morphogenetic protein (BMP)-2 in hepatocytes in the regenerating liver in rats after partial hepatectomy (PH), and examine the effects of BMP-2 on proliferation of human Huh7 hepatoma cells. Fifty-four adult male Wistar rats were randomly

  11. Bone morphogenetic protein-2 is a negative regulator of hepatocyte proliferation downregulated in the regenerating liver

    NARCIS (Netherlands)

    Xu, Cui-Ping; Ji, Wen-Min; van den Brink, Gijs R.; Peppelenbosch, Maikel P.

    2006-01-01

    AIM: To characterize the expression and dynamic changes of bone morphogenetic protein (BMP)-2 in hepatocytes in the regenerating liver in rats after partial hepatectomy (PH), and examine the effects of BMP-2 on proliferation of human Huh7 hepatoma cells. METHODS: Fifty-four adult male Wistar rats

  12. Development of MAPC derived induced endodermal progenitors : Generation of pancreatic beta cells and hepatocytes

    NARCIS (Netherlands)

    Sambathkumar, Rangarajan

    2017-01-01

    Multipotent Adult Progenitor Cells (MAPCs) are one potential stem cell source to generate functional hepatocytes or β-cells. However, human MAPCs have less plasticity than pluripotent stem cells (PSCs), as their ability to generate endodermal cells is not robust. Here we studied the role of 14

  13. 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...

  14. Generation of hepatocyte- and endocrine pancreatic-like cells from human induced endodermal progenitor cells

    NARCIS (Netherlands)

    Sambathkumar, Rangarajan; Akkerman, Renate; Dastidar, Sumitava; Roelandt, Philip; Kumar, Manoj; Bajaj, Manmohan; Mestre Rosa, Ana Rita; Helsen, Nicky; Vanslembrouck, Veerle; Kalo, Eric; Khurana, Satish; Laureys, Jos; Gysemans, Conny; Faas, Marijke M; de Vos, Paul; Verfaillie, Catherine M

    2018-01-01

    Multipotent Adult Progenitor Cells (MAPCs) are one potential stem cell source to generate functional hepatocytes or β-cells. However, human MAPCs have less plasticity than pluripotent stem cells (PSCs), as their ability to generate endodermal cells is not robust. Here we studied the role of 14

  15. 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

  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. 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

  18. 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.

  19. Assessing the use of immersive virtual reality, mouse and touchscreen in pointing and dragging-and-dropping tasks among young, middle-aged and older adults.

    Science.gov (United States)

    Chen, Jiayin; Or, Calvin

    2017-11-01

    This study assessed the use of an immersive virtual reality (VR), a mouse and a touchscreen for one-directional pointing, multi-directional pointing, and dragging-and-dropping tasks involving targets of smaller and larger widths by young (n = 18; 18-30 years), middle-aged (n = 18; 40-55 years) and older adults (n = 18; 65-75 years). A three-way, mixed-factorial design was used for data collection. The dependent variables were the movement time required and the error rate. Our main findings were that the participants took more time and made more errors in using the VR input interface than in using the mouse or the touchscreen. This pattern applied in all three age groups in all tasks, except for multi-directional pointing with a larger target width among the older group. Overall, older adults took longer to complete the tasks and made more errors than young or middle-aged adults. Larger target widths yielded shorter movement times and lower error rates in pointing tasks, but larger targets yielded higher rates of error in dragging-and-dropping tasks. Our study indicated that any other virtual environments that are similar to those we tested may be more suitable for displaying scenes than for manipulating objects that are small and require fine control. Although interacting with VR is relatively difficult, especially for older adults, there is still potential for older adults to adapt to that interface. Furthermore, adjusting the width of objects according to the type of manipulation required might be an effective way to promote performance. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. Expression of C4.4A, a structural uPAR homolog, reflects squamous epithelial differentiation in the adult mouse and during embryogenesis

    DEFF Research Database (Denmark)

    Kriegbaum, Mette Camilla; Jacobsen, Benedikte; Hald, Andreas

    2011-01-01

    by a comprehensive immunohistochemical mapping. This task was accomplished by staining paraffin-embedded tissues with a specific rabbit polyclonal anti-C4.4A antibody. In the adult mouse, C4.4A was predominantly expressed in the suprabasal layers of the squamous epithelia of the oral cavity, esophagus, non...... expression first appears in the developing squamous epithelium at embryonic day 13.5. This anatomical location of C4.4A is thus concordant with a possible functional role in early differentiation of stratified squamous epithelia....

  1. Differential Structural Development of Adult-Born Septal Hippocampal Granule Cells in the Thy1-GFP Mouse, Nuclear Size as a New Index of Maturation.

    Directory of Open Access Journals (Sweden)

    Tijana Radic

    Full Text Available Adult neurogenesis is frequently studied in the mouse hippocampus. We examined the morphological development of adult-born, immature granule cells in the suprapyramidal blade of the septal dentate gyrus over the period of 7-77 days after mitosis with BrdU-labeling in 6-weeks-old male Thy1-GFP mice. As Thy1-GFP expression was restricted to maturated granule cells, it was combined with doublecortin-immunolabeling of immature granule cells. We developed a novel classification system that is easily applicable and enables objective and direct categorization of newborn granule cells based on the degree of dendritic development in relation to the layer specificity of the dentate gyrus. The structural development of adult-generated granule cells was correlated with age, albeit with notable differences in the time course of development between individual cells. In addition, the size of the nucleus, immunolabeled with the granule cell specific marker Prospero-related homeobox 1 gene, was a stable indicator of the degree of a cell's structural maturation and could be used as a straightforward parameter of granule cell development. Therefore, further studies could employ our doublecortin-staging system and nuclear size measurement to perform investigations of morphological development in combination with functional studies of adult-born granule cells. Furthermore, the Thy1-GFP transgenic mouse model can be used as an additional investigation tool because the reporter gene labels granule cells that are 4 weeks or older, while very young cells could be visualized through the immature marker doublecortin. This will enable comparison studies regarding the structure and function between young immature and older matured granule cells.

  2. 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

  3. H3 and H4 Lysine Acetylation Correlates with Developmental and Experimentally Induced Adult Experience-Dependent Plasticity in the Mouse Visual Cortex

    Directory of Open Access Journals (Sweden)

    Gabriela Vierci

    2016-01-01

    Full Text Available Histone posttranslational modifications play a fundamental role in orchestrating gene expression. In this work, we analyzed the acetylation of H3 and H4 histones (AcH3-AcH4 and its modulation by visual experience in the mouse visual cortex (VC during normal development and in two experimental conditions that restore juvenile-like plasticity levels in adults (fluoxetine treatment and enriched environment. We found that AcH3-AcH4 declines with age and is upregulated by treatments restoring plasticity in the adult. We also found that visual experience modulates AcH3-AcH4 in young and adult plasticity-restored mice but not in untreated ones. Finally, we showed that the transporter vGAT is downregulated in adult plasticity-restored models. In summary, we identified a dynamic regulation of AcH3-AcH4, which is associated with high plasticity levels and enhanced by visual experience. These data, along with recent ones, indicate H3-H4 acetylation as a central hub in the control of experience-dependent plasticity in the VC.

  4. 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.

  5. Hepatocytes polyploidization and cell cycle control in liver physiopathology.

    Science.gov (United States)

    Gentric, Géraldine; Desdouets, Chantal; Celton-Morizur, Séverine

    2012-01-01

    Most cells in mammalian tissues usually contain a diploid complement of chromosomes. However, numerous studies have demonstrated a major role of "diploid-polyploid conversion" during physiopathological processes in several tissues. In the liver parenchyma, progressive polyploidization of hepatocytes takes place during postnatal growth. Indeed, at the suckling-weaning transition, cytokinesis failure events induce the genesis of binucleated tetraploid liver cells. Insulin signalling, through regulation of the PI3K/Akt signalling pathway, is essential in the establishment of liver tetraploidization by controlling cytoskeletal organisation and consequently mitosis progression. Liver cell polyploidy is generally considered to indicate terminal differentiation and senescence, and both lead to a progressive loss of cell pluripotency associated to a markedly decreased replication capacity. Although adult liver is a quiescent organ, it retains a capacity to proliferate and to modulate its ploidy in response to various stimuli or aggression (partial hepatectomy, metabolic overload (i.e., high copper and iron hepatic levels), oxidative stress, toxic insult, and chronic hepatitis etc.). Here we review the mechanisms and functional consequences of hepatocytes polyploidization during normal and pathological liver growth.

  6. Hepatocytes Polyploidization and Cell Cycle Control in Liver Physiopathology

    Directory of Open Access Journals (Sweden)

    Géraldine Gentric

    2012-01-01

    Full Text Available Most cells in mammalian tissues usually contain a diploid complement of chromosomes. However, numerous studies have demonstrated a major role of “diploid-polyploid conversion” during physiopathological processes in several tissues. In the liver parenchyma, progressive polyploidization of hepatocytes takes place during postnatal growth. Indeed, at the suckling-weaning transition, cytokinesis failure events induce the genesis of binucleated tetraploid liver cells. Insulin signalling, through regulation of the PI3K/Akt signalling pathway, is essential in the establishment of liver tetraploidization by controlling cytoskeletal organisation and consequently mitosis progression. Liver cell polyploidy is generally considered to indicate terminal differentiation and senescence, and both lead to a progressive loss of cell pluripotency associated to a markedly decreased replication capacity. Although adult liver is a quiescent organ, it retains a capacity to proliferate and to modulate its ploidy in response to various stimuli or aggression (partial hepatectomy, metabolic overload (i.e., high copper and iron hepatic levels, oxidative stress, toxic insult, and chronic hepatitis etc.. Here we review the mechanisms and functional consequences of hepatocytes polyploidization during normal and pathological liver growth.

  7. 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

  8. 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.

  9. Swelling of rat hepatocytes stimulates glycogen synthesis

    NARCIS (Netherlands)

    Baquet, A.; Hue, L.; Meijer, A. J.; van Woerkom, G. M.; Plomp, P. J.

    1990-01-01

    In hepatocytes from fasted rats, several amino acids are known to stimulate glycogen synthesis via activation of glycogen synthase. The hypothesis that an increase in cell volume resulting from amino acid uptake may be involved in the stimulation of glycogen synthesis is supported by the following

  10. Protease-activated receptor-1 negatively regulates proliferation of neural stem/progenitor cells derived from the hippocampal dentate gyrus of the adult mouse

    Directory of Open Access Journals (Sweden)

    Masayuki Tanaka

    2016-07-01

    Full Text Available Thrombin-activated protease-activated receptor (PAR-1 regulates the proliferation of neural cells following brain injury. To elucidate the involvement of PAR-1 in the neurogenesis that occurs in the adult hippocampus, we examined whether PAR-1 regulated the proliferation of neural stem/progenitor cells (NPCs derived from the murine hippocampal dentate gyrus. NPC cultures expressed PAR-1 protein and mRNA encoding all subtypes of PAR. Direct exposure of the cells to thrombin dramatically attenuated the cell proliferation without causing cell damage. This thrombin-induced attenuation was almost completely abolished by the PAR antagonist RWJ 56110, as well as by dabigatran and 4-(2-aminoethylbenzenesulfonyl fluoride (AEBSF, which are selective and non-selective thrombin inhibitors, respectively. Expectedly, the PAR-1 agonist peptide (AP SFLLR-NH2 also attenuated the cell proliferation. The cell proliferation was not affected by the PAR-1 negative control peptide RLLFT-NH2, which is an inactive peptide for PAR-1. Independently, we determined the effect of in vivo treatment with AEBSF or AP on hippocampal neurogenesis in the adult mouse. The administration of AEBSF, but not that of AP, significantly increased the number of newly-generated cells in the hippocampal subgranular zone. These data suggest that PAR-1 negatively regulated adult neurogenesis in the hippocampus by inhibiting the proliferative activity of the NPCs.

  11. Maternal high-protein diet during pregnancy, but not during suckling, induced altered expression of an increasing number of hepatic genes in adult mouse offspring.

    Science.gov (United States)

    Vanselow, Jens; Kucia, Marzena; Langhammer, Martina; Koczan, Dirk; Metges, Cornelia C

    2016-04-01

    Indirect effects of a high-protein maternal diet are not well understood. In this study, we analyzed short-term and sustainable effects of a prenatal versus early postnatal maternal high-protein diet on growth and hepatic gene expression in mouse offspring. Dams were exposed to an isoenergetic high-protein (HP, 40 % w/w) diet during pregnancy or lactation. Growth and hepatic expression profiles of male offspring were evaluated directly after weaning and 150 days after birth. Offspring from two dietary groups, high-protein diet during pregnancy and control diet during lactation (HPC), and control diet during pregnancy and high-protein diet during lactation (CHP), were compared with offspring (CC) from control-fed dams. Maternal CHP treatment was associated with sustained offspring growth retardation, but decreased numbers of affected hepatic genes in adults compared to weanlings. In contrast, offspring of the HPC group did not show persistent effects on growth parameters, but the number of affected hepatic genes was even increased at adult age. In both dietary groups, however, only a small subset of genes was affected in weanlings as well as in adults. We conclude that (1) prenatal and early postnatal maternal HP diet caused persistent, but (2) different effects and partially complementary trends on growth characteristics and on the hepatic transcriptome and associated pathways and that (3) only a small number of genes and associated upstream regulators might be involved in passing early diet-induced imprints to adulthood.

  12. Adult mouse motor units develop almost all of their force in the subprimary range: a new all-or-none strategy for force recruitment?

    Science.gov (United States)

    Manuel, Marin; Heckman, C J

    2011-10-19

    Classical studies of the mammalian neuromuscular system have shown an impressive adaptation match between the intrinsic properties of motoneurons and the contractile properties of their motor units. In these studies, the rate at which motoneurons start to fire repetitively corresponds to the rate at which individual twitches start to sum, and the firing rate increases linearly with the amount of excitation ("primary range") up to the point where the motor unit develops its maximal force. This allows for the gradation of the force produced by a motor unit by rate modulation. In adult mouse motoneurons, however, we recently described a regime of firing ("subprimary range") that appears at lower excitation than what is required for the primary range, a finding that might challenge the classical conception. To investigate the force production of mouse motor units, we simultaneously recorded, for the first time, the motoneuron discharge elicited by intracellular ramps of current and the force developed by its motor unit. We showed that the motor unit developed nearly its maximal force during the subprimary range. This was found to be the case regardless of the input resistance of the motoneuron, the contraction speed, or the tetanic force of the motor unit. Our work suggests that force modulation in small mammals mainly relies on the number of motor units that are recruited rather than on rate modulation of individual motor units.

  13. Alteration of Gene Expression, DNA Methylation, and Histone Methylation in Free Radical Scavenging Networks in Adult Mouse Hippocampus following Fetal Alcohol Exposure.

    Directory of Open Access Journals (Sweden)

    Eric J Chater-Diehl

    Full Text Available The molecular basis of Fetal Alcohol Spectrum Disorders (FASD is poorly understood; however, epigenetic and gene expression changes have been implicated. We have developed a mouse model of FASD characterized by learning and memory impairment and persistent gene expression changes. Epigenetic marks may maintain expression changes over a mouse's lifetime, an area few have explored. Here, mice were injected with saline or ethanol on postnatal days four and seven. At 70 days of age gene expression microarray, methylated DNA immunoprecipitation microarray, H3K4me3 and H3K27me3 chromatin immunoprecipitation microarray were performed. Following extensive pathway analysis of the affected genes, we identified the top affected gene expression pathway as "Free radical scavenging". We confirmed six of these changes by droplet digital PCR including the caspase Casp3 and Wnt transcription factor Tcf7l2. The top pathway for all methylation-affected genes was "Peroxisome biogenesis"; we confirmed differential DNA methylation in the Acca1 thiolase promoter. Altered methylation and gene expression in oxidative stress pathways in the adult hippocampus suggests a novel interface between epigenetic and oxidative stress mechanisms in FASD.

  14. Alteration of Gene Expression, DNA Methylation, and Histone Methylation in Free Radical Scavenging Networks in Adult Mouse Hippocampus following Fetal Alcohol Exposure.

    Science.gov (United States)

    Chater-Diehl, Eric J; Laufer, Benjamin I; Castellani, Christina A; Alberry, Bonnie L; Singh, Shiva M

    2016-01-01

    The molecular basis of Fetal Alcohol Spectrum Disorders (FASD) is poorly understood; however, epigenetic and gene expression changes have been implicated. We have developed a mouse model of FASD characterized by learning and memory impairment and persistent gene expression changes. Epigenetic marks may maintain expression changes over a mouse's lifetime, an area few have explored. Here, mice were injected with saline or ethanol on postnatal days four and seven. At 70 days of age gene expression microarray, methylated DNA immunoprecipitation microarray, H3K4me3 and H3K27me3 chromatin immunoprecipitation microarray were performed. Following extensive pathway analysis of the affected genes, we identified the top affected gene expression pathway as "Free radical scavenging". We confirmed six of these changes by droplet digital PCR including the caspase Casp3 and Wnt transcription factor Tcf7l2. The top pathway for all methylation-affected genes was "Peroxisome biogenesis"; we confirmed differential DNA methylation in the Acca1 thiolase promoter. Altered methylation and gene expression in oxidative stress pathways in the adult hippocampus suggests a novel interface between epigenetic and oxidative stress mechanisms in FASD.

  15. In vitro differentiation of mouse embryonic stem cells into functional ...

    African Journals Online (AJOL)

    Studies have shown that embryonic stem (ES) cells can be successfully differentiated into liver cells, which offer the potential unlimited cell source for a variety of end-stage liver disease. In our study, in order to induce mouse ES cells to differentiate into hepatocyte-like cells under chemically defined conditions, ES cells ...

  16. Regional localization of activin-βA, activin-βC, follistatin, proliferation, and apoptosis in adult and developing mouse prostate ducts.

    Science.gov (United States)

    Gold, Elspeth; Zellhuber-McMillan, Sylvia; Risbridger, Gail; Marino, Francesco Elia

    2017-01-01

    Activins and inhibins, members of the TGF-β superfamily, are growth and differentiation factors involved in the regulation of several biological processes, including reproduction, development, and fertility. Previous studies have shown that the activin-β A subunit plays a pivotal role in prostate development. Activin-A inhibits branching morphogenesis in the developing prostate, and its expression is associated with increased apoptosis in the adult prostate. Follistatin, a structurally unrelated protein to activins, is an antagonist of activin-A. A balance between endogenous activin-A and follistatin is required to maintain prostatic branching morphogenesis. Deregulation of this balance leads to branching inhibition or excessive branching and increased maturation of the stroma surrounding the differentiating epithelial ducts. Recent work identified another member of the TGF-β superfamily, the activin-β C subunit, as a novel antagonist of activin-A. Over-expression of activin-C (β C -β C ) alters prostate homeostasis, by interfering with the activin-A signaling. The current study characterized the spatiotemporal localization of activin-A, activin-C and follistatin in the adult and developing mouse prostate using immunohistochemical analysis. Results showed activin-C and follistatin are differentially expressed during prostate development and suggested that the antagonistic property of follistatin is secondary to the action of activin-C. In conclusion, the present study provides evidence to support a role of activin-C in prostate development and provides new insights in the spatiotemporal localization of activins and their antagonists during mouse prostate development. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. 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

  18. 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

  19. Adult Brtl/+ mouse model of osteogenesis imperfecta demonstrates anabolic response to sclerostin antibody treatment with increased bone mass and strength.

    Science.gov (United States)

    Sinder, B P; White, L E; Salemi, J D; Ominsky, M S; Caird, M S; Marini, J C; Kozloff, K M

    2014-08-01

    Treatments to reduce fracture rates in adults with osteogenesis imperfecta are limited. Sclerostin antibody, developed for treating osteoporosis, has not been explored in adults with OI. This study demonstrates that treatment of adult OI mice respond favorably to sclerostin antibody therapy despite retention of the OI-causing defect. Osteogenesis imperfecta (OI) is a heritable collagen-related bone dysplasia, characterized by brittle bones with increased fracture risk. Although OI fracture risk is greatest before puberty, adults with OI remain at risk of fracture. Antiresorptive bisphosphonates are commonly used to treat adult OI, but have shown mixed efficacy. New treatments which consistently improve bone mass throughout the skeleton may improve patient outcomes. Neutralizing antibodies to sclerostin (Scl-Ab) are a novel anabolic therapy that have shown efficacy in preclinical studies by stimulating bone formation via the canonical wnt signaling pathway. The purpose of this study was to evaluate Scl-Ab in an adult 6 month old Brtl/+ model of OI that harbors a typical heterozygous OI-causing Gly > Cys substitution on Col1a1. Six-month-old WT and Brtl/+ mice were treated with Scl-Ab (25 mg/kg, 2×/week) or Veh for 5 weeks. OCN and TRACP5b serum assays, dynamic histomorphometry, microCT and mechanical testing were performed. Adult Brtl/+ mice demonstrated a strong anabolic response to Scl-Ab with increased serum osteocalcin and bone formation rate. This anabolic response led to improved trabecular and cortical bone mass in the femur. Mechanical testing revealed Scl-Ab increased Brtl/+ femoral stiffness and strength. Scl-Ab was successfully anabolic in an adult Brtl/+ model of OI.

  20. Tet2 Rescues Age-Related Regenerative Decline and Enhances Cognitive Function in the Adult Mouse Brain

    Directory of Open Access Journals (Sweden)

    Geraldine Gontier

    2018-02-01

    Full Text Available Restoring adult stem cell function provides an exciting approach for rejuvenating the aging brain. However, molecular mechanisms mediating neurogenic rejuvenation remain elusive. Here we report that the enzyme ten eleven translocation methylcytosine dioxygenase 2 (Tet2, which catalyzes the production of 5-hydroxymethylcytosine (5hmC, rescues age-related decline in adult neurogenesis and enhances cognition in mice. We detected a decrease in Tet2 expression and 5hmC levels in the aged hippocampus associated with adult neurogenesis. Mimicking an aged condition in young adults by abrogating Tet2 expression within the hippocampal neurogenic niche, or adult neural stem cells, decreased neurogenesis and impaired learning and memory. In a heterochronic parabiosis rejuvenation model, hippocampal Tet2 expression was restored. Overexpressing Tet2 in the hippocampal neurogenic niche of mature adults increased 5hmC associated with neurogenic processes, offset the precipitous age-related decline in neurogenesis, and enhanced learning and memory. Our data identify Tet2 as a key molecular mediator of neurogenic rejuvenation.

  1. Mitochondrial protein adducts formation and mitochondrial dysfunction during N-acetyl-m-aminophenol (AMAP)-induced hepatotoxicity in primary human hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Xie, Yuchao; McGill, Mitchell R.; Du, Kuo; Dorko, Kenneth [Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS 66160 (United States); Kumer, Sean C.; Schmitt, Timothy M. [Department of Surgery, University of Kansas Medical Center, Kansas City, KS 66160 (United States); Ding, Wen-Xing [Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS 66160 (United States); Jaeschke, Hartmut, E-mail: hjaeschke@kumc.edu [Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS 66160 (United States)

    2015-12-01

    3′-Hydroxyacetanilide or N-acetyl-meta-aminophenol (AMAP) is generally regarded as a non-hepatotoxic analog of acetaminophen (APAP). Previous studies demonstrated the absence of toxicity after AMAP in mice, hamsters, primary mouse hepatocytes and several cell lines. In contrast, experiments with liver slices suggested that it may be toxic to human hepatocytes; however, the mechanism of toxicity is unclear. To explore this, we treated primary human hepatocytes (PHH) with AMAP or APAP for up to 48 h and measured several parameters to assess metabolism and injury. Although less toxic than APAP, AMAP dose-dependently triggered cell death in PHH as indicated by alanine aminotransferase (ALT) release and propidium iodide (PI) staining. Similar to APAP, AMAP also significantly depleted glutathione (GSH) in PHH and caused mitochondrial damage as indicated by glutamate dehydrogenase (GDH) release and the JC-1 assay. However, unlike APAP, AMAP treatment did not cause relevant c-jun-N-terminal kinase (JNK) activation in the cytosol or phospho-JNK translocation to mitochondria. To compare, AMAP toxicity was assessed in primary mouse hepatocytes (PMH). No cytotoxicity was observed as indicated by the lack of lactate dehydrogenase release and no PI staining. Furthermore, there was no GSH depletion or mitochondrial dysfunction after AMAP treatment in PMH. Immunoblotting for arylated proteins suggested that AMAP treatment caused extensive mitochondrial protein adduct formation in PHH but not in PMH. In conclusion, AMAP is hepatotoxic in PHH and the mechanism involves the formation of mitochondrial protein adducts and mitochondrial dysfunction. - Highlights: • AMAP induces cell death in primary human hepatocytes (PHH). • AMAP does not cause cell death in primary mouse hepatocytes (PMH). • AMAP leads to mitochondria dysfunction in PHH but not PMH. • Protein adduct formation and dysfunction in mitochondria correlate with toxicity.

  2. Mitochondrial protein adducts formation and mitochondrial dysfunction during N-acetyl-m-aminophenol (AMAP)-induced hepatotoxicity in primary human hepatocytes

    International Nuclear Information System (INIS)

    Xie, Yuchao; McGill, Mitchell R.; Du, Kuo; Dorko, Kenneth; Kumer, Sean C.; Schmitt, Timothy M.; Ding, Wen-Xing; Jaeschke, Hartmut

    2015-01-01

    3′-Hydroxyacetanilide or N-acetyl-meta-aminophenol (AMAP) is generally regarded as a non-hepatotoxic analog of acetaminophen (APAP). Previous studies demonstrated the absence of toxicity after AMAP in mice, hamsters, primary mouse hepatocytes and several cell lines. In contrast, experiments with liver slices suggested that it may be toxic to human hepatocytes; however, the mechanism of toxicity is unclear. To explore this, we treated primary human hepatocytes (PHH) with AMAP or APAP for up to 48 h and measured several parameters to assess metabolism and injury. Although less toxic than APAP, AMAP dose-dependently triggered cell death in PHH as indicated by alanine aminotransferase (ALT) release and propidium iodide (PI) staining. Similar to APAP, AMAP also significantly depleted glutathione (GSH) in PHH and caused mitochondrial damage as indicated by glutamate dehydrogenase (GDH) release and the JC-1 assay. However, unlike APAP, AMAP treatment did not cause relevant c-jun-N-terminal kinase (JNK) activation in the cytosol or phospho-JNK translocation to mitochondria. To compare, AMAP toxicity was assessed in primary mouse hepatocytes (PMH). No cytotoxicity was observed as indicated by the lack of lactate dehydrogenase release and no PI staining. Furthermore, there was no GSH depletion or mitochondrial dysfunction after AMAP treatment in PMH. Immunoblotting for arylated proteins suggested that AMAP treatment caused extensive mitochondrial protein adduct formation in PHH but not in PMH. In conclusion, AMAP is hepatotoxic in PHH and the mechanism involves the formation of mitochondrial protein adducts and mitochondrial dysfunction. - Highlights: • AMAP induces cell death in primary human hepatocytes (PHH). • AMAP does not cause cell death in primary mouse hepatocytes (PMH). • AMAP leads to mitochondria dysfunction in PHH but not PMH. • Protein adduct formation and dysfunction in mitochondria correlate with toxicity.

  3. GLP-1 analogs reduce hepatocyte steatosis and improve survival by enhancing the unfolded protein response and promoting macroautophagy.

    Directory of Open Access Journals (Sweden)

    Shvetank Sharma

    Full Text Available Nonalcoholic fatty liver disease (NAFLD is a known outcome of hepatosteatosis. Free fatty acids (FFA induce the unfolded protein response (UPR or endoplasmic reticulum (ER stress that may induce apoptosis. Recent data indicate ER stress to be a major player in the progression of fatty liver to more aggressive lesions. Autophagy on the other hand has been demonstrated to be protective against ER stress-induced cell death. We hypothesized that exendin-4 (GLP-1 analog treatment of fat loaded hepatocytes can reduce steatosis by autophagy which leads to reduced ER stress-related hepatocyte apoptosis.Primary human hepatocytes were loaded with saturated, cis- and trans-unsaturated fatty acids (palmitic, oleic and elaidic acid respectively. Steatosis, induced with all three fatty acids, was significantly resolved after exendin-4 treatment. Exendin-4 sustained levels of GRP78 expression in fat-loaded cells when compared to untreated fat-loaded cells alone. In contrast, CHOP (C/EBP homologous protein; the penultimate protein that leads to ER stress-related cell death was significantly decreased by exendin-4 in hepatocytes loaded with fatty acids. Finally, exendin-4 in fat loaded hepatocytes clearly promoted gene products associated with macroautophagy as measured by enhanced production of both Beclin-1 and LC3B-II, markers for autophagy; and visualized by transmission electron microscopy (TEM. Similar observations were made in mouse liver lysates after mice were fed with high fat high fructose diet and treated with a long acting GLP-1 receptor agonist, liraglutide.GLP-1 proteins appear to protect hepatocytes from fatty acid-related death by prohibition of a dysfunctional ER stress response; and reduce fatty acid accumulation, by activation of both macro-and chaperone-mediated autophagy. These findings provide a novel role for GLP-1 proteins in halting the progression of more aggressive lesions from underlying steatosis in humans afflicted with NAFLD.

  4. Adult-Onset Fluoxetine Treatment Does Not Improve Behavioral Impairments and May Have Adverse Effects on the Ts65Dn Mouse Model of Down Syndrome

    Directory of Open Access Journals (Sweden)

    Markus Heinen

    2012-01-01

    Full Text Available Down syndrome is caused by triplication of chromosome 21 and is associated with neurocognitive phenotypes ranging from severe intellectual disability to various patterns of more selective neuropsychological deficits, including memory impairments. In the Ts65Dn mouse model of Down syndrome, excessive GABAergic neurotransmission results in local over-inhibition of hippocampal circuits, which dampens hippocampal synaptic plasticity and contributes to cognitive impairments. Treatments with several GABAA receptor antagonists result in increased plasticity and improved memory deficits in Ts65Dn mice. These GABAA receptor antagonists are, however, not suitable for clinical applications. The selective serotonin reuptake inhibitor fluoxetine, in contrast, is a widely prescribed antidepressant that can also enhance plasticity in the adult rodent brain by lowering GABAergic inhibition. For these reasons, we wondered if an adult-onset 4-week oral fluoxetine treatment restores spatial learning and memory impairments in Ts65Dn mice. Fluoxetine did not measurably improve behavioral impairments of Ts65Dn mice. On the contrary, we observed seizures and mortality in fluoxetine-treated Ts65Dn mice, raising the possibility of a drug × genotype interaction with respect to these adverse treatment outcomes. Future studies should re-address this in larger animal cohorts and determine if fluoxetine treatment is associated with adverse treatment effects in individuals with Down syndrome.

  5. Selective Deletion of Sodium Salt Taste during Development Leads to Expanded Terminal Fields of Gustatory Nerves in the Adult Mouse Nucleus of the Solitary Tract.

    Science.gov (United States)

    Sun, Chengsan; Hummler, Edith; Hill, David L

    2017-01-18

    Neuronal activity plays a key role in the development of sensory circuits in the mammalian brain. In the gustatory system, experimental manipulations now exist, through genetic manipulations of specific taste transduction processes, to examine how specific taste qualities (i.e., basic tastes) impact the functional and structural development of gustatory circuits. Here, we used a mouse knock-out model in which the transduction component used to discriminate sodium salts from other taste stimuli was deleted in taste bud cells throughout development. We used this model to test the hypothesis that the lack of activity elicited by sodium salt taste impacts the terminal field organization of nerves that carry taste information from taste buds to the nucleus of the solitary tract (NST) in the medulla. The glossopharyngeal, chorda tympani, and greater superficial petrosal nerves were labeled to examine their terminal fields in adult control mice and in adult mice in which the α-subunit of the epithelial sodium channel was conditionally deleted in taste buds (αENaC knockout). The terminal fields of all three nerves in the NST were up to 2.7 times greater in αENaC knock-out mice compared with the respective field volumes in control mice. The shapes of the fields were similar between the two groups; however, the density and spread of labels were greater in αENaC knock-out mice. Overall, our results show that disruption of the afferent taste signal to sodium salts disrupts the normal age-dependent "pruning" of all terminal fields, which could lead to alterations in sensory coding and taste-related behaviors. Neural activity plays a major role in the development of sensory circuits in the mammalian brain. To date, there has been no direct test of whether taste-elicited neural activity has a role in shaping central gustatory circuits. However, recently developed genetic tools now allow an assessment of how specific taste stimuli, in this case sodium salt taste, play a role

  6. The role of long-term label-retaining cells in the regeneration of adult mouse kidney after ischemia/reperfusion injury.

    Science.gov (United States)

    Liu, Xiangchun; Liu, Haiying; Sun, Lina; Chen, Zhixin; Nie, Huibin; Sun, Aili; Liu, Gang; Guan, Guangju

    2016-04-30

    Label-retaining cells (LRCs) have been recognized as rare stem and progenitor-like cells, but their complex biological features in renal repair at the cellular level have never been reported. This study was conducted to evaluate whether LRCs in kidney are indeed renal stem/progenitor cells and to delineate their potential role in kidney regeneration. We utilized a long-term pulse chase of 5-bromo-2'-deoxyuridine (BrdU)-labeled cells in C57BL/6J mice to identify renal LRCs. We tracked the precise morphological characteristics and locations of BrdU(+)LRCs by both immunohistochemistry and immunofluorescence. To examine whether these BrdU(+)LRCs contribute to the repair of acute kidney injury, we analyzed biological characteristics of BrdU(+)LRCs in mice after ischemia/reperfusion (I/R) injury. The findings revealed that the nuclei of BrdU(+) LRCs exhibited different morphological characteristics in normal adult kidneys, including nuclei in pairs or scattered, fragmented or intact, strongly or weakly positive. Only 24.3 ± 1.5 % of BrdU(+) LRCs co-expressed with Ki67 and 9.1 ± 1.4 % of BrdU(+) LRCs were positive for TUNEL following renal I/R injury. Interestingly, we found that newly regenerated cells formed a niche-like structure and LRCs in pairs tended to locate in this structure, but the number of those LRCs was very low. We found a few scattered LRCs co-expressed Lotus tetragonolobus agglutinin (LTA) in the early phase of injury, suggesting differentiation of those LRCs in mouse kidney. Our findings suggest that LRCs are not a simple type of slow-cycling cells in adult kidneys, indicating a limited role of these cells in the regeneration of I/R injured kidney. Thus, LRCs cannot reliably be considered stem/progenitor cells in the regeneration of adult mouse kidney. When researchers use this technique to study the cellular basis of renal repair, these complex features of renal LRCs and the purity of real stem cells among renal LRCs should be considered.

  7. Metabolism of lipoproteins by human fetal hepatocytes

    International Nuclear Information System (INIS)

    Carr, B.R.

    1987-01-01

    The rate of clearance of lipoproteins from plasma appears to play a role in the development of atherogenesis. The liver may account for as much as two thirds of the removal of low-density lipoprotein and one third of the clearance of high-density lipoprotein in certain animal species and humans, mainly by receptor-mediated pathways. The purpose of the present investigation was to determine if human fetal hepatocytes maintained in vitro take up and degrade lipoproteins. We first determined that the maximal binding capacity of iodine 125-iodo-LDL was approximately 300 ng of low-density lipoprotein protein/mg of membrane protein and an apparent dissociation constant of approximately 60 micrograms low-density lipoprotein protein/ml in membranes prepared from human fetal liver. We found that the maximal uptake of [ 125 I]iodo-LDL and [ 125 I]iodo-HDL by fetal hepatocytes occurred after 12 hours of incubation. Low-density lipoprotein uptake preceded the appearance of degradation products by 4 hours, and thereafter the degradation of low-density lipoprotein increased linearly for at least 24 hours. In contrast, high-density lipoprotein was not degraded to any extent by fetal hepatocytes. [ 125 I]Iodo-LDL uptake and degradation were inhibited more than 75% by preincubation with low-density lipoprotein but not significantly by high-density lipoprotein, whereas [ 125 I]iodo-HDL uptake was inhibited 70% by preincubation with high-density lipoprotein but not by low-density lipoprotein. In summary, human fetal hepatocytes take up and degrade low-density lipoprotein by a receptor-mediated process similar to that described for human extrahepatic tissues

  8. 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

  9. 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.

  10. Hepatocyte membrane injury and bleb formation following low dose comfrey toxicity in rats.

    Science.gov (United States)

    Yeong, M L; Wakefield, S J; Ford, H C

    1993-04-01

    Comfrey, a popular herbal remedy, contains hepatotoxic pyrrolizidine alkaloids and has been implicated in recent human toxicity. Although alkaloids from other plant sources have been extensively researched, studies on the hepatotoxic effects of comfrey alkaloids are scant. The effects of high dose comfrey toxicity have been studied and the present investigation was undertaken to identify changes associated with relatively low dose toxicity. Eight young adult rats were dosed weekly for six weeks with 50 mg/kg of comfrey derived alkaloids. The animals were dissected one week after the last dose and the livers examined by light and electron microscopy. Changes at the light microscopic level showed vascular congestion, mild zone 3 necrosis and loss of definition of hepatocyte cellular membranes. Extensive ultrastructural abnormalities were identified in the form of endothelial sloughing and the loss of hepatocyte microvilli. A striking finding was florid bleb formation on the sinusoidal borders of hepatocytes. Many blebs were shed into the space of Disse and extruded to fill, and sometimes occlude, sinusoidal lumina. Platelets were frequently found in areas of bleb formation. There was evidence of late damage in collagenization of Disse's space. Hepatocyte bleb formation is known to occur under a variety of pathological conditions but there is little to no information in the literature on the effects, if any, of bleb formation on fibrogenesis and the microcirculation and its role in the pathogenesis of liver disease. The pyrrolizidine alkaloids of comfrey may serve as an experimental tool to study the process of bleb formation and the intimate relationship between hepatocyte and sinusoidal injury in the liver.

  11. Amplification of R-spondin1 signaling induces granulosa cell fate defects and cancers in mouse adult ovary

    NARCIS (Netherlands)

    De Cian, M-C; Pauper, E.; Bandiera, R.; Vidal, V. P. I.; Sacco, S.; Gregoire, E. P.; Chassot, A-A; Panzolini, C.; WILHELM, D; Pailhoux, E.; Youssef, Sameh A.; de Bruin, A.; Teerds, K.; Schedl, A.; Gillot, I.; Chaboissier, M-C

    2017-01-01

    R-spondin1 is a secreted regulator of WNT signaling, involved in both embryonic development and homeostasis of adult organs. It can have a dual role, acting either as a mitogen or as a tumor suppressor. During ovarian development, Rspo1 is a key factor required for sex determination and

  12. Hepatocyte growth factor in renal failure: promise and reality.

    Science.gov (United States)

    Vargas, G A; Hoeflich, A; Jehle, P M

    2000-04-01

    Can science discover some secrets of Greek mythology? In the case of Prometheus, we can now suppose that his amazing hepatic regeneration was caused by a peptide growth factor called hepatocyte growth factor (HGF). Increasing evidence indicates that HGF acts as a multifunctional cytokine on different cell types. This review addresses the molecular mechanisms that are responsible for the pleiotropic effects of HGF. HGF binds with high affinity to its specific tyrosine kinase receptor c-met, thereby stimulating not only cell proliferation and differentiation, but also cell migration and tumorigenesis. The three fundamental principles of medicine-prevention, diagnosis, and therapy-may be benefited by the rational use of HGF. In renal tubular cells, HGF induces mitogenic and morphogenetic responses. In animal models of toxic or ischemic acute renal failure, HGF acts in a renotropic and nephroprotective manner. HGF expression is rapidly up-regulated in the remnant kidney of nephrectomized rats, inducing compensatory growth. In a mouse model of chronic renal disease, HGF inhibits the progression of tubulointerstitial fibrosis and kidney dysfunction. Increased HGF mRNA transcripts were detected in mesenchymal and tubular epithelial cells of rejecting kidney. In transplanted patients, elevated HGF levels may indicate renal rejection. When HGF is considered as a therapeutic agent in human medicine, for example, to stimulate kidney regeneration after acute injury, strategies need to be developed to stimulate cell regeneration and differentiation without an induction of tumorigenesis.

  13. Mechanism of free radical generation in platelets and primary hepatocytes: A novel electron spin resonance study.

    Science.gov (United States)

    Wang, Chiun-Lang; Yang, Po-Sheng; Tsao, Jeng-Ting; Jayakumar, Thanasekaran; Wang, Meng-Jiy; Sheu, Joen-Rong; Chou, Duen-Suey

    2018-01-01

    Oxygen free radicals have been implicated in the pathogenesis of toxic liver injury and are thought to be involved in cardiac dysfunction in the cirrhotic heart. Therefore, direct evidence for the electron spin resonance (ESR) detection of how D‑galactosamine (GalN), an established experimental hepatotoxic substance, induced free radicals formation in platelets and primary hepatocytes is presented in the present study. ESR results demonstrated that GalN induced hydroxyl radicals (OH•) in a resting human platelet suspension; however, radicals were not produced in a cell free Fenton reaction system. The GalN‑induced OH• formation was significantly inhibited by the cyclooxygenase (COX) inhibitor indomethasin, though it was not affected by the lipoxygenase (LOX) or cytochrome P450 inhibitors, AA861 and 1‑aminobenzotriazole (ABT), in platelets. In addition, the present study demonstrated that baicalein induced semiquinone free radicals in platelets, which were significantly reduced by the COX inhibitor without affecting the formed OH•. In the mouse primary hepatocytes, the formation of arachidonic acid (AA) induced carbon‑centered radicals that were concentration dependently enhanced by GalN. These radicals were inhibited by AA861, though not affected by indomethasin or ABT. In addition, GalN did not induce platelet aggregation prior to or following collagen pretreatment in human platelets. The results of the present study indicated that GalN and baicalein may induce OH• by COX and LOX in human platelets. GalN also potentiated AA induced carbon‑centered radicals in hepatocytes via cytochrome P450. The present study presented the role of free radicals in the pathophysiological association between platelets and hepatocytes.

  14. Augmentation of DHCR24 expression by hepatitis C virus infection facilitates viral replication in hepatocytes.

    Science.gov (United States)

    Takano, Takashi; Tsukiyama-Kohara, Kyoko; Hayashi, Masahiro; Hirata, Yuichi; Satoh, Masaaki; Tokunaga, Yuko; Tateno, Chise; Hayashi, Yukiko; Hishima, Tsunekazu; Funata, Nobuaki; Sudoh, Masayuki; Kohara, Michinori

    2011-09-01

    We characterized the role of 24-dehydrocholesterol reductase (DHCR24) in hepatitis C virus infection (HCV). DHCR24 is a cholesterol biosynthetic enzyme and cholesterol is a major component of lipid rafts, which is reported to play an important role in HCV replication. Therefore, we examined the potential of DHCR24 as a target for novel HCV therapeutic agents. We examined DHCR24 expression in human hepatocytes in both the livers of HCV-infected patients and those of chimeric mice with human hepatocytes. We targeted DHCR24 with siRNA and U18666A which is an inhibitor of both DHCR24 and cholesterol synthesis. We measured the level of HCV replication in these HCV replicon cell lines and HCV infected cells. U18666A was administrated into chimeric mice with humanized liver, and anti-viral effects were assessed. Expression of DHCR24 was induced by HCV infection in human hepatocytes in vitro, and in human hepatocytes of chimeric mouse liver. Silencing of DHCR24 by siRNA decreased HCV replication in replicon cell lines and HCV JFH-1 strain-infected cells. Treatment with U18666A suppressed HCV replication in the replicon cell lines. Moreover, to evaluate the anti-viral effect of U18666A in vivo, we administrated U18666A with or without pegylated interferon to chimeric mice and observed an inhibitory effect of U18666A on HCV infection and a synergistic effect with interferon. DHCR24 is an essential host factor which augmented its expression by HCV infection, and plays a significant role in HCV replication. DHCR24 may serve as a novel anti-HCV drug target. Copyright © 2010 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

  15. Glucose metabolism and recycling by hepatocytes of OB/OB and ob/ob mice

    International Nuclear Information System (INIS)

    Lahtela, J.T.; Wals, P.A.; Katz, J.

    1990-01-01

    Hepatocytes were prepared from livers of ob/ob (obese diabetic) mice and their lean (OB/OB) siblings that had been fasted for 24 h. The hepatocytes were incubated with [U-14C, 2-3H]-, [U-14C, 3-3H]-, and [U-14C, 6-3H]glucose at concentrations from 20 to 120 mM. 14C was recovered mainly in CO2, glycogen, and lactate. Tritium was recovered in water and glycogen. The yield in labeled products from [2-3H]glucose ranged from two to three times that from [U-14C]glucose. The yields from [3-3H]- and [6-3H]glucose were similar, and 1.3-1.7 times that from [U-14C]glucose. At 40 mM, total utilization of glucose by obese mice was about twice that for lean mice, but there was little difference at 120 mM. The rate of recycling between glucose and glucose 6-phosphate was calculated. An equation to calculate the rate of recycling of glucose from the 2-3H/U-14C ratio in glycogen is derived in the APPENDIX. Our results show that (1) the utilization of glucose by hepatocytes from obese diabetic mice exceeds that of their lean controls, (2) the rate of glucose phosphorylation in both groups greatly exceeds glucose uptake and the rate of glycogen synthesis, (3) glucose phosphorylation represents a difference between a high glucokinase rate and hydrolysis of glucose 6-phosphate, and (4) recycling of glucose carbon between glucose 6-phosphate and pyruvate occurs within mouse hepatocytes

  16. Hepatocyte transplantation improves early survival after partial hepatic resection and irradiation

    International Nuclear Information System (INIS)

    Guha, C.; Sharma, A.; Alfieri, A.; Guha, U.; Sokhi, R.; Gagandeep, S.; Gupta, S.; Vikram, B.; RoyChowdhury, J.

    1997-01-01

    Purpose: Radiation therapy (RT) is limited in its role as an adjuvant therapy of intrahepatic malignancies because of lower tolerance of human liver to irradiation (TD (5(5)) -TD (50(5)) ∼ 30-40 Gy). Although, surgical resection of primary or metastatic hepatic tumors has been shown to prolong survival, it is often limited by the presence of residual disease. RT could potentially improve survival of patients with positive surgical margins. However, radiation damage to the liver may be enhanced by hepatocellular proliferation induced by partial hepatic (PH) resection. We hypothesize that hepatocyte transplantation would be able to provide metabolic support and modulate the development of radiation-induced liver disease post-resection. The present study was designed to test the potential of hepatocyte transplantation in modifying the outcome of hepatocellular damage induced by PH and RT. Methods: Adult male Fischer 344 rats (Charles River) received hepatic irradiation of 50 Gy in a single fraction, after surgical exposure and shielding of the stomach and intestine, using a 320 MGC Philips orthovoltage unit. Immediately following irradiation, a two-third partial hepatectomy was performed. Four days post-radiation, the treatment group was injected with 5 x 10 6 syngeneic hepatocytes into the splenic pulp after a left subcostal incision, which allows homogeneous liver engraftment of the transplanted hepatocytes. Hematoxylin and eosin stains of liver biopsies, performed at various time points (3 days, 1, 2, 3 weeks or, anytime when animals died) were used for histologic evaluation. Time-adjusted survival was calculated from the date of irradiation by the product-limit Kaplan-Meier method, adjusting the denominator at every time point for the number of rats at risk. Results: Eight weeks after RT, 30% (n = 11) of the control animals (PH + 50 Gy) were alive compared to 100% (n = 9) of the transplant recipients (p <0.05). The median survival of the control group was 15

  17. Dose of Phenobarbital and Age of Treatment at Early Life are Two Key Factors for the Persistent Induction of Cytochrome P450 Enzymes in Adult Mouse Liver.

    Science.gov (United States)

    Tien, Yun-Chen; Liu, Ke; Pope, Chad; Wang, Pengcheng; Ma, Xiaochao; Zhong, Xiao-bo

    2015-12-01

    Drug treatment of neonates and infants and its long-term consequences on drug responses have emerged in recent years as a major challenge for health care professionals. In the current study, we use phenobarbital as a model drug and mouse as an in vivo model to demonstrate that the dose of phenobarbital and age of treatment are two key factors for the persistent induction of gene expression and consequential increases of enzyme activities of Cyp2b, Cyp2c, and Cyp3a in adult livers. We show that phenobarbital treatment at early life of day 5 after birth with a low dose (phenobarbital treatment with a high dose (>200 mg/kg) significantly increases expression and enzyme activities of these P450s in adult liver. We also demonstrate that phenobarbital treatment before day 10 after birth, but not at later ages, significantly increases mRNAs, proteins, and enzyme activities of the tested P450s. Such persistent induction of P450 gene expression and enzyme activities in adult livers by phenobarbital treatment only occurs within a sensitive age window early in life. The persistent induction in gene expression and enzyme activities is higher in female mice than in male mice for Cyp2b10 but not for Cyp2c29 and Cyp3a11. These results will stimulate studies to evaluate the long-term impacts of drug treatment with different doses at neonatal and infant ages on drug metabolism, therapeutic efficacy, and drug-induced toxicity throughout the rest of life. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

  18. 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

  19. Lead induces similar gene expression changes in brains of gestationally exposed adult mice and in neurons differentiated from mouse embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Francisco Javier Sánchez-Martín

    Full Text Available Exposure to environmental toxicants during embryonic life causes changes in the expression of developmental genes that may last for a lifetime and adversely affect the exposed individual. Developmental exposure to lead (Pb, an ubiquitous environmental contaminant, causes deficits in cognitive functions and IQ, behavioral effects, and attention deficit hyperactivity disorder (ADHD. Long-term effects observed after early life exposure to Pb include reduction of gray matter, alteration of myelin structure, and increment of criminal behavior in adults. Despite growing research interest, the molecular mechanisms responsible for the effects of lead in the central nervous system are still largely unknown. To study the molecular changes due to Pb exposure during neurodevelopment, we exposed mice to Pb in utero and examined the expression of neural markers, neurotrophins, transcription factors and glutamate-related genes in hippocampus, cortex, and thalamus at postnatal day 60. We found that hippocampus was the area where gene expression changes due to Pb exposure were more pronounced. To recapitulate gestational Pb exposure in vitro, we differentiated mouse embryonic stem cells (ESC into neurons and treated ESC-derived neurons with Pb for the length of the differentiation process. These neurons expressed the characteristic neuronal markers Tubb3, Syp, Gap43, Hud, Ngn1, Vglut1 (a marker of glutamatergic neurons, and all the glutamate receptor subunits, but not the glial marker Gafp. Importantly, several of the changes observed in Pb-exposed mouse brains in vivo were also observed in Pb-treated ESC-derived neurons, including those affecting expression of Ngn1, Bdnf exon IV, Grin1, Grin2D, Grik5, Gria4, and Grm6. We conclude that our ESC-derived model of toxicant exposure during neural differentiation promises to be a useful model to analyze mechanisms of neurotoxicity induced by Pb and other environmental agents.

  20. Characterization of Aromatase Expression in the Adult Male and Female Mouse Brain. I. Coexistence with Oestrogen Receptors α and β, and Androgen Receptors

    Science.gov (United States)

    Stanić, Davor; Dubois, Sydney; Chua, Hui Kheng; Tonge, Bruce; Rinehart, Nicole; Horne, Malcolm K.; Boon, Wah Chin

    2014-01-01

    Aromatase catalyses the last step of oestrogen synthesis. There is growing evidence that local oestrogens influence many brain regions to modulate brain development and behaviour. We examined, by immunohistochemistry, the expression of aromatase in the adult male and female mouse brain, using mice in which enhanced green fluorescent protein (EGFP) is transcribed following the physiological activation of the Cyp19A1 gene. EGFP-immunoreactive processes were distributed in many brain regions, including the bed nucleus of the stria terminalis, olfactory tubercle, medial amygdaloid nucleus and medial preoptic area, with the densest distributions of EGFP-positive cell bodies in the bed nucleus and medial amygdala. Differences between male and female mice were apparent, with the density of EGFP-positive cell bodies and fibres being lower in some brain regions of female mice, including the bed nucleus and medial amygdala. EGFP-positive cell bodies in the bed nucleus, lateral septum, medial amygdala and hypothalamus co-expressed oestrogen receptor (ER) α and β, or the androgen receptor (AR), although single-labelled EGFP-positive cells were also identified. Additionally, single-labelled ERα−, ERβ- or AR-positive cell bodies often appeared to be surrounded by EGFP-immunoreactive nerve fibres/terminals. The widespread distribution of EGFP-positive cell bodies and fibres suggests that aromatase signalling is common in the mouse brain, and that locally synthesised brain oestrogens could mediate biological effects by activating pre- and post-synaptic oestrogen α and β receptors, and androgen receptors. The higher number of EGFP-positive cells in male mice may indicate that the autocrine and paracrine effects of oestrogens are more prominent in males than females. PMID:24646567

  1. Hepatocyte Hyperproliferation upon Liver-Specific Co-disruption of Thioredoxin-1, Thioredoxin Reductase-1, and Glutathione Reductase

    Directory of Open Access Journals (Sweden)

    Justin R. Prigge

    2017-06-01

    Full Text Available Energetic nutrients are oxidized to sustain high intracellular NADPH/NADP+ ratios. NADPH-dependent reduction of thioredoxin-1 (Trx1 disulfide and glutathione disulfide by thioredoxin reductase-1 (TrxR1 and glutathione reductase (Gsr, respectively, fuels antioxidant systems and deoxyribonucleotide synthesis. Mouse livers lacking both TrxR1 and Gsr sustain these essential activities using an NADPH-independent methionine-consuming pathway; however, it remains unclear how this reducing power is distributed. Here, we show that liver-specific co-disruption of the genes encoding Trx1, TrxR1, and Gsr (triple-null causes dramatic hepatocyte hyperproliferation. Thus, even in the absence of Trx1, methionine-fueled glutathione production supports hepatocyte S phase deoxyribonucleotide production. Also, Trx1 in the absence of TrxR1 provides a survival advantage to cells under hyperglycemic stress, suggesting that glutathione, likely via glutaredoxins, can reduce Trx1 disulfide in vivo. In triple-null livers like in many cancers, deoxyribonucleotide synthesis places a critical yet relatively low-volume demand on these reductase systems, thereby favoring high hepatocyte turnover over sustained hepatocyte integrity.

  2. Sex-comparative study of mouse cerebellum physiology under adult-onset hypothyroidism: The significance of GC-MS metabolomic data normalization in meta-analysis.

    Science.gov (United States)

    Maga-Nteve, Christoniki; Vasilopoulou, Catherine G; Constantinou, Caterina; Margarity, Marigoula; Klapa, Maria I

    2017-01-15

    A systematic data quality validation and normalization strategy is an important component of the omic profile meta-analysis, ensuring comparability of the profiles and exclusion of experimental biases from the derived biological conclusions. In this study, we present the normalization methodology applied on the sets of cerebellum gas chromatography-mass spectrometry metabolic profiles of 124days old male and female animals in an adult-onset-hypothyroidism (AOH) mouse model before combining them into a sex-comparative analysis. The employed AOH model concerns the monitoring of the brain physiology of Balb/cJ mice after eight-week administration of 1%w/v KClO 4 in the drinking water, initiated on the 60th day of their life. While originating from the same animal study, the tissues of the two sexes were processed and their profiles acquired and analyzed at different time periods. Hence, the previously published profile set of male mice was first re-annotated based on the presently available resources. Then, after being validated as acquired under the same analytical conditions, both profiles sets were corrected for derivatization biases and filtered for low-confidence measurements based on the same criteria. The final normalized 73-metabolite profiles contribute to the currently few available omic datasets of the AOH effect on brain molecular physiology, especially with respect to sex differentiation. Multivariate statistical analysis indicated one (unknown) and three (succinate, benzoate, myristate) metabolites with significantly higher and lower, respectively, cerebellum concentration in the hypothyroid compared to the euthyroid female mice. The respective numbers for the males were two and 24. Comparison of the euthyroid cerebellum metabolic profiles between the two sexes indicated 36 metabolites, including glucose, myo- and scyllo-inositol, with significantly lower concentration in the females versus the males. This implies that the female mouse cerebellum has

  3. Adult Mouse DRG Explant and Dissociated Cell Models to Investigate Neuroplasticity and Responses to Environmental Insults Including Viral Infection.

    Science.gov (United States)

    Fornaro, Michele; Sharthiya, Harsh; Tiwari, Vaibhav

    2018-03-09

    This protocol describes an ex vivo model of mouse-derived dorsal root ganglia (DRG) explant and in vitro DRG-derived co-culture of dissociated sensory neurons and glial satellite cells. These are useful and versatile models to investigate a variety of biological responses associated with physiological and pathological conditions of the peripheral nervous system (PNS) ranging from neuron-glial interaction, neuroplasticity, neuroinflammation, and viral infection. The usage of DRG explant is scientifically advantageous compared to simplistic single cells models for multiple reasons. For instance, as an organotypic culture, the DRG explant allows ex vivo transfer of an entire neuronal network including the extracellular microenvironment that play a significant role in all the neuronal and glial functions. Further, DRG explants can also be maintained ex vivo for several days and the culture conditions can be perturbed as desired. In addition, the harvested DRG can be further dissociated into an in vitro co-culture of primary sensory neurons and satellite glial cells to investigate neuronal-glial interaction, neuritogenesis, axonal cone interaction with the extracellular microenvironment, and more general, any aspect associated with the neuronal metabolism. Therefore, the DRG-explant system offers a great deal of flexibility to study a wide array of events related to biological, physiological, and pathological conditions in a cost-effective manner.

  4. Electrophysiological and gene expression characterization of the ontogeny of nestin-expressing cells in the adult mouse midbrain

    Directory of Open Access Journals (Sweden)

    Anupama Dey

    2017-08-01

    Full Text Available The birth of new neurons, or neurogenesis, in the adult midbrain is important for progressing dopamine cell-replacement therapies for Parkinson's disease. Most studies suggest newborn cells remain undifferentiated or differentiate into glia within the adult midbrain. However, some studies suggest nestin + neural precursor cells (NPCs have a propensity to generate new neurons here. We sought to confirm this by administering tamoxifen to adult NesCreERT2/R26eYFP transgenic mice, which permanently labelled adult nestin-expressing cells and their progeny with enhanced yellow fluorescent protein (eYFP. eYFP+ midbrain cells were then characterized 1–32 weeks later in acutely prepared brain slices using whole-cell patch clamp electrophysiology combined with single-cell RT-qPCR. Most eYFP+ cells exhibited a mature neuronal phenotype with large amplitude fast action potentials (APs, spontaneous post-synaptic currents (sPSCs, and expression of ‘mature’ neuronal genes (NeuN, Gad1, Gad2 and/or VGLUT2. This was the case even at the earliest time-point following tamoxifen (i.e. 1 week. In comparison to neighboring eYFP− (control cells, eYFP+ cells discharged more APs per unit current injection, and had faster AP time-to-peak, hyperpolarized resting membrane potential, smaller membrane capacitance and shorter duration sPSCs. eYFP+ cells were also differentiated from eYFP− cells by increased expression of ‘immature’ pro-neuronal genes (Pax6, Ngn2 and/or Msx1. However, further analyses failed to reveal evidence of a place of birth, neuronal differentiation, maturation and integration indicative of classical neurogenesis. Thus our findings do not support the notion that nestin + NPCs in the adult SNc and midbrain generate new neurons via classical neurogenesis. Rather, they raise the possibility that mature neurons express nestin under unknown circumstances, and that this is associated with altered physiology and gene expression.

  5. 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.

  6. Late gestational hypoxia and a postnatal high salt diet programs endothelial dysfunction and arterial stiffness in adult mouse offspring.

    Science.gov (United States)

    Walton, Sarah L; Singh, Reetu R; Tan, Tiffany; Paravicini, Tamara M; Moritz, Karen M

    2016-03-01

    Gestational hypoxia and high dietary salt intake have both been associated with impaired vascular function in adulthood. Using a mouse model of prenatal hypoxia, we examined whether a chronic high salt diet had an additive effect in promoting vascular dysfunction in offspring. Pregnant CD1 dams were placed in a hypoxic chamber (12% O2) or housed under normal conditions (21% O2) from embryonic day 14.5 until birth. Gestational hypoxia resulted in a reduced body weight for both male and female offspring at birth. This restriction in body weight persisted until weaning, after which the animals underwent catch-up growth. At 10 weeks of age, a subset of offspring was placed on a high salt diet (5% NaCl). Pressurized myography of mesenteric resistance arteries at 12 months of age showed that both male and female offspring exposed to maternal hypoxia had significantly impaired endothelial function, as demonstrated by impaired vasodilatation to ACh but not sodium nitroprusside. Endothelial dysfunction caused by prenatal hypoxia was not exacerbated by postnatal consumption of a high salt diet. Prenatal hypoxia increased microvascular stiffness in male offspring. The combination of prenatal hypoxia and a postnatal high salt diet caused a leftward shift in the stress-strain relationship in both sexes. Histopathological analysis of aortic sections revealed a loss of elastin integrity and increased collagen, consistent with increased vascular stiffness. These results demonstrate that prenatal hypoxia programs endothelial dysfunction in both sexes. A chronic high salt diet in postnatal life had an additive deleterious effect on vascular mechanics and structural characteristics in both sexes. © 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.

  7. Human mesenchymal stem cells towards non-alcoholic steatohepatitis in an immunodeficient mouse model

    International Nuclear Information System (INIS)

    Winkler, Sandra; Borkham-Kamphorst, Erawan; Stock, Peggy; Brückner, Sandra; Dollinger, Matthias; Weiskirchen, Ralf; Christ, Bruno

    2014-01-01

    Non-alcoholic steatohepatitis (NASH) is a frequent clinical picture characterised by hepatic inflammation, lipid accumulation and fibrosis. When untreated, NASH bears a high risk of developing liver cirrhosis and consecutive hepatocellular carcinoma requiring liver transplantation in its end-stage. However, donor organ scarcity has prompted the search for alternatives, of which hepatocyte or stem cell-derived hepatocyte transplantation are regarded auspicious options of treatment. Mesenchymal stem cells (MSC) are able to differentiate into hepatocyte-like cells and thus may represent an alternative cell source to primary hepatocytes. In addition these cells feature anti-inflammatory and pro-regenerative characteristics, which might favour liver recovery from NASH. The aim of this study was to investigate the potential benefit of hepatocyte-like cells derived from human bone marrow MSC in a mouse model of diet-induced NASH. Seven days post-transplant, human hepatocyte-like cells were found in the mouse liver parenchyma. Triglyceride depositions were lowered in the liver but restored to normal in the blood. Hepatic inflammation was attenuated as verified by decreased expression of the acute phase protein serum amyloid A, inflammation-associated markers (e.g. lipocalin 2), as well as the pro-inflammatory cytokine TNFα. Moreover, the proliferation of host hepatocytes that indicate the regenerative capacity in livers receiving cell transplants was enhanced. Transplantation of MSC-derived human hepatocyte-like cells corrects NASH in mice by restoring triglyceride depositions, reducing inflammation and augmenting the regenerative capacity of the liver. - Highlights: • First time to show NASH in an immune-deficient mouse model. • Human MSC attenuate NASH and improve lipid homeostasis. • MSC act anti-fibrotic and augment liver regeneration by stimulation of proliferation. • Pre-clinical assessment of human MSC for stem cell-based therapy of NASH

  8. Human mesenchymal stem cells towards non-alcoholic steatohepatitis in an immunodeficient mouse model

    Energy Technology Data Exchange (ETDEWEB)

    Winkler, Sandra, E-mail: sandra.pelz@medizin.uni-leipzig.de [Applied Molecular Hepatology Laboratory, Department of Visceral, Transplantation, Thoracic and Vascular Surgery, University Hospital Leipzig, Liebigstraße 21, D-04103 Leipzig (Germany); Borkham-Kamphorst, Erawan, E-mail: ekamphorst@ukaachen.de [Institute of Clinical Chemistry and Pathobiochemistry, RWTH University Hospital Aachen, Pauwelsstraße 30, D-52074 Aachen (Germany); Stock, Peggy, E-mail: peggy.stock@medizin.uni-leipzig.de [Applied Molecular Hepatology Laboratory, Department of Visceral, Transplantation, Thoracic and Vascular Surgery, University Hospital Leipzig, Liebigstraße 21, D-04103 Leipzig (Germany); Brückner, Sandra, E-mail: sandra.brueckner@medizin.uni-leipzig.de [Applied Molecular Hepatology Laboratory, Department of Visceral, Transplantation, Thoracic and Vascular Surgery, University Hospital Leipzig, Liebigstraße 21, D-04103 Leipzig (Germany); Dollinger, Matthias, E-mail: matthias.dollinger@uniklinik-ulm.de [Department for Internal Medicine I, University Hospital Ulm, Albert-Einstein-Allee 23, D-89081 Ulm (Germany); Weiskirchen, Ralf, E-mail: rweiskirchen@ukaachen.de [Institute of Clinical Chemistry and Pathobiochemistry, RWTH University Hospital Aachen, Pauwelsstraße 30, D-52074 Aachen (Germany); Christ, Bruno, E-mail: bruno.christ@medizin.uni-leipzig.de [Applied Molecular Hepatology Laboratory, Department of Visceral, Transplantation, Thoracic and Vascular Surgery, University Hospital Leipzig, Liebigstraße 21, D-04103 Leipzig (Germany); Translational Centre for Regenerative Medicine (TRM), University of Leipzig, Leipzig (Germany)

    2014-08-15

    Non-alcoholic steatohepatitis (NASH) is a frequent clinical picture characterised by hepatic inflammation, lipid accumulation and fibrosis. When untreated, NASH bears a high risk of developing liver cirrhosis and consecutive hepatocellular carcinoma requiring liver transplantation in its end-stage. However, donor organ scarcity has prompted the search for alternatives, of which hepatocyte or stem cell-derived hepatocyte transplantation are regarded auspicious options of treatment. Mesenchymal stem cells (MSC) are able to differentiate into hepatocyte-like cells and thus may represent an alternative cell source to primary hepatocytes. In addition these cells feature anti-inflammatory and pro-regenerative characteristics, which might favour liver recovery from NASH. The aim of this study was to investigate the potential benefit of hepatocyte-like cells derived from human bone marrow MSC in a mouse model of diet-induced NASH. Seven days post-transplant, human hepatocyte-like cells were found in the mouse liver parenchyma. Triglyceride depositions were lowered in the liver but restored to normal in the blood. Hepatic inflammation was attenuated as verified by decreased expression of the acute phase protein serum amyloid A, inflammation-associated markers (e.g. lipocalin 2), as well as the pro-inflammatory cytokine TNFα. Moreover, the proliferation of host hepatocytes that indicate the regenerative capacity in livers receiving cell transplants was enhanced. Transplantation of MSC-derived human hepatocyte-like cells corrects NASH in mice by restoring triglyceride depositions, reducing inflammation and augmenting the regenerative capacity of the liver. - Highlights: • First time to show NASH in an immune-deficient mouse model. • Human MSC attenuate NASH and improve lipid homeostasis. • MSC act anti-fibrotic and augment liver regeneration by stimulation of proliferation. • Pre-clinical assessment of human MSC for stem cell-based therapy of NASH.

  9. Maternal Active Mastication during Prenatal Stress Ameliorates Prenatal Stress-Induced Lower Bone Mass in Adult Mouse Offspring.

    Science.gov (United States)

    Azuma, Kagaku; Ogura, Minori; Kondo, Hiroko; Suzuki, Ayumi; Hayashi, Sakurako; Iinuma, Mitsuo; Onozuka, Minoru; Kubo, Kin-Ya

    2017-01-01

    Chronic psychological stress is a risk factor for osteoporosis. Maternal active mastication during prenatal stress attenuates stress response. The aim of this study is to test the hypothesis that maternal active mastication influences the effect of prenatal stress on bone mass and bone microstructure in adult offspring. Pregnant ddY mice were randomly divided into control, stress, and stress/chewing groups. Mice in the stress and stress/chewing groups were placed in a ventilated restraint tube for 45 minutes, 3 times a day, and was initiated on day 12 of gestation and continued until delivery. Mice in the stress/chewing group were allowed to chew a wooden stick during the restraint stress period. The bone response of 5-month-old male offspring was evaluated using quantitative micro-CT, bone histomorphometry, and biochemical markers. Prenatal stress resulted in significant decrease of trabecular bone mass in both vertebra and distal femur of the offspring. Maternal active mastication during prenatal stress attenuated the reduced bone formation and increased bone resorption, improved the lower trabecular bone volume and bone microstructural deterioration induced by prenatal stress in the offspring. These findings indicate that maternal active mastication during prenatal stress can ameliorate prenatal stress-induced lower bone mass of the vertebra and femur in adult offspring. Active mastication during prenatal stress in dams could be an effective coping strategy to prevent lower bone mass in their offspring.

  10. Effects of perinatal daidzein exposure on subsequent behavior and central estrogen receptor α expression in the adult male mouse.

    Science.gov (United States)

    Yu, Chengjun; Tai, Fadao; Zeng, Shuangyan; Zhang, Xia

    2013-06-03

    Daidzein is one of the most important isoflavones present in soy and it is unique as it can be further metabolized to equol, a compound with greater estrogenic activity than other isoflavones. The potential role of daidzein in the prevention of some chronic diseases has drawn public attention and increased its consumption in human, including in pregnant women and adolescent. It is unclear whether perinatal exposure to daidzein through maternal diets affects subsequent behavior and central estrogen receptor α (ERα) expression in male adults. Following developmental exposure to daidzein through maternal diets during perinatal period, subsequent anxiety-like behavior, social behavior, spatial learning and memory of male mice at adulthood were assessed using a series of tests. The levels of central ER α expression were also examined using immunocytochemistry. Compared with the controls, adult male mice exposed to daidzein during the perinatal period showed significantly less exploration, higher levels of anxiety and aggression. They also displayed more social investigation for females and a tendency to improve spatial learning and memory. The mice with this early daidzein treatment demonstrated significantly higher levels of ERα expression in several brain regions such as the bed nucleus of the stria terminalis, medial preoptic, arcuate hypothalamic nucleus and central amygdaloid mucleus, but decreased it in the lateral septum. Our results indicated that perinatal exposure to daidzein enhanced masculinization on male behaviors which is assocciated with alterations in ERα expression levels led by perinatal daidzein exposure. Copyright © 2012 Elsevier Inc. All rights reserved.

  11. Pituitary adenylate cyclase activating polypeptide reduces A-type K+ currents and caspase activity in cultured adult mouse olfactory neurons.

    Science.gov (United States)

    Han, P; Lucero, M T

    2005-01-01

    Pituitary adenylate cyclase activating polypeptide has been shown to reduce apoptosis in neonatal cerebellar and olfactory receptor neurons, however the underlying mechanisms have not been elucidated. In addition, the neuroprotective effects of pituitary adenylate cyclase activating polypeptide have not been examined in adult tissues. To study the effects of pituitary adenylate cyclase activating polypeptide on neurons in apoptosis, we measured caspase activation in adult olfactory receptor neurons in vitro. Interestingly, we found that the protective effects of pituitary adenylate cyclase activating polypeptide were related to the absence of a 4-aminopyridine (IC50=144 microM) sensitive rapidly inactivating potassium current often referred to as A-type current. In the presence of 40 nM pituitary adenylate cyclase activating polypeptide 38, both A-type current and activated caspases were significantly reduced. A-type current reduction by pituitary adenylate cyclase activating polypeptide was blocked by inhibiting the phospholipase C pathway, but not the adenylyl cyclase pathway. Our observation that 5 mM 4-aminopyridine mimicked the caspase inhibiting effects of pituitary adenylate cyclase activating polypeptide indicates that A-type current is involved in apoptosis. This work contributes to our growing understanding that potassium currents are involved with the activation of caspases to affect the balance between cell life and death.

  12. 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.

  13. Long-Term Adult Feline Liver Organoid Cultures for Disease Modeling of Hepatic Steatosis

    Directory of Open Access Journals (Sweden)

    Hedwig S. Kruitwagen

    2017-04-01

    Full Text Available Summary: Hepatic steatosis is a highly prevalent liver disease, yet research is hampered by the lack of tractable cellular and animal models. Steatosis also occurs in cats, where it can cause severe hepatic failure. Previous studies demonstrate the potential of liver organoids for modeling genetic diseases. To examine the possibility of using organoids to model steatosis, we established a long-term feline liver organoid culture with adult liver stem cell characteristics and differentiation potential toward hepatocyte-like cells. Next, organoids from mouse, human, dog, and cat liver were provided with fatty acids. Lipid accumulation was observed in all organoids and interestingly, feline liver organoids accumulated more lipid droplets than human organoids. Finally, we demonstrate effects of interference with β-oxidation on lipid accumulation in feline liver organoids. In conclusion, feline liver organoids can be successfully cultured and display a predisposition for lipid accumulation, making them an interesting model in hepatic steatosis research. : In this study Kruitwagen and colleagues establish and characterize a feline liver organoid culture, which has adult stem cell properties and can be differentiated toward hepatocyte-like cells. They propose liver organoids as a tool to model hepatic steatosis and show that feline liver organoids accumulate more lipids than human organoids when provided with excess fatty acids. Keywords: feline liver organoids, adult liver stem cells, hepatic steatosis, disease modeling, feline hepatic lipidosis, species differences

  14. Long-chain n-3 PUFAs from fish oil enhance resting state brain glucose utilization and reduce anxiety in an adult nonhuman primate, the grey mouse lemur.

    Science.gov (United States)

    Pifferi, Fabien; Dorieux, Olène; Castellano, Christian-Alexandre; Croteau, Etienne; Masson, Marie; Guillermier, Martine; Van Camp, Nadja; Guesnet, Philippe; Alessandri, Jean-Marc; Cunnane, Stephen; Dhenain, Marc; Aujard, Fabienne

    2015-08-01

    Decreased brain content of DHA, the most abundant long-chain n-3 polyunsaturated fatty acid (n-3 LCPUFA) in the brain, is accompanied by severe neurosensorial impairments linked to impaired neurotransmission and impaired brain glucose utilization. In the present study, we hypothesized that increasing n-3 LCPUFA intake at an early age may help to prevent or correct the glucose hypometabolism observed during aging and age-related cognitive decline. The effects of 12 months' supplementation with n-3 LCPUFA on brain glucose utilization assessed by positron emission tomography was tested in young adult mouse lemurs (Microcebus murinus). Cognitive function was tested in parallel in the same animals. Lemurs supplemented with n-3 LCPUFA had higher brain glucose uptake and cerebral metabolic rate of glucose compared with controls in all brain regions. The n-3 LCPUFA-supplemented animals also had higher exploratory activity in an open-field task and lower evidence of anxiety in the Barnes maze. Our results demonstrate for the first time in a nonhuman primate that n-3 LCPUFA supplementation increases brain glucose uptake and metabolism and concomitantly reduces anxiety. Copyright © 2015 by the American Society for Biochemistry and Molecular Biology, Inc.

  15. The lncRNA Malat1 Is Dispensable for Mouse Development but Its Transcription Plays a cis-Regulatory Role in the Adult

    Directory of Open Access Journals (Sweden)

    Bin Zhang

    2012-07-01

    Full Text Available Genome-wide studies have identified thousands of long noncoding RNAs (lncRNAs lacking protein-coding capacity. However, most lncRNAs are expressed at a very low level, and in most cases there is no genetic evidence to support their in vivo function. Malat1 (metastasis associated lung adenocarcinoma transcript 1 is among the most abundant and highly conserved lncRNAs, and it exhibits an uncommon 3′-end processing mechanism. In addition, its specific nuclear localization, developmental regulation, and dysregulation in cancer are suggestive of it having a critical biological function. We have characterized a Malat1 loss-of-function genetic model that indicates that Malat1 is not essential for mouse pre- and postnatal development. Furthermore, depletion of Malat1 does not affect global gene expression, splicing factor level and phosphorylation status, or alternative pre-mRNA splicing. However, among a small number of genes that were dysregulated in adult Malat1 knockout mice, many were Malat1 neighboring genes, thus indicating a potential cis-regulatory role of Malat1 gene transcription.

  16. Effects of Chronic Sleep Restriction during Early Adolescence on the Adult Pattern of Connectivity of Mouse Secondary Motor Cortex123

    Science.gov (United States)

    Billeh, Yazan N.; Bernard, Amy; de Vivo, Luisa; Honjoh, Sakiko; Mihalas, Stefan; Ng, Lydia; Koch, Christof

    2016-01-01

    Abstract Cortical circuits mature in stages, from early synaptogenesis and synaptic pruning to late synaptic refinement, resulting in the adult anatomical connection matrix. Because the mature matrix is largely fixed, genetic or environmental factors interfering with its establishment can have irreversible effects. Sleep disruption is rarely considered among those factors, and previous studies have focused on very young animals and the acute effects of sleep deprivation on neuronal morphology and cortical plasticity. Adolescence is a sensitive time for brain remodeling, yet whether chronic sleep restriction (CSR) during adolescence has long-term effects on brain connectivity remains unclear. We used viral-mediated axonal labeling and serial two-photon tomography to measure brain-wide projections from secondary motor cortex (MOs), a high-order area with diffuse projections. For each MOs target, we calculated the projection fraction, a combined measure of passing fibers and axonal terminals normalized for the size of each target. We found no homogeneous differences in MOs projection fraction between mice subjected to 5 days of CSR during early adolescence (P25–P30, ≥50% decrease in daily sleep, n=14) and siblings that slept undisturbed (n=14). Machine learning algorithms, however, classified animals at significantly above chance levels, indicating that differences between the two groups exist, but are subtle and heterogeneous. Thus, sleep disruption in early adolescence may affect adult brain connectivity. However, because our method relies on a global measure of projection density and was not previously used to measure connectivity changes due to behavioral manipulations, definitive conclusions on the long-term structural effects of early CSR require additional experiments. PMID:27351022

  17. 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.

  18. 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.

  19. Scintigraphic evidence of transplanted hepatocytes in spleen and liver

    International Nuclear Information System (INIS)

    Henne-Bruns, D.; Kremer, B.; Gramminger, K.; Broelsch, C.

    1986-01-01

    In rats suffering from hepatic enzymatic deficiency transplanted hepatocytes could be evidenced scintigraphically in liver, spleen and granulomas. In pigs, however, it is very difficult to demonstrate transplanted hepatocytes by scintiscanning because of the thickness of the tissues and the high background radiation in large animals

  20. Targeted deletion of hepatocyte Ikkβ confers growth advantages

    International Nuclear Information System (INIS)

    Koch, Katherine S.; Maeda, Shin; He, Guobin; Karin, Michael; Leffert, Hyam L.

    2009-01-01

    Mice lacking hepatocyte IKKβ (Ikkβ Δhep ) are defective in TNFα-activation of hepatocellular transcription factor NF-κB, and highly susceptible to hepatotoxicity. Following diethylnitrosamine (DEN) exposure, Ikkβ Δhep mice develop more hepatocellular carcinoma (HCC) than control mice due partly to enhanced DEN-induced hepatocyte death. Here we show that Ikkβ Δhep hepatocytes display growth advantages over normal hepatocytes consisting of precocious PCNA and cyclin D1 expression during liver regeneration (shortened hepatocyte G 0 → G 1 transitions), and enhanced recovery efficiency, cyclin D1 expression and cell proliferation after plating. Ex vivo deletion of Ikkβ also accelerates hepatocyte growth. Ikkβ Δhep hepatocyte proliferative responses show heightened sensitivity to TGFα and TNFα, and heightened expression of fibronectin, collagens I/III, nidogen, β-actin and integrin β1 mRNAs. These findings suggest that altered mitogen signaling and expression of extracellular matrix and its associated components underlie growth advantages. Increased HCC development in Ikkβ Δhep mice may also be caused by growth advantages of surviving Ikkβ-deleted hepatocytes.

  1. Cell swelling and glycogen metabolism in hepatocytes from fasted rats

    NARCIS (Netherlands)

    Gustafson, L. A.; Jumelle-Laclau, M. N.; van Woerkom, G. M.; van Kuilenburg, A. B.; Meijer, A. J.

    1997-01-01

    Cell swelling is known to increase net glycogen production from glucose in hepatocytes from fasted rats by activating glycogen synthase. Since both active glycogen synthase and phosphorylase are present in hepatocytes, suppression of flux through phosphorylase may also contribute to the net increase

  2. Phenotypic screening of hepatocyte nuclear factor (HNF) 4-γ receptor knockout mice

    International Nuclear Information System (INIS)

    Gerdin, Anna Karin; Surve, Vikas V.; Joensson, Marie; Bjursell, Mikael; Bjoerkman, Maria; Edenro, Anne; Schuelke, Meint; Saad, Alaa; Bjurstroem, Sivert; Lundgren, Elisabeth Jensen; Snaith, Michael; Fransson-Steen, Ronny; Toernell, Jan; Berg, Anna-Lena; Bohlooly-Y, Mohammad

    2006-01-01

    Using the mouse as a model organism in pharmaceutical research presents unique advantages as its physiology in many ways resembles the human physiology, it also has a relatively short generation time, low breeding and maintenance costs, and is available in a wide variety of inbred strains. The ability to genetically modify mouse embryonic stem cells to generate mouse models that better mimic human disease is another advantage. In the present study, a comprehensive phenotypic screening protocol is applied to elucidate the phenotype of a novel mouse knockout model of hepatocyte nuclear factor (HNF) 4-γ. HNF4-γ is expressed in the kidneys, gut, pancreas, and testis. First level of the screen is aimed at general health, morphologic appearance, normal cage behaviour, and gross neurological functions. The second level of the screen looks at metabolic characteristics and lung function. The third level of the screen investigates behaviour more in-depth and the fourth level consists of a thorough pathological characterisation, blood chemistry, haematology, and bone marrow analysis. When compared with littermate wild-type mice (HNF4-γ +/+ ), the HNF4-γ knockout (HNF4-γ -/- ) mice had lowered energy expenditure and locomotor activity during night time that resulted in a higher body weight despite having reduced intake of food and water. HNF4-γ -/- mice were less inclined to build nest and were found to spend more time in a passive state during the forced swim test

  3. Comparison of Species and Cell-Type Differences in Fraction Unbound of Liver Tissues, Hepatocytes, and Cell Lines.

    Science.gov (United States)

    Riccardi, Keith; Ryu, Sangwoo; Lin, Jian; Yates, Phillip; Tess, David; Li, Rui; Singh, Dhirender; Holder, Brian R; Kapinos, Brendon; Chang, George; Di, Li

    2018-04-01

    Fraction unbound ( f u ) of liver tissue, hepatocytes, and other cell types is an essential parameter used to estimate unbound liver drug concentration and intracellular free drug concentration. f u,liver and f u,cell are frequently measured in multiple species and cell types in drug discovery and development for various applications. A comparison study of 12 matrices for f u,liver and f u,cell of hepatocytes in five different species (mouse, rat, dog, monkey, and human), as well as f u,cell of Huh7 and human embryonic kidney 293 cell lines, was conducted for 22 structurally diverse compounds with the equilibrium dialysis method. Using an average bioequivalence approach, our results show that the average difference in binding to liver tissue, hepatocytes, or different cell types was within 2-fold of that of the rat f u,liver Therefore, we recommend using rat f u,liver as a surrogate for liver binding in other species and cell types in drug discovery. This strategy offers the potential to simplify binding studies and reduce cost, thereby enabling a more effective and practical determination of f u for liver tissues, hepatocytes, and other cell types. In addition, f u under hepatocyte stability incubation conditions should not be confused with f u,cell , as one is a diluted f u and the other is an undiluted f u Cell density also plays a critical role in the accurate measurement of f u,cell . Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.

  4. Trifluoperazine inhibits acetaminophen-induced hepatotoxicity and hepatic reactive nitrogen formation in mice and in freshly isolated hepatocytes

    Directory of Open Access Journals (Sweden)

    Sudip Banerjee

    Full Text Available The hepatotoxicity of acetaminophen (APAP occurs by initial metabolism to N-acetyl-p-benzoquinone imine which depletes GSH and forms APAP-protein adducts. Subsequently, the reactive nitrogen species peroxynitrite is formed from nitric oxide (NO and superoxide leading to 3-nitrotyrosine in proteins. Toxicity occurs with inhibited mitochondrial function. We previously reported that in hepatocytes the nNOS (NOS1 inhibitor NANT inhibited APAP toxicity, reactive nitrogen and oxygen species formation, and mitochondrial dysfunction. In this work we examined the effect of trifluoperazine (TFP, a calmodulin antagonist that inhibits calcium induced nNOS activation, on APAP hepatotoxicity and reactive nitrogen formation in murine hepatocytes and in vivo. In freshly isolated hepatocytes TFP inhibited APAP induced toxicity, reactive nitrogen formation (NO, GSNO, and 3-nitrotyrosine in protein, reactive oxygen formation (superoxide, loss of mitochondrial membrane potential, decreased ATP production, decreased oxygen consumption rate, and increased NADH accumulation. TFP did not alter APAP induced GSH depletion in the hepatocytes or the formation of APAP protein adducts which indicated that reactive metabolite formation was not inhibited. Since we previously reported that TFP inhibits the hepatotoxicity of APAP in mice without altering hepatic APAP-protein adduct formation, we examined the APAP treated mouse livers for evidence of reactive nitrogen formation. 3-Nitrotyrosine in hepatic proteins and GSNO were significantly increased in APAP treated mouse livers and decreased in the livers of mice treated with APAP plus TFP. These data are consistent with a hypothesis that APAP hepatotoxicity occurs with altered calcium metabolism, activation of nNOS leading to increased reactive nitrogen formation, and mitochondrial dysfunction. Keywords: Acetaminophen, Neuronal nitric oxide, Oxidative stress, Mitochondria

  5. Ketose induced respiratory inhibition in isolated hepatocytes.

    Science.gov (United States)

    Martínez, P; Carrascosa, J M; Núñez de Castro, I

    1987-06-01

    The addition of 10 mM fructose or 10 mM tagatose to a suspension of hepatocytes caused respiratory inhibition, whereas no change in oxygen uptake was observed following the addition of glucose. However, incubations in the presence of fructose showed a high, aerobic glycolytic activity. Tagatose is phosphorylated to tagatose 1-phosphate but is not further metabolized by cell free liver extract. Moreover, the addition of fructose to glucagon treated cells also caused the Crabtree-like effect. The concentration of adenine nucleotides and inorganic phosphate (Pi) in the mitochondrial and cytosolic compartments during incubation (time 30 min) was determined by the digitonin fractionation procedure. In the presence of 10 mM fructose or tagatose, the total adenine nucleotide pools decreased by 40%; however, glucose produced no change. The addition of ketoses diminished the asymmetric distribution of extramitochondrial (ATP/ADP)e ratio and intramitochondrial (ATP/ADP)i ratio. At the same time the total mitochondrial Pi fell from 17 mM to 6-7 mM. The mitochondrial membrane potential (-161 mV) in the presence of fructose showed no changes during the 30 min experimental period. An increase in the NADH/NAD+ ratio was observed. These results suggest that in hepatocytes the inhibition of respiration is not necessarily linked with the enhanced aerobic glycolysis, by competition for common substrates.

  6. Insulin internalization in isolated rat hepatocytes

    International Nuclear Information System (INIS)

    Galan, J.; Trankina, M.; Noel, R.; Ward, W.

    1990-01-01

    This project was designed to determine whether neomycin, an aminoglycoside antibiotic, has a significant effect upon the pathways of ligand endocytosis in isolated rat hepatocytes. The pathways studied include receptor-mediated endocytosis and fluid-phase endocytosis. Neomycin causes a dose-dependent acceleration of 125 I-insulin internalization. Since fluid-phase endocytosis can also be a significant factor in 125 I-insulin internalization, lucifer yellow (LY), a marker for fluid-phase endocytosis, was incorporated into an assay similar to the 125 I-insulin internalization procedure. In the presence of 5 mM neomycin, a significant increase in LY uptake was evident at 0.2 and 0.4 mg/ml of LY. At 0.8 mg/ml, a decrease in LY uptake was observed. The increased rate of 125 I-insulin internalization in the presence of neomycin was intriguing. Since one action of neomycin is to inhibit phosphoinositidase C, it suggests that the phosphotidylinositol cycle may be involved in ligand internalization by hepatocytes. At low insulin concentrations, receptor-mediated uptake predominates. Fluid-phase uptake can become an important uptake route as insulin concentrations are increased. Since neomycin stimulates fluid-phase endocytosis, it must also be taken into account when measuring ligand internalization

  7. Selective activation of microglia in spinal cord but not higher cortical regions following nerve injury in adult mouse

    Directory of Open Access Journals (Sweden)

    Shang Yuze

    2008-04-01

    Full Text Available Abstract Neuronal plasticity along the pathway for sensory transmission including the spinal cord and cortex plays an important role in chronic pain, including inflammatory and neuropathic pain. While recent studies indicate that microglia in the spinal cord are involved in neuropathic pain, a systematic study has not been performed in other regions of the central nervous system (CNS. In the present study, we used heterozygous Cx3cr1GFP/+mice to characterize the morphological phenotypes of microglia following common peroneal nerve (CPN ligation. We found that microglia showed a uniform distribution throughout the CNS, and peripheral nerve injury selectively activated microglia in the spinal cord dorsal horn and related ventral horn. In contrast, microglia was not activated in supraspinal regions of the CNS, including the anterior cingulate cortex (ACC, prefrontal cortex (PFC, primary and secondary somatosensory cortex (S1 and S2, insular cortex (IC, amygdala, hippocampus, periaqueductal gray (PAG and rostral ventromedial medulla (RVM. Our results provide strong evidence that nerve injury primarily activates microglia in the spinal cord of adult mice, and pain-related cortical plasticity is likely mediated by neurons.

  8. Adenosine Receptors in Developing and Adult Mouse Neuromuscular Junctions and Functional Links With Other Metabotropic Receptor Pathways.

    Science.gov (United States)

    Tomàs, Josep; Garcia, Neus; Lanuza, Maria A; Santafé, Manel M; Tomàs, Marta; Nadal, Laura; Hurtado, Erica; Simó-Ollé, Anna; Cilleros-Mañé, Víctor; Just-Borràs, Laia

    2018-01-01

    In the last few years, we have studied the presence and involvement in synaptogenesis and mature transmitter release of the adenosine autoreceptors (AR) in the mammalian neuromuscular junction (NMJ). Here, we review and bring together the previously published data to emphasize the relevance of these receptors for developmental axonal competition, synaptic loss and mature NMJ functional modulation. However, in addition to AR, activity-dependent mediators originating from any of the three cells that make the synapse (nerve, muscle, and glial cells) cross the extracellular cleft to generate signals in target metabotropic receptors. Thus, the integrated interpretation of the complementary function of all these receptors is needed. We previously studied, in the NMJ, the links of AR with mAChR and the neurotrophin receptor TrkB in the control of synapse elimination and transmitter release. We conclude that AR cooperate with these receptors through synergistic and antagonistic effects in the developmental synapse elimination process. In the adult NMJ, this cooperation is manifested so as that the functional integrity of a given receptor group depends on the other receptors operating normally (i.e., the functional integrity of mAChR depends on AR operating normally). These observations underlie the relevance of AR in the NMJ function.

  9. Adenosine Receptors in Developing and Adult Mouse Neuromuscular Junctions and Functional Links With Other Metabotropic Receptor Pathways

    Directory of Open Access Journals (Sweden)

    Josep Tomàs

    2018-04-01

    Full Text Available In the last few years, we have studied the presence and involvement in synaptogenesis and mature transmitter release of the adenosine autoreceptors (AR in the mammalian neuromuscular junction (NMJ. Here, we review and bring together the previously published data to emphasize the relevance of these receptors for developmental axonal competition, synaptic loss and mature NMJ functional modulation. However, in addition to AR, activity-dependent mediators originating from any of the three cells that make the synapse (nerve, muscle, and glial cells cross the extracellular cleft to generate signals in target metabotropic receptors. Thus, the integrated interpretation of the complementary function of all these receptors is needed. We previously studied, in the NMJ, the links of AR with mAChR and the neurotrophin receptor TrkB in the control of synapse elimination and transmitter release. We conclude that AR cooperate with these receptors through synergistic and antagonistic effects in the developmental synapse elimination process. In the adult NMJ, this cooperation is manifested so as that the functional integrity of a given receptor group depends on the other receptors operating normally (i.e., the functional integrity of mAChR depends on AR operating normally. These observations underlie the relevance of AR in the NMJ function.

  10. Single cell electroporation for longitudinal imaging of synaptic structure and function in the adult mouse neocortex in vivo

    Directory of Open Access Journals (Sweden)

    Stephane ePages

    2015-04-01

    Full Text Available Longitudinal imaging studies of neuronal structures in vivo have revealed rich dynamics in dendritic spines and axonal boutons. Spines and boutons are considered to be proxies for synapses. This implies that synapses display similar dynamics. However, spines and boutons do not always bear synapses, some may contain more than one, and dendritic shaft synapses have no clear structural proxies. In addition, synaptic strength is not always accurately revealed by just the size of these structures. Structural and functional dynamics of synapses could be studied more reliably using fluorescent synaptic proteins as markers for size and function. These proteins are often large and possibly interfere with circuit development, which renders them less suitable for conventional transfection or transgenesis methods such as viral vectors, in utero electroporation and germline transgenesis. Single cell electroporation has been shown to be a potential alternative for transfection of recombinant fluorescent proteins in adult cortical neurons. Here we provide proof of principle for the use of single cell electroporation to express and subsequently image fluorescently tagged synaptic proteins over days to weeks in vivo.

  11. Transcriptional profiling reveals gland-specific differential expression in the three major salivary glands of the adult mouse.

    Science.gov (United States)

    Gao, Xin; Oei, Maria S; Ovitt, Catherine E; Sincan, Murat; Melvin, James E

    2018-04-01

    RNA-Seq was used to better understand the molecular nature of the biological differences among the three major exocrine salivary glands in mammals. Transcriptional profiling found that the adult murine parotid, submandibular, and sublingual salivary glands express greater than 14,300 protein-coding genes, and nearly 2,000 of these genes were differentially expressed. Principle component analysis of the differentially expressed genes revealed three distinct clusters according to gland type. The three salivary gland transcriptomes were dominated by a relatively few number of highly expressed genes (6.3%) that accounted for more than 90% of transcriptional output. Of the 912 transcription factors expressed in the major salivary glands, greater than 90% of them were detected in all three glands, while expression for ~2% of them was enriched in an individual gland. Expression of these unique transcription factors correlated with sublingual and parotid specific subsets of both highly expressed and differentially expressed genes. Gene ontology analyses revealed that the highly expressed genes common to all glands were associated with global functions, while many of the genes expressed in a single gland play a major role in the function of that gland. In summary, transcriptional profiling of the three murine major salivary glands identified a limited number of highly expressed genes, differentially expressed genes, and unique transcription factors that represent the transcriptional signatures underlying gland-specific biological properties.

  12. Novel insulin from the bullfrog: its structure and function in protein secretion by hepatocytes

    International Nuclear Information System (INIS)

    Hulsebus, J.J.

    1987-01-01

    Bullfrog insulin was extracted and purified from the pancreas of Rana catesbeiana adults using gel filtration and reverse phase high performance liquid chromatography. Amino acid analysis of bullfrog insulin revealed 52 amino acids instead of the most common number of 51. The most unique features of bullfrog insulin is a two amino acid extension on the amino terminus (A1) of the A chain. This is the only insulin to date that has an extension at this position. Bullfrog and porcine insulin increase protein secretion from bullfrog adult and three developmental stages of tadpole hepatocytes in a totally defined, serum-free culture system. The hormone slightly stimulates protein secretion by premetamorphic and early prometamorphic tadpoles. Late prometamorphic tadpoles respond to bullfrog and porcine insulin with higher concentrations of secreted protein than either of the two previous developmental stages. Insulin treated adult hepatocytes secrete significantly higher concentrations of protein than any of the tadpole stages. 35 S-methionine and 35 S-cysteine were added to the culture medium for twelve hours. Proteins secreted into the medium were separated using SDS polyacrylamide linear gradient gels. Densitometer scans of autoradiograms did not show an increases in any specific proteins, but did show a generalized increase in all secreted proteins for both adults, and tadpoles

  13. Subchronic inhalation of soluble manganese induces expression of hypoxia-associated angiogenic genes in adult mouse lungs

    International Nuclear Information System (INIS)

    Bredow, Sebastian; Falgout, Melanie M.; March, Thomas H.; Yingling, Christin M.; Malkoski, Stephen P.; Aden, James; Bedrick, Edward J.; Lewis, Johnnye L.; Divine, Kevin K.

    2007-01-01

    Although the lung constitutes the major exposure route for airborne manganese (Mn), little is known about the potential pulmonary effects and the underlying molecular mechanisms. Transition metals can mimic a hypoxia-like response, activating the hypoxia inducible factor-1 (HIF-1) transcription factor family. Through binding to the hypoxia-response element (HRE), these factors regulate expression of many genes, including vascular endothelial growth factor (VEGF). Increases in VEGF, an important biomarker of angiogenesis, have been linked to respiratory diseases, including pulmonary hypertension. The objective of this study was to evaluate pulmonary hypoxia-associated angiogenic gene expression in response to exposure of soluble Mn(II) and to assess the genes' role as intermediaries of potential pulmonary Mn toxicity. In vitro, 0.25 mM Mn(II) altered morphology and slowed the growth of human pulmonary epithelial cell lines. Acute doses between 0.05 and 1 mM stimulated VEGF promoter activity up to 3.7-fold in transient transfection assays. Deletion of the HRE within the promoter had no effect on Mn(II)-induced VEGF expression but decreased cobalt [Co(II)]-induced activity 2-fold, suggesting that HIF-1 may not be involved in Mn(II)-induced VEGF gene transcription. Nose-only inhalation to 2 mg Mn(II)/m 3 for 5 days at 6 h/day produced no significant pulmonary inflammation but induced a 2-fold increase in pulmonary VEGF mRNA levels in adult mice and significantly altered expression of genes associated with murine angiogenesis. These findings suggest that even short-term exposures to soluble, occupationally relevant Mn(II) concentrations may alter pulmonary gene expression in pathways that ultimately could affect the lungs' susceptibility to respiratory disease

  14. A Western diet ecological module identified from the 'humanized' mouse microbiota predicts diet in adults and formula feeding in children.

    Science.gov (United States)

    Siddharth, Jay; Holway, Nicholas; Parkinson, Scott J

    2013-01-01

    The interplay between diet and the microbiota has been implicated in the growing frequency of chronic diseases associated with the Western lifestyle. However, the complexity and variability of microbial ecology in humans and preclinical models has hampered identification of the molecular mechanisms underlying the association of the microbiota in this context. We sought to address two key questions. Can the microbial ecology of preclinical models predict human populations? And can we identify underlying principles that surpass the plasticity of microbial ecology in humans? To do this, we focused our study on diet; perhaps the most influential factor determining the composition of the gut microbiota. Beginning with a study in 'humanized' mice we identified an interactive module of 9 genera allied with Western diet intake. This module was applied to a controlled dietary study in humans. The abundance of the Western ecological module correctly predicted the dietary intake of 19/21 top and 21/21 of the bottom quartile samples inclusive of all 5 Western and 'low-fat' diet subjects, respectively. In 98 volunteers the abundance of the Western module correlated appropriately with dietary intake of saturated fatty acids, fat-soluble vitamins and fiber. Furthermore, it correlated with the geographical location and dietary habits of healthy adults from the Western, developing and third world. The module was also coupled to dietary intake in children (and piglets) correlating with formula (vs breast) feeding and associated with a precipitous development of the ecological module in young children. Our study provides a conceptual platform to translate microbial ecology from preclinical models to humans and identifies an ecological network module underlying the association of the gut microbiota with Western dietary habits.

  15. A Western diet ecological module identified from the 'humanized' mouse microbiota predicts diet in adults and formula feeding in children.

    Directory of Open Access Journals (Sweden)

    Jay Siddharth

    Full Text Available The interplay between diet and the microbiota has been implicated in the growing frequency of chronic diseases associated with the Western lifestyle. However, the complexity and variability of microbial ecology in humans and preclinical models has hampered identification of the molecular mechanisms underlying the association of the microbiota in this context. We sought to address two key questions. Can the microbial ecology of preclinical models predict human populations? And can we identify underlying principles that surpass the plasticity of microbial ecology in humans? To do this, we focused our study on diet; perhaps the most influential factor determining the composition of the gut microbiota. Beginning with a study in 'humanized' mice we identified an interactive module of 9 genera allied with Western diet intake. This module was applied to a controlled dietary study in humans. The abundance of the Western ecological module correctly predicted the dietary intake of 19/21 top and 21/21 of the bottom quartile samples inclusive of all 5 Western and 'low-fat' diet subjects, respectively. In 98 volunteers the abundance of the Western module correlated appropriately with dietary intake of saturated fatty acids, fat-soluble vitamins and fiber. Furthermore, it correlated with the geographical location and dietary habits of healthy adults from the Western, developing and third world. The module was also coupled to dietary intake in children (and piglets correlating with formula (vs breast feeding and associated with a precipitous development of the ecological module in young children. Our study provides a conceptual platform to translate microbial ecology from preclinical models to humans and identifies an ecological network module underlying the association of the gut microbiota with Western dietary habits.

  16. 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

  17. Single-cell analysis of ploidy and centrosomes underscores the peculiarity of normal hepatocytes.

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    Francesca Faggioli

    Full Text Available Polyploidization is the most well recognized feature of the liver. Yet, a quantitative and behavioral analysis of centrosomes and DNA content in normal hepatocytes has been limited by the technical challenges of methods available. By using a novel approach employing FISH for chromosomes 18, X and Y we provide, for the first time, a detailed analysis of DNA copies during physiological development in the liver at single cell level. We demonstrate that aneuploidy and unbalanced DNA content in binucleated hepatocytes are common features in normal adult liver. Despite the common belief that hepatocytes contain 1, 2 or no more than 4 centrosomes, our double staining for centrosome associated proteins reveals extranumerary centrosomes in a high percentage of cells as early as 15 days of age. We show that in murine liver the period between 15 days and 1.5 months marks the transition from a prevalence of mononucleated cells to up to 75% of binucleated cells. Our data demonstrate that this timing correlates with a switch in centrosomes number. At 15 days the expected 1 or 2 centrosomes converge with several hepatocytes that contain 3 centrosomes; at 1.5 months the percentage of cells with 3 centrosomes decreases concomitantly with the increase of cells with more than 4 centrosomes. Our analysis shows that the extranumerary centrosomes emerge in concomitance with the process of binucleation and polyploidization and maintain α-tubulin nucleation activity. Finally, by integrating interphase FISH and immunofluorescent approaches, we detected an imbalance between centrosome number and DNA content in liver cells that deviates from the equilibrium expected in normal cells. We speculate that these unique features are relevant to the peculiar biological function of liver cells which are continuously challenged by stress, a condition that could predispose to genomic instability.

  18. Involvement of Bcl-xL degradation and mitochondrial-mediated apoptotic pathway in pyrrolizidine alkaloids-induced apoptosis in hepatocytes

    International Nuclear Information System (INIS)

    Ji Lili; Chen Ying; Liu Tianyu; Wang Zhengtao

    2008-01-01

    Pyrrolizidine alkaloids (PAs) are natural hepatotoxins with worldwide distribution in more than 6000 high plants including medicinal herbs or teas. The aim of this study is to investigate the signal pathway involved in PAs-induced hepatotoxicity. Our results showed that clivorine, isolated from Ligularia hodgsonii Hook, decreased cell viability and induced apoptosis in L-02 cells and mouse hepatocytes. Western-blot results showed that clivorine induced caspase-3/-9 activation, mitochondrial release of cytochrome c and decreased anti-apoptotic Bcl-xL in a time (8-48 h)- and concentration (1-100 μM)-dependent manner. Furthermore, inhibitors of pan-caspase, caspase-3 and caspase-9 significantly inhibited clivorine-induced apoptosis and rescued clivorine-decreased cell viability. Polyubiquitination of Bcl-xL was detected after incubation with 100 μM clivorine for 40 h in the presence of proteasome specific inhibitor MG132, indicating possible degradation of Bcl-xL protein. Furthermore, pretreatment with MG132 or calpain inhibitor I for 2 h significantly enhanced clivorine-decreased Bcl-xL level and cell viability. All the other tested PAs such as senecionine, isoline and monocrotaline decreased mouse hepatocytes viability in a concentration-dependent manner. Clivorine (10 μM) induced caspase-3 activation and decreased Bcl-xL was also confirmed in mouse hepatocytes. Meanwhile, another PA senecionine isolated from Senecio vulgaris L also induced apoptosis, caspase-3 activation and decreased Bcl-xL in mouse hepatocytes. In conclusion, our results suggest that PAs may share the same hepatotoxic signal pathway, which involves degradation of Bcl-xL protein and thus leading to the activation of mitochondrial-mediated apoptotic pathway

  19. Noggin and BMP4 co-modulate adult hippocampal neurogenesis in the APPswe/PS1ΔE9 transgenic mouse model of Alzheimer's disease

    International Nuclear Information System (INIS)

    Tang, Jun; Song, Min; Wang, Yanyan; Fan, Xiaotang; Xu, Haiwei; Bai, Yun

    2009-01-01

    In addition to the subventricular zone, the dentate gyrus of the hippocampus is one of the few brain regions in which neurogenesis continues into adulthood. Perturbation of neurogenesis can alter hippocampal function, and previous studies have shown that neurogenesis is dysregulated in Alzheimer disease (AD) brain. Bone morphogenetic protein-4 (BMP4) and its antagonist Noggin have been shown to play important roles both in embryonic development and in the adult nervous system, and may regulate hippocampal neurogenesis. Previous data indicated that increased expression of BMP4 mRNA within the dentate gyrus might contribute to decreased hippocampal cell proliferation in the APP swe /PS1 ΔE9 mouse AD model. However, it is not known whether the BMP antagonist Noggin contributes to the regulation of neurogenesis. We therefore studied the relative expression levels and localization of BMP4 and its antagonist Noggin in the dentate gyrus and whether these correlated with changes in neurogenesis in 6-12 mo old APP swe /PS1 ΔE9 transgenic mice. Bromodeoxyuridine (BrdU) was used to label proliferative cells. We report that decreased neurogenesis in the APP/PS1 transgenic mice was accompanied by increased expression of BMP4 and decreased expression of Noggin at both the mRNA and protein levels; statistical analysis showed that the number of proliferative cells at different ages correlated positively with Noggin expression and negatively with BMP4 expression. Intraventricular administration of a chimeric Noggin/Fc protein was used to block the action of endogenous BMP4; this resulted in a significant increase in the number of BrdU-labeled cells in dentate gyrus subgranular zone and hilus in APP/PS1 mice. These results suggest that BMP4 and Noggin co-modulate neurogenesis.

  20. Marrow-isolated adult multilineage inducible cells embedded within a biologically-inspired construct promote recovery in a mouse model of peripheral vascular disease.

    Science.gov (United States)

    Grau-Monge, Cristina; Delcroix, Gaëtan J-R; Bonnin-Marquez, Andrea; Valdes, Mike; Awadallah, Ead Lewis Mazen; Quevedo, Daniel F; Armour, Maxime R; Montero, Ramon B; Schiller, Paul C; Andreopoulos, Fotios M; D'Ippolito, Gianluca

    2017-02-17

    Peripheral vascular disease is one of the major vascular complications in individuals suffering from diabetes and in the elderly that is associated with significant burden in terms of morbidity and mortality. Stem cell therapy is being tested as an attractive alternative to traditional surgery to prevent and treat this disorder. The goal of this study was to enhance the protective and reparative potential of marrow-isolated adult multilineage inducible (MIAMI) cells by incorporating them within a bio-inspired construct (BIC) made of two layers of gelatin B electrospun nanofibers. We hypothesized that the BIC would enhance MIAMI cell survival and engraftment, ultimately leading to a better functional recovery of the injured limb in our mouse model of critical limb ischemia compared to MIAMI cells used alone. Our study demonstrated that MIAMI cell-seeded BIC resulted in a wide range of positive outcomes with an almost full recovery of blood flow in the injured limb, thereby limiting the extent of ischemia and necrosis. Functional recovery was also the greatest when MIAMI cells were combined with BICs, compared to MIAMI cells alone or BICs in the absence of cells. Histology was performed 28 days after grafting the animals to explore the mechanisms at the source of these positive outcomes. We observed that our critical limb ischemia model induces an extensive loss of muscular fibers that are replaced by intermuscular adipose tissue (IMAT), together with a highly disorganized vascular structure. The use of MIAMI cells-seeded BIC prevented IMAT infiltration with some clear evidence of muscular fibers regeneration.

  1. Unrestricted Hepatocyte Transduction with Adeno-Associated Virus Serotype 8 Vectors in Mice

    Science.gov (United States)

    Nakai, Hiroyuki; Fuess, Sally; Storm, Theresa A.; Muramatsu, Shin-ichi; Nara, Yuko; Kay, Mark A.

    2005-01-01

    Recombinant adeno-associated virus (rAAV) vectors can mediate long-term stable transduction in various target tissues. However, with rAAV serotype 2 (rAAV2) vectors, liver transduction is confined to only a small portion of hepatocytes even after administration of extremely high vector doses. In order to investigate whether rAAV vectors of other serotypes exhibit similar restricted liver transduction, we performed a dose-response study by injecting mice with β-galactosidase-expressing rAAV1 and rAAV8 vectors via the portal vein. The rAAV1 vector showed a blunted dose-response similar to that of rAAV2 at high doses, while the rAAV8 vector dose-response remained unchanged at any dose and ultimately could transduce all the hepatocytes at a dose of 7.2 × 1012 vector genomes/mouse without toxicity. This indicates that all hepatocytes have the ability to process incoming single-stranded vector genomes into duplex DNA. A single tail vein injection of the rAAV8 vector was as efficient as portal vein injection at any dose. In addition, intravascular administration of the rAAV8 vector at a high dose transduced all the skeletal muscles throughout the body, including the diaphragm, the entire cardiac muscle, and substantial numbers of cells in the pancreas, smooth muscles, and brain. Thus, rAAV8 is a robust vector for gene transfer to the liver and provides a promising research tool for delivering genes to various target organs. In addition, the rAAV8 vector may offer a potential therapeutic agent for various diseases affecting nonhepatic tissues, but great caution is required for vector spillover and tight control of tissue-specific gene expression. PMID:15596817

  2. Hepatocyte growth factor profile with breast cancer

    Directory of Open Access Journals (Sweden)

    Hoda A EL-Attar

    2011-01-01

    Full Text Available Background: The multifunctional hepatocyte growth factor (HGF is the ligand of c-Met receptor; it plays important role in mammary differentiation. HGF-Met signaling is a critical downstream function of c-Src-Stat3 pathway in mammalian tumorigenesis. Aim: Evaluation of tissue c-Met receptor hepatocyte growth factor receptor (HGFR and serum level of HGF in female breast ductal carcinoma. Materials and Methods: Sixty-eight premenopausal females were divided as 30 control females subdivided into: [Group 1] 15 healthy volunteer females and [Group 2] five with fibrocystic disease and 10 having fibroadenoma of the breast and patients group [Group 3] consisted of 38 female patients with breast ductal carcinoma. Thorough clinical examination, preoperative fine needle aspiration cytology, estimation of fasting serum glucose, urea, creatinine, and uric acid levels, alanine aminotransferase activities, C-reactive protein, HGF level, before surgery and histopathological examination of the breast masses, and immunohistochemical detection of HGFR were done. Results and Conclusions: Significant increase in serum HGF levels were found in patients with breast cancer as compared with controls. Significant increase was also seen in patients with breast cancer with and without lymph node metastasis when each subgroup was compared with controls. Serum level of HGF is an independent prognostic indicator of breast cancer. Fibrocystic disease of the breast showed weak HGFR expression, while in normal tissue, HGFR was scanty; meanwhile, breast invasive ductal carcinoma showed homogenous strong reaction to HGFR. HGF is only one of a number of key factors involved in breast cancer and preoperative high serum HGF levels and malignancy occur usually together.

  3. Metabolism of oxycodone in human hepatocytes from different age groups and prediction of hepatic plasma clearance

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    Timo eKorjamo

    2012-01-01

    Full Text Available Oxycodone is commonly used to treat severe pain in adults and children. It is extensively metabolized in the liver in adults, but the maturation of metabolism is not well understood. Our aim was to study the metabolism of oxycodone in cryopreserved human hepatocytes from different age groups (3 days, 2 and 5 months, 4 years, adult pool and predict hepatic plasma clearance of oxycodone using these data. Oxycodone (0.1, 1 and 10 µM was incubated with hepatocytes for 4 hours, and 1 µM oxycodone also with CYP3A inhibitor ketoconazole (1 µM. Oxycodone and noroxycodone concentrations were determined at several time points with liquid chromatography-mass spectrometry. In vitro clearance of oxycodone was used to predict hepatic plasma clearance, using the well-stirred model and published physiological parameters. Noroxycodone was the major metabolite in all batches and ketoconazole inhibited the metabolism markedly in most cases. A clear correlation between in vitro oxycodone clearance and CYP3A4 activity was observed. The predicted hepatic plasma clearances were typically much lower than the published median total plasma clearance from pharmacokinetic studies. In general, this in vitro to in vivo extrapolation method provides valuable information on the maturation of oxycodone metabolism that can be utilized in the design of clinical pharmacokinetic studies in infants and young children.

  4. Reversibility of hepatocyte nuclear modifications in mice fed on genetically modified soybean

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    M Malatesta

    2009-06-01

    Full Text Available In the literature, the reports on the effects of a genetically modified (GM diet are scanty and heterogeneous; in particular, no direct evidence has so far been reported that GM food may affect human or animal health. Hepatocytes represent a suitable model for monitoring the effects of a GM diet, the liver potentially being a primary target. In a previous study, we demonstrated that some modifications occur in hepatocyte nuclei of mice fed on GM soybean. In order to elucidate whether such modifications can be reversed, in the present study, 3 months old mice fed on GM soybean since their weaning were submitted to a diet containing wild type soybean only, for one month. In parallel, to investigate the influence of GM soybean on adult individuals, mice fed on wild type soybean were changed to a GM diet, for the same time. Using immunoelectron microscopy, we demonstrated that a one-month diet reversion can influence some nuclear features in adult mice, restoring typical characteristics of controls in GM-fed animals, and inducing in control mice modifications similar to those observed in animals fed on GM soybean from weaning. This suggests that the modifications related to GM soybean are potentially reversible, but also that some modifications are inducible in adult organisms in a short time.

  5. Effect of brain-derived neurotrophic factor (BDNF) on hepatocyte metabolism.

    Science.gov (United States)

    Genzer, Yoni; Chapnik, Nava; Froy, Oren

    2017-07-01

    Brain-derived neurotrophic factor (BDNF) plays crucial roles in the development, maintenance, plasticity and homeostasis of the central and peripheral nervous systems. Perturbing BDNF signaling in mouse brain results in hyperphagia, obesity, hyperinsulinemia and hyperglycemia. Currently, little is known whether BDNF affects liver tissue directly. Our aim was to determine the metabolic signaling pathways activated after BDNF treatment in hepatocytes. Unlike its effect in the brain, BDNF did not lead to activation of the liver AKT pathway. However, AMP protein activated kinase (AMPK) was ∼3 times more active and fatty acid synthase (FAS) ∼2-fold less active, suggesting increased fatty acid oxidation and reduced fatty acid synthesis. In addition, cAMP response element binding protein (CREB) was ∼3.5-fold less active together with its output the gluconeogenic transcript phosphoenolpyruvate carboxykinase (Pepck), suggesting reduced gluconeogenesis. The levels of glycogen synthase kinase 3b (GSK3b) was ∼3-fold higher suggesting increased glycogen synthesis. In parallel, the expression levels of the clock genes Bmal1 and Cry1, whose protein products play also a metabolic role, were ∼2-fold increased and decreased, respectively. In conclusion, BDNF binding to hepatocytes leads to activation of catabolic pathways, such as fatty acid oxidation. In parallel gluconeogenesis is inhibited, while glycogen storage is triggered. This metabolic state mimics that of after breakfast, in which the liver continues to oxidize fat, stops gluconeogenesis and replenishes glycogen stores. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. 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.

  7. Transcriptome Analysis Uncovers a Growth-Promoting Activity of Orosomucoid-1 on Hepatocytes

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    Xian-Yang Qin

    2017-10-01

    Full Text Available The acute phase protein orosomucoid-1 (Orm1 is mainly expressed by hepatocytes (HPCs under stress conditions. However, its specific function is not fully understood. Here, we report a role of Orm1 as an executer of HPC proliferation. Increases in serum levels of Orm1 were observed in patients after surgical resection for liver cancer and in mice undergone partial hepatectomy (PH. Transcriptome study showed that Orm1 became the most abundant in HPCs isolated from regenerating mouse liver tissues after PH. Both in vitro and in vivo siRNA-induced knockdown of Orm1 suppressed proliferation of mouse regenerating HPCs and human hepatic cells. Microarray analysis in regenerating mouse livers revealed that the signaling pathways controlling chromatin replication, especially the minichromosome maintenance protein complex genes were uniformly down-regulated following Orm1 knockdown. These data suggest that Orm1 is induced in response to hepatic injury and executes liver regeneration by activating cell cycle progression in HPCs.

  8. 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

  9. Tbx3 Promotes Liver Bud Expansion During Mouse Development by Suppression of Cholangiocyte Differentiation

    NARCIS (Netherlands)

    Lüdtke, Timo H.-W.; Christoffels, Vincent M.; Petry, Marianne; Kispert, Andreas

    2009-01-01

    After specification of the hepatic endoderm, mammalian liver organogenesis progresses through a series of morphological stages that culminate in the migration of hepatocytes into the underlying mesenchyme to populate the hepatic lobes. Here, we show that in the mouse the transcriptional repressor

  10. 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.

  11. Histopathologic characterization of the BTBR mouse model of autistic-like behavior reveals selective changes in neurodevelopmental proteins and adult hippocampal neurogenesis

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    Stephenson Diane T

    2011-05-01

    Full Text Available Abstract Background The inbred mouse strain BTBR T+ tf/J (BTBR exhibits behavioral deficits that mimic the core deficits of autism. Neuroanatomically, the BTBR strain is also characterized by a complete absence of the corpus callosum. The goal of this study was to identify novel molecular and cellular changes in the BTBR mouse, focusing on neuronal, synaptic, glial and plasticity markers in the limbic system as a model for identifying putative molecular and cellular substrates associated with autistic behaviors. Methods Forebrains of 8 to 10-week-old male BTBR and age-matched C57Bl/6J control mice were evaluated by immunohistochemistry using free-floating and paraffin embedded sections. Twenty antibodies directed against antigens specific to neurons, synapses and glia were used. Nissl, Timm and acetylcholinesterase (AchE stains were performed to assess cytoarchitecture, mossy fibers and cholinergic fiber density, respectively. In the hippocampus, quantitative stereological estimates for the mitotic marker bromodeoxyuridine (BrdU were performed to determine hippocampal progenitor proliferation, survival and differentiation, and brain-derived neurotrophic factor (BDNF mRNA was quantified by in situ hybridization. Quantitative image analysis was performed for NG2, doublecortin (DCX, NeuroD, GAD67 and Poly-Sialic Acid Neural Cell Adhesion Molecule (PSA-NCAM. Results In midline structures including the region of the absent corpus callosum of BTBR mice, the myelin markers 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase and myelin basic protein (MBP were reduced, and the oligodendrocyte precursor NG2 was increased. MBP and CNPase were expressed in small ectopic white matter bundles within the cingulate cortex. Microglia and astrocytes showed no evidence of gliosis, yet orientations of glial fibers were altered in specific white-matter areas. In the hippocampus, evidence of reduced neurogenesis included significant reductions in the number of

  12. Mouse adhalin

    DEFF Research Database (Denmark)

    Liu, L; Vachon, P H; Kuang, W

    1997-01-01

    . To analyze the biological roles of adhalin, we cloned the mouse adhalin cDNA, raised peptide-specific antibodies to its cytoplasmic domain, and examined its expression and localization in vivo and in vitro. The mouse adhalin sequence was 80% identical to that of human, rabbit, and hamster. Adhalin...... was specifically expressed in striated muscle cells and their immediate precursors, and absent in many other cell types. Adhalin expression in embryonic mouse muscle was coincident with primary myogenesis. Its expression was found to be up-regulated at mRNA and protein levels during myogenic differentiation...

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

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    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. Metabolism of para-aminophenol by rat hepatocytes.

    Science.gov (United States)

    Yan, Z; Nikelly, J G; Killmer, L; Tarloff, J B

    2000-08-01

    Autoxidation of para-aminophenol (PAP) has been proposed to account for the selective nephrotoxicity of this compound. However, other studies suggest that hepatic metabolites of PAP rather than the parent compound may be responsible for renal damage. These studies were designed to investigate PAP metabolism in isolated hepatocytes. We synthesized several proposed metabolites for analysis by HPLC/mass spectrometry and compared those results with HPLC/mass spectrometric analyses of metabolites found after incubating hepatocytes with PAP. Hepatocytes prepared from male Sprague-Dawley rats were incubated in Krebs-Henseleit buffer at 37 degrees C for 5 h with 2.3 mM PAP under an atmosphere of 5% CO2/95% O2. Aliquots were withdrawn at 0.1 h of incubation and then hourly through 5 h of incubation. Reactions were terminated by the addition of acetonitrile. Hepatocyte viability was unaltered with PAP present in the incubation medium. We found that hepatocytes converted PAP to two major metabolites (PAP-GSH conjugates and PAP-N-acetylcysteine conjugates) and several minor metabolites [PAP-O-glucuronide, acetaminophen (APAP), APAP-O-glucuronide, APAP-GSH conjugates, and 4-hydroxyformanilide]. Preincubating hepatoyctes with 1-aminobenzotriazole, an inhibitor of cytochromes P450, did not alter the pattern of PAP metabolism. In conclusion, we found that PAP was metabolized in hepatocytes predominantly to PAP-GSH conjugates and PAP-N-acetylcysteine conjugates in sufficient quantities to account for the nephrotoxicity of PAP.

  15. Functional assessment of hepatocytes after transplantation into rat spleen

    International Nuclear Information System (INIS)

    Woods, R.J.; Fuller, B.J.; Attenburrow, V.D.; Nutt, L.H.; Hobbs, K.E.

    1982-01-01

    The retention of structural integrity and metabolic function by isolated hepatocytes after ectopic transplantation has been investigated in autografted rats. Rats were partially hepatectomized and isolated hepatocytes prepared from the excised liver lobes were implanted into their spleens. Histological examination of the spleens 7 or more weeks after implantation revealed aggregates of hepatocytes in the red pulp. Two tests of biochemical function were applied to the hepatocytes after transplantation. In the first the hepatobiliary imaging agent technetium-99m N-[N'-(2, 6-dimethylphenyl)carbamoylmethyl]iminodiacetic acid (99mTc HIDA), which was shown to be avidly taken up by isolated hepatocytes in vitro, was infused into the tail veins of autograft and control rats. Radioactivity accumulating in the spleens of autografted rats was markedly greater than that in controls implanted with lethally damaged cells or in nontransplanted rats. In the second the presence of bilirubin metabolites was sought in autograft spleens after intravenous infusion of bilirubin. Both mono- and diglucuronides of bilirubin were recovered from the spleens of autograft rats but no conjugates were recovered from the spleens of unoperated controls. We conclude that after autotransplantation isolated hepatocytes retain their morphology and at least some of their functional activities

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

    Directory of Open Access Journals (Sweden)

    Adam Szelag

    2010-06-01

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

  17. Overexpression of hepatocyte growth factor in SBMA model mice has an additive effect on combination therapy with castration

    International Nuclear Information System (INIS)

    Ding, Ying; Adachi, Hiroaki; Katsuno, Masahisa; Huang, Zhe; Jiang, Yue-Mei; Kondo, Naohide; Iida, Madoka; Tohnai, Genki; Nakatsuji, Hideaki; Funakoshi, Hiroshi; Nakamura, Toshikazu; Sobue, Gen

    2015-01-01

    Spinal and bulbar muscular atrophy (SBMA) is an inherited motor neuron disease caused by the expansion of a polyglutamine (polyQ)-encoding tract within the androgen receptor (AR) gene. The pathologic features of SBMA are motor neuron loss in the spinal cord and brainstem and diffuse nuclear accumulation and nuclear inclusions of mutant AR in residual motor neurons and certain visceral organs. Hepatocyte growth factor (HGF) is a polypeptide growth factor which has neuroprotective properties. To investigate whether HGF overexpression can affect disease progression in a mouse model of SBMA, we crossed SBMA transgenic model mice expressing an AR gene with an expanded CAG repeat with mice overexpressing HGF. Here, we report that high expression of HGF induces Akt phosphorylation and modestly ameliorated motor symptoms in an SBMA transgenic mouse model treated with or without castration. These findings suggest that HGF overexpression can provide a potential therapeutic avenue as a combination therapy with disease-modifying therapies in SBMA. - Highlights: • HGF overexpression ameliorates the motor phenotypes of the SBMA mouse model. • HGF overexpression induces Akt phosphorylation in the SBMA mouse model. • This is the first report of combination therapy in a mouse model of polyQ diseases.

  18. Overexpression of hepatocyte growth factor in SBMA model mice has an additive effect on combination therapy with castration

    Energy Technology Data Exchange (ETDEWEB)

    Ding, Ying [Department of Neurology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550 (Japan); Adachi, Hiroaki, E-mail: hadachi-ns@umin.org [Department of Neurology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550 (Japan); Department of Neurology, University of Occupational and Environmental Health School of Medicine, 1-1 Iseigaoka, Yahata-nishi-ku, Kitakyushu 807-8555 (Japan); Katsuno, Masahisa [Department of Neurology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550 (Japan); Huang, Zhe [Department of Neurology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550 (Japan); Department of Neurology, University of Occupational and Environmental Health School of Medicine, 1-1 Iseigaoka, Yahata-nishi-ku, Kitakyushu 807-8555 (Japan); Jiang, Yue-Mei; Kondo, Naohide; Iida, Madoka; Tohnai, Genki; Nakatsuji, Hideaki [Department of Neurology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550 (Japan); Funakoshi, Hiroshi [Center for Advanced Research and Education, Asahikawa Medical University, 1-1-1- Higashinijo Midorigaoka, Asahikawa 078-8510 (Japan); Nakamura, Toshikazu [Neurogen Inc., 1-1-52-201 Nakahozumi, Ibaraki 567-0034 (Japan); Sobue, Gen, E-mail: sobueg@med.nagoya-u.ac.jp [Department of Neurology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550 (Japan); Research Division of Dementia and Neurodegenerative Disease, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550 (Japan)

    2015-12-25

    Spinal and bulbar muscular atrophy (SBMA) is an inherited motor neuron disease caused by the expansion of a polyglutamine (polyQ)-encoding tract within the androgen receptor (AR) gene. The pathologic features of SBMA are motor neuron loss in the spinal cord and brainstem and diffuse nuclear accumulation and nuclear inclusions of mutant AR in residual motor neurons and certain visceral organs. Hepatocyte growth factor (HGF) is a polypeptide growth factor which has neuroprotective properties. To investigate whether HGF overexpression can affect disease progression in a mouse model of SBMA, we crossed SBMA transgenic model mice expressing an AR gene with an expanded CAG repeat with mice overexpressing HGF. Here, we report that high expression of HGF induces Akt phosphorylation and modestly ameliorated motor symptoms in an SBMA transgenic mouse model treated with or without castration. These findings suggest that HGF overexpression can provide a potential therapeutic avenue as a combination therapy with disease-modifying therapies in SBMA. - Highlights: • HGF overexpression ameliorates the motor phenotypes of the SBMA mouse model. • HGF overexpression induces Akt phosphorylation in the SBMA mouse model. • This is the first report of combination therapy in a mouse model of polyQ diseases.

  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. Caspase 1 activation is protective against hepatocyte cell death by up-regulating beclin 1 protein and mitochondrial autophagy in the setting of redox stress.

    Science.gov (United States)

    Sun, Qian; Gao, Wentao; Loughran, Patricia; Shapiro, Rick; Fan, Jie; Billiar, Timothy R; Scott, Melanie J

    2013-05-31

    Caspase 1 activation can be induced by oxidative stress, which leads to the release of the proinflammatory cytokines IL1β and IL18 in myeloid cells and a potentially damaging inflammatory response. However, little is known about the role of caspase 1 in non-immune cells, such as hepatocytes, that express and activate the inflammasome but do not produce a significant amount of IL1β/IL18. Here we demonstrate that caspase 1 activation protects against cell death after redox stress induced by hypoxia/reoxygenation in hepatocytes. Mechanistically, we show that caspase 1 reduces mitochondrial respiration and reactive oxygen species by increasing mitochondrial autophagy and subsequent clearance of mitochondria in hepatocytes after hypoxia/reoxygenation. Caspase 1 increases autophagic flux through up-regulating autophagy initiator beclin 1 during redox stress and is an important cell survival factor in hepatocytes. We find that during hemorrhagic shock with resuscitation, an in vivo mouse model associated with severe hepatic redox stress, caspase 1 activation is also protective against liver injury and excessive oxidative stress through the up-regulation of beclin 1. Our findings suggest an alternative role for caspase 1 activation in promoting adaptive responses to oxidative stress and, more specifically, in limiting reactive oxygen species production and damage in cells and tissues where IL1β/IL18 are not highly expressed.

  1. Caspase 1 Activation Is Protective against Hepatocyte Cell Death by Up-regulating Beclin 1 Protein and Mitochondrial Autophagy in the Setting of Redox Stress*

    Science.gov (United States)

    Sun, Qian; Gao, Wentao; Loughran, Patricia; Shapiro, Rick; Fan, Jie; Billiar, Timothy R.; Scott, Melanie J.

    2013-01-01

    Caspase 1 activation can be induced by oxidative stress, which leads to the release of the proinflammatory cytokines IL1β and IL18 in myeloid cells and a potentially damaging inflammatory response. However, little is known about the role of caspase 1 in non-immune cells, such as hepatocytes, that express and activate the inflammasome but do not produce a significant amount of IL1β/IL18. Here we demonstrate that caspase 1 activation protects against cell death after redox stress induced by hypoxia/reoxygenation in hepatocytes. Mechanistically, we show that caspase 1 reduces mitochondrial respiration and reactive oxygen species by increasing mitochondrial autophagy and subsequent clearance of mitochondria in hepatocytes after hypoxia/reoxygenation. Caspase 1 increases autophagic flux through up-regulating autophagy initiator beclin 1 during redox stress and is an important cell survival factor in hepatocytes. We find that during hemorrhagic shock with resuscitation, an in vivo mouse model associated with severe hepatic redox stress, caspase 1 activation is also protective against liver injury and excessive oxidative stress through the up-regulation of beclin 1. Our findings suggest an alternative role for caspase 1 activation in promoting adaptive responses to oxidative stress and, more specifically, in limiting reactive oxygen species production and damage in cells and tissues where IL1β/IL18 are not highly expressed. PMID:23589298

  2. Chemical Cocktails Enable Hepatic Reprogramming of Mouse Fibroblasts with a Single Transcription Factor

    Directory of Open Access Journals (Sweden)

    Ren Guo

    2017-08-01

    Full Text Available Liver or hepatocytes transplantation is limited by the availability of donor organs. Functional hepatocytes independent of the donor sources may have wide applications in regenerative medicine and the drug industry. Recent studies have demonstrated that chemical cocktails may induce reprogramming of fibroblasts into a range of functional somatic cells. Here, we show that mouse fibroblasts can be transdifferentiated into the hepatocyte-like cells (iHeps using only one transcription factor (TF (Foxa1, Foxa2, or Foxa3 plus a chemical cocktail. These iHeps show typical epithelial morphology, express multiple hepatocyte-specific genes, and acquire hepatocyte functions. Genetic lineage tracing confirms the fibroblast origin of these iHeps. More interestingly, these iHeps are expandable in vitro and can reconstitute the damaged hepatic tissues of the fumarylacetoacetate hydrolase-deficient (Fah−/− mice. Our study provides a strategy to generate functional hepatocyte-like cells by using a single TF plus a chemical cocktail and is one step closer to generate the full-chemical iHeps.

  3. 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

  4. Effects of edaravone, a radical scavenger, on hepatocyte transplantation.

    Science.gov (United States)

    Hayashi, Chihiro; Ito, Masahiro; Ito, Ryoutaro; Murakumo, Akiko; Yamamoto, Naoki; Hiramatsu, Noriko; Fox, Ira J; Horiguchi, Akihiko

    2014-12-01

    Hepatocyte transplantation (HTx) has yielded significant improvements in liver function and survival in experimentally induced acute liver failure and liver-based metabolic disease. However, transplantation is inefficient, and it is thought that transplanted hepatocytes have a shortened lifespan because of inflammation involving excess nitric oxide (NO). The present study aimed to clarify whether edaravone, a free radical scavenger used to treat ischemic stroke, could reduce ischemic changes in hepatocyte-transplanted livers. Edaravone (3 mg/kg) was administered intravenously 24 h before HTx to Nagase analbuminemic rats (NARs). Hepatocytes were isolated, and 30 × 10(6) cells were injected in a 1.0-ml volume directly into the spleens of NARs. All experimental groups studied received FK506 to control rejection. Animals in Group A received medium-only; Group B received HTx only; and Group C received HTx and edaravone. Forty-eight hours after transplantation, the hepatocytes from animals were isolated and analyzed for staining with propidium iodide- and annexin-V using flow cytometry. Liver sections were also studied by immunostaining for albumin, and TUNEL. Peripheral blood serum albumin levels were measured on post-transplant days 0, 3, 5, 7, 10 and 14 using ELISA. The edaravone-treated animals demonstrated an increased number of engrafted donor hepatocytes in the liver. The edaravone-treated liver sections also contained fewer TUNEL-positive cells and animals that received edaravone had higher serum albumin levels post-transplantation. Hepatocytes were also found to have increased in numbers 2 weeks following treatment with edaravone. Edaravone administration during HTx can suppress apoptosis near the transplanted cells, increasing engraftment. These studies indicate its potential usefulness for future clinical application. © 2014 Japanese Society of Hepato-Biliary-Pancreatic Surgery.

  5. Water and nonelectrolyte permeability of isolated rat hepatocytes

    International Nuclear Information System (INIS)

    Alpini, G.; Garrick, R.A.; Jones, M.J.; Nunes, R.; Tavoloni, N.

    1986-01-01

    We have measured the diffusive permeability coefficients of isolated rat hepatocytes to 3 H 2 O, [ 14 C]urea, [ 14 C]erythritol, [ 14 C]mannitol, [ 3 H]sucrose, and [ 3 H]inulin, employing a technique previously developed for erythrocytes (Redwood et al., J. Gen. Physiol 64:706-729, 1974). Diffusion coefficients for the tracer molecules were measured in packed hepatocytes, supernatant fluid, and intracellular medium (lysed hepatocytes) and were calculated assuming one-dimensional semi-infinite diffusion through a homogeneous medium. By applying the series-parallel pathway model, the following permeability coefficients (10(-5) cm/sec) for the hepatocyte plasma membrane were obtained. 3 H 2 O, 98.6 +/- 18.4; [ 14 C]urea, 18.2 +/- 5.3; [ 14 C]erythritol, 4.8 +/- 1.6; [ 14 C]mannitol, 3.1 +/- 1.4; [ 3 H]sucrose, 0; [ 3 H]inulin, 0. These results indicate that isolated rat hepatocytes are highly permeable to water and polar nonelectrolytes, when compared with other transporting epithelia. This relatively high cellular permeability is consistent with a model in which nonelectrolyte permeation is via an aqueous pathway of equivalent pore diameter of 8-12 A. The finding that [ 14 C]erythritol and [ 14 C]mannitol cross the hepatocyte plasma membrane indicates that these molecules enter the bile canaliculus through the transcellular route. Conversely, the failure of [ 3 H]sucrose and [ 3 H]inulin to permeate the hepatocyte in the isolated condition supports the concept that biliary entry of these large carbohydrates, at least that fraction which cannot be accounted for by a vesicular mechanism, must occur via the transjunctional shunt pathway

  6. Hepatocyte-specific deletion of the keap1 gene activates Nrf2 and confers potent resistance against acute drug toxicity

    International Nuclear Information System (INIS)

    Okawa, Hiromi; Motohashi, Hozumi; Kobayashi, Akira; Aburatani, Hiroyuki; Kensler, Thomas W.; Yamamoto, Masayuki

    2006-01-01

    Nrf2 is a key regulator of many detoxifying enzyme genes, and cytoplasmic protein Keap1 represses the Nrf2 activity under quiescent conditions. Germ line deletion of the keap1 gene results in constitutive activation of Nrf2, but the pups unexpectedly died before weaning. To investigate how constitutive activation of Nrf2 influences the detoxification system in adult mice, we generated mice bearing a hepatocyte-specific disruption of the keap1 gene. Homozygous mice were viable and their livers displayed no apparent abnormalities, but nuclear accumulation of Nrf2 is elevated. Microarray analysis revealed that, while many detoxifying enzyme genes are highly expressed, some of the typical Nrf2-dependent genes are only marginally increased in the Keap1-deficient liver. The mutant mice were significantly more resistant to toxic doses of acetaminophen than control animals. These results demonstrate that chronic activation of Nrf2 confers animals with resistance to xenobiotics without affecting the morphological and physiological integrity of hepatocytes

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

    Directory of Open Access Journals (Sweden)

    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

  8. Whole-body γ-irradiation decelerates rat hepatocyte polyploidization.

    Science.gov (United States)

    Ikhtiar, Adnan M

    2015-07-01

    To characterize hepatocyte polyploidization induced by intermediate dose of γ-ray. Male Wistar strain rats were whole-body irradiated (WBI) with 2 Gy of γ-ray at the age of 1 month, and 5-6 rats were sacrificed monthly at 0-25 months after irradiation. The nuclear DNA content of individual hepatocytes was measured by flow cytometry, then hepatocytes were classified into various ploidy classes. Survival percentage, after exposure up to the end of the study, did not indicate any differences between the irradiated groups and controls. The degree of polyploidization in hepatocytes of irradiated rats, was significantly lower than that for the control after 1 month of exposure, and it continued to be lower after up to 8 months. Thereafter, the degree of polyploidization in the irradiated group slowly returned to the control level when the irradiated rats reached the age of 10 months. Intermediate dose of ionizing radiation, in contrast to high doses, decelerate hepatocyte polyploidization, which may coincides with the hypothesis of the beneficial effects of low doses of ionizing radiation.

  9. Ultrastructural study of Rift Valley fever virus in the mouse model

    Energy Technology Data Exchange (ETDEWEB)

    Reed, Christopher; Steele, Keith E.; Honko, Anna; Shamblin, Joshua; Hensley, Lisa E. [United States Army Medical Research Institute of Infectious Diseases (USAMRIID), Fort Detrick, MD (United States); Smith, Darci R., E-mail: darci.smith1@us.army.mil [United States Army Medical Research Institute of Infectious Diseases (USAMRIID), Fort Detrick, MD (United States)

    2012-09-15

    Detailed ultrastructural studies of Rift Valley fever virus (RVFV) in the mouse model are needed to develop and characterize a small animal model of RVF for the evaluation of potential vaccines and therapeutics. In this study, the ultrastructural features of RVFV infection in the mouse model were analyzed. The main changes in the liver included the presence of viral particles in hepatocytes and hepatic stem cells accompanied by hepatocyte apoptosis. However, viral particles were observed rarely in the liver; in contrast, particles were extremely abundant in the CNS. Despite extensive lymphocytolysis, direct evidence of viral replication was not observed in the lymphoid tissue. These results correlate with the acute-onset hepatitis and delayed-onset encephalitis that are dominant features of severe human RVF, but suggest that host immune-mediated mechanisms contribute significantly to pathology. The results of this study expand our knowledge of RVFV-host interactions and further characterize the mouse model of RVF.

  10. Ultrastructural study of Rift Valley fever virus in the mouse model

    International Nuclear Information System (INIS)

    Reed, Christopher; Steele, Keith E.; Honko, Anna; Shamblin, Joshua; Hensley, Lisa E.; Smith, Darci R.

    2012-01-01

    Detailed ultrastructural studies of Rift Valley fever virus (RVFV) in the mouse model are needed to develop and characterize a small animal model of RVF for the evaluation of potential vaccines and therapeutics. In this study, the ultrastructural features of RVFV infection in the mouse model were analyzed. The main changes in the liver included the presence of viral particles in hepatocytes and hepatic stem cells accompanied by hepatocyte apoptosis. However, viral particles were observed rarely in the liver; in contrast, particles were extremely abundant in the CNS. Despite extensive lymphocytolysis, direct evidence of viral replication was not observed in the lymphoid tissue. These results correlate with the acute-onset hepatitis and delayed-onset encephalitis that are dominant features of severe human RVF, but suggest that host immune-mediated mechanisms contribute significantly to pathology. The results of this study expand our knowledge of RVFV–host interactions and further characterize the mouse model of RVF.

  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. Determination of metabolic stability using cryopreserved hepatocytes from rainbow trout (Oncorhynchus mykiss)

    Science.gov (United States)

    Standard protocols for isolating, cryopreserving, and thawing rainbow trout hepatocytes are described, along with procedures for using fresh or cryopreserved hepatocytes to assess chemical metabolic stability in fish by means of a substrate depletion approach. Variations on thes...

  13. Microencapsulation of Hepatocytes and Mesenchymal Stem Cells for Therapeutic Applications.

    Science.gov (United States)

    Meier, Raphael P H; Montanari, Elisa; Morel, Philippe; Pimenta, Joël; Schuurman, Henk-Jan; Wandrey, Christine; Gerber-Lemaire, Sandrine; Mahou, Redouan; Bühler, Leo H

    2017-01-01

    Encapsulated hepatocyte transplantation and encapsulated mesenchymal stem cell transplantation are newly developed potential treatments for acute and chronic liver diseases, respectively. Cells are microencapsulated in biocompatible semipermeable alginate-based hydrogels. Microspheres protect cells against antibodies and immune cells, while allowing nutrients, small/medium size proteins and drugs to diffuse inside and outside the polymer matrix. Microencapsulated cells are assessed in vitro and designed for experimental transplantation and for future clinical applications.Here, we describe the protocol for microencapsulation of hepatocytes and mesenchymal stem cells within hybrid poly(ethylene glycol)-alginate hydrogels.

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

    Directory of Open Access Journals (Sweden)

    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

  15. Unraveling the Molecular Mechanism(s) Underlying Er+/PR-Breast Tumorigenesis Using a Novel Genetically Engineered Mouse Model

    Science.gov (United States)

    2011-11-01

    and subjected to silver stain, immunoblot, or LC-MS/MS spec- tral analyses (The MSU Proteomics Facility, Michigan State University). Mouse Hepatocyte... flotation gradient essentially as described [29]. Briefly, cells were incubated with 10 ng/ml EGF for 10 min at 37uC. After several washings, cells were

  16. 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

  17. 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.

  18. File list: His.Liv.10.AllAg.Hepatocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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  11. File list: Oth.Liv.50.AllAg.Hepatocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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  14. File list: ALL.Liv.10.AllAg.Hepatocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  15. File list: Pol.Liv.05.AllAg.Hepatocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  16. File list: Oth.Liv.20.AllAg.Hepatocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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  11. Purification of Oogonial Stem Cells From Adult Mouse and Human Ovaries: An Assessment of the Literature and a View Toward the Future

    OpenAIRE

    Woods, Dori C.; White, Yvonne A. R.; Tilly, Jonathan L.

    2013-01-01

    Contemporary claims that mitotically active female germ line or oogonial stem cells (OSCs) exist and support oogenesis during postnatal life in mammals have been debated in the field of reproductive biology since March 2004, when a mouse study posed the first serious challenge to the dogma of a fixed pool of oocytes being endowed at birth in more than 50 years. Other studies have since been put forth that further question the validity of this dogma, including the isolation of OSCs from neonat...

  12. Genome-wide binding and transcriptome analysis of human farnesoid X receptor in primary human hepatocytes.

    Directory of Open Access Journals (Sweden)

    Le Zhan

    Full Text Available Farnesoid X receptor (FXR, NR1H4 is a ligand-activated transcription factor, belonging to the nuclear receptor superfamily. FXR is highly expressed in the liver and is essential in regulating bile acid homeostasis. FXR deficiency is implicated in numerous liver diseases and mice with modulation of FXR have been used as animal models to study liver physiology and pathology. We have reported genome-wide binding of FXR in mice by chromatin immunoprecipitation - deep sequencing (ChIP-seq, with results indicating that FXR may be involved in regulating diverse pathways in liver. However, limited information exists for the functions of human FXR and the suitability of using murine models to study human FXR functions.In the current study, we performed ChIP-seq in primary human hepatocytes (PHHs treated with a synthetic FXR agonist, GW4064 or DMSO control. In parallel, RNA deep sequencing (RNA-seq and RNA microarray were performed for GW4064 or control treated PHHs and wild type mouse livers, respectively.ChIP-seq showed similar profiles of genome-wide FXR binding in humans and mice in terms of motif analysis and pathway prediction. However, RNA-seq and microarray showed more different transcriptome profiles between PHHs and mouse livers upon GW4064 treatment.In summary, we have established genome-wide human FXR binding and transcriptome profiles. These results will aid in determining the human FXR functions, as well as judging to what level the mouse models could be used to study human FXR functions.

  13. Immortalization of Human Fetal Hepatocyte by Ectopic Expression of Human Telomerase Reverse Transcriptase, Human Papilloma Virus (E7) and Simian Virus 40 Large T (SV40 T) Antigen Towards Bioartificial Liver Support.

    Science.gov (United States)

    Giri, Shibashish; Bader, Augustinus

    2014-09-01

    Generation of genetically stable and non-tumoric immortalization cell line from primary cells would be enormously useful for research and therapeutic purposes, but progress towards this goal has so far been limited. It is now universal acceptance that immortalization of human fetal hepatocytes based on recent advances of telomerase biology and oncogene, lead to unlimited population doubling could be the possible source for bioartificial liver device. Immortalization of human fetal hepatocytes cell line by ectopic expression of human telomerase reverse transcriptase (hTERT), human papilloma virus gene (E7) and simian virus 40 large T (SV40 T) antigens is main goal of present study. We used an inducible system containing human telomerase and E7, both of which are cloned into responder constructs controlled by doxycycline transactivator. We characterized the immortalized human fetal hepatocyte cells by analysis of green fluorescent cells (GFP) positive cells using flow cytometry (FACs) cell sorting and morphology, proliferative rate and antigen expression by immunohistochemical analysis. In addition to we analysized lactate formation, glucose consumption, albumin secretion and urea production of immortalized human fetal hepatocyte cells. After 25 attempts for transfection of adult primary hepatocytes by human telomerase and E7 to immortalize them, none of the transfection systems resulted in the production of a stable, proliferating cell line. Although the transfection efficiency was more than 70% on the first day, the vast majority of the transfected hepatocytes lost their signal within the first 5-7 days. The remaining transfected hepatocytes persisted for 2-4 weeks and divided one or two times without forming a clone. After 10 attempts of transfection human fetal hepatocytes using the same transfection system, we obtained one stable human fetal hepatocytes cell line which was able albumin secretion urea production and glucose consumption. We established a

  14. 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

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

    International Nuclear Information System (INIS)

    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

  16. Insulin infusion reduces hepatocyte growth factor in lean humans

    DEFF Research Database (Denmark)

    de Courten, Barbora; de Courten, Maximilian; Dougherty, Sonia

    2013-01-01

    OBJECTIVE: Plasma Hepatocyte Growth Factor (HGF) is significantly elevated in obesity and may contribute to vascular disease, metabolic syndrome or cancer in obese individuals. The current studies were done to determine if hyperinsulinemia increases plasma HGF. MATERIALS/METHODS: Twenty-two parti...

  17. 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.

  18. The use of pig hepatocytes for biotransformation and toxicity studies

    NARCIS (Netherlands)

    Hoogenboom, L.A.P.

    1991-01-01

    The three main objectives of this study were, (1) to investigate the possibility to isolate viable hepatocytes from liver samples of pigs, (2) to study their use for biotransformation and toxicity studies, and (3) to demonstrate the value of this model, in particular in the field of residue

  19. Hepatocytes in the development of liver support systems

    NARCIS (Netherlands)

    I.H.M. Borel Rinkes (Inne)

    1993-01-01

    textabstractThis thesis focuses on the development of alternative strategies in the treatment of patients with acute fulminant hepatic failure and inborn errors of metabolism, using hepatocytes as the basis of liver support. When compared with transplantation of the liver as an organ, the

  20. Hepatocyte heterogeneity in the metabolism of amino acids and ammonia

    NARCIS (Netherlands)

    Häussinger, D.; Lamers, W. H.; Moorman, A. F.

    1992-01-01

    With respect to hepatocyte heterogeneity in ammonia and amino acid metabolism, two different patterns of sublobular gene expression are distinguished: 'gradient-type' and 'strict- or compartment-type' zonation. An example for strict-type zonation is the reciprocal distribution of carbamoylphosphate

  1. Induction of hepatocyte polyploidization in rats of different age by ionizing radiation of different LET

    International Nuclear Information System (INIS)

    Gil'yano, N.Ya.; Malinovskij, O.V.; Khair, M.B.

    1992-01-01

    A decrease in the effectiveness of neutron-irradiation with respect to fusion of nonproliferating hepatocytes of animals with age was shown by the method of flow cytometry. There was an inverse relationship between the effectiveness of induction of non-proliferating hepatocytes fusion and neutron energy. The process of hepatocyte fusion induced by neutrons was inhibited by uranyl acetate. No age-dependent changes were noted in the induction of polyploidization of proliferating hepatocytes by sparsely ionizing radiation. A hypothesis is proposed concerning a membrane nature of the target responsible for hepatocyte polyploidization induced by densely ionizing radiation. (authors). 8 refs., 4 figs., 5 tabs

  2. Induction of hepatocyte polyploidization in rats of different age by ionizing radiation of different LET

    International Nuclear Information System (INIS)

    Gil'yano, N.Ya.; Malinovskij, O.V.; Khair, M.B.

    1990-01-01

    A decrease in the effectiveness of neutron-irradiation with respect to fusion of nonproliferating hepatocytes of animals with age was shown by the method of flow cytometry. There was an inverse relationship between the effectiveness of induction of non-proliferating hepatocytes fusion and neutron energy. The process of hepatocyte fusion induced by neutrons was inhibited by uranyl acetate. No age-dependent changes were noted in the induction of polyploidization of proliferating hepatocytes by sparsely ionizing radiation. A hypothesis is proposed concerning a membrane nature of the target responsible for hepatocyte polyploidization induced by densely ionizing radiation

  3. Localization of brain-derived neurotrophic factor, neurotrophin-4, tropomyosin-related kinase b receptor, and p75 NTR receptor by high-resolution immunohistochemistry on the adult mouse neuromuscular junction.

    Science.gov (United States)

    Garcia, Neus; Tomàs, Marta; Santafe, Manel M; Lanuza, M Angel; Besalduch, Nuria; Tomàs, Josep

    2010-03-01

    Neurotrophins and their receptors, the trk receptor tyrosine kinases (trks) and p75(NTR), are differentially expressed among the cell types that make up synapses. It is important to determine the precise location of these molecules involved in neurotransmission. Here we use immunostaining and Western blotting to study the localization and expression of neurotrophin brain-derived neurotrophic factor (BDNF) and neurotrophin-4 (NT-4) and the receptors tropomyosin-related kinase b (trkB) and p75(NTR) at the adult neuromuscular junction. Our confocal immunofluorescence results on the whole mounts of the mouse Levator auris longus muscle and on semithin cross-sections showed that BDNF, NT-4, trkB, and p75(NTR) were localized on the three cells in the neuromuscular synapse (motor axons, post-synaptic muscle and Schwann cells).

  4. Radio-deoxynucleoside Analogs used for Imaging tk Expression in a Transgenic Mouse Model of Induced Hepatocellular Carcinoma

    Directory of Open Access Journals (Sweden)

    Haibin Tian, Xincheng Lu, Hong Guo, David Corn, Joseph Molter, Bingcheng Wang, Guangbin Luo, Zhenghong Lee

    2012-01-01

    Full Text Available Purpose: A group of radiolabeled thymidine analogs were developed as radio-tracers for imaging herpes viral thymidine kinase (HSV1-tk or its variants used as reporter gene. A transgenic mouse model was created to express tk upon liver injury or naturally occurring hepatocellular carcinoma (HCC. The purpose of this study was to use this unique animal model for initial testing with radio-labeled thymidine analogs, mainly a pair of newly emerging nucleoside analogs, D-FMAU and L-FMAU.Methods: A transgeneic mouse model was created by putting a fused reporter gene system, firefly luciferase (luc and HSV1-tk, under the control of mouse alpha fetoprotein (Afp promoter. Initial multimodal imaging, which was consisted of bioluminescent imaging (BLI and planar gamma scintigraphy with [125I]-FIAU, was used for examining the model creation in the new born and liver injury in the adult mice. Carcinogen diethylnitrosamine (DEN was then administrated to induce HCC in these knock-in mice such that microPET imaging could be used to track the activity of Afp promoter during tumor development and progression by imaging tk expression first with [18F]-FHBG. Dynamic PET scans with D-[18F]-FMAU and L-[18F]-FMAU were then performed to evaluate this pair of relatively new tracers. Cells were derived from these liver tumors for uptake assays using H-3 labeled version of PET tracers.Results: The mouse model with dual reporters: HSV1-tk and luc placed under the transcriptional control of an endogenous Afp promoter was used for imaging studies. The expression of the Afp gene was highly specific in proliferative hepatocytes, in regenerative liver, and in developing fetal liver, and thus provided an excellent indicator for liver injury and cancer development in adult mice. Both D-FMAU and L-FMAU showed stable liver tumor uptake where the tk gene was expressed under the Afp promoter. The performance of this pair of tracers was slightly different in terms of signal

  5. 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.

  6. 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.

  7. A methionine-choline-deficient diet elicits NASH in the immunodeficient mouse featuring a model for hepatic cell transplantation.

    Science.gov (United States)

    Pelz, Sandra; Stock, Peggy; Brückner, Sandra; Christ, Bruno

    2012-02-01

    Non-alcoholic staetohepatitis (NASH) is associated with fat deposition in the liver favoring inflammatory processes and development of fibrosis, cirrhosis and finally hepatocellular cancer. In Western lifestyle countries, NASH has reached a 20% prevalence in the obese population with escalating tendency in the future. Very often, liver transplantation is the only therapeutic option. Recently, transplantation of hepatocyte-like cells differentiated from mesenchymal stem cells was suggested a feasible alternative to whole organ transplantation to ameliorate donor organ shortage. Hence, in the present work an animal model of NASH was established in immunodeficient mice to investigate the feasibility of human stem cell-derived hepatocyte-like cell transplantation. NASH was induced by feeding a methionine/choline-deficient diet (MCD-diet) for up to 5 weeks. Animals developed a fatty liver featuring fibrosis and elevation of the proinflammatory markers serum amyloid A (SAA) and tumor necrosis factor alpha (TNFα). Hepatic triglycerides were significantly increased as well as alanine aminotransferase demonstrating inflammation-linked hepatocyte damage. Elevation of αSMA mRNA and collagen I as well as liver architecture deterioation indicated massive fibrosis. Both short- and long-term post-transplantation human hepatocyte-like cells resided in the mouse host liver indicating parenchymal penetration and most likely functional engraftment. Hence, the NASH model in the immunodeficient mouse is the first to allow for the assessment of the therapeutic impact of human stem cell-derived hepatocyte transplantation. Copyright © 2011 Elsevier Inc. All rights reserved.

  8. E4orf1 improves lipid and glucose metabolism in hepatocytes: a template to improve steatosis & hyperglycemia.

    Directory of Open Access Journals (Sweden)

    Emily J Dhurandhar

    Full Text Available Hepatic steatosis often accompanies obesity and insulin resistance. The cornerstones of steatosis treatment include reducing body weight and dietary fat intake, which are marginally successful over the long term. Ad36, a human adenovirus, may offer a template to overcome these limitations. In vitro and in vivo studies collectively indicate that via its E4orf1 protein, Ad36 improves hyperglycemia, and attenuates hepatic steatosis, despite a high fat diet and without weight loss. Considering that hepatic insulin sensitivity, or the synthesis, oxidation, or export of fatty acid by hepatocytes are the key determinant of hepatic lipid storage, we determined the role of E4orf1 protein in modulating these physiological pathways. For this study, HepG2 cells, or mouse primary hepatocytes were transfected with E4orf1 or the null vector. Glucose output by hepatocytes was determined under gluconeogenic conditions (cAMP and dexamethasone, or glucagon exposure. Also, de-novo lipogenesis, palmitate oxidation, and lipid export as determined by apoB secretion were measured 48 h post transfection. Results show that compared to null vector transfected cells, E4orf1 significantly reduced glucose output in basal and gluconeogenic conditions. E4orf1 reduced de-novo lipogenesis by about 35%, increased complete fatty acid oxidation 2-fold (p<0.0001, and apoB secretion 1.5 fold(p<0.003. Response of key signaling molecules to E4orf1 transfection was in agreement with these findings. Thus, E4orf1 offers a valuable template to exogenously modulate hepatic glucose and lipid metabolism. Elucidating the underlying molecular mechanism may help develop therapeutic approaches for treating diabetes or non-alcoholic fatty liver disease(NAFLD.

  9. E4orf1 improves lipid and glucose metabolism in hepatocytes: a template to improve steatosis & hyperglycemia.

    Science.gov (United States)

    Dhurandhar, Emily J; Krishnapuram, Rashmi; Hegde, Vijay; Dubuisson, Olga; Tao, Rongya; Dong, X Charlie; Ye, Jianping; Dhurandhar, Nikhil V

    2012-01-01

    Hepatic steatosis often accompanies obesity and insulin resistance. The cornerstones of steatosis treatment include reducing body weight and dietary fat intake, which are marginally successful over the long term. Ad36, a human adenovirus, may offer a template to overcome these limitations. In vitro and in vivo studies collectively indicate that via its E4orf1 protein, Ad36 improves hyperglycemia, and attenuates hepatic steatosis, despite a high fat diet and without weight loss. Considering that hepatic insulin sensitivity, or the synthesis, oxidation, or export of fatty acid by hepatocytes are the key determinant of hepatic lipid storage, we determined the role of E4orf1 protein in modulating these physiological pathways. For this study, HepG2 cells, or mouse primary hepatocytes were transfected with E4orf1 or the null vector. Glucose output by hepatocytes was determined under gluconeogenic conditions (cAMP and dexamethasone, or glucagon exposure). Also, de-novo lipogenesis, palmitate oxidation, and lipid export as determined by apoB secretion were measured 48 h post transfection. Results show that compared to null vector transfected cells, E4orf1 significantly reduced glucose output in basal and gluconeogenic conditions. E4orf1 reduced de-novo lipogenesis by about 35%, increased complete fatty acid oxidation 2-fold (pE4orf1 transfection was in agreement with these findings. Thus, E4orf1 offers a valuable template to exogenously modulate hepatic glucose and lipid metabolism. Elucidating the underlying molecular mechanism may help develop therapeutic approaches for treating diabetes or non-alcoholic fatty liver disease(NAFLD).

  10. Regulation of the G1/S Transition in Hepatocytes: Involvement of the Cyclin-Dependent Kinase Cdk1 in the DNA Replication

    Directory of Open Access Journals (Sweden)

    Anne Corlu

    2012-01-01

    Full Text Available A singular feature of adult differentiated hepatocytes is their capacity to proliferate allowing liver regeneration. This review emphasizes the literature published over the last 20 years that established the most important pathways regulating the hepatocyte cell cycle. Our article also aimed at illustrating that many discoveries in this field benefited from the combined use of in vivo models of liver regeneration and in vitro models of primary cultures of human and rodent hepatocytes. Using these models, our laboratory has contributed to decipher the different steps of the progression into the G1 phase and the commitment to S phase of proliferating hepatocytes. We identified the mitogen dependent restriction point located at the two-thirds of the G1 phase and the concomitant expression and activation of both Cdk1 and Cdk2 at the G1/S transition. Furthermore, we demonstrated that these two Cdks contribute to the DNA replication. Finally, we provided strong evidences that Cdk1 expression and activation is correlated to extracellular matrix degradation upon stimulation by the pro-inflammatory cytokine TNFα leading to the identification of a new signaling pathway regulating Cdk1 expression at the G1/S transition. It also further confirms the well-orchestrated regulation of liver regeneration via multiple extracellular signals and pathways.

  11. Postnatal liver growth and regeneration are independent of c-myc in a mouse model of conditional hepatic c-myc deletion

    Directory of Open Access Journals (Sweden)

    Sanders Jennifer A

    2012-03-01

    Full Text Available Abstract Background The transcription factor c-myc regulates genes involved in hepatocyte growth, proliferation, metabolism, and differentiation. It has also been assigned roles in liver development and regeneration. In previous studies, we made the unexpected observation that c-Myc protein levels were similar in proliferating fetal liver and quiescent adult liver with c-Myc displaying nucleolar localization in the latter. In order to investigate the functional role of c-Myc in adult liver, we have developed a hepatocyte-specific c-myc knockout mouse, c-mycfl/fl;Alb-Cre. Results Liver weight to body weight ratios were similar in control and c-myc deficient mice. Liver architecture was unaffected. Conditional c-myc deletion did not result in compensatory induction of other myc family members or in c-Myc's binding partner Max. Floxed c-myc did have a negative effect on Alb-Cre expression at 4 weeks of age. To explore this relationship further, we used the Rosa26 reporter line to assay Cre activity in the c-myc floxed mice. No significant difference in Alb-Cre activity was found between control and c-mycfl/fl mice. c-myc deficient mice were studied in a nonproliferative model of liver growth, fasting for 48 hr followed by a 24 hr refeeding period. Fasting resulted in a decrease in liver mass and liver protein, both of which recovered upon 24 h of refeeding in the c-mycfl/fl;Alb-Cre animals. There was also no effect of reducing c-myc on recovery of liver mass following 2/3 partial hepatectomy. Conclusions c-Myc appears to be dispensable for normal liver growth during the postnatal period, restoration of liver mass following partial hepatectomy and recovery from fasting.

  12. Selective alteration of adult hippocampal neurogenesis and impaired spatial pattern separation performance in the RSK2-deficient mouse model of Coffin-Lowry syndrome.

    Science.gov (United States)

    Castillon, Charlotte; Lunion, Steeve; Desvignes, Nathalie; Hanauer, André; Laroche, Serge; Poirier, Roseline

    2018-07-01

    Adult neurogenesis is involved in certain hippocampus-dependent cognitive functions and is linked to psychiatric diseases including intellectual disabilities. The Coffin-Lowry syndrome (CLS) is a developmental disorder caused by mutations in the Rsk2 gene and characterized by intellectual disabilities associated with growth retardation. How RSK2-deficiency leads to cognitive dysfunctions in CLS is however poorly understood. Here, using Rsk2 Knock-Out mice, we characterized the impact of RSK2 deficiency on adult hippocampal neurogenesis in vivo. We report that the absence of RSK2 does not affect basal proliferation, differentiation and survival of dentate gyrus adult-born neurons but alters the maturation progression of young immature newborn neurons. Moreover, when RSK2-deficient mice were submitted to spatial learning, in contrast to wild-type mice, proliferation of adult generated neurons was decreased and no pro-survival effect of learning was observed. Thus, learning failed to recruit a selective population of young newborn neurons in association with deficient long-term memory recall. Given the proposed role of the dentate gyrus and of adult-generated newborn neurons in hippocampal-dependent pattern separation function, we explored this function in a delayed non-matching to place task and in an object-place pattern separation task and report severe deficits in spatial pattern separation in Rsk2-KO mice. Together, this study reveals a previously unknown role for RSK2 in the early stages of maturation and learning-dependent involvement of adult-born dentate gyrus neurons. These alterations associated with a deficit in the ability of RSK2-deficient mice to finely discriminate relatively similar spatial configurations, may contribute to cognitive dysfunction in CLS. Copyright © 2018 Elsevier Inc. All rights reserved.

  13. Keratin impact on PKCδ- and ASMase-mediated regulation of hepatocyte lipid raft size – implication for FasR-associated apoptosis

    Science.gov (United States)

    Gilbert, Stéphane; Loranger, Anne; Omary, M. Bishr

    2016-01-01

    ABSTRACT Keratins are epithelial cell intermediate filament (IF) proteins that are expressed as pairs in a cell-differentiation-regulated manner. Hepatocytes express the keratin 8 and 18 pair (denoted K8/K18) of IFs, and a loss of K8 or K18, as in K8-null mice, leads to degradation of the keratin partner. We have previously reported that a K8/K18 loss in hepatocytes leads to altered cell surface lipid raft distribution and more efficient Fas receptor (FasR, also known as TNFRSF6)-mediated apoptosis. We demonstrate here that the absence of K8 or transgenic expression of the K8 G62C mutant in mouse hepatocytes reduces lipid raft size. Mechanistically, we find that the lipid raft size is dependent on acid sphingomyelinase (ASMase, also known as SMPD1) enzyme activity, which is reduced in absence of K8/K18. Notably, the reduction of ASMase activity appears to be caused by a less efficient redistribution of surface membrane PKCδ toward lysosomes. Moreover, we delineate the lipid raft volume range that is required for an optimal FasR-mediated apoptosis. Hence, K8/K18-dependent PKCδ- and ASMase-mediated modulation of lipid raft size can explain the more prominent FasR-mediated signaling resulting from K8/K18 loss. The fine-tuning of ASMase-mediated regulation of lipid rafts might provide a therapeutic target for death-receptor-related liver diseases. PMID:27422101

  14. Developmental exposure to 50 parts-per-billion arsenic influences histone modifications and associated epigenetic machinery in a region- and sex-specific manner in the adult mouse brain

    International Nuclear Information System (INIS)

    Tyler, Christina R.; Hafez, Alexander K.; Solomon, Elizabeth R.; Allan, Andrea M.

    2015-01-01

    Epidemiological studies report that arsenic exposure via drinking water adversely impacts cognitive development in children and, in adults, can lead to greater psychiatric disease susceptibility, among other conditions. While it is known that arsenic toxicity has a profound effect on the epigenetic landscape, very few studies have investigated its effects on chromatin architecture in the brain. We have previously demonstrated that exposure to a low level of arsenic (50 ppb) during all three trimesters of fetal/neonatal development induces deficits in adult hippocampal neurogenesis in the dentate gyrus (DG), depressive-like symptoms, and alterations in gene expression in the adult mouse brain. As epigenetic processes control these outcomes, here we assess the impact of our developmental arsenic exposure (DAE) paradigm on global histone posttranslational modifications and associated chromatin-modifying proteins in the dentate gyrus and frontal cortex (FC) of adult male and female mice. DAE influenced histone 3 K4 trimethylation with increased levels in the male DG and FC and decreased levels in the female DG (no change in female FC). The histone methyltransferase MLL exhibited a similar sex- and region-specific expression profile as H3K4me3 levels, while histone demethylase KDM5B expression trended in the opposite direction. DAE increased histone 3 K9 acetylation levels in the male DG along with histone acetyltransferase (HAT) expression of GCN5 and decreased H3K9ac levels in the male FC along with decreased HAT expression of GCN5 and PCAF. DAE decreased expression of histone deacetylase enzymes HDAC1 and HDAC2, which were concurrent with increased H3K9ac levels but only in the female DG. Levels of H3 and H3K9me3 were not influenced by DAE in either brain region of either sex. These findings suggest that exposure to a low, environmentally relevant level of arsenic during development leads to long-lasting changes in histone methylation and acetylation in the adult

  15. Developmental exposure to 50 parts-per-billion arsenic influences histone modifications and associated epigenetic machinery in a region- and sex-specific manner in the adult mouse brain

    Energy Technology Data Exchange (ETDEWEB)

    Tyler, Christina R.; Hafez, Alexander K.; Solomon, Elizabeth R.; Allan, Andrea M., E-mail: aallan@salud.unm.edu

    2015-10-01

    Epidemiological studies report that arsenic exposure via drinking water adversely impacts cognitive development in children and, in adults, can lead to greater psychiatric disease susceptibility, among other conditions. While it is known that arsenic toxicity has a profound effect on the epigenetic landscape, very few studies have investigated its effects on chromatin architecture in the brain. We have previously demonstrated that exposure to a low level of arsenic (50 ppb) during all three trimesters of fetal/neonatal development induces deficits in adult hippocampal neurogenesis in the dentate gyrus (DG), depressive-like symptoms, and alterations in gene expression in the adult mouse brain. As epigenetic processes control these outcomes, here we assess the impact of our developmental arsenic exposure (DAE) paradigm on global histone posttranslational modifications and associated chromatin-modifying proteins in the dentate gyrus and frontal cortex (FC) of adult male and female mice. DAE influenced histone 3 K4 trimethylation with increased levels in the male DG and FC and decreased levels in the female DG (no change in female FC). The histone methyltransferase MLL exhibited a similar sex- and region-specific expression profile as H3K4me3 levels, while histone demethylase KDM5B expression trended in the opposite direction. DAE increased histone 3 K9 acetylation levels in the male DG along with histone acetyltransferase (HAT) expression of GCN5 and decreased H3K9ac levels in the male FC along with decreased HAT expression of GCN5 and PCAF. DAE decreased expression of histone deacetylase enzymes HDAC1 and HDAC2, which were concurrent with increased H3K9ac levels but only in the female DG. Levels of H3 and H3K9me3 were not influenced by DAE in either brain region of either sex. These findings suggest that exposure to a low, environmentally relevant level of arsenic during development leads to long-lasting changes in histone methylation and acetylation in the adult

  16. Similarities in the immunoglobulin response and VH gene usage in rhesus monkeys and humans exposed to porcine hepatocytes

    Directory of Open Access Journals (Sweden)

    Borie Dominic C

    2006-03-01

    Full Text Available Abstract Background The use of porcine cells and organs as a source of xenografts for human patients would vastly increase the donor pool; however, both humans and Old World primates vigorously reject pig tissues due to xenoantibodies that react with the polysaccharide galactose α (1,3 galactose (αGal present on the surface of many porcine cells. We previously examined the xenoantibody response in patients exposed to porcine hepatocytes via treatment(s with bioartficial liver devices (BALs, composed of porcine cells in a support matrix. We determined that xenoantibodies in BAL-treated patients are predominantly directed at porcine αGal carbohydrate epitopes, and are encoded by a small number of germline heavy chain variable region (VH immunoglobulin genes. The studies described in this manuscript were designed to identify whether the xenoantibody responses and the IgVH genes encoding antibodies to porcine hepatocytes in non-human primates used as preclinical models are similar to those in humans. Adult non-immunosuppressed rhesus monkeys (Macaca mulatta were injected intra-portally with porcine hepatocytes or heterotopically transplanted with a porcine liver lobe. Peripheral blood leukocytes and serum were obtained prior to and at multiple time points after exposure, and the immune response was characterized, using ELISA to evaluate the levels and specificities of circulating xenoantibodies, and the production of cDNA libraries to determine the genes used by B cells to encode those antibodies. Results Xenoantibodies produced following exposure to isolated hepatocytes and solid organ liver grafts were predominantly encoded by genes in the VH3 family, with a minor contribution from the VH4 family. Immunoglobulin heavy-chain gene (VH cDNA library screening and gene sequencing of IgM libraries identified the genes as most closely-related to the IGHV3-11 and IGHV4-59 germline progenitors. One of the genes most similar to IGHV3-11, VH3-11cyno, has

  17. CRISPR/Cas9 Engineering of Adult Mouse Liver Demonstrates That the Dnajb1-Prkaca Gene Fusion is Sufficient to Induce Tumors Resembling Fibrolamellar Hepatocellular Carcinoma

    DEFF Research Database (Denmark)

    Engelholm, Lars H; Riaz, Anjum; Serra, Denise

    2017-01-01

    BACKGROUND & AIMS: Fibrolamellar hepatocellular carcinoma (FL-HCC) is a primary liver cancer that predominantly affects young adults with no underlying liver disease. A somatic, 400 Kb deletion on chromosome 19 that fuses part of the DnaJ heat shock protein family (Hsp40) member B1 gene (DNAJB1...

  18. MRI-based assessment of liver perfusion and hepatocyte injury in the murine model of acute hepatitis.

    Science.gov (United States)

    Byk, Katarzyna; Jasinski, Krzysztof; Bartel, Zaneta; Jasztal, Agnieszka; Sitek, Barbara; Tomanek, Boguslaw; Chlopicki, Stefan; Skorka, Tomasz

    2016-12-01

    To assess alterations in perfusion and liver function in the concanavalin A (ConA)-induced mouse model of acute liver failure (ALF) using two magnetic resonance imaging (MRI)-based methods: dynamic contrast-enhanced MRI (DCE-MRI) with Gd-EOB-DTPA contrast agent and arterial spin labelling (ASL). BALB/c mice were studied using a 9.4 T MRI system. The IntraGateFLASH TM and FAIR-EPI pulse sequences were used for optimum mouse abdomen imaging. The average perfusion values for the liver of the control and ConA group were equal to 245 ± 20 and 200 ± 32 ml/min/100 g (p = 0.008, respectively). DCE-MRI showed that the time to the peak of the image enhancement was 6.14 ± 1.07 min and 9.72 ± 1.69 min in the control and ConA group (p < 0.001, respectively), while the rate of the contrast wash-out in the control and ConA group was 0.037 ± 0.008 and 0.021 ± 0.008 min -1 (p = 0.004, respectively). These results were consistent with hepatocyte injury in the ConA-treated mice as confirmed by histopathological staining. Both the ASL and DCE-MRI techniques represent a reliable methodology to assess alterations in liver perfusion and hepatocyte integrity in murine hepatitis.

  19. Hepatic tissue environment in NEMO-deficient mice critically regulates positive selection of donor cells after hepatocyte transplantation.

    Directory of Open Access Journals (Sweden)

    Michaela Kaldenbach

    Full Text Available BACKGROUND: Hepatocyte transplantation (HT is a promising alternative treatment strategy for end-stage liver diseases compared with orthotopic liver transplantation. A limitation for this approach is the low engraftment of donor cells. The deletion of the I-kappa B kinase-regulatory subunit IKKγ/NEMO in hepatocytes prevents nuclear factor (NF-kB activation and triggers spontaneous liver apoptosis, chronic hepatitis and the development of liver fibrosis and hepatocellular carcinoma. We hypothesized that NEMOΔhepa mice may therefore serve as an experimental model to study HT. METHODS: Pre-conditioned NEMOΔhepa mice were transplanted with donor-hepatocytes from wildtype (WT and mice deficient for the pro-apoptotic mediator Caspase-8 (Casp8Δhepa. RESULTS: Transplantation of isolated WT-hepatocytes into pre-conditioned NEMOΔhepa mice resulted in a 6-7 fold increase of donor cells 12 weeks after HT, while WT-recipients showed no liver repopulation. The use of apoptosis-resistant Casp8Δhepa-derived donor cells further enhanced the selection 3-fold after 12-weeks and up to 10-fold increase after 52 weeks compared with WT donors. While analysis of NEMOΔhepa mice revealed strong liver injury, HT-recipient NEMOΔhepa mice showed improved liver morphology and decrease in serum transaminases. Concomitant with these findings, the histological examination elicited an improved liver tissue architecture associated with significantly lower levels of apoptosis, decreased proliferation and a lesser amount of liver fibrogenesis. Altogether, our data clearly support the therapeutic benefit of the HT procedure into NEMOΔhepa mice. CONCLUSION: This study demonstrates the feasibility of the NEMOΔhepa mouse as an in vivo tool to study liver repopulation after HT. The improvement of the characteristic phenotype of chronic liver injury in NEMOΔhepa mice after HT suggests the therapeutic potential of HT in liver diseases with a chronic inflammatory phenotype and

  20. The cytoskeleton of digitonin-treated rat hepatocytes.

    Science.gov (United States)

    Fiskum, G; Craig, S W; Decker, G L; Lehninger, A L

    1980-06-01

    Treatment of isolated rat hepatocptes with low concentrations of digitonin increases the permeability of the plsma membrane to cytosolic proteins without causing release of organelles such as mitochondria into the surrounding medium. Electron microscopy showed that treatment of the cells with increasing concentations of digitonin results in a progressive loss in the continuity of the plasma membrane, while most other aspects of cellular morphology remain normal. Depletion of background staining material from the cytosol by digitonin treatment of the cells greatly enhances the visualization of the cytoskeleton. The use of this technique, together with immunofluorescent light microscopy, has verified the presence of an actin-containing filamentous network at the hepatocyte cortex as well as intermediate filaments distributed throughout the cell. Digitonin is thus useful both for selectively permeabilizing the plasma membrane and for intensifying the appearance of intracellular structures such as microfilaments that are normally difficult to observe in cells such as hepatocytes.

  1. [Current status and future perspectives of hepatocyte transplantation].

    Science.gov (United States)

    Pareja, Eugenia; Cortés, Miriam; Gómez-Lechón, M José; Maupoey, Javier; San Juan, Fernando; López, Rafael; Mir, Jose

    2014-02-01

    The imbalance between the number of potential beneficiaries and available organs, originates the search for new therapeutic alternatives, such as Hepatocyte transplantation (HT).Even though this is a treatment option for these patients, the lack of unanimity of criteria regarding indications and technique, different cryopreservation protocols, as well as the different methodology to assess the response to this therapy, highlights the need of a Consensus Conference to standardize criteria and consider future strategies to improve the technique and optimize the results.Our aim is to review and update the current state of hepatocyte transplantation, emphasizing the future research attempting to solve the problems and improve the results of this treatment. Copyright © 2013 AEC. Published by Elsevier Espana. All rights reserved.

  2. In vivo effects of Faizol Ubat Batuk, a herbal product on aminopyrine metabolism in rat hepatocytes

    Directory of Open Access Journals (Sweden)

    Abas Hj Hussin

    2011-09-01

    Full Text Available Traditional medicines, in particular herbal products, have been used abundantly over the years in curing several diseases. Pharmacological interactions of herbal products with modern drugs, however, remain to some extent unknown. Herein, we examined whether co-administration of Faizol Ubat Batuk (FUB, a mixture of aqueous extract of different plants, modifies the metabolism of aminopyrine, a conventional analgesic drug, in rat liver. We used rat hepatocytes outfitted by collagenase perfusion technique. Determination of aminopyrine n-demethylase activity was performed using the Nash colorimetric method, by measuring the amount of formaldehyde produced. Compared to control treatment, FUB significantly increased the hepatic metabolism of aminopyrine in healthy adult male rats. In contrast, the hepatic metabolism of aminopyrine in adult female rats was decreased. Besides, a biphasic effect in n-demethylase activity was observed in young male rats treated with FUB. In a subsequent experiment, FUB did not change the metabolism of aminopyrine in streptozotocin (STZ-diabetic adult male rats. In conclusion, administration of FUB could affect phase I aminopyrine metabolism in rat heptocytes. In addition, the effects of FUB on hepatic n-demethylase activity were gender and disease dependent.

  3. Ultrastructure of hepatocyte nuclei in irradiated, adrenalectomized rats

    Energy Technology Data Exchange (ETDEWEB)

    Orkisz, S.; Bartel, H.; Kmiec, B. (Military Medical Academy, Lodz (Poland))

    1984-01-01

    A cytochemical study of hepatocyte nuclei of adrenalectomized and irradiated rats was performed. After irradiation alone, the behaviour of the ribonucleoprotein components was studied according to Bernhard. The findings suggest that a delay occurs in the synthesis of preribosomal RNA in the nucleoli and in the transport of messenger RNA to the cytoplasm. The indirect effect of ionizing radiation on nuclear RNA synthesis is assumed to occur through the influence of cortical steroid hormones on the transcription process.

  4. Insulin-induced CARM1 upregulation facilitates hepatocyte proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Yeom, Chul-gon; Kim, Dong-il; Park, Min-jung; Choi, Joo-hee [College of Veterinary Medicine, Chonnam National University, Gwangju 500-757 (Korea, Republic of); Jeong, Jieun; Wi, Anjin; Park, Whoashig [Jeollanamdo Forest Resources Research Institute, Naju 520-833 (Korea, Republic of); Han, Ho-jae [College of Veterinary Medicine, Seoul National University, Seoul 151-741 (Korea, Republic of); Park, Soo-hyun, E-mail: parksh@chonnam.ac.kr [College of Veterinary Medicine, Chonnam National University, Gwangju 500-757 (Korea, Republic of)

    2015-06-05

    Previously, we reported that CARM1 undergoes ubiquitination-dependent degradation in renal podocytes. It was also reported that CARM1 is necessary for fasting-induced hepatic gluconeogenesis. Based on these reports, we hypothesized that treatment with insulin, a hormone typically present under the ‘fed’ condition, would inhibit gluconeogenesis via CARM1 degradation. HepG2 cells, AML-12 cells, and rat primary hepatocytes were treated with insulin to confirm CARM1 downregulation. Surprisingly, insulin treatment increased CARM1 expression in all cell types examined. Furthermore, treatment with insulin increased histone 3 methylation at arginine 17 and 26 in HepG2 cells. To elucidate the role of insulin-induced CARM1 upregulation, the HA-CARM1 plasmid was transfected into HepG2 cells. CARM1 overexpression did not increase the expression of lipogenic proteins generally increased by insulin signaling. Moreover, CARM1 knockdown did not influence insulin sensitivity. Insulin is known to facilitate hepatic proliferation. Like insulin, CARM1 overexpression increased CDK2 and CDK4 expression. In addition, CARM1 knockdown reduced the number of insulin-induced G2/M phase cells. Moreover, GFP-CARM1 overexpression increased the number of G2/M phase cells. Based on these results, we concluded that insulin-induced CARM1 upregulation facilitates hepatocyte proliferation. These observations indicate that CARM1 plays an important role in liver pathophysiology. - Highlights: • Insulin treatment increases CARM1 expression in hepatocytes. • CARM1 overexpression does not increase the expression of lipogenic proteins. • CARM1 knockdown does not influence insulin sensitivity. • Insulin-induced CARM1 upregulation facilitates hepatocyte proliferation.

  5. Insulin-induced CARM1 upregulation facilitates hepatocyte proliferation

    International Nuclear Information System (INIS)

    Yeom, Chul-gon; Kim, Dong-il; Park, Min-jung; Choi, Joo-hee; Jeong, Jieun; Wi, Anjin; Park, Whoashig; Han, Ho-jae; Park, Soo-hyun

    2015-01-01

    Previously, we reported that CARM1 undergoes ubiquitination-dependent degradation in renal podocytes. It was also reported that CARM1 is necessary for fasting-induced hepatic gluconeogenesis. Based on these reports, we hypothesized that treatment with insulin, a hormone typically present under the ‘fed’ condition, would inhibit gluconeogenesis via CARM1 degradation. HepG2 cells, AML-12 cells, and rat primary hepatocytes were treated with insulin to confirm CARM1 downregulation. Surprisingly, insulin treatment increased CARM1 expression in all cell types examined. Furthermore, treatment with insulin increased histone 3 methylation at arginine 17 and 26 in HepG2 cells. To elucidate the role of insulin-induced CARM1 upregulation, the HA-CARM1 plasmid was transfected into HepG2 cells. CARM1 overexpression did not increase the expression of lipogenic proteins generally increased by insulin signaling. Moreover, CARM1 knockdown did not influence insulin sensitivity. Insulin is known to facilitate hepatic proliferation. Like insulin, CARM1 overexpression increased CDK2 and CDK4 expression. In addition, CARM1 knockdown reduced the number of insulin-induced G2/M phase cells. Moreover, GFP-CARM1 overexpression increased the number of G2/M phase cells. Based on these results, we concluded that insulin-induced CARM1 upregulation facilitates hepatocyte proliferation. These observations indicate that CARM1 plays an important role in liver pathophysiology. - Highlights: • Insulin treatment increases CARM1 expression in hepatocytes. • CARM1 overexpression does not increase the expression of lipogenic proteins. • CARM1 knockdown does not influence insulin sensitivity. • Insulin-induced CARM1 upregulation facilitates hepatocyte proliferation

  6. [Crabtree effect caused by ketoses in isolated rat hepatocytes].

    Science.gov (United States)

    Martínez, P; Carrascosa, J M; Núñez de Castro, I

    1982-01-01

    Oxygen uptake and glycolytic activity were studied in hepatocytes isolated from fed rats. The addition of fructose or tagatose resulted in a 38% and 31% inhibition of cellular respiration respectively. The addition of 10 mM D-glyceraldehyde caused a slight Crabtree effect. Glucose, L-sorbose, or glycerol failed to modify oxygen consumption. Only incubation in the presence of fructose showed a high aerobic glycolysis measured by lactate production.

  7. Hepatocyte polyploidization and its association with pathophysiological processes

    OpenAIRE

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

    2017-01-01

    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 ...

  8. Functional activity of the rats’ hepatocytes under cancerogenesis

    Directory of Open Access Journals (Sweden)

    V. V. Ivchuk

    2007-10-01

    Full Text Available Enzymatic activity in rat’s hepatocytes under carcinoma Geuren T8 development as well as after introduction of rhenium compounds and cis-platin were studied. It has been determined that the decrease of enzymatic activity contrary to the control animals has been observed under simultaneous injection of cis-platin and cluster rhenium compounds in a liposome form. That confirms possible hepatoprotective properties of the rhenium compounds.

  9. 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

  10. Cholangiocarcinoma in Cirrhosis: Value of Hepatocyte Specific Magnetic Resonance Imaging.

    Science.gov (United States)

    Piscaglia, Fabio; Iavarone, Massimo; Galassi, Marzia; Vavassori, Sara; Renzulli, Matteo; Forzenigo, Laura Virginia; Granito, Alessandro; Salvatore, Veronica; Sangiovanni, Angelo; Golfieri, Rita; Colombo, Massimo; Bolondi, Luigi

    2015-10-01

    The diagnosis of intrahepatic cholangiocellular carcinoma (ICC) remains elusive at imaging, which is a critical issue in cirrhotic patients in whom a diagnosis of hepatocellular carcinoma (HCC) can be established only by imaging. The aim of the study was to evaluate the potential of MRI in the diagnosis of ICC in cirrhosis using 'hepatocyte-specific' Gadolinium (Gd)-based contrast agents. Sixteen histologically proven and retrospectively identified ICCs on cirrhosis were investigated with hepatocyte-specific magnetic resonance contrast agents (6 in Bologna with Gd-EOB-DTPA and 10 in Milan with Gd-BOPTA). The control group consisted of 41 consecutively and prospectively collected nodules (31 HCCs) imaged with Gd-EOB-DTPA. Fifteen ICC nodules (94%) displayed hypointensity in the hepatobiliary phase, suggesting malignancy. Thirteen cholangiocarcinomas (81%) showed hyperenhancement in the venous phase. Only 2 cholangiocarcinoma nodules showed hypoenhancement in the venous phase, corresponding to washout, in both cases preceded by rim enhancement in arterial phase. All the hepatocarcinomas showed hypointensity in hepatobiliary phase, but was always preceded by hypointensity in the venous phase; arterial rim enhancement was never observed in any hepatocarcinoma or regenerative nodule. MRI with hepatocyte-specific Gd-based contrast agents showed a pattern of malignancy in almost all the ICCs, concurrently avoiding misdiagnosis with hepatocarcinoma. These findings suggest a greater diagnostic capacity for this technique compared with the results of MRI with conventional contrast agents reported in the literature in this setting. © 2015 S. Karger AG, Basel.

  11. A fast and robust hepatocyte quantification algorithm including vein processing

    Directory of Open Access Journals (Sweden)

    Homeyer André

    2010-03-01

    Full Text Available Abstract Background Quantification of different types of cells is often needed for analysis of histological images. In our project, we compute the relative number of proliferating hepatocytes for the evaluation of the regeneration process after partial hepatectomy in normal rat livers. Results Our presented automatic approach for hepatocyte (HC quantification is suitable for the analysis of an entire digitized histological section given in form of a series of images. It is the main part of an automatic hepatocyte quantification tool that allows for the computation of the ratio between the number of proliferating HC-nuclei and the total number of all HC-nuclei for a series of images in one processing run. The processing pipeline allows us to obtain desired and valuable results for a wide range of images with different properties without additional parameter adjustment. Comparing the obtained segmentation results with a manually retrieved segmentation mask which is considered to be the ground truth, we achieve results with sensitivity above 90% and false positive fraction below 15%. Conclusions The proposed automatic procedure gives results with high sensitivity and low false positive fraction and can be applied to process entire stained sections.

  12. 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.

  13. Homocysteine inhibits hepatocyte proliferation via endoplasmic reticulum stress.

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    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.

  14. Hemolytic anemia repressed hepcidin level without hepatocyte iron overload: lesson from Günther disease model.

    Science.gov (United States)

    Millot, Sarah; Delaby, Constance; Moulouel, Boualem; Lefebvre, Thibaud; Pilard, Nathalie; Ducrot, Nicolas; Ged, Cécile; Lettéron, Philippe; de Franceschi, Lucia; Deybach, Jean Charles; Beaumont, Carole; Gouya, Laurent; De Verneuil, Hubert; Lyoumi, Saïd; Puy, Hervé; Karim, Zoubida

    2017-02-01

    Hemolysis occurring in hematologic diseases is often associated with an iron loading anemia. This iron overload is the result of a massive outflow of hemoglobin into the bloodstream, but the mechanism of hemoglobin handling has not been fully elucidated. Here, in a congenital erythropoietic porphyria mouse model, we evaluate the impact of hemolysis and regenerative anemia on hepcidin synthesis and iron metabolism. Hemolysis was confirmed by a complete drop in haptoglobin, hemopexin and increased plasma lactate dehydrogenase, an increased red blood cell distribution width and osmotic fragility, a reduced half-life of red blood cells, and increased expression of heme oxygenase 1. The erythropoiesis-induced Fam132b was increased, hepcidin mRNA repressed, and transepithelial iron transport in isolated duodenal loops increased. Iron was mostly accumulated in liver and spleen macrophages but transferrin saturation remained within the normal range. The expression levels of hemoglobin-haptoglobin receptor CD163 and hemopexin receptor CD91 were drastically reduced in both liver and spleen, resulting in heme- and hemoglobin-derived iron elimination in urine. In the kidney, the megalin/cubilin endocytic complex, heme oxygenase 1 and the iron exporter ferroportin were induced, which is reminiscent of significant renal handling of hemoglobin-derived iron. Our results highlight ironbound hemoglobin urinary clearance mechanism and strongly suggest that, in addition to the sequestration of iron in macrophages, kidney may play a major role in protecting hepatocytes from iron overload in chronic hemolysis. Copyright© Ferrata Storti Foundation.

  15. Hepatocyte growth factor, a determinant of airspace homeostasis in the murine lung.

    Directory of Open Access Journals (Sweden)

    Carla Calvi

    Full Text Available The alveolar compartment, the fundamental gas exchange unit in the lung, is critical for tissue oxygenation and viability. We explored hepatocyte growth factor (HGF, a pleiotrophic cytokine that promotes epithelial proliferation, morphogenesis, migration, and resistance to apoptosis, as a candidate mediator of alveolar formation and regeneration. Mice deficient in the expression of the HGF receptor Met in lung epithelial cells demonstrated impaired airspace formation marked by a reduction in alveolar epithelial cell abundance and survival, truncation of the pulmonary vascular bed, and enhanced oxidative stress. Administration of recombinant HGF to tight-skin mice, an established genetic emphysema model, attenuated airspace enlargement and reduced oxidative stress. Repair in the TSK/+ mouse was punctuated by enhanced akt and stat3 activation. HGF treatment of an alveolar epithelial cell line not only induced proliferation and scattering of the cells but also conferred protection against staurosporine-induced apoptosis, properties critical for alveolar septation. HGF promoted cell survival was attenuated by akt inhibition. Primary alveolar epithelial cells treated with HGF showed improved survival and enhanced antioxidant production. In conclusion, using both loss-of-function and gain-of-function maneuvers, we show that HGF signaling is necessary for alveolar homeostasis in the developing lung and that augmentation of HGF signaling can improve airspace morphology in murine emphysema. Our studies converge on prosurvival signaling and antioxidant protection as critical pathways in HGF-mediated airspace maintenance or repair. These findings support the exploration of HGF signaling enhancement for diseases of the airspace.

  16. 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.

  17. 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.

  18. 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.

  19. Activation of the constitutive androstane receptor inhibits gluconeogenesis without affecting lipogenesis or fatty acid synthesis in human hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Lynch, Caitlin; Pan, Yongmei; Li, Linhao [Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, MD 21201 (United States); Heyward, Scott; Moeller, Timothy [Bioreclamation In Vitro Technologies, Baltimore, MD 21227 (United States); Swaan, Peter W. [Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, MD 21201 (United States); Wang, Hongbing, E-mail: hwang@rx.umaryland.edu [Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, MD 21201 (United States)

    2014-08-15

    Objective: Accumulating evidence suggests that activation of mouse constitutive androstane receptor (mCAR) alleviates type 2 diabetes and obesity by inhibiting hepatic gluconeogenesis, lipogenesis, and fatty acid synthesis. However, the role of human (h) CAR in energy metabolism is largely unknown. The present study aims to investigate the effects of selective hCAR activators on hepatic energy metabolism in human primary hepatocytes (HPH). Methods: Ligand-based structure–activity models were used for virtual screening of the Specs database ( (www.specs.net)) followed by biological validation in cell-based luciferase assays. The effects of two novel hCAR activators (UM104 and UM145) on hepatic energy metabolism were evaluated in HPH. Results: Real-time PCR and Western blotting analyses reveal that activation of hCAR by UM104 and UM145 significantly repressed the expression of glucose-6-phosphatase and phosphoenolpyruvate carboxykinase, two pivotal gluconeogenic enzymes, while exerting negligible effects on the expression of genes associated with lipogenesis and fatty acid synthesis. Functional experiments show that UM104 and UM145 markedly inhibit hepatic synthesis of glucose but not triglycerides in HPH. In contrast, activation of mCAR by 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene, a selective mCAR activator, repressed the expression of genes associated with gluconeogenesis, lipogenesis, and fatty acid synthesis in mouse primary hepatocytes, which were consistent with previous observations in mouse model in vivo. Conclusion: Our findings uncover an important species difference between hCAR and mCAR in hepatic energy metabolism, where hCAR selectively inhibits gluconeogenesis without suppressing fatty acid synthesis. Implications: Such species selectivity should be considered when exploring CAR as a potential therapeutic target for metabolic disorders. - Highlights: • Novel hCAR activators were identified by computational and biological approaches. • The role

  20. Activation of the constitutive androstane receptor inhibits gluconeogenesis without affecting lipogenesis or fatty acid synthesis in human hepatocytes

    International Nuclear Information System (INIS)

    Lynch, Caitlin; Pan, Yongmei; Li, Linhao; Heyward, Scott; Moeller, Timothy; Swaan, Peter W.; Wang, Hongbing

    2014-01-01

    Objective: Accumulating evidence suggests that activation of mouse constitutive androstane receptor (mCAR) alleviates type 2 diabetes and obesity by inhibiting hepatic gluconeogenesis, lipogenesis, and fatty acid synthesis. However, the role of human (h) CAR in energy metabolism is largely unknown. The present study aims to investigate the effects of selective hCAR activators on hepatic energy metabolism in human primary hepatocytes (HPH). Methods: Ligand-based structure–activity models were used for virtual screening of the Specs database ( (www.specs.net)) followed by biological validation in cell-based luciferase assays. The effects of two novel hCAR activators (UM104 and UM145) on hepatic energy metabolism were evaluated in HPH. Results: Real-time PCR and Western blotting analyses reveal that activation of hCAR by UM104 and UM145 significantly repressed the expression of glucose-6-phosphatase and phosphoenolpyruvate carboxykinase, two pivotal gluconeogenic enzymes, while exerting negligible effects on the expression of genes associated with lipogenesis and fatty acid synthesis. Functional experiments show that UM104 and UM145 markedly inhibit hepatic synthesis of glucose but not triglycerides in HPH. In contrast, activation of mCAR by 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene, a selective mCAR activator, repressed the expression of genes associated with gluconeogenesis, lipogenesis, and fatty acid synthesis in mouse primary hepatocytes, which were consistent with previous observations in mouse model in vivo. Conclusion: Our findings uncover an important species difference between hCAR and mCAR in hepatic energy metabolism, where hCAR selectively inhibits gluconeogenesis without suppressing fatty acid synthesis. Implications: Such species selectivity should be considered when exploring CAR as a potential therapeutic target for metabolic disorders. - Highlights: • Novel hCAR activators were identified by computational and biological approaches. • The role

  1. 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.

  2. Differential Impacts of Soybean and Fish Oils on Hepatocyte Lipid Droplet Accumulation and Endoplasmic Reticulum Stress in Primary Rabbit Hepatocytes

    OpenAIRE

    Zhu, Xueping; Xiao, Zhihui; Xu, Yumin; Zhao, Xingli; Cheng, Ping; Cui, Ningxun; Cui, Mingling; Li, Jie; Zhu, Xiaoli

    2016-01-01

    Parenteral nutrition-associated liver disease (PNALD) is a severe ailment associated with long-term parenteral nutrition. Soybean oil-based lipid emulsions (SOLE) are thought to promote PNALD development, whereas fish oil-based lipid emulsions (FOLE) are thought to protect against PNALD. This study aimed to investigate the effects of SOLE and FOLE on primary rabbit hepatocytes. The results reveal that SOLE caused significant endoplasmic reticulum (ER) and mitochondrial damage, ultimately resu...

  3. Iron may induce both DNA synthesis and repair in rat hepatocytes stimulated by EGF/pyruvate

    Energy Technology Data Exchange (ETDEWEB)

    Chenoufi, N.; Loreal, O.; Cariou, S.; Hubert, N.; Lescoat, G. [Univ. Hospital Pontchaillou, Unite de Recherches Hepatologiques, INSERM U 49, Rennes (France); Drenou, B. [Univ. Hospital Pontchaillou, Lab. d`Hematologie et d`Immunologie, Rennes (France); Leroyer, P.; Brissot, P. [Univ. Hospital Pontchaillou, Clinique des Maladies du Foie, Rennes (France)

    1997-03-01

    Background/Aims: Hepatocellular carcinoma develops frequently in the course of genetic hemochromatosis, and a role of iron overload in hepatic carcinogenesis is strongly suggested. Methods: The aim of our study was to investigate the effect of iron exposure on DNA synthesis of adult rat hepatocytes maintained in primary culture stimulated or not by EGF/pyruvate and exposed to iron-citrate complex. Results: In EGF/pyruvate-stimulated cultures, the level of [{sup 3}H] methyl thymidine incorporation was strongly increased as compared to unstimulated cultures. The addition of iron to stimulated cultures increased [{sup 3}H] methyl thymidine incorporation. The mitotic index was also significantly higher at 72 h. However,the number of cells found in the cell layer was not significantly different from iron-citrate free culture. By flow cytometry, no difference in cell ploidy was found between iron-treated and untreated EGF/pyruvate-stimulated cultures. A significant increase in LDH leakage reflecting a toxic effect of iron was found in the cell medium 48 h after cell seeding. In addition, [{sup 3}H] methyl thymidine incorporation in the presence of hydroxyurea was increased in iron-treated compared to untreated cultures. Conclusions: Our results show that DNA synthesis is increased in the presence of iron in rat hepatocyte cultures stimulated by EGF/pyruvate, and they suggest that DNA synthesis is likely to be related both to cell proliferation and to DNA repair. These observations may allow better understanding of the role of iron overload in the development of hepatocellular carcinoma. (au) 61 refs.

  4. Differential Impacts of Soybean and Fish Oils on Hepatocyte Lipid Droplet Accumulation and Endoplasmic Reticulum Stress in Primary Rabbit Hepatocytes

    Directory of Open Access Journals (Sweden)

    Xueping Zhu

    2016-01-01

    Full Text Available Parenteral nutrition-associated liver disease (PNALD is a severe ailment associated with long-term parenteral nutrition. Soybean oil-based lipid emulsions (SOLE are thought to promote PNALD development, whereas fish oil-based lipid emulsions (FOLE are thought to protect against PNALD. This study aimed to investigate the effects of SOLE and FOLE on primary rabbit hepatocytes. The results reveal that SOLE caused significant endoplasmic reticulum (ER and mitochondrial damage, ultimately resulting in lipid droplets accumulation and ER stress. While these deleterious events induce hepatocyte injury, FOLE at high doses cause only minor ER and mitochondrial damage, which has no effect on hepatic function. SOLE also significantly upregulated glucose-regulated protein 94 mRNA and protein expression. These data indicate that SOLE, but not FOLE, damage the ER and mitochondria, resulting in lipid droplets accumulation and ER stress and, finally, hepatocyte injury. This likely contributes to the differential impacts of SOLE and FOLE on PNALD development and progression.

  5. CD147 promotes liver fibrosis progression via VEGF-A/VEGFR2 signalling-mediated cross-talk between hepatocytes and sinusoidal endothelial cells.

    Science.gov (United States)

    Yan, Zhaoyong; Qu, Kai; Zhang, Jing; Huang, Qichao; Qu, Ping; Xu, Xinsen; Yuan, Peng; Huang, Xiaojun; Shao, Yongping; Liu, Chang; Zhang, Hongxin; Xing, Jinliang

    2015-10-01

    Although previous evidence indicates close involvement of CD147 in the pathogenesis of liver fibrosis, the underlying molecular mechanisms and its therapeutic value remain largely unknown. In the present study, we investigated the biological roles of CD147 in liver fibrosis and assessed its therapeutic value as a target molecule in the CCl4-induced liver fibrosis mouse model. We found that CD147 was highly expressed in both hepatocytes and SECs (sinusoidal endothelial cells) in fibrotic liver tissues. Additionally, it was significantly associated with the fibrosis stage. TGF-β1 (transforming growth factor β1) was found to be mainly responsible for the up-regulation of CD147. Bioinformatic and experimental data suggest a functional link between CD147 expression and VEGF-A (vascular endothelial growth factor A)/VEGR-2 (VEGF receptor 2) signalling-mediated angiogenesis in fibrotic liver tissues. Furthermore, we observed that the CD147-induced activation of the PI3K (phosphoinositide 3-kinase)/Akt signalling pathway promotes the production of VEGF-A in hepatocytes and expression of VEGFR-2 in SECs, which was found to enhance the angiogenic capability of SECs. Finally, our data indicate that blocking of CD147 using an mAb (monoclonal antibody) attenuated liver fibrosis progression via inhibition of VEGF-A/VEGFR-2 signalling and subsequent amelioration of microvascular abnormality in the CCl4-induced mouse model. Our findings suggest a novel functional mechanism that CD147 may promote liver fibrosis progression via inducing the VEGF-A/VEGFR-2 signalling pathway-mediated cross-talk between hepatocytes and SECs. New strategies based on the intervention of CD147 can be expected for prevention of liver fibrosis. © 2015 Authors; published by Portland Press Limited.

  6. Hepatocyte and keratinocyte growth factors and their receptors in human lung emphysema

    Directory of Open Access Journals (Sweden)

    Marchal Joëlle

    2005-10-01

    Full Text Available Abstract Background Hepatocyte and keratinocyte growth factors are key growth factors in the process of alveolar repair. We hypothesized that excessive alveolar destruction observed in lung emphysema involves impaired expression of hepatocyte and keratinocyte growth factors or their respective receptors, c-met and keratinocyte growth factor receptor. The aim of our study was to compare the expression of hepatocyte and keratinocyte growth factors and their receptors in lung samples from 3 groups of patients: emphysema; smokers without emphysema and non-smokers without emphysema. Methods Hepatocyte and keratinocyte growth factor proteins were analysed by immunoassay and western blot; mRNA expression was measured by real time quantitative polymerase chain reaction. Results Hepatocyte and keratinocyte growth factors, c-met and keratinocyte growth factor receptor mRNA levels were similar in emphysema and non-emphysema patients. Hepatocyte growth factor mRNA correlated negatively with FEV1 and the FEV1/FVC ratio both in emphysema patients and in smokers with or without emphysema. Hepatocyte and keratinocyte growth factor protein concentrations were similar in all patients' groups. Conclusion The expression of hepatocyte and keratinocyte growth factors and their receptors is preserved in patients with lung emphysema as compared to patients without emphysema. Hepatocyte growth factor mRNA correlates with the severity of airflow obstruction in smokers.

  7. DNA synthesis in periportal and perivenous hepatocytes of intact and hepatectomized young mice.

    Science.gov (United States)

    Fernández-Blanco, A; Inda, A M; Errecalde, A L

    2015-01-01

    DNA synthesis of hepatocytes in two areas of Intact and Hepatectomized young mice liver along a circadian period was studied. DNA synthesis was significantly different at all analyzed time points in Intact and Hepatectomized animals. Differences between periportal and perivenous hepatocytes were found in hepatectomized animals at 04/42 and 08/46 hr of day/hour post-hepatectomy. DNAs peak in periportal hepatocytes regenerating liver occurs 4 hr earlier than in perivenous hepatocytes, probably reflecting their shorter G1 phase. Besides, daily mean values of regenerating livers were higher than those observed in Intact animals, as a consequence of surgical removal.

  8. 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

  9. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  11. Perinatal exposure to environmental tobacco smoke is associated with changes in DNA methylation that precede the adult onset of lung disease in a mouse model.

    Science.gov (United States)

    Cole, Elizabeth; Brown, Traci A; Pinkerton, Kent E; Postma, Britten; Malany, Keegan; Yang, Mihi; Kim, Yang Jee; Hamilton, Raymond F; Holian, Andrij; Cho, Yoon Hee

    2017-08-01

    Prenatal and early-life environmental tobacco smoke (ETS) exposure can induce epigenetic alterations associated with inflammation and respiratory disease. The objective of this study was to address the long-term epigenetic consequences of perinatal ETS exposure on latent respiratory disease risk, which are still largely unknown. C57BL/6 mice were exposed to prenatal and early-life ETS; offspring lung pathology, global DNA, and gene-specific methylation were measured at two adult ages. Significant alterations in global DNA methylation and promoter methylation of IFN-γ and Thy-1 were found in ETS-exposed offspring at 10-12 and 20 weeks of age. These sustained epigenetic alterations preceded the onset of significant pulmonary pathologies observed at 20 weeks of age. This study suggests that perinatal ETS exposure induces persistent epigenetic alterations in global DNA, as well as IFN-γ and Thy-1 promoter methylation that precede the adult onset of fibrotic lung pathology. These epigenetic findings could represent potential biomarkers of latent respiratory disease risk.

  12. Effect of maternal protein restriction during pregnancy and postweaning high-fat feeding on diet-induced thermogenesis in adult mouse offspring.

    Science.gov (United States)

    Sellayah, Dyan; Dib, Lea; Anthony, Frederick W; Watkins, Adam J; Fleming, Tom P; Hanson, Mark A; Cagampang, Felino R

    2014-10-01

    Prenatal undernutrition followed by postweaning feeding of a high-fat diet results in obesity in the adult offspring. In this study, we investigated whether diet-induced thermogenesis is altered as a result of such nutritional mismatch. Female MF-1 mice were fed a normal protein (NP, 18% casein) or a protein-restricted (PR, 9% casein) diet throughout pregnancy and lactation. After weaning, male offspring of both groups were fed either a high-fat diet (HF; 45% kcal fat) or standard chow (C, 7% kcal fat) to generate the NP/C, NP/HF, PR/C and PR/HF adult offspring groups (n = 7-11 per group). PR/C and NP/C offspring have similar body weights at 30 weeks of age. Postweaning HF feeding resulted in significantly heavier NP/HF offspring (P protein-1 and β-3 adrenergic receptor in the interscapular brown adipose tissue (iBAT) compared with the NP/C mice (both at P diet during pregnancy and lactation, and the postweaning diet of the offspring, can attenuate diet-induced thermogenesis in the iBAT, resulting in the development of obesity in adulthood.

  13. The impact of long-term exposure to space environment on adult mammalian organisms: a study on mouse thyroid and testis.

    Directory of Open Access Journals (Sweden)

    Maria Angela Masini

    Full Text Available Hormonal changes in humans during spaceflight have been demonstrated but the underlying mechanisms are still unknown. To clarify this point thyroid and testis/epididymis, both regulated by anterior pituitary gland, have been analyzed on long-term space-exposed male C57BL/10 mice, either wild type or pleiotrophin transgenic, overexpressing osteoblast stimulating factor-1. Glands were submitted to morphological and functional analysis.In thyroids, volumetric ratios between thyrocytes and colloid were measured. cAMP production in 10(-7M and 10(-8M thyrotropin-treated samples was studied. Thyrotropin receptor and caveolin-1 were quantitized by immunoblotting and localized by immunofluorescence. In space-exposed animals, both basal and thyrotropin-stimulated cAMP production were always higher. Also, the structure of thyroid follicles appeared more organized, while thyrotropin receptor and caveolin-1 were overexpressed. Unlike the control samples, in the space samples thyrotropin receptor and caveolin-1 were both observed at the intracellular junctions, suggesting their interaction in specific cell membrane microdomains.In testes, immunofluorescent reaction for 3β- steroid dehydrogenase was performed and the relative expressions of hormone receptors and interleukin-1β were quantified by RT-PCR. Epididymal sperm number was counted. In space-exposed animals, the presence of 3β and 17β steroid dehydrogenase was reduced. Also, the expression of androgen and follicle stimulating hormone receptors increased while lutenizing hormone receptor levels were not affected. The interleukin 1 β expression was upregulated. The tubular architecture was altered and the sperm cell number was significantly reduced in spaceflight mouse epididymis (approx. -90% vs. laboratory and ground controls, indicating that the space environment may lead to degenerative changes in seminiferous tubules.Space-induced changes of structure and function of thyroid and testis

  14. Disruption of the ErbB signaling in adolescence increases striatal dopamine levels and affects learning and hedonic-like behavior in the adult mouse.

    Science.gov (United States)

    Golani, Idit; Tadmor, Hagar; Buonanno, Andres; Kremer, Ilana; Shamir, Alon

    2014-11-01

    The ErbB signaling pathway has been genetically and functionally implicated in schizophrenia. Numerous findings support the dysregulation of Neuregulin (NRG) and epidermal growth factor (EGF) signaling in schizophrenia. However, it is unclear whether alterations of these pathways in the adult brain or during development are involved in the pathophysiology of schizophrenia. Herein we characterized the behavioral profile and molecular changes resulting from pharmacologically blocking the ErbB signaling pathway during a critical period in the development of decision making, planning, judgments, emotions, social cognition and cognitive skills, namely adolescence. We demonstrate that chronic administration of the pan-ErbB kinase inhibitor JNJ-28871063 (JNJ) to adolescent mice elevated striatal dopamine levels and reduced preference for sucrose without affecting locomotor activity and exploratory behavior. In adulthood, adolescent JNJ-treated mice continue to consume less sucrose and needed significantly more correct-response trials to reach the learning criterion during the discrimination phase of the T-maze reversal learning task than their saline-injected controls. In addition, JNJ mice exhibited deficit in reference memory but not in working memory as measured in the radial arm maze. Inhibition of the pathway during adolescence did not affect exploratory behavior and locomotor activity in the open field, social interaction, social memory, and reversal learning in adult mice. Our data suggest that alteration of ErbB signaling during adolescence resulted in changes in the dopaminergic systems that emerge in pathological learning and hedonic behavior in adulthood, and pinpoints the possible role of the pathway in the development of cognitive skills and motivated behavior. Copyright © 2014 Elsevier B.V. and ECNP. All rights reserved.

  15. Manganese Transport and Toxicity in Polarized WIF-B Hepatocytes.

    Science.gov (United States)

    Thompson, Khristy J; Hein, Jennifer; Baez, Andrew; Sosa, Jose Carlo; Wessling-Resnick, Marianne

    2018-05-24

    Mn toxicity arises from nutritional problems, community and occupational exposures, and genetic risks. Mn blood levels are controlled by hepatobiliary clearance. The goals of this study were to determine the cellular distribution of Mn transporters in polarized hepatocytes, to establish an in vitro assay for hepatocyte Mn efflux, and to examine possible roles the Mn transporters would play in metal import and export. For these experiments, hepatocytoma WIF-B cells were grown for 12-14 days to achieve maximal polarity. Immunoblots showed that Mn transporters ZIP8, ZnT10, ferroportin (Fpn), and ZIP14 were present. Indirect immunofluorescence microscopy localized Fpn and ZIP14 to WIF-B cell basolateral domains while ZnT10 and ZIP8 associated with intracellular vesicular compartments. ZIP8-positive structures were distributed uniformly throughout the cytoplasm, but ZnT10-positive vesicles were adjacent to apical bile compartments. WIF-B cells were sensitive to Mn toxicity, showing decreased viability after 16 h exposure to > 250 M MnCl2. However, the hepatocytes were resistant to 4 h exposures of up to 500 M MnCl2 despite 50-fold increased Mn content. Washout experiments showed time-dependent efflux with 80% Mn released after a 4 h chase period. Hepcidin reduced levels of Fpn in WIF-B cells, clearing Fpn from the cell surface, but Mn efflux was unaffected. The secretory inhibitor brefeldin A did block release of Mn from WIF-B cells, suggesting vesicle fusion may be involved in export. These results point to a possible role of ZnT10 to import Mn into vesicles that subsequently fuse with the apical membrane and empty their contents into bile.

  16. Curcumin inhibits activation of TRPM2 channels in rat hepatocytes

    Directory of Open Access Journals (Sweden)

    E. Kheradpezhouh

    2016-04-01

    Full Text Available Oxidative stress is a hallmark of many liver diseases including viral and drug-induced hepatitis, ischemia-reperfusion injury, and non-alcoholic steatohepatitis. One of the consequences of oxidative stress in the liver is deregulation of Ca2+ homeostasis, resulting in a sustained elevation of the free cytosolic Ca2+ concentration ([Ca2+]c in hepatocytes, which leads to irreversible cellular damage. Recently it has been shown that liver damage induced by paracetamol and subsequent oxidative stress is, in large part, mediated by Ca2+ entry through Transient Receptor Potential Melastatin 2 (TRPM2 channels. Involvement of TRPM2 channels in hepatocellular damage induced by oxidative stress makes TRPM2 a potential therapeutic target for treatment of a range of oxidative stress-related liver diseases. We report here the identification of curcumin ((1E,6E-1,7-bis(4-hydroxy-3-methoxyphenyl-1,6-heptadiene-3,5-dione, a natural plant-derived polyphenol in turmeric spice, as a novel inhibitor of TRPM2 channel. Presence of 5 µM curcumin in the incubation medium prevented the H2O2- and paracetamol-induced [Ca2+]c rise in rat hepatocytes. Furthermore, in patch clamping experiments incubation of hepatocytes with curcumin inhibited activation of TRPM2 current by intracellular ADPR with IC50 of approximately 50 nM. These findings enhance understanding of the actions of curcumin and suggest that the known hepatoprotective properties of curcumin are, at least in part, mediated through inhibition of TRPM2 channels.

  17. Centralized mouse repositories.

    Science.gov (United States)

    Donahue, Leah Rae; Hrabe de Angelis, Martin; Hagn, Michael; Franklin, Craig; Lloyd, K C Kent; Magnuson, Terry; McKerlie, Colin; Nakagata, Naomi; Obata, Yuichi; Read, Stuart; Wurst, Wolfgang; Hörlein, Andreas; Davisson, Muriel T

    2012-10-01

    Because the mouse is used so widely for biomedical research and the number of mouse models being generated is increasing rapidly, centralized repositories are essential if the valuable mouse strains and models that have been developed are to be securely preserved and fully exploited. Ensuring the ongoing availability of these mouse strains preserves the investment made in creating and characterizing them and creates a global resource of enormous value. The establishment of centralized mouse repositories around the world for distributing and archiving these resources has provided critical access to and preservation of these strains. This article describes the common and specialized activities provided by major mouse repositories around the world.

  18. CYP1A1 and CYP1A2 expression: Comparing 'humanized' mouse lines and wild-type mice; comparing human and mouse hepatoma-derived cell lines

    International Nuclear Information System (INIS)

    Uno, Shigeyuki; Endo, Kaori; Ishida, Yuji; Tateno, Chise; Makishima, Makoto; Yoshizato, Katsutoshi; Nebert, Daniel W.

    2009-01-01

    Human and rodent cytochrome P450 (CYP) enzymes sometimes exhibit striking species-specific differences in substrate preference and rate of metabolism. Human risk assessment of CYP substrates might therefore best be evaluated in the intact mouse by replacing mouse Cyp genes with human CYP orthologs; however, how 'human-like' can human gene expression be expected in mouse tissues? Previously a bacterial-artificial-chromosome-transgenic mouse, carrying the human CYP1A1 C YP1A2 locus and lacking the mouse Cyp1a1 and Cyp1a2 orthologs, was shown to express robustly human dioxin-inducible CYP1A1 and basal versus inducible CYP1A2 (mRNAs, proteins, enzyme activities) in each of nine mouse tissues examined. Chimeric mice carrying humanized liver have also been generated, by transplanting human hepatocytes into a urokinase-type plasminogen activator(+/+) s evere-combined-immunodeficiency (uPA/SCID) line with most of its mouse hepatocytes ablated. Herein we compare basal and dioxin-induced CYP1A mRNA copy numbers, protein levels, and four enzymes (benzo[a]pyrene hydroxylase, ethoxyresorufin O-deethylase, acetanilide 4-hydroxylase, methoxyresorufin O-demethylase) in liver of these two humanized mouse lines versus wild-type mice; we also compare these same parameters in mouse Hepa-1c1c7 and human HepG2 hepatoma-derived established cell lines. Most strikingly, mouse liver CYP1A1-specific enzyme activities are between 38- and 170-fold higher than human CYP1A1-specific enzyme activities (per unit of mRNA), whereas mouse versus human CYP1A2 enzyme activities (per unit of mRNA) are within 2.5-fold of one another. Moreover, both the mouse and human hepatoma cell lines exhibit striking differences in CYP1A mRNA levels and enzyme activities. These findings are relevant to risk assessment involving human CYP1A1 and CYP1A2 substrates, when administered to mice as environmental toxicants or drugs.

  19. High throughput micro-well generation of hepatocyte micro-aggregates for tissue engineering.

    Directory of Open Access Journals (Sweden)

    Elien Gevaert

    Full Text Available The main challenge in hepatic tissue engineering is the fast dedifferentiation of primary hepatocytes in vitro. One successful approach to maintain hepatocyte phenotype on the longer term is the cultivation of cells as aggregates. This paper demonstrates the use of an agarose micro-well chip for the high throughput generation of hepatocyte aggregates, uniform in size. In our study we observed that aggregation of hepatocytes had a beneficial effect on the expression of certain hepatocyte specific markers. Moreover we observed that the beneficial effect was dependent on the aggregate dimensions, indicating that aggregate parameters should be carefully considered. In a second part of the study, the selected aggregates were immobilized by encapsulation in methacrylamide-modified gelatin. Phenotype evaluations revealed that a stable hepatocyte phenotype could be maintained during 21 days when encapsulated in the hydrogel. In conclusion we have demonstrated the beneficial use of micro-well chips for hepatocyte aggregation and the size-dependent effects on hepatocyte phenotype. We also pointed out that methacrylamide-modified gelatin is suitable for the encapsulation of these aggregates.

  20. High Throughput Micro-Well Generation of Hepatocyte Micro-Aggregates for Tissue Engineering

    NARCIS (Netherlands)

    Gevaert, Elien; Dollé, Laurent; Billiet, Thomas; Dubruel, Peter; van Grunsven, Leo; van Apeldoorn, Aart A.; Cornelissen, Ria

    2014-01-01

    The main challenge in hepatic tissue engineering is the fast dedifferentiation of primary hepatocytes in vitro. One successful approach to maintain hepatocyte phenotype on the longer term is the cultivation of cells as aggregates. This paper demonstrates the use of an agarose micro-well chip for the

  1. High efficient differentiation of functional hepatocytes from porcine induced pluripotent stem cells.

    Directory of Open Access Journals (Sweden)

    Ying Ao

    Full Text Available Hepatocyte transplantation is considered to be a promising therapy for patients with liver diseases. Induced pluripotent stem cells (iPSCs provide an unlimited source for the generation of functional hepatocytes. In this study, we generated iPSCs from porcine ear fibroblasts (PEFs by overexpressing Sox2, Klf4, Oct4, and c-Myc (SKOM, and developed a novel strategy for the efficient differentiation of hepatocyte-like cells from porcine iPSCs by following the processes of early liver development. The differentiated cells displayed the phenotypes of hepatocytes, exhibited classic hepatocyte-associated bio-functions, such as LDL uptake, glycogen storage and urea secretion, as well as possessed the metabolic activities of cytochrome P-450 (CYP 3A and 2C. Furthermore, we compared the hepatocyte differentiation efficacy of our protocol with another published method, and the results demonstrated that our differentiation strategy could significantly improve the generation of morphological and functional hepatocyte-like cells from porcine iPSCs. In conclusion, this study establishes an efficient method for in vitro generation of functional hepatocytes from porcine iPSCs, which could represent a promising cell source for preclinical testing of cell-based therapeutics for liver failure and for pharmacological applications.

  2. 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

  3. High throughput micro-well generation of hepatocyte micro-aggregates for tissue engineering.

    Science.gov (United States)

    Gevaert, Elien; Dollé, Laurent; Billiet, Thomas; Dubruel, Peter; van Grunsven, Leo; van Apeldoorn, Aart; Cornelissen, Ria

    2014-01-01

    The main challenge in hepatic tissue engineering is the fast dedifferentiation of primary hepatocytes in vitro. One successful approach to maintain hepatocyte phenotype on the longer term is the cultivation of cells as aggregates. This paper demonstrates the use of an agarose micro-well chip for the high throughput generation of hepatocyte aggregates, uniform in size. In our study we observed that aggregation of hepatocytes had a beneficial effect on the expression of certain hepatocyte specific markers. Moreover we observed that the beneficial effect was dependent on the aggregate dimensions, indicating that aggregate parameters should be carefully considered. In a second part of the study, the selected aggregates were immobilized by encapsulation in methacrylamide-modified gelatin. Phenotype evaluations revealed that a stable hepatocyte phenotype could be maintained during 21 days when encapsulated in the hydrogel. In conclusion we have demonstrated the beneficial use of micro-well chips for hepatocyte aggregation and the size-dependent effects on hepatocyte phenotype. We also pointed out that methacrylamide-modified gelatin is suitable for the encapsulation of these aggregates.

  4. Computational Topology Based Quantification Of Hepatocytes Nuclei In Lipopolysaccharide-Induced Liver Injury In Mice

    Directory of Open Access Journals (Sweden)

    Rodrigo Rojas Moraleda

    2016-06-01

    The computational topology approach proposed successfully detected hepatocyte cells under several natural variations. We evaluated on a per-pixel basis how the segmentation performs on: i all nuclei in the images, ii big round nuclei considered belonging to hepatocytes cells (accuracy 87.2%, recall 80.3%, and iii nuclei regarded to non-parenchymal cells.  

  5. Lineage tracing in the adult mouse corneal epithelium supports the limbal epithelial stem cell hypothesis with intermittent periods of stem cell quiescence

    Directory of Open Access Journals (Sweden)

    Natalie J. Dorà

    2015-11-01

    Full Text Available The limbal epithelial stem cell (LESC hypothesis proposes that LESCs in the corneal limbus maintain the corneal epithelium both during normal homeostasis and wound repair. The alternative corneal epithelial stem cell (CESC hypothesis proposes that LESCs are only involved in wound repair and CESCs in the corneal epithelium itself maintain the corneal epithelium during normal homeostasis. We used tamoxifen-inducible, CreER-loxP lineage tracing to distinguish between these hypotheses. Clones of labelled cells were induced in adult CAGG-CreER;R26R-LacZ reporter mice and their distributions analysed after different chase periods. Short-lived clones, derived from labelled transient amplifying cells, were shed during the chase period and long-lived clones, derived from stem cells, expanded. At 6 weeks, labelled clones appeared at the periphery, extended centripetally as radial stripes and a few reached the centre by 14 weeks. Stripe numbers depended on the age of tamoxifen treatment. Stripes varied in length, some were discontinuous, few reached the centre and almost half had one end at the limbus. Similar stripes extended across the cornea in CAGG-CreER;R26R-mT/mG reporter mice. The distributions of labelled clones are inconsistent with the CESC hypothesis and support the LESC hypothesis if LESCs cycle between phases of activity and quiescence, each lasting several weeks.

  6. A Fab fragment directed against the neural cell adhesion molecule L1 enhances functional recovery after injury of the adult mouse spinal cord.

    Science.gov (United States)

    Loers, Gabriele; Cui, Yi-Fang; Neumaier, Irmgard; Schachner, Melitta; Skerra, Arne

    2014-06-15

    Lack of permissive mechanisms and abundance of inhibitory molecules in the lesioned central nervous system of adult mammals contribute to the failure of functional recovery, which leads to severe disabilities in motor functions or pain. Previous studies have indicated that the neural cell adhesion molecule L1 constitutes a viable target to promote regeneration. In the present study, we describe the cloning, functional expression in Escherichia coli cells and purification of a recombinant αL1 Fab fragment that binds to L1 with comparable activity as the function-triggering monoclonal antibody 557.B6 and induces neurite outgrowth and neuronal survival in cultured neurons, despite its monovalent function. Infusion of αL1 Fab into the lesioned spinal cord of mice enhanced functional recovery after thoracic spinal cord compression injury. αL1 Fab treatment resulted in reduced scar volume, enhanced number of tyrosine hydroxylase-positive axons and increased linear density of VGLUT1 (vesicular glutamate transporter 1) on motoneurons. Furthermore, the number and soma size of ChAT (choline acetyltransferase)-positive motoneurons and the linear density of ChAT-positive boutons on motoneurons as well as parvalbumin-positive interneurons in the lumbar spinal cord were elevated. Stimulation of endogenous L1 by application of the αL1 Fab opens new avenues for recombinant antibody technology, offering prospects for therapeutic applications after traumatic nervous system lesions.

  7. CRISPR/Cas9 Engineering of Adult Mouse Liver Demonstrates That the Dnajb1–Prkaca Gene Fusion Is Sufficient to Induce Tumors Resembling Fibrolamellar Hepatocellular Carcinoma

    DEFF Research Database (Denmark)

    Engelholm, Lars H.; Riaz, Anjum; Serra, Denise

    2017-01-01

    Background & Aims Fibrolamellar hepatocellular carcinoma (FL-HCC) is a primary liver cancer that predominantly affects children and young adults with no underlying liver disease. A somatic, 400 Kb deletion on chromosome 19 that fuses part of the DnaJ heat shock protein family (Hsp40) member B1 gene...... (DNAJB1) to the protein kinase cAMP-activated catalytic subunit alpha gene (PRKACA) has been repeatedly identified in patients with FL-HCC. However, the DNAJB1–PRKACA gene fusion has not been shown to induce liver tumorigenesis. We used the CRISPR/Cas9 technique to delete in mice the syntenic region...... on chromosome 8 to create a Dnajb1–Prkaca fusion and monitored the mice for liver tumor development. Methods We delivered CRISPR/Cas9 vectors designed to juxtapose exon 1 of Dnajb1 with exon 2 of Prkaca to create the Dnajb1–Prkaca gene fusion associated with FL-HCC, or control Cas9 vector, via hydrodynamic tail...

  8. 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

  9. The role of hepatocyte nuclear factor 4 alpha in development and progression of liver diseases

    Directory of Open Access Journals (Sweden)

    YANG Jinlian

    2016-02-01

    Full Text Available Hepatocyte nuclear factor 4 alpha (HNF4α, a member of the nuclear receptor superfamily, has a high expression level in mature hepatocytes. HNF4α can regulate hepatocyte-specific gene expression at a transcriptional level, promote hepatocyte development and differentiation, participate in establishment and maintenance of hepatocyte polarity, and enhance the synthetic, metabolic, and detoxifying functions of the liver. Through inhibiting the activation of hepatic stellate cells, reversing epithelial-mesenchymal transition, and inhibiting the proliferation, invasion, and metastasis of hepatoma cells, HNF4α may be involved in the development and progression of various liver diseases including liver fibrosis, liver cirrhosis, and hepatocellular carcinoma. This paper elaborates on the biological functions of HNF4α, and summarizes and analyzes the research advances in the mechanisms of action of HNF4α in the pathological process of liver diseases, in order to provide references for further investigation of the potential targeted therapies for liver diseases.

  10. Morphological and Functional Analysis of Hepatocyte Spheroids Generated on Poly-HEMA-Treated Surfaces under the Influence of Fetal Calf Serum and Nonparenchymal Cells

    Directory of Open Access Journals (Sweden)

    Augustinus Bader

    2013-03-01

    Full Text Available Poly (2-hydroxyethyl methacrylate (HEMA has been used as a clinical material, in the form of a soft hydrogel, for various surgical procedures, including endovascular surgery of liver. It is a clear liquid compound and, as a soft, flexible, water-absorbing material, has been used to make soft contact lenses from small, concave, spinning molds. Primary rat hepatocyte spheroids were created on a poly-HEMA-coated surface with the intention of inducing hepatic tissue formation and improving liver functions. We investigated spheroid formation of primary adult rat hepatocyte cells and characterized hepatic-specific functions under the special influence of fetal calf serum (FCS and nonparencymal cells (NPC up to six days in different culture systems (e.g., hepatocytes + FCS, hepatocytes – FCS, NPC + FCS, NPC – FCS, co-culture + FCS, co-culture – FCS in both the spheroid model and sandwich model. Immunohistologically, we detected gap junctions, Ito cell/Kupffer cells, sinusoidal endothelial cells and an extracellular matrix in the spheroid model. FCS has no positive effect in the sandwich model, but has a negative effect in the spheroid model on albumin production, and no influence in urea production in either model. We found more cell viability in smaller diameter spheroids than larger ones by using the apoptosis test. Furthermore, there is no positive influence of the serum or NPC on spheroid formation, suggesting that it may only depend on the physical condition of the culture system. Since the sandwich culture has been considered a “gold standard” in vitro culture model, the hepatocyte spheroids generated on the poly-HEMA-coated surface were compared with those in the sandwich model. Major liver-specific functions, such as albumin secretion and urea synthesis, were evaluated in both the spheroid and sandwich model. The synthesis performance in the spheroid compared to the sandwich culture increases approximately by a factor of 1

  11. Galactosylated DNA lipid nanocapsules for efficient hepatocyte targeting.

    Science.gov (United States)

    Morille, M; Passirani, C; Letrou-Bonneval, E; Benoit, J-P; Pitard, B

    2009-09-11

    The main objective of gene therapy via a systemic pathway is the development of a stable and non-toxic gene vector that can encapsulate and deliver foreign genetic materials into specific cell types with the transfection efficiency of viral vectors. With this objective, DNA complexed with cationic lipids of DOTAP/DOPE was encapsulated into lipid nanocapsules (LNCs) forming nanocarriers (DNA LNCs) with a size suitable for systemic injection (109+/-6 nm). With the goal of increasing systemic delivery, LNCs were stabilised with long chains of poly(ethylene glycol) (PEG), either from a PEG lipid derivative (DSPE-mPEG(2000)) or from an amphiphilic block copolymer (F108). In order to overcome internalisation difficulties encountered with PEG shield, a specific ligand (galactose) was covalently added at the distal end of the PEG chains, in order to provide active targeting of the asialoglycoprotein-receptor present on hepatocytes. This study showed that DNA LNCs were as efficient as positively charged DOTAP/DOPE lipoplexes for transfection. In primary hepatocytes, when non-galactosylated, the two polymers significantly decreased the transfection, probably by creating a barrier around the DNA LNCs. Interestingly, galactosylated F108 coated DNA LNCs led to a 18-fold increase in luciferase expression compared to non-galactosylated ones.

  12. Study of Valproic Acid-Enhanced Hepatocyte Steatosis

    Science.gov (United States)

    Chang, Renin; Chou, Mei-Chia; Hung, Li-Ying; Wang, Mu-En; Hsu, Meng-Chieh; Chiu, Chih-Hsien

    2016-01-01

    Valproic acid (VPA) is one of the most widely used antiepilepsy drugs. However, several side effects, including weight gain and fatty liver, have been reported in patients following VPA treatment. In this study, we explored the molecular mechanisms of VPA-induced hepatic steatosis using FL83B cell line-based in vitro model. Using fluorescent lipid staining technique, we found that VPA enhanced oleic acid- (OLA-) induced lipid accumulation in a dose-dependent manner in hepatocytes; this may be due to upregulated lipid uptake, triacylglycerol (TAG) synthesis, and lipid droplet formation. Real-time PCR results showed that, following VPA treatment, the expression levels of genes encoding cluster of differentiation 36 (Cd36), low-density lipoprotein receptor-related protein 1 (Lrp1), diacylglycerol acyltransferase 2 (Dgat2), and perilipin 2 (Plin2) were increased, that of carnitine palmitoyltransferase I a (Cpt1a) was not affected, and those of acetyl-Co A carboxylase α (Acca) and fatty acid synthase (Fasn) were decreased. Furthermore, using immunofluorescence staining and flow cytometry analyses, we found that VPA also induced peroxisome proliferator-activated receptor γ (PPARγ) nuclear translocation and increased levels of cell-surface CD36. Based on these results, we propose that VPA may enhance OLA-induced hepatocyte steatosis through the upregulation of PPARγ- and CD36-dependent lipid uptake, TAG synthesis, and lipid droplet formation. PMID:27034954

  13. Study of Valproic Acid-Enhanced Hepatocyte Steatosis

    Directory of Open Access Journals (Sweden)

    Renin Chang

    2016-01-01

    Full Text Available Valproic acid (VPA is one of the most widely used antiepilepsy drugs. However, several side effects, including weight gain and fatty liver, have been reported in patients following VPA treatment. In this study, we explored the molecular mechanisms of VPA-induced hepatic steatosis using FL83B cell line-based in vitro model. Using fluorescent lipid staining technique, we found that VPA enhanced oleic acid- (OLA- induced lipid accumulation in a dose-dependent manner in hepatocytes; this may be due to upregulated lipid uptake, triacylglycerol (TAG synthesis, and lipid droplet formation. Real-time PCR results showed that, following VPA treatment, the expression levels of genes encoding cluster of differentiation 36 (Cd36, low-density lipoprotein receptor-related protein 1 (Lrp1, diacylglycerol acyltransferase 2 (Dgat2, and perilipin 2 (Plin2 were increased, that of carnitine palmitoyltransferase I a (Cpt1a was not affected, and those of acetyl-Co A carboxylase α (Acca and fatty acid synthase (Fasn were decreased. Furthermore, using immunofluorescence staining and flow cytometry analyses, we found that VPA also induced peroxisome proliferator-activated receptor γ (PPARγ nuclear translocation and increased levels of cell-surface CD36. Based on these results, we propose that VPA may enhance OLA-induced hepatocyte steatosis through the upregulation of PPARγ- and CD36-dependent lipid uptake, TAG synthesis, and lipid droplet formation.

  14. Transport of heparan sulfate into the nuclei of hepatocytes

    International Nuclear Information System (INIS)

    Ishihara, M.; Fedarko, N.S.; Conrad, H.E.

    1986-01-01

    A rat hepatocyte cell line which accumulates free heparan sulfate (HS) chains enriched in GlcA-2-SO 4 residues in the nucleus was labeled with 35 SO 4 2- and the rate of appearance of [ 35 SO 4 ]HS in the nucleus was measured. [ 35 SO 4 ]HS began to accumulate in the nucleus 2 h after the addition of 35 SO 42- and reached a steady state level after 20 h. HS was lost from the nuclei of prelabeled cells with a t/sub 1/2/ of 8 h. Chloroquine did not inhibit the transport of HS into the nucleus, but increased the t/sub 1/2/ for the exit of HS from the nucleus to 20 h. At both 37 0 C and 16 0 C exogenous [ 35 SO 4 ]proteoHS was taken up by the cells and converted to free chains and about 10% of the internalized [ 35 SO 4 ]HS was transported into the nucleus. The [ 35 SO 4 ]HS isolated from the nucleus was enriched in GlcA-2-SO 4 residues, whereas the [ 35 SO 4 ]HS remaining in the rest of the intra-cellular pool showed a corresponding depletion in GlcA-2-SO 4 residues. The results show that nuclear HS is derived from the pool of a secreted proteoHS and that metabolism of exogenous HS by hepatocytes does not involve lysosomal processing of the internalized HS

  15. 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

  16. Rapid and sensitive measure of gluconeogenesis in isolated bovine hepatocytes

    International Nuclear Information System (INIS)

    Azain, M.J.; Kasser, T.R.; Atwell, C.A.; Baile, C.A.

    1986-01-01

    Available methods for determining glucose synthesis from radiolabelled precursors using ion exchange column chromatography limit the number of samples that can be processed. To facilitate this process, a rapid method for determining glucose synthesis from 3-carbon precursors was developed using suspensions of anion and cation exchange resins. Hepatocytes were prepared from calf liver by collagenase perfusion of the caudate lobe. Isolated cells were incubated with 14 C-labelled lactate or propionate in the presence or absence of glucagen and/or palmitate. Glucose synthesis was determined by vortexing an aliquot of cell suspension with a 50% slurry of anion exchange resin (acetate form), followed by cation exchange resin. After centrifugation 14 C-glucose was recovered in the supernatant and measured by scintillation counting. Using this method, more than 95% of unused labelled precursor was bound to the ion exchange resin and essentially 100% of 14 C-glucose tracer was recovered in the supernatant. In hepatocyte suspensions, radioactivity recovered in the supernatants was confirmed to be glucose by pre-incubating aliquots of media with glucose oxidase prior to addition of ion exchange resins. The present system allows determination of hepatic gluconeogenesis, is sensitive to substrate and hormonal manipulations and has the capacity for processing several hundred samples per day

  17. Stimulation of glucose phosphorylation by fructose in isolated rat hepatocytes.

    Science.gov (United States)

    Van Schaftingen, E; Vandercammen, A

    1989-01-15

    The phosphorylation of glucose was measured by the formation of [3H]H2O from [2-3H]glucose in suspensions of freshly isolated rat hepatocytes. Fructose (0.2 mM) stimulated 2-4-fold the rate of phosphorylation of 5 mM glucose although not of 40 mM glucose, thus increasing the apparent affinity of the glucose phosphorylating system. A half-maximal stimulatory effect was observed at about 50 microM fructose. Stimulation was maximal 5 min after addition of the ketose and was stable for at least 40 min, during which period 60% of the fructose was consumed. The effect of fructose was reversible upon removal of the ketose. Sorbitol and tagatose were as potent as fructose in stimulating the phosphorylation of 5 mM glucose. D-Glyceraldehyde also had a stimulatory effect but at tenfold higher concentrations. In contrast, dihydroxyacetone had no significant effect and glycerol inhibited the detritiation of glucose. Oleate did not affect the phosphorylation of glucose, even in the presence of fructose, although it stimulated the formation of ketone bodies severalfold, indicating that it was converted to its acyl-CoA derivative. These results allow the conclusion that fructose stimulates glucokinase in the intact hepatocyte. They also suggest that this effect is mediated through the formation of fructose 1-phosphate, which presumably interacts with a competitive inhibitor of glucokinase other than long-chain acyl-CoAs.

  18. Metformin protects rat hepatocytes against bile acid-induced apoptosis.

    Directory of Open Access Journals (Sweden)

    Titia E Woudenberg-Vrenken

    Full Text Available BACKGROUND: Metformin is used in the treatment of Diabetes Mellitus type II and improves liver function in patients with non-alcoholic fatty liver disease (NAFLD. Metformin activates AMP-activated protein kinase (AMPK, the cellular energy sensor that is sensitive to changes in the AMP/ATP-ratio. AMPK is an inhibitor of mammalian target of rapamycin (mTOR. Both AMPK and mTOR are able to modulate cell death. AIM: To evaluate the effects of metformin on hepatocyte cell death. METHODS: Apoptotic cell death was induced in primary rat