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Sample records for body forming hepatocytes

  1. Differentiation of embryoid-body cells derived from embryonic stem cells into hepatocytes in alginate microbeads in vitro

    Institute of Scientific and Technical Information of China (English)

    Sheng FANG; Yu-dong QIU; Liang MAO; Xiao-lei SHI; De-cai YU; Yi-tao DING

    2007-01-01

    Aim: Embryonic stem (ES) cells are being widely investigated as a promising source of hepatocytes with their proliferative, renewable, and pluripotent capacities. However, controlled and scalable ES cell differentiation culture into functional hepatocytes is challenging. In this study, we examined the differentiat- ing potential of embryoid-body cells derived from ES cells into hepatocytes in alginate microbeads containing exogenous growth factors in vitro. Methods: Embryoid bodies were formed from ES cells by suspension methods. Embryoid bodies cultured for 5 d were treated with trypsin-EDTA. The disaggregated cells were encapsulated in alginate microbeads and stimulated with exogenous growth factors to induce hepatic differentiation. In the course of cell differentiation, cell morphology and viability were observed, and the expression patterns of some genes of the hepatocyte were confirmed by RT-PCR. An immunofluorescence analysis revealed the expression of albumin (ALB) and cytokeratin-18 (CK18). Hepatocyte functional assays were confirmed by the secretion of ALB and urea. Results: We showed that embryoid-body cells could maintain cell viability in alginate microbeads in vitro. We also found that directed differentiated cells expressed several hepatocyte genes including α-fetoprotein (AFP), ALB, Cyp7al, CK18, transthyretin (TTR) and tyrosine aminotransferase (TAT) and produced ALB and urea in alginate microbeads. The directed differentiated cells expressed ALB and CK18 proteins on d 14. However, embryoid-body cells could not form hepatocytes without exogenous growth factors in alginate microbeads. Conclusion: The differentiation of embryoid-body cells into hepatocytes con- taining exogenous growth factors in alginate microbeads gives rise to functional hepatocytes and may develop scalable stem cell differentiation strategies for bioartificial livers and hepatocyte transplantation.

  2. Hepatocyte-derived cultured cells with unusual cytoplasmic keratin-rich spheroid bodies

    Energy Technology Data Exchange (ETDEWEB)

    Delavalle, Pierre-Yves; Alsaleh, Khaled; Pillez, Andre; Cocquerel, Laurence [INSERM U1019, CNRS UMR 8204, CIIL, F-59021 Lille (France); Universite Lille Nord de France, F-59000 Lille (France); Institut Pasteur de Lille, F-59019 Lille (France); Allet, Cecile [INSERM U837-JPARC, 59045 Lille (France); Dumont, Patrick [Universite Lille Nord de France, F-59000 Lille (France); Institut Pasteur de Lille, F-59019 Lille (France); CNRS UMR 8161, F-59021 Lille (France); Loyens, Anne [INSERM U837-JPARC, 59045 Lille (France); Leteurtre, Emmanuelle [Service d' Anatomie et de Cytologie Pathologiques, Centre de Biologie Pathologie, CHRU de Lille, Avenue Oscar-Lambret, Lille cedex (France); Omary, M. Bishr [Department of Molecular and Integrative Physiology, University of Michigan Medical School, Ann Arbor, Michigan, United States of America (United States); Dubuisson, Jean; Rouille, Yves [INSERM U1019, CNRS UMR 8204, CIIL, F-59021 Lille (France); Universite Lille Nord de France, F-59000 Lille (France); Institut Pasteur de Lille, F-59019 Lille (France); Wychowski, Czeslaw, E-mail: czeslaw.wychowski@ibl.fr [INSERM U1019, CNRS UMR 8204, CIIL, F-59021 Lille (France); Universite Lille Nord de France, F-59000 Lille (France); Institut Pasteur de Lille, F-59019 Lille (France)

    2011-11-01

    Cytoplasmic inclusions are found in a variety of diseases that are characteristic morphological features of several hepatic, muscular and neurodegenerative disorders. They display a predominantly filamentous ultrastructure that is also observed in malignant rhabdoid tumor (MRT). A cellular clone containing an intracytoplasmic body was isolated from hepatocyte cell culture, and in the present study we examined whether this body might be related or not to Mallory-Denk body (MDB), a well characterized intracytoplasmic inclusion, or whether this cellular clone was constituted by malignant rhabdoid tumor cells. The intracytoplasmic body was observed in electron microscopy (EM), confocal immunofluorescence microscopy and several proteins involved in the formation of its structure were identified. Using light microscopy, a spheroid body (SB) described as a single regular-shaped cytoplasmic body was observed in cells. During cytokinesis, the SB was disassembled and reassembled in a way to reconstitute a unique SB in each progeny cell. EM examination revealed that the SB was not surrounded by a limiting membrane. However, cytoplasmic filaments were concentrated in a whorled array. These proteins were identified as keratins 8 and 18 (K8/K18), which formed the central core of the SB surrounded by a vimentin cage-like structure. This structure was not related to Mallory-Denk body or aggresome since no aggregated proteins were located in SB. Moreover, the structure of SB was not due to mutations in the primary sequence of K8/K18 and vimentin since no difference was observed in the mRNA sequence of their genes, isolated from Huh-7 and Huh-7w7.3 cells. These data suggested that cellular factor(s) could be responsible for the SB formation process. Aggregates of K18 were relocated in the SB when a mutant of K18 inducing disruption of K8/K18 IF network was expressed in the cellular clone. Furthermore, the INI1 protein, a remodeling-chromatin factor deficient in rhabdoid cells, which

  3. Hepatocyte-derived cultured cells with unusual cytoplasmic keratin-rich spheroid bodies.

    Science.gov (United States)

    Delavalle, Pierre-Yves; Alsaleh, Khaled; Pillez, André; Cocquerel, Laurence; Allet, Cécile; Dumont, Patrick; Loyens, Anne; Leteurtre, Emmanuelle; Omary, M Bishr; Dubuisson, Jean; Rouillé, Yves; Wychowski, Czeslaw

    2011-11-01

    Cytoplasmic inclusions are found in a variety of diseases that are characteristic morphological features of several hepatic, muscular and neurodegenerative disorders. They display a predominantly filamentous ultrastructure that is also observed in malignant rhabdoid tumor (MRT). A cellular clone containing an intracytoplasmic body was isolated from hepatocyte cell culture, and in the present study we examined whether this body might be related or not to Mallory-Denk body (MDB), a well characterized intracytoplasmic inclusion, or whether this cellular clone was constituted by malignant rhabdoid tumor cells. The intracytoplasmic body was observed in electron microscopy (EM), confocal immunofluorescence microscopy and several proteins involved in the formation of its structure were identified. Using light microscopy, a spheroid body (SB) described as a single regular-shaped cytoplasmic body was observed in cells. During cytokinesis, the SB was disassembled and reassembled in a way to reconstitute a unique SB in each progeny cell. EM examination revealed that the SB was not surrounded by a limiting membrane. However, cytoplasmic filaments were concentrated in a whorled array. These proteins were identified as keratins 8 and 18 (K8/K18), which formed the central core of the SB surrounded by a vimentin cage-like structure. This structure was not related to Mallory-Denk body or aggresome since no aggregated proteins were located in SB. Moreover, the structure of SB was not due to mutations in the primary sequence of K8/K18 and vimentin since no difference was observed in the mRNA sequence of their genes, isolated from Huh-7 and Huh-7w7.3 cells. These data suggested that cellular factor(s) could be responsible for the SB formation process. Aggregates of K18 were relocated in the SB when a mutant of K18 inducing disruption of K8/K18 IF network was expressed in the cellular clone. Furthermore, the INI1 protein, a remodeling-chromatin factor deficient in rhabdoid cells, which

  4. PCNA Expression and Electron Microscopic Study of Acinus-Forming Hepatocytes in Chronic Hepatitis B

    Science.gov (United States)

    Han, Nam Ik; Lee, Young Sok; Choi, Hwang; Choi, Jong Young; Yun, Seung Kyu; Cho, Se Hyun; Han, Jun Youl; Yang, Jin Mo; Ahn, Byung Min; Choi, Sang Wook; Lee, Chang Don; Cha, Sang Bok; Sun, Hee Sik; Park, Doo Ho

    2002-01-01

    Background One of the major morphologic characteristics of hepatitis B is a hepatocellular regeneration which is induced by massive hepatocyte necrosis and associated with proliferative activity of hepatocytes. The purpose of this study is to document the proliferative activity of hepatocytes in various types of hepatitis B by immunohistochemical staining for proliferative cell nuclear antigen-labelling index (PCNA-LI) and electron microscopy. Methods We studied 83 patients with hepatitis B; 11 cases of acute viral hepatitis, 24 cases of mild chronic hepatitis, 34 cases of severe chronic hepatitis with early cirrhosis and 14 cases of severe chronic hepatitis. The PCNA was tested by immunohistochemical staining using anti-PCNA antibody. Furthermore we evaluated the ultrastructure of acinus-forming hepatocytes (AFH) by electron microscopy. Results The expression rate and labelling index of PCNA were 27.3% and 5.3±0.9% in acute viral hepatitis, 62.5% and 22.9±31.7% in mild chronic hepatits, and then 47.1% and 14.7±24.2% in severe chronic hepatitis with early cirrhosis, respectively (Figure 1). By contrast, no detectable PCNA expression was noted in AFH. Electron microscopic findings showed that hepatocytes forming a rosette underwent marked degenerative changes with sinusoidal capillarization and increased fine strands of collagen fiber in portal area. Conclusion The proliferative acitivity of hepatitis B was significantly decreased in severe chronic hepatitis containing AFH. This result suggested that differences in proliferative activity was associated with hepatic cell necrosis and AFH. PMID:12164086

  5. Effect of butylated hydroxytoluene, curcumin, propyl gallate and thiabendazole on cytochrome P450 forms in cultured human hepatocytes

    NARCIS (Netherlands)

    Price, R.J.; Scott, M.P.; Giddings, A.M.; Walters, D.G.; Stierum, R.H.; Meredith, C.; Lake, B.G.

    2008-01-01

    1. The objective of this study was to investigate the effects of four food chemicals, namely butylated hydroxytoluene (BHT), curcumin (CC), propyl gallate (PG) and thiabendazole (TB), on cytochrome P450 (CYP) forms in cultured human hepatocytes. 2. Treatment of human hepatocytes for 72 h with 2-200

  6. Effect of butylated hydroxytoluene, curcumin, propyl gallate and thiabendazole on cytochrome P450 forms in cultured human hepatocytes

    NARCIS (Netherlands)

    Price, R.J.; Scott, M.P.; Giddings, A.M.; Walters, D.G.; Stierum, R.H.; Meredith, C.; Lake, B.G.

    2008-01-01

    1. The objective of this study was to investigate the effects of four food chemicals, namely butylated hydroxytoluene (BHT), curcumin (CC), propyl gallate (PG) and thiabendazole (TB), on cytochrome P450 (CYP) forms in cultured human hepatocytes. 2. Treatment of human hepatocytes for 72 h with 2-200

  7. The neural basis of body form and body action agnosia.

    Science.gov (United States)

    Moro, Valentina; Urgesi, Cosimo; Pernigo, Simone; Lanteri, Paola; Pazzaglia, Mariella; Aglioti, Salvatore Maria

    2008-10-23

    Visual analysis of faces and nonfacial body stimuli brings about neural activity in different cortical areas. Moreover, processing body form and body action relies on distinct neural substrates. Although brain lesion studies show specific face processing deficits, neuropsychological evidence for defective recognition of nonfacial body parts is lacking. By combining psychophysics studies with lesion-mapping techniques, we found that lesions of ventromedial, occipitotemporal areas induce face and body recognition deficits while lesions involving extrastriate body area seem causatively associated with impaired recognition of body but not of face and object stimuli. We also found that body form and body action recognition deficits can be double dissociated and are causatively associated with lesions to extrastriate body area and ventral premotor cortex, respectively. Our study reports two category-specific visual deficits, called body form and body action agnosia, and highlights their neural underpinnings.

  8. Perfused human hepatocyte microtissues identify reactive metabolite-forming and mitochondria-perturbing hepatotoxins.

    Science.gov (United States)

    Rowe, Cliff; Shaeri, Mohsen; Large, Emma; Cornforth, Terri; Robinson, Angela; Kostrzewski, Tomasz; Sison-Young, Rowena; Goldring, Christopher; Park, Kevin; Hughes, David

    2017-09-15

    Hepatotoxins cause liver damage via many mechanisms but the formation of reactive metabolites and/or damage to liver mitochondria are commonly implicated. We assess 3D human primary hepatocyte microtissues as a platform for hepatotoxicity studies with reactive metabolite-forming and mitochondria-perturbing compounds. We show that microtissues formed from cryopreserved human hepatocytes had bile canaliculi, transcribed mRNA from genes associated with xenobiotic metabolism and expressed functional cytochrome P450 enzymes. Hierarchical clustering was used to distinguish dose-dependent hepatotoxicity elicited by clozapine, fialuridine and acetaminophen (APAP) from control cultures and less liver-damaging compounds, olanzapine and entecavir. The regio-isomer of acetaminophen, N-acetyl-meta-aminophenol (AMAP) clustered with the hepatotoxic compounds. The principal metabolites of APAP were formed and dose-dependent changes in metabolite profile similar to those seen in patient overdose was observed. The toxicological profile of APAP was indistinguishable from that of AMAP, confirming AMAP as a human hepatotoxin. Tissue oxygen consumption rate was significantly decreased within 2h of exposure to APAP or AMAP, concomitant with glutathione depletion. These data highlight the potential utility of perfused metabolically functional human liver microtissues in drug development and mechanistic toxicology. Copyright © 2017. Published by Elsevier Ltd.

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

    Science.gov (United States)

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

    2009-04-05

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

  10. body shape versus body form: a comparison of the body shapes of ...

    African Journals Online (AJOL)

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  11. A new splice variant of the major subunit of human asialoglycoprotein receptor encodes a secreted form in hepatocytes.

    Directory of Open Access Journals (Sweden)

    Jia Liu

    Full Text Available BACKGROUND: The human asialoglycoprotein receptor (ASGPR is composed of two polypeptides, designated H1 and H2. While variants of H2 have been known for decades, the existence of H1 variants has never been reported. PRINCIPAL FINDINGS: We identified two splice variants of ASGPR H1 transcripts, designated H1a and H1b, in human liver tissues and hepatoma cells. Molecular cloning of ASGPR H1 variants revealed that they differ by a 117 nucleotide segment corresponding to exon 2 in the ASGPR genomic sequence. Thus, ASGPR variant H1b transcript encodes a protein lacking the transmembrane domain. Using an H1b-specific antibody, H1b protein and a functional soluble ASGPR (sASGPR composed of H1b and H2 in human sera and in hepatoma cell culture supernatant were identified. The expression of ASGPR H1a and H1b in Hela cells demonstrated the different cellular loctions of H1a and H1b proteins at cellular membranes and in intracellular compartments, respectively. In vitro binding assays using fluorescence-labeled sASGPR or the substract ASOR revealed that the presence of sASGPR reduced the binding of ASOR to cells. However, ASOR itself was able to enhance the binding of sASGPR to cells expressing membrane-bound ASGPR. Further, H1b expression is reduced in liver tissues from patients with viral hepatitis. CONCLUSIONS: We conclude that two naturally occurring ASGPR H1 splice variants are produced in human hepatocytes. A hetero-oligomeric complex sASGPR consists of the secreted form of H1 and H2 and may bind to free substrates in circulation and carry them to liver tissue for uptake by ASGPR-expressing hepatocytes.

  12. Cajal bodies: where form meets function.

    Science.gov (United States)

    Machyna, Martin; Heyn, Patricia; Neugebauer, Karla M

    2013-01-01

    The cell nucleus contains dozens of subcompartments that separate biochemical processes into confined spaces. Cajal bodies (CBs) were discovered more than 100 years ago, but only extensive research in the past decades revealed the surprising complexity of molecular and cellular functions taking place in these structures. Many protein and RNA species are modified and assembled within CBs, which have emerged as a meeting place and factory for ribonucleoprotein (RNP) particles involved in splicing, ribosome biogenesis and telomere maintenance. Recently, a distinct structure near histone gene clusters--the Histone locus body (HLB)--was discovered. Involved in histone mRNA 3'-end formation, HLBs can share several components with CBs. Whether the appearance of distinct HLBs is simply a matter of altered affinity between these structures or of an alternate mode of CB assembly is unknown. However, both structures share basic assembly properties, in which transcription plays a decisive role in initiation. After this seeding event, additional components associate in random order. This appears to be a widespread mechanism for body assembly. CB assembly encompasses an additional layer of complexity, whereby a set of pre-existing substructures can be integrated into mature CBs. We propose this as a multi-seeding model of CB assembly.

  13. A monocarboxylate transporter required for hepatocyte secretion of ketone bodies during fasting.

    Science.gov (United States)

    Hugo, Sarah E; Cruz-Garcia, Lourdes; Karanth, Santhosh; Anderson, Ryan M; Stainier, Didier Y R; Schlegel, Amnon

    2012-02-01

    To find new genes that influence liver lipid mass, we performed a genetic screen for zebrafish mutants with hepatic steatosis, a pathological accumulation of fat. The red moon (rmn) mutant develops hepatic steatosis as maternally deposited yolk is depleted. Conversely, hepatic steatosis is suppressed in rmn mutants by adequate nutrition. Adult rmn mutants show increased liver neutral lipids and induction of hepatic lipid biosynthetic genes when fasted. Positional cloning of the rmn locus reveals a loss-of-function mutation in slc16a6a (solute carrier family 16a, member 6a), a gene that we show encodes a transporter of the major ketone body β-hydroxybutyrate. Restoring wild-type zebrafish slc16a6a expression or introducing human SLC16A6 in rmn mutant livers rescues the mutant phenotype. Radiotracer analysis confirms that loss of Slc16a6a function causes diversion of liver-trapped ketogenic precursors into triacylglycerol. Underscoring the importance of Slc16a6a to normal fasting physiology, previously fed rmn mutants are more sensitive to death by starvation than are wild-type larvae. Our unbiased, forward genetic approach has found a heretofore unrecognized critical step in fasting energy metabolism: hepatic ketone body transport. Since β-hydroxybutyrate is both a major fuel and a signaling molecule in fasting, the discovery of this transporter provides a new direction for modulating circulating levels of ketone bodies in metabolic diseases.

  14. Form Factors of Few-Body Systems: Point Form Versus Front Form

    CERN Document Server

    Gómez-Rocha, Maria; Schweiger, Wolfgang

    2011-01-01

    We present a relativistic point-form approach for the calculation of electroweak form factors of few-body bound states that leads to results which resemble those obtained within the covariant light-front formalism of Carbonell et al. Our starting points are the physical processes in which such form factors are measured, i.e. electron scattering off the bound state, or the semileptonic weak decay of the bound state. These processes are treated by means of a coupled-channel framework for a Bakamjian-Thomas type mass operator. A current with the correct covariance properties is then derived from the pertinent leading-order electroweak scattering or decay amplitude. As it turns out, the electromagnetic current is affected by unphysical contributions which can be traced back to wrong cluster properties inherent in the Bakamjian-Thomas construction. These spurious contributions, however, can be separated uniquely, as in the covariant light-front approach. In this way we end up with form factors which agree with tho...

  15. The pore-forming toxin listeriolysin O mediates a novel entry pathway of L. monocytogenes into human hepatocytes.

    Directory of Open Access Journals (Sweden)

    Stephen Vadia

    2011-11-01

    Full Text Available Intracellular pathogens have evolved diverse strategies to invade and survive within host cells. Among the most studied facultative intracellular pathogens, Listeria monocytogenes is known to express two invasins-InlA and InlB-that induce bacterial internalization into nonphagocytic cells. The pore-forming toxin listeriolysin O (LLO facilitates bacterial escape from the internalization vesicle into the cytoplasm, where bacteria divide and undergo cell-to-cell spreading via actin-based motility. In the present study we demonstrate that in addition to InlA and InlB, LLO is required for efficient internalization of L. monocytogenes into human hepatocytes (HepG2. Surprisingly, LLO is an invasion factor sufficient to induce the internalization of noninvasive Listeria innocua or polystyrene beads into host cells in a dose-dependent fashion and at the concentrations produced by L. monocytogenes. To elucidate the mechanisms underlying LLO-induced bacterial entry, we constructed novel LLO derivatives locked at different stages of the toxin assembly on host membranes. We found that LLO-induced bacterial or bead entry only occurs upon LLO pore formation. Scanning electron and fluorescence microscopy studies show that LLO-coated beads stimulate the formation of membrane extensions that ingest the beads into an early endosomal compartment. This LLO-induced internalization pathway is dynamin-and F-actin-dependent, and clathrin-independent. Interestingly, further linking pore formation to bacteria/bead uptake, LLO induces F-actin polymerization in a tyrosine kinase-and pore-dependent fashion. In conclusion, we demonstrate for the first time that a bacterial pathogen perforates the host cell plasma membrane as a strategy to activate the endocytic machinery and gain entry into the host cell.

  16. The pore-forming toxin listeriolysin O mediates a novel entry pathway of L. monocytogenes into human hepatocytes.

    Science.gov (United States)

    Vadia, Stephen; Arnett, Eusondia; Haghighat, Anne-Cécile; Wilson-Kubalek, Elisabeth M; Tweten, Rodney K; Seveau, Stephanie

    2011-11-01

    Intracellular pathogens have evolved diverse strategies to invade and survive within host cells. Among the most studied facultative intracellular pathogens, Listeria monocytogenes is known to express two invasins-InlA and InlB-that induce bacterial internalization into nonphagocytic cells. The pore-forming toxin listeriolysin O (LLO) facilitates bacterial escape from the internalization vesicle into the cytoplasm, where bacteria divide and undergo cell-to-cell spreading via actin-based motility. In the present study we demonstrate that in addition to InlA and InlB, LLO is required for efficient internalization of L. monocytogenes into human hepatocytes (HepG2). Surprisingly, LLO is an invasion factor sufficient to induce the internalization of noninvasive Listeria innocua or polystyrene beads into host cells in a dose-dependent fashion and at the concentrations produced by L. monocytogenes. To elucidate the mechanisms underlying LLO-induced bacterial entry, we constructed novel LLO derivatives locked at different stages of the toxin assembly on host membranes. We found that LLO-induced bacterial or bead entry only occurs upon LLO pore formation. Scanning electron and fluorescence microscopy studies show that LLO-coated beads stimulate the formation of membrane extensions that ingest the beads into an early endosomal compartment. This LLO-induced internalization pathway is dynamin-and F-actin-dependent, and clathrin-independent. Interestingly, further linking pore formation to bacteria/bead uptake, LLO induces F-actin polymerization in a tyrosine kinase-and pore-dependent fashion. In conclusion, we demonstrate for the first time that a bacterial pathogen perforates the host cell plasma membrane as a strategy to activate the endocytic machinery and gain entry into the host cell.

  17. CD73 (ecto-5′-nucleotidase) hepatocyte levels differ across mouse strains and contribute to Mallory-Denk body formation

    OpenAIRE

    2013-01-01

    The formation of hepatocyte Mallory-Denk bodies (MDBs), which are aggregates of keratins 8 and 18 (K8/K18), ubiquitin, and the ubiquitin-binding protein p62, has a genetic predisposition component in humans and mice. We tested the hypothesis that metabolomic profiling of the MDB-susceptible C57BL and the MDB-resistant C3H mouse strains can illuminate MDB-associated pathways. Using both targeted and unbiased metabolomic analyses we demonstrated significant differences in intermediates of purin...

  18. Viewpoint and pose in body-form adaptation.

    Science.gov (United States)

    Sekunova, Alla; Black, Michael; Parkinson, Laura; Barton, Jason J S

    2013-01-01

    Faces and bodies are complex structures, perception of which can play important roles in person identification and inference of emotional state. Face representations have been explored using behavioural adaptation: in particular, studies have shown that face aftereffects show relatively broad tuning for viewpoint, consistent with origin in a high-level structural descriptor far removed from the retinal image. Our goals were to determine first, if body aftereffects also showed a degree of viewpoint invariance, and second if they also showed pose invariance, given that changes in pose create even more dramatic changes in the 2-D retinal image. We used a 3-D model of the human body to generate headless body images, whose parameters could be varied to generate different body forms, viewpoints, and poses. In the first experiment, subjects adapted to varying viewpoints of either slim or heavy bodies in a neutral stance, followed by test stimuli that were all front-facing. In the second experiment, we used the same front-facing bodies in neutral stance as test stimuli, but compared adaptation from bodies in the same neutral stance to adaptation with the same bodies in different poses. We found that body aftereffects were obtained over substantial viewpoint changes, with no significant decline in aftereffect magnitude with increasing viewpoint difference between adapting and test images. Aftereffects also showed transfer across one change in pose but not across another. We conclude that body representations may have more viewpoint invariance than faces, and demonstrate at least some transfer across pose, consistent with a high-level structural description.

  19. [Inclusion Bodies are Formed in SFTSV-infected Human Macrophages].

    Science.gov (United States)

    Jin, Cong; Song, Jingdong; Han, Ying; Li, Chuan; Qiu, Peihong; Liang, Mifang

    2016-01-01

    The severe fever with thrombocytopenia syndrome virus (SFTSV) is a new member in the genus Phlebovirus of the family Bunyaviridae identified in China. The SFTSV is also the causative pathogen of an emerging infectious disease: severe fever with thrombocytopenia syndrome. Using immunofluorescent staining and confocal microscopy, the intracellular distribution of nucleocapsid protein (NP) in SFTSV-infected THP-1 cells was investigated with serial doses of SFTSV at different times after infection. Transmission electron microscopy was used to observe the ultrafine intracellular structure of SFTSV-infected THP-1 cells at different times after infection. SFTSV NP could form intracellular inclusion bodies in infected THP-1 cells. The association between NP-formed inclusion bodies and virus production was analyzed: the size of the inclusion body formed 3 days after infection was correlated with the viral load in supernatants collected 7 days after infection. These findings suggest that the inclusion bodies formed in SFTSV-infected THP-1 cells could be where the SFTSV uses host-cell proteins and intracellular organelles to produce new viral particles.

  20. Artificial fish schools : Collective effects of school size, body size, and body form

    NARCIS (Netherlands)

    Kunz, H.; Hemelrijk, C.K.

    2003-01-01

    Individual-based models of schooling in fish have demonstrated that, via processes of self-organization. artificial fish may school in the absence of a leader or external stimuli, using local information only. We study for the first time how body size and body form of artificial fish affect school f

  1. High pressure sheet metal forming of large scale body structures

    Energy Technology Data Exchange (ETDEWEB)

    Trompeter, M.; Krux, R.; Homberg, W.; Kleiner, M. [Dortmund Univ. (Germany). Inst. of Forming Technology and Lightweight Construction

    2005-07-01

    An important trend in the automotive industry is the weight reduction of car bodies by lightweight construction. One approach to realise lightweight structures is the use of load optimised sheet metal parts (e.g. tailored blanks), especially for crash relevant car body structures. To form such parts which are mostly complex and primarily made of high strength steels, the use of working media based forming processes is favorable. The paper presents the manufacturing of a large scale structural component made of tailor rolled blanks (TRB) by high pressure sheet metal forming (HBU). The paper focuses mainly on the tooling system, which is integrated into a specific 100 MN hydroform press at the IUL. The HBU tool basically consists of a multipoint blankholder, a specially designed flange draw-in sensor, which is necessary to determine the material flow, and a sealing system. Furthermore, the paper presents a strategy for an effective closed loop flange draw-in control. (orig.)

  2. Yeast prions form infectious amyloid inclusion bodies in bacteria

    Directory of Open Access Journals (Sweden)

    Espargaró Alba

    2012-06-01

    Full Text Available Abstract Background Prions were first identified as infectious proteins associated with fatal brain diseases in mammals. However, fungal prions behave as epigenetic regulators that can alter a range of cellular processes. These proteins propagate as self-perpetuating amyloid aggregates being an example of structural inheritance. The best-characterized examples are the Sup35 and Ure2 yeast proteins, corresponding to [PSI+] and [URE3] phenotypes, respectively. Results Here we show that both the prion domain of Sup35 (Sup35-NM and the Ure2 protein (Ure2p form inclusion bodies (IBs displaying amyloid-like properties when expressed in bacteria. These intracellular aggregates template the conformational change and promote the aggregation of homologous, but not heterologous, soluble prionogenic molecules. Moreover, in the case of Sup35-NM, purified IBs are able to induce different [PSI+] phenotypes in yeast, indicating that at least a fraction of the protein embedded in these deposits adopts an infectious prion fold. Conclusions An important feature of prion inheritance is the existence of strains, which are phenotypic variants encoded by different conformations of the same polypeptide. We show here that the proportion of infected yeast cells displaying strong and weak [PSI+] phenotypes depends on the conditions under which the prionogenic aggregates are formed in E. coli, suggesting that bacterial systems might become useful tools to generate prion strain diversity.

  3. Matriptase is required for the active form of hepatocyte growth factor induced Met, focal adhesion kinase and protein kinase B activation on neural stem/progenitor cell motility.

    Science.gov (United States)

    Fang, Jung-Da; Lee, Sheau-Ling

    2014-07-01

    Hepatocyte growth factor (HGF) is a chemoattractant and inducer for neural stem/progenitor (NS/P) cell migration. Although the type II transmembrane serine protease, matriptase (MTP) is an activator of the latent HGF, MTP is indispensable on NS/P cell motility induced by the active form of HGF. This suggests that MTP's action on NS/P cell motility involves mechanisms other than proteolytic activation of HGF. In the present study, we investigate the role of MTP in HGF-stimulated signaling events. Using specific inhibitors of phosphatidylinositol-3-kinase (PI3K), protein kinase B (Akt) or focal adhesion kinase (FAK), we demonstrated that in NS/P cells HGF-activated c-Met induces PI3k-Akt signaling which then leads to FAK activation. This signaling pathway ultimately induces MMP2 expression and NS/P cell motility. Knocking down of MTP in NS/P cells with specific siRNA impaired HGF-stimulation of c-Met, Akt and FAK activation, blocked HGF-induced production of MMP2 and inhibited HGF-stimulated NS/P cell motility. MTP-knockdown NS/P cells cultured in the presence of recombinant protein of MTP protease domain or transfected with the full-length wild-type but not the protease-defected MTP restored HGF-responsive events in NS/P cells. In addition to functioning as HGF activator, our data revealed novel function of MTP on HGF-stimulated c-Met signaling activation.

  4. DNA adducts of the tobacco carcinogens 2-amino-9H-pyrido[2,3-b]indole and 4-aminobiphenyl are formed at environmental exposure levels and persist in human hepatocytes.

    Science.gov (United States)

    Nauwelaërs, Gwendoline; Bellamri, Medjda; Fessard, Valérie; Turesky, Robert J; Langouët, Sophie

    2013-09-16

    Aromatic amines and structurally related heterocyclic aromatic amines (HAAs) are produced during the combustion of tobacco or during the high-temperature cooking of meat. Exposure to some of these chemicals may contribute to the etiology of several common types of human cancers. 2-Amino-9H-pyrido[2,3-b]indole (AαC) is the most abundant HAA formed in mainstream tobacco smoke: it arises in amounts that are 25-100 times greater than the levels of the arylamine, 4-aminobiphenyl (4-ABP), a human carcinogen. 2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) is a prevalent HAA formed in cooked meats. AαC and MeIQx are rodent carcinogens; however, their carcinogenic potency in humans is unknown. A preliminary assessment of the carcinogenic potential of these HAAs in humans was conducted by examining the capacity of primary human hepatocytes to form DNA adducts of AαC and MeIQx, in comparison to 4-ABP, followed by the kinetics of DNA adduct removal by cellular enzyme repair systems. The principal DNA adducts formed were N-(deoxyguanosin-8-yl) (dG-C8) adducts. Comparable levels of DNA adducts were formed with AαC and 4-ABP, whereas adduct formation was ∼5-fold lower for MeIQx. dG-C8-AαC and dG-C8-4-ABP were formed at comparable levels in a concentration-dependent manner in human hepatocytes treated with procarcinogens over a 10,000-fold concentration range (1 nM-10 μM). Pretreatment of hepatocytes with furafylline, a selective inhibitor of cytochrome P450 1A2, resulted in a strong diminution of DNA adducts signifying that P450 1A2 is a major P450 isoform involved in bioactivation of these procarcinogens. The kinetics of adduct removal varied for each hepatocyte donor. Approximately half of the DNA adducts were removed within 24 h of treatment; however, the remaining lesions persisted over 5 days. The high levels of AαC present in tobacco smoke and its propensity to form persistent DNA adducts in human hepatocytes suggest that AαC can contribute to DNA damage

  5. INTEGRATED DISINFECTION BY-PRODUCTS (DBP) MIXTURES RESEARCH: GENE EXPRESSION ALTERATIONS IN PRIMARY RAT HEPATOCYTE CULTURES EXPOSED TO DBP MIXTURES FORMED BY CHLORINATION AND OZONATION/POSTCHLORINATION

    Science.gov (United States)

    What is the study? This study was designed to provide data on the in vitro toxicity of water concentrates containing complex mixtures of DBPs. Rat hepatocytes in primary culture were exposed for 24 hr to full strength, 1:10 or 1:20 dilutions of chlorination or ozonation/chl...

  6. Phosphoproteomic analysis of the Chlamydia caviae elementary body and reticulate body forms.

    Science.gov (United States)

    Fisher, Derek J; Adams, Nancy E; Maurelli, Anthony T

    2015-08-01

    Chlamydia are Gram-negative, obligate intracellular bacteria responsible for significant diseases in humans and economically important domestic animals. These pathogens undergo a unique biphasic developmental cycle transitioning between the environmentally stable elementary body (EB) and the replicative intracellular reticulate body (RB), a conversion that appears to require extensive regulation of protein synthesis and function. However, Chlamydia possess a limited number of canonical mechanisms of transcriptional regulation. Ser/Thr/Tyr phosphorylation of proteins in bacteria has been increasingly recognized as an important mechanism of post-translational control of protein function. We utilized 2D gel electrophoresis coupled with phosphoprotein staining and MALDI-TOF/TOF analysis to map the phosphoproteome of the EB and RB forms of Chlamydia caviae. Forty-two non-redundant phosphorylated proteins were identified (some proteins were present in multiple locations within the gels). Thirty-four phosphorylated proteins were identified in EBs, including proteins found in central metabolism and protein synthesis, Chlamydia-specific hypothetical proteins and virulence-related proteins. Eleven phosphorylated proteins were identified in RBs, mostly involved in protein synthesis and folding and a single virulence-related protein. Only three phosphoproteins were found in both EB and RB phosphoproteomes. Collectively, 41 of 42 C. caviae phosphoproteins were present across Chlamydia species, consistent with the existence of a conserved chlamydial phosphoproteome. The abundance of stage-specific phosphoproteins suggests that protein phosphorylation may play a role in regulating the function of developmental-stage-specific proteins and/or may function in concert with other factors in directing EB-RB transitions.

  7. What women like: influence of motion and form on esthetic body perception

    Directory of Open Access Journals (Sweden)

    Valentina eCazzato

    2012-07-01

    Full Text Available Several studies have shown the distinct contribution of motion and form to the esthetic evaluation of female bodies. Here, we investigated how variations of implied motion and body size interact in the esthetic evaluation of female and male bodies in a sample of young healthy women. Participants provided attractiveness, beauty, and liking ratings for the shape and posture of virtual renderings of human bodies with variable body size and implied motion. The esthetic judgments for both shape and posture of human models were influenced by body size and implied motion, with a preference for thinner and more dynamic stimuli. Implied motion, however, attenuated the impact of extreme body size on the esthetic evaluation of body postures, and body size variations did not affect the preference for more dynamic stimuli. Results show that body form and action cues interact in esthetic perception, but the final esthetic appreciation of human bodies is predicted by a mixture of perceptual and affective evaluative components.

  8. Cadmium toxicity in perinatal rat hepatocytes: Electron microscopy, X-ray microanalysis, and morphometric analysis

    Energy Technology Data Exchange (ETDEWEB)

    Kawahara, A.; Yoshizuka, M.; Hirano, T.; Ohsato, K.; Fujimoto, S. (Univ. of Occupational and Environmental Health, Kitakyushu (Japan))

    1990-10-01

    Effects of cadmium on the fetal and postnatal rat hepatocytes were studied with an electron microscope and an X-ray microanalyzer. Pregnant and lactating Wistar rat dams at 15 and 21 days of pregnancy and at 3 days after delivery received intraperitoneal injections of cadmium sulfate (1 mg/kg body weight) for 3 days. On the day following the last injection, the livers were isolated from the fetal and suckling rats and provided for electron microscopy. The livers from the untreated fetal and newborn rats served as control. Large bile canaliculi, which were formed by five or more hepatocytes, were frequently observed in the cadmium-treated perinatal rat livers. The intercellular space between each adjacent hepatocyte was widened. By X-ray microanalysis, cadmium peaks were preferentially detected out from intramitochondrial granules of the cadmium-treated hepatocytes. By morphometric analysis, the increase both in the mitochondria volume and in the number of intramitochondrial granules was evident in the cadmium-treated hepatocytes when compared to those of control. These data suggest the preferential accumulation of cadmium in mitochondria of the hepatocytes interferes with the morphogenesis of the perinatal rat liver.

  9. Composite materials and bodies including silicon carbide and titanium diboride and methods of forming same

    Science.gov (United States)

    Lillo, Thomas M.; Chu, Henry S.; Harrison, William M.; Bailey, Derek

    2013-01-22

    Methods of forming composite materials include coating particles of titanium dioxide with a substance including boron (e.g., boron carbide) and a substance including carbon, and reacting the titanium dioxide with the substance including boron and the substance including carbon to form titanium diboride. The methods may be used to form ceramic composite bodies and materials, such as, for example, a ceramic composite body or material including silicon carbide and titanium diboride. Such bodies and materials may be used as armor bodies and armor materials. Such methods may include forming a green body and sintering the green body to a desirable final density. Green bodies formed in accordance with such methods may include particles comprising titanium dioxide and a coating at least partially covering exterior surfaces thereof, the coating comprising a substance including boron (e.g., boron carbide) and a substance including carbon.

  10. THE TEACHERS ROLE IN FORMING PROPER BODY POSTURE

    Directory of Open Access Journals (Sweden)

    Zoran Bogdanović

    2007-05-01

    Full Text Available Being acquainted and well aware of the presence of physical deformation in school population, this study is based on the research of postural deformity of the pupils of the 5th grade of elementary school and determination of dependance of deformations appearance in relation to frequency of remonstration and indication to correct sitting position from proffesors’ perspective. The complete program content is conducted in the territory of the city of Kragujevac in several elementary schools comprising 299 students of the 5th grade. The object was to determine the number of students with kyphotic and lordotic deformity, to determine the presence of deformation in depandance of gender and to determine the presence of kyphotic and lordotic deformity in depandance of the frequency of proffesors indication to improper sitting. We can notice higher presence of kyphotic deformity at the probationers of male population that it is the case with female population while the higher presence of lordotic deformity is at female population.The highest number of probationers have reported that none of the proffesors warn them about proper sitting. The measures inside the groups sorted by gender qualifi cation, indicate on high percentage of both boys and girls who are not warned on proper sitting. Also, inside the groups of improper body holders, we can notice the most signifi cant kyphotic and lordotic deformity in the category of students who are never warned to sit properly. These indicators report us that is necessary to invest much more work on the education of parents and children as well as school stuff at the preschool and school institutions which would result in reducing the appearence and development of postural deformity at the population who is more liable to transformations of such kind.

  11. Primary 3-dimensional culture of mouse hepatocytes

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Complex 3-dimensional structures with good functions have been obtained under the primary mixcoculture of mouse hepatocytes with mouse liver fibroblasts without serum. Albumin secretion is kept above 10 μg/106 cells and urea synthesis reaches 25 μg/106 on the 7th day of culture. Avoiding serum affection, liver fibroblasts' effects on hepatocytes' viability, functions and 3-dimensional structure forming in primary serum-free culture have been studied. Important effects of the mesenchyma, especially the direct adherence of fibroblasts to hepatocytes, are shown.

  12. 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.

  13. Form or function: Does focusing on body functionality protect women from body dissatisfaction when viewing media images?

    Science.gov (United States)

    Mulgrew, Kate E; Tiggemann, Marika

    2016-07-04

    We examined whether shifting young women's (N =322) attention toward functionality components of media-portrayed idealized images would protect against body dissatisfaction. Image type was manipulated via images of models in either an objectified body-as-object form or active body-as-process form; viewing focus was manipulated via questions about the appearance or functionality of the models. Social comparison was examined as a moderator. Negative outcomes were most pronounced within the process-related conditions (body-as-process images or functionality viewing focus) and for women who reported greater functionality comparison. Results suggest that functionality-based depictions, reflections, and comparisons may actually produce worse outcomes than those based on appearance.

  14. Numerical study on forming complex fitting body on end of integrated stainless steel pipe without welds

    Institute of Scientific and Technical Information of China (English)

    张大伟; 赵升吨

    2016-01-01

    It is necessary to use the integrated stainless steel pipe having two fitting bodies without welds while train travelling at high speed. In order to form this type of integrated stainless steel pipe, the method of preforming combined finish forming process is developed. The preforming process is characterized by flaring combined upsetting for left fitting body which is like a flange, and is characterized by tube axial compressive process under die constraint for right fitting body which is like a double-wall pipe. The finite element simulations of the processes are carried out by software package DEFORM, and the results indicate that: 1) left or right fitting body can be formed by a two-step forming process without folding and under-filling defects; 2) by using two-step forming, strain and stress in left fitting body are larger than those in right fitting body, and deformation in right fitting body is more homogenous than the deformation in left fitting body; 3) two or more preforming steps may be needed for left fitting body considering the distributions of strain and stress.

  15. Con-forming bodies: the interplay of machines and bodies and the implications of agency in medical imaging.

    Science.gov (United States)

    Wood, Lisa A

    2016-06-01

    Attending to the material discursive constructions of the patient body within cone beam computed tomography (CBCT) imaging in radiotherapy treatments, in this paper I describe how bodies and machines co-create images. Using an analytical framework inspired by Science and Technology Studies and Feminist Technoscience, I describe the interplay between machines and bodies and the implications of materialities and agency. I argue that patients' bodies play a part in producing scans within acceptable limits of machines as set out through organisational arrangements. In doing so I argue that bodies are fabricated into the order of work prescribed and embedded within and around the CBCT system, becoming, not only the subject of resulting images, but part of that image. The scan is not therefore a representation of a passive subject (a body) but co-produced by the work of practitioners and patients who actively control (and contort) and discipline their body according to protocols and instructions and the CBCT system. In this way I suggest they are 'con-forming' the CBCT image. A Virtual Abstract of this paper can be found at: https://youtu.be/qysCcBGuNSM.

  16. Study on Body Form and Garment Size Series of the Middle Age and Aged People

    Institute of Scientific and Technical Information of China (English)

    刘瑜; 郁进明

    2003-01-01

    This paper was designed to analyze on the data,which was obtained from "National Physique Fitness Investigation Report (2000)".In order to get the typical body form and figure type of the middle age and aged people,it was focused on the body form data of this group (age 4060)[1].After calculation and analyzing,the distinguishing feature of body form and the distribution of figure type were deduced.Finally,the re-classification of body form for Chinese middle age and aged people was suggested.It as also suggested that a new garment size series especially for the middle age and aged should be built to fit for these people.This conclusion would be useful and significant to design and production for clothing company,especially that who take the aged people as their target consumer.

  17. The fibrous form of intracellular inclusion bodies in recombinant variant fibrinogen-producing cells is specific to the hepatic fibrinogen storage disease-inducible variant fibrinogen.

    Science.gov (United States)

    Arai, Shinpei; Ogiwara, Naoko; Mukai, Saki; Takezawa, Yuka; Sugano, Mitsutoshi; Honda, Takayuki; Okumura, Nobuo

    2017-06-01

    Fibrinogen storage disease (FSD) is a rare disorder that is characterized by the accumulation of fibrinogen in hepatocytes and induces liver injury. Six mutations in the γC domain (γG284R, γT314P, γD316N, the deletion of γG346-Q350, γG366S, and γR375W) have been identified for FSD. Our group previously established γ375W fibrinogen-producing Chinese hamster ovary (CHO) cells and observed aberrant large granular and fibrous forms of intracellular inclusion bodies. The aim of this study was to investigate whether fibrous intracellular inclusion bodies are specific to FSD-inducible variant fibrinogen. Thirteen expression vectors encoding the variant γ-chain were stably or transiently transfected into CHO cells expressing normal fibrinogen Aα- and Bβ-chains or HuH-7 cells, which were then immunofluorescently stained. Six CHO and HuH-7 cell lines that transiently produced FSD-inducible variant fibrinogen presented the fibrous (3.2-22.7 and 2.1-24.5%, respectively) and large granular (5.4-25.5 and 7.7-23.9%) forms of intracellular inclusion bodies. Seven CHO and HuH-7 cell lines that transiently produced FSD-non-inducible variant fibrinogen only exhibit the large granular form. These results demonstrate that transiently transfected variant fibrinogen-producing CHO cells and inclusion bodies of the fibrous form may be useful in non-invasive screening for FSD risk factors for FSD before its onset.

  18. Primary hepatocyte culture in collagen gel mixture and collagen sandwich

    Institute of Scientific and Technical Information of China (English)

    Ying-Jie Wang; Hong-Ling Liu; Hai-Tao Guo; Hong-Wei Wen; Jun Liu

    2004-01-01

    AIM: To explore the methods of hepatocytes culture in a collagen gel mixture or between double layers of collagen sandwich configuration and to examine the functional and cytomorphological characteristics of cultured hepatocytes.METHODS: A two-step collagenase perfusion technique was used to isolate the hepatocytes from Wistar rats or newborn Chinese experimental piglets. The isolated hepatocytes were cultured in a collagen gel mixture or between double layers of collagen sandwich configuration respectively. The former was that rat hepatocytes were mixed with type I rat tail collagen solution till gelled, and the medium was added onto the gel. The latter was that swine hepatocytes were seeded on a plate precoated with collagen gel for 24 h, then another layer of collagen gel was overlaid, resulting in a sandwich configuration. The cytomorphological characteristics, albumin secretion, and LDH-release of the hepatocytes cultured in these two models were examined.RESULTS: Freshly isolated rat hepatocytes were successfully mixed and fixed in collagen gel, and cultured in the gel condition. During the culture period, the urea synthesized and secreted by rat hepatocytes was detected throughout the period. Likewise, newborn experimental piglet hepatocytes were successfully fixed between the double layers of collagen gel, forming a sandwich configuration.Within a week of culture, the albumin secreted by swine hepatocytes was detected by SDS/PAGE analysis. The typical cytomorphological characteristics of the hepatocytes cultured by the above two culture models were found under a phasecontrast microscope. There was little LDH-release during the culture period.CONCLUSION: Both collagen gel mixture and double layers of collagen sandwich configuration can provide cultural conditions much closer to in vivoenvironment, and are helpful for maintaining specific hepatic fiJnctions and cytomorphological characteristics. A collagen gel mixture culture may be more eligible for the

  19. Aesthetic skin branding: a novel form of body art with adverse clinical sequela.

    Science.gov (United States)

    Karamanoukian, Raffy; Ukatu, Chidi; Lee, Edward; Hyman, Josh; Sundine, Michael; Kobayashi, Mark; Evans, Gregory R D

    2006-01-01

    Branding is a form of body art wherein third-degree burns are inflicted on the skin to produce permanent scars. This method of scarification is a common practice among many indigenous cultures and has become exceedingly common in western societies. As with other forms of body art, branding is not a manifestation of a psychiatric disorder but, rather, a method of self-expression. The process can be performed through the use of electrocautery, laser, chemicals, freezing, and hot metal. Complications arising from the procedure include acute infection, transmission of blood-borne pathogens, allergic reactions, and sequelae arising from third-degree burns. In addition, skin branding has been shown to be associated with substance abuse and high-risk behaviors among adolescents. The purpose of this article is to present the following case report and review to familiarize clinicians with this dangerous method of body art.

  20. The Charge Form Factors of the Three- and Four-Body Nuclei

    Energy Technology Data Exchange (ETDEWEB)

    R. Schiavilla; V.R. Pandharipande; D.O. Riska

    1990-01-01

    The charge form factors of 3H, 3He, and 4He are calculated using the Monte Carlo method and variational ground-state wave functions obtained for the Argonne two-nucleon and Urbana-VII three-nucleon interactions. The model for the charge density operator contains the two-body exchange contributions of longest range. With some spread due to the uncertainty in the electromagnetic form factors of the nucleon the calculated charge form factors are in good agreement with the empirical values over the whole experimentally covered range of momentum transfer.

  1. The parametric orbits and the form invariance of three-body in one-dimension

    Institute of Scientific and Technical Information of China (English)

    Lou Zhi-Mei

    2005-01-01

    In this paper, the differential equations of motion of a three-body interacting pairwise by inverse cubic forces("centrifugal potential") in addition to linear forces ("harmonical potential") are expressed in Ermakov formalism in two-dimension polar coordinates, and the Ermakov invariant is obtained. By rescaling of the time variable and the space coordinates, the parametric orbits of the three bodies are expressed in terms of relative energy H1 and Ermakov invariant. The form invariance of the transformations of two conserved quantities are also studied.

  2. Morphological and functional behaviors of rat hepatocytes cultured on single-walled carbon nanotubes.

    Science.gov (United States)

    Koga, Haruka; Fujigaya, Tsuyohiko; Nakashima, Naotoshi; Nakazawa, Kohji

    2011-09-01

    This study describes the morphological and functional behaviors of rat hepatocytes on single-walled carbon nanotube (CNT)-coated surfaces. Although the hydrophobic characteristics of CNT-coated surfaces increased with increasing CNT density, hepatocyte adhesion decreased, indicating that the interaction between hepatocytes and CNTs is weak. We found that hepatocytes on a CNT-coated surface gradually gather together and form spheroids (spherical multicellular aggregates). These spheroids exhibit compact spherical morphology with a smooth surface and express connexin-32, an intracellular communication molecule. In contrast, collagen treatment in conjunction with the CNT-coated surface improved hepatocyte adhesion, and the cells maintained a monolayer configuration throughout the culture period. The albumin secretion and ammonia removal activities of hepatocyte spheroids were maintained at elevated levels for at least 15 days of culturing as compared with hepatocyte monolayers. These results indicate that CNTs can be used for the formation and long-term culture of hepatocyte spheroids.

  3. 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

  4. Mechanisms of the statins cytotoxicity in freshly isolated rat hepatocytes.

    Science.gov (United States)

    Abdoli, Narges; Heidari, Reza; Azarmi, Yadollah; Eghbal, Mohammad Ali

    2013-06-01

    Statins are potent drugs, used as lipid-lowering agents in cardiovascular diseases. Hepatotoxicity is one of the serious adverse effects of statins, and the exact mechanism of hepatotoxicity is not yet clear. In this study, the cytotoxic effects of the most commonly used statins, that is, atorvastatin, lovastatin, and simvastatin toward isolated rat hepatocytes, were evaluated. Markers, such as cell death, reactive oxygen species (ROS) formation, lipid peroxidation, mitochondrial membrane potential, and the amount of reduced and oxidized glutathione in the statin-treated hepatocytes, were investigated. It was found that the statins caused cytotoxicity toward rat hepatocytes dose dependently. An elevation in ROS formation, accompanied by a significant amount of lipid peroxidation and mitochondrial depolarization, was observed. Cellular glutathione reservoirs were decreased, and a significant amount of oxidized glutathione was formed. This study suggests that the adverse effect of statins toward hepatocytes is mediated through oxidative stress and the hepatocytes mitochondria play an important role in the statin-induced toxicity.

  5. Hyperbolic Error Analysis and Parametric Optimization of Round Body Form Tool

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    with the merits of the easy manufacture and the long service life and the processing the inside or outside form surface, round body form tool is extensive use in large scales production. Its main demerit is the big hyperbolic error which is caused in the process of processing cone, but about the discussion of hyperbolic error, there are two drawbacks in the current books and documents: (1) The error measuring plane is established on the rake face of tool, which doesn't coincide with the actual measuring pl...

  6. Bulge formed cooling channels with a variable lead helix on a hollow body of revolution

    Science.gov (United States)

    McAninch, Michael D. (Inventor); Holbrook, Richard L. (Inventor); Lacount, Dale F. (Inventor); Kawashige, Chester M. (Inventor); Crapuchettes, John M. (Inventor); Scala, James (Inventor)

    1993-01-01

    A method of constructing a nozzle having cooling channels comprises a shell and a liner which are formed into a body of revolution having an axis of revolution. Helical welds are formed to hold the liner and shell to each other with a channel position being defined between each pair of helical welds. Pressurized fluid which may be a gas or a liquid, is introduced between the weld pairs to outwardly bulge the material of at least one of the liner and shell to define the channels.

  7. Evolution of the snake body form reveals homoplasy in amniote Hox gene function.

    Science.gov (United States)

    Head, Jason J; Polly, P David

    2015-04-02

    Hox genes regulate regionalization of the axial skeleton in vertebrates, and changes in their expression have been proposed to be a fundamental mechanism driving the evolution of new body forms. The origin of the snake-like body form, with its deregionalized pre-cloacal axial skeleton, has been explained as either homogenization of Hox gene expression domains, or retention of standard vertebrate Hox domains with alteration of downstream expression that suppresses development of distinct regions. Both models assume a highly regionalized ancestor, but the extent of deregionalization of the primaxial domain (vertebrae, dorsal ribs) of the skeleton in snake-like body forms has never been analysed. Here we combine geometric morphometrics and maximum-likelihood analysis to show that the pre-cloacal primaxial domain of elongate, limb-reduced lizards and snakes is not deregionalized compared with limbed taxa, and that the phylogenetic structure of primaxial morphology in reptiles does not support a loss of regionalization in the evolution of snakes. We demonstrate that morphometric regional boundaries correspond to mapped gene expression domains in snakes, suggesting that their primaxial domain is patterned by a normally functional Hox code. Comparison of primaxial osteology in fossil and modern amniotes with Hox gene distributions within Amniota indicates that a functional, sequentially expressed Hox code patterned a subtle morphological gradient along the anterior-posterior axis in stem members of amniote clades and extant lizards, including snakes. The highly regionalized skeletons of extant archosaurs and mammals result from independent evolution in the Hox code and do not represent ancestral conditions for clades with snake-like body forms. The developmental origin of snakes is best explained by decoupling of the primaxial and abaxial domains and by increases in somite number, not by changes in the function of primaxial Hox genes.

  8. Uranium(VI) Binding Forms in Selected Human Body Fluids: Thermodynamic Calculations versus Spectroscopic Measurements.

    Science.gov (United States)

    Osman, Alfatih A A; Geipel, Gerhard; Barkleit, Astrid; Bernhard, Gert

    2015-02-16

    Human exposure to uranium increasingly becomes a subject of interest in many scientific disciplines such as environmental medicine, toxicology, and radiation protection. Knowledge about uranium chemical binding forms(speciation) in human body fluids can be of great importance to understand not only its biokinetics but also its relevance in risk assessment and in designing decorporation therapy in the case of accidental overexposure. In this study, thermodynamic calculations of uranium speciation in relevant simulated and original body fluids were compared with spectroscopic data after ex-situ uranium addition. For the first time, experimental data on U(VI) speciation in body fluids (saliva, sweat, urine) was obtained by means of cryogenic time-resolved laser-induced fluorescence spectroscopy (cryo-TRLFS) at 153 K. By using the time dependency of fluorescence decay and the band positions of the emission spectra, various uranyl complexes were demonstrated in the studied samples. The variations of the body fluids in terms of chemical composition, pH, and ionic strength resulted in different binding forms of U(VI). The speciation of U(VI) in saliva and in urine was affected by the presence of bioorganic ligands, whereas in sweat, the distribution depends mainly on inorganic ligands. We also elucidated the role of biological buffers, i.e., phosphate (H(2)PO(4−)/HPO(4)(2−)) on U(VI) distribution, and the system Ca(2+)/UO(2)(2+)/PO(4)(3−) was discussed in detail in both saliva and urine. The theoretical speciation calculations of the main U(VI) species in the investigated body fluids were significantly consistent with the spectroscopic data. Laser fluorescence spectroscopy showed success and reliability for direct determination of U(VI) in such biological matrices with the possibility for further improvement.

  9. Fate tracing of mature hepatocytes in mouse liver homeostasis and regeneration.

    Science.gov (United States)

    Malato, Yann; Naqvi, Syed; Schürmann, Nina; Ng, Raymond; Wang, Bruce; Zape, Joan; Kay, Mark A; Grimm, Dirk; Willenbring, Holger

    2011-12-01

    Recent evidence has contradicted the prevailing view that homeostasis and regeneration of the adult liver are mediated by self duplication of lineage-restricted hepatocytes and biliary epithelial cells. These new data suggest that liver progenitor cells do not function solely as a backup system in chronic liver injury; rather, they also produce hepatocytes after acute injury and are in fact the main source of new hepatocytes during normal hepatocyte turnover. In addition, other evidence suggests that hepatocytes are capable of lineage conversion, acting as precursors of biliary epithelial cells during biliary injury. To test these concepts, we generated a hepatocyte fate-tracing model based on timed and specific Cre recombinase expression and marker gene activation in all hepatocytes of adult Rosa26 reporter mice with an adenoassociated viral vector. We found that newly formed hepatocytes derived from preexisting hepatocytes in the normal liver and that liver progenitor cells contributed minimally to acute hepatocyte regeneration. Further, we found no evidence that biliary injury induced conversion of hepatocytes into biliary epithelial cells. These results therefore restore the previously prevailing paradigms of liver homeostasis and regeneration. In addition, our new vector system will be a valuable tool for timed, efficient, and specific loop out of floxed sequences in hepatocytes.

  10. Apatite-forming ability of polymers with carboxy groups in simulated body fluid

    Energy Technology Data Exchange (ETDEWEB)

    Kawashita, M.; Nakao, M.; Kim, H.M.; Kokubo, T. [Kyoto Univ. (Japan). Dept. of Material Chemistry; Minoda, M. [Kyoto Inst. of Tech. (Japan). Dept. of Chemistry and Materials Technology; Miyamoto, T. [Matsue National Coll. of Technology (Japan); Nakamura, T. [Kyoto Univ. (Japan). Dept. of Orthopaedic Surgery

    2001-07-01

    Apatite-polymer composites with analogous structure to that of the natural bone are desired to be developed, since such composites are believed to show biological and mechanical properties similar to those of the natural bone. In the present study, apatite-forming ability of various kinds of polymers with or without carboxy (COOH) groups in simulated body fluid (SBF) was investigated. Carboxymethyl- (CM)- chitin and gellan gum gels, which have COOH groups, formed apatite on their surfaces within 3 d, when they were previously treated with saturated Ca(OH){sub 2} solution. Calcium alginate gel with COOH groups formed apatite on its surface within 7 d without the Ca(OH){sub 2}-treatment, since calcium ions are previously incorporated into the gel structure in the gel-forming process. In contrast, curdlan gel without COOH groups did not form that the COOH groups on a polymer surface are effective for the apatite nucleation and the apatite-forming ability of the polymer can be improved by incorporation of the calcium ions. In conclusion, these types of polymers are promising candidates for obtaining apatite-polymer composites with bonelike structure by a biomimetic process. (orig.)

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

    DEFF Research Database (Denmark)

    Wrzesinski, Krzysztof; Fey, S. J.

    2011-01-01

    hepatocytes using the classical approaches (in “2D”) and using a system which leads to the generation of spheroids of cells held in suspension (“3D”). Both approaches gave rise to cultures where the large majority of cells were viable, produced similar amounts of ATP, incorporated similar amounts......It is widely expected that cells grown in 3D environments (in suspension, on scaffolds etc.) will be superior to growing cells in classical 2D culture flasks. These expectations include the belief that cells grown in 3D culture will possess physiological characteristics that resemble more closely...... 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...

  12. Calculating two- and three-body decays with FeynArts and FormCalc

    Energy Technology Data Exchange (ETDEWEB)

    Klasen, M. [Hamburg Univ. (Germany). 2. Inst. fuer Theoretische Physik

    2002-10-01

    The Feynman diagram generator FeynArts and the computer algebra program FormCalc allow for an automatic computation of 2{yields}2 and 2{yields}3 scattering processes in High Energy Physics. We have extended this package by four new kinematical routines and adapted one existing routine in order to accomodate also two- and three-body decays of massive particles. This makes it possible to compute automatically tow- and three-body particle decay widths and decay energy distributions as well as resonant particle production within the Standard Model and the Minimal Supersymmetric Standard Model at the tree- and loop-level. The use of the program is illustrated with three standard examples: h{yields}bb, {mu} {yields} e{nu}{sub e}{nu}{sub {mu}}, and Z{yields} {nu}{sub e}{nu}{sub e}. (orig.)

  13. Hepatocyte differentiation of mesenchymalstemcells

    Institute of Scientific and Technical Information of China (English)

    Xu-Bo Wu; Ran Tao

    2012-01-01

    BACKGROUND: Liver cell transplantation and bioartiifcial liver may provide metabolic support of liver function temporary and are prospective treatments for patients with liver failure. Mesenchymal stem cells (MSCs) are expected to be an ideal cell source for transplantation or liver tissue engineering, however the hepatic differentiation of MSCs is still insufifcient for clinical application. DATA  SOURCES: A PubMed search on "mesenchymal stem cells","liver cell"and"hepatocyte differentiation"was performed on the topic, and the relevant articles published in the past ten years were reviewed. RESULTS:Hepatocyte-like cells differentiated from MSCs are a promising cell source for liver regeneration or tissue engineering. Although it is still a matter of debate as to whether MSC-derived hepatocytes may efifciently repopulate a host liver to provide adequate functional substitution, the majority of animal studies support that MSCs can become key players in liver-directed regenerative medicine. However the clinical application of human stem cells in the treatment of liver diseases is still in its infancy. CONCLUSIONS: Future studies are required to improve the efifcacy and consistency of hepatic differentiation from MSCs. It is necessary to better understand the mechanism to achieve transdifferentiation with high efifciency. More clinical trials are warranted to prove their efifcacy in the management of patients with liver failure.

  14. In Vivo Osteogenic Differentiation of Human Embryoid Bodies in an Injectable in Situ-Forming Hydrogel

    Directory of Open Access Journals (Sweden)

    Moon Suk Kim

    2013-07-01

    Full Text Available In this study, we examined the in vivo osteogenic differentiation of human embryoid bodies (hEBs by using an injectable in situ-forming hydrogel. A solution containing MPEG-b-(polycaprolactone-ran-polylactide (MCL and hEBs was easily prepared at room temperature. The MCL solution with hEBs and osteogenic factors was injected into nude mice and developed into in situ-forming hydrogels at the injection sites; these hydrogels maintained their shape even after 12 weeks in vivo, thereby indicating that the in situ-forming MCL hydrogel was a suitable scaffold for hEBs. The in vivo osteogenic differentiation was observed only in the in situ gel-forming MCL hydrogel in the presence of hEBs and osteogenic factors. In conclusion, this preliminary study suggests that hEBs and osteogenic factors embedded in an in situ-forming MCL hydrogel may provide numerous benefits as a noninvasive alternative for allogeneic tissue engineering applications.

  15. Mass preparation of primary porcine hepatocytes and the design of a hybrid artificial liver module using spheroid culture for a clinical trial.

    Science.gov (United States)

    Fukuda, J; Sakiyama, R; Nakazawa, K; Ijima, H; Yamashita, Y; Shimada, M; Shirabe, K; Tsujita, E; Sugimachi, K; Funatsu, K

    2001-11-01

    To isolate a large number of porcine hepatocytes, we originally developed a mass preparation method that combined the usual collagenase perfusion method of a whole liver with a collagenase redigestion method of tissue fragments after liver perfusion. Using a pig of 10kg, collagenase perfusion only resulted in a yield of 63+/-78 x 10(8) total cells with a viability of 69.2+/-25.3 %, but our combined method had a yield of 167+/-31 x 10(8) total cells with a viability of 87.9+/-4.4% (mean +/- SD). Also, the combined method was applied to two pigs of 10kg body weight at the same time, and isolated 387+/-89 x 10(8) hepatocytes with a viability of 87.1+/-6.9% and a purity of 93.6+/-2.8 % in 11 experiments. We designed a large multi-capillary polyurethane foam (MC-PUF) packed-bed module containing 1 x 10(10) porcine hepatocytes on a clinical trial scale. The porcine hepatocytes in the module formed spherical multicellular aggregates (spheroids) of 200 - 500 microm diameter. Most hepatocytes forming spheroids were viable judged by fluorescein diacetate and ethidium bromide staining. The activities of ammonia removal, albumin secretion and oxygen consumption of the large MC-PUF module were the same as for a small MC-PUF module containing 2 x 10(8) porcine hepatocytes, and were maintained for at least 9 days of culture. These results show that a large MC-PUF module is successfully scaled up 50 times. In conclusion, we succeeded in developing a mass preparation method of porcine hepatocytes and a large hybrid artificial liver module on a clinical trial scale.

  16. Culture of cryopreserved rat hepatocyte

    Institute of Scientific and Technical Information of China (English)

    Haitao Yin; Gaojun Teng; Lifeng Wang; Baorui Liu; Xiaoping Qian

    2006-01-01

    Objective: To study the method of cryopreserving rat hepatocytes and double collagen gel culture measurement after its cryopreservation. Methods: Rat hepatocytes, isolated by two-step perfusion with collagenase using an extra corporeal perfusion apparatus, were cryopreserved in double collagen gel with culture medium added by epidermal growth factor(EGF).The expression of cell function and cellular morphology were examined during culture. Results: The hepatocytes cryopreserved in double collagen gel concluding EGF showed good morphology and biological characteristics. After thawing, the MTT metabolism and protein synthesis of hepatocytes in sandwich ± EGF groups were better than those in control group. And the morphology and function of hepatocytes in sandwich group was better than that in EGF group(P < 0.05). Conclusion: Double collagen gel culture can keep hepatocyte's activities. Thawed hepatocytes can be cultivated with collagenous matrix, which provides an environment that more closely resembles that in vivo and maintain the expression of certain liver-specific function of hepatocytes.

  17. Quantifying Model Form Uncertainty in RANS Simulation of Wing-Body Junction Flow

    CERN Document Server

    Wu, Jin-Long; Xiao, Heng

    2016-01-01

    Wing-body junction flows occur when a boundary layer encounters an airfoil mounted on the surface. The corner flow near the trailing edge is challenging for the linear eddy viscosity Reynolds Averaged Navier-Stokes (RANS) models, due to the interaction of two perpendicular boundary layers which leads to highly anisotropic Reynolds stress at the near wall region. Recently, Xiao et al. proposed a physics-informed Bayesian framework to quantify and reduce the model-form uncertainties in RANS simulations by utilizing sparse observation data. In this work, we extend this framework to incorporate the use of wall function in RANS simulations, and apply the extended framework to the RANS simulation of wing-body junction flow. Standard RANS simulations are performed on a 3:2 elliptic nose and NACA0020 tail cylinder joined at their maximum thickness location. Current results show that both the posterior mean velocity and the Reynolds stress anisotropy show better agreement with the experimental data at the corner regio...

  18. In vivo localization of antibodies raised against Eimeria maxima wall forming bodies during sexual intracellular development.

    Science.gov (United States)

    Frölich, Sonja; Shahparee, Annisha; Wasinger, Valerie C; Wallach, Michael

    2014-11-01

    SUMMARY Apicomplexan parasites cause devastating diseases in humans and livestock. Previously we demonstrated that antibodies targeting transmissible forms of the apicomplexan parasite, Eimeria, are effective at reducing parasite shedding thus preventing the transmission of the disease. However, the mechanisms responsible have not been fully defined. Moreover, there is no direct evidence that the parasite-specific IgG antibodies can reach the parasite developing in the enterocytes of the infected chicken host. This study summarizes our efforts using host immunity, parasite proteomics and 3D microscopy to provide a step forward in our understanding of how this immune response works. Eimeria maxima is an important pathogen of poultry and used as a surrogate for a number of human pathogens including Toxoplasma and Plasmodium. Our studies demonstrate that immunization with the purified wall forming bodies (WFBs) results in a production of parasite-specific IgG antibodies, which have the ability to reach in situ gametocytes in the intestinal lumen and permeate the enterocyte/parasite membranes in order to bind to the cytoplasmic Type 1 and Type 2 WFBs. This raises the intriguing possibility that via this process antibodies block the development of Eimeria maxima in vivo.

  19. Study on radioprotection of alliin and damage mechanism in hepatocyte after irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Ji, Tae Jeong; Kim, Won Tae [Dept, of Radiological Science, Kaya University, Kimhae (Korea, Republic of)

    2016-12-15

    Liver tissue damage by a radiation exposure caused a jaundice and ascitic fluid e form harden atrophy. The reason for this lies in morphological damage of a liver cells. This study tried that observe damage mechanism of the cell organelles. It was especially observed mitochondria, endoplasmic reticulum and nuclear membrane associated with energy metabolizable. also, This study had with a radio-protector development research at the same time. Radio-protector was used to alliin that has an blood flow increase. Cell observation make used of transmission electron microscope(TEM). The result of an experiment, 7Gy of whole body irradiation was caused an inflammation in cell organelles and hypertrophy of the nucleus membrane. After 20 days, The hepatocyte has been observed in a damaged membrane on peroxisome, mitochondria and vacuole of the cell organelles. After 30 days, The hepatocyte has been observed in disconnected ribosomes on a rough endoplasmic reticulum. There was looked a giant lipoblast. There was clearly normal observed a mitochondria and nucleus membrane in the hepatocyte after alliin injection. aslo, It was no damaged the nucleus membrane. Therefore, It was identified portion a radio-protector effect from alliin.

  20. Evidence for Distinct Contributions of Form and Motion Information to the Recognition of Emotions from Body Gestures

    Science.gov (United States)

    Atkinson, Anthony P.; Tunstall, Mary L.; Dittrich, Winand H.

    2007-01-01

    The importance of kinematics in emotion perception from body movement has been widely demonstrated. Evidence also suggests that the perception of biological motion relies to some extent on information about spatial and spatiotemporal form, yet the contribution of such form-related cues to emotion perception remains unclear. This study reports, for…

  1. [Mortality among able-bodied population in industrial cities in accordance with specific enterprise forming a company city].

    Science.gov (United States)

    Tikhonova, G I; Gorchakova, T Iu; Churanova, A N

    2013-01-01

    The article covers comparative analysis of mortality causes and levels among male able-bodied population in small and medium industrial cities of Murmansk region in accordance with specific enterprise forming a company city. Findings are that, if compared to Murmansk having no enterprise forming a company, other industrial cities in the region, situated in the same climate area, demonstrated higher levels of mortality among the male able-bodied population with the death causes associated etiologically to occupational hazards on the enterprises forming a company city.

  2. Wave-free floating body forms for a shallow sea area; Senkaiiki no naminashi futai keijo ni tsuite

    Energy Technology Data Exchange (ETDEWEB)

    Kyozuka, Y.; Nariai, Y. [Kyushu University, Fukuoka (Japan)

    1997-10-01

    In column footing or semi-submergible type marine structures, a vertical wave force vanishes at a specific period of waves. This phenomenon is called wave-free characteristics. This wave-free characteristics make it possible to design marine structures superior in oscillation performance in waves. Since Bessho`s wave-free theory is useful only for an infinite water depth, this paper studied the wave-free theory for a shallow sea area. On a wave-free singularity and required floating body form, 2-D and 3-D axisymmetric floating body forms were determined, and vertical wave force characteristics of the obtained body forms were calculated and verified experimentally. Since the source term of the wave-free singularity was weaker in a shallow sea area than an infinite deep water area, resulting in the narrow width of the obtained wave-free body forms in a shallow sea area. The wave-free theory for a shallow sea area was verified by both numerical calculation based on a singularity distribution method and model experiment for these floating body forms. 3 refs., 10 figs.

  3. Analysis tools for precision studies of hadronic three-body decays and transition form factors

    Energy Technology Data Exchange (ETDEWEB)

    Schneider, Sebastian Philipp

    2013-02-14

    Due to the running coupling constant of Quantum Chromodynamics one of the pillars of the Standard Model, the strong interactions, is still insufficiently understood at low energies. In order to describe the interactions of hadrons that form in this physical regime, one has to devise methods that are non-perturbative in the strong coupling constant. In particular hadronic three-body decays and transition form factors present a great challenge due to the complex analytic structure ensued by strong final-state interactions. In this thesis we present two approaches to tackle these processes. In the first part we use a modified version of non-relativistic effective field theory to analyze the decay {eta}{yields}3{pi}. This perturbative low-energy expansion is ideally suited to study the effects of {pi}{pi} rescattering and contributes greatly to the understanding of the slope parameter of the {eta}{yields}3{pi}{sup 0} Dalitz plot, a quantity that is strongly influenced by final-state interactions and has presented a long-standing puzzle for theoretical approaches. In the second part we present dispersion relations as a non-perturbative means to study three-particle decays. Using the example of {eta}'{yields}{eta}{pi}{pi} we give a detailed introduction to the framework and its numerical implementation. We confront our findings with recent experimental data from the BES-III and VES collaborations and discuss whether the extraction of {pi}{eta} scattering parameters, one of the prime motives to study this decay channel, is feasible in such an approach. A more clear-cut application is given in our study of the decays {omega}/{phi}{yields}3{pi} due to the relative simplicity of this decay channel: our results are solely dependent on the {pi}{pi} P-wave scattering phase shift. We give predictions for the Dalitz plot distributions and compare our findings to very precise data on {phi}{yields}3{pi} by the KLOE and CMD-2 collaborations. We also predict Dalitz plot

  4. Current status of hepatocyte xenotransplantation.

    Science.gov (United States)

    Meier, Raphael P H; Navarro-Alvarez, Nalu; Morel, Philippe; Schuurman, Henk-Jan; Strom, Stephen; Bühler, Leo H

    2015-11-01

    The treatment of acute liver failure, a condition with high mortality, comprises optimal clinical care, and in severe cases liver transplantation. However, there are limitations in availability of organ donors. Hepatocyte transplantation is a promising alternative that could fill the medical need, in particular as the bridge to liver transplantation. Encapsulated porcine hepatocytes represent an unlimited source that could function as a bioreactor requiring minimal immunosuppression. Besides patients with acute liver failure, patients with alcoholic hepatitis who are unresponsive to a short course of corticosteroids are a target for hepatocyte transplantation. In this review we present an overview of the innate immune barriers in hepatocyte xenotransplantation, including the role of complement and natural antibodies; the role of phagocytic cells and ligands like CD47 in the regulation of phagocytic cells; and the role of Natural Killer cells. We present also some illustrations of physiological species incompatibilities in hepatocyte xenotransplantation, such as incompatibilities in the coagulation system. An overview of the methodology for cell microencapsulation is presented, followed by proof-of-concept studies in rodent and nonhuman primate models of fulminant liver failure: these studies document the efficacy of microencapsulated porcine hepatocytes which warrants progress towards clinical application. Lastly, we present an outline of a provisional clinical trial, that upon completion of preclinical work could start within the upcoming 2-3 years.

  5. CHARACTERISTICS OF BODY POSTURE IN THE SAGITTAL PLANE AND FITNESS OF FIRST-FORM PUPILS FROM RURAL AREAS

    OpenAIRE

    Żukowska Hanna; Szark-Eckardt Mirosława; Muszkieta Radosław; Iermakova T.S.

    2014-01-01

    Purpose: to find correlations between characteristics of body posture in the sagittal plane and fitness and endurance of first-form children from rural areas. Material: an analysis of more than 30 sources of scientific and educational literature. Results: the study involved 209 children, including 102 girls and 107 boys. They were children who lived in the country since they were born. To assess particular characteristics of body posture, the children were studied by means of the measuring eq...

  6. TUNING IN TO FISH SWIMMING WAVES - BODY FORM, SWIMMING MODE AND MUSCLE FUNCTION

    NARCIS (Netherlands)

    WARDLE, CS; VIDELER, JJ; ALTRINGHAM, JD

    1995-01-01

    Most fish species swim with lateral body undulations running from head to tail, These waves run more slowly than the waves of muscle activation causing them, reflecting the effect of the interaction between the fish's body and the reactive forces from the water, The coupling between both waves depen

  7. TUNING IN TO FISH SWIMMING WAVES - BODY FORM, SWIMMING MODE AND MUSCLE FUNCTION

    NARCIS (Netherlands)

    WARDLE, CS; VIDELER, JJ; ALTRINGHAM, JD

    1995-01-01

    Most fish species swim with lateral body undulations running from head to tail, These waves run more slowly than the waves of muscle activation causing them, reflecting the effect of the interaction between the fish's body and the reactive forces from the water, The coupling between both waves depen

  8. TUNING IN TO FISH SWIMMING WAVES - BODY FORM, SWIMMING MODE AND MUSCLE FUNCTION

    NARCIS (Netherlands)

    WARDLE, CS; VIDELER, JJ; ALTRINGHAM, JD

    Most fish species swim with lateral body undulations running from head to tail, These waves run more slowly than the waves of muscle activation causing them, reflecting the effect of the interaction between the fish's body and the reactive forces from the water, The coupling between both waves

  9. Maternal Body-Mass Index and Cord Blood Circulating Endothelial Colony-Forming Cells

    Science.gov (United States)

    Lin, Ruei-Zeng; Miranda, Maria L.; Vallejo-Vaz, Antonio J.; Stiefel, Pablo; Praena-Fernández, Juan M.; Bernal-Bermejo, Jose; Jimenez-Jimenez, Luis M.; Villar, Jose; Melero-Martin, Juan M.

    2013-01-01

    Objective Endothelial colony-forming cells (ECFCs) are a subset of circulating endothelial progenitor cells that are particularly abundant in umbilical cord blood. We sought to determine whether ECFC abundance in cord blood is associated with maternal body-mass index (BMI) in non-pathological pregnancies. Study design We measured the level of ECFCs in the cord blood of neonates (n=27) born from non-obese healthy mothers with non-pathological pregnancies and examined whether ECFC abundance correlated with maternal BMI. We also examined the effect of maternal BMI on ECFC phenotype and function using angiogenic and vasculogenic assays. Results We observed variation in ECFC abundance among subjects and found a positive correlation between pre-pregnancy maternal BMI and ECFC content (r=0.51, P=0.007), which was independent of other obstetric factors. Despite this variation, ECFC phenotype and functionality were deemed normal and highly similar between subjects with maternal BMI <25 kg/m2 and BMI between 25–30 kg/m2, including the ability to form vascular networks in vivo. Conclusions This study underlines the need to consider maternal BMI as a potential confounding factor for cord blood levels of ECFCs in future comparative studies between healthy and pathological pregnancies. Endothelial colony-forming cells (ECFCs) are a subset of progenitor cells that circulate in peripheral blood and can give rise to endothelial cells (1,2), contributing to the formation of new vasculature and the maintenance of vascular integrity (3–5). The mechanisms that regulate the abundance of these cells in vivo remain poorly understood. ECFCs are rare in adult peripheral blood (1,2,10). In contrast, there is an elevated number of these cells in fetal blood during the third trimester of pregnancy (11–13). Emerging evidence indicates that deleterious conditions during fetal life can impair ECFC content and function. For instance, offspring of diabetic mothers have been shown to have

  10. Effects of the physical form of the diet on food intake, growth, and body composition changes in mice.

    Science.gov (United States)

    Yan, Lin; Combs, Gerald F; DeMars, Lana C; Johnson, LuAnn K

    2011-07-01

    The present study investigated effects of the physical form of the diet on food intake, growth, and body composition in male C57BL/6 mice. Three-week-old mice were fed isocaloric diets (AIN93G or a modification containing 25% wheat) in powdered or pelleted form. In experiment 1, mice were assigned into 4 groups offered the AIN93G or the wheat-modified diet in powdered or pelleted form. In experiment 2, mice were pair-fed the powdered diets to the ad libitum level of food intake of those fed the pelleted form of the respective diets. Body weight, food intake, and fecal excretion were recorded, and body composition was assessed on mice 1 wk before termination of the experiment. Mice fed the powdered diets showed greater increases in body weight in 2 wk of feeding than did mice fed the pelleted diets. Compared with the pelleted diets, the powdered diets supported an approximately 85% increase in the fat-mass:body-mass ratio and a 2-fold increase in the abdominal-fat-weight:carcass-weight ratio. In addition, mice fed the powdered diet showed significantly greater plasma concentrations of insulin and leptin and significantly lower plasma adiponectin, compared with their pellet-fed counterparts. Food intake of mice fed the powdered diet was 11% greater for the AIN93G and 16% greater for the wheat diet compared with that of the respective pelleted diet. These results demonstrate that C57BL/6 mice responded to the physical form of these diets in terms of food intake, which affected their growth, body composition, and plasma concentrations of insulin and adipocytokines.

  11. Hepatocytes isolated from neoplastic liver-immunomagnetic purging as a new source for transplantation

    Institute of Scientific and Technical Information of China (English)

    Aravin Gunasegaram; Javed Akhter; Peng Yao; Loreena A Johnson; Stephen M Riodan; David L Morris

    2008-01-01

    AIM: To investigate whether hepatocytes isolated from macroscopically normal liver during hepatic resection for neoplasia could provide a novel source of healthy hepatocytes, including the development of reliable protocols for malignant cells removal from the hepatocyte preparation.METHODS: Hepatocytes were procured from resected liver of 18 patients with liver tumors using optimised digestion and cell-enrichment protocols. Suspensions of various known quantities of the HT-29 tumor cell line and patient hepatocytes were treated or not with Ep-CAM-antibody-coated immunomagnetic beads in order to investigate the efficacy of tumor-purging by immunomagnetic depletion, using a semi-quantitative RT-PCR method developed to detect tumor cells.Immunomagnetic bead-treated or bead-untreated tumor cell-hepatocyte suspensions were transplanted intra-peritoneally in Balb/C nude mice to assess the rates of tumor development.RESULTS: Mean viable hepatocyte yield was 9.3 x 106 cells per gram of digested liver with mean viability of 70.5%. Immunomagnetic depletion removed tumor cells to below the RT-PCR detection-threshold of 1 tumor cell in 106 hepatocytes, representing a maximum tumor purging efficacy of greater than 400000-fold.Transplanted, immunomagnetic bead-purged tumor cell-hepatocyte suspensions did not form peritoneal tumors in Balb/C nude mice. Co-transplantation of hepatocytes with tumor cells did not increase tumorigenesis of the tumor cells.CONCLUSION: Immunomagnetic depletion appears to be an effective method of purging contaminating tumor cells to below threshold for likely tumorigenesis.Along with improved techniques for isolation of large numbers of viable hepatocytes, normal liver resected for neoplasia has potential as another clinically useful source of hepatocytes for transplantation.

  12. Genetic abolishment of hepatocyte proliferation activates hepatic stem cells.

    Directory of Open Access Journals (Sweden)

    Yoko Endo

    Full Text Available Quiescent hepatic stem cells (HSCs can be activated when hepatocyte proliferation is compromised. Chemical injury rodent models have been widely used to study the localization, biomarkers, and signaling pathways in HSCs, but these models usually exhibit severe promiscuous toxicity and fail to distinguish damaged and non-damaged cells. Our goal is to establish new animal models to overcome these limitations, thereby providing new insights into HSC biology and application. We generated mutant mice with constitutive or inducible deletion of Damaged DNA Binding protein 1 (DDB1, an E3 ubiquitin ligase, in hepatocytes. We characterized the molecular mechanism underlying the compensatory activation and the properties of oval cells (OCs by methods of mouse genetics, immuno-staining, cell transplantation and gene expression profiling. We show that deletion of DDB1 abolishes self-renewal capacity of mouse hepatocytes in vivo, leading to compensatory activation and proliferation of DDB1-expressing OCs. Partially restoring proliferation of DDB1-deficient hepatocytes by ablation of p21, a substrate of DDB1 E3 ligase, alleviates OC proliferation. Purified OCs express both hepatocyte and cholangiocyte markers, form colonies in vitro, and differentiate to hepatocytes after transplantation. Importantly, the DDB1 mutant mice exhibit very minor liver damage, compared to a chemical injury model. Microarray analysis reveals several previously unrecognized markers, including Reelin, enriched in oval cells. Here we report a genetic model in which irreversible inhibition of hepatocyte duplication results in HSC-driven liver regeneration. The DDB1 mutant mice can be broadly applied to studies of HSC differentiation, HSC niche and HSCs as origin of liver cancer.

  13. Why does a trait evolve multiple times within a clade? Repeated evolution of snakelike body form in squamate reptiles.

    Science.gov (United States)

    Wiens, John J; Brandley, Matthew C; Reeder, Tod W

    2006-01-01

    Why does a trait evolve repeatedly within a clade? When examining the evolution of a trait, evolutionary biologists typically focus on the selective advantages it may confer and the genetic and developmental mechanisms that allow it to vary. Although these factors may be necessary to explain why a trait evolves in a particular instance, they may not be sufficient to explain phylogenetic patterns of repeated evolution or conservatism. Instead, other factors may also be important, such as biogeography and competitive interactions. In squamate reptiles (lizards and snakes) a dramatic transition in body form has occurred repeatedly, from a fully limbed, lizardlike body form to a limb-reduced, elongate, snakelike body form. We analyze this trait in a phylogenetic and biogeographic context to address why this transition occurred so frequently. We included 261 species for which morphometric data and molecular phylogenetic information were available. Among the included species, snakelike body form has evolved about 25 times. Most lineages of snakelike squamates belong to one of two "ecomorphs," either short-tailed burrowers or long-tailed surface dwellers. The repeated origins of snakelike squamates appear to be associated with the in situ evolution of these two ecomorphs on different continental regions (including multiple origins of the burrowing morph within most continents), with very little dispersal of most limb-reduced lineages between continental regions. Overall, the number of repeated origins of snakelike morphology seems to depend on large-scale biogeographic patterns and community ecology, in addition to more traditional explanations (e.g., selection, development).

  14. Effects of the Physical Form of the Diet on Food Intake, Growth, and Body Composition Changes in Mice

    OpenAIRE

    Yan, Lin; Combs, Gerald F.; Lana C DeMars; LuAnn K. Johnson

    2011-01-01

    The present study investigated effects of the physical form of the diet on food intake, growth, and body composition in male C57BL/6 mice. Three-week-old mice were fed isocaloric diets (AIN93G or a modification containing 25% wheat) in powdered or pelleted form. In experiment 1, mice were assigned into 4 groups offered the AIN93G or the wheat-modified diet in powdered or pelleted form. In experiment 2, mice were pair-fed the powdered diets to the ad libitum level of food intake of those fed t...

  15. Molecular cytotoxic mechanisms of chlorpromazine in isolated rat hepatocytes.

    Science.gov (United States)

    MacAllister, Stephanie L; Young, Cheryl; Guzdek, Anna; Zhidkov, Nickholas; O'Brien, Peter J

    2013-01-01

    Chlorpromazine (CPZ), a member of the largest class of first-generation antipsychotic agents, is known to cause hepatotoxicity in the form of cholestasis and hepatocellular necrosis in some patients. The mechanism of CPZ hepatotoxicity is unclear, but is thought to result from reactive metabolite formation. The goal of this research was to assess potential cytotoxic mechanisms of CPZ using the accelerated cytotoxicity mechanism screening (ACMS) technique with freshly isolated rat hepatocytes. This study identified CPZ cytotoxicity and inhibition of mitochondrial membrane potential (MMP) to be concentration-dependent. Furthermore, inhibition of cytochrome P450s (CYPs), including CYP2D1 and 1A2, delayed CPZ cytotoxicity, suggesting a role for CYP activation of CPZ to a toxic metabolite(s) in this model. Metabolism studies also demonstrated glucuronide and glutathione (GSH) requirement for CPZ detoxification in hepatocytes. Inactivating the 2-electron reduction pathway, NAD(P)H quinone oxidoreductase (NQO1), caused a significant increase in hepatocyte susceptibility to CPZ, indicating quinoneimine contribution to CPZ cytotoxicity. Nontoxic concentrations of peroxidase/H(2)O(2) (inflammatory model) increased cytotoxicity in CPZ-treated hepatocytes and caused additional mitochondrial toxicity. Inflammation further depleted GSH and increased oxidized glutathione (GSSG) levels. Results suggest activation of CPZ to reactive metabolites by 2 pathways in hepatocytes: (i) a CYP-catalyzed quinoneimine pathway, and (ii) a peroxidase-catalyzed oxidation of CPZ to CPZ radicals.

  16. Morphology and function of isolated hepatocytes transplanted into rat spleen.

    Science.gov (United States)

    Mito, M; Ebata, H; Kusano, M; Onishi, T; Saito, T; Sakamoto, S

    1979-12-01

    Hepatocytes isolated by the collagenase digestive method were transplanted into the spleens of syngeneic rats. Morphology and function of the hepatocytes in the spleen were investigated for 12 to 17 months after transplantation. The transplanted hepatocytes proliferated and reconfigured in the spleen without direct perfusion of portal venous blood and with the presence of an intact host liver. Fourteen to 17 months after transplantation, the hepatocytes which had formed a demarcated nodule occupied approximately 40% of the area of the splenic parenchyma without undifferentiation on microscopic examination. However, the weight of the hepatized spleen did not increase beyond the weight of a normal spleen and the weight of the host liver that had normal morphology also did not differ from a normal liver. Light and electron microscopic studies demonstrated differentiated cord structure and normal architecture for each heptocyte. Furthermore, the hepatized spleen synthesized albumin and glycogen as demonstrated by immunofluorescence and histochemical studies. Ammonia tolerance and indocyanine green clearance tests revealed functioning hepatocytes in the spleen proper. These results indicate that our experimental model lends itself well to investigations in cell growth mechanism and that hepatocellular transplantation has potential clinical application to compensate for impaired hepatic function.

  17. An unusual pattern of tattooing in the form of body designing: A case report

    Directory of Open Access Journals (Sweden)

    Laxman Gangadhar Phad

    2016-12-01

    In the present case the patient was brought to our hospital for eliciting consumption of alcohol and any other injuries over the body. The person examined had decorated his left arm and forearm by fine designs by a blue ball point pen. He also had multiple fresh incised cuts on the forearms and wrist. Such a presentation with a somewhat risk taking, masochistic behavior and the chronic nature of drawing designs over the body by a ball point pen was very rare and not noted as per best of our knowledge.

  18. 10-(6'-Plastoquinonyl)decyltriphenylphosphonium (SkQ1) Does Not Increase the Level of Cytochromes P450 in Rat Liver and Human Hepatocyte Cell Culture.

    Science.gov (United States)

    Myasoedova, K N; Silachev, D N; Petrov, A D

    2016-12-01

    Mitochondria-targeted antioxidant SkQ1 did not increase the content of cytochromes P450 in livers of rats that were given SkQ1 in drinking water for 5 days in a dose (2.5 µmol per kg body weight) that exceeded 10 times the SkQ1 therapeutic dose. SkQ1 did not affect the levels of cytochrome P450 forms CYP1A2, CYP2B6, and CYP3A4 in monolayer cultures of freshly isolated human hepatocytes, while specific inducers of these forms (omeprazole, phenobarbital, and rifampicin, respectively) significantly increased expression of the cytochromes P450 under the same conditions. We conclude that therapeutic doses of SkQ1 do not induce cytochromes P450 in liver, and the absence of the inducing effect cannot be explained by poor availability of hepatocytes to SkQ1 in vivo.

  19. Form and performance: body shape and prey-capture success in four drift-feeding minnows

    Science.gov (United States)

    Pedro A. Rincón; Markus Bastir; Gary D. Grossman

    2008-01-01

    Identifying links between morphology and performance for ecologically relevant tasks will help elucidate the relationships between organismal design and fitness. We conducted a laboratory study to quantify the relationship between variation in body shape and prey-capture success in four drift-feeding minnow species. We offered drifting prey to individual fish in a test...

  20. Requirements of glycerol and fatty acid for triglyceride synthesis and ketogenesis by hepatocytes from normal and triiodothyronine-treated rats

    Energy Technology Data Exchange (ETDEWEB)

    Olubadewo, J.O.; Heimberg, M.

    1985-11-15

    Hepatocytes from T3-treated rats synthesized less triglyceride and more ketone bodies from (1-/sup 14/C)oleate at all concentrations from 0-2 mM, than did hepatocytes from euthyroid animals; addition of 1.0 mM glycerol increased triglyceride synthesis and reduced ketogenesis in hepatocytes from T3-treated rats to the rates observed in euthyroid hepatocytes in the absence of added glycerol. Glycerol did not alter triglyceride synthesis, but reduced ketogenesis genesis by euthyroid hepatocytes. It is probable from these and other data that, in the hyperthyroid rat, glycero-3-P, and not fatty acid, is rate limiting for synthesis of triglyceride, and, secondarily for reducing rates of ketogenesis in the hepatocyte.

  1. Role of Nuclear Constitutive Androstane Receptor in Regulation of Hepatocyte Proliferation and Hepatocarcinogenesis.

    Science.gov (United States)

    Kazantseva, Y A; Pustylnyak, Y A; Pustylnyak, V O

    2016-04-01

    Activation of the constitutive androstane receptor (CAR) in hepatocytes occurs as a body adaptation in response to a number of external influences, and its functional activity is primarily related to induction of enzymes detoxifying xenobiotics. However, special attention was recently given to CAR due to the fact that its key role becomes unveiled in various physiological and pathophysiological processes occurring in the liver: gluconeogenesis, metabolism of fatty acids and bilirubin, hormonal regulation, proliferation of hepatocytes, and hepatocarcinogenesis. Here we review the main pathways and mechanisms that elevate hepatocyte proliferative activity related to CAR and whose disturbance may be a pivotal factor in hepatocarcinogenesis.

  2. EFFECT OF THE ORTHOSIPHON STAMINEUS, BENTH ON AMINOPYRINE METABOLISM IN RAT HEPATOCYTES

    Directory of Open Access Journals (Sweden)

    CHIN JIN HAN1

    2007-01-01

    Full Text Available One of the commonly used herbal medicines in Malaysia is Orthosiphon stamineus, Benth (family: Lamiaceae or locally known as Misai Kucing. This experiment was undertaken to evaluate possible interaction of methanol extract of O. stamineus with aminopyrine, a model drug, in different age groups (young, adult and old of Sprague-Dawley (SD female rat hepatocytes. Hepatocytes were prepared by collagenase perfusion technique. Determination of aminopyrine N-demethylase activity was done by measuring formaldehyde formed. From these findings, only normal young female rat hepatocytes in the presence of 0.001 mg/ml of methanol extract of O. stamineus showed significant increase in aminopyrine N-demethylase activity. However, aminopyrine N-demethylase activity was not affected in hepatocytes of normal adult and old female SD rats. In conclusion, exposure of methanol extract of O. stamineus could affect phase I aminopyrine metabolism in normal young female SD rat hepatocytes but this effect was age-dependent.

  3. Metabolism of dicarboxylic acids in rat hepatocytes.

    Science.gov (United States)

    Bergseth, S; Poisson, J P; Bremer, J

    1990-02-06

    [carboxyl-14C]Dodecanedioic acid (DC12) is metabolized in hepatocytes at a rate about two thirds that of [1-14C]palmitate. Shorter dicarboxylates (sebacic (DC10), suberic (DC8), and adipic (DC6) acid) are formed, mainly DC6, less DC8 and only a little DC10. In hepatocytes from clofibrate-treated rats, more polar products account for most of the breakdown products, presumably because the beta-oxidation proceeds all the way to succinate and acetyl-CoA. [carboxyl-14C]Suberic acid (DC8) is oxidized at a rate only one fifth that of dodecanedioic acid. (+)-Decanoylcarnitine inhibits palmitate oxidation but not the oxidation of dodecanedioic acid. At low concentrations of [carboxyl-14C]dodecanedioic acid or of [1-14C]palmitate, acetylsulfanilamide is more efficiently labeled by the former. High concentrations of dodecanedioic acid inhibit palmitate oxidation and the acetylation of sulfanilamide, presumably because their CoA-esters accumulate in the cytosol. These results indicate that medium-chain dicarboxylic acids are beta-oxidized mainly in the peroxisomes.

  4. Mesenchymal stem cells support hepatocyte function in engineered liver grafts.

    Science.gov (United States)

    Kadota, Yoshie; Yagi, Hiroshi; Inomata, Kenta; Matsubara, Kentaro; Hibi, Taizo; Abe, Yuta; Kitago, Minoru; Shinoda, Masahiro; Obara, Hideaki; Itano, Osamu; Kitagawa, Yuko

    2014-01-01

    Recent studies suggest that organ decellularization is a promising approach to facilitate the clinical application of regenerative therapy by providing a platform for organ engineering. This unique strategy uses native matrices to act as a reservoir for the functional cells which may show therapeutic potential when implanted into the body. Appropriate cell sources for artificial livers have been debated for some time. The desired cell type in artificial livers is primary hepatocytes, but in addition, other supportive cells may facilitate this stem cell technology. In this context, the use of mesenchymal stem cells (MSC) is an option meeting the criteria for therapeutic organ engineering. Ideally, supportive cells are required to (1) reduce the hepatic cell mass needed in an engineered liver by enhancing hepatocyte function, (2) modulate hepatic regeneration in a paracrine fashion or by direct contact, and (3) enhance the preservability of parenchymal cells during storage. Here, we describe enhanced hepatic function achieved using a strategy of sequential infusion of cells and illustrate the advantages of co-cultivating bone marrow-derived MSCs with primary hepatocytes in the engineered whole-liver scaffold. These co-recellularized liver scaffolds colonized by MSCs and hepatocytes were transplanted into live animals. After blood flow was established, we show that expression of adhesion molecules and proangiogenic factors was upregulated in the graft.

  5. Discovering multiple transcripts of human hepatocytes using massively parallel signature sequencing (MPSS

    Directory of Open Access Journals (Sweden)

    Li Yi-Xue

    2007-07-01

    Full Text Available Abstract Background The liver is the largest human internal organ – it is composed of multiple cell types and plays a vital role in fulfilling the body's metabolic needs and maintaining homeostasis. Of these cell types the hepatocytes, which account for three-quarters of the liver's volume, perform its main functions. To discover the molecular basis of hepatocyte function, we employed Massively Parallel Signature Sequencing (MPSS to determine the transcriptomic profile of adult human hepatocytes obtained by laser capture microdissection (LCM. Results 10,279 UniGene clusters, representing 7,475 known genes, were detected in human hepatocytes. In addition, 1,819 unique MPSS signatures matching the antisense strand of 1,605 non-redundant UniGene clusters (such as APOC1, APOC2, APOB and APOH were highly expressed in hepatocytes. Conclusion Apart from a large number of protein-coding genes, some of the antisense transcripts expressed in hepatocytes could play important roles in transcriptional interference via a cis-/trans-regulation mechanism. Our result provided a comprehensively transcriptomic atlas of human hepatocytes using MPSS technique, which could be served as an available resource for an in-depth understanding of human liver biology and diseases.

  6. In vitro induction of human embryonic stem cells into hepatocyte-like cells

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Human embryonic stem cells (hES cells) are pluripotent and provide a unique, unlimited resource for human hepatocytes, which can serve as a novel cell source for cell transplantation and bioartificial liver (BAL). Here, we have developed a procedure by which hES cells can differentiate into hepatocyte-like cells. After being cultured in suspension in bacteriological petri dishes for 7 d, hES cells developed into cystic embryoid bodies (EBs). The EBs were then cultured in conditional medium containing dexamethasone and insulin in collagen type I-coated tissue culture dishes for two weeks. The hES cell-derived hepatocyte-like cells (HLCs) displayed some morphologic characteristics of hepatocytes. Reverse-transcription polymerase chain reaction (RT-PCR) and immunofluorescence cell staining proved that the induced HLCs expressed several hepatocyte specific genes including AFP, ALB, CYP1B1 and cytokeratins CK18 and CK19. Furthermore, the induced cells executed a range of hepatocyte functions, such as ICG uptake/excretion, glycogen deposits, albumin production and ammonium metabolism. Taken together, our results show that HLCs exhibit similar morphologic, phenotypic, and functional characteristics to hepatocytes.

  7. Hepatocytes: critical for glucose homeostasis.

    Science.gov (United States)

    Klover, Peter J; Mooney, Robert A

    2004-05-01

    Maintaining blood glucose levels within a narrow range is a critical physiological function requiring multiple metabolic pathways and involving several cell types, including a prominent role for hepatocytes. Under hormonal control, hepatocytes can respond to either feeding or fasting conditions by storing or producing glucose as necessary. In the fasting state, the effects of glucagon avoid hypoglycemia by stimulating glucogenesis and glycogenolysis and initiating hepatic glucose release. Postprandially, insulin prevents hyperglycemia, in part, by suppressing hepatic gluconeogenesis and glycogenolysis and facilitating hepatic glycogen synthesis. Both transcriptional regulation of rate limiting enzymes and modulation of enzyme activity through phosphorylation and allosteric regulation are involved. Type 2 diabetes mellitus is the most common serious metabolic condition in the world, and results from a subnormal response of tissues to insulin (insulin resistance) and a failure of the insulin-secreting beta cells to compensate. In type 2 diabetes, glucose is overproduced by the hepatocyte and is ineffectively metabolized by other organs. Impairments in the insulin signal transduction pathway appear to be critical lesions contributing to insulin resistance and type 2 diabetes.

  8. Strategies for immortalization of primary hepatocytes

    Science.gov (United States)

    Eva, Ramboer; Bram, De Craene; Joery, De Kock; Tamara, Vanhaecke; Geert, Berx; Vera, Rogiers; Mathieu, Vinken

    2014-01-01

    The liver has the unique capacity to regenerate in response to a damaging event. Liver regeneration is hereby largely driven by hepatocyte proliferation, which in turn relies on cell cycling. The hepatocyte cell cycle is a complex process that is tightly regulated by several well-established mechanisms. In vitro, isolated hepatocytes do not longer retain this proliferative capacity. However, in vitro cell growth can be boosted by immortalization of hepatocytes. Well-defined immortalization genes can be artificially overexpressed in hepatocytes or the cells can be conditionally immortalized leading to controlled cell proliferation. This paper discusses the current immortalization techniques and provides a state-of-the-art overview of the actually available immortalized hepatocyte-derived cell lines and their applications. PMID:24911463

  9. Asialoglycoprotein receptor mediated hepatocyte targeting - strategies and applications.

    Science.gov (United States)

    D'Souza, Anisha A; Devarajan, Padma V

    2015-04-10

    Hepatocyte resident afflictions continue to affect the human population unabated. The asialoglycoprotein receptor (ASGPR) is primarily expressed on hepatocytes and minimally on extra-hepatic cells. This makes it specifically attractive for receptor-mediated drug delivery with minimum concerns of toxicity. ASGPR facilitates internalization by clathrin-mediated endocytosis and exhibits high affinity for carbohydrates specifically galactose, N-acetylgalactosamine and glucose. Isomeric forms of sugar, galactose density and branching, spatial geometry and galactose linkages are key factors influencing ligand-receptor binding. Popular ligands for ASGPR mediated targeting are carbohydrate polymers, arabinogalactan and pullulan. Other ligands include galactose-bearing glycoproteins, glycopeptides and galactose modified polymers and lipids. Drug-ligand conjugates provide a viable strategy; nevertheless ligand-anchored nanocarriers provide an attractive option for ASGPR targeted delivery and are widely explored. The present review details various ligands and nanocarriers exploited for ASGPR mediated delivery of drugs to hepatocytes. Nanocarrier properties affecting ASGPR mediated uptake are discussed at length. The review also highlights the clinical relevance of ASGPR mediated targeting and applications in diagnostics. ASGPR mediated hepatocyte targeting provides great promise for improved therapy of hepatic afflictions.

  10. Pluripotent stem cells isolated from umbilical cord form embryonic like bodies in a mesenchymal layer culture.

    Science.gov (United States)

    Tsagias, Nikos; Kouzi-Koliakos, Kokkona; Karagiannis, Vasileios; Tsikouras, P; Koliakos, George G

    2015-03-01

    Recently the matrix of umbilical cord began to use as an alternative source of stem cells additionally to the blood of umbilical cord. Umbilical cord has been used mainly for mesenchymal stem cell banking. The immunological characteristics of mesenchymal stem cells in combination with their ability to avoid rejection make them an attractive biological material for transplantations. In this study the isolation of small in size pluripotent stem cells from umbilical cord expressing early transcription factors with characteristics that resemble to embryonic stem cells is investigated. Pluripotent stem cells were isolated from human umbilical cords, by a new strategy method based on unique characteristics such as the small size and the positivity on early transcription factors OCT and Nanog. An enriched population of CXCR4(+) OCT(+) Nanog(+) CD45(-) small stem cells from the cord was isolated. This fraction was able to create alkaline phosphatase positive like spheres forms in a mesenchymal layer with multilineage differentiation capacity. Our results were assessed by RT PCR and electophoresis for the pluripotent genes. These data suggest that umbilical cord provides an attractive source not only of mesenchymal stem cells but moreover of pluripotent stem cells. The method described herein should be applied in the field of stem cell banking in addition to the classical umbilical cord harvesting method. Isolation of a population of cells with pluripotent characteristics from umbilical cord. Adoption of a second centrifugation step for the pluripotent stem isolation. Increasing the value of the cord and explaining the pluripotency. This work will enhance the value of umbilical cord harvesting.

  11. Hepatocyte IKK2 protects Mdr2-/- mice from chronic liver failure.

    Directory of Open Access Journals (Sweden)

    Hanno Ehlken

    Full Text Available Mice lacking the Abc4 protein encoded by the multidrug resistance-2 gene (Mdr2(-/- develop chronic periductular inflammation and cholestatic liver disease resulting in the development of hepatocellular carcinoma (HCC. Inhibition of NF-κB by expression of an IκBα super-repressor (IκBαSR transgene in hepatocytes was shown to prevent HCC development in Mdr2(-/- mice, suggesting that NF-κB acts as a tumour promoter in this model of inflammation-associated carcinogenesis. On the other hand, inhibition of NF-κB by hepatocyte specific ablation of IKK2 resulted in increased liver tumour development induced by the chemical carcinogen DEN. To address the role of IKK2-mediated NF-κB activation in hepatocytes in the pathogenesis of liver disease and HCC in Mdr2(-/- mice, we generated Mdr2-deficient animals lacking IKK2 specifically in hepatocytes using the Cre-loxP system. Mdr2(-/- mice lacking IKK2 in hepatocytes developed spontaneously a severe liver disease characterized by cholestasis, major hyperbilirubinemia and severe to end-stage fibrosis, which caused muscle wasting, loss of body weight, lethargy and early spontaneous death. Cell culture experiments showed that primary hepatocytes lacking IKK2 were more sensitive to bile acid induced death, suggesting that hepatocyte-specific IKK2 deficiency sensitized hepatocytes to the toxicity of bile acids under conditions of cholestasis resulting in greatly exacerbated liver damage. Mdr2(-/-IKK2(Hep-KO mice remarkably recapitulate chronic liver failure in humans and might be of special importance for the study of the mechanisms contributing to the pathogenesis of end-stage chronic liver disease or its implications on other organs.IKK2-mediated signaling in hepatocytes protects the liver from damage under conditions of chronic inflammatory cholestasis and prevents the development of severe fibrosis and liver failure.

  12. Hepatic Stellate Cells Improve Engraftment of Human Primary Hepatocytes: A Preclinical Transplantation Study in an Animal Model.

    Science.gov (United States)

    Dusabineza, Ange-Clarisse; Najimi, Mustapha; van Hul, Noémi; Legry, Vanessa; Khuu, Dung Ngoc; van Grunsven, Leo A; Sokal, Etienne; Leclercq, Isabelle A

    2015-01-01

    Human hepatocytes are used for liver cell therapy, but the small number of engrafting cells limits the benefit of cell transplantation. We tested whether cotransplantation of hepatocytes with hepatic stellate cells (HSCs) could improve hepatocyte engraftment in vivo. Human primary hepatocytes were transplanted into SCID mice either alone or in a mixture with HSCs (quiescent or after culture activation) or LX-2 cells (ratio 20:1). Four weeks after transplantation into mouse livers, human albumin-positive (huAlb(+)) hepatocytes were found scattered. When cotransplanted in a mixture with HSCs or LX-2 cells, huAlb(+) hepatocytes formed clusters and were more numerous occupying 2- to 5.9-fold more surface on the tissue section than in livers transplanted with hepatocytes alone. Increased huAlb mRNA expression in livers transplanted with the cell mixtures confirmed those results. The presence of HSCs increased the number of hepatocytes entrapped in the host liver at an early time point posttransplantation but not their proliferation in situ as assessed by cumulative incorporation of BrdU. Importantly, 4 weeks posttransplantation, we found no accumulation of αSMA(+)-activated HSCs or collagen deposition. To follow the fate of transplanted HSCs, HSCs derived from GFP(+) mice were injected into GFP(-) littermates: 17 h posttransplant, GFP(+) HSCs were found in the sinusoids, without proliferating or actively producing ECM; they were undetectable at later time points. Coculture with HSCs improved the number of adherent hepatocytes, with best attachment obtained when hepatocytes were seeded in contact with activated HSCs. In vivo, cotransplantation of hepatocytes with HSCs into a healthy liver recipient does not generate fibrosis, but significantly improves the engraftment of hepatocytes, probably by ameliorating cell homing.

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

    Science.gov (United States)

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

    2016-01-01

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

  14. Content delivery to newly forming Weibel-Palade bodies is facilitated by multiple connections with the Golgi apparatus.

    Science.gov (United States)

    Mourik, Marjon J; Faas, Frank G A; Zimmermann, Hans; Voorberg, Jan; Koster, Abraham J; Eikenboom, Jeroen

    2015-05-28

    Weibel-Palade bodies (WPBs) comprise an on-demand storage organelle within vascular endothelial cells. It's major component, the hemostatic protein von Willebrand factor (VWF), is known to assemble into long helical tubules and is hypothesized to drive WPB biogenesis. However, electron micrographs of WPBs at the Golgi apparatus show that these forming WPBs contain very little tubular VWF compared with mature peripheral WPBs, which raises questions on the mechanisms that increase the VWF content and facilitate vesicle growth. Using correlative light and electron microscopy and electron tomography, we investigated WPB biogenesis in time. We reveal that forming WPBs maintain multiple connections to the Golgi apparatus throughout their biogenesis. Also by volume scanning electron microscopy, we confirmed the presence of these connections linking WPBs and the Golgi apparatus. From electron tomograms, we provided evidence that nontubular VWF is added to WPBs, which suggested that tubule formation occurs in the WPB lumen. During this process, the Golgi membrane and clathrin seem to provide a scaffold to align forming VWF tubules. Overall, our data show that multiple connections with the Golgi facilitate content delivery and indicate that the Golgi appears to provide a framework to determine the overall size and dimensions of newly forming WPBs.

  15. Asialoglycoprotein Receptor-Mediated Gene Delivery to Hepatocytes Using Galactosylated Polymers.

    Science.gov (United States)

    Thapa, Bindu; Kumar, Piyush; Zeng, Hongbo; Narain, Ravin

    2015-09-14

    Highly efficient, specific, and nontoxic gene delivery vector is required for gene therapy to the liver. Hepatocytes exclusively express asialoglycoprotein receptor (ASGPR), which can recognize and bind to galactose or N-acetylgalactosamine. Galactosylated polymers are therefore explored for targeted gene delivery to the liver. A library of safe and stable galactose-based glycopolymers that can specifically deliver genes to hepatocytes were synthesized having different architectures, compositions, and molecular weights via the reversible addition-fragmentation chain transfer process. The physical and chemical properties of these polymers have a great impact on gene delivery efficacy into hepatocytes, as such block copolymers are found to form more stable complexes with plasmid and have high gene delivery efficiency into ASGPR expressing hepatocytes. Transfection efficiency and uptake of polyplexes with these polymers decreased significantly by preincubation of hepatocytes with free asialofetuin or by adding free asialofetuin together with polyplexes into hepatocytes. The results confirmed that polyplexes with these polymers were taken up specifically by hepatocytes via ASGPR-mediated endocytosis. The results from transfection efficiency and uptake of these polymers in cells without ASGPR, such as SK Hep1 and HeLa cells, further support this mechanism. Since in vitro cytotoxicity assays prove these glycopolymers to be nontoxic, they may be useful for delivery of clinically important genes specifically to the liver.

  16. Mechanisms and prevention of hepatocyte cell death

    NARCIS (Netherlands)

    Vrenken, Titia Eveline

    2008-01-01

    In many liver diseases, hepatocyte damage occurs upon exposure to toxic bile acids, inflammatory cytokines and increased levels of reactive oxygen species (ROS). Detailed information of the signaling pathways involved in hepatocyte damage will facilitate the discovery of new targets for intervention

  17. Pleomorphism of the myelin-like bodies in the hepatocytes of patients with Dubin-Johnson syndrome complicated with chronic hepatitis B%Dubin-Johnson综合征合并慢性乙型肝炎患者脂褐素小体的超微结构特点

    Institute of Scientific and Technical Information of China (English)

    鲁晓擘; 刘浩; 徐琴; 张跃新; 邓泽润; 胡安华; 刘文杰; 吕荣福

    2011-01-01

    Objective To explore characteristics of the myelin-like bodies in the hepatocytes of patients with Dubin-Johnson syndrome (DJS) complicated with chronic hepatitis B (CHB). Methods 11 cases of DJS complicated with CHB and 5 cases DJS without CHB were studied clinicopathologically. The hepatocyte ultrastructure was observed with transmission electron microscope and taken photos. The data were compared and analyzed using Fisher's Exact Test. Results Deposition of myelin-like bodies can be observed in the hepatocytes of DJS patients with CHB but can not in DJS patients without CHB. The morphology of pigment varys. The electron density and volume of pigment in DJS patients with CHB can be classified into five types: brights (2/11,18.2%), reticulation (1/11, 9.1%), punctiform (6/11, 54.5%), abnormiry (1 / 11, 9.1%) and primary type (1 / 11, 9.1%). The myelin-like bodies in the hepatocytes of patients with DJS are high density and round with membrance (we named it as primary type) (5/5, 100%). Conclusions The myelin-like bodies in the hepatocytes of DJS patients with CHB possess special pleomorphism and may have important diagnostic value.%目的 研究Dubin-Johnson综合征合并慢性乙型肝炎患者肝细胞脂褐素小体的超微结构特点.方法用透射电子显微镜(简称电镜)对11例Dubin-Johnson综合征合并慢性乙型肝炎(轻度)患者、5例Dubin-Johnson综合征患者的肝细胞超微结构进行观察,重点观察脂褐素小体的形态及密度.对数据的统计学分析采用Fisher's精确概率法.结果 11例Dubin-Johnson综合征合并慢性乙型肝炎(轻度)患者的肝细胞脂褐素小体形态呈现多形性,电镜下至少有5种形态:"明亮型"(18.2%,2/11)、"网眼型"(9.1%,1/11)、"点状型"(54.5%,6/11)、"不规则型"(9.1%,1/11)和"基本型"(9.1%,1/11);而未合并慢性乙型肝炎的Dubin-Johnson综合征患者的肝细胞脂褐素小体形态相对较为单一,为"基本型"(100.0%,5/5)."基本型"

  18. Non-canonical Cajal bodies form in the nucleus of late stage avian oocytes lacking functional nucleolus.

    Science.gov (United States)

    Khodyuchenko, Tatiana; Gaginskaya, Elena; Krasikova, Alla

    2012-07-01

    In the somatic cell nucleus, there are several universal domains such as nucleolus, SC35-domains, Cajal bodies (CBs) and histone locus bodies (HLBs). Among them, CBs were described more than 100 years ago; however, we still do not have a final understanding of their nature and biological significance. The giant nucleus of avian and amphibian growing oocytes represents an advantageous model for analysis of functions and biogenesis of various nuclear domains. Nevertheless, in large-sized avian oocytes that contain transcriptionally active lampbrush chromosomes, CB-like organelles have not been identified yet. Here we demonstrate that in the pigeon (Columba livia) oocyte nucleus, characterized by absence of any functional nucleoli, extrachromosomal spherical bodies contain TMG-capped spliceosomal snRNAs, core proteins of Sm snRNPs and the protein coilin typical for CBs, but not splicing factor SC35 nor the histone pre-mRNA 3'-end processing factor symplekin. The results establish that coilin-rich nuclear organelles in pigeon late-stage oocyte are not the equivalents of HLBs but belong to a group of CBs. At the same time, they do not contain the snoRNP/scaRNP protein fibrillarin involved in 2'-O-methylation of snoRNAs and snRNAs. Thus, the nucleus of late-stage pigeon oocytes houses CB-like organelles that have an unusual molecular composition and are implicated in the snRNP biogenesis pathway. These data demonstrate that snRNP-rich non-canonical CBs can form in the absence of nucleolus. We argue that pigeon oocytes represent a new promising model to investigate CB modular organization, functions and formation mechanism.

  19. An Analysis of Form Changes of a Toroidal Body with a Meridional Arrangement of Fibers on the Basis of the Two-Level Carcass Theory and of a Homogeneous Body Congruent to It

    Science.gov (United States)

    Akhundov, V. M.

    2017-01-01

    Results of an analysis of form changes of a toroidal body highly filled with fibers at large torsional deformations and rotational motions are presented. The body is reinforced in the meridional direction. The investigation was carried out by using the two-level carcass theory of fibrous media at large deformations, according to which the macroscopic fields of a reinforced body are determined by its internal fields. These fields are represented by the material configurations of nodal material blocks of the body, for which, on the basis of the model of a piecewise homogeneous medium, boundary-values problems of the micromechanical level of the theory are solved. The results obtained are compared with those for a homogeneous body. The congruent deformation of the homogeneous body at which its initial form and dimensions are practically restored upon superposition of torsion and rotation is determined.

  20. Cultivation of adult rat hepatocytes on 3T3 cells: expression of various liver differentiated functions.

    Science.gov (United States)

    Kuri-Harcuch, W; Mendoza-Figueroa, T

    1989-08-01

    Adult rat hepatocytes were maintained in culture for at least 1 month without losing the expression of their differentiated functions; they were cultured on lethally treated 3T3 fibroblasts inoculated at 35,000 cells/cm2 with medium containing 10-25 micrograms/ml hydrocortisone. Hepatocytes showed their typical morphology; they formed bile canaliculi, microvilli, and intercellular junctions with desmosomes and nexus; some formed structures that may resemble the perisinusoidal space of Disse. In addition, they showed DNA synthesis and expressed some liver-specific functions. They synthesized albumin and other proteins, which were exported to the culture medium. Like parenchymal liver cells in vivo, de novo fatty acid synthesis and esterification took place, and more than 80% of the lipids synthesized by the hepatocytes were secreted into the medium as triglycerides; they also showed cytochrome-P450 activity that was inducible with phenobarbital, suggesting that the hepatocytes have the capacity to metabolize drugs. These culture conditions allow the study of various hepatocyte differentiated functions, and they may provide the means to analyze the effect on liver of hormones, viruses and hepatotoxic chemicals and drugs; they may also indicate conditions adequate for serial growth of hepatocytes.

  1. Fabrication of nanotube arrays on commercially pure titanium and their apatite-forming ability in a simulated body fluid

    Energy Technology Data Exchange (ETDEWEB)

    Hsu, Hsueh-Chuan; Wu, Shih-Ching; Hsu, Shih-Kuang [Department of Dental Technology and Materials Science, Central Taiwan University of Science and Technology, Taiwan, ROC (China); Institute of Biomedical Engineering and Materials Science, Central Taiwan University of Science and Technology, Taiwan, ROC (China); Chang, Yu-Chen [Department of Mechanical and Automation Engineering, Da-Yeh University, Taiwan, ROC (China); Ho, Wen-Fu, E-mail: fujii@nuk.edu.tw [Department of Chemical and Materials Engineering, National University of Kaohsiung, Kaohsiung, Taiwan, ROC (China)

    2015-02-15

    In this study, we investigated self-organized TiO{sub 2} nanotubes that were grown using anodization of commercially pure titanium at 5 V or 10 V in NH{sub 4}F/NaCl electrolyte. The nanotube arrays were annealed at 450 °C for 3 h to convert the amorphous nanotubes to anatase and then they were immersed in simulated body fluid at 37 °C for 0.5, 1, and 14 days. The purpose of this experiment was to evaluate the apatite-formation abilities of anodized Ti nanotubes with different tube diameters and lengths. The nanotubes that formed on the surfaces of Ti were examined using a field emission scanning electron microscope, X-ray diffraction, and X-ray photoelectron spectroscope. When the anodizing potential was increased from 5 V to 10 V, the pore diameter of the nanotube increased from approximately 24–30 nm to 35–53 nm, and the tube length increased from approximately 590 nm to 730 nm. In vitro testing of the heat-treated nanotube arrays indicated that Ca-P formation occurred after only 1 day of immersion in simulated body fluid. This result was particularly apparent in the samples that were anodized at 10 V. It was also found that the thickness of the Ca-P layer increases as the applied potential for anodized c.p. Ti increases. The average thickness of the Ca-P layer on Ti that was anodized at 5 V and 10 V was approximately 170 nm and 190 nm, respectively, after immersion in simulated body fluid for 14 days. - Highlights: • TiO{sub 2} nanotube on Ti surface was formed by anodic oxidation in a NaCl/NH{sub 4}F solution. • TiO{sub 2} layers show a tube length of 590 nm and 730 nm at 5 V and 10 V, respectively. • After soaking in SBF, Ca-P layer completely covered the entire nanotubular surfaces. • The Ca-P layer was thicker on the Ti surface anodized at 10 V.

  2. Higher body mass index in adults at diagnosis of the slowly progressive form of type 1 diabetes mellitus is associated with lower risk HLA genes.

    Science.gov (United States)

    Fourlanos, S; Elkassaby, S; Varney, M D; Colman, P G; Harrison, L C

    2014-06-01

    We hypothesised that higher body weight, a proposed risk factor for type 1 diabetes mellitus, would be associated with increased penetrance of lower risk genes. In adults at diagnosis of the slowly progressive form of type 1 diabetes mellitus we found that higher body mass index was associated with the absence of the highest risk HLA genes.

  3. Granulocytes of sea anemone Actinia equina (Linnaeus, 1758 body fluid contain and release cytolysins forming plaques of lysis

    Directory of Open Access Journals (Sweden)

    MG Parisi

    2014-01-01

    Full Text Available The Cnidaria phylum includes organisms that are among the most poisonous animals. The exact composition of cnidarian bioactive molecules is not known in detail, but little is known on the cells that produce the toxins. Here we have shown that the presence of cytolysins is not exclusive of nematocysts. A plaque-forming assay was carried out with cell populations extracted from the percoled body fluid showed for the first time that anthozoan granulocytes are able to form plaque of lysis. We have partitioned the total population of free cells into three distinct discrete bands by discontinuous Percoll gradient, and we have identified six small different types cells: morular granulocytes; cells with large or small peripherical granules, granulocytes with irregular shape containing blue and red granules, cells showing one fine red granule of uniform size and, finally, cells with elongated shape and small dispersed granules. Cell lysate of each cellular band resulted cytolytic toward different erythrocytes types. SDS page analysis of the lysate cell fraction showed a predominant of 20 kDa that corresponds to the weight of the cytolytic equinatoxin. The nature of equinatoxins-related activity was demonstrated by inhibition experiments using bovine sphingomyelin.

  4. Do nuclear bodies in oocytes of Tenebrio molitor (Coleoptera: Polyphaga, Tenebrionidae) contain two forms of RNA polymerase II?

    Science.gov (United States)

    Bogolyubov, D; Parfenov, V

    2004-02-01

    Late vitellogenic oocytes of the mealworm beetle, Tenebrio molitor, which are transcriptionally inert, contain numerous fibrogranular nuclear bodies (NBs). Previously, we have shown that these NBs contain both unphosphorylated and phosphorylated forms of RNA polymerase II (pol II) [Tissue Cell 33 (2001) 549]. The conclusion on the presence of phosphorylated pol II was based on our immunoelectron experiments with monoclonal antibody (mAb) H5 against the phosphorylated serine-2 of the carboxy-terminal domain (CTD) of pol II. Because the specificity of mAb H5 was recently questioned by demonstration of its cross-reaction with SR-proteins [J. Struct. Biol. 140 (2002) 154], we re-examined here the occurence of pol II in T. molitor oocyte NBs using other appropriate antibodies. We confirm the presence of phosphorylated pol II in NBs using the affinity-purified polyclonal antibody against the phosphorylated CTD. Using double immunogold labeling with this antibody plus mAb 8WG16 against the unphosphorylated CTD, we confirm the presence of two forms of pol II in NBs. Additionally, the presence of pol II in NBs was verified here using mAb ARNA3 against the epitope outside CTD. We suggest that at the transcriptionally inactive stage, T. molitor oocyte NBs represent storage domains for pol II disengaged from the transcription.

  5. [Cytoskeletal reorganization in hepatocytes of the regenerating mouse liver].

    Science.gov (United States)

    Gleĭberman, A S; Troianovskiĭ, S M; Bannikov, G A

    1984-12-01

    The intracellular pattern of prekeratin and actin filaments has been studied on sections of mouse livers regenerating after CCl4 injury. Monoclonal antibodies against one of liver prekeratins and monospecific polyclonal actin antibodies were used in the indirect immunofluorescent test. The presence of alpha-fetoprotein and bile canaliculi antigen was also monitored during regeneration. In control livers, prekeratin and actin filaments formed thick bundles adjacent to plasma membranes. The cytoplasmic prekeratin network was unmarked. In contrast to the latter, the bright well developed intracytoplasmic prekeratin network and intracytoplasmic actin fibers were identified in the perinecrotic hepatocytes by the 3d-4th day of regeneration. This rearrangement of the cytoskeleton coincided in time with the appearance of alpha-fetoprotein and the loss of the bile canaliculi antigen in the perinecrotic hepatocytes.

  6. Clinical applications of hepatocyte transplantation

    Institute of Scientific and Technical Information of China (English)

    Giada Pietrosi; Giovanni Battista Vizzini; Salvatore Gruttadauria; Bruno Gridelli

    2009-01-01

    The shortage of organ donors is a problem worldwide, with approximately 15% of adult patients with lifethreatening liver diseases dying while on the waiting list. The use of cell transplantation for liver disease is an attempt to correct metabolic defects, or to support liver function as a bridge to liver transplantation and, as such, has raised a number of expectations. Most of the available studies briefly reported here focus on adult hepatocyte transplantation (HT), and the results are neither reproducible nor comparable, because the means of infusion, amount of injected cells and clinical variability differ among the studies. To better understand the specific role of HT in the management of end-stage liver disease, it is important that controlled studies, designed on the principles of evidence-based medicine, be done in order to guarantee the reproducibility of results.

  7. TDP-43 inclusion bodies formed in bacteria are structurally amorphous, non-amyloid and inherently toxic to neuroblastoma cells.

    Directory of Open Access Journals (Sweden)

    Claudia Capitini

    Full Text Available Accumulation of ubiquitin-positive, tau- and α-synuclein-negative intracellular inclusions of TDP-43 in the central nervous system represents the major hallmark correlated to amyotrophic lateral sclerosis and frontotemporal lobar degeneration with ubiquitin-positive inclusions. Such inclusions have variably been described as amorphous aggregates or more structured deposits having an amyloid structure. Following the observations that bacterial inclusion bodies generally consist of amyloid aggregates, we have overexpressed full-length TDP-43 and C-terminal TDP-43 in E. coli, purified the resulting full-length and C-terminal TDP-43 containing inclusion bodies (FL and Ct TDP-43 IBs and subjected them to biophysical analyses to assess their structure/morphology. We show that both FL and Ct TDP-43 aggregates contained in the bacterial IBs do not bind amyloid dyes such as thioflavin T and Congo red, possess a disordered secondary structure, as inferred using circular dichroism and infrared spectroscopies, and are susceptible to proteinase K digestion, thus possessing none of the hallmarks for amyloid. Moreover, atomic force microscopy revealed an irregular structure for both types of TDP-43 IBs and confirmed the absence of amyloid-like species after proteinase K treatment. Cell biology experiments showed that FL TDP-43 IBs were able to impair the viability of cultured neuroblastoma cells when added to their extracellular medium and, more markedly, when transfected into their cytosol, where they are at least in part ubiquitinated and phosphorylated. These data reveal an inherently high propensity of TDP-43 to form amorphous aggregates, which possess, however, an inherently high ability to cause cell dysfunction. This indicates that a gain of toxic function caused by TDP-43 deposits is effective in TDP-43 pathologies, in addition to possible loss of function mechanisms originating from the cellular mistrafficking of the protein.

  8. TDP-43 inclusion bodies formed in bacteria are structurally amorphous, non-amyloid and inherently toxic to neuroblastoma cells.

    Science.gov (United States)

    Capitini, Claudia; Conti, Simona; Perni, Michele; Guidi, Francesca; Cascella, Roberta; De Poli, Angela; Penco, Amanda; Relini, Annalisa; Cecchi, Cristina; Chiti, Fabrizio

    2014-01-01

    Accumulation of ubiquitin-positive, tau- and α-synuclein-negative intracellular inclusions of TDP-43 in the central nervous system represents the major hallmark correlated to amyotrophic lateral sclerosis and frontotemporal lobar degeneration with ubiquitin-positive inclusions. Such inclusions have variably been described as amorphous aggregates or more structured deposits having an amyloid structure. Following the observations that bacterial inclusion bodies generally consist of amyloid aggregates, we have overexpressed full-length TDP-43 and C-terminal TDP-43 in E. coli, purified the resulting full-length and C-terminal TDP-43 containing inclusion bodies (FL and Ct TDP-43 IBs) and subjected them to biophysical analyses to assess their structure/morphology. We show that both FL and Ct TDP-43 aggregates contained in the bacterial IBs do not bind amyloid dyes such as thioflavin T and Congo red, possess a disordered secondary structure, as inferred using circular dichroism and infrared spectroscopies, and are susceptible to proteinase K digestion, thus possessing none of the hallmarks for amyloid. Moreover, atomic force microscopy revealed an irregular structure for both types of TDP-43 IBs and confirmed the absence of amyloid-like species after proteinase K treatment. Cell biology experiments showed that FL TDP-43 IBs were able to impair the viability of cultured neuroblastoma cells when added to their extracellular medium and, more markedly, when transfected into their cytosol, where they are at least in part ubiquitinated and phosphorylated. These data reveal an inherently high propensity of TDP-43 to form amorphous aggregates, which possess, however, an inherently high ability to cause cell dysfunction. This indicates that a gain of toxic function caused by TDP-43 deposits is effective in TDP-43 pathologies, in addition to possible loss of function mechanisms originating from the cellular mistrafficking of the protein.

  9. From lizard body form to serpentiform morphology: The atlas-axis complex in African cordyliformes and their relatives.

    Science.gov (United States)

    Čerňanský, Andrej

    2016-04-01

    The comparative vertebral morphology of the atlas-axis complex in cordyliforms, xantusiid and several skinks is studied here. These lizards are particularly interesting because of their different ecological adaptations and anti-predation strategies, where conformation ranges from the lizard-like body to a snake-like body. This transition to serpentiform morphology shows several evolutionary patterns in the atlas-axis complex: 1) the zygapophyseal articulations are lost in the early stage of the transition. In contrast to mammals, the atlas is more or less locked to the axis in lepidosaurs, but the absence of zygapophyseal articulation releases this locking for rotation. However despite its serpentiform morphology, Chamaesaura is different, in possessing this articulation; 2) the first intercentrum of Chamaesaura and Tetradactylus africanus (serpentiform grass-swimmers) is fully curved anteriorly, underlying the occipital condyle. While this limits ventral skull rotation beyond a certain angle, it locks the skull, which is a crucial adaptation for a sit-and-wait position in grassland habitats that needs to keep the head stabilized; and 3) in Acontias, most of the atlas articular surface with the occipital condyle is formed by the lateral aspect of the articulation area relative to the area located in the dorsal region of the slightly reduced intercentrum. A similar state occurs in amphisbaenians, most likely reflecting a fossorial lifestyle of the limbless lizards. Although Chamaesaura and Tetradactylus live sympatrically in grasslands, Chamaesaura differs in several ways in atlas-axis complex: for example, aforementioned presence of the atlas-axis zygapophyseal articulation, and long posterodorsal processes. Its occipital condyle protrudes further posteriorly, placing the atlas-axis complex further from the endocranium than in Tetradactylus. Hence, adaptation in the same niche, even among sister clades, can lead to different atlas-axis morphology due to different

  10. Deformable registration for image-guided spine surgery: preserving rigid body vertebral morphology in free-form transformations

    Science.gov (United States)

    Reaungamornrat, S.; Wang, A. S.; Uneri, A.; Otake, Y.; Zhao, Z.; Khanna, A. J.; Siewerdsen, J. H.

    2014-03-01

    Purpose: Deformable registration of preoperative and intraoperative images facilitates accurate localization of target and critical anatomy in image-guided spine surgery. However, conventional deformable registration fails to preserve the morphology of rigid bone anatomy and can impart distortions that confound high-precision intervention. We propose a constrained registration method that preserves rigid morphology while allowing deformation of surrounding soft tissues. Method: The registration method aligns preoperative 3D CT to intraoperative cone-beam CT (CBCT) using free-form deformation (FFD) with penalties on rigid body motion imposed according to a simple intensity threshold. The penalties enforced 3 properties of a rigid transformation - namely, constraints on affinity (AC), orthogonality (OC), and properness (PC). The method also incorporated an injectivity constraint (IC) to preserve topology. Physical experiments (involving phantoms, an ovine spine, and a human cadaver) as well as digital simulations were performed to evaluate the sensitivity to registration parameters, preservation of rigid body morphology, and overall registration accuracy of constrained FFD in comparison to conventional unconstrained FFD (denoted uFFD) and Demons registration. Result: FFD with orthogonality and injectivity constraints (denoted FFD+OC+IC) demonstrated improved performance compared to uFFD and Demons. Affinity and properness constraints offered little or no additional improvement. The FFD+OC+IC method preserved rigid body morphology at near-ideal values of zero dilatation (D = 0.05, compared to 0.39 and 0.56 for uFFD and Demons, respectively) and shear (S = 0.08, compared to 0.36 and 0.44 for uFFD and Demons, respectively). Target registration error (TRE) was similarly improved for FFD+OC+IC (0.7 mm), compared to 1.4 and 1.8 mm for uFFD and Demons. Results were validated in human cadaver studies using CT and CBCT images, with FFD+OC+IC providing excellent preservation

  11. Wheat proteins enhance stability and function of adhesion molecules in cryopreserved hepatocytes.

    Science.gov (United States)

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

    2009-01-01

    Cryopreserved hepatocytes with good hepatospecific functions upon thawing are important for clinical transplantation and for in vitro drug toxicity testing. However, cryopreservation reduces viability and certain hepatospecific functions, but the most pronounced change is diminished attachment efficiency of hepatocytes. Adhesion of cells to the extracellular matrix and cell-cell contacts are crucial for many aspects of cellular function. These processes are partly mediated and controlled by cellular adhesion molecules. The mechanisms responsible for reduced attachment efficiency of cryopreserved hepatocytes are not well understood. To address this question, we investigated the effect of a new cryopreservation procedure, using wheat proteins (WPs) or mixtures of recombinant forms of wheat freezing tolerance-associated proteins, on the stability of three important adhesion molecules (beta1-integrin, E-cadherin, and beta-catenin). Immunoblot analyses revealed that the levels of beta1-integrin, E-cadherin, and beta-catenin were much lower in cryopreserved rat hepatocytes, when compared to fresh cells. Protein expression of the adhesion molecules was generally lower in cells cryopreserved with DMSO, compared to WPs. Moreover, the stability of the adhesion molecules was not affected by cryopreservation to the same degree, with more pronounced decreases occurring for beta1-integrin (62-74%) > beta-catenin (51-58%) > E-cadherin (21-37%). However, when hepatocytes were cryopreserved with partially purified WPs (SulWPE, AcWPE) or with mixtures of recombinant wheat proteins, there was a clear protective effect against the loss of protein expression of beta1-integrin, E-cadherin, and beta-catenin. Protein expression was only 10-20% lower than that observed in fresh hepatocytes. These findings clearly demonstrate that WPs, and more particularly, partially purified WPs and recombinant wheat proteins, were more efficient for cryopreservation of rat hepatocytes by maintaining good

  12. Genome Analysis of the Fruiting Body-Forming Myxobacterium Chondromyces crocatus Reveals High Potential for Natural Product Biosynthesis

    Science.gov (United States)

    Zaburannyi, Nestor; Bunk, Boyke; Maier, Josef; Overmann, Jörg

    2016-01-01

    Here, we report the complete genome sequence of the type strain of the myxobacterial genus Chondromyces, Chondromyces crocatus Cm c5. It presents one of the largest prokaryotic genomes featuring a single circular chromosome and no plasmids. Analysis revealed an enlarged set of tRNA genes, along with reduced pressure on preferred codon usage compared to that of other bacterial genomes. The large coding capacity and the plethora of encoded secondary metabolite biosynthetic gene clusters are in line with the capability of Cm c5 to produce an arsenal of antibacterial, antifungal, and cytotoxic compounds. Known pathways of the ajudazol, chondramide, chondrochloren, crocacin, crocapeptin, and thuggacin compound families are complemented by many more natural compound biosynthetic gene clusters in the chromosome. Whole-genome comparison of the fruiting-body-forming type strain (Cm c5, DSM 14714) to an accustomed laboratory strain which has lost this ability (nonfruiting phenotype, Cm c5 fr−) revealed genetic changes in three loci. In addition to the low synteny found with the closest sequenced representative of the same family, Sorangium cellulosum, extensive genetic information duplication and broad application of eukaryotic-type signal transduction systems are hallmarks of this 11.3-Mbp prokaryotic genome. PMID:26773087

  13. Hepatocyte ABCA1 Deletion Impairs Liver Insulin Signaling and Lipogenesis

    Directory of Open Access Journals (Sweden)

    Chia-Chi C. Key

    2017-06-01

    Full Text Available Plasma membrane (PM free cholesterol (FC is emerging as an important modulator of signal transduction. Here, we show that hepatocyte-specific knockout (HSKO of the cellular FC exporter, ATP-binding cassette transporter A1 (ABCA1, leads to decreased PM FC content and defective trafficking of lysosomal FC to the PM. Compared with controls, chow-fed HSKO mice had reduced hepatic (1 insulin-stimulated Akt phosphorylation, (2 activation of the lipogenic transcription factor Sterol Regulatory Element Binding Protein (SREBP-1c, and (3 lipogenic gene expression. Consequently, Western-type diet-fed HSKO mice were protected from steatosis. Surprisingly, HSKO mice had intact glucose metabolism; they showed normal gluconeogenic gene suppression in response to re-feeding and normal glucose and insulin tolerance. We conclude that: (1 ABCA1 maintains optimal hepatocyte PM FC, through intracellular FC trafficking, for efficient insulin signaling; and (2 hepatocyte ABCA1 deletion produces a form of selective insulin resistance so that lipogenesis is suppressed but glucose metabolism remains normal.

  14. 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.

  15. Genetic tracing of hepatocytes in liver homeostasis, injury, and regeneration.

    Science.gov (United States)

    Wang, Yue; Huang, XiuZhen; He, Lingjuan; Pu, Wenjuan; Li, Yan; Liu, Qiaozhen; Li, Yi; Zhang, Libo; Yu, Wei; Zhao, Huan; Zhou, Yingqun; Zhou, Bin

    2017-05-26

    The liver possesses a remarkable capacity to regenerate after damage. There is a heated debate on the origin of new hepatocytes after injuries in adult liver. Hepatic stem/progenitor cells have been proposed to produce functional hepatocytes after injury. Recent studies have argued against this model and suggested that pre-existing hepatocytes, rather than stem cells, contribute new hepatocytes. This hepatocyte-to-hepatocyte model is mainly based on labeling of hepatocytes with Cre-recombinase delivered by the adeno-associated virus. However, the impact of virus infection on cell fate determination, consistency of infection efficiency, and duration of Cre-virus in hepatocytes remain confounding factors that interfere with the data interpretation. Here, we generated a new genetic tool Alb-DreER to label almost all hepatocytes (>99.5%) and track their contribution to different cell lineages in the liver. By "pulse-and-chase" strategy, we found that pre-existing hepatocytes labeled by Alb-DreER contribute to almost all hepatocytes during normal homeostasis and after liver injury. Virtually all hepatocytes in the injured liver are descendants of pre-existing hepatocytes through self-expansion. We concluded that stem cell differentiation is unlikely to be responsible for the generation of a substantial number of new hepatocytes in adult liver. Our study also provides a new mouse tool for more precise in vivo genetic study of hepatocytes in the field. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. Selective insulin resistance in hepatocyte senescence

    Energy Technology Data Exchange (ETDEWEB)

    Aravinthan, Aloysious [Division of Gastroenterology and Hepatology, Department of Medicine, University of Cambridge, Cambridge (United Kingdom); Challis, Benjamin [Institute of Metabolic Sciences, University of Cambridge, Cambridge (United Kingdom); Shannon, Nicholas [Cancer Research UK Cambridge Institute, Cambridge (United Kingdom); Hoare, Matthew [Division of Gastroenterology and Hepatology, Department of Medicine, University of Cambridge, Cambridge (United Kingdom); Cancer Research UK Cambridge Institute, Cambridge (United Kingdom); Heaney, Judith [Division of Gastroenterology and Hepatology, Department of Medicine, University of Cambridge, Cambridge (United Kingdom); Foundation for Liver Research, Institute of Hepatology, London (United Kingdom); Alexander, Graeme J.M., E-mail: gja1000@doctors.org.uk [Division of Gastroenterology and Hepatology, Department of Medicine, University of Cambridge, Cambridge (United Kingdom)

    2015-02-01

    Insulin resistance has been described in association with chronic liver disease for decades. Hepatocyte senescence has been demonstrated in chronic liver disease and as many as 80% of hepatocytes show a senescent phenotype in advanced liver disease. The aim of this study was to understand the role of hepatocyte senescence in the development of insulin resistance. Senescence was induced in HepG2 cells via oxidative stress. The insulin metabolic pathway was studied in control and senescent cells following insulin stimulation. GLUT2 and GLUT4 expressions were studied in HepG2 cells and human liver tissue. Further, GLUT2 and GLUT4 expressions were studied in three independent chronic liver disease cohorts. Signalling impairment distal to Akt in phosphorylation of AS160 and FoxO1 was evident in senescent HepG2 cells. Persistent nuclear localisation of FoxO1 was demonstrated in senescent cells despite insulin stimulation. Increased GLUT4 and decreased GLUT2 expressions were evident in senescent cells, human cirrhotic liver tissue and publically available liver disease datasets. Changes in GLUT expressions were associated with a poor clinical prognosis. In conclusion, selective insulin resistance is evident in senescent HepG2 cells and changes in GLUT expressions can be used as surrogate markers of hepatocyte senescence. - Highlights: • Senescent hepatocytes demonstrate selective insulin resistance. • GLUT changes act as markers of hepatocyte senescence and have prognostic value. • Study offers insight into long noticed intimacy of cirrhosis and insulin resistance.

  17. Acute Toxicity Test of Polysaccharides Krestin from Coriolus versicolor Extract with Parameters of Hepatocyte Damages, SGPT and SGOT Enzyme in Mice

    National Research Council Canada - National Science Library

    Andita Ayu Mandasari; Sri Puji Astuti Wahyuningsih; Win Darmanto

    2015-01-01

    .... However, all substances entering in body could change become toxic depending on dosage. Therefore, this research was aimed to know the effects of PSK on hepatocyte damages, SGPT and SGOT enzymes...

  18. 78 FR 40442 - Submission for OMB Review; Comment Request-Third Party Conformity Assessment Body Registration Form

    Science.gov (United States)

    2013-07-05

    ..., the number of new third party conformity assessment bodies (53) accepted by the CPSC has remained stable. Based on these historical levels of acceptance, the estimated number of third party conformity... COMMISSION Submission for OMB Review; Comment Request--Third Party Conformity Assessment Body Registration...

  19. Generation of human pluripotent stem cell-derived hepatocyte-like cells for drug toxicity screening.

    Science.gov (United States)

    Takayama, Kazuo; Mizuguchi, Hiroyuki

    2017-02-01

    Because drug-induced liver injury is one of the main reasons for drug development failures, it is important to perform drug toxicity screening in the early phase of pharmaceutical development. Currently, primary human hepatocytes are most widely used for the prediction of drug-induced liver injury. However, the sources of primary human hepatocytes are limited, making it difficult to supply the abundant quantities required for large-scale drug toxicity screening. Therefore, there is an urgent need for a novel unlimited, efficient, inexpensive, and predictive model which can be applied for large-scale drug toxicity screening. Human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells are able to replicate indefinitely and differentiate into most of the body's cell types, including hepatocytes. It is expected that hepatocyte-like cells generated from human ES/iPS cells (human ES/iPS-HLCs) will be a useful tool for drug toxicity screening. To apply human ES/iPS-HLCs to various applications including drug toxicity screening, homogenous and functional HLCs must be differentiated from human ES/iPS cells. In this review, we will introduce the current status of hepatocyte differentiation technology from human ES/iPS cells and a novel method to predict drug-induced liver injury using human ES/iPS-HLCs.

  20. Comparison of Growth Performance and Whole-body Amino Acid Composition in Red Seabream (Pagrus major) Fed Free or Dipeptide Form of Phenylalanine.

    Science.gov (United States)

    Kim, Sung-Sam; Rahimnejad, Samad; Song, Jin-Woo; Lee, Kyeong-Jun

    2012-08-01

    This study was conducted to evaluate the efficacy of the dipeptide form of phenylalanine as a new source of amino acid in terms of growth performance and whole-body amino acid composition in comparison to the free form for red seabream (Pagrus major). Fish (1.46±0.001 g) were fed four isonitrogenous and isocaloric experimental diets containing 0.7 or 1.4% phenylalanine either in free or dipeptide form. A feeding trial was carried out in three replicates and the fish were fed to apparent satiation for six weeks. At the end of the feeding trial, feed intake of fish was influenced by both phenylalanine form and level and significantly higher values were obtained at an inclusion level of 0.7% and by the use of dipeptide form. However, the other growth parameters did not significantly differ among treatments. Whole-body amino acid compositions revealed no significant changes in concentrations of both essential and non-essential amino acids regardless of the increase in phenylalanine levels or the use of its different forms. The finding in this study indicates that juvenile red seabream can utilize dipeptide phenylalanine as efficiently as free form without any undesirable effects on growth performance or whole-body amino acid composition.

  1. Conserved balance of hepatocyte nuclear DNA content in mononuclear and binuclear hepatocyte populations during the course of chronic viral hepatitis

    Institute of Scientific and Technical Information of China (English)

    Hidenori Toyoda; Takashi Kumada; Olivier Bregerie; Christian Brechot; Chantal Desdouets

    2006-01-01

    AIM: To analyze the percentages of hepatocytes with increased nuclear DNA content, i.e., tetraploid (4n) and octoploid (8n) nuclei, and then compared mononuclear and binuclear hepatocyte populations:METHODS: The percentages of mononuclear diploid(2n), 4n, and 8n hepatocytes and those of binuclear 2× 2n, 2 × 4n, and 2 × 8n hepatocytes were determined with a method that can simultaneously measure hepatocyte nuclear DNA content and binuclearity in 62patients with chronic hepatitis B or C. The percentage of 4n and 8n hepatocytes in the mononuclear hepatocyte population was compared with the percentage of 2 ×4n and 2 × 8n hepatocytes in the binuclear hepatocyte population.RESULTS: The percentages of 4n and 8n hepatocytes in mononuclear hepatocytes and 2 × 4n and 2 × 8n hepatocytes in binuclear hepatocytes were similar,regardless of the activity or fibrosis grade of chronic hepatitis and regardless of the infecting virus.CONCLUSION: The distribution of nuclear DNA content within mononuclear and binuclear hepatocyte populations was conserved during the course of chronic viral hepatitis.

  2. The mitochondrial import gene tomm22 is specifically required for hepatocyte survival and provides a liver regeneration model

    Science.gov (United States)

    Curado, Silvia; Ober, Elke A.; Walsh, Susan; Cortes-Hernandez, Paulina; Verkade, Heather; Koehler, Carla M.; Stainier, Didier Y. R.

    2010-01-01

    SUMMARY Understanding liver development should lead to greater insights into liver diseases and improve therapeutic strategies. In a forward genetic screen for genes regulating liver development in zebrafish, we identified a mutant – oliver – that exhibits liver-specific defects. In oliver mutants, the liver is specified, bile ducts form and hepatocytes differentiate. However, the hepatocytes die shortly after their differentiation, and thus the resulting mutant liver consists mainly of biliary tissue. We identified a mutation in the gene encoding translocase of the outer mitochondrial membrane 22 (Tomm22) as responsible for this phenotype. Mutations in tomm genes have been associated with mitochondrial dysfunction, but most studies on the effect of defective mitochondrial protein translocation have been carried out in cultured cells or unicellular organisms. Therefore, the tomm22 mutant represents an important vertebrate genetic model to study mitochondrial biology and hepatic mitochondrial diseases. We further found that the temporary knockdown of Tomm22 levels by morpholino antisense oligonucleotides causes a specific hepatocyte degeneration phenotype that is reversible: new hepatocytes repopulate the liver as Tomm22 recovers to wild-type levels. The specificity and reversibility of hepatocyte ablation after temporary knockdown of Tomm22 provides an additional model to study liver regeneration, under conditions where most hepatocytes have died. We used this regeneration model to analyze the signaling commonalities between hepatocyte development and regeneration. PMID:20483998

  3. Fenofibrate: metabolism and species differences for peroxisome proliferation in cultured hepatocytes.

    Science.gov (United States)

    Cornu-Chagnon, M C; Dupont, H; Edgar, A

    1995-06-01

    The hypolipidemic agent fenofibrate, which is a peroxisome proliferator in some rodents in vivo, was studied in cultured hepatocytes for its metabolism and effects on enzymatic induction related to peroxisome proliferation so as to lead to a better understanding of the mechanisms involved in peroxisome proliferation. [14C]-Fenofibrate was completely metabolized within 24 hr by primary cultures of rat hepatocytes and the metabolic pattern corresponded to that found in vivo. The main products were fenofibric acid and its glucuronidated form. Carbonyl reduction of fenofibric acid also occurred. The metabolic pattern of [14C]fenofibric acid was nearly the same as that of fenofibrate. Fenofibrate, fenofibric acid, and its reduced metabolite all induced peroxisomal (cyanide-insensitive) palmitoyl-CoA oxidation activity (PCOA) in rat hepatocytes, whereas derivatives lacking the carboxyl group were nearly inactive. The known species differences with respect to sensitivity to peroxisome proliferators in vivo was mirrored in cultured cells because fenofibric acid did not induce peroxisomal PCOA in primary culture of guinea pig hepatocytes nor in the human hepatoma cell line HepG2. The mechanistic association between the induction of CYP4A1-catalyzed lauric acid omega-hydroxylase (LAH) activity and peroxisomal PCOA induction was investigated. Fenofibric acid concomitantly induced LAH activity and peroxisomal PCOA in rat hepatocytes. Specific inhibition of LAH activity (-52%) by 10-undecynoic acid partially prevented induction of peroxisomal PCOA (-32%). The putative role of dicarboxylic acids, the oxidation product of omega-hydroxymonocarboxylic acids, in PCOA induction was further substantiated by the observed induction of peroxisomal PCOA by 1-12-dodecanedioic acid. We conclude that (1) fenofibric acid is the possible proximate peroxisome proliferator of fenofibrate in rat hepatocytes, (2) cultured hepatocytes reflect in vivo sensitivity to fenofibrate with respect to

  4. Distribution of different phosphorylated forms of RNA polymerase II in relation to Cajal and PML bodies in human cells: an ultrastructural study.

    Science.gov (United States)

    Xie, Sheila Q; Pombo, Ana

    2006-01-01

    The mammalian nucleus is a highly organised organelle that contains many subcompartments with roles in DNA replication and repair, gene expression and RNA processing. Cajal and promyelocytic leukaemia (PML) bodies are discrete nuclear structures with specific molecular signatures. RNA polymerase II and many transcription factors have been identified within these compartments by immunofluorescence microscopy, suggesting a role in polymerase II assembly or transcriptional activity. Here, we have examined the presence of different phosphorylated forms of polymerase II and newly made RNA in Cajal and PML bodies using high-resolution imaging of ultrathin cryosections (approximately 120 nm thick) with fluorescence and electron microscopes. We show that Cajal bodies contain polymerase II phosphorylated on Ser5, and not the Ser2-phosphorylated (active) form or newly made RNA. The presence of polymerase II in the absence of transcriptional activity suggests that Cajal bodies have roles in polymerase assembly or transport, but not in gene transcription. PML bodies contain no detectable polymerase II or nascent RNA in HeLa cells, at the resolution achieved by electron microscopy, but are often surrounded by these markers at distances>25 nm. These results support the view that although PML bodies are present in transcriptionally active areas of the nucleus, they are not generally sites of polymerase II assembly, transport or activity.

  5. 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.

  6. Four waves of hepatocyte proliferation linked with three waves of hepatic fat accumulation during partial hepatectomy-induced liver regeneration.

    Directory of Open Access Journals (Sweden)

    Yuhong Zou

    Full Text Available Partial hepatectomy (PH triggers hepatocyte proliferation-mediated liver repair and is widely used to study the mechanisms governing liver regeneration in mice. However, the dynamics of the hepatocyte proliferative response to PH remain unclear. We found that PH-induced mouse liver regrowth was driven by four consecutive waves of hepatocyte replication. The first wave exhibited the highest magnitude followed by two moderate waves and one minor wave. Underlying this continuous hepatocyte replication was persistent activation of cell cycle components throughout the period of liver regeneration. Hepatocyte mitotic activity in the first three proliferative cycles showed a circadian rhythm manifested by three corresponding mitosis peaks, which were always observed at Zeitgeber time 0. The Bmal1-Clock/Wee1/Cdc2 pathway has been proposed by others to govern the circadian rhythm of hepatocyte mitosis during liver regeneration. However, we did not observe the correlations in the expression or phosphorylation of these proteins in regenerating livers. Notably, Bmal1 protein displayed frequent changes in hepatic distribution and cellular localization as the liver regrowth progressed. Further, three waves of hepatic fat accumulation occurred during hepatic regeneration. The first started before and lasted through the first round of hepatocyte proliferation, whereas the second and third occurred concomitantly with the second and third mitotic peaks, respectively.PH-induced liver regeneration consists of four continuous waves of hepatocyte proliferation coupled with three waves of hepatic fat accumulation. Bmal1, Wee1, and Cdc2 may not form a pathway regulating the circadian rhythm of hepatocyte mitosis during liver regeneration.

  7. Cholangiocarcinomas can originate from hepatocytes in mice.

    Science.gov (United States)

    Fan, Biao; Malato, Yann; Calvisi, Diego F; Naqvi, Syed; Razumilava, Nataliya; Ribback, Silvia; Gores, Gregory J; Dombrowski, Frank; Evert, Matthias; Chen, Xin; Willenbring, Holger

    2012-08-01

    Intrahepatic cholangiocarcinomas (ICCs) are primary liver tumors with a poor prognosis. The development of effective therapies has been hampered by a limited understanding of the biology of ICCs. Although ICCs exhibit heterogeneity in location, histology, and marker expression, they are currently thought to derive invariably from the cells lining the bile ducts, biliary epithelial cells (BECs), or liver progenitor cells (LPCs). Despite lack of experimental evidence establishing BECs or LPCs as the origin of ICCs, other liver cell types have not been considered. Here we show that ICCs can originate from fully differentiated hepatocytes. Using a mouse model of hepatocyte fate tracing, we found that activated NOTCH and AKT signaling cooperate to convert normal hepatocytes into biliary cells that act as precursors of rapidly progressing, lethal ICCs. Our findings suggest a previously overlooked mechanism of human ICC formation that may be targetable for anti-ICC therapy.

  8. Successful mouse hepatocyte culture with sandwich collagen gel formation

    National Research Council Canada - National Science Library

    Choi, Hyun Jung; Choi, Dongho

    2013-01-01

    .... However, long-term cultures of functional hepatocytes are difficult to establish. To increase the longevity and maintain the differentiated functions of hepatocytes in primary culture, cells can be cultured in a sandwich configuration of collagen...

  9. A novel oral form of salmon calcitonin improves glucose homeostasis and reduces body weight in diet-induced obese rats

    DEFF Research Database (Denmark)

    Feigh, M; Henriksen, K; Andreassen, K V

    2011-01-01

    To investigate the effects of acute and chronic administration of a novel oral formulation of salmon calcitonin (sCT) on glycaemic control, glucose homeostasis and body weight regulation in diet-induced obese (DIO) rats-an animal model of obesity-related insulin resistance and type 2 diabetes....

  10. [Cytotoxic effects of etoposide at different stages of differentiation of embryoid bodies formed by mouse embryonic stem cells].

    Science.gov (United States)

    Gordeeva, O F

    2013-01-01

    The initial stages of in vitro differentiation of embryonic stem cells are considered as unique three-dimensional models of early development of mammals for basic, pharmacological, and toxicological studies. It has been previously shown (Gordeeva, 2012) that the assessment of embryotoxicity in the model of undifferentiated embryonic stem cells can be insufficiently accurate in predicting toxic effects on mammalian embryos. In view of this, we performed a comparative study of the damaging effects of the cytostatic etoposide in undifferentiated embryonic stem cells and embryoid bodiesof different stages of differentiation that have similar three-dimensional structures with early embryos. The analysis of growth, cell death, and dynamics of differentiation of embryonic stem cells and embryoid bodies exposed to etoposide showed that the cytostatic and cytotoxic effects of etoposide are stage-specific. The damaging effects of etoposide were maximum in the undifferentiated embryonic stem cells and decreased with growth and differentiation of embryoid bodies. We assume that the increase in the cell volume of embryoid bodies and the development of the hypertrophic we suggest that the increase of embryoid body volume and overgrowth of extraembryonic endoderm layer lead to a decrease in the diffusion, transport, and metabolism of chemical and bioactive substances and prevent the damaging effects.

  11. 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

  12. 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 expressi

  13. Forming limit prediction using a self-consistent crystal plasticity framework: a case study for body-centered cubic materials

    Science.gov (United States)

    Jeong, Youngung; Pham, Minh-Son; Iadicola, Mark; Creuziger, Adam; Foecke, Timothy

    2016-06-01

    A rate-dependent self-consistent crystal plasticity model was incorporated with the Marciniak-Kuczyński model in order to study the effects of anisotropy on the forming limits of BCC materials. The computational speed of the model was improved by a factor of 24 when running the simulations for several strain paths in parallel. This speed-up enabled a comprehensive investigation of the forming limits of various BCC textures, such as γ , σ , α , η and ɛ fibers and a uniform (random) texture. These simulations demonstrate that the crystallographic texture has significant (both positive and negative) effects on the resulting forming limit diagrams. For example, the γ fiber texture, which is often sought through thermo-mechanical processing due to a high r-value, had the highest forming limit in the balanced biaxial strain path but the lowest forming limit under the plane strain path among the textures under consideration. A systematic investigation based on the results produced by the current model, referred to as ‘VPSC-FLD’, suggests that the r-value does not serve as a good measure of forming limit strain. However, model predictions show a degree of correlation between the r-value and the forming limit stress.

  14. 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.

  15. Hepatocyte Growth Factor Reduces Free Cholesterol-Mediated Lipotoxicity in Primary Hepatocytes by Countering Oxidative Stress

    Science.gov (United States)

    Domínguez-Pérez, Mayra; Nuño-Lámbarri, Natalia; Clavijo-Cornejo, Denise; Luna-López, Armando; Souza, Verónica; Bucio, Leticia; Miranda, Roxana U.; Muñoz, Linda; Gomez-Quiroz, Luis Enrique; Uribe-Carvajal, Salvador; Gutiérrez-Ruiz, María Concepción

    2016-01-01

    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. PMID:27143995

  16. 53BP1 nuclear bodies form around DNA lesions generated by mitotic transmission of chromosomes under replication stress

    DEFF Research Database (Denmark)

    Lukas, Claudia; Savic, Velibor; Bekker-Jensen, Simon

    2011-01-01

    Completion of genome duplication is challenged by structural and topological barriers that impede progression of replication forks. Although this can seriously undermine genome integrity, the fate of DNA with unresolved replication intermediates is not known. Here, we show that mild replication...... bodies shield chromosomal fragile sites sequestered in these compartments against erosion. Together, these data indicate that restoration of DNA or chromatin integrity at loci prone to replication problems requires mitotic transmission to the next cell generations....... increases after genetic ablation of BLM, a DNA helicase associated with dissolution of entangled DNA. Conversely, 53BP1 nuclear bodies are partially suppressed by knocking down SMC2, a condensin subunit required for mechanical stability of mitotic chromosomes. Finally, we provide evidence that 53BP1 nuclear...

  17. Coupling dynamical and collisional evolution of small bodies II Forming the Kuiper Belt, the Scattered Disk and the Oort Cloud

    CERN Document Server

    Morbidelli, S C A

    2006-01-01

    The Oort Cloud, the Kuiper Belt and the Scattered Disk are dynamically distinct populations of small bodies evolving in the outer regions of the Solar System. Whereas their collisional activity is now quiet, gravitational interactions with giant planets may have shaped these populations both dynamically and collisionally during their formation. Using a hybrid approach (Charnoz & Morbidelli 2003), the present paper tries to couple the primordial collisional and dynamical evolution of these three populations in a self-consistent way. A critical parameter is the primordial size-distribution. We show that the initial planetesimal size distribution that allows an effective mass depletion of the Kuiper belt by collisional grinding, would decimate also the population of comet-size bodies that end in the Oort Cloud and, in particular, in the Scattered Disk. As a consequence, the Scattered Disk and the Oort Cloud would be too anemic, by a factor 20 to 100, relative to the estimates achieved from the observation of...

  18. Ultrastructure of the intercalated body, a novel organelle associated with fluid forming cells in the organ of Corti.

    Science.gov (United States)

    Sobkowicz, H M; Holy, J; Scott, G L

    1990-07-01

    The intercalated body is a newly discovered organelle in the inner and outer spiral sulcus cells of the mouse organ of Corti. The organelle was found in the cochleas of 14-day and older intact mice and in organs in culture of corresponding ages. The organelle consists of a stack of interconnected cisternae of endoplasmic reticulum and of membrane bound rodlets that are intercalated between, and run parallel to, the cisternae. The cisternal membranes are predominantly smooth, but some may display ribosomes. Most rodlets are from 1 to 2 microns long, about 0.1 micron wide, and contain electron dense material. Mitochondria are commonly associated with or incorporated into the organelle. Some electron micrographs suggest that the rodlets may originate from modified mitochondria. It is our impression that the formation of the organelle begins with the apposition of cisternae and mitochondria. Cisternal-associated mitochondria appear to constrict, elongate, and lose their inner membranes. In both the intact animal and in culture, the cells of the inner and outer spiral sulci display microvilli, apical junctional complexes, lateral intercellular spaces containing interdigitating cell processes, and appear to be involved in fluid formation. Moreover, in culture, the cells of inner and outer spiral sulci as well as some cells proliferating in the outgrowth zone participate in fluid formation, producing large fluid pockets. All these cells commonly contain intercalated bodies. It is possible that in the intact animal, as in culture, intercalated bodies may play a role in fluid regulation in the immediate vicinity of the hair cells.

  19. Constrained spheroids for prolonged hepatocyte culture.

    Science.gov (United States)

    Tong, Wen Hao; Fang, Yu; Yan, Jie; Hong, Xin; Hari Singh, Nisha; Wang, Shu Rui; Nugraha, Bramasta; Xia, Lei; Fong, Eliza Li Shan; Iliescu, Ciprian; Yu, Hanry

    2016-02-01

    Liver-specific functions in primary hepatocytes can be maintained over extended duration in vitro using spheroid culture. However, the undesired loss of cells over time is still a major unaddressed problem, which consequently generates large variations in downstream assays such as drug screening. In static culture, the turbulence generated by medium change can cause spheroids to detach from the culture substrate. Under perfusion, the momentum generated by Stokes force similarly results in spheroid detachment. To overcome this problem, we developed a Constrained Spheroids (CS) culture system that immobilizes spheroids between a glass coverslip and an ultra-thin porous Parylene C membrane, both surface-modified with poly(ethylene glycol) and galactose ligands for optimum spheroid formation and maintenance. In this configuration, cell loss was minimized even when perfusion was introduced. When compared to the standard collagen sandwich model, hepatocytes cultured as CS under perfusion exhibited significantly enhanced hepatocyte functions such as urea secretion, and CYP1A1 and CYP3A2 metabolic activity. We propose the use of the CS culture as an improved culture platform to current hepatocyte spheroid-based culture systems.

  20. In-body optical stimulation formed connective tissue vascular grafts, "biotubes," with many capillaries and elastic fibers.

    Science.gov (United States)

    Oie, Tomonori; Yamanami, Masashi; Ishibashi-Ueda, Hatsue; Kanda, Keiichi; Yaku, Hitoshi; Nakayama, Yasuhide

    2010-12-01

    The autologous biotube, developed by using in-body tissue architecture technology, is one of the most promising small-diameter vascular grafts in regenerative medicine. The walls of the biotubes obtained by a traditional silicone mold-based method were very thin, and this is still the primary obstacle while handling anastomosis, even though these biotubes have adequate pressure resistance ability. This pilot study showed the effect of optical stimulation of subcutaneous tissue formation in the body during the preparation of the biotubes. A blue light-emitting diode (LED) was embedded into a silicone rod as a mold. The biotube was prepared by placing the luminescent molds into the dorsal subcutaneous pouches of a pair of beagles (each weighing ~10 kg) for 2 weeks under photoirradiation. The wall thickness of the obtained biotubes was 506.9 ± 185.7 μm, which was remarkably more than that of the previous biotubes prepared by 2 months of embedding similarly in beagles' subcutaneous pouches (thickness, 77.2 ± 14.8 μm). Many capillaries with smooth muscle cells were infiltrated into the wall and concentrated in the internal layer. Interestingly, the formation of elastic fibers had already started along with collagen fibers, mostly with a regular circumferential orientation. The short-term in-body optical stimulation resulted in the rapid formation of a biotube. These phenomena will allow easy surgical handling and may induce vascular maturation in histology during the acute phase after implantation.

  1. Primary exploration of conditions for hypothermic preservation of rat hepatocyte spheroids

    Directory of Open Access Journals (Sweden)

    Hong-ling LIU

    2015-01-01

    Full Text Available Objective To optimize conditions for hypothermic preservation of rat hepatocyte spheroids without freezing in order to facilitate the application of biological artificial liver. Methods Rat hepatic cells were isolated by a two-step perfusion method, and hepatocyte spheroids formed after 48 hours of rocking culture in serum free medium (SFM. Spheroids were then maintained in rocking culture at 37℃ (control condition, or cold stored at 4℃ for 24 or 48 hours in four different cold storage solutions: SFM alone; SFM+1mmol/L deferoxamine (Def ; SFM+1μmol/L cyclosporin A (CsA; and SFM+1mmol/L Def+1μmol/L CsA. After culturing for another 4 or 5 days, survival rate, changes in ultrastructure, and the production of albumin and urea were observed. Results Cold-induced injury could be reduced significantly by the addition of the iron chelators Def and CsA. The function and structure of hepatocyte spheroids stored in SFM+Def+CsA or SFM+Def for 24 hours were similar to those in control conditions. But the function was significantly reduced after hypothermic preservation in SFM alone. After cold storage for 48 hours, the ultrastructure of hepatocyte spheroids obviously changed and the number of dead cells increased. The survival rate of hepatocyte spheroids stored in SFM+Def+CsA or SFM+Def was significantly higher than that stored in SFM or SFM+CsA(P0.05. Conclusions Hepatocyte spheroids tolerate 24 hours of cold storage with stable viability and function. Hypothermic preservation increases the availability of cell-based therapy for liver diseases. DOI: 10.11855/j.issn.0577-7402.2014.12.02

  2. Membrane secretory component is cleaved on the cell surface of rat hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Musil, L.S.; Baenziger, J.U.

    1986-03-05

    Transcellular transport of polymeric IgA from serum to bile by rat hepatocytes is mediated by a 105Kd membranous form of secretory component (mSC). In the presence or absence of IgA, mSC is cleaved and released into bile as a soluble 80Kd protein (fSC). They used monolayer cultures of rat hepatocytes, which synthesize mSC and efficiently cleave it to fSC, to determine the site of this conversion. (/sup 35/S)Cys-mSC accumulated in hepatocytes in the presence of leupeptin and was released as fSC when hepatocytes were placed in leupeptin-free media at 37/sup 0/. Small amounts of fSC were also produced when leupeptin was removed at 4/sup 0/, suggesting cleavage might occur on the cell membrane. Lactoperoxidase-catalyzed iodination of hepatocytes at 4/sup 0/ selectively labeled surface mSC which remained trypsin sensitive at 4/sup 0/. Hepatocytes maintained at 4/sup 0/ released significant amounts of /sup 125/I-mSC as fSC. Anti-SC antiserum reduced fSC generation at 4/sup 0/ by 70%. Following incubation at 37/sup 0/ for 10 min, /sup 125/I-mSC became resistant to degradation by trypsin and no production of fSC was seen if the cells were returned to 4/sup 0/. /sup 125/I-mSC was also cleaved to fSC following disruption by Dounce homogenization if cells were maintained at 4/sup 0/ following iodination but not if they were incubated at 37/sup 0/ for 10 min. They propose that mSC is cleaved to fSC at the plasma membrane but not intracellularly. This may reflect localization of the protease at the cell surface in a bile canalicular-like domain.

  3. The use of microencapsulated hepatocytes transplantation reduces mortality and liver alterations in Schistosoma mansoni infected hamsters.

    Science.gov (United States)

    Sherif, Soad A; Moharib, Mona N; El-Lakkany, Naglaa M; Hammam, Olfat A; Salman, Fatma H; El-Naggar, Mohamed M

    2014-04-01

    Hepatocyte transplantation is an attractive therapeutic modality for liver disease as an alternative for orthotropic liver transplantation. The goal of this work was to study the adequacy of intrasplenic hepatocyte transplantation (HCTx) in fresh and microencapsulated forms, in a hamster model of liver fibrosis by Schistosoma mansoni infected hamsters were divided into 6 groups; untreated for 11 weeks (GI) and for 15 weeks (GII), treated with praziquantel (PZQ) 7 weeks PI, and killed 4 weeks (GIII) and 8 weeks (GIV) post-treatment. Treated with PZQ 7 weeks PI, and then treated orally with immunosuppressive drug "cyclosporine (4 weeks post PZQ treatment), 24 hr. before interasplenic injection with fresh hepatocytes (V). Treated with PZQ 7 weeks PI, and then injected interasplenically (4 weeks post-treatment) with microencapsulated hepatocytes (GVI). GI & GIII were killed 11 weeks PI for assessment the anti-schistosomal efficacy of PZQ. The other four groups were killed 15 weeks PI for investigation of liver and spleen histology, serum liver enzymes and hepatic oxidative markers before and after HCTx. Freshly isolated hepatocytes with a mean viability 92.97 +/- 1.2% were used for microencapsulation and transplantation. Histological study showed the presence of transplanted hepatocytes in spleen of recipient. PZQ accelerated healing of hepatic granulomatous lesions as evidenced parasitologically by the increase in the percentage of dead eggs and histologically showing more granuloma circumscription with more ova degeneration and less inflammatory cells. The 25-day survival rates in GII, GIV, GV& GVI were 5/15 (33.3%), 8/15 (53.3%), 10/15 (66.7%) and 9/15 (60%) respectively. In addition, there were significantly better outcomes in serum biochemical indexes such as ALT, AST, gamma-GT, ALP, and hepatic SOD and MDA in the fresh and microencapsulated groups than in PZQ-treated group, without great differences between the microencapsulated and the fresh transplanted groups

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1986-03-05

    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 /sup 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 /sup 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 /sup 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.

  5. Characterization of liver-specific structure and function during hepatocyte spheroid self-assembly: Implications for a bioartificial liver device

    Science.gov (United States)

    Friend, Julie Renee

    A hollow fiber bioreactor containing collagen-entrapped hepatocytes has been developed as a bioartificial liver device. For clinical application, further scale-up of the device is desirable. This may be achieved through the use of hepatocyte spheroids, which are compacted aggregates that exhibit prolonged viability, higher liver-specific function and a more tissue-like ultrastructure compared to hepatocytes cultured as monolayers. In order to gain a better understanding of structural changes in spheroids over the course of their self-assembly, confocal microscopy was used to optically section spheroids and monitor changes in situ. Channels within spheroids hypothesized to be bile canaliculi were first evaluated by monitoring the diffusion of a fluorescent tracer, FITC-dextran, into spheroids. Three-dimensional reconstruction of spheroids showed that a continuous network of channels was forming within spheroids. Functionality of these channels as bile canaliculi was demonstrated by monitoring secretion of a fluorescently tagged bile acid, FITC-glycocholate, by hepatocytes in spheroids. Secretion of FITC-glycocholate could be seen in both rat and porcine hepatocyte spheroids. To elucidate changes in metabolism occurring during spheroid self-assembly, metabolic flux analysis was applied to hepatocyte spinner cultures. Glucose, lactate, amino acid, albumin and urea concentration in culture medium were measured and used to estimate intracellular fluxes within hepatocytes. Metabolism before and after spheroid formation was compared. Overall, little difference was seen in metabolism before and after spheroid self-assembly. As the BAL approaches clinical trials, methods of bioreactor storage for shipping and inventory purposed need to be developed. Storage conditions were tested in various hepatocyte culture systems. A protocol for storing reactors for 24 hours without significant loss in function was developed. Further optimization will be necessary for storage for longer

  6. Relationship between autophagy and the intracellular degradation of asialoglycoproteins in cultured rat hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Kindberg, G.M.; Refsnes, M.; Christoffersen, T.; Norum, K.R.; Berg, T.

    1987-05-25

    The relationship between autophagy and the intracellular distribution of endocytosed asialoorosomucoid was studied in cultured rat hepatocytes. Overt autophagy was induced by shifting the cells to a minimal salt medium. Incubation in minimal salt medium led to the formation of buoyant lysosomes at the expense of denser lysosomes manifested as a dual distribution of these organelles in Nycodenz gradients. Asialoorosomucoid was labeled with /sup 125/I-tyramine cellobiose. The labeled degradation products formed from this ligand are trapped at the site of degradation and may therefore serve as markers for the subgroup of lysosomes involved in the degradation. In control cells the degradation of the ligand was initiated in a light prelysosomal compartment and continued in denser lysosomes. In cells with high autophagic activity, the degradation of labeled asialoorosomucoid took place exclusively in a buoyant group of lysosomes. These results suggest that degradation of endocytosed ligand takes place in the same secondary lysosomes as substrate sequestered by autophagic mechanisms. These light lysosomes represent a subgroup of active lysosomes which are gradually recruited from dense bodies. Data are also presented that indicate that insulin may prevent the change in buoyant density brought about by incubation in deficient medium.

  7. Transplantation of human thioredoxin gene-modified hepatocytes for treatment of acute liver failure in rat model

    Institute of Scientific and Technical Information of China (English)

    LI Hua; JIANG Nan; ZHANG Jian; WANG Gen-shu; YANG Yang; CHEN Gui-hua

    2009-01-01

    Background Mostly because of the limited number and proliferative ability of the transplanted hepatocytes,hepatocyte transplantation offers only temporary support to the hepatic function with rather poor functional replacement of the damaged liver parenchyma.This study aimed to observe the therapeutic effect of human thioredoxin(hTrx)gene-modified hepatocytes on experimental acute liver failure in rats.Methods hTrx cDNA was obtained by reverse transcription-polymerase chain reaction(RT-PCR)from human osteosercoma 143(TK-)cells to construct the recombinant retrovirus vector pLEGFP/hTrx,which was packaged into PA317 cells to collect the recombinant retrovirus containing hTrx gene.After titration and characterization,the recombinant retrovirus was applied to primary cultured rat hepatocyte for infection to generate hTrx gene-modified rat hepatocytes,whose viability and antioxidative capacity were examined by immunohistochemistry and MIF assay,respectively.In a Sprague-Dawley(SD)rat model of acute liver failure,the modified hepatocytes were injected into the spleen,and the hepatic function and survival rate of the recipient rats were evaluated at different time points after the transplantation.Results NIH3T3 cells infected by the recombinant retrovirus were capable of expressing bioactive hTrx in the form of fusion proteins.Immunohistochemistry demonstrated normal function of the hTrx gene-modified hepatocytes,which possessed strong antioxidative capacity as shown by MTT assay.Transplantation of the modified hepatocytes in rats with acute liver failure resulted in significantly lowered serum alanine aminotransferase(ALT)and total bilirubin(TBIL)levels(P<0.05).The hepatocytes exhibited long-term survival and efficient proliferation after transplantation.Fourteen days after the operation,the rat models receiving hTrx gene-modified hepatocytes had significantly higher survival rate than those without the transplantation.Conclusion hTrx gene-modifled hepatocyte

  8. EXPERIMENTAL HEPATOCYTE XENOTRANSPLANTATION – A COMPREHENSIVE REVIEW OF THE LITERATURE

    Science.gov (United States)

    Zhou, Huidong; Liu, Hong; Ezzelarab, Mohamed; Schmelzer, Eva; Wang, Yi; Gerlach, Jörg; Gridelli, Bruno; Cooper, David K. C.

    2015-01-01

    Background Hepatocyte transplantation is a potential therapy for certain diseases of the liver, including hepatic failure. However, there is a limited supply of human livers as a source of cells and, after isolation, human hepatocytes can be difficult to expand in culture, limiting the number available for transplantation. Hepatocytes from other species, e.g., the pig, have therefore emerged as a potential alternative source. We searched the literature through the end of 2014 to assess the current status of experimental research into hepatocyte xenotransplantation. Literature search and results The literature search identified 51 reports of in vivo cross-species transplantation of hepatocytes in a variety of experimental models. Most studies investigated the transplantation of human (n=23) or pig (n=19) hepatocytes. No studies explored hepatocytes from genetically-engineered pigs. The spleen was the most common site of transplantation (n=23), followed by the liver (through the portal vein [n=6]) and peritoneal cavity (n=19). In 47 studies (92%), there was evidence of hepatocyte engraftment and function across a species barrier. Conclusions The data provided by this literature search strengthen the hypothesis that xenotransplantation of hepatocytes is feasible and potentially successful as a clinical therapy for certain liver diseases, including hepatic failure. By excluding vascular structures, hepatocytes isolated from genetically-engineered pig livers may address some of the immunological problems of xenotransplantation. PMID:25950141

  9. Experimental hepatocyte xenotransplantation--a comprehensive review of the literature.

    Science.gov (United States)

    Zhou, Huidong; Liu, Hong; Ezzelarab, Mohamed; Schmelzer, Eva; Wang, Yi; Gerlach, Jörg; Gridelli, Bruno; Cooper, David K C

    2015-01-01

    Hepatocyte transplantation (Tx) is a potential therapy for certain diseases of the liver, including hepatic failure. However, there is a limited supply of human livers as a source of cells and, after isolation, human hepatocytes can be difficult to expand in culture, limiting the number available for Tx. Hepatocytes from other species, for example, the pig, have therefore emerged as a potential alternative source. We searched the literature through the end of 2014 to assess the current status of experimental research into hepatocyte xenoTx. The literature search identified 51 reports of in vivo cross-species Tx of hepatocytes in a variety of experimental models. Most studies investigated the Tx of human (n = 23) or pig (n = 19) hepatocytes. No studies explored hepatocytes from genetically engineered pigs. The spleen was the most common site of Tx (n = 23), followed by the liver (through the portal vein [n = 6]) and peritoneal cavity (n = 19). In 47 studies (92%), there was evidence of hepatocyte engraftment and function across a species barrier. The data provided by this literature search strengthen the hypothesis that xenoTx of hepatocytes is feasible and potentially successful as a clinical therapy for certain liver diseases, including hepatic failure. By excluding vascular structures, hepatocytes isolated from genetically engineered pig livers may address some of the immunological problems of xenoTx.

  10. Hydrothermal calcium modification of 316L stainless steel and its apatite forming ability in simulated body fluid.

    Science.gov (United States)

    Valanezahad, Alireza; Ishikawa, Kunio; Tsuru, Kanji; Maruta, Michito; Matsuya, Shigeki

    2011-01-01

    To understand the feasibility of calcium (Ca) modification of type 316L stainless steel (316L SS) surface using hydrothermal treatment, 316L SS plates were treated hydrothermally in calcium chloride (CaCl(2)) solution. X-ray photoelectron spectroscopic analysis revealed that the surface of 316L SS plate was modified with Ca after hydrothermal treatment at 200°C. And the immobilized Ca increased with CaCl(2) concentration. However no Ca-modification was occurred for 316L SS plates treated at 100°C. When Ca-modified 316L SS plate was immersed in simulated body fluid (SBF) with ion concentrations nearly equal to those of human blood plasma, low crystalline apatite was precipitated on its surface whereas no precipitate was observed on non Ca-modified 316L SS. The results obtained in the present study indicated that hydrothermal treatment at 200°C in CaCl(2) solution is useful for Ca-modification of 316L SS, and Ca-modification plays important role for apatite precipitation in SBF.

  11. Bile acid formation in primary human hepatocytes

    Institute of Scientific and Technical Information of China (English)

    Curt Einarsson; Ewa Ellis; Anna Abrahamsson; Bo-G6ran Ericzon; Ingemar Bj rkhem; Magnus Axelson

    2000-01-01

    AIM To evaluate a culture system for bile acid formation in primary human hepatocytes in comparison with HepG2 cells. METHODS Hepatocytes were isolated from normal human liver tissue and were cultured in serum-free William's E medium. The medium was collected and renewed every 24 h. Bile acids and their precursors in media were finally analysed by gas chromatography-mass spectrometry. RESULTS Cholic acid ( CA ) andchenodeoxycholic acid (CDCA) conjugated with glycine or taurine accounted for 70% and 25% of total steroids. A third of CDCA was also conjugated with sulphuric acid. Dexamathasone and thyroid hormorm alone or in combination did not significantly effect bile acid formation. The addition of cyclosporin A (10 μmol/L) inhibited the synthesis of CA and CDCA by about 13% and 30%, respectively. CONCLUSION Isolated human hepatocytes in primary culture behave as in the intact liver by converting cholesterol to conjugated CA and CDCA. This is in contrast to cultured HepG2 cells, which release large amounts of bile acid precursors and unconjugated bile acids into the medium.

  12. Bile acid formation in primary human hepatocytes

    Institute of Scientific and Technical Information of China (English)

    Curt Einarsson; Ewa Ellis; Anna Abrahamsson; Bo-G ran Ericzon; Ingemar Bj rkhem; Magnus Axelson

    2000-01-01

    AIM To evaluate a system for bile acid formation in human hepatocytes in comparison with HepG2 cells.METHODS Hepatocytes were isolated from normal human liver tissue and were cultured in serum-freeWilliam's E medium. The medium was collected and renewed every 24 h. Bile acids and their precursors inmedia were finally analysed by gas chromatography-mass spectrometry.RESULTS Cholic acid (CA) and chenodeoxycholic acid (CDCA) conjugated with glycine or taurineaccounted for 70% and 25% of total steroids. One third of CDCA was also conjugated with sulphuric acid.Dexamethasone and thyroid hormone alone or in combination did not significantly affect bile acid formation.The addition of cyclosporin A (10 tm) inhibited the synthesis of CA and CDCA by about 13% and 30%,respectively.CONCLUSION Isolated human hepatocytes in primary culture behave as in the intact liver by convertingalmost quantitatively cholesterol to conjugated CA and CDCA. This is in contrast to cultured HepG2 cells,which release large amounts of bile acid precursors and unconjugated bile acids into the medium.

  13. Hepatocyte xenotransplantation for treating liver disease.

    Science.gov (United States)

    Bonavita, André Gustavo; Quaresma, Kátia; Cotta-de-Almeida, Vinícius; Pinto, Marcelo Alves; Saraiva, Roberto Magalhães; Alves, Luiz Anastácio

    2010-01-01

    The treatment of acute and chronic liver failure is still a challenge despite modern therapeutic innovations. While liver transplantation can restore liver function and improve patient survival, donor shortages limit this treatment to a small number of patients. Cellular xenotransplantation has emerged as an alternative for treating liver failure. Xenohepatocytes could be readily available in sufficient quantities to treat patients in critical condition and thereby reduce the donor shortage. The use of isolated encapsulated or non-encapsulated cells can reduce the immunorejection response. Several studies using animal models of acute or chronic liver failure have demonstrated improved survival and recovery of liver function after xenotransplantation of adult hepatocytes. Porcine liver cells are a potential source of xenohepatocytes due to similarities with human physiology and the great number of hepatocytes that can be obtained. The recent development of less immunogenic transgenic pigs, new immunosuppressive drugs, and cellular encapsulation systems represents important advances in the field of cellular xenotransplantation. In this study, we review the work carried out in animal models that deals with the advantages and limitations of hepatocyte xenotransplantation, and we propose new studies needed in this field.

  14. Conditional genetic elimination of hepatocyte growth factor in mice compromises liver regeneration after partial hepatectomy.

    Directory of Open Access Journals (Sweden)

    Kari Nejak-Bowen

    Full Text Available Hepatocyte growth factor (HGF has been shown to be indispensable for liver regeneration because it serves as a main mitogenic stimulus driving hepatocytes toward proliferation. We hypothesized that ablating HGF in adult mice would have a negative effect on the ability of hepatocytes to regenerate. Deletion of the HGF gene was achieved by inducing systemic recombination in mice lacking exon 5 of HGF and carrying the Mx1-cre or Cre-ER(T transgene. Analysis of liver genomic DNA from animals 10 days after treatment showed that a majority (70-80% of alleles underwent cre-induced genetic recombination. Intriguingly, however, analysis by RT-PCR showed the continued presence of both unrecombined and recombined forms of HGF mRNA after treatment. Separation of liver cell populations into hepatocytes and non-parenchymal cells showed equal recombination of genomic HGF in both cell types. The presence of the unrecombined form of HGF mRNA persisted in the liver in significant amounts even after partial hepatectomy (PH, which correlated with insignificant changes in HGF protein and hepatocyte proliferation. The amount of HGF produced by stellate cells in culture was indirectly proportional to the concentration of HGF, suggesting that a decrease in HGF may induce de novo synthesis of HGF from cells with residual unrecombined alleles. Carbon tetrachloride (CCl4-induced regeneration resulted in a substantial decrease in preexisting HGF mRNA and protein, and subsequent PH led to a delayed regenerative response. Thus, HGF mRNA persists in the liver even after genetic recombination affecting most cells; however, PH subsequent to CCl4 treatment is associated with a decrease in both HGF mRNA and protein and results in compromised liver regeneration, validating an important role of this mitogen in hepatic growth.

  15. Primary Hepatocytes Cultured on a Fiber-Embedded PDMS Chip to Study Drug Metabolism

    Directory of Open Access Journals (Sweden)

    Yaowen Liu

    2017-06-01

    Full Text Available In vitro drug screening using reliable and predictable liver models remains a challenge. The identification of an ideal biological substrate is essential to maintain hepatocyte functions during in vitro culture. Here, we developed a fiber-embedded polydimethylsiloxane (PDMS chip to culture hepatocytes. Hepatocyte spheroids formed in this device were subjected to different flow rates, of which a flow rate of 50 μL/min provided the optimal microenvironment for spheroid formation, maintained significantly higher rates of albumin and urea synthesis, yielded higher CYP3A1 (cytochrome P450 3A1 and CYP2C11 (cytochrome P450 2C11 enzyme activities for metabolism, and demonstrated higher expression levels of liver-specific genes. In vitro metabolism tests on tolbutamide and testosterone by hepatocytes indicated predicted clearance rates of 1.98 ± 0.43 and 40.80 ± 10.13 mL/min/kg, respectively, which showed a good in vitro–in vivo correspondence. These results indicate that this system provides a strategy for the construction of functional engineered liver tissue that can be used to study drug metabolism.

  16. Hyperinsulinemia is Associated with Increased Soluble Insulin Receptors Release from Hepatocytes

    Science.gov (United States)

    Hiriart, Marcia; Sanchez-Soto, Carmen; Diaz-Garcia, Carlos Manlio; Castanares, Diana T.; Avitia, Morena; Velasco, Myrian; Mas-Oliva, Jaime; Macias-Silva, Marina; González-Villalpando, Clicerio; Delgado-Coello, Blanca; Sosa-Garrocho, Marcela; Vidaltamayo, Román; Fuentes-Silva, Deyanira

    2014-01-01

    It has been generally assumed that insulin circulates freely in blood. However it can also interact with plasma proteins. Insulin receptors are located in the membrane of target cells and consist of an alpha and beta subunits with a tyrosine kinase cytoplasmic domain. The ectodomain, called soluble insulin receptor (SIR) has been found elevated in patients with diabetes mellitus. We explored if insulin binds to SIRs in circulation under physiological conditions and hypothesize that this SIR may be released by hepatocytes in response to high insulin concentrations. The presence of SIR in rat and human plasmas and the culture medium of hepatocytes was explored using Western blot analysis. A purification protocol was performed to isolated SIR using affinity, gel filtration, and ion exchange chromatographies. A modified reverse hemolytic plaque assay was used to measure SIR release from cultured hepatocytes. Incubation with 1 nmol l−1 insulin induces the release of the insulin receptor ectodomains from normal rat hepatocytes. This effect can be partially prevented by blocking protease activity. Furthermore, plasma levels of SIR were higher in a model of metabolic syndrome, where rats are hyperinsulinemic. We also found increased SIR levels in hyperinsulinemic humans. SIR may be an important regulator of the amount of free insulin in circulation. In hyperinsulinemia, the amount of this soluble receptor increases and this could lead to higher amounts of insulin bound to this receptor, rather than free insulin, which is the biologically active form of the hormone. This observation could enlighten the mechanisms of insulin resistance. PMID:24995000

  17. Hyperinsulinemia is associated with increased soluble insulin receptors release from hepatocytes

    Directory of Open Access Journals (Sweden)

    Marcia eHiriart

    2014-06-01

    Full Text Available It has been generally assumed that insulin circulates freely in blood. However it can also interact with plasma proteins. Insulin receptors are located in the membrane of target cells and consist of an alpha and beta subunits with a tyrosine kinase cytoplasmic domain. The ectodomain, called soluble insulin receptor (SIR has been found elevated in patients with diabetes mellitus. We explored if insulin binds to SIRs in circulation under physiological conditions and hypothesize that this SIR may be released by hepatocytes in response to high insulin concentrations. The presence of SIR in rat and human plasmas and the culture medium of hepatocytes was explored using Western blot analysis. A purification protocol was performed to isolated SIR using affinity, gel filtration and ion exchange chromatographies. A modified reverse hemolytic plaque assay was used to measure SIR release from cultured hepatocytes. Incubation with 1 nmol l-1 insulin induces the release of the insulin receptor ectodomains from normal rat hepatocytes. This effect can be partially prevented by blocking protease activity. Furthermore, plasma levels of SIR were higher in a model of metabolic syndrome, where rats are hyperinsulinemic. We also found increased SIR levels in hyperinsulinemic humans. SIR may be an important regulator of the amount of free insulin in circulation. In hyperinsulinemia the amount of this soluble receptor increases, this could lead to higher amounts of insulin bound to this receptor, rather than free insulin, which is the biologically active form of the hormone. This observation could enlighten the mechanisms of insulin resistance.

  18. Claudin-2 promotes breast cancer liver metastasis by facilitating tumor cell interactions with hepatocytes.

    Science.gov (United States)

    Tabariès, Sébastien; Dupuy, Fanny; Dong, Zhifeng; Monast, Anie; Annis, Matthew G; Spicer, Jonathan; Ferri, Lorenzo E; Omeroglu, Atilla; Basik, Mark; Amir, Eitan; Clemons, Mark; Siegel, Peter M

    2012-08-01

    We previously identified claudin-2 as a functional mediator of breast cancer liver metastasis. We now confirm that claudin-2 levels are elevated in liver metastases, but not in skin metastases, compared to levels in their matched primary tumors in patients with breast cancer. Moreover, claudin-2 is specifically expressed in liver-metastatic breast cancer cells compared to populations derived from bone or lung metastases. The increased liver tropism exhibited by claudin-2-expressing breast cancer cells requires claudin-2-mediated interactions between breast cancer cells and primary hepatocytes. Furthermore, the reduction of the claudin-2 expression level, either in cancer cells or in primary hepatocytes, diminishes these heterotypic cell-cell interactions. Finally, we demonstrate that the first claudin-2 extracellular loop is essential for mediating tumor cell-hepatocyte interactions and the ability of breast cancer cells to form liver metastases in vivo. Thus, during breast cancer liver metastasis, claudin-2 shifts from acting within tight-junctional complexes to functioning as an adhesion molecule between breast cancer cells and hepatocytes.

  19. Role of apoptotic hepatocytes in HCV dissemination: regulation by acetaldehyde.

    Science.gov (United States)

    Ganesan, Murali; Natarajan, Sathish Kumar; Zhang, Jinjin; Mott, Justin L; Poluektova, Larisa I; McVicker, Benita L; Kharbanda, Kusum K; Tuma, Dean J; Osna, Natalia A

    2016-06-01

    Alcohol consumption exacerbates hepatitis C virus (HCV) pathogenesis and promotes disease progression, although the mechanisms are not quite clear. We have previously observed that acetaldehyde (Ach) continuously produced by the acetaldehyde-generating system (AGS), temporarily enhanced HCV RNA levels, followed by a decrease to normal or lower levels, which corresponded to apoptosis induction. Here, we studied whether Ach-induced apoptosis caused depletion of HCV-infected cells and what role apoptotic bodies (AB) play in HCV-alcohol crosstalk. In liver cells exposed to AGS, we observed the induction of miR-122 and miR-34a. As miR-34a has been associated with apoptotic signaling and miR-122 with HCV replication, these findings may suggest that cells with intensive viral replication undergo apoptosis. Furthermore, when AGS-induced apoptosis was blocked by a pan-caspase inhibitor, the expression of HCV RNA was not changed. AB from HCV-infected cells contained HCV core protein and the assembled HCV particle that infect intact hepatocytes, thereby promoting the spread of infection. In addition, AB are captured by macrophages to switch their cytokine profile to the proinflammatory one. Macrophages exposed to HCV(+) AB expressed more IL-1β, IL-18, IL-6, and IL-10 mRNAs compared with those exposed to HCV(-) AB. The generation of AB from AGS-treated HCV-infected cells even enhanced the induction of aforementioned cytokines. We conclude that HCV and alcohol metabolites trigger the formation of AB containing HCV particles. The consequent spread of HCV to neighboring hepatocytes via infected AB, as well as the induction of liver inflammation by AB-mediated macrophage activation potentially exacerbate the HCV infection course by alcohol and worsen disease progression.

  20. Role of 4-bromophenol and 4-bromocatechol in bromobenzene covalent binding and toxicity in isolated rat hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Dankovic, D.A.; Billings, R.E.

    1985-06-30

    4-Bromophenol and 4-bromocatechol are formed as metabolites of bromobenzene in vivo and in isolated rat hepatocytes. Both of these metabolites may potentially contribute to the hepatotoxicity of bromobenzene. Bromobenzene metabolism in hepatocytes isolated from phenobarbital-treated rats forms 0.12 to 0.17 mM 4-bromophenol and 4-bromocatechol in 2 hr, with 1 to 3 mM bromobenzene. The role of activated metabolites derived from 4-bromophenol and 4-bromocatechol in bromobenzene covalent binding and toxicity was investigated with isolated hepatocytes in suspension. The covalent binding of the phenol and the catechol was increased four- to eightfold by the addition of unlabeled bromobenzene. Two-hour incubations of 0.25 mM /sup 14/C-labeled 4-bromophenol or 4-bromocatechol with hepatocytes isolated from phenobarbital-treated rats resulted, under these conditions, in no significant toxicity, and approximately 4 and 25%, respectively, of the covalent binding associated with bromobenzene itself. Two- and six-hour incubations with higher 4-bromophenol and 4-bromocatechol concentrations demonstrated that 1 to 3 mM substrate concentrations were required for cytotoxicity. These results show that metabolically produced 4-bromophenol and 4-bromocatechol do not play significant roles in the production of bromobenzene cytotoxicity in isolated hepatocytes, and that they contribute only modestly to bromobenzene covalent binding.

  1. Stem cell-derived hepatocytes for functional liver replacement

    Directory of Open Access Journals (Sweden)

    Bruno eChrist

    2012-06-01

    Full Text Available Mesenchymal stem cells (MSC represent an alternate cell source to substitute for primary hepatocytes in hepatocyte transplantation because of their multiple differentiation potential and nearly unlimited availability. They may differentiate into hepatocyte-like cells in vitro and maintain specific hepatocyte functions also after transplantation into the regenerating livers of mice or rats both under injury and non-injury conditions. Depending on the underlying liver disease their mode of action is either to replace the diseased liver tissue or to support liver regeneration through their anti-inflammatory and anti-apoptotic as well as their pro-proliferative action.

  2. Heterogeneity of renin substrate released from hepatocytes and in brain extracts

    Energy Technology Data Exchange (ETDEWEB)

    Murakami, E.; Eggena, P.; Barrett, J.D.; Sambhi, M.P.

    1984-01-23

    Renin substrate was characterized in incubation medium of isolated hepatocytes, plasma, and brain extracts of the rat by isoelectric focusing and polyacrylamide gel electrophoresis. The isoelectric focusing (IEF) profile of renin substrate released into incubation medium of rat hepatocytes demonstrated two peaks with isoelectric points (pI) of 4.1 (minor peak) and 4.6 (major peak). Extracts of normal rat brain also showed two forms (pI 4.6 major form, and pI 5.1 minor form). In contrast, normal rat plasma contained a single broad peak of substrate with pI 4.5. Intraperitoneal injection of 17..beta.. estradiol (1 mg) or bilateral nephrectomy significantly elevated renin substrate levels in plasma and increased its release from hepatocytes, however, no change in the IEF and PAGE profiles was evident. Molecular weights of renin substrate were 60,000-65,000 from all preparations. The presence of distinct forms of brain renin substrate and the lack of an increase in brain renin substrate following nephrectomy or estrogen treatment suggest local synthesis and support the postulate of an independent renin-angiotensin system in the central nervous system.

  3. Concise Review: Advances in Generating Hepatocytes from Pluripotent Stem Cells for Translational Medicine.

    Science.gov (United States)

    Szkolnicka, Dagmara; Hay, David C

    2016-06-01

    The liver is one of the major organs in the human body. Severe or prolonged exposure of the liver to different factors may cause life-threatening disease, which necessitates donor organ transplantation. While orthotopic liver transplantation can be used to effectively treat liver failure, it is an invasive procedure, which is severely limited by organ donation. Therefore, alternative sources of liver support have been proposed and studied. This includes the use of pluripotent stem cell-derived hepatocytes as a renewable source of cells for therapy. In addition to cell-based therapies, in vitro engineered liver tissue provides powerful models for human drug discovery and disease modeling. This review focuses on the generation of hepatocyte-like cells from pluripotent stem cells and their application in translational medicine. Stem Cells 2016;34:1421-1426.

  4. 四大象形文字中的身体运动形态研究%A study of body movement forms in four major hieroglyphs

    Institute of Scientific and Technical Information of China (English)

    张洁; 胡小明

    2014-01-01

    通过对世界四大象形文字体系:古埃及圣书字、苏美尔楔形文字、中美洲玛雅文字和中国甲骨文中以人及其行为构形的文字进行统计与分析,探讨人类早期生活中的身体运动情况。经研究发现在人类的早期生活中存在有丰富的身体运动形态,这些身体运动是早期先民认知活动的主要途径和推动力之一,对文字的形成具有重要的推动作用。从象形文字中所见的人类早期身体运动的内容,对体育与文化的萌生具有发生学意义上的重要研究价值。%By collating and analyzing 4 major hieroglyphic systems in the world: ancient Egyptian hieroglyph, Sumerian cuneiform, central American Maya hieroglyph and Chinese oracle bone inscription as well as their be-havior depicting characters, the authors probed into body movements in the life of human beings in the early peri-ods, and found that in the life of human beings in the early periods, there were a lot of body movement forms, these body movements were one of primary ways and driving forces for the cognitive activities of the ancestors in the early periods, playing an important role in boosting the formation of characters. The content of body movements of human beings in the early periods, as seen in hieroglyphs, has an embryologically important value in studying the birth of sports and cultures.

  5. The origin of biliary ductular cells that appear in the spleen after transplantation of hepatocytes.

    Science.gov (United States)

    Fukuda, Kenji; Sugihara, Ayako; Nakasho, Keiji; Tsujimura, Tohru; Yamada, Naoko; Okaya, Atsuhito; Sakagami, Masafumi; Terada, Nobuyuki

    2004-01-01

    Transplantation of rat hepatocytes into the syngeneic rat spleen results in the appearance of cytokeration (CK)-19-positive biliary cells that form ductules. The exact origin of CK-19-positive cells is not known and the possibility that they are derived from biliary cells or precursors of oval cells in transplanted hepatocyte preparations has been raised. In the present study, we found that the number of CK-19-positive biliary cells increased rapidly after transplantation of hepatocytes, reached the maximum at 4 weeks, and then gradually decreased. However, a Ki-67 labeling index of CK-19-positive biliary cells was low and showed no significant changes throughout the experimental period. In addition, no or few CK-19-positive cells appeared in the spleen after transplantation of nonparenchymal liver cells enriched with biliary cells. These results showed that biliary cells were not the source of CK-19-positive cells in the spleen. Impairment of precursors of oval cells in the liver by administration of 4,4'-diaminodiphenylmethane 24 h before transplantation of hepatocytes did not prevent the appearance of CK-19-positive biliary cells in the spleen. Moreover, transplantation of nonparenchymal cells carrying an increased number of oval cells by means of treatment with 2-acetylaminofluorene and partial hepatectomy resulted in no appearance of CK-19-positive biliary cells in the spleen. These results ruled out oval cells as the origin of CK-19-positive biliary cells in the spleen. Because CK-19-positive biliary cells appeared in the spleen only when hepatocyte fractions were transplanted, we suggest transdifferentiation of heptocytes may be the mechanism by which CK-19-positive biliary cells are generated.

  6. Subculture of proliferating adult rat hepatocytes in medium supplemented with nicotinamide and EGF.

    Science.gov (United States)

    Mitaka, T; Kojima, T; Mizuguchi, T; Mochizuki, Y

    1996-09-01

    To establish parenchymal hepatocyte cell lines, we tried to subculture the primary hepatocytes isolated from adult rats. The hepatocytes were cultured in serum-free modified Dulbecco's modified Eagle's medium supplemented with 10 mM nicotinamide and 10 ng/ml epidermal growth factor. When 6 x 10(5) cells were plated on 35-mm dishes coated with rat tail collagen, the cells proliferated and reached confluence at Day 6 to Day 8. The first subculture was carried out at Day 8 using 0.005% collagenase and gentle pipettings. Most cells were recovered and plated on the new dishes coated with the collagen (first passage). The attached cells could proliferate and reached near confluence when the cells occupied more than two-thirds of the dish surface. About a week after the first subculture, the second one was conducted. Although the number of the recovered cells was smaller than at the first passage, the cells could attach and proliferate to a certain extent. Thereafter, they were maintained for more than 2 mo, but they never overgrew. Albumin secretion into the culture medium was confirmed in the subcultured cells. Ultrastructurally, these subcultured cells possessed hepatic characteristics such as peroxisomes with a crystalline nucleiod and bile-canaliculus structures. When 10% fetal bovine serum and ascorbic acid 2-phosphate were added to the cells of the second passage, they began to proliferate very slowly. These proliferating cells were mainly mononucleate and had a small cytoplasm. In addition, some of them could differentitate into typical mature hepatocytes by forming a three-dimensional structure interacting with nonparenchymal cells. In this experiment, we showed the successful subculturing of parenchymal hepatocytes isolated from adult rats and provided evidence that the subcultured cells still have the potential to proliferate and to differentiate.

  7. 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.

  8. Cryopreservation and gel collagen culture of porcine hepatocytes

    Institute of Scientific and Technical Information of China (English)

    Hong-Ling Liu; Ying-Jie Wang; Hai-Tao Guo; Yu-Ming Wang; Jun Liu; Yue-Cheng Yu

    2004-01-01

    AIM: To study the method of cryopreserving porcine hepatocytes and gel collagen culture measure after its cryopreservation.METHODS: Hepatocytes, isolated from Chinese experimental suckling mini-pigs by two-step perfusion with collagenase using an extra corporeal perfusion apparatus, were cryopreserved with 50 mL/L to 200 mL/L DMSO in liquid nitrogen for 4 mo, then thawed and seeded in 1 or between 2 layers of gel collagen. The expression of porcine albumin message RNA, cellular morphology and content of aspartate aminotransferase (AST) and urea nitrogen (UN) were examined during culture in gel.RESULTS: Viability of 150 mL/L DMSO group thawed hepatocytes was (83±4)%, but after purification, its viability was (90±5)%, attachment efficiency was (86±7)%, the viability of thawed hepatocytes was near to fresh cells. When the thawed hepatocytes were cultivated in gel collagen with culture medium adding epidermal growth factor, the hepatocytes grew in various administrative levels in mixed collagen gel, and bunchy in the sandwich configuration cultures. For up to 10 days' culture, the typical cellular morphological characteristics of cultivated hepatocytes could be observed. The leakage of AST was lower during culture in gel than that in common culture. At the same time, the UN synthesized by cells cultivated in mixed gel collagen was higher than that in other groups.CONCLUSION: Storage in liquid nitrogen can long keep hepatocytes' activities, the concentration of 150 mL/L DMSO is fit for porcine hepatocytes' cryopreservation. Thawed hepatocytes can be cultivated with coliagenous matrix, which provides an environment that more closely resembles that in vivo and maintain the expression of certain liver-specific function of hepatocytes.

  9. Body contact and body language

    DEFF Research Database (Denmark)

    Winther, Helle Dagmar

    2008-01-01

    Body contact and body language are unique and existential and, although culturally dependent and socially embodied, they are also universal communication forms. For small children all over the world, warm, close and nourishing body contact is fundamental to their embodied experi­ence of themselve...

  10. Scavenger receptor BI boosts hepatocyte permissiveness to Plasmodium infection.

    NARCIS (Netherlands)

    Yalaoui, S.; Huby, T.; Franetich, J.F.; Gego, A.; Rametti, A.; Moreau, M.; Collet, X.; Siau, A.; Gemert, G.J.A. van; Sauerwein, R.W.; Luty, A.J.F.; Vaillant, J.C.; Hannoun, L.; Chapman, J.; Mazier, D.; Froissard, P.

    2008-01-01

    Infection of hepatocytes by Plasmodium falciparum sporozoites requires the host tetraspanin CD81. CD81 is also predicted to be a coreceptor, along with scavenger receptor BI (SR-BI), for hepatitis C virus. Using SR-BI-knockout, SR-BI-hypomorphic and SR-BI-transgenic primary hepatocytes, as well as s

  11. Pluripotent stem cell-derived hepatocyte-like cells.

    Science.gov (United States)

    Schwartz, R E; Fleming, H E; Khetani, S R; Bhatia, S N

    2014-01-01

    Liver disease is an important clinical problem, impacting over 30 million Americans and over 600 million people worldwide. It is the 12th leading cause of death in the United States and the 16th worldwide. Due to a paucity of donor organs, several thousand Americans die yearly while waiting for liver transplantation. Unfortunately, alternative tissue sources such as fetal hepatocytes and hepatic cell lines are unreliable, difficult to reproduce, and do not fully recapitulate hepatocyte phenotype and functions. As a consequence, alternative cell sources that do not have these limitations have been sought. Human embryonic stem (hES) cell- and induced pluripotent stem (iPS) cell-derived hepatocyte-like cells may enable cell based therapeutics, the study of the mechanisms of human disease and human development, and provide a platform for screening the efficacy and toxicity of pharmaceuticals. iPS cells can be differentiated in a step-wise fashion with high efficiency and reproducibility into hepatocyte-like cells that exhibit morphologic and phenotypic characteristics of hepatocytes. In addition, iPS-derived hepatocyte-like cells (iHLCs) possess some functional hepatic activity as they secrete urea, alpha-1-antitrypsin, and albumin. However, the combined phenotypic and functional traits exhibited by iHLCs resemble a relatively immature hepatic phenotype that more closely resembles that of fetal hepatocytes rather than adult hepatocytes. Specifically, iHLCs express fetal markers such as alpha-fetoprotein and lack key mature hepatocyte functions, as reflected by drastically reduced activity (~0.1%) of important detoxification enzymes (i.e. CYP2A6, CYP3A4). These key differences between iHLCs and primary adult human hepatocytes have limited the use of stem cells as a renewable source of functional adult hepatocytes for in vitro and in vivo applications. Unfortunately, the developmental pathways that control hepatocyte maturation from a fetal into an adult hepatocyte are

  12. Combining a Laboratory Practical Class with a Computer Simulation: Studies on the Synthesis of Urea in Isolated Hepatocytes.

    Science.gov (United States)

    Bender, David A.

    1986-01-01

    Describes how a computer simulation is used with a laboratory experiment on the synthesis of urea in isolated hepatocytes. The simulation calculates the amount of urea formed and the amount of ammonium remaining as the concentrations of ornithine, citrulline, argininosuccinate, arginine, and aspartate are altered. (JN)

  13. Trabalho, corpo e subjetividade: toyotismo e formas de precariedade no capitalismo global Labor, body and subjectivity: toyotism and forms of precarization in global capitalism

    Directory of Open Access Journals (Sweden)

    Giovanni Alves

    2005-09-01

    Full Text Available Nos últimos trinta anos de desenvolvimento capitalista, ocorreram transformações significativas nas diversas instâncias do ser social, com destaque para o mundo do trabalho e da reprodução social. Desenvolve-se o toyotismo, ideologia orgânica da nova produção capitalista, 'momento predominante' da reestruturação produtiva do capital. Sob o toyotismo, tende a constituir-se, pelo menos como 'promessa frustrada' do capital, o que iremos denominar 'compressão psicocorporal'. Esta constitui-se como um elemento da nova disposição sócio-subjetiva instaurada pelo toyotismo que caracteriza uma nova experiência do corpo, tanto no processo de trabalho quanto no processo sócio-reprodutivo.Many significant transformations in various instances of the social being took place over the past thirty years of capitalist development, especially in the sphere of labor and of social reproduction. That is the period when toyotism, the organic ideology of the new form of capitalist production and the most important moment in the productive reorganization of capitalism, was developed. Under the ideology of toyotism, something we will name 'mind-body compression' tends to take place (at least as capitalism's 'unkept promise'. This is a new element of the social and subjective arrangement instituted by toyotism that characterizes a new way of experiencing the body both in labor and social reproduction processes.

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

    Science.gov (United States)

    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 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 mice and hepatocytes. Taken together

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

    Science.gov (United States)

    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.

    2012-01-01

    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 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 mice and hepatocytes. Taken together

  16. 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.

  17. Temperature shift and host cell contact up-regulate sporozoite expression of Plasmodium falciparum genes involved in hepatocyte infection.

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    Anthony Siau

    proteins involved in hepatocyte invasion, while the other two were predominantly expressed during hepatic parasite development. The genome-wide up-regulation of expression observed supports the hypothesis that the shift from the mosquito to the mammalian host contributes to activate quiescent salivary gland sporozoites into a state of readiness for the hepatic stages. Functional studies on four of the up-regulated genes validated our approach as one means to determine the repertoire of proteins implicated during the early events of the Plasmodium infection, and in this case that of P. falciparum, the species responsible for the severest forms of malaria.

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

    Science.gov (United States)

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

    2013-01-01

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

  19. An Explicit Formulation of Singularity-Free Dynamic Equations of Mechanical Systems in Lagrangian Form---Part one: Single Rigid Bodies

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    Pål Johan From

    2012-04-01

    differential geometry to implement the dynamic equations of rigid bodies without the presence of singularities. Presenting the explicit dynamic equations also allows for more insight into the dynamic structure of the system. Another motivation is to correct some errors commonly found in the literature. Unfortunately, the formulation of the Boltzmann-Hamel equations used here are presented incorrectly. This has been corrected by the authors, but we present here, for the first time, the detailed mathematical details on how to arrive at the correct equations. We also show through examples that using the equations presented here, the dynamics of a single rigid body is reduced to the standard equations on a Lagrangian form, for example Euler's equations for rotational motion and Euler--Lagrange equations for free motion.

  20. A buffered form of creatine does not promote greater changes in muscle creatine content, body composition, or training adaptations than creatine monohydrate

    Directory of Open Access Journals (Sweden)

    Jagim Andrew R

    2012-09-01

    Full Text Available Abstract Background Creatine monohydrate (CrM has been consistently reported to increase muscle creatine content and improve high-intensity exercise capacity. However, a number of different forms of creatine have been purported to be more efficacious than CrM. The purpose of this study was to determine if a buffered creatine monohydrate (KA that has been purported to promote greater creatine retention and training adaptations with fewer side effects at lower doses is more efficacious than CrM supplementation in resistance-trained individuals. Methods In a double-blind manner, 36 resistance-trained participants (20.2 ± 2 years, 181 ± 7 cm, 82.1 ± 12 kg, and 14.7 ± 5% body fat were randomly assigned to supplement their diet with CrM (Creapure® AlzChem AG, Trostberg, Germany at normal loading (4 x 5 g/d for 7-days and maintenance (5 g/d for 21-days doses; KA (Kre-Alkalyn®, All American Pharmaceutical, Billings, MT, USA at manufacturer’s recommended doses (KA-L, 1.5 g/d for 28-days; or, KA with equivalent loading (4 x 5 g/d for 7-days and maintenance (5 g/d doses of CrM (KA-H. Participants were asked to maintain their current training programs and record all workouts. Muscle biopsies from the vastus lateralis, fasting blood samples, body weight, DEXA determined body composition, and Wingate Anaerobic Capacity (WAC tests were performed at 0, 7, and 28-days while 1RM strength tests were performed at 0 and 28-days. Data were analyzed by a repeated measures multivariate analysis of variance (MANOVA and are presented as mean ± SD changes from baseline after 7 and 28-days, respectively. Results Muscle free creatine content obtained in a subgroup of 25 participants increased in all groups over time (1.4 ± 20.7 and 11.9 ± 24.0 mmol/kg DW, p = 0.03 after 7 and 28-days, respectively, with no significant differences among groups (KA-L −7.9 ± 22.3, 4.7 ± 27.0; KA-H 1.0 ± 12.8, 9.1

  1. The extracellular redox state modulates mitochondrial function, gluconeogenesis, and glycogen synthesis in murine hepatocytes.

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    Laura Nocito

    Full Text Available Circulating redox state changes, determined by the ratio of reduced/oxidized pairs of different metabolites, have been associated with metabolic diseases. However, the pathogenic contribution of these changes and whether they modulate normal tissue function is unclear. As alterations in hepatic gluconeogenesis and glycogen metabolism are hallmarks that characterize insulin resistance and type 2 diabetes, we tested whether imposed changes in the extracellular redox state could modulate these processes. Thus, primary hepatocytes were treated with different ratios of the following physiological extracellular redox couples: β-hydroxybutyrate (βOHB/acetoacetate (Acoc, reduced glutathione (GSH/oxidized glutathione (GSSG, and cysteine/cystine. Exposure to a more oxidized ratio via extracellular βOHB/Acoc, GSH/GSSG, and cysteine/cystine in hepatocytes from fed mice increased intracellular hydrogen peroxide without causing oxidative damage. On the other hand, addition of more reduced ratios of extracellular βOHB/Acoc led to increased NAD(PH and maximal mitochondrial respiratory capacity in hepatocytes. Greater βOHB/Acoc ratios were also associated with decreased β-oxidation, as expected with enhanced lipogenesis. In hepatocytes from fasted mice, a more extracellular reduced state of βOHB/Acoc led to increased alanine-stimulated gluconeogenesis and enhanced glycogen synthesis capacity from added glucose. Thus, we demonstrated for the first time that the extracellular redox state regulates the major metabolic functions of the liver and involves changes in intracellular NADH, hydrogen peroxide, and mitochondrial respiration. Because redox state in the blood can be communicated to all metabolically sensitive tissues, this work confirms the hypothesis that circulating redox state may be an important regulator of whole body metabolism and contribute to alterations associated with metabolic diseases.

  2. Down-regulation of microRNA-26a promotes mouse hepatocyte proliferation during liver regeneration.

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    Jian Zhou

    Full Text Available BACKGROUND: Inadequate liver regeneration (LR is still an unsolved problem in major liver resection and small-for-size syndrome post-living donor liver transplantation. A number of microRNAs have been shown to play important roles in cell proliferation. Herein, we investigated the role of miR-26a as a pivotal regulator of hepatocyte proliferation in LR. METHODOLOGY/PRINCIPAL FINDINGS: Adult male C57BL/6J mice, undergoing 70% partial hepatectomy (PH, were treated with Ad5-anti-miR-26a-LUC or Ad5-miR-26a-LUC or Ad5-LUC vector via portal vein. The animals were subjected to in vivo bioluminescence imaging. Serum and liver samples were collected to test liver function, calculate liver-to-body weight ratio (LBWR, document hepatocyte proliferation (Ki-67 staining, and investigate potential targeted gene expression of miR-26a by quantitative real-time PCR and Western blot. The miR-26a level declined during LR after 70% PH. Down-regulation of miR-26a by anti-miR-26a expression led to enhanced proliferation of hepatocytes, and both LBWR and hepatocyte proliferation (Ki-67(+ cells % showed an increased tendency, while liver damage, indicated by aspartate aminotransferase (AST, alanine aminotransferase (ALT and total bilirubin (T-Bil, was reduced. Furthermore, CCND2 and CCNE2, as possible targeted genes of miR-26a, were up-regulated. In addition, miR-26a over-expression showed converse results. CONCLUSIONS/SIGNIFICANCE: MiR-26a plays crucial role in regulating the proliferative phase of LR, probably by repressing expressions of cell cycle proteins CCND2 and CCNE2. The current study reveals a novel miRNA-mediated regulation pattern during the proliferative phase of LR.

  3. Isolation and Primary Culture of Rat Hepatocytes Using Kiwifruit Actinidin

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    Z. Shirvani Farsani

    2007-07-01

    Full Text Available Introduction & Objective: Isolation of cells from different tissues rely on proteolytic enzymes mainly collagenases that selectively digest collagen fibers of extra-cellular matrix. It is important to find new and proper collagenases from plant sources. In the present research actinidin, a cysteine protease abundant in Kiwifruit, was used to isolate and culture of rat hepatocytes. Materials & Methods: Different concentrations of actinidin was used to isolate rat hepatocytes by one or two-step perfusion method. The viability of the separated cells was examined by the trypan blue test. The isolated rat hepatocytes were cultured on collagen coated plates in William´s E medium. The morphology of hepatocytes was examined microscopically after staining with the Papanicolaou method.Results: Actinidin in the concentration of 0.4 mg/ml in two-step perfusion method properly isolated hepatocytes from rat liver. The viability of isolated hepatocytes that successfully cultured in collagen coated plates was 90-95 percent.Conclusion: These results showed that actinidin is a proper protease for isolation of hepatocytes. In addition, purification of this enzyme is simpler than the collagenases.

  4. Effect of dithiotheritol on viability of cryopreserved rat hepatocytes

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    A Jamshidzadeh

    2009-10-01

    Full Text Available Background: Prolonged storage of cryopreserved isolated hepatocytes is now possible and such cells have been used with success for drug metabolism and toxicity studies. Quality of the cells before cryopreservation is important for viability and function after freezing. Methods: In this study, fresh rat hepatocytes were incubated with the thiol-containing compounds N-acetylcysteine (NAC, dithiotheritol (DTT and fructose as ATP supplier, at incubation times 1 and 3 hrs. The preincubated hepatocytes cryopreserved for 24 hour, and 1 and 3 months. Hepatocytes, viability were determined immediately postthaw by Trypan Blue exclusion. Results: Fructose preincubation improved the viability of hepatocytes at a concentration of 300 mM. Preincubation with DTT (50, 100 and 200 μM prior to cryopreservation had beneficial effects on viability of hepatocytes. The postthaw viability of hepatocytes preincubated with NAC was uniformly poor. Conclusion: Preincubation with DTT, improved GSH levels before freezing which could be responsible for the reduction in membrane damage during cryopreservation. It may be concluded that the intracellular

  5. Obesity affects the liver - the link between adipocytes and hepatocytes.

    Science.gov (United States)

    Wree, Alexander; Kahraman, Alisan; Gerken, Guido; Canbay, Ali

    2011-01-01

    The incidence of obesity has dramatically increased in recent years. Consequently, obesity and associated disorders such as nonalcoholic fatty liver disease (NAFLD) constitute a serious threat. Therefore, the contribution of visceral adipose tissue to metabolic homeostasis has become a focus of interest. Visceral adipose tissue secretes free fatty acids (FFAs) and hormones, known as adipokines, and thus seems to play a major role in the development of NAFLD. Apoptotic cell death is a prominent feature in nonalcoholic steatohepatitis (NASH). Indeed, toxic FFAs can activate the intrinsic apoptosis pathway in hepatocytes via c-Jun N-terminal kinase (JNK). JNK activates the proapoptotic protein Bim, resulting in Bax activation and enhanced apoptosis, termed 'lipoapoptosis'. Reduced adiponectin levels may establish a proinflammatory milieu, thus increasing vulnerability to lipotoxicity, which promotes progression from simple steatosis to NASH and even advanced hepatic fibrosis. Moreover, obesity seems to be a risk factor for hepatocellular carcinoma, the most frequent liver cancer subtype. Even in acute liver failure, a high body mass index is associated with poor outcome, and recent data suggest a major role of obesity in the progression of chronic hepatitis C and B. This review summarizes current knowledge - highlighting the inflammatory and cytokine view - of the intimate relationship between adipose and liver tissue. Copyright © 2010 S. Karger AG, Basel.

  6. Alginate microencapsulated hepatocytes optimised for transplantation in acute liver failure.

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    Suttiruk Jitraruch

    Full Text Available BACKGROUND AND AIM: Intraperitoneal transplantation of alginate-microencapsulated human hepatocytes is an attractive option for the management of acute liver failure (ALF providing short-term support to allow native liver regeneration. The main aim of this study was to establish an optimised protocol for production of alginate-encapsulated human hepatocytes and evaluate their suitability for clinical use. METHODS: Human hepatocyte microbeads (HMBs were prepared using sterile GMP grade materials. We determined physical stability, cell viability, and hepatocyte metabolic function of HMBs using different polymerisation times and cell densities. The immune activation of peripheral blood mononuclear cells (PBMCs after co-culture with HMBs was studied. Rats with ALF induced by galactosamine were transplanted intraperitoneally with rat hepatocyte microbeads (RMBs produced using a similar optimised protocol. Survival rate and biochemical profiles were determined. Retrieved microbeads were evaluated for morphology and functionality. RESULTS: The optimised HMBs were of uniform size (583.5±3.3 µm and mechanically stable using 15 min polymerisation time compared to 10 min and 20 min (p<0.001. 3D confocal microscopy images demonstrated that hepatocytes with similar cell viability were evenly distributed within HMBs. Cell density of 3.5×10(6 cells/ml provided the highest viability. HMBs incubated in human ascitic fluid showed better cell viability and function than controls. There was no significant activation of PBMCs co-cultured with empty or hepatocyte microbeads, compared to PBMCs alone. Intraperitoneal transplantation of RMBs was safe and significantly improved the severity of liver damage compared to control groups (empty microbeads and medium alone; p<0.01. Retrieved RMBs were intact and free of immune cell adherence and contained viable hepatocytes with preserved function. CONCLUSION: An optimised protocol to produce GMP grade alginate

  7. Ploidy reductions in murine fusion-derived hepatocytes.

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    Andrew W Duncan

    2009-02-01

    Full Text Available We previously showed that fusion between hepatocytes lacking a crucial liver enzyme, fumarylacetoacetate hydrolase (FAH, and wild-type blood cells resulted in hepatocyte reprogramming. FAH expression was restored in hybrid hepatocytes and, upon in vivo expansion, ameliorated the effects of FAH deficiency. Here, we show that fusion-derived polyploid hepatocytes can undergo ploidy reductions to generate daughter cells with one-half chromosomal content. Fusion hybrids are, by definition, at least tetraploid. We demonstrate reduction to diploid chromosome content by multiple methods. First, cytogenetic analysis of fusion-derived hepatocytes reveals a population of diploid cells. Secondly, we demonstrate marker segregation using ss-galactosidase and the Y-chromosome. Approximately 2-5% of fusion-derived FAH-positive nodules were negative for one or more markers, as expected during ploidy reduction. Next, using a reporter system in which ss-galactosidase is expressed exclusively in fusion-derived hepatocytes, we identify a subpopulation of diploid cells expressing ss-galactosidase and FAH. Finally, we track marker segregation specifically in fusion-derived hepatocytes with diploid DNA content. Hemizygous markers were lost by >or=50% of Fah-positive cells. Since fusion-derived hepatocytes are minimally tetraploid, the existence of diploid hepatocytes demonstrates that fusion-derived cells can undergo ploidy reduction. Moreover, the high degree of marker loss in diploid daughter cells suggests that chromosomes/markers are lost in a non-random fashion. Thus, we propose that ploidy reductions lead to the generation of genetically diverse daughter cells with about 50% reduction in nuclear content. The generation of such daughter cells increases liver diversity, which may increase the likelihood of oncogenesis.

  8. Raman spectroscopic identification of normal and malignant hepatocytes

    Institute of Scientific and Technical Information of China (English)

    Jianyu Guo; Bing Du; Min Qian; Weiying Cai; Zugeng Wang; Zhenrong Sun

    2009-01-01

    Raman spectroscopy has strong potential for providing non-invasion diagnosis of cancers. In this paper, micro-Raman spectroscopy is used to diagnose one most common liver cancer, hepatocellular carcinoma (HCC). The statistical analyzes, including i-test, principal component analysis (PCA), and linear discrim inant analysis (LDA), are performed on the Raman spectra of malignant and normal hepatocytes. The t-test-LDA results show that the 786- and 1004-cm-1 bands of the malignant and normal hepatocytes are significantly different, and PCA-LDA results show an overall accuracy of 100% for the Raman spectro scopic identification of normal and malignant hepatocytes in our experiment.

  9. Higher protein kinase C ζ in fatty rat liver and its effect on insulin actions in primary hepatocytes.

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    Wei Chen

    Full Text Available We previously showed the impairment of insulin-regulated gene expression in the primary hepatocytes from Zucker fatty (ZF rats, and its association with alterations of hepatic glucose and lipid metabolism. However, the molecular mechanism is unknown. A preliminary experiment shows that the expression level of protein kinase C ζ (PKCζ, a member of atypical PKC family, is higher in the liver and hepatocytes of ZF rats than that of Zucker lean (ZL rats. Herein, we intend to investigate the roles of atypical protein kinase C in the regulation of hepatic gene expression. The insulin-regulated hepatic gene expression was evaluated in ZL primary hepatocytes treated with atypical PKC recombinant adenoviruses. Recombinant adenovirus-mediated overexpression of PKCζ, or the other atypical PKC member PKCι/λ, alters the basal and impairs the insulin-regulated expressions of glucokinase, sterol regulatory element-binding protein 1c, the cytosolic form of phosphoenolpyruvate carboxykinase, the catalytic subunit of glucose 6-phosphatase, and insulin like growth factor-binding protein 1 in ZL primary hepatocytes. PKCζ or PKCι/λ overexpression also reduces the protein level of insulin receptor substrate 1, and the insulin-induced phosphorylation of AKT at Ser473 and Thr308. Additionally, PKCι/λ overexpression impairs the insulin-induced Prckz expression, indicating the crosstalk between PKCζ and PKCι/λ. We conclude that the PKCζ expression is elevated in hepatocytes of insulin resistant ZF rats. Overexpressions of aPKCs in primary hepatocytes impair insulin signal transduction, and in turn, the down-stream insulin-regulated gene expression. These data suggest that elevation of aPKC expression may contribute to the hepatic insulin resistance at gene expression level.

  10. 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 drugs showed this strength of inhibition in primary human hepatocytes and these drugs are associated only with cholestatic and mixed hepatocellular cholestatic (mixed) injuries. The sDILI drugs, which did not show substantial inhibition of bile salt transport activity, are likely to be associated with immune-mediated liver injury. Twenty-four drugs were also tested in 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

  11. Toxicity monitoring with primary cultured hepatocytes underestimates the acetaminophen-induced inflammatory responses of the mouse liver.

    Science.gov (United States)

    Tachibana, Shinjiro; Shimomura, Akiko; Inadera, Hidekuni

    2011-01-01

    In vitro gene expression profiling with isolated hepatocytes has been used to assess the hepatotoxicity of certain chemicals because of animal welfare issues. However, whether an in vitro system can completely replace the in vivo system has yet to be elucidated in detail. Using a focused microarray established in our laboratory, we examined gene expression profiles in the mouse liver and primary cultured hepatocytes after treatment with different doses of acetaminophen, a widely used analgesic that frequently causes liver injury. The acute hepatotoxicity of acetaminophen was confirmed by showing the induction of an oxidative stress marker, heme oxygenase-1, elevated levels of serum transaminase, and histopathological findings. In vivo microarray and network analysis showed that acetaminophen treatment provoked alterations in relation to the inflammatory response, and that tumor necrosis factor-α plays a central role in related pathway alterations. By contrast, pathway analyses in in vitro isolated hepatocytes did not find such prominent changes in the inflammation-related networks compared with the in vivo situation. Thus, although in vitro gene expression profiles are useful for evaluating the direct toxicity of chemicals, indirect toxicities including inflammatory responses mediated by cell-cell interactions or secondary toxicity due to pathophysiological changes in the whole body may be overlooked. Our results indicate that the in vitro hepatotoxicity prediction system using isolated hepatocytes does not fully reflect the in vivo cellular response. An in vitro system may be appropriate, therefore, for high throughput screening to detect the direct hepatotoxicity of a test compound.

  12. Hepatocyte growth factor and alternative splice variants - expression, regulation and implications in osteogenesis and bone health and repair.

    Science.gov (United States)

    Frisch, Rachel N; Curtis, Kevin M; Aenlle, Kristina K; Howard, Guy A

    2016-09-01

    Bone marrow-derived mesenchymal stem cells (MSCs) can differentiate into multiple cell types, including osteoblasts, chondrocytes, and adipocytes. These pluripotent cells secrete hepatocyte growth factor (HGF), which regulates cell growth, survival, motility, migration, mitogenesis and is important for tissue development/regeneration. HGF has four splice variants, NK1, NK2, NK3, and NK4 which have varying functions and affinities for the HGF receptor, cMET. HGF promotes osteoblastic differentiation of MSCs into bone forming cells, playing a role in bone development, health and repair. This review will focus on the effects of HGF in osteogenesis, bone repair and bone health, including structural and functional insights into the role of HGF in the body. Approximately 6.2 million Americans experience a fracture annually, with 5-10% being mal- or non-union fractures. HGF is important in priming MSCs for osteogenic differentiation in vitro and is currently being studied to assess its role during bone repair in vivo. Due to the high turnover rate of systemic HGF, non-classic modes of HGF-treatment, including naked-plasmid HGF delivery and the use of HGF splice variants (NK1 & NK2) are being studied to find safe and efficacious treatments for bone disorders, such as mal- or non-union fractures.

  13. Hepatocyte Turnover in Chronic HCV-Induced Liver Injury and Cirrhosis

    Directory of Open Access Journals (Sweden)

    Nikolaos P. Karidis

    2015-01-01

    Full Text Available Chronic hepatitis C virus (HCV infection may eventually lead to progressive liver fibrosis and cirrhosis through a complex, multistep process involving hepatocyte death and regeneration. Despite common pathogenetic pathways present in all forms of liver cirrhosis irrespective of etiology, hepatocyte turnover and related molecular events in HCV-induced cirrhosis are increasingly being distinguished from even “similar” causes, such as hepatitis B virus- (HBV- related cirrhosis. New insights in HCV-induced hepatocellular injury, differential gene expression, and regenerative pathways have recently revealed a different pattern of progression to irreversible parenchymal liver damage. A shift to the significant role of the host immune response rather than the direct effect of HCV on hepatocytes and the imbalance between antiapoptotic and proapoptotic signals have been investigated in several studies but need to be further elucidated. The present review aims to comprehensively summarize the current evidence on HCV-induced hepatocellular turnover with a view to outline the significant trends of ongoing research.

  14. Hepatocyte gene transfer mediated by stable polyplexes based on MPP-containing DNA complexes

    Institute of Scientific and Technical Information of China (English)

    Bao-Feng Yu; Wan-I Li; Xiao-Nian Hu; Yue-Hong Zhang; Bo Niu; Jun Xie

    2009-01-01

    BACKGROUND: In the field of gene therapy, viral vectors as delivery tools have a number of disadvantages for medical application. This study aimed to explore a novel nonviral vector as a vehicle for gene therapy. METHODS: Transvector-rpE-MPP and EGFP (enhanced green fluorescent protein) were used as the gene transfer carrier and the reporter gene, respectively. Polyplexes which integrate transvector-rpE-MPP, the object gene, and EGFP were formed. The optimal charge ratio, stability, and transduction capacity of the polyplexes in mouse hepatocytes in vitro and in mouse liver in vivo were investigated. The polyplexes of transvector-rpE-MPP and pcDNA3-EGFP, with charge ratios of 0, 0.25, 0.5, 0.75, 1 and 1.5 were compared to determine the optimal charge ratio. RESULTS:  Polyplexes with charge ratios of 1∶1 were most stable; pcDNA3-EGFP in these complexes resisted digestion by DNase Ⅰ and blood plasma. On the other hand, pcDNA3-EGFP alone was digested. Fluorescence analysis indicated that transvector-rpE-MPP successfully delivered the reporter gene EGFP into hepatocytes and that EGFP expression was detected in hepatocyte cultures and in liver tissue. CONCLUSION: These results have laid a foundation for further study of a novel nonviral gene delivery system.

  15. Machine perfusion enhances hepatocyte isolation yields from ischemic livers.

    Science.gov (United States)

    Izamis, Maria-Louisa; Perk, Sinem; Calhoun, Candice; Uygun, Korkut; Yarmush, Martin L; Berthiaume, François

    2015-10-01

    High-quality human hepatocytes form the basis of drug safety and efficacy tests, cell-based therapies, and bridge-to-transplantation devices. Presently the only supply of cells derives from an inadequate pool of suboptimal disqualified donor livers. Here we evaluated whether machine perfusion could ameliorate ischemic injury that many of these livers experience prior to hepatocyte isolation. Non-heparinized female Lewis rat livers were exposed to an hour of warm ischemia (34°C) and then perfused for 3h. Five different perfusion conditions that utilized the cell isolation apparatus were investigated, namely: (1) modified Williams Medium E and (2) Lifor, both with active oxygenation (95%O(2)/5%CO(2)), as well as (3) Lifor passively oxygenated with ambient air (21%O(2)/0.04%CO(2)), all at ambient temperatures (20 ± 2°C). At hypothermic temperatures (5 ± 1°C) and under passive oxygenation were (4) University of Wisconsin solution (UW) and (5) Vasosol. Negative and positive control groups comprised livers that had ischemia (WI) and livers that did not (Fresh) prior to cell isolation, respectively. Fresh livers yielded 32 ± 9 million cells/g liver while an hour of ischemia reduced the cell yield to 1.6 ± 0.6 million cells/g liver. Oxygenated Williams Medium E and Lifor recovered yields of 39 ± 11 and 31 ± 2.3 million cells/g liver, respectively. The passively oxygenated groups produced 15 ± 7 (Lifor), 13 ± 7 (Vasosol), and 10 ± 6 (UW)million cells/g liver. Oxygenated Williams Medium E was most effective at sustaining pH values, avoiding the accumulation of lactate, minimizing edematous weight gain and producing bile during perfusion. Machine perfusion results in a dramatic increase in cell yields from livers that have had up to an hour of warm ischemia, but perfusate choice significantly impacts the extent of recovery. Oxygenated Williams Medium E at room temperature is superior to Lifor, UW and Vasosol, largely facilitated by its high oxygen content and low

  16. General review on in vitro hepatocyte models and their applications.

    Science.gov (United States)

    Guguen-Guillouzo, Christiane; Guillouzo, Andre

    2010-01-01

    In vitro hepatocyte models represent very useful systems in both fundamental research and various application areas. Primary hepatocytes appear as the closest model for the liver in vivo. However, they are phenotypically unstable, have a limited life span and in addition, exhibit large interdonor variability when of human origin. Hepatoma cell lines appear as an alternative but only the HepaRG cell line exhibits various functions, including major cytochrome P450 activities, at levels close to those found in primary hepatocytes. In vitro hepatocyte models have brought a substantial contribution to the understanding of the biochemistry, physiology, and cell biology of the normal and diseased liver and in various application domains such as xenobiotic metabolism and toxicity, virology, parasitology, and more generally cell therapies. In the future, new well-differentiated hepatocyte cell lines derived from tumors or from either embryonic or adult stem cells might be expected and although hepatocytes will continue to be used in various fields, these in vitro liver models should allow marked advances, especially in cell-based therapies and predictive and mechanistic hepatotoxicity of new drugs and other chemicals. All models will benefit from new developments in throughput screening based on cell chips coupled with high-content imaging and in toxicogenomics technologies.

  17. Biomechanics and functionality of hepatocytes in liver cirrhosis.

    Science.gov (United States)

    Sun, Shan; Song, Zhenyuan; Cotler, Scott J; Cho, Michael

    2014-06-27

    Cirrhosis is a life-threatening condition that is generally attributed to overproduction of collagen fibers in the extracellular matrix that mechanically stiffens the liver. Chronic liver injury due to causes including viral hepatitis, inherited and metabolic liver diseases and external factors such as alcohol abuse can result in the development of cirrhosis. Progression of cirrhosis leads to hepatocellular dysfunction. While extensive studies to understand the complexity underlying liver fibrosis have led to potential application of anti-fibrotic drugs, no such FDA-approved drugs are currently available. Additional studies of hepatic fibrogenesis and cirrhosis primarily have focused on the extracellular matrix, while hepatocyte biomechanics has received limited attention. The role of hepatocyte biomechanics in liver cirrhosis remains elusive, and how the cell stiffness is correlated with biological functions of hepatocytes is also unknown. In this study, we demonstrate that the biomechanical properties of hepatocytes are correlated with their functions (e.g., glucose metabolism), and that hepatic dysfunction can be restored through modulation of the cellular biomechanics. Furthermore, our results indicate the hepatocyte functionality appears to be regulated through a crosstalk between the Rho and Akt signaling. These novel findings may lead to biomechanical intervention of hepatocytes and the development of innovative tissue engineering for clinical treatment to target liver cells rather than exclusively focusing on the extracellular matrix alone in liver cirrhosis.

  18. 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.

  19. Pediatric non-alcoholic steatohepatitis: the first report on the ultrastructure of hepatocyte mitochondria.

    Science.gov (United States)

    Lotowska, Joanna M; Sobaniec-Lotowska, Maria E; Bockowska, Sylwia B; Lebensztejn, Dariusz M

    2014-04-21

    longitudinally, or as an evenly spaced matrix in cross section, and frequently caused mitochondrial deformation. The average diameter of these linear structures was 10 nm and the average space between them 20 nm. Sometimes enlarged intramitochondrial granules were seen in their vicinity. Foamy cytoplasm of hepatocytes was found, resulting from the proliferation of smooth endoplasmic reticulum and glycogen accumulation. The perivascular space of Disse was frequently dilated, and contained transitional hepatic stellate cells, as well as mature and/or newly forming collagen fiber bundles. Marked ultrastructural abnormalities observed in hepatocyte mitochondria, especially their polymorphism in the form of MMC and loss of mitochondrial cristae, accompanied by foamy cytoplasm, clearly indicate a major role of these organelles in the morphogenesis of pediatric NASH. Our findings seem to prove the high effectiveness of electron microscopy in the diagnosis of the disease.

  20. Dicer-dependent production of microRNA221 in hepatocytes inhibits p27 and is required for liver regeneration in mice.

    Science.gov (United States)

    Oya, Yuki; Masuzaki, Ryota; Tsugawa, Daisuke; Ray, Kevin C; Dou, Yongchao; Karp, Seth J

    2017-05-01

    Dicer processes microRNAs (miRs) into active forms in a wide variety of tissues, including the liver. To determine the role of Dicer in liver regeneration, we performed a series of in vivo and in vitro studies in a murine 2/3 hepatectomy model. Dicer was downregulated after 2/3 hepatectomy, and loss of Dicer inhibited liver regeneration associated with decreased cyclin A2 and miR-221, as well as increased levels of the cell cycle inhibitor p27. In vitro, miR-221 inhibited p27 production in primary hepatocytes and increased hepatocyte proliferation. Specific reconstitution of miR-221 in hepatocyte-specific Dicer-null mice inhibited p27 and restored liver regeneration. In wild type mice, targeted inhibition of miR-221 using a cholesterol-conjugated miR-221 inhibited hepatocyte proliferation after 2/3 hepatectomy. These results identify Dicer production of miR-221 as an essential component of a miRNA-dependent mechanism for suppression of p27 that controls the rate of hepatocyte proliferation after partial hepatectomy.NEW & NOTEWORTHY Our findings demonstrate a direct role for microRNAs in controlling the rate of liver regeneration after injury. By deleting Dicer, an enzyme responsible for processing microRNAs into mature forms, we determined miR-221 is a critical microRNA in the physiological process of restoration of liver mass after injury. miR-221 suppresses p27, releasing its inhibitory effects on hepatocyte proliferation. Pharmaceuticals based on miR-221 may be useful to modulate hepatocyte proliferation in the setting of liver injury. Copyright © 2017 the American Physiological Society.

  1. Troglitazone, but not rosiglitazone, damages mitochondrial DNA and induces mitochondrial dysfunction and cell death in human hepatocytes.

    Science.gov (United States)

    Rachek, Lyudmila I; Yuzefovych, Larysa V; Ledoux, Susan P; Julie, Neil L; Wilson, Glenn L

    2009-11-01

    Thiazolidinediones (TZDs), such as troglitazone (TRO) and rosiglitazone (ROSI), improve insulin resistance by acting as ligands for the nuclear receptor peroxisome proliferator-activated receptor-gamma (PPARgamma). TRO was withdrawn from the market because of reports of serious hepatotoxicity. A growing body of evidence suggests that TRO caused mitochondrial dysfunction and induction of apoptosis in human hepatocytes but its mechanisms of action remain unclear. We hypothesized that damage to mitochondrial DNA (mtDNA) is an initiating event involved in TRO-induced mitochondrial dysfunction and hepatotoxicity. Primary human hepatocytes were exposed to TRO and ROSI. The results obtained revealed that TRO, but not ROSI at equimolar concentrations, caused a substantial increase in mtDNA damage and decreased ATP production and cellular viability. The reactive oxygen species (ROS) scavenger, N-acetyl cystein (NAC), significantly diminished the TRO-induced cytotoxicity, suggesting involvement of ROS in TRO-induced hepatocyte cytotoxicity. The PPARgamma antagonist (GW9662) did not block the TRO-induced decrease in cell viability, indicating that the TRO-induced hepatotoxicity is PPARgamma-independent. Furthermore, TRO induced hepatocyte apoptosis, caspase-3 cleavage and cytochrome c release. Targeting of a DNA repair protein to mitochondria by protein transduction using a fusion protein containing the DNA repair enzyme Endonuclease III (EndoIII) from Escherichia coli, a mitochondrial translocation sequence (MTS) and the protein transduction domain (PTD) from HIV-1 TAT protein protected hepatocytes against TRO-induced toxicity. Overall, our results indicate that significant mtDNA damage caused by TRO is a prime initiator of the hepatoxicity caused by this drug.

  2. A search for H-chondritic chromite grains in sediments that formed immediately after the breakup of the L-chondrite parent body 470 Ma ago

    Science.gov (United States)

    Heck, Philipp R.; Schmitz, Birger; Rout, Surya S.; Tenner, Travis; Villalon, Krysten; Cronholm, Anders; Terfelt, Fredrik; Kita, Noriko T.

    2016-03-01

    A large abundance of L-chondritic material, mainly in the form of fossil meteorites and chromite grains from micrometeorites, has been found in mid-Ordovician 470 Ma old sediments globally. The material has been determined to be ejecta from the L chondrite parent body breakup event, a major collision in the asteroid belt 470 Ma ago. In this study we search the same sediments for H-chondritic chromite grains in order to improve our understanding of the extraterrestrial flux to Earth after the asteroid breakup event. We have used SIMS in conjunction with quantitative SEM/EDS to determine the three oxygen isotopic and elemental compositions, respectively, of a total of 120 randomly selected, sediment-dispersed extraterrestrial chromite grains mainly representing micrometeorites from 470 Ma old post-breakup limestone from the Thorsberg quarry in Sweden and the Lynna River site in Russia. We show that 99% or more of the grains are L-chondritic, whereas the H-chondritic fraction is 1% or less. The L-/H-chondrite ratio after the breakup thus was >99 compared to 1.1 in today's meteoritic flux. This represents independent evidence, in agreement with previous estimates based on sediment-dispersed extraterrestrial chromite grain abundances and sedimentation rates, of a two orders of magnitude higher post-breakup flux of L-chondritic material in the micrometeorite fraction. Finally, we confirm the usefulness of three oxygen isotopic SIMS analyses of individual extraterrestrial chromite grains for classification of equilibrated ordinary chondrites. The H- and L-chondritic chromites differ both in their three oxygen isotopic and elemental compositions, but there is some overlap between the groups. In chromite, TiO2 is the oxide most resistant to diagenesis, and the combined application of TiO2 and oxygen three-isotope analysis can resolve uncertainties arising from the compositional overlaps.

  3. LALF32-51 -E7, a HPV-16 therapeutic vaccine candidate, forms protein body-like structures when expressed in Nicotiana benthamiana leaves.

    Science.gov (United States)

    Yanez, Romana J R; Lamprecht, Renate; Granadillo, Milaid; Torrens, Isis; Arcalís, Elsa; Stöger, Eva; Rybicki, Edward P; Hitzeroth, Inga I

    2017-07-22

    High-risk human papillomaviruses (HPVs) cause cervical cancer, and while there are good prophylactic vaccines on the market, these are ineffective against established infections, creating a clear need for therapeutic vaccines. The HPV E7 protein is one of the essential oncoproteins for the onset and maintenance of malignancy and is therefore an ideal therapeutic vaccine target. We fused the HPV-16 E7 protein to the Limulus polyphemus antilipopolysaccharide factor (LALF32-51 ), a small hydrophobic peptide that can penetrate cell membranes and that has immunomodulatory properties. LALF32-51 -E7 was transiently expressed in Nicotiana benthamiana, and we previously determined that it accumulated better when targeted to chloroplasts compared to being localized in the cytoplasm. Subsequently, we aimed to prove whether LALF32-51 -E7 was indeed associated with the chloroplasts by determining its subcellular localization. The LALF32-51 -E7 gene was fused to one encoding enhanced GFP to generate a LG fusion protein, and localization was determined by confocal laser scanning microscopy and transmission electron microscopy (TEM). The fluorescence observed from chloroplast-targeted LG was distinctively different from that of the cytoplasmic LG. Small spherical structures resembling protein bodies (PBs) were seen that clearly localized with the chloroplasts. Larger but less abundant PB-like structures were also seen for the cytoplasmic LG. PB-like structure formation was confirmed for both LG and LALF32-51 -E7 by TEM. LALF32-51 -E7 was indeed targeted to the chloroplasts by the chloroplast transit peptide used in this study, and it formed aggregated PB-like structures. This study could open a new avenue for the use of LALF32-51 as a PB-inducing peptide. © 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

  4. 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.

  5. Inhibitors and pathways of hepatocytic protein degradation.

    Science.gov (United States)

    Seglen, P O; Gordon, P B; Grinde, B; Solheim, A; Kovács, A L; Poli, A

    1981-01-01

    On the basis of experiments using amino acids and various inhibitors (lysosomotropic amines, leupeptin, chymostatin, vanadate, vinblastine, anoxia, methylaminopurines), five different modes of endogenous protein degradation in isolated rat hepatocytes can be distinguished. The two non-lysosomal (amine-resistant) mechanisms preferentially degrade relatively labile (short-lived) proteins: one of these mechanisms is energy-dependent and chymostatin-sensitive, the other is not. Of the three lysosomal (amine-sensitive) mechanisms, one--quantitatively minor--is amino acid-resistant and preferentially degrades labile proteins. The two amino acid-sensitive mechanisms each seen account for about one-half of the degradation of relatively stable (long-lived) proteins; one of them is suppressed by leucine and apparently corresponds to the formation of electron microscopically visible autophagosomes; the other may represent a different type of autophagy, inhibited by asparagine and glutamine. A new class of inhibitors, the purine derivatives (methylated 6-aminopurines, and 6-mercaptopurines) appear to specifically suppress autophagic/lysosomal protein degradation, and may help to further elucidate the mechanisms of autophagy.

  6. HEMETβ: improvement of hepatocyte metabolism mathematical model.

    Science.gov (United States)

    Orsi, G; De Maria, C; Guzzardi, M; Vozzi, F; Vozzi, G

    2011-10-01

    This article describes hepatocyte metabolism mathematical model (HEMETβ), which is an improved version of HEMET, an effective and versatile virtual cell model based on hepatic cell metabolism. HEMET is based on a set of non-linear differential equations, implemented in Simulink®, which describes the biochemical reactions and energetic cell state, and completely mimics the principal metabolic pathways in hepatic cells. The cell energy function and modular structure are the core of this model. HEMETβ as HEMET model describes hepatic cellular metabolism in standard conditions (cell culture in a plastic multi-well placed in an incubator at 37° C with 5% of CO2) and with excess substrates concentration. The main improvements in HEMETβ are the introductions of Michaelis-Menten models for reversible reactions and enzymatic inhibition. In addition, we eliminated hard non-linearities and modelled cell proliferation and every single aminoacid degradation pathway. All these innovations, combined with a user-friendly aspect, allow researchers to create new cell types and validate new experimental protocols just varying 'peripheral' pathways or model inputs.

  7. In vitro culture of isolated primary hepatocytes and stem cell-derived hepatocyte-like cells for liver regeneration.

    Science.gov (United States)

    Hu, Chenxia; Li, Lanjuan

    2015-08-01

    Various liver diseases result in terminal hepatic failure, and liver transplantation, cell transplantation and artificial liver support systems are emerging as effective therapies for severe hepatic disease. However, all of these treatments are limited by organ or cell resources, so developing a sufficient number of functional hepatocytes for liver regeneration is a priority. Liver regeneration is a complex process regulated by growth factors (GFs), cytokines, transcription factors (TFs), hormones, oxidative stress products, metabolic networks, and microRNA. It is well-known that the function of isolated primary hepatocytes is hard to maintain; when cultured in vitro, these cells readily undergo dedifferentiation, causing them to lose hepatocyte function. For this reason, most studies focus on inducing stem cells, such as embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), hepatic progenitor cells (HPCs), and mesenchymal stem cells (MSCs), to differentiate into hepatocyte-like cells (HLCs) in vitro. In this review, we mainly focus on the nature of the liver regeneration process and discuss how to maintain and enhance in vitro hepatic function of isolated primary hepatocytes or stem cell-derived HLCs for liver regeneration. In this way, hepatocytes or HLCs may be applied for clinical use for the treatment of terminal liver diseases and may prolong the survival time of patients in the near future.

  8. 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

  9. Induced pluripotent stem cell-derived hepatocytes and endothelial cells in multi-component hydrogel fibers for liver tissue engineering.

    Science.gov (United States)

    Du, Chan; Narayanan, Karthikeyan; Leong, Meng Fatt; Wan, Andrew C A

    2014-07-01

    Liver tissue engineering requires a suitable cell source, methodologies to assemble the cells within their niche microenvironments in a spatially defined manner, and vascularization of the construct in vivo for maintenance of hepatocyte viability and function. Recently, we have developed methods of encapsulating cells within separate domains in multi-component hydrogel fibers and methods of assembling fibers to form 3D-patterned tissue constructs. In the present work, we have combined these approaches to encapsulate hepatocytes and endothelial cells within their specific niches, and to assemble them into endothelialized liver tissue constructs. The hepatocytes and endothelial cells were obtained in parallel by differentiating human recombinant protein-induced human pluripotent stem cells, resulting in a construct which contained genetically identical endothelial and parenchymal elements. We were able to demonstrate that the presence of endothelial cells in the scaffold significantly improved hepatocyte function in vitro and facilitated vascularization of the scaffold when implanted in a mouse partial hepatectomy model. The in vivo studies further asserted that integration of the scaffold with host vasculature had occurred, as demonstrated by the presence of human albumin in the mouse serum. Copyright © 2014 Elsevier Ltd. All rights reserved.

  10. Fluorometric assessment of acetaminophen-induced toxicity in rat hepatocyte spheroids seeded on micro-space cell culture plates.

    Science.gov (United States)

    Sanoh, Seigo; Santoh, Masataka; Takagi, Masashi; Kanayama, Tatsuya; Sugihara, Kazumi; Kotake, Yaichiro; Ejiri, Yoko; Horie, Toru; Kitamura, Shigeyuki; Ohta, Shigeru

    2014-09-01

    Hepatotoxicity induced by the metabolic activation of drugs is a major concern in drug discovery and development. Three-dimensional (3-D) cultures of hepatocyte spheroids may be superior to monolayer cultures for evaluating drug metabolism and toxicity because hepatocytes in spheroids maintain the expression of various metabolizing enzymes and transporters, such as cytochrome P450 (CYP). In this study, we examined the hepatotoxicity due to metabolic activation of acetaminophen (APAP) using fluorescent indicators of cell viability and intracellular levels of glutathione (GSH) in rat hepatocyte spheroids grown on micro-space cell culture plates. The mRNA expression levels of some drug-metabolizing enzymes were maintained during culture. Additionally, this culture system was compatible with microfluorometric imaging under confocal laser scanning microscopy. APAP induced a decrease in intracellular ATP at 10mM, which was blocked by the CYP inhibitor 1-aminobenzotriazole (ABT). APAP (10mM, 24h) decreased the levels of both intracellular ATP and GSH, and GSH-conjugated APAP (APAP-GSH) were formed. All three effects were blocked by ABT, confirming a contribution of APAP metabolic activation by CYP to spheroid toxicity. Fluorometric imaging of hepatocyte spheroids on micro-space cell culture plates may allow the screening of drug-induced hepatotoxicity during pharmaceutical development.

  11. IN VITRO EFFECT OF MITRAGYNINE (A MAJOR ALKALOID OF MITRAGYNA SPECIOSA KORTH ON AMINOPYRINE METABOLISM IN RAT HEPATOCYTES

    Directory of Open Access Journals (Sweden)

    Rukhsana Anwar, Abas Hj Hussin , Sabariah Ismail* and Sharif Mahsufi Mansor

    2012-07-01

    Full Text Available Mitragyna speciosa Korth. is a member of the Rubiaceae family. More than 25 alkaloids have been isolated from Mitragyna speciosa. Mitragynine is the major alkaloid of this plant and is responsible for antinociceptive action. No single study is available about the effect of mitragynine on aminopyrine N-demethylase activity in rat hepatocytes. Experiments were undertaken to evaluate the effect of mitragynine in different age groups (adult & old of Sprague- Dawley (SD male and female rat hepatocytes. In vitro this evaluation was assessed by different concentration of mitragynine (0.0025µM-250µM. Hepatocytes were prepared by collagenase perfusion technique. Aminopyrine N-demethylase activity was determined by measuring the quantity of formaldehyde formed. Results showed that a significant increase in aminopyrine N-demethylase activity was observed in the adult male, female and old female SD rat hepatocytes treated with 250µM mitragynine (p< 0.05. However, the old male rat did not show any significant change at any concentration of mitragynine. In conclusion this study indicates the induction of hepatic drug metabolizing enzymes by mitragynine is affected by the aging process in male but unaffected in female.

  12. Caffeine induces CYP1A2 expression in rat hepatocytes but not in human hepatocytes.

    Science.gov (United States)

    Vaynshteyn, David; Jeong, Hyunyoung

    2012-06-01

    Caffeine is the active constituent in coffee. Continual consumption of caffeine can lead to an attenuated response also known as tolerance. Results from rat studies have shown that caffeine is an inducer of CYP1A2, the enzyme responsible for caffeine's metabolism. This suggests that CYP1A2 induction by caffeine may be in part responsible for caffeine tolerance. However, whether caffeine induces CYP1A2 expression in humans remains unknown. Our results from luciferase assays performed in HepG2 cells showed that caffeine is not an activator of the aromatic hydrocarbon receptor (AhR), a major transcription factor involved in upregulation of CYP1A2. Furthermore, caffeine did not induce CYP1A2 expression in primary human hepatocytes at a concentration attained by ordinary coffee drinking. On the other hand, caffeine enhanced CYP1A2 expression by 9-fold in rat hepatocytes. Our results suggest that caffeine from ordinary coffee drinking does not induce CYP1A2 expression in humans and that factors other than CYP1A2 induction by caffeine likely contribute to development of caffeine tolerance in humans.

  13. Keratin 8 modulation of desmoplakin deposition at desmosomes in hepatocytes.

    Science.gov (United States)

    Loranger, Anne; Gilbert, Stéphane; Brouard, Jean-Simon; Magin, Thomas M; Marceau, Normand

    2006-12-10

    Keratins, the intermediate filament proteins of epithelial cells, connect to desmosomes, the cell-cell adhesion structures at the surface membrane. The building elements of desmosomes include desmoglein and desmocollin, which provide the actual cell adhesive properties, and desmoplakins, which anchor the keratin intermediate filaments to desmosomes. In the work reported here, we address the role of keratin 8 in modulating desmoplakin deposition at surface membrane in mouse hepatocytes. The experimental approach is based on the use of keratin 8- and keratin 18-null mouse hepatocytes as cell models. In wild-type mouse hepatocytes, desmoplakin is aligned with desmoglein and keratin 8 at the surface membrane. In keratin 8-null hepatocytes, the intermediate filament loss leads to alterations in desmoplakin distribution at the surface membrane, but not of desmoglein. Intriguingly, a significant proportion of keratin 18-null hepatocytes express keratin 8 at the surface membrane, associated with a proper desmoplakin alignment with desmoglein at desmosomes. A Triton treatment of the monolayer reveals that most of the desmoplakin present in either wild-type, keratin 8- or keratin 18-null hepatocytes is insoluble. Deletion analysis of keratin 8 further suggests that the recovery of desmoplakin alignment requires the keratin 8 rod domain. In addition, similarly to other works revealing a key role of desmoplakin phosphorylation on its interaction with intermediate filaments, we find that the phosphorylation status of the keratin 8 head domain affects desmoplakin distribution at desmosomes. Together, the data indicate that a proper alignment/deposition of desmoplakin with keratins and desmoglein in hepatocytes requires keratin 8, through a reciprocal phosphoserine-dependent process.

  14. Hepatitis B antigen in hepatocytes of chronic active liver disease.

    Science.gov (United States)

    Kawanishi, H

    1979-04-01

    To study the morphologic interrelation of hepatocytes with the replication of hepatitis B vius (HBV) and immunocompetent cells in chronic active liver disease(CALD), organ cultures were prepared from liver biopsy specimens. Replication of hepatitis B core antigen (HBcAg) appears to occur in the nucleus of the hepatocyte in close association with intranuclear electron-dense strands and sometimes intranucleolar matrixes (likely HBcAg genomes), and cytoplasmic maturation of the HBcAg takes place in the preautolytic condition of host hepatocytes. Immunocompetent cells became progressively autolyzed in the early period of cultures. No difference in progression of hepatocyte injury in tissues from normal subjects and from hepatitis B surface antigen (HBsAg)-positive and HBsAg-negative patients with CALD may suggest that intracellular synthesis of HBV alone is not cytopathic to host hepatocytes. This model is promising for the study of HBV replication and development, and also for testing the efficacy of new antiviral agents against the virus.

  15. The effects of human umbilical cord perivascular cells on rat hepatocyte structure and functional polarity.

    Science.gov (United States)

    Gómez-Aristizábal, Alejandro; Davies, John Edward

    2013-06-01

    Hepatocyte culture is a useful tool for the study of their biology and the development of bioartificial livers. However, many challenges have to be overcome since hepatocytes rapidly lose their normal phenotype in vitro. We have recently demonstrated that human umbilical cord perivascular cells (HUCPVCs) are able to provide support to hepatocytes. In the present study we go further into exploring the effects that HUCPVCs have in the functional polarization, and both the internal and external organization, of hepatocytes. Also, we investigate HUCPVC-hepatocyte crosstalk by tracking both the effects of HUCPVCs on hepatocyte transcription factors and those of hepatocytes on the expression of hepatotrophic factors in HUCPVCs. Our results show that HUCPVCs maintain the functional polarity of hepatocytes ex vivo, as judged by the secretion of fluorescein into bile canaliculi, for at least 40 days. Transmission electron microscopy revealed that hepatocytes in coculture organize in an organoid-like structure embedded in extracellular matrix surrounded by HUCPVCs. In coculture, hepatocytes displayed a higher expression of C/EBPα, implicated in maintenance of the mature hepatocyte phenotype, and HUCPVCs upregulated hepatocyte growth factor and Jagged1 indicating that these genes may play important roles in HUCPVC-hepatocyte interactions.

  16. Embryonic stem cells develop into hepatocytes after intrasplenic transplantation in CCl4-treated mice

    Institute of Scientific and Technical Information of China (English)

    Kei Moriya; Masahide Yoshikawa; Ko Saito; Yukiteru Ouji; Mariko Nishiofuku; Noriko Hayashi; Shigeaki Ishizaka; Hiroshi Fukui

    2007-01-01

    AIM: To transplant undifferentiated embryonic stem (ES) cells into the spleens of carbon tetrachloride (CCl4)-treated mice to determine their ability to differentiate into hepatocytes in the liver.METHODS: CCU, 0.5 mL/kg body weight, was injected into the peritoneum of C57BL/6 mice twice a week for 5 wk. In group 1 (n = 12), 1 x 105 undifferentiated ES cells (0.1 mL of 1 x 106/mL solution), genetically labeled with GFP, were transplanted into the spleens 1 d after the second injection. Group 2 mice (n = 12) were injected with 0.2 mL of saline twice a week, instead of CCU, and the same amount of ES cells was transplanted into the spleens. Group 3 mice (n = 6) were treated with CCU and injected with 0.1 mL of saline into the spleen, instead of ES cells. Histochemical analyses of the livers were performed on post-transplantation d (PD) 10, 20, and 30.RESULTS: Considerable numbers of GFP-immunopositive cells were found in the periportal regions in group 1 mice (CCl4-treated) on PD 10, however, not in those untreated with CCl4 (group 2). The GFP-positive cells were also immunopositive for albumin (ALB), alpha-1 antitrypsin, cytokeratin 18, and hepatocyte nuclear factor 4 alpha on PD 20. Interestingly, most of the GFP-positive cells were immunopositive for DLK, a hepatoblast marker, on PD 10. Although very few ES-derived cells were demonstrated immunohistologically in the livers of group 1 mice on PD 30, improvements in liver fibrosis were observed. Unexpectedly, liver tumor formation was not observed in any of the mice that received ES cell transplantation during the experimental period.CONCLUSION: Undifferentiated ES cells developed into hepatocyte-like cells with appropriate integration into tissue, without uncontrolled cell growth.

  17. 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.

  18. Protective effect of liver ischemic preconditioning on rat hepatocytes

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    Ischemic preconditioning (IPC) protects liver graft function following ischemia in liver transplantation and liver resection. The aim of this study was to assess the advantages and any potential disadvantages of liver IPC prior to isolation for rat hepatocytes during isolation and cryopreservation. After isolating and thawing the cryopreserved hepatocytes after 14 and 28 d,cell viability,efficiency,and lactate dehydrogenase (LDH) levels in preserve solution were examined for every group. Groups treated with IPC had better cell viability determination,assessment of plating efficiency and lactate dehydrogenase (LDH) assay than Group without IPC,suggesting that IPC prior to isolation may have a significant protective effect on hepatocytes subjected to isolation and short period cryopreservation.

  19. Acetaminophen metabolism, cytotoxicity, and genotoxicity in rat primary hepatocyte cultures

    Energy Technology Data Exchange (ETDEWEB)

    Milam, K.M.; Byard, J.L.

    1985-06-30

    Acetaminophen (APAP) metabolism, cytotoxicity, and genotoxicity were measured in primary cultures of rat hepatocytes. Although 3 mM APAP caused a slight increase in cellular release of lactate dehydrogenase into the culture medium, cellular glutathione concentration (an index of APAP metabolism) was reduced by 50%. APAP at 7 mM was significantly more toxic to these hepatocytes and had a similar but more marked effect on glutathione concentrations. In spite of its cytotoxicity, neither dose of APAP stimulated DNA repair synthesis when monitored by the rate of incorporation of (/sup 3/H)thymidine into DNA following exposure to APAP. Thus, although APAP has been shown to be both hepato- and nephrotoxic in several in vivo and in vitro systems, the reactive toxic metabolite of APAP is not genotoxic in rat primary hepatocyte cultures.

  20. Early membrane depressions in hepatocytes cultured on Primaria supports.

    Science.gov (United States)

    Griffiths, B J; Evans, P J

    2001-01-01

    The topography of spreading hepatocytes on positively charged Primaria plates was examined using scanning electron microscopy. The cells acquired a uniform morphology within 2 h. The spreading was rapid and the surface of the cells showed early prominent depressions or dips. The hepatocytes had either one or two of these structures which corresponded to the frequency of mononuclear and binuclear cells, respectively. The nuclear origin of the dips was strengthened after 6 h. They contained solid structures, whose number, size and shape were the same as nucleoli. The membrane dipping was independent of cell density and took place under conditions where phenotypic changes can occur. Kidney proximal tubule cells had no dips. Co-cultures of hepatocytes and kidney proximal tubule cells showed that the cell types behave differently.

  1. Self-assembled drug delivery systems. Part 7: hepatocyte-targeted nanoassemblies of an adefovir lipid derivative with cytochrome P450-triggered drug release.

    Science.gov (United States)

    Du, Lina; Wu, Lailong; Jin, Yiguang; Jia, Junwei; Li, Miao; Wang, Yu

    2014-09-10

    A novel strategy was used in the design of self-assembled drug delivery systems (SADDSs) in this study. The nanoassemblies of an amphiphilic adefovir lipid derivative were prepared and demonstrated to have the functions of hepatocyte targeting, enzyme-triggered drug release and high anti-hepatitis effect. An amphiphilic adefovir lipid derivative, N-lauroyl-1-(3-chlorophenyl)-1,3-propanyl phosphonyl adefovir (LCPA) was prepared and formed the nanoassemblies by injecting the mixture of LCPA and another amphiphilic polymer, d-galactide polyoxyethylene (20) cetyl ether (GPCE) (ca. 20:1, mol/mol) into water. The nanoassemblies were very stable and showed negative charge. LCPA was sensitive to the cytochrome P450 isozymes that were expressed predominantly in the hepatocytes to produce adefovir. GPCE contained a long hydrophilic chain and a galactose ligand targeting the asialoglycoprotein receptors overexpressed on the surface of hepatocytes. The nanoassemblies showed the long-circulating and liver targeting effects according to the results of pharmacokinetics, tissue distribution and fluorescence imagination after bolus intravenous administration of the nanoassemblies to the mice. The highly efficient hepatitis B treatment was achieved by 10 day continuous administration of the nanoassemblies to the HBV-infected mice. Many functions were combined in the nanoassemblies, including prodrug, molecular self-assembly, nanotechnology, long-circulating, hepatocyte targeting and hepatocyte over expressing enzyme-triggered drug release.

  2. Metabolic activation capacity by primary hepatocytes expands the applicability of the embryonic stem cell test as alternative to experimental animal testing.

    Science.gov (United States)

    Hettwer, Michael; Reis-Fernandes, Marcos A; Iken, Marcus; Ott, Michael; Steinberg, Pablo; Nau, Heinz

    2010-08-01

    The murine embryonic stem cell test (EST) represents a validated alternative method for in vivo embryotoxicity testing. In the present study, primary hepatocytes were combined with the EST by a preincubation approach to improve its predictivity on bioactivation caused teratogenicity. As substances the well-known proteratogens cyclophosphamide (CPA) and valpromide (VPD) were used. The embryotoxic potential of CPA was detected by a strong decrease of the resulting ID(50)-concentration (50% inhibition of ES cell differentiation) after incubation with murine hepatocytes. Interspecies variation in metabolism was detected by testing VPD. After incubation of VPD with murine hepatocytes no inhibition of ES cell differentiation was observed, since hardly any teratogenic VPD metabolites were formed. In contrast, with human hepatocytes a significant conversion of VPD into the teratogen valproic acid (VPA) was observed. In summary we developed a co-culture approach for embryotoxicity testing, whereby the test compounds were incubated with hepatocytes and the supernatant was added to the ES cell culture to obtain a dose dependency of the preincubated test substances. Copyright 2010 Elsevier Inc. All rights reserved.

  3. In vivo hepatocyte MR imaging using lactose functionalized magnetoliposomes.

    Science.gov (United States)

    Ketkar-Atre, Ashwini; Struys, Tom; Dresselaers, Tom; Hodenius, Michael; Mannaerts, Inge; Ni, Yicheng; Lambrichts, Ivo; Van Grunsven, Leo A; De Cuyper, Marcel; Himmelreich, Uwe

    2014-01-01

    The aim of this study was to assess a novel lactose functionalized magnetoliposomes (MLs) as an MR contrast agent to target hepatocytes as well as to evaluate the targeting ability of MLs for in vivo applications. In the present work, 17 nm sized iron oxide cores functionalized with anionic MLs bearing lactose moieties were used for targeting the asialoglycoprotein receptor (ASGP-r), which is highly expressed in hepatocytes. Non-functionalized anionic MLs were tested as negative controls. The size distribution of lactose and anionic MLs was determined by transmission electron microscopy (TEM) and dynamic light scattering (DLS). After intravenous administration of both MLs, contrast enhancement in the liver was observed by magnetic resonance imaging (MRI). Label retention was monitored non-invasively by MRI and validated with Prussian blue staining and TEM for up to eight days post MLs administration. Although the MRI signal intensity did not show significant differences between functionalized and non-functionalized particles, iron-specific Prussian blue staining and TEM analysis confirmed the uptake of lactose MLs mainly in hepatocytes. In contrast, non-functionalized anionic MLs were mainly taken up by Kupffer and sinusoidal cells. Target specificity was further confirmed by high-resolution MR imaging of phantoms containing isolated hepatocytes, Kupffer cell (KCs) and hepatic stellate cells (HSCs) fractions. Hypointense signal was observed for hepatocytes isolated from animals which received lactose MLs but not from animals which received anionic MLs. These data demonstrate that galactose-functionalized MLs can be used as a hepatocyte targeting MR contrast agent to potentially aid in the diagnosis of hepatic diseases if the non-specific uptake by KCs is taken into account.

  4. Intrasplenic transplantation of allogeneic hepatocytes prolongs survival in anhepatic rats.

    Science.gov (United States)

    Arkadopoulos, N; Lilja, H; Suh, K S; Demetriou, A A; Rozga, J

    1998-11-01

    To examine whether hepatocytes transplanted in the spleen can function as an ectopic liver, we performed hepatocyte transplantation in rats that were rendered anhepatic. Total hepatectomy was performed by using a novel single-stage technique. Following hepatectomy, Group 1 rats (n = 16) were monitored until death to determine survival time without prior intervention. Group 2 anhepatic rats (n = 20) were sacrificed at various times to measure blood hepatocyte growth factor (HGF) and transforming growth factor beta1 (TGF-beta1) levels. Group 3 (n = 16) rats received intrasplenic injection of isolated hepatocytes (2.5 x 10(7) cells/rat) followed by total hepatectomy after 3 days. Group 4 (n = 12) sham-transplanted rats received intrasplenic saline infusion, and after 3 days they were rendered anhepatic. Group 2, 3, and 4 rats were maintained on daily Cyclosporine A (10 mg/kg; intramuscularly). Group 1 anhepatic rats survived for 22.4 +/- 5.2 hours (standard deviation). The anhepatic state was associated with a progressive and statistically significant rise in blood HGF and TGF-beta1 levels. Rats that received hepatocyte transplantation before total hepatectomy had a significantly longer survival time than sham-transplanted anhepatic controls (34.1 +/- 8.5 vs. 15.5 +/- 4.8 hrs, P ammonia, prothrombin time, international normalized ratio, and TGF-beta1 levels when compared with sham-transplanted controls. In conclusion, intrasplenic transplantation of allogeneic hepatocytes prolonged survival, improved blood chemistry, and lowered blood TGF-beta1 levels in rats rendered anhepatic.

  5. Hepatocyte autophagy model established by physical method

    Directory of Open Access Journals (Sweden)

    ZHU Xuemin

    2016-08-01

    Full Text Available ObjectiveTo establish the autophagy model of normal human liver cell line 7702 induced by hypoxia and starvation, and to lay a foundation for further studies on the influence of autophagy on liver function. MethodsThe 7702 cells were selected and incubated with 95% air and 5% CO2 at a temperature of 37 ℃(normal control group. The Binder three-gas incubator was used, with a temperature of 37 ℃, a CO2 concentration of 5%, and an O2 concentration of 0.3% to provide a hypoxic environment, and the serum-free DMEM was used to induce starvation. These cells were divided into 6-, 12-, 18-, and 24-hour hypoxia-starvation groups. Western blot was used to measure the protein expression of Beclin 1, Atg5, and LC3 in the normal control group and experimental groups, RT-qPCR was used to measure the mRNA expression of Beclin 1 and Atg5 in each group, and after transfection of LC3 plasmid, immunofluorescence assay was used to observe autophagy in each group. An analysis of variance was used for comparison of continuous data between groups, and the least significant difference t-test was used for further comparison between any two groups; the chi-square test was used for comparison of categorical data between groups. ResultsThe 6-hour hypoxia-starvation groups had higher protein expression of Beclin 1, Atg5, and LC3 than the normal control group or other treated groups. Compared with all the other groups, the 6-hour hypoxia-starvation group showed significantly increased mRNA expression of Beclin 1 and Atg5, as well as significantly greater increases in the mRNA expression of Beclin 1 and Atg5 (all P<0.05. The hypoxia-starvation groups had significantly lower numbers of autophagosomes than the normal control group, and the 6-hour hypoxia-starvation group had the highest number of autophagosomes (all P<0.05. ConclusionHypoxia and starvation established by physical methods can successfully induce hepatocyte autophagy, which is the most remarkable at 6

  6. 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

  7. Teaching Form as Form

    DEFF Research Database (Denmark)

    Keiding, Tina Bering

    2012-01-01

    understanding of form per se, or, to use an expression from this text, of form as form. This challenge can be reduced to one question: how can design teaching support students in achieving not only the ability to recognize and describe different form-related concepts in existing design (i.e. analytical...... means that form serves both as the connective value and as the concept for reflection. In other words, form is observed as form, not anything else. The didactical challenge of teaching form as form is accentuated by students’ everyday-based pre-orientation towards function at the expense of form....... In general, students enter design education as far more skilled observers with regards to function than form. They are, in other words, predisposed to observe objects asking ‘what is?’, rather than ‘how is?’. This habit has not only cognitive implications. It is closely intertwined with a rudimentary...

  8. Teaching Form as Form

    DEFF Research Database (Denmark)

    Keiding, Tina Bering

    2012-01-01

    understanding of form per se, or, to use an expression from this text, of form as form. This challenge can be reduced to one question: how can design teaching support students in achieving not only the ability to recognize and describe different form-related concepts in existing design (i.e. analytical...... means that form serves both as the connective value and as the concept for reflection. In other words, form is observed as form, not anything else. The didactical challenge of teaching form as form is accentuated by students’ everyday-based pre-orientation towards function at the expense of form...... vocabulary of form. Even in cases in which teaching uses terms and phrases from everyday life (for instance, ‘intersection’), the meaning of the word cannot necessarily be transmitted directly from an ordinary vocabulary into a design context. And it is clearly a common issue for the contributions...

  9. Study of endophytic Xylariaceae in Thailand: diversity and taxonomy inferred from rDNA sequence analyses with saprobes forming fruit bodies in the field

    DEFF Research Database (Denmark)

    Okane, Izumi; Srikitikulchai, Prasert; Toyama, Kyoko;

    2008-01-01

    ). In addition, strains deposited beforehand were selected in which both endophytic strains isolated from living plant tissues and saprobic strains from fruit bodies were included. Consequently, 405 strains of Xylariaceae (273 endophytic and 132 saprobic strains, including identified strains) were studied...

  10. A Nonhuman Primate Model of Human Radiation-Induced Venocclusive Liver Disease and Hepatocyte Injury

    Energy Technology Data Exchange (ETDEWEB)

    Yannam, Govardhana Rao [Department of Surgery, University of Nebraska Medical Center, Omaha, Nebraska (United States); Han, Bing [Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania (United States); Department of Hepatobiliary Surgery, First Affiliated Hospital of Xi' an Jiaotong University, Xi' an, Shaanxi (China); Setoyama, Kentaro [Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania (United States); Yamamoto, Toshiyuki [Department of Surgery, University of Nebraska Medical Center, Omaha, Nebraska (United States); Ito, Ryotaro; Brooks, Jenna M. [Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania (United States); Guzman-Lepe, Jorge [Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania (United States); Department of Pathology, Children' s Hospital of Pittsburgh, Pittsburgh, Pennsylvania (United States); Galambos, Csaba [Department of Pathology, Children' s Hospital of Pittsburgh, Pittsburgh, Pennsylvania (United States); Fong, Jason V. [Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania (United States); Deutsch, Melvin; Quader, Mubina A. [Department of Radiation Oncology, Children' s Hospital of Pittsburgh, Pittsburgh, Pennsylvania (United States); Yamanouchi, Kosho [Department of Radiation Oncology, Albert Einstein College of Medicine, Bronx, New York (United States); Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, New York (United States); Kabarriti, Rafi; Mehta, Keyur [Department of Radiation Oncology, Albert Einstein College of Medicine, Bronx, New York (United States); Soto-Gutierrez, Alejandro [Department of Pathology, Children' s Hospital of Pittsburgh, Pittsburgh, Pennsylvania (United States); McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania (United States); and others

    2014-02-01

    Background: Human liver has an unusual sensitivity to radiation that limits its use in cancer therapy or in preconditioning for hepatocyte transplantation. Because the characteristic veno-occlusive lesions of radiation-induced liver disease do not occur in rodents, there has been no experimental model to investigate the limits of safe radiation therapy or explore the pathogenesis of hepatic veno-occlusive disease. Methods and Materials: We performed a dose-escalation study in a primate, the cynomolgus monkey, using hypofractionated stereotactic body radiotherapy in 13 animals. Results: At doses ≥40 Gy, animals developed a systemic syndrome resembling human radiation-induced liver disease, consisting of decreased albumin, elevated alkaline phosphatase, loss of appetite, ascites, and normal bilirubin. Higher radiation doses were lethal, causing severe disease that required euthanasia approximately 10 weeks after radiation. Even at lower doses in which radiation-induced liver disease was mild or nonexistent, latent and significant injury to hepatocytes was demonstrated by asialoglycoprotein-mediated functional imaging. These monkeys developed hepatic failure with encephalopathy when they received parenteral nutrition containing high concentrations of glucose. Histologically, livers showed central obstruction via an unusual intimal swelling that progressed to central fibrosis. Conclusions: The cynomolgus monkey, as the first animal model of human veno-occlusive radiation-induced liver disease, provides a resource for characterizing the early changes and pathogenesis of venocclusion, for establishing nonlethal therapeutic dosages, and for examining experimental therapies to minimize radiation injury.

  11. Non-viral FoxM1 gene delivery to hepatocytes enhances liver repopulation.

    Science.gov (United States)

    Xiang, D; Liu, C-C; Wang, M-J; Li, J-X; Chen, F; Yao, H; Yu, B; Lu, L; Borjigin, U; Chen, Y-X; Zhong, L; Wangensteen, K J; He, Z-Y; Wang, X; Hu, Y-P

    2014-05-22

    Hepatocyte transplantation as a substitute strategy of orthotopic liver transplantation is being studied for treating end-stage liver diseases. Several technical hurdles must be overcome in order to achieve the therapeutic liver repopulation, such as the problem of insufficient expansion of the transplanted hepatocytes in recipient livers. In this study, we analyzed the application of FoxM1, a cell-cycle regulator, to enhance the proliferation capacity of hepatocytes. The non-viral sleeping beauty (SB) transposon vector carrying FoxM1 gene was constructed for delivering FoxM1 into the hepatocytes. The proliferation capacities of hepatocytes with FoxM1 expression were examined both in vivo and in vitro. Results indicated that the hepatocytes with FoxM1 expression had a higher proliferation rate than wild-type (WT) hepatocytes in vitro. In comparison with WT hepatocytes, the hepatocytes with FoxM1 expression had an enhanced level of liver repopulation in the recipient livers at both sub-acute injury (fumaryl acetoacetate hydrolase (Fah)(-/-) mice model) and acute injury (2/3 partial hepatectomy mice model). Importantly, there was no increased risk of tumorigenicity with FoxM1 expression in recipients even after serial transplantation. In conclusion, expression of FoxM1 in hepatocytes enhanced the capacity of liver repopulation without inducing tumorigenesis. FoxM1 gene delivered by non-viral SB vector into hepatocytes may be a viable approach to promote therapeutic repopulation after hepatocyte transplantation.

  12. File list: His.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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  17. File list: NoD.Liv.20.AllAg.Hepatocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  18. File list: ALL.Liv.20.AllAg.Hepatocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  19. File list: NoD.Liv.10.AllAg.Hepatocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  20. File list: His.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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    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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  5. File list: Pol.Liv.50.AllAg.Hepatocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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  8. File list: ALL.Liv.50.AllAg.Hepatocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  9. File list: ALL.Liv.05.AllAg.Hepatocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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  12. File list: Oth.Liv.05.AllAg.Hepatocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  13. File list: ALL.Liv.50.AllAg.Hepatocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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

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

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

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  1. File list: InP.Liv.05.AllAg.Hepatocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  2. File list: NoD.Liv.50.AllAg.Hepatocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  4. File list: NoD.Liv.05.AllAg.Hepatocytes [Chip-atlas[Archive

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  8. File list: InP.Liv.50.AllAg.Hepatocytes [Chip-atlas[Archive

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  9. File list: InP.Liv.20.AllAg.Hepatocytes [Chip-atlas[Archive

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  10. File list: InP.Liv.50.AllAg.Hepatocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  11. In vitro proliferation of primary rat hepatocytes expressing ureogenesis activity by coculture with STO cells.

    Science.gov (United States)

    Takagi, Mutsumi; Kondo, Hiroki; Yoshida, Toshiomi

    2002-01-01

    A coculture system for in vitro proliferation of rat primary hepatocytes with feeder cells was investigated with the view of constructing a hybrid artificial liver module using human hepatocytes. A mouse cell line, STO, was apparently effective compared with other kinds of feeder cells such as FLS5 and MRC5 in increasing the total cell number in the coculture of rat primary hepatocytes. The parenchymal hepatocyte, which are polygonal cells, spread well as the cultivation progressed. Monitoring of parenchymal hepatocyte colonies under a microscope showed that the area of each colony increased as the single culture and cocultures progressed. The adhesion area per hepatocyte increased little during the coculture with STO cells, while the adhesion area increased markedly in the single culture of hepatocytes. The density of hepatocytes increased more than two times during the coculture for 5 d, while it decreased during the single culture. The production rate of urea by hepatocytes markedly increased during the coculture, while the rate in the single culture was lower than the coculture and increased only slightly. The specific rate of urea production by hepatocytes in the coculture did not decrease even as the hepatocytes grew. Consequently, rat primary hepatocytes could proliferate in vitro by coculture with STO cells without a decrease in urea production activity.

  12. cAMP prevents TNF-induced apoptosis through inhibiting DISC complex formation in rat hepatocytes.

    Science.gov (United States)

    Bhattacharjee, Rajesh; Xiang, Wenpei; Wang, Yinna; Zhang, Xiaoying; Billiar, Timothy R

    2012-06-22

    Tumor necrosis factor α (TNF) is a pleiotropic proinflammatory cytokine that plays a role in immunity and the control of cell proliferation, cell differentiation, and apoptosis. The pleiotropic nature of TNF is due to the formation of different signaling complexes upon the binding of TNF to its receptor, TNF receptor type 1 (TNFR1). TNF induces apoptosis in various mammalian cells when the cells are co-treated with a transcription inhibitor like actinomycin D (ActD). When TNFR1 is activated, it recruits an adaptor protein, TNF receptor-associated protein with death domain (TRADD), through its cytoplasmic death effector domain (DED). TRADD, in turn, recruits other signaling proteins, including TNF receptor-associated protein 2 (TRAF2) and receptor-associated protein kinase (RIPK) 1, to form a complex. Subsequently, this complex combines with FADD and procaspase-8, converts into a death-inducing signaling complex (DISC) to induce apoptosis. Cyclic AMP (cAMP) is a second messenger that regulates various cellular processes such as cell proliferation, gene expression, and apoptosis. cAMP analogues are reported to act as anti-apoptotic agents in various cell types, including hepatocytes. We found that a cAMP analogue, dibutyryl cAMP (db-cAMP), inhibits TNF+ActD-induced apoptosis in rat hepatocytes. The protein kinase A (PKA) inhibitor KT-5720 reverses this inhibitory effect of cAMP on apoptosis. Cytoprotection by cAMP involves down-regulation of various apoptotic signal regulators like TRADD and FADD and inhibition of caspase-8 and caspase-3 cleavage. We also found that cAMP exerts its affect at the proximal level of TNF signaling by inhibiting the formation of the DISC complex upon the binding of TNF to TNFR1. In conclusion, our study shows that cAMP prevents TNF+ActD-induced apoptosis in rat hepatocytes by inhibiting DISC complex formation.

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

    Science.gov (United States)

    Kohno, Y; Shiraki, K; Mura, T

    1991-03-01

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

  14. Potential inhibition of cytochrome P450 3A4 by propofol in human primary hepatocytes

    Institute of Scientific and Technical Information of China (English)

    Li-Qun Yang; Wei-Feng Yu; Yun-Fei Cao; Bin Gong; Qing Chang; Guang-Shun Yang

    2003-01-01

    AIM: Hepatic cytochrome P450 isoenzymes constitute a superfamily of hemoproteins that play a major role in the metabolism of endogenous compounds and in the detoxification of xenobiotic molecules. P450 3A4 is one of the most important forms in human being, and mediates the metabolism of around 70% of therapeutic drugs and endogenous compounds. Propofol, a widely used intravenous anesthetic drug, is known to inhibit cytochrome P450activities in isolated rat hepatocytes. The goal of this study was to evaluate the potential efficacy of propofol on P4503A4 in a dose-dependent manner to understand its drugdrug interaction.METHODS: Hepatocytes were isolated from liver specimens from hepatic angioma patients undergone hepatic surgery.Primary incubated hepatocytes were treated with 0, 0.01,0.05, 0.1, 0.5, and 1.0 mM propofol for 24 hours. P450 3A4activity was measured with Nash′s colorimetry. The protein expression was assessed by Western blot analysis.RESULTS: A dose-dependent inhibitory effect of propofol was observed in cytochrome P450 3A4 activity. A minimal dosage of propofol (0.01 mM) induced a significant inhibition of P450 3A4 activity, although its regular dosages (0.01-0.1mM) showed no inhibitory effect on the cellular protein expression of P450 3A4.CONCLUSION: Propofol may be a potential CYP3A4 inhibitor as this anesthetic can inhibit isoenzyme activity significantly and reduce the metabolic rate of CYP3A4 substrates. This inhibition occurs at post-expression level, and concentration of propofol used clinically does not affect CYP3A4 protein expression. propofol may thus induce drug interaction of cytochrome P450 3A4 activity at the dosage used clinically.

  15. Hemin Improves Insulin Sensitivity and Lipid Metabolism in Cultured Hepatocytes and Mice Fed a High-Fat Diet

    Directory of Open Access Journals (Sweden)

    Yi Luan

    2017-07-01

    Full Text Available Hemin is a breakdown product of hemoglobin. It has been reported that the injection of hemin improves lipid metabolism and insulin sensitivity in various genetic models. However, the effect of hemin supplementation in food on lipid metabolism and insulin sensitivity is still unclear, and whether hemin directly affects cellular insulin sensitivity is yet to be elucidated. Here we show that hemin enhances insulin-induced phosphorylation of insulin receptors, Akt, Gsk3β, FoxO1 and cytoplasmic translocation of FoxO1 in cultured primary hepatocytes under insulin-resistant conditions. Furthermore, hemin diminishes the accumulation of triglyceride and increases in free fatty acid content in primary hepatocytes induced by palmitate. Oral administration of hemin decreases body weight, energy intake, blood glucose and triglyceride levels, and improves insulin and glucose tolerance as well as hepatic insulin signaling and hepatic steatosis in male mice fed a high-fat diet. In addition, hemin treatment decreases the mRNA and protein levels of some hepatic genes involved in lipogenic regulation, fatty acid synthesis and storage, and increases the mRNA level and enzyme activity of CPT1 involved in fatty acid oxidation. These data demonstrate that hemin can improve lipid metabolism and insulin sensitivity in both cultured hepatocytes and mice fed a high-fat diet, and show the potential beneficial effects of hemin from food on lipid and glucose metabolism.

  16. Accretion and differentiation of the terrestrial planets with implications for the compositions of early-formed Solar System bodies and accretion of water

    CERN Document Server

    Rubie, David C; Morbidelli, Alessandro; O'Brien, Dave P; Young, Ed D; de Vries, Jellie; Palme, Herbert; Frost, Daniel J

    2014-01-01

    In order to test planetary accretion and differentiation scenarios, we integrated a multistage core-mantle differentiation model with N-body accretion simulations. Impacts between embryos and planetesimals result in magma ocean formation and episodes of core formation. The core formation model combines rigorous chemical mass balance with metal-silicate element partitioning data. The primary constraint on the combined model is the composition of the Earth's primitive mantle, the composition of the Martian mantle, and the mass fractions of the metallic cores of Earth and Mars. The model is refined by least squares minimization with up to five fitting parameters that consist of the metal-silicate equilibrium pressure and 1-4 parameters that define the starting compositions of primitive bodies. This integrated model has been applied to 6 Grand Tack simulations. Investigations of a broad parameter space indicate that: accretion of Earth was heterogeneous, metal-silicate equilibration pressures increase as accretio...

  17. The Protection of Hepatocyte Cells from the Effects of Oxidative Stress by Treatment with Vitamin E in Conjunction with DTT

    Directory of Open Access Journals (Sweden)

    Jen-Hsiang Tsai

    2010-01-01

    Full Text Available We investigated the effect of vitamin E on membrane protein thiols under oxidative stress, which we induced by treating hepatocytes with tert-butyl hydroperoxide (TBH for 60 mins. Those cells which we pretreated with vitamin E formed fewer blebs (22.3% compared to 60.0% in nonvitamin E-treated cells and maintained cytosolic calcium concentration and the number of membrane protein thiols instead of showing the usual symptoms in cells undergoing oxidative stress. Dithiothreitol (DTT also commonly reduces bleb formation in hepatocytes affected by TBH. However, our experiments clearly demonstrate that DTT does not prevent the changes in cytosolic calcium and membrane protein thiols in the blebbing cells. Consequently, we decided to pretreat cells with both DTT and vitamin E and found that the influence of TBH was entirely prevented. These findings may provide us with a new aspect for investigating the mechanism of bleb formation under oxidative stress.

  18. Stem cell-derived hepatocytes and their use in toxicology.

    Science.gov (United States)

    Guguen-Guillouzo, Christiane; Corlu, Anne; Guillouzo, Andre

    2010-03-30

    Better prediction of safety risk and understanding of mechanism of action of drug candidates remain a major challenge in order to prevent late stage attrition. Continuous efforts are made to improve and develop new models, especially in some areas such as hepatotoxicity. Besides primary hepatocytes and transformed liver cell lines, stem cells either isolated from embryos or adult tissues or obtained by reprogramming somatic cells are emerging as a new potential source of unlimited numbers of hepatocytes. Presently, only hepatocyte-like cells expressing low levels of liver-specific markers, especially drug metabolizing and detoxifying enzymes, are usually obtained, making them still unsuitable as metabolically competent cells for toxicity studies. The only exceptions are some hepatoma cell lines, particularly the HepaRG cell line that can differentiate from a bipotent progenitor stage to attain the functional capacity of normal adult hepatocytes in primary culture without losing the indefinite growth property of transformed cells. Since the research field on stem cells is growing fast marked advances might be expected in the next future.

  19. Metformin stimulates FGF21 expression in primary hepatocytes

    DEFF Research Database (Denmark)

    Nygaard, Eva B; Vienberg, Sara G; Ørskov, Cathrine

    2012-01-01

    incubated with Compound C (an AMPK inhibitor) in the presence of metformin. A strong dose-dependent increase in FGF21 expression was observed in both rat and human hepatocytes treated with metformin. This effect was blocked by addition of the AMPK-inhibitor Compound C. The study shows that metformin...

  20. 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

  1. 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 toxicolo

  2. 3D Cultivation Techniques for Primary Human Hepatocytes

    Directory of Open Access Journals (Sweden)

    Anastasia Bachmann

    2015-02-01

    Full Text Available One of the main challenges in drug development is the prediction of in vivo toxicity based on in vitro data. The standard cultivation system for primary human hepatocytes is based on monolayer cultures, even if it is known that these conditions result in a loss of hepatocyte morphology and of liver-specific functions, such as drug-metabolizing enzymes and transporters. As it has been demonstrated that hepatocytes embedded between two sheets of collagen maintain their function, various hydrogels and scaffolds for the 3D cultivation of hepatocytes have been developed. To further improve or maintain hepatic functions, 3D cultivation has been combined with perfusion. In this manuscript, we discuss the benefits and drawbacks of different 3D microfluidic devices. For most systems that are currently available, the main issues are the requirement of large cell numbers, the low throughput, and expensive equipment, which render these devices unattractive for research and the drug-developing industry. A higher acceptance of these devices could be achieved by their simplification and their compatibility with high-throughput, as both aspects are of major importance for a user-friendly device.

  3. Inhibitory effect of picroside Ⅱ on hepatocyte apoptosis

    Institute of Scientific and Technical Information of China (English)

    Hua GAO; Ya-wei ZHOU

    2005-01-01

    Aim: To investigate the influence of picroside Ⅱ on hepatocyte apoptosis and its mechanism. Methods: Morphological changes and quantification of apoptotic cells were determined under transmission electron microscopy and flow cytometry respectively. DNA fragmentation was visualized by agarose gel electrophoresis.Semi-quantitative reverse transcription-PCR (RT-PCR) was used to analyze the expression of bcl-2 and bax genes. The content of manganese-superoxide dismutase (SOD) in liver mitochondria was detected by the Marland method. The content of malonic aldehyde (MDA) and the protein level in liver tissue were determined by thiobarbituric acid colorimetry and Lowry method. Results:Picroside Ⅱ decreased the levels of alanine aminotransferase and aspartate aminotransferase in the serum resulting from acute-liver injured mice induced with D-GalN and LPS; it also reduced the content of MDA, and thus, enhanced the activity of SOD. Picroside Ⅱ 10 mg/kg was found to protect hepatocytes against apoptosis in a dose-dependent manner; it up-regulated the expression of bcl-2 genes,thus increased the bcl-2/bax ratio. Conclusion: Picroside Ⅱ can protect hepatocytes against injury and prevent hepatocytes from apoptosis. It might by upregulating the bcl-2 gene expression and antioxidation.

  4. Effect of matrine on primary human hepatocytes in vitro.

    Science.gov (United States)

    Gong, Xiaobing; Gao, Yuan; Guo, Guoqing; Vondran, Florian W R; Schwartlander, Ruth; Efimova, Ekaterina; Pless, Gesine; Sauera, Igor M; Neuhaus, Peter

    2015-03-01

    Matrine is a bioactive component of the traditional Chinese medical herb Sophora flavescens that has been used in China to treat various kinds of diseases including virus hepatitis. However, the molecular mechanisms underlying its hepatoprotective effects remains elusive. In the present study, primary human hepatocytes were employed to elucidate the protective effects and molecular mechanisms of matrine. We observed that low concentrations of matrine had no significant impact on albumin secretion, but high concentrations (>140 mg/L) of matrine decreased the albumin secretion in hepatocytes. Western blot data indicated that matrine at 140 mg/L at 72 h induced protein expression of CYP2A6, CYP2B6 and CYP3A4. Furthermore, high concentrations of matrine reduced LDH and AST levels and were cytotoxic to hepatocytes, leading to a decreased cell viability and total protein amount. Moreover, low concentrations of matrine, enhanced the ECOD activity and decreased the level of NO2 (-) induced by cytokines in human hepatocytes. Taken together, the present study sheds novel light on the molecular mechanisms of matrine and potential application of matrine in hepatic diseases.

  5. Transplantation of human hepatocytes into tolerized genetically immunocompetent rats

    Institute of Scientific and Technical Information of China (English)

    Edwin C. Ouyang; Catherine H. Wu; Cherie Walton; Kittichai Promrat; George Y. Wu

    2001-01-01

    AIM To determine whether normal geneticallyirnmunocornpetent rodent hosts could bemanipulated to accept human hepatocytetransplants with long term survival withoutirnrnunosuppression.METHODS Tolerance towards humanhepatocytes was established by injection ofprimary human hepatocytes or Huh7 humanhepatoma cells into the peritoneal cavities offetal rats. Corresponding cells weresubsequently transplanted into newborn rats viaintrasplenic injection within 24 h after birth.RESULTS Mixed lymphocyte assays showedthat spleen cells from non-tolerized rats werestimulated to proliferate when exposed to humanhepatocytes, while cells from tolerized ratswere not. Injections made between 15 d and 17 dof gestation produced optimal tolerizaton.Transplanted human hepatocytes in rat liverswere visualized by immunohistochemicalstaining of human albumin. By dot blotting ofgenomic DNA in livers of tolerized rats 16 weeksafter hepatocyte transplantation, it was foundthat approximately 2.5 × 105 human hepatocytessurvived per rat liver. Human albumin mRNA wasdetected in rat livers by RT-PCR for 15 wk, andhuman albumin protein was also detectable in ratserum.CONCLUSION Tolerization of an immuno-competent rat can permit transplantation, andsurvival of functional human hepatocytes.

  6. A Microfabricated Platform for Generating Physiologically-Relevant Hepatocyte Zonation

    Science.gov (United States)

    McCarty, William J.; Usta, O. Berk; Yarmush, Martin L.

    2016-05-01

    In vitro liver models have been important tools for more than 40 years for academic research and preclinical toxicity screening by the pharmaceutical industry. Hepatocytes, the highly metabolic parenchymal cells of the liver, are efficient at different metabolic chemistries depending on their relative spatial location along the sinusoid from the portal triad to the central vein. Although replicating hepatocyte metabolic zonation is vitally important for physiologically-relevant in vitro liver tissue and organ models, it is most often completely overlooked. Here, we demonstrate the creation of spatially-controlled zonation across multiple hepatocyte metabolism levels through the application of precise concentration gradients of exogenous hormone (insulin and glucagon) and chemical (3-methylcholanthrene) induction agents in a microfluidic device. Observed gradients in glycogen storage via periodic acid-Schiff staining, urea production via carbamoyl phosphatase synthetase I staining, and cell viability after exposure to allyl alcohol and acetaminophen demonstrated the in vitro creation of hepatocyte carbohydrate, nitrogen, alcohol degradation, and drug conjugation metabolic zonation. This type of advanced control system will be crucial for studies evaluating drug metabolism and toxicology using in vitro constructs.

  7. 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.

  8. Preservation of hepatocyte suspensions at 4 degrees C.

    Science.gov (United States)

    Lund, P; Wiggins, D

    1988-10-01

    Suspensions of rat hepatocytes are resistant to hypoxia for at least 24 hr at 4 degrees C. After storage for 24 hr with xylitol, sorbitol, or glycerol, their subsequent capacity for glucogenesis from these substrates is increased. Even storage under N2/CO2 as the gas phase has little deleterious effect.

  9. Processes for forming exoergic structures with the use of a plasma and for producing dense refractory bodies of arbitrary shape therefrom

    Science.gov (United States)

    Holt, J. Birch; Kelly, Michael D.

    1990-01-01

    Plasma spraying methods of forming exoergic structures and coatings, as well as exoergic structures produced by such methods, are provided. The methods include the plasma spraying of reactive exoergic materials that are capable of sustaining a combustion synthesis reaction onto a flat substrate or into molds of arbitrary shape and igniting said plasma sprayed materials, either under an inert gas pressure or not, to form refractory materials of varying densities and of varying shapes.

  10. The Oncogenic Fusion Proteins SET-Nup214 and Sequestosome-1 (SQSTM1)-Nup214 Form Dynamic Nuclear Bodies and Differentially Affect Nuclear Protein and Poly(A)+ RNA Export.

    Science.gov (United States)

    Port, Sarah A; Mendes, Adélia; Valkova, Christina; Spillner, Christiane; Fahrenkrog, Birthe; Kaether, Christoph; Kehlenbach, Ralph H

    2016-10-28

    Genetic rearrangements are a hallmark of several forms of leukemia and can lead to oncogenic fusion proteins. One example of an affected chromosomal region is the gene coding for Nup214, a nucleoporin that localizes to the cytoplasmic side of the nuclear pore complex (NPC). We investigated two such fusion proteins, SET-Nup214 and SQSTM1 (sequestosome)-Nup214, both containing C-terminal portions of Nup214. SET-Nup214 nuclear bodies containing the nuclear export receptor CRM1 were observed in the leukemia cell lines LOUCY and MEGAL. Overexpression of SET-Nup214 in HeLa cells leads to the formation of similar nuclear bodies that recruit CRM1, export cargo proteins, and certain nucleoporins and concomitantly affect nuclear protein and poly(A)(+) RNA export. SQSTM1-Nup214, although mostly cytoplasmic, also forms nuclear bodies and inhibits nuclear protein but not poly(A)(+) RNA export. The interaction of the fusion proteins with CRM1 is RanGTP-dependent, as shown in co-immunoprecipitation experiments and binding assays. Further analysis revealed that the Nup214 parts mediate the inhibition of nuclear export, whereas the SET or SQSTM1 part determines the localization of the fusion protein and therefore the extent of the effect. SET-Nup214 nuclear bodies are highly mobile structures, which are in equilibrium with the nucleoplasm in interphase and disassemble during mitosis or upon treatment of cells with the CRM1-inhibitor leptomycin B. Strikingly, we found that nucleoporins can be released from nuclear bodies and reintegrated into existing NPC. Our results point to nuclear bodies as a means of preventing the formation of potentially insoluble and harmful protein aggregates that also may serve as storage compartments for nuclear transport factors.

  11. Repression of allo-cell transplant rejection through CIITA ribonuclease P+ hepatocyte

    Institute of Scientific and Technical Information of China (English)

    Rong Guo; Ping Zou; Hua-Hua Fan; Feng Gao; Qing-Xin Shang; Yi-Lin Cao; Hua-Zhong Lu

    2003-01-01

    AIM: Allo-cell transplant rejection and autoimmune responses were associated with the presence of class Ⅱmajor histocompatibility complex (MHC Ⅱ) molecules on cells.This paper studied the effect of Ribonuclease P (RNase P)against CIITA, which was a major regulator of MHCII molecules, on repressing the expression of MHCII molecules on hepatocyte.METHODS: M1-RNA is the catalytic RNA subunit of RNase P from Escherichia coli. It were constructed that M1-RNA with guide sequences (GS) recognizing the 452, 3408 site of CIITA by PCR from pTK117 plasmid, then were cloned into the EcoRI/Bg/II or EcoR//SalIsite of vector psNAV (osNAV-M1-452-GS, psNAV-M1-3408-GS) respectively. The target mould plate (3176-3560) of CIITA was obtained from Raji cell by RT-PCR, and then inserted into the XhoI/EcoRIof pGEM-7zf(+) plasmid (pGEM-3176). These recombinant plasmids were screened out by sequence analysis. psNAV-M 1-452-GS, psNAV-M1-3408-GS and its target RNA pGEM-3176 were transcribed and then mixed up and incubated in vitro. It showed that M1-3408-GS could exclusively cleave target RNA that formed a base pair with the GS. Stable transfectants of hepatocyte cell line with psNAVl-M1-3408-GS were tested for expression of class Ⅱ MHC through FCM, for mRNA abundance of MHCII, Ii and CIITA by RTPCR., for the level of IL-2 mRNA on T cell by mixed lymphocyte reaction.RESULTS: When induced with recombinant human interferon-gamma (IFN-γ), the expression of HLA-DR, -DP,-DQ on psNAV-M1-3408-GS+ hepatocyte was reduced 83.27 %, 88.93 %, 58.82 % respectively, the mRNA contents of CIITA, HLA-DR, -DP, -DQ and Ii decreased significantly.While T cell expressed less IL-2 mRNA in the case of psNAV-M1-3408-GS+ hepatocyte.CONCLUSION: The Ribonuclease P against CIITA-M1-3408-GS could effectively induce antigen-specific tolerance through cleaving CIITA. These results provided insight into the future application of M1-3408-GS as a new nucleic acid drug against allo-transplantation rejection and

  12. cAMP prevents TNF-induced apoptosis through inhibiting DISC complex formation in rat hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Bhattacharjee, Rajesh [Department of Surgery, University of Pittsburgh Medical Center, Pittsburgh, PA 15213 (United States); Xiang, Wenpei [Department of Surgery, University of Pittsburgh Medical Center, Pittsburgh, PA 15213 (United States); Family Planning Research Institute, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, People' s Republic of China (China); Wang, Yinna [Vascular Medicine Institute, University of Pittsburgh School of Medicine, 10051-5A BST 3, 3501 Fifth Avenue, Pittsburgh, PA 15261 (United States); Zhang, Xiaoying [Department of Medicine/Endocrinology Division, University of Pittsburgh Medical Center, 200 Lothrop St., Pittsburgh, PA 15213 (United States); Billiar, Timothy R., E-mail: billiartr@upmc.edu [Department of Surgery, University of Pittsburgh Medical Center, Pittsburgh, PA 15213 (United States)

    2012-06-22

    Highlights: Black-Right-Pointing-Pointer cAMP blocks cell death induced by TNF and actinomycin D in cultured hepatocytes. Black-Right-Pointing-Pointer cAMP blocks NF-{kappa}B activation induced by TNF and actinomycin D. Black-Right-Pointing-Pointer cAMP blocks DISC formation following TNF and actinomycin D exposure. Black-Right-Pointing-Pointer cAMP blocks TNF signaling at a proximal step. -- Abstract: Tumor necrosis factor {alpha} (TNF) is a pleiotropic proinflammatory cytokine that plays a role in immunity and the control of cell proliferation, cell differentiation, and apoptosis. The pleiotropic nature of TNF is due to the formation of different signaling complexes upon the binding of TNF to its receptor, TNF receptor type 1 (TNFR1). TNF induces apoptosis in various mammalian cells when the cells are co-treated with a transcription inhibitor like actinomycin D (ActD). When TNFR1 is activated, it recruits an adaptor protein, TNF receptor-associated protein with death domain (TRADD), through its cytoplasmic death effector domain (DED). TRADD, in turn, recruits other signaling proteins, including TNF receptor-associated protein 2 (TRAF2) and receptor-associated protein kinase (RIPK) 1, to form a complex. Subsequently, this complex combines with FADD and procaspase-8, converts into a death-inducing signaling complex (DISC) to induce apoptosis. Cyclic AMP (cAMP) is a second messenger that regulates various cellular processes such as cell proliferation, gene expression, and apoptosis. cAMP analogues are reported to act as anti-apoptotic agents in various cell types, including hepatocytes. We found that a cAMP analogue, dibutyryl cAMP (db-cAMP), inhibits TNF + ActD-induced apoptosis in rat hepatocytes. The protein kinase A (PKA) inhibitor KT-5720 reverses this inhibitory effect of cAMP on apoptosis. Cytoprotection by cAMP involves down-regulation of various apoptotic signal regulators like TRADD and FADD and inhibition of caspase-8 and caspase-3 cleavage. We also found

  13. Reinforced Airfoil Shaped Body

    DEFF Research Database (Denmark)

    2011-01-01

    The present invention relates to an airfoil shaped body with a leading edge and a trailing edge extending along the longitudinal extension of the body and defining a profile chord, the airfoil shaped body comprising an airfoil shaped facing that forms the outer surface of the airfoil shaped body...

  14. Dual delivery of vascular endothelial growth factor and hepatocyte growth factor coacervate displays strong angiogenic effects.

    Science.gov (United States)

    Awada, Hassan K; Johnson, Noah R; Wang, Yadong

    2014-05-01

    Controlled delivery of multiple growth factors (GFs) holds great potential for the clinical treatment of ischemic diseases and might be more therapeutically effective to reestablish vasculature than the provision of a single GF. Vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) are two potent angiogenic factors. However, due to rapid degradation and dilution in the body, their clinical potential will rely on an effective mode of delivery. A coacervate, composed of heparin and a biodegradable polycation, which protects GFs from proteolysis and potentiates their bioactivities, is developed. Here, the coacervate incorporates VEGF and HGF and sustains their release for at least three weeks. Their strong angiogenic effects on endothelial cell proliferation and tube formation in vitro are confirmed. Furthermore, it is demonstrated that coacervate-based delivery of these factors has stronger effects than free application of both factors and to coacervate delivery of each GF separately.

  15. Effects of macroalgae on the recruitment, growth, and body condition of an invasive reef forming polychaete in a south-western Atlantic coastal lagoon

    Science.gov (United States)

    Bazterrica, María Cielo; Bruschetti, Carlos Martín; Alvarez, María Fernanda; Iribarne, Oscar; Botto, Florencia

    2014-04-01

    Species interactions could mediate species invasive processes. In Mar Chiquita coastal lagoon (37° 40‧S, 57° 23´W, Argentine), the invasive reef building polychaete Ficopomatus enigmaticus (Fauvel 1923) enhances the biomass of the red alga Polysiphonia subtilissima Montagne 1840 on reef surfaces, and excludes green macroalgae (mainly Cladophora sp. Kützing, 1843) from sediment between reefs. In turn, macroalgae could have several community structuring effects (e.g., as food or by competing for space). Therefore, macroalgae may affect F. enigmaticus. To evaluate this hypothesis we studied (1) the interaction between macroalgae and F. enigmaticus during the colonization of new substrates and (2) the effects of macroalgae on the recruitment, growth, and body condition of F. enigmaticus. Field sampling and experiments suggested a lack of competition on new substrates. However, there was a positive effect of macroalgae on F. enigmaticus during the warm season, since its recruitment, tube length, and body condition were higher in areas with macroalgae on reef surfaces. Considering that previous studies showed that reefs positively affect macroalgae, our results suggest that there is a positive feedback on F. enigmaticus created by macroalgae on established reefs and during the reefs' growing season. This interaction may contribute to the maintenance and growth of established reefs.

  16. Chemically induced hepatotoxicity in human stem cell-induced hepatocytes compared with primary hepatocytes and HepG2.

    Science.gov (United States)

    Kang, Seok-Jin; Lee, Hyuk-Mi; Park, Young-Il; Yi, Hee; Lee, Hunjoo; So, ByungJae; Song, Jae-Young; Kang, Hwan-Goo

    2016-10-01

    Stem cell-induced hepatocytes (SC-iHeps) have been suggested as a valuable model for evaluating drug toxicology. Here, human-induced pluripotent stem cells (QIA7) and embryonic stem cells (WA01) were differentiated into hepatocytes, and the hepatotoxic effects of acetaminophen (AAP) and aflatoxin B1 (AFB1) were compared with primary hepatocytes (p-Heps) and HepG2. In a cytotoxicity assay, the IC50 of SC-iHeps was similar to that in p-Heps and HepG2 in the AAP groups but different from that in p-Heps of the AFB1 groups. In a multi-parameter assay, phenotypic changes in mitochondrial membrane potential, calcium influx and oxidative stress were similar between QIA7-iHeps and p-Heps following AAP and AFB1 treatment but relatively low in WA01-iHeps and HepG2. Most hepatic functional markers (hepatocyte-specific genes, albumin/urea secretion, and the CYP450 enzyme activity) were decreased in a dose-dependent manner following AAP and AFB1 treatment in SC-iHeps and p-Heps but not in HepG2. Regarding CYP450 inhibition, the cell viability of SC-iHeps and p-Heps was increased by ketoconazole, a CYP3A4 inhibitor. Collectively, SC-iHeps and p-Heps showed similar cytotoxicity and hepatocyte functional effects for AAP and AFB1 compared with HepG2. Therefore, SC-iHeps have phenotypic characteristics and sensitivity to cytotoxic chemicals that are more similar to p-Heps than to HepG2 cells.

  17. Scalable spheroid model of human hepatocytes for hepatitis C infection and replication.

    Science.gov (United States)

    Ananthanarayanan, Abhishek; Nugraha, Bramasta; Triyatni, Miriam; Hart, Stefan; Sankuratri, Suryanarayana; Yu, Hanry

    2014-07-07

    Developing effective new drugs against hepatitis C (HCV) virus has been challenging due to the lack of appropriate small animal and in vitro models recapitulating the entire life cycle of the virus. Current in vitro models fail to recapitulate the complexity of human liver physiology. Here we present a method to study HCV infection and replication on spheroid cultures of Huh 7.5 cells and primary human hepatocytes. Spheroid cultures are constructed using a galactosylated cellulosic sponge with homogeneous macroporosity, enabling the formation and maintenance of uniformly sized spheroids. This facilitates easy handling of the tissue-engineered constructs and overcomes limitations inherent of traditional spheroid cultures. Spheroids formed in the galactosylated cellulosic sponge show enhanced hepatic functions in Huh 7.5 cells and maintain liver-specific functions of primary human hepatocytes for 2 weeks in culture. Establishment of apical and basolateral polarity along with the expression and localization of all HCV specific entry proteins allow for a 9-fold increase in viral entry in spheroid cultures over conventional monolayer cultures. Huh 7.5 cells cultured in the galactosylated cellulosic sponge also support replication of the HCV clone, JFH (Japanese fulminant hepatitis)-1 at higher levels than in monolayer cultures. The advantages of our system in maintaining liver-specific functions and allowing HCV infection together with its ease of handling make it suitable for the study of HCV biology in basic research and pharmaceutical R&D.

  18. [Influence of specification on chemical composition of dissolution and hepatocytes toxicity of Polygonum multiflorum].

    Science.gov (United States)

    lI, Yu-Meng; Li, Rui-Yu; Niu, Ming; Li, Chun-Yu; Bai, Zhao-Fang; Feng, Wu-Wen; Zhang, Cong-En; Tan, Peng; Huang, Zhi-Pu; Ma, Wei-Guang; Wang, Jia-Bo; Xiao, Xiao-He

    2016-03-01

    According to different toxicities of various aqueous extracts of Polygonum multiflorum on hepatocyte, the impacts of chemical composition on the safety of P. multiforum was studied. In this study, 8 main chemical compositions in aqueous extracts of P. multiflorum were determined by the established HPLC method; at the same time, the inhibition ratios of different aqueous extracts of P. multiflorum on L02 cell were determined. Afterwards, the potential compounds related to the toxicity of P. multiforum were tentatively found through a multiple correlation analysis. The results showed that P. multiforum with different chemical compositions exhibited great differences in dissolution. The hepatocyte toxicity of P. multiflorum powder was much greater than P. multiflorum lumps. In addition, three constituents closely related to toxicity of P. multiflorum were found by multiple correlation analysis. The study revealed that chemical composition of P. multiflorum is closely related to the hepatotoxicity, and the hepatotoxicity of P. multiflorum powder is greater than that of other dosage forms. This study indicates that P. multiflorum with different chemical compositions show varying toxicity, which therefore shall be given high attention. Copyright© by the Chinese Pharmaceutical Association.

  19. Self-assembly model, hepatocytes attachment and inflammatory response for silk fibroin/chitosan scaffolds

    Energy Technology Data Exchange (ETDEWEB)

    She Zhending; Feng Qingling [State Key Laboratory of New Ceramics and Fine Processing, Department of Materials Science and Engineering, Tsinghua University, Beijing 100084 (China); Liu Weiqiang, E-mail: biomater@mail.tsinghua.edu.c [Center for Advanced Materials and Biotechnology, Research Institute of Tsinghua University in Shenzhen, Shenzhen 518057 (China)

    2009-08-15

    Silk fibroin is an attractive natural fibrous protein for biomedical application due to its good biocompatibility and high tensile strength. Silk fibroin is apt to form a sheet-like structure during the freeze-drying process, which is not suitable for the scaffold of tissue engineering. In our former study, the adding of chitosan promoted the self-assembly of silk fibroin/chitosan (SFCS) into a three-dimensional (3D) homogeneous porous structure. In this study, a model of the self-assembly is proposed; furthermore, hepatocytes attachment and inflammatory response for the SFCS scaffold were examined. The rigid chain of chitosan may be used as a template for beta-sheet formation of silk fibroin, and this may break the sheet structure of the silk fibroin scaffold and promote the formation of a 3D porous structure of the SFCS scaffold. Compared with the polylactic glycolic acid scaffold, the SFCS scaffold further facilitates the attachment of hepatocytes. To investigate the inflammatory response, SFCS scaffolds were implanted into the greater omentum of rats. From the results of implantation, we could demonstrate in vivo that the implantation of SFCS scaffolds resulted in only slight inflammation. Keeping the good histocompatibility and combining the advantages of both fibroin and chitosan, the SFCS scaffold could be a prominent candidate for soft tissue engineering, for example, in the liver.

  20. Zooplankton body composition

    DEFF Research Database (Denmark)

    Kiørboe, Thomas

    2013-01-01

    I compiled literature on zooplankton body composition, from protozoans to gelatinous plankton, and report allometric relations and average body composition. Zooplankton segregate into gelatinous and non-gelatinous forms, with few intermediate taxa (chaetognaths, polychaetes, and pteropods). In most...

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-11-01

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

  2. Acute Toxicity Test of Polysaccharides Krestin from Coriolus versicolor Extract with Parameters of Hepatocyte Damages, SGPT and SGOT Enzyme in Mice

    OpenAIRE

    Andita Ayu Mandasari; Sri Puji Astuti Wahyuningsih; Win Darmanto

    2015-01-01

    Coriolus versicolor is a mushroom that has polysaccharopeptide krestin (PSK) and polysaccharopeptide (PSP). Many reports showed that polysaccharide krestin as a compound which could reduce mutagen induction, radiation, and development of cancer spontaneusly. However, all substances entering in body could change become toxic depending on dosage. Therefore, this research was aimed to know the effects of PSK on hepatocyte damages, SGPT and SGOT enzymes. Polysaccharide krestin was given by intrap...

  3. The involvement of cytochrome P450 peroxidase in the metabolic bioactivation of cumene hydroperoxide by isolated rat hepatocytes.

    Science.gov (United States)

    Anari, M R; Khan, S; O'Brien, P J

    1996-09-01

    Organic hydroperoxides are believed to be primarily detoxified in cells by the GSH peroxidase/GSSG reductase system and activated to cytotoxic radical species by non-heme iron. However, organic hydroperoxides seem to be bioactivated by cytochrome P450 (P450) in isolated hepatocytes as various P450 (particularly P450 2E1) inhibitors inhibited cumene hydroperoxide (CumOOH) metabolism and attenuated subsequent cytotoxic effects including antimycin A-resistant respiration, lipid peroxidation, iron mobilization, ATP depletion, and cell membrane disruption. CumOOH metabolism was also faster in P450 1A-induced hepatocytes and was inhibited by the P450 1A inhibitor alpha-naphthoflavone. The ferric chelator deferoxamine also prevented cytotoxicity even after CumOOH had been metabolized but had no effect on CumOOH metabolism. This emphasizes the toxicological significance of the iron released following hydroperoxide metabolic activation by cytochrome P450. The radical trap, 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO), had no effect on CumOOH metabolism but prevented CumOOH-induced antimycin A-resistant respiration, lipid peroxidation, iron mobilization, and loss of membrane integrity. These results suggest that CumOOH is metabolically activated by some P450 enzymes (e.g., P450 2E1) in hepatocytes to form reactive radical metabolites or oxidants that cause lipid peroxidation and cytotoxicity.

  4. Vitamin A and feeding statuses modulate the insulin-regulated gene expression in Zucker lean and fatty primary rat hepatocytes.

    Directory of Open Access Journals (Sweden)

    Wei Chen

    Full Text Available Unattended hepatic insulin resistance predisposes individuals to dyslipidemia, type 2 diabetes and many other metabolic complications. The mechanism of hepatic insulin resistance at the gene expression level remains unrevealed. To examine the effects of vitamin A (VA, total energy intake and feeding conditions on the insulin-regulated gene expression in primary hepatocytes of Zucker lean (ZL and fatty (ZF rats, we analyze the expression levels of hepatic model genes in response to the treatments of insulin and retinoic acid (RA. We report that the insulin- and RA-regulated glucokinase, sterol regulatory element-binding protein-1c and cytosolic form of phosphoenolpyruvate carboxykinase expressions are impaired in hepatocytes of ZF rats fed chow or a VA sufficient (VAS diet ad libitum. The impairments are partially corrected when ZF rats are fed a VA deficient (VAD diet ad libitum or pair-fed a VAS diet to the intake of their VAD counterparts in non-fasting conditions. Interestingly in the pair-fed ZL and ZF rats, transient overeating on the last day of pair-feeding regimen changes the expression levels of some VA catabolic genes, and impairs the insulin- and RA-regulated gene expression in hepatocytes. These results demonstrate that VA and feeding statuses modulate the hepatic insulin sensitivity at the gene expression level.

  5. Microcystin-LR induces anoikis resistance to the hepatocyte uptake transporter OATP1B3-expressing cell lines.

    Science.gov (United States)

    Takano, Hiroyuki; Takumi, Shota; Ikema, Satoshi; Mizoue, Nozomi; Hotta, Yuki; Shiozaki, Kazuhiro; Sugiyama, Yasumasa; Furukawa, Tatsuhiko; Komatsu, Masaharu

    2014-12-04

    Microcystin-LR is a cyclic peptide released by several bloom-forming cyanobacteria. Understanding the mechanism of microcystin-LR toxicity is important, because of the both potencies of its acute cytotoxicity and tumor-promoting activity in hepatocytes of animals and humans. Recently, we have reported that the expression of human hepatocyte uptake transporter OATP1B3 was critical for the selective uptake of microcystin-LR into hepatocytes and for induction of its fatal cytotoxicity. In this study, we demonstrated a novel function of microcystin-LR which induced bipotential changes including anoikis resistance and cytoskeleton reorganization to OATP1B3-transfected HEK293 cells (HEK293-OATP1B3). After exposure to microcystin-LR, HEK293-OATP1B3 cells were divided to the floating cells and remaining adherent cells. After collection and reseeding the floating cells into a fresh flask, cells were confluently proliferated (HEK293-OATP1B3-FL) under the microcystin-LR-free condition. Both the proliferated HEK293-OATP1B3-FL and remaining adherent HEK293-OATP1B3-AD cells changed the character with down- and up-regulation of E-cadherin, respectively. Additionally, these cells acquired resistance to microcystin-LR. These results suggest that microcystin-LR could be associated with not only tumor promotion, but also epithelial-mesenchymal transition-mediated cancer metastasis. Furthermore, microcystin-LR might induce the cytoskeleton reorganization be accompanied epithelial-mesenchymal transition.

  6. Cytotoxicity of mequindox and its metabolites in HepG2 cells in vitro and murine hepatocytes in vivo.

    Science.gov (United States)

    Liu, Yingchun; Jiang, Wei; Chen, Yongjun; Liu, Yanyan; Zeng, Peng; Xue, Feiqun; Wang, Quan

    2016-02-01

    Mequindox, a quinoxaline 1,4-dioxide, is widely used as a feed additive in the Chinese livestock industry because of its effective antibacterial properties. Many recent studies have found that mequindox is rapidly metabolized to numerous metabolites following administration to animals. There have, however, been few reports describing the cytotoxicity of mequindox metabolites. In this study, HepG2 cells were treated with mequindox (0, 2, 10, 50 or 100 μg/ml) or its major metabolites (0, 40, 100, 250 or 500 μg/ml) for 24h. Mice were administrated with mequindox (0, 50, 200 or 500 mg/kg.bw) for five days. DNA damage in the HepG2 cells and mouse hepatocytes was then assessed using an SCGE assay. The cell cycle of the HepG2 cells was also determined by flow cytometry. Mequindox was found to induce cell cycle arrest to the G2/M phase and cause dose-dependent DNA damage in HepG2 cells in vitro and in murine hepatocytes in vivo. Compared with mequindox, the major metabolites had much smaller effects on the cell cycle and caused much less DNA damage in HepG2 cells. And the results indicated that the process of metabolites formed by reduction of the MEQ acetyl group or reduction of the N → O groups could contribute to DNA damage in murine hepatocytes in vivo. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. Affinity labeling of the galactose/N-acetylgalactosamine-specific receptor of rat hepatocytes: preferential labeling of one of the subunits

    Energy Technology Data Exchange (ETDEWEB)

    Lee, R.T.; Lee, Y.C.

    1987-10-06

    The galactose/N-acetylgalactosamine-specific receptor (also known as asialoglycoprotein receptor) of rat hepatocytes consists of three subunits, one of which (43 kilodalton (kDa)) exists in a greater abundance (up to 70% of total protein) over the two minor species (52 and 60 kDa). When the receptor on the hepatocyte membranes was photoaffinity labeled with an /sup 125/I-labeled high-affinity reagent the labeling occurred mainly (51-80%) on one of the minor bands (52 kDa). Similarly, affinity-bound, N-acetylgalactosamine-modified lactoperoxidase radioiodinated the same 52-kDa band preferentially. In contrast, both the photoaffinity labeling and lactoperoxidase-catalyzed iodination of the purified, detergent-solubilized receptor resulted in a distribution of the label that is comparable to the Coomassie blue staining pattern of the three bands; i.e., the 43-kDa band was the major band labeled. These and other experimental results suggest that the preferential labeling of the minor band and inefficient labeling of the major band on the hepatocyte membrane resulted from a specific topological arrangement of these subunits on the membranes. The authors postulate that in the native, membrane-bound state of the receptor, the 52-kDa minor band is topologically prominent, while the major (43 kDa) band is partially masked. This partial masking may result from a tight packing of the receptor subunits on the membranes to form a lattice work.

  8. Deuterium D(V/K) isotope effects on ethanol oxidation in hepatocytes; Importance of the reverse ADH-reaction

    Energy Technology Data Exchange (ETDEWEB)

    Lundquist, F.; Iversen, H.L.; Hansen, L.L. (Department of Biochemistry A, The Panum Institute, University of Copenhagen (Denmark))

    1990-01-01

    The kinetic deuterium isotope effect, D(V/K), on ethanol oxidation was measured on hepatocytes from rat and pig by the radiometric competitive method using {sup 14}C-labelled ethanol containing deuterium in the (a-R)-position. The corrected D(V/K) values of 2.68 and 2.80 for rat and pig hepatocytes respectively were significantly different, suggesting differences in the amount of non-ADH ethanol oxidizing activity. The apparent isotope effects declined repidly with time when acetaldehyde was present in the medium as a result of the reduction to ethanol of the ({sup 14}C)-acetaldehyde formed from the double labelled ethanol by alcohol dehydrogenase (ADH). Fructose and cynamide caused the acetaldehyde concentration during ethanol oxidation to increase by entirely different mechanisms, and the isotope effect to decrease with time, as did also the addition of acetaldehyde. The apparent first order rate constant for the reverse ADH reaction, assuming the reactants to be acetaldehyde and the ADH-NADH complex, was determined by two metohods giving comparable results. In the presence of semicarbazide, which removes acetaldehyde, the isotope effect was nearly constant. This was the case also when the acetaldehyde concentration was very low (<1 {mu}M) for other reasons, as in hepatocytes from starved animals. A mathematical formula describing the expected decrease of the apparent isotope effect with time was derived. The different response of pig and rat hepatocytes to addition of fructose (the 'fructose effect') is suggested to be caused by differences in activity of aldehyde dehydrogenases in the two species. (author).

  9. Challenges in using cultured primary rodent hepatocytes or cell lines to study hepatic HDL receptor SR-BI regulation by its cytoplasmic adaptor PDZK1.

    Directory of Open Access Journals (Sweden)

    Kosuke Tsukamoto

    Full Text Available BACKGROUND: PDZK1 is a four PDZ-domain containing cytoplasmic protein that binds to a variety of membrane proteins via their C-termini and can influence the abundance, localization and/or function of its target proteins. One of these targets in hepatocytes in vivo is the HDL receptor SR-BI. Normal hepatic expression of SR-BI protein requires PDZK1 - <5% of normal hepatic SR-BI is seen in the livers of PDZK1 knockout mice. Progress has been made in identifying features of PDZK1 required to control hepatic SR-BI in vivo using hepatic expression of wild-type and mutant forms of PDZK1 in wild-type and PDZK1 KO transgenic mice. Such in vivo studies are time consuming and expensive, and cannot readily be used to explore many features of the underlying molecular and cellular mechanisms. METHODOLOGY/PRINCIPAL FINDINGS: Here we have explored the potential to use either primary rodent hepatocytes in culture using 2D collagen gels with newly developed optimized conditions or PDZK1/SR-BI co-transfected cultured cell lines (COS, HEK293 for such studies. SR-BI and PDZK1 protein and mRNA expression levels fell rapidly in primary hepatocyte cultures, indicating this system does not adequately mimic hepatocytes in vivo for analysis of the PDZK1 dependence of SR-BI. Although PDZK1 did alter SR-BI protein expression in the cell lines, its influence was independent of SR-BI's C-terminus, and thus is not likely to occur via the same mechanism as that which occurs in hepatocytes in vivo. CONCLUSIONS/SIGNIFICANCE: Caution must be exercised in using primary hepatocytes or cultured cell lines when studying the mechanism underlying the regulation of hepatic SR-BI by PDZK1. It may be possible to use SR-BI and PDZK1 expression as sensitive markers for the in vivo-like state of hepatocytes to further improve primary hepatocyte cell culture conditions.

  10. Signifying Bodies

    DEFF Research Database (Denmark)

     In our everyday lives we strive to stay healthy and happy, while we live as our selves, engage with each other, and discover an infinite world of possibilities. Health arises and diminishes as human beings draw on a vibrant ecology of actions, interactions and coactions. Intricate processes...... of biosemiosis connect signifying bodies with their natural surroundings, cultural activities and subjective experiences. Health stretches all the way from the ecosocial surroundings, through the skin and into the self-organizing processes of every living cell. Signifying Bodies lays out a new approach to health...... and health care. Eschewing all forms of dualism, the authors emphasise the interdependency of how we act, think, feel and function. They advocate a relational turn in health care, in which bodies live and learn from suffering and care. In this view, health is inseparable from both living beings...

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

    NARCIS (Netherlands)

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

    1995-01-01

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

  12. Transplantation of human stem cell-derived hepatocytes in an animal model of acute liver failure.

    Science.gov (United States)

    Ramanathan, Rajesh; Pettinato, Giuseppe; Beeston, John T; Lee, David D; Wen, Xuejun; Mangino, Martin J; Fisher, Robert A

    2015-08-01

    Hepatocyte cell transplantation can be life-saving in patients with acute liver failure (ALF); however, primary human hepatocyte transplantation is limited by the scarcity of donor hepatocytes. We investigated the effect of stem cell-derived, hepatocyte-like cells in an animal xenotransplant model of ALF. Intraperitoneal d-galactosamine was used to develop a lethal model of ALF in the rat. Human induced pluripotent stem cells (iPSC), human mesenchymal stem cells, and human iPSC combined with human endothelial cells (iPSC + EC) were differentiated into hepatocyte-like cells and transplanted into the spleens of athymic nude rats with ALF. A reproducible lethal model of ALF was achieved with nearly 90% death within 3 days. Compared with negative controls, rats transplanted with stem cell-derived, hepatocyte-like cells were associated with increased survival. Human albumin was detected in the rat serum 3 days after transplantation in more than one-half the animals transplanted with hepatocyte-like cells. Only animals transplanted with iPSC + EC-derived hepatocytes had serum human albumin at 14 days posttransplant. Transplanted hepatocyte-like cells homed to the injured rat liver, whereas the ECs were only detected in the spleen. Transplantation of stem cell-derived, hepatocyte-like cells improved survival with evidence of in vivo human albumin production. Combining ECs may prolong cell function after transplantation. Copyright © 2015 Elsevier Inc. All rights reserved.

  13. Effects of electroporation on primary rat hepatocytes in vitro

    Institute of Scientific and Technical Information of China (English)

    Yun-Qing Yao; Ding-Feng Zhang; Ai-Long Huang; Yun Luo; Da-Zhi Zhang; Bo Wang; Wei-Ping Zhou; Hong Ren; Shu-Hua Guo

    2002-01-01

    AIM: To investigate the effects of electroporation on primaryrat hepatocyte and to optimize the electroporation conditionsintroducing foreign genes into primary hepatocytes.METHODS: A single-pulse procedure was performed at Iowvoltage (220-400 V) but with high capacitance (500-950 μF).Hepatocytes were divided into 4 groups according to theelectroporation conditions: group Ⅰ, 220 V and 500 μF;group Ⅱ, 220 Vand 950 μF; group Ⅲ, 400 V and 950 μF,and group Ⅳ.The control group was freshly isolatedhepatocytes and directly cultured under the same conditionsas those of electroporation groups. The effects ofelectroporation on primary rat hepatocytes were detectedby trypan blue exclusion (TBE) and MTT analysis. Besides,albumin (AIb), alanine transaminase (ALT) and lactatedehydrogenase (LDH) in the supernatants of culturedhepatocytes were measured by biochemical assay.RESULTS: Between day 1 and day 15 after incubation,primary rat hepatocytes of each electroporation groupappeared normal, being the same with those of controlgroup. TBE staining showed that slight hepatocyte damageand high survival rate were found in the electroporationgroups and the control group. Cultured for 3, 7, 11 and 15days, hepatocyte viability was approximatly 92.6±2.5 %,89.5±3.3 %, 82.0±3.5 % and 74.3±1.2 %, respectively.MTT analysis indicated that the viabilities of hepatocyteshad no significant difference between each electroporationgroup, and those were similar to that of control group. Atthe 36th hour after electroporation, AIb, ALT and LDH in thesupernatants of control group were 5.3±0.1 g. L-1, 183.7±8.4 nkat. L-1 and 896.8±58.5 nkat. L-1; those of group Ⅱwere 5.7±0.1 g. L-1, 215.4±16.7 nkat. L-1 and 1063.8±51.8 nkat. L-1; and those of group Ⅲ were 5.80.2 g. L-1,217.1 ± 8.4 nkat. L-1 and 1063.8± 10.0 nkat. L-1 . Statistically,the proteins of group Ⅱ and group Ⅲ were significantlyhigher than those of control group (P<0.05), whereas theprotein production of group

  14. [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.

  15. 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.

  16. [Autotelic activities aimed at the alteration of the human body from socially accepted to pathological forms: about non-suicidal self-injury].

    Science.gov (United States)

    Kalmar, Sandor

    2016-03-01

    The author lays down that non-suicidal self-injury (NSSI) constitutes an increasingly more common and serious public health problem, especially during the age of adolescence. In spite of the fact that the phenomenon has been known since the beginning of human mankind even in animals, we have not been able to either give a clear explanation or prevent its spreading yet. The author reviews the conceptual disturbances, behavioural phenotypes, cultural-historical and mythological antecedents related to self-injury, just as the controversial concepts, reasons of unclearness of the concepts, and clinical classification of self-injuries, and he outlines a new categorisation/ classification of the explanation of autotelic activities aimed at the alteration of the human body. He reviews the relationship between self-injuries and other psychological signs and symptoms and psychiatric illnesses, the explanations of developing self-injurious behaviour and further research directions. Besides the different models of self-injury he presents a holistic model. Besides the therapeutic guidelines of self-injurious behaviour, he calls the attention to the importance of genetic and nervous system researches, psychological and spiritual research, the importance of mental education and prevention, and he also lists some more essential questions future researchers have to find the answers for if we would like all children to be allowed to enter the adults' world in a healthy and sound state.

  17. Discs large 1 (Dlg1) scaffolding protein participates with clathrin and adaptator protein complex 1 (AP-1) in forming Weibel-Palade bodies of endothelial cells.

    Science.gov (United States)

    Philippe, Monique; Léger, Thibaut; Desvaux, Raphaëlle; Walch, Laurence

    2013-05-03

    Weibel-Palade bodies (WPBs) are specific cigar-shaped granules that store von Willebrand factor (VWF) for its regulated secretion by endothelial cells. The first steps of the formation of these granules at the trans-Golgi network specifically require VWF aggregation and an external scaffolding complex that contains the adaptator protein complex 1 (AP-1) and clathrin. Discs large 1 (Dlg1) is generally considered to be a modular scaffolding protein implicated in the control of cell polarity in a large variety of cells by specific recruiting of receptors, channels, or signaling proteins to specialized zones of the plasma membrane. We propose here that in endothelial cells, Dlg1, in a complex with AP-1 and clathrin, participates in the biogenesis of WPBs. Supporting data show that Dlg1 colocalizes with microtubules, intermediate filaments, and Golgi markers. Tandem mass spectrometry experiments led to the identification of clathrin as an Dlg1-interacting partner. Interaction was confirmed by in situ proximity ligation assays. Furthermore, AP-1 and VWF immunoprecipitate and colocalize with Dlg1 in the juxtanuclear zone. Finally, Dlg1 depletion by siRNA duplexes disrupts trans-Golgi network morphology and WPB formation. Our results provide the first evidence for an unexpected role of Dlg1 in controlling the formation of specific secretory granules involved in VWF exocytosis in endothelial cells.

  18. Inhibitory effects of berberine on ion channels of rat hepatocytes

    Institute of Scientific and Technical Information of China (English)

    Fang Wang; Hong-Yi Zhou; Gang Zhao; Li-Ying Fu; Lan Cheng; Jian-Guo Chen; Wei-Xing Yao

    2004-01-01

    AIM: To examine the effects of berberine, an isoquinoline alkaloid with a long history used as a tonic remedy for liver and heart, on ion channels of isolated rat hepatocytes.METHODS: Tight-seal whole-cell patch-clamp techniques were performed to investigate the effects of berberine on the delayed outward potassium currents (IK), inward rectifier potassium currents (IK1) and Ca2+ release-activated Ca2+currents (ICRAC) in enzymatically isolated rat hepatocytes.RESULTS: Berberine 1-300 nmol/L reduced IK in a concentration dependent manner with EC50 of 38.86±5.37 μmol/L and nH of 0.82±0.05 (n = 8). When the bath solution was changed to tetraethylammonium (TEA) 8 mmol/L, IK was inhibited.Berberine 30 μmol/L reduced IK at all examined membrane potentials, especially at potentials positive to +60 mV (n = 8,P<0.05 or P<0.01 vs control). Berberine had mild inhibitory effects on IK1 in rat hepatocytes. Berberine 1-300 μmol/L also inhibited ICRAC in a concentration-dependent fashion.The fitting parameters were EC50 = 47.20±10.86 μmol/L,nH = 0.71±0.09 (n = 8). The peak value of ICRAC in the Ⅰ-Ⅴrelationship was decreased by berberine 30 μmol/L at potential negative to -80 mV (n = 8, P<0.05 vscontrol). But the reverse potential of ICRAC occurred at voltage 0 mV in all cells.CONCLUSION: Berberine has inhibitory effects on potassium and calcium currents in isolated rat hepatocytes, which may be involved in hepatoprotection.

  19. Detection of Dichlorvos Adducts in a Hepatocyte Cell Line

    Science.gov (United States)

    2014-06-30

    G6019) was obtained from Sigma (St. Louis, MS), human actin gamma recombinant protein (ACTG1, H00000071) and lactate dehydrogenase A recombi - nant...microRNA and mRNA expression profiling analysis of dichlorvos cytotoxicity in porcine kidney epithelial PK15 cells. DNA Cell Biol. 2011, 30 (12), 1073...Dissociation of DDVP-induced DNA strand breaks from oxidative damage in isolated rat hepatocytes. Toxicology 1996, 108 (1−2), 49−56. (40) Clapp, C.; Portt, L

  20. 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.

  1. Hepatocyte and Sertoli Cell Aquaporins, Recent Advances and Research Trends

    Directory of Open Access Journals (Sweden)

    Raquel L. Bernardino

    2016-07-01

    Full Text Available Aquaporins (AQPs are proteinaceous channels widespread in nature where they allow facilitated permeation of water and uncharged through cellular membranes. AQPs play a number of important roles in both health and disease. This review focuses on the most recent advances and research trends regarding the expression and modulation, as well as physiological and pathophysiological functions of AQPs in hepatocytes and Sertoli cells (SCs. Besides their involvement in bile formation, hepatocyte AQPs are involved in maintaining energy balance acting in hepatic gluconeogenesis and lipid metabolism, and in critical processes such as ammonia detoxification and mitochondrial output of hydrogen peroxide. Roles are played in clinical disorders including fatty liver disease, diabetes, obesity, cholestasis, hepatic cirrhosis and hepatocarcinoma. In the seminiferous tubules, particularly in SCs, AQPs are also widely expressed and seem to be implicated in the various stages of spermatogenesis. Like in hepatocytes, AQPs may be involved in maintaining energy homeostasis in these cells and have a major role in the metabolic cooperation established in the testicular tissue. Altogether, this information represents the mainstay of current and future investigation in an expanding field.

  2. Hepatocyte and Sertoli Cell Aquaporins, Recent Advances and Research Trends.

    Science.gov (United States)

    Bernardino, Raquel L; Marinelli, Raul A; Maggio, Anna; Gena, Patrizia; Cataldo, Ilaria; Alves, Marco G; Svelto, Maria; Oliveira, Pedro F; Calamita, Giuseppe

    2016-07-09

    Aquaporins (AQPs) are proteinaceous channels widespread in nature where they allow facilitated permeation of water and uncharged through cellular membranes. AQPs play a number of important roles in both health and disease. This review focuses on the most recent advances and research trends regarding the expression and modulation, as well as physiological and pathophysiological functions of AQPs in hepatocytes and Sertoli cells (SCs). Besides their involvement in bile formation, hepatocyte AQPs are involved in maintaining energy balance acting in hepatic gluconeogenesis and lipid metabolism, and in critical processes such as ammonia detoxification and mitochondrial output of hydrogen peroxide. Roles are played in clinical disorders including fatty liver disease, diabetes, obesity, cholestasis, hepatic cirrhosis and hepatocarcinoma. In the seminiferous tubules, particularly in SCs, AQPs are also widely expressed and seem to be implicated in the various stages of spermatogenesis. Like in hepatocytes, AQPs may be involved in maintaining energy homeostasis in these cells and have a major role in the metabolic cooperation established in the testicular tissue. Altogether, this information represents the mainstay of current and future investigation in an expanding field.

  3. Homocysteine inhibits hepatocyte proliferation via endoplasmic reticulum stress.

    Directory of Open Access Journals (Sweden)

    Xue Yu

    Full Text Available Homocysteine is an independent risk factor for coronary, cerebral, and peripheral vascular diseases. Recent studies have shown that levels of homocysteine are elevated in patients with impaired hepatic function, but the precise role of homocysteine in the development of hepatic dysfunction is unclear. In this study, we examined the effect of homocysteine on hepatocyte proliferation in vitro. Our results demonstrated that homocysteine inhibited hepatocyte proliferation by up-regulating protein levels of p53 as well as mRNA and protein levels of p21(Cip1 in primary cultured hepatocytes. Homocysteine induced cell growth arrest in p53-positive hepatocarcinoma cell line HepG2, but not in p53-null hepatocarcinoma cell line Hep3B. A p53 inhibitor pifithrin-α inhibited the expression of p21(Cip1 and attenuated homocysteine-induced cell growth arrest. Homocysteine induced TRB3 expression via endoplasmic reticulum stress pathway, resulting in Akt dephosphorylation. Knock-down of endogenous TRB3 significantly suppressed the inhibitory effect of homocysteine on cell proliferation and the phosphorylation of Akt. LiCl reversed homocysteine-mediated cell growth arrest by inhibiting TRB3-mediated Akt dephosphorylation. These results demonstrate that both TRB3 and p21(Cip1 are critical molecules in the homocysteine signaling cascade and provide a mechanistic explanation for impairment of liver regeneration in hyperhomocysteinemia.

  4. Biomaterial Scaffolds with Biomimetic Fluidic Channels for Hepatocyte Culture

    Institute of Scientific and Technical Information of China (English)

    Xiao Li; Jiankang He; Yaxiong Liu; Qian Zhao; Wanquan Wu; Dichen Li; Zhongmin Jin

    2013-01-01

    Biomaterial scaffolds play an important role in maintaining the viability and biological functions of highly metabolic hepatocytes in liver tissue engineering.One of the major challenges involves building a complex microchannel network inside three-dimensional (3D) scaffolds for efficient mass transportation.Here we presented a biomimetic strategy to generate a microchannel network within porous biomaterial scaffolds by mimicking the vascular tree of rat liver.The typical parameters of the blood vessels were incorporated into the biomimetic design of the microchannel network such as branching angle and diameter.Silk fibroin-gelatin scaffolds with biomimetic vascular tree were fabricated by combining micromolding,freeze drying and 3D rolling techniques.The relationship between the micro-channeled design and flow pattern was revealed by a flow experiment,which indicated that the scaffolds with biomimetic vascular tree exhibited unique capability in improving mass transportation inside the 3D scaffold.The 3D scaffolds,preseeded with primary hepatocytes,were dynamically cultured in a bioreactor system.The results confirmed that the pre-designed biomimetic microchannel network facilitated the generation and expansion of hepatocytes.

  5. Hepatocyte and Sertoli Cell Aquaporins, Recent Advances and Research Trends

    Science.gov (United States)

    Bernardino, Raquel L.; Marinelli, Raul A.; Maggio, Anna; Gena, Patrizia; Cataldo, Ilaria; Alves, Marco G.; Svelto, Maria; Oliveira, Pedro F.; Calamita, Giuseppe

    2016-01-01

    Aquaporins (AQPs) are proteinaceous channels widespread in nature where they allow facilitated permeation of water and uncharged through cellular membranes. AQPs play a number of important roles in both health and disease. This review focuses on the most recent advances and research trends regarding the expression and modulation, as well as physiological and pathophysiological functions of AQPs in hepatocytes and Sertoli cells (SCs). Besides their involvement in bile formation, hepatocyte AQPs are involved in maintaining energy balance acting in hepatic gluconeogenesis and lipid metabolism, and in critical processes such as ammonia detoxification and mitochondrial output of hydrogen peroxide. Roles are played in clinical disorders including fatty liver disease, diabetes, obesity, cholestasis, hepatic cirrhosis and hepatocarcinoma. In the seminiferous tubules, particularly in SCs, AQPs are also widely expressed and seem to be implicated in the various stages of spermatogenesis. Like in hepatocytes, AQPs may be involved in maintaining energy homeostasis in these cells and have a major role in the metabolic cooperation established in the testicular tissue. Altogether, this information represents the mainstay of current and future investigation in an expanding field. PMID:27409609

  6. 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.

  7. Gene expression changes after hypoxic preconditioning in rat hepatocytes

    Institute of Scientific and Technical Information of China (English)

    Wei Chen; Jiang-Feng Qiu; Zhi-Qi Zhang; Hai-Feng Luo; Joan Rosello-Catafau; Zhi-Yong Wu

    2006-01-01

    BACKGROUND: Hypoxic preconditioning can protect hepatocytes against hypoxic injury, but its mechanism has not been elucidated. The aim of this study was to proifle gene expression patterns involved in hypoxic preconditioning and probable mechanism at the level of gene expression. METHODS: Hepatocytes were divided into 2 groups:control group and hypoxic preconditioning group. Biotin-labeled cRNA from the control group and the hypoxic preconditioning group was hybridized by oligonucleotide microarray. Genes that were signiifcantly associated with hypoxic preconditioning were ifltered, and validated at the level of transcript expression. RESULTS: Forty-three genes with signiifcantly altered expression patterns were discovered and most of them had not been previously reported. Among these genes, genes encoding superoxide dismutase 2 (SOD2)and interleukin 10 (IL-10) in the hypoxic preconditioning group were conifrmed to be up-regulated with real-time quantitative PCR. CONCLUSIONS:Many cytokines are involved in hypoxic preconditioning and protect hepatocytes from hypoxia-reoxygenation injury, and the increase of oxygen free-radical scavengers and anti-inlfammatory factors may play a key role in this phenomenon. Diverse signal pathways are probably involved.

  8. Silver nanoparticles stimulate glycogenolysis in rainbow trout (Oncorhynchus mykiss) hepatocytes.

    Science.gov (United States)

    Massarsky, Andrey; Labarre, Justine; Trudeau, Vance L; Moon, Thomas W

    2014-02-01

    Silver nanoparticles (AgNPs) are found in many consumer products yet their biological effects on non-target aquatic organisms are yet to be fully understood. This research aimed to investigate the effects of AgNPs on cell signaling in rainbow trout (Oncorhynchus mykiss) hepatocytes. We focused on the β-adrenoreceptor (AR), which mediates glycogenolysis, and the glucocorticoid receptor (GCR), which mediates gluconeogenesis. These two receptors have been extensively studied in trout hepatocytes due to their key roles during the stress response to increase glucose availability (among other things), allowing the organisms to cope with the stressor. We show for the first time that AgNPs at a concentration of 1 μg/mL did not interfere with the function of either the β-AR or the GCR systems in rainbow trout hepatocytes, but at the concentration of 10 μg/mL AgNPs stimulated glycogenolysis which was apparently receptor-independent. This study suggests that AgNPs could affect hormone-regulated cell signaling pathways at a concentration of 10 μg/mL.

  9. Hepatocyte growth factor is crucial for development of the carapace in turtles.

    Science.gov (United States)

    Kawashima-Ohya, Yoshie; Narita, Yuichi; Nagashima, Hiroshi; Usuda, Ryo; Kuratani, Shigeru

    2011-01-01

    Turtles are characterized by their shell, composed of a dorsal carapace and a ventral plastron. The carapace first appears as the turtle-specific carapacial ridge (CR) on the lateral aspect of the embryonic flank. Accompanying the acquisition of the shell, unlike in other amniotes, hypaxial muscles in turtle embryos appear as thin threads of fibrous tissue. To understand carapacial evolution from the perspective of muscle development, we compared the development of the muscle plate, the anlage of hypaxial muscles, between the Chinese soft-shelled turtle, Pelodiscus sinensis, and chicken embryos. We found that the ventrolateral lip (VLL) of the thoracic dermomyotome of P. sinensis delaminates early and produces sparse muscle plate in the lateral body wall. Expression patterns of the regulatory genes for myotome differentiation, such as Myf5, myogenin, Pax3, and Pax7 have been conserved among amniotes, including turtles. However, in P. sinensis embryos, the gene hepatocyte growth factor (HGF), encoding a regulatory factor for delamination of the dermomyotomal VLL, was uniquely expressed in sclerotome and the lateral body wall at the interlimb level. Implantation of COS-7 cells expressing a HGF antagonist into the turtle embryo inhibited CR formation. We conclude that the de novo expression of HGF in the turtle mesoderm would have played an innovative role resulting in the acquisition of the turtle-specific body plan. © 2011 Wiley Periodicals, Inc.

  10. ß-Hydroxybutyrate Activates the NF-κB Signaling Pathway to Promote the Expression of Pro-Inflammatory Factors in Calf Hepatocytes

    Directory of Open Access Journals (Sweden)

    Xiaoxia Shi

    2014-01-01

    Full Text Available Background/Aims: ß-hydroxybutyrate (BHBA is the major component of ketone bodies in ketosis. Dairy cows with ketosis often undergo oxidative stress. BHBA is related to the inflammation involved in other diseases of dairy cattle. However, whether BHBA can induce inflammatory injury in dairy cow hepatocytes and the potential mechanism of this induction are not clear. The NF-κB pathway plays a vital role in the inflammatory response. Methods: Therefore, this study evaluated the oxidative stress, pro-inflammatory factors and NF-κB pathway in cultured calf hepatocytes treated with different concentrations of BHBA, pyrrolidine dithiocarbamate (PDTC, an NF-κB pathway inhibitor and N-acetylcysteine (NAC, antioxidant. Results: The results showed that BHBA could significantly increase the levels of oxidation indicators (MDA, NO and iNOS, whereas the levels of antioxidation indicators (GSH-Px, CAT and SOD were markedly decreased in hepatocytes. The IKKß activity and phospho-IκBa (p-IκBa contents were increased in BHBA-treated hepatocytes. This increase was accompanied by the increased expression level and transcription activity of p65. The expression levels of NF-κB-regulated inflammatory cytokines, namely TNF-a, IL-6 and IL-1ß, were markedly increased after BHBA treatment, while significantly decreased after NAC treatment. However, the p-IκBa level and the expression and activity of p65 and its target genes were markedly decreased in the PDTC + BHBA group compared with the BHBA (1.8 mM group. Moreover, immunocytofluorescence of p65 showed a similar trend. Conclusion: The present data indicate that higher concentrations of BHBA can induce cattle hepatocyte inflammatory injury through the NF-κB signaling pathway, which may be activated by oxidative stress.

  11. Written on the Body

    DEFF Research Database (Denmark)

    Christiansen, Steen Ledet

    may choose to alter how we are perceived and to at least some extent control the discontent we may project onto our own body. Through body modification, we can alter the impression of our personality and express a cultural solidarity, as Chris Rojek points out. Tattoos, piercings and other body...... modifications become ways to express a difference from or identification with, a particular cultural segment. Body modification marks a personal subjectivity, just as it marks a border around those who participate. A distinctive bodily border is formed through the use of body modifications, and it can be viewed......Our bodies define a border between ourselves and the world around us. However we might feel about our body, it is what we present to the world. Victoria L. Blum in her book Flesh Wounds discusses how bodies are a form of inkblots, where discontent is projected onto. As bodies can be modified, we...

  12. Freshly isolated hepatocyte transplantation in acetaminophen-induced hepatotoxicity model in rats

    Directory of Open Access Journals (Sweden)

    Daniela Rodrigues

    2012-12-01

    Full Text Available CONTEXT: Hepatocyte transplantation is an attractive therapeutic modality for liver disease as an alternative for orthotopic liver transplantation. OBJECTIVE: The aim of the current study was to investigate the feasibility of freshly isolated rat hepatocyte transplantation in acetaminophen-induced hepatotoxicity model. METHODS: Hepatocytes were isolated from male Wistar rats and transplanted 24 hours after acetaminophen administration in female recipients. Female rats received either 1x10(7 hepatocytes or phosphate buffered saline through the portal vein or into the spleen and were sacrificed after 48 hours. RESULTS: Alanine aminotransferase levels measured within the experiment did not differ between groups at any time point. Molecular analysis and histology showed presence of hepatocytes in liver of transplanted animals injected either through portal vein or spleen. CONCLUSION: These data demonstrate the feasibility and efficacy of hepatocyte transplantation in the liver or spleen in a mild acetaminophen-induced hepatotoxicity model.

  13. Effects of quercetin on hepatocyte stimulating factor production by mouse peritoneal macrophases

    Institute of Scientific and Technical Information of China (English)

    周斌; 张俊平; 刘宏; 殷明; 钱定华

    2003-01-01

    Objective: To study the effects of quercetin on hepatocyte stimulating factor production from mouse peritoneal macrophages. Methods: Hepatocyte stimulating factor was evaluated by the amount of fibrinogen synthesized in Hep3B cells. Interleukin-6 activity was measured by B9 cell proliferation methyl thiazolyl tetrazolium colorimetric method. Hep3B cell supernatant fibrinogen was quantitated with ELISA. Results: LPS induced the synthesis of hepatocyte stimulating factor in mouse peritoneal macrophages, and hepatocyte stimulating factor promotes the synthesis of fibrinogen from Hep3B cells. Quercetin(5 to 40 μmol/ L)inhibited the synthesis of hepatocyte stimulating factor stimulated by LPS. Quercetin(5 to 20 μmol/ L) inhibited release of interleukin-6 from mouse peritoneal macrophages induced by 0.5 g/ L fibrin fibrinogen degradation products. Conclusion: Quercetin inhibits the synthesis of hepatocyte stimulating factor in macrophages.

  14. Effect of Concentrated Fibroblast-Conditioned Media on In Vitro Maintenance of Rat Primary Hepatocyte.

    Directory of Open Access Journals (Sweden)

    Dayeong Jeong

    Full Text Available The effects of concentrated fibroblast-conditioned media were tested to determine whether hepatocyte function can be maintained without direct contact between hepatocytes and fibroblasts. Primary rat hepatocytes cultured with a concentrated conditioned media of NIH-3T3 J2 cell line (final concentration of 55 mg/ml showed significantly improved survival and functions (albumin and urea compared to those of control groups. They also showed higher expression levels of mRNA, albumin and tyrosine aminotransferase compared to hepatocyte monoculture. The results suggest that culture with concentrated fibroblast-conditioned media could be an easy method for in vitro maintenance of primary hepatocytes. They also could be contribute to understand and analyze co-culture condition of hepatocyte with stroma cells.

  15. Piceatannol increases the expression of hepatocyte growth factor and IL-10 thereby protecting hepatocytes in thioacetamide-induced liver fibrosis.

    Science.gov (United States)

    Abd-Elgawad, Hazem; Abu-Elsaad, Nashwa; El-Karef, Amr; Ibrahim, Tarek

    2016-07-01

    Piceatannol is a polyphenolic analog of resveratrol that selectively inhibits the non-receptor tyrosine kinase-Syk. This study investigates the potential ability of piceatannol to attenuate liver fibrosis and protect hepatocytes from injury. Thioacetamide was injected in adult male mice (100 mg/kg, i.p., 3 times/week) for 8 weeks. Piceatannol (1 or 5 mg/kg per day) was administered by oral gavage during the last 4 weeks. Liver function biomarkers, tissue malondialdehyde (MDA), cytokeratin-18 (CK18), hepatocyte growth factor (HGF), and interleukin-10 (IL-10) were measured. Necroinflammation, fibrosis, expression of transforming growth factor (TGF)-β1, and α-smooth muscle actin (SMA) were scored by histopathological examination and immunohistochemistry. Obtained results showed ability of piceatannol (1 mg/kg) to restore liver function and reduce inflammation. It significantly (p IL-10. It can be concluded that piceatannol at low dose can inhibit TGF-β1 induced hepatocytes apoptosis and exerts an anti-inflammatory effect attenuating fibrosis progression.

  16. Species-specific toxicity of troglitazone on rats and human by gel entrapped hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Shen, Chong [College of Materials Science and Chemical Engineering, Zhejiang University, Zhejiang 310027 (China); Meng, Qin, E-mail: mengq@zju.edu.cn [College of Materials Science and Chemical Engineering, Zhejiang University, Zhejiang 310027 (China); Zhang, Guoliang [Institute of Biological and Environmental Engineering, Zhejiang University of Technology, Zhejiang 310012 (China)

    2012-01-01

    Troglitazone, despite passing preclinical trials on animals, was shortly withdrawn from market due to its severe hepatotoxicity in clinic. As rat hepatocyte monolayer consistently showed sensitive troglitazone toxicity as human hepatocyte monolayer in contrast to the species-specific toxicity in vivo, this paper utilized both hepatocytes in three-dimensional culture of gel entrapment to reflect the species difference on hepatotoxicity. Rat hepatocytes in gel entrapment did not show obvious cellular damage even under a long-term exposure for 21 days while gel entrapped human hepatocytes significantly displayed oxidative stress, steatosis, mitochondrial damage and cell death at a short exposure for 4 days. As a result, the detected species-specific toxicity of troglitazone between gel entrapped rat and human hepatocytes consisted well with the situation in vivo but was in a sharp contrast to the performance of two hepatocytes by monolayer culture. Such contradictory toxicity of rat hepatocytes between monolayer and gel entrapment culture could be explained by the fact that troglitazone was cleared more rapidly in gel entrapment than in monolayer culture. Similarly, the differential clearance of troglitazone in rat and human might also explain its species-specific toxicity. Therefore, gel entrapment of hepatocytes might serve as a platform for evaluation of drug toxicity at early stage of drug development by reducing costs, increasing the likelihood of clinical success and limiting human exposure to unsafe drugs. -- Highlights: ► Species-specific toxicity of troglitazone reflected by rat/human hepatocytes ► 3D hepatocytes in 21 days’ long-term culture used for drug hepatotoxicity ► Oversensitive toxicity in hepatocyte monolayer by slow troglitazone clearance.

  17. TRANSPLANTATION OF HEPATOCYTES AS THE METHOD OF TREATMENT OF LIVER FAILURE: EXPERIMENTAL AND CLINICAL EXPERIENCE

    Directory of Open Access Journals (Sweden)

    M. Y. Shagidulin

    2010-01-01

    Full Text Available For correction and treatment of liver failure before liver transplantation were proposed severe methods such as: extracorporal devices, transplantation of hepatocytes and implanted tissue-engineering units. The function of healthy hepatocytes presumes to stabilize the state of patients with chronic liver diseases and to wait a donor organ transplantation. In this review the results of experimental and clinical therapy of liver diseases by method of hepatocyte transplantation were summarized. 

  18. Heparanase enhances early hepatocyte inclusion in the recipient liver after transplantation in partially hepatectomized rats.

    Science.gov (United States)

    Tsiperson, Vladislav; Goldshmidt, Orit; Ilan, Neta; Shoshany, Gideon; Vlodavsky, Israel; Veitsman, Ella; Baruch, Yaacov

    2008-03-01

    Hepatocyte transplantation is an emerging approach for the treatment of liver diseases. However, broad clinical application of this method has been limited by restricted source of cells and low efficiency of cell integration within the recipient liver. Heparanase cleaves heparan sulfate proteoglycans in the extracellular matrix and basement membrane, activity that affects cellular invasion associated with cancer metastasis and inflammation. This activity has a multifunctional effect on cell-cell interaction, cell adhesion, and angiogenesis. All these factors are important for successful integration of transplanted hepatocytes. Male donor hepatocytes pretreated with heparanase or untreated were transplanted into recipient female rat spleen following partial hepatectomy. Engraftment efficacy was evaluated by PCR for Y chromosome, histology and PCNA, and heparanase immunohistochemistry. In addition, proliferative activity of hepatocytes in vitro was determined by bromodeoxyuridine immunostaining. The number of heparanase-treated cells detected in the recipient liver was significantly increased three- to fivefold within 24-48 h posttransplantation and twofold at 14 days compared with untreated cells. The transplanted hepatocytes treated with heparanase were clearly seen inside portal vein radicles as cell aggregates up to 72 h posttransplantation. The number of portal radicles filled with heparanase-treated hepatocytes was increased compared to control early after transplantation. Heparanase treatment enhanced hepatocyte and sinusoidal endothelial cell proliferation in the liver, and hepatocyte proliferation within the spleen tissue. Preliminary in vitro studies with isolated hepatocytes treated with heparanase showed increased proliferative activity within 24-48 h of cell culture. These results suggest that preincubation of hepatocytes with heparanase increases the presence of hepatocytes within the recipient liver early following cell transplantation and stimulates

  19. Freshly isolated hepatocyte transplantation in acetaminophen-induced hepatotoxicity model in rats

    OpenAIRE

    Daniela Rodrigues; Themis Reverbel Da Silveira; Ursula Matte

    2012-01-01

    CONTEXT: Hepatocyte transplantation is an attractive therapeutic modality for liver disease as an alternative for orthotopic liver transplantation. OBJECTIVE: The aim of the current study was to investigate the feasibility of freshly isolated rat hepatocyte transplantation in acetaminophen-induced hepatotoxicity model. METHODS: Hepatocytes were isolated from male Wistar rats and transplanted 24 hours after acetaminophen administration in female recipients. Female rats received either 1x10(7) ...

  20. FGF19-induced Hepatocyte Proliferation Is Mediated through FGFR4 Activation

    OpenAIRE

    Wu, Xinle; Ge, Hongfei; Lemon, Bryan; Vonderfecht, Steven; Weiszmann, Jennifer; Hecht, Randy; Gupte, Jamila; Hager, Todd; Wang, Zhulun; Lindberg, Richard; Li, Yang

    2009-01-01

    FGF19 and FGF21, unique members of the fibroblast growth factor (FGF) family, are hormones that regulate glucose, lipid, and energy homeostasis. Increased hepatocyte proliferation and liver tumor formation have also been observed in FGF19 transgenic mice. Here, we report that, in contrast to FGF19, FGF21 does not induce hepatocyte proliferation in vivo. To identify the mechanism for FGF19-induced hepatocyte proliferation, we explored similarities and differences in receptor specificity betwee...

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

    Directory of Open Access Journals (Sweden)

    Anayelly López-Islas

    2016-01-01

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

  2. Three-dimensional (3D) printing of mouse primary hepatocytes to generate 3D hepatic structure

    Science.gov (United States)

    Kim, Yohan; Kang, Kyojin; Jeong, Jaemin; Paik, Seung Sam; Kim, Ji Sook; Park, Su A; Kim, Wan Doo; Park, Jisun

    2017-01-01

    Purpose The major problem in producing artificial livers is that primary hepatocytes cannot be cultured for many days. Recently, 3-dimensional (3D) printing technology draws attention and this technology regarded as a useful tool for current cell biology. By using the 3D bio-printing, these problems can be resolved. Methods To generate 3D bio-printed structures (25 mm × 25 mm), cells-alginate constructs were fabricated by 3D bio-printing system. Mouse primary hepatocytes were isolated from the livers of 6–8 weeks old mice by a 2-step collagenase method. Samples of 4 × 107 hepatocytes with 80%–90% viability were printed with 3% alginate solution, and cultured with well-defined culture medium for primary hepatocytes. To confirm functional ability of hepatocytes cultured on 3D alginate scaffold, we conducted quantitative real-time polymerase chain reaction and immunofluorescence with hepatic marker genes. Results Isolated primary hepatocytes were printed with alginate. The 3D printed hepatocytes remained alive for 14 days. Gene expression levels of Albumin, HNF-4α and Foxa3 were gradually increased in the 3D structures. Immunofluorescence analysis showed that the primary hepatocytes produced hepatic-specific proteins over the same period of time. Conclusion Our research indicates that 3D bio-printing technique can be used for long-term culture of primary hepatocytes. It can therefore be used for drug screening and as a potential method of producing artificial livers. PMID:28203553

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

    Science.gov (United States)

    López-Islas, Anayelly; Chagoya-Hazas, Victoria; Pérez-Aguilar, Benjamin; Palestino-Domínguez, Mayrel; Souza, Verónica; Miranda, Roxana U; Bucio, Leticia; Gómez-Quiroz, Luis Enrique; Gutiérrez-Ruiz, María-Concepción

    2016-01-01

    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.

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

    Science.gov (United States)

    López-Islas, Anayelly; Chagoya-Hazas, Victoria; Pérez-Aguilar, Benjamin; Palestino-Domínguez, Mayrel; Souza, Verónica; Miranda, Roxana U.; Bucio, Leticia; Gómez-Quiroz, Luis Enrique; Gutiérrez-Ruiz, María-Concepción

    2016-01-01

    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. PMID:26788255

  5. Hepatitis B virus infection and replication in primarily cultured human fetal hepatocytes

    Institute of Scientific and Technical Information of China (English)

    Min Lin; Qun Chen; Li-Ye Yang; Wen-Yu Li; Xi-Biao Cao; Jiao-Ren Wu; You-Peng Peng; Mo-Rui Chen

    2007-01-01

    AIM:To investigate the infection and replication of hepatitis B virus(HBV)in primarily cultured human fetal hepatocytes(HFHs).METHODS:The human fetal hepatocytes were cultured in serum-free medium,HBV-positive serum was added into the medium to study the susceptibility of hepatocytes to HBV infection.The supernatant was collected for ELISA assay of HBsAg and HBeAg,and quantitative fluorescence PCR for HBV-DNA assay daily.Albumin and HBcAg,CK8 and CK18 expressions were detected by immunohistochemistry in cultured hepatocytes.Content of lactate dehydrogenate(LDH)was measured to find out the integrity of the cell membrane.RESULTS:A stable hepatocyte culture system was established.HBV could infect the hepatocytes and replicate,and HBcAg expression could be detected by immunohistochemistry in hepatocyte-like cells.HBV-DNA in the supernatant could be detected from d 2 to d 18 and HBsAg and HBeAg were positive on d 3-d 18 after HBV infection.HBV in medium increased from d 0 to d 6 and subsequently decreased as the cells were progressively loosing their hepatocyte phenotypes.CONCLUSION:HBV could infect human fetal hepatocytes and replicate.This in vitro model allowed a detailed Study on early events associated with human HBV entry into cells and subsequent replication.

  6. Three-dimensional (3D) printing of mouse primary hepatocytes to generate 3D hepatic structure.

    Science.gov (United States)

    Kim, Yohan; Kang, Kyojin; Jeong, Jaemin; Paik, Seung Sam; Kim, Ji Sook; Park, Su A; Kim, Wan Doo; Park, Jisun; Choi, Dongho

    2017-02-01

    The major problem in producing artificial livers is that primary hepatocytes cannot be cultured for many days. Recently, 3-dimensional (3D) printing technology draws attention and this technology regarded as a useful tool for current cell biology. By using the 3D bio-printing, these problems can be resolved. To generate 3D bio-printed structures (25 mm × 25 mm), cells-alginate constructs were fabricated by 3D bio-printing system. Mouse primary hepatocytes were isolated from the livers of 6-8 weeks old mice by a 2-step collagenase method. Samples of 4 × 10(7) hepatocytes with 80%-90% viability were printed with 3% alginate solution, and cultured with well-defined culture medium for primary hepatocytes. To confirm functional ability of hepatocytes cultured on 3D alginate scaffold, we conducted quantitative real-time polymerase chain reaction and immunofluorescence with hepatic marker genes. Isolated primary hepatocytes were printed with alginate. The 3D printed hepatocytes remained alive for 14 days. Gene expression levels of Albumin, HNF-4α and Foxa3 were gradually increased in the 3D structures. Immunofluorescence analysis showed that the primary hepatocytes produced hepatic-specific proteins over the same period of time. Our research indicates that 3D bio-printing technique can be used for long-term culture of primary hepatocytes. It can therefore be used for drug screening and as a potential method of producing artificial livers.

  7. Collaborative form(s)

    DEFF Research Database (Denmark)

    Gunn, Wendy

    Gunn asks us to consider beauty as collaborative forms of action generated by moving between design by means of anthropology and anthropology by means of design. Specifically, she gives focus to play-like reflexions on practices of designing energy products, systems and infrastructure. Design...

  8. Mapping the Cell-Surface N-Glycoproteome of Human Hepatocytes Reveals Markers for Selecting a Homogeneous Population of iPSC-Derived Hepatocytes.

    Science.gov (United States)

    Mallanna, Sunil K; Cayo, Max A; Twaroski, Kirk; Gundry, Rebekah L; Duncan, Stephen A

    2016-09-13

    When comparing hepatic phenotypes between iPSC-derived hepatocyte-like cells from different liver disease patients, cell heterogeneity can confound interpretation. We proposed that homogeneous cell populations could be generated by fluorescence-activated cell sorting (FACS). Using cell-surface capture proteomics, we identified a total of 300 glycoproteins on hepatocytes. Analyses of the expression profiles during the differentiation of iPSCs revealed that SLC10A1, CLRN3, and AADAC were highly enriched during the final stages of hepatocyte differentiation. FACS purification of hepatocyte-like cells expressing SLC10A1, CLRN3, or AADAC demonstrated enrichment of cells with hepatocyte characteristics. Moreover, transcriptome analyses revealed that cells expressing the liver gene regulatory network were enriched while cells expressing a pluripotent stem cell network were depleted. In conclusion, we report an extensive catalog of cell-surface N-linked glycoproteins expressed in primary hepatocytes and identify cell-surface proteins that facilitate the purification of homogeneous populations of iPSC-derived hepatocyte-like cells.

  9. Mapping the Cell-Surface N-Glycoproteome of Human Hepatocytes Reveals Markers for Selecting a Homogeneous Population of iPSC-Derived Hepatocytes

    Directory of Open Access Journals (Sweden)

    Sunil K. Mallanna

    2016-09-01

    Full Text Available When comparing hepatic phenotypes between iPSC-derived hepatocyte-like cells from different liver disease patients, cell heterogeneity can confound interpretation. We proposed that homogeneous cell populations could be generated by fluorescence-activated cell sorting (FACS. Using cell-surface capture proteomics, we identified a total of 300 glycoproteins on hepatocytes. Analyses of the expression profiles during the differentiation of iPSCs revealed that SLC10A1, CLRN3, and AADAC were highly enriched during the final stages of hepatocyte differentiation. FACS purification of hepatocyte-like cells expressing SLC10A1, CLRN3, or AADAC demonstrated enrichment of cells with hepatocyte characteristics. Moreover, transcriptome analyses revealed that cells expressing the liver gene regulatory network were enriched while cells expressing a pluripotent stem cell network were depleted. In conclusion, we report an extensive catalog of cell-surface N-linked glycoproteins expressed in primary hepatocytes and identify cell-surface proteins that facilitate the purification of homogeneous populations of iPSC-derived hepatocyte-like cells.

  10. A fusion between domains of the human bone morphogenetic protein-2 and maize 27 kD gamma-zein accumulates to high levels in the endoplasmic reticulum without forming protein bodies in transgenic tobacco

    Directory of Open Access Journals (Sweden)

    Valentina eCeresoli

    2016-03-01

    Full Text Available Human Bone Morphogenetic Protein-2 (hBMP2 is an osteoinductive agent physiologically involved in bone remodelling processes. A commercialized recombinant hBMP2 produced in mammalian cell lines is available in different clinical applications where bone regeneration is needed, but widespread use has been hindered due to an unfavorable cost/effective ratio. Protein bodies are very large insoluble protein polymers that originate within the endoplasmic reticulum by prolamine accumulation during the cereal seed development. The N-terminal domain of the maize prolamin 27 kD -zein is able to promote protein body biogenesis when fused to other proteins. To produce high yield of recombinant hBMP2 active domain (ad in stably transformed tobacco plants we have fused it to the γ-zein domain. We show that this zein-hBMP2ad fusion is retained in the endoplasmic reticulum without forming insoluble protein bodies. The accumulation levels are above 1% of total soluble leaf proteins, indicating that it could be a rapid and suitable strategy to produce hBMP2ad at affordable costs.

  11. A Fusion between Domains of the Human Bone Morphogenetic Protein-2 and Maize 27 kD γ-Zein Accumulates to High Levels in the Endoplasmic Reticulum without Forming Protein Bodies in Transgenic Tobacco.

    Science.gov (United States)

    Ceresoli, Valentina; Mainieri, Davide; Del Fabbro, Massimo; Weinstein, Roberto; Pedrazzini, Emanuela

    2016-01-01

    Human Bone Morphogenetic Protein-2 (hBMP2) is an osteoinductive agent physiologically involved in bone remodeling processes. A commercialized recombinant hBMP2 produced in mammalian cell lines is available in different clinical applications where bone regeneration is needed, but widespread use has been hindered due to an unfavorable cost/effective ratio. Protein bodies are very large insoluble protein polymers that originate within the endoplasmic reticulum by prolamine accumulation during the cereal seed development. The N-terminal domain of the maize prolamin 27 kD γ-zein is able to promote protein body biogenesis when fused to other proteins. To produce high yield of recombinant hBMP2 active domain (ad) in stably transformed tobacco plants we have fused it to the γ-zein domain. We show that this zein-hBMP2ad fusion is retained in the endoplasmic reticulum without forming insoluble protein bodies. The accumulation levels are above 1% of total soluble leaf proteins, indicating that it could be a rapid and suitable strategy to produce hBMP2ad at affordable costs.

  12. Accurate quantitation for in vitro refolding of single domain antibody fragments expressed as inclusion bodies by referring the concomitant expression of a soluble form in the periplasms of Escherichia coli.

    Science.gov (United States)

    Noguchi, Tomoaki; Nishida, Yuichi; Takizawa, Keiji; Cui, Yue; Tsutsumi, Koki; Hamada, Takashi; Nishi, Yoshisuke

    2017-03-01

    Single domain antibody fragments from two species, a camel VHH (PM1) and a shark VNAR (A6), were derived from inclusion bodies of E. coli and refolded in vitro following three refolding recipes for comparing refolding efficiencies: three-step cold dialysis refolding (TCDR), one-step hot dialysis refolding (OHDR), and one-step cold dialysis refolding (OCDR), as these fragments were expressed as 'a soluble form' either in cytoplasm or periplasm, but the amount were much less than those expressed as 'an insoluble form (inclusion body)' in cytoplasm and periplasm. In order to verify the refolding efficiencies from inclusion bodies correctly, proteins purified from periplasmic soluble fractions were used as reference samples. These samples showed far-UV spectra of a typical β-sheet-dominant structure in circular dichroism (CD) spectroscopy and so did the refolded samples as well. As the maximal magnitude of ellipticity in millidegrees (θmax) observed at a given wave length was proportional to the concentrations of the respective reference samples, we could draw linear regression lines for the magnitudes vs. sample concentrations. By using these lines, we measured the concentrations for the refolded PM1 and A6 samples purified from solubilized cytoplasmic insoluble fractions. The refolding efficiency of PM1 was almost 50% following TCDR and 40% and 30% following OHDR and OCDR, respectively, whereas the value of A6 was around 30% following TCDR, and out of bound for quantitation following the other two recipes. The ELISA curves, which were derived from the refolded samples, coincided better with those obtained from the reference samples after converting the values from the protein-concentrations at recovery to the ones of refolded proteins using recovery ratios, indicating that such a correction gives better results for the accurate measure of the ELISA curves than those without correction. Our method require constructing a dual expression system, expressed both in

  13. The role of insulin, glucagon, dexamethasone, and leptin in the regulation of ketogenesis and glycogen storage in primary cultures of porcine hepatocytes prepared from 60 kg pigs.

    Science.gov (United States)

    Fernández-Fígares, I; Shannon, A E; Wray-Cahen, D; Caperna, T J

    2004-08-01

    A study was conducted to elucidate hormonal control of ketogenesis and glycogen deposition in primary cultures of porcine hepatocytes. Hepatocytes were isolated from pigs (54-68 kg) by collagenase perfusion and seeded into collagen-coated T-25 flasks. Monolayers were established in medium containing fetal bovine serum for 1 day and switched to a serum-free medium for the remainder of the culture period. Hepatocytes were maintained in DMEM/M199 containing 1% DMSO, dexamethasone (10(-6) or 10(-7) M), linoleic acid (3.4 x 10(-5) M), and carnitine (10(-3) M) for 3 days. On the first day of serum-free culture, insulin was added at 1 or 100 ng/ml and glucagon was added at 0, 1, or 100 ng/ml. Recombinant human leptin (200 ng/ml) was added during the final 24 h; medium and all cells were harvested on the third day. Concentrations of acetoacetate and beta-hydroxybutyrate (ketone bodies) in media and glycogen deposition in the cellular compartment were determined. Ketogenesis was highly stimulated by glucagon (1 and 100 ng/ml) and inhibited by insulin. In contrast, glycogen deposition was stimulated by insulin and attenuated by glucagon; high insulin was also associated with a reduction in the ketone body ratio (acetoacetate:beta-hydroxybutyrate). High levels of dexamethasone stimulated ketogenesis, but inhibited glycogen deposition at low insulin. Culture of cells with leptin for 24 h, over the range of insulin, glucagon, and dexamethasone concentrations had no effect on either glycogen deposition or ketogenesis. These data suggest that while adult porcine hepatocytes are indeed sensitive to hormonal manipulation, leptin has no direct influence on hepatic energy metabolism in swine.

  14. Induction of foci of altered, γ-glutamyltranspeptidase-positive hepatocytes in carcinogen-treated rats fed a choline-deficient diet

    Science.gov (United States)

    Sells, M. A.; Katyal, S. L.; Sell, S.; Shinozuka, H.; Lombardi, B.

    1979-01-01

    A series of experiments was performed to investigate whether, after exposure of rats to a chemical hepatocarcinogen, feeding a choline-deficient (CD) diet would promote the proliferation of initiated liver cells, and their evolution to foci of altered γ-glutamyltranspeptidase (GGT)-positive hepatocytes, without subjecting the animals to further experimental manipulations. Diethylnitrosamine (DEN), in single doses of 15-150 mg/kg body weight, was injected into male, Sprague-Dawley rats, either intact or 18 h after a partial hepatectomy (PH). The animals were then fed either a CD or a choline-supplemented (CS) diet for 2-8 weeks. Emergence in the liver of foci of altered, GGT+ hepatocytes was studied by histological and histochemical techniques. Foci, in varying numbers, developed in the liver of all rats fed the CD diet. The number of foci induced was larger when DEN was administered after PH rather than to intact rats. Foci developed in none of the livers of rats fed the CS diet, except in one experiment in which 30 mg DEN/kg body weight was injected after a PH. In all cases, foci of altered, GGT+ hepatocytes were shown to be α-foetoprotein after immunofluorescence staining of liver sections. It is concluded that feeding a CD diet exerts a strong promoting action on the proliferation and further evolution of liver cells initiated by a chemical carcinogen, providing the basis for a new and efficient procedure for the induction of foci of altered hepatocytes in rat liver. ImagesFig. 2Fig. 3Fig. 4Fig. 5 PMID:89859

  15. Contextualizing Hepatocyte Functionality of Cryopreserved HepaRG Cell Cultures.

    Science.gov (United States)

    Jackson, Jonathan P; Li, Linhou; Chamberlain, Erica D; Wang, Hongbing; Ferguson, Stephen S

    2016-09-01

    Over the last decade HepaRG cells have emerged as a promising alternative to primary human hepatocytes (PHH) and have been featured in over 300 research publications. Most of these reports employed freshly differentiated HepaRG cells that require time-consuming culture (∼28 days) for full differentiation. Recently, a cryopreserved, predifferentiated format of HepaRG cells (termed here "cryo-HepaRG") has emerged as a new model that improves global availability and experimental flexibility; however, it is largely unknown whether HepaRG cells in this format fully retain their hepatic characteristics. Therefore, we systematically investigated the hepatocyte functionality of cryo-HepaRG cultures in context with the range of interindividual variation observed with PHH in both sandwich-culture and suspension formats. These evaluations uncovered a novel adaptation period for the cryo-HepaRG format and demonstrated the impact of extracellular matrix on cryo-HepaRG functionality. Pharmacologically important drug-metabolizing alleles were genotyped in HepaRG cells and poor metabolizer alleles for CYP2D6, CYP2C9, and CYP3A5 were identified and consistent with higher frequency alleles found in individuals of Caucasian decent. We observed liver enzyme inducibility with aryl hydrocarbon receptor, constitutive androstane receptor (CAR), and pregnane X receptor activators comparable to that of sandwich-cultured PHH. Finally, we show for the first time that cryo-HepaRG supports proper CAR cytosolic sequestration and translocation to hepatocyte nuclei in response to phenobarbital treatment. Taken together, these data reveal important considerations for the use of this cell model and demonstrate that cryo-HepaRG are suitable for metabolism and toxicology screening.

  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. Effects of ethanol on antioxidant capacity in isolated rat hepatocytes

    Institute of Scientific and Technical Information of China (English)

    Sien-Sing Yang; Chi-Chang Huang; Jiun-Rong Chen; Che-Lin Chiu; Ming-Jer Shieh; Su-Jiun Lin; Suh-Ching Yang

    2005-01-01

    AIM: To investigate dose-response and time-course of the effects of ethanol on the cell viability and antioxidant capacity in isolated rat hepatocytes.METHODS: Hepatocytes were isolated from male adult Wistar rats and seeded into 100-mm dishes. Hepatocytes were treated with ethanol at concentrations between 0 (C), 10 (E10), 50 (E50), and 100 (E100) mmol/L (dose response) for 12, 24, and 36 h (time course). Then,lactate dehydrogenase (LDH) leakage, malondialdehyde (MDA) concentration, glutathione (GSH) level, and activities of glutathione peroxidase (GPX), glutathione reductase (GRD), superoxide dismutase (SOD), and catalase (CAT) were measured.RESULTS: Our data revealed that LDH leakage was significantly increased by about 30% in group E100 over those in groups C and E10 at 24 and 36 h, The MDA concentration in groups C, E10 and E50 were significantly lower than that in group E100 at 36 h. Furthermore,the concentration of MDA in group E100 at 36 h was significantly higher by 4.5- and 1.7-fold, respectively,than that at 12 and 24 h. On the other hand, the GSH level in group E100 at 24 and 36 h was significantly decreased, by 32% and 28%, respectively, compared to that at 12 h. The activities of GRD and CAT in group E100 at 36 h were significantly less than those in groups C and E10. However, The GPX and SOD activities showed no significant change in each group.CONCLUSION: These results suggest that longtime incubation with higher concentration of ethanol (100 mmol/L) decreased the cell viability by means of reducing GRD and CAT activities and increasing lipid peroxidation.

  18. 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 participants (10 women/12 men, BMI 20.6-34.5 kg/m(2), age 18-49 years) underwent a hyperinsulinemic euglycemic clamp with measurement of HGF at baseline and steady state. Relationships between baseline HGF, anthropometrics, triglycerides, liver enzymes, c-reactive protein and adiponectin were also evaluated...

  19. N-deacetyl ketoconazole-induced hepatotoxicity in a primary culture system of rat hepatocytes.

    Science.gov (United States)

    Rodriguez, R J; Acosta, D

    1997-02-28

    Ketoconazole (KT) is an azole antifungal agent that has been associated with hepatotoxicity. The mechanism of its hepatotoxicity has not yet been resolved. It has been suggested that a reactive metabolite may be the cause of toxicity because the hepatic injury does not appear to be mediated through an immunoallergic mechanism. Several metabolites of KT have been reported in the literature of which the deacetylated metabolite, N-deacetyl ketoconazole (DAK), is the major metabolite which undergoes further metabolism by the flavin-containing monooxygenases (FMO) to form a potentially toxic dialdehyde. The objective of this study was to evaluate DAK's cytotoxicity and the role of FMO in a primary culture system of rat hepatocytes. Cytotoxicity was evaluated by measuring the leakage of the cytosolic enzyme, lactate dehydrogenase (LDH), into the medium and by assessing mitochondrial reduction of 3-(4,5-dimethythiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT). The cultures were exposed to various concentrations of DAK (20-160 microM) for 0.5-4 h. There was a significant increase (P DAK at 0.5, 1.0, 2.0 and 4.0 h, respectively). The concentrations to observe 50% LDH leakage from the hepatocytes were 155, 133, 100, 70 microM DAK at 0.5, 1.0, 2.0 and 4.0 h, respectively. Moreover, co-treatment with methimazole, a competitive substrate for FMO, produced a significant decrease (P DAK. Also, the toxicity was significantly (P DAK is a more potent cytotoxicant than its parent compound, KT, as reported previously by our laboratory (Rodriguez and Acosta, Toxicology, 96: 83-92, 1995) and its toxicity was expressed in a dose- and time-dependent manner. Furthermore, DAK's cytotoxicity was enhanced with n-octylamine and suppressed with methimazole, suggesting a role for FMO in the toxicity of the metabolite.

  20. Targeted in vivo delivery of siRNA and an endosome-releasing agent to hepatocytes.

    Science.gov (United States)

    Sebestyén, Magdolna G; Wong, So C; Trubetskoy, Vladimir; Lewis, David L; Wooddell, Christine I

    2015-01-01

    The discoveries of RNA interference (RNAi) and short interfering RNAs (siRNAs) have provided the opportunity to treat diseases in a fundamentally new way: by co-opting a natural process to inhibit gene expression at the mRNA level. Given that siRNAs must interact with the cells' natural RNAi machinery in order to exert their silencing effect, one of the most fundamental requirements for their use is efficient delivery to the desired cell type and, specifically, into the cytoplasm of those cells. Numerous research efforts involving the testing of a large number of delivery approaches using various carrier molecules and inventing several distinct formulation technologies during the past decade illustrate the difficulty and complexity of this task. We have developed synthetic polymer formulations for in vivo siRNA delivery named Dynamic PolyConjugates™ (DPCs) that are designed to mimic the features viruses possess for efficient delivery of their nucleic acids. These include small size, long half-life in circulation, capability of displaying distinct host cell tropism, efficient receptor binding and cell entry, disassembly in the endosome and subsequent release of the nucleic acid cargo to the cytoplasm. Here we present an example of this delivery platform composed of a hepatocyte-targeted endosome-releasing agent and a cholesterol-conjugated siRNA (chol-siRNA). This delivery platform forms the basis of ARC-520, an siRNA-based therapeutic for the treatment of chronic hepatitis B virus (HBV) infection. In this chapter, we provide a general overview of the steps in developing ARC-520 and detailed protocols for two critical stages of the discovery process: (1) verifying targeted in vivo delivery to hepatocytes and (2) evaluating in vivo drug efficacy using a mouse model of chronic HBV infection.

  1. Phosphatidylinositol 3-Kinase and Protein Kinase C Contribute to the Inhibition by Interleukin 6 of Phosphoenolpyruvate Carboxykinase Gene Expression in Cultured Rat Hepatocytes

    DEFF Research Database (Denmark)

    Christ, Bruno; Yazici, Emine; Nath, Annegret

    2000-01-01

    Gluconeogenesis, hepatocytes, interleukin 6, liver, phosphoinositide 3-kinase, phosphoenolpyruvate carboxykinase......Gluconeogenesis, hepatocytes, interleukin 6, liver, phosphoinositide 3-kinase, phosphoenolpyruvate carboxykinase...

  2. Beyond packing of hard spheres: The effects of core softness, non-additivity, intermediate-range repulsion, and many-body interactions on the glass-forming ability of bulk metallic glasses

    Science.gov (United States)

    Zhang, Kai; Fan, Meng; Liu, Yanhui; Schroers, Jan; Shattuck, Mark D.; O'Hern, Corey S.

    2015-11-01

    When a liquid is cooled well below its melting temperature at a rate that exceeds the critical cooling rate Rc, the crystalline state is bypassed and a metastable, amorphous glassy state forms instead. Rc (or the corresponding critical casting thickness dc) characterizes the glass-forming ability (GFA) of each material. While silica is an excellent glass-former with small Rc alloys are typically poor glass-formers with large Rc > 1010 K/s. Only in the past thirty years have bulk metallic glasses (BMGs) been identified with Rc approaching that for silica. Recent simulations have shown that simple, hard-sphere models are able to identify the atomic size ratio and number fraction regime where BMGs exist with critical cooling rates more than 13 orders of magnitude smaller than those for pure metals. However, there are a number of other features of interatomic potentials beyond hard-core interactions. How do these other features affect the glass-forming ability of BMGs? In this manuscript, we perform molecular dynamics simulations to determine how variations in the softness and non-additivity of the repulsive core and form of the interatomic pair potential at intermediate distances affect the GFA of binary alloys. These variations in the interatomic pair potential allow us to introduce geometric frustration and change the crystal phases that compete with glass formation. We also investigate the effect of tuning the strength of the many-body interactions from zero to the full embedded atom model on the GFA for pure metals. We then employ the full embedded atom model for binary BMGs and show that hard-core interactions play the dominant role in setting the GFA of alloys, while other features of the interatomic potential only change the GFA by one to two orders of magnitude. Despite their perturbative effect, understanding the detailed form of the intermetallic potential is important for designing BMGs with cm or greater casting thickness.

  3. Is body-weight-supported treadmill training or robotic-assisted gait training superior to overground gait training and other forms of physiotherapy in people with spinal cord injury? A systematic review.

    Science.gov (United States)

    Mehrholz, J; Harvey, L A; Thomas, S; Elsner, B

    2017-08-01

    Systematic review about randomised trials comparing different training strategies to improve gait in people with spinal cord injuries (SCI). The aim of this systematic review was to compare the effectiveness of body-weight-supported treadmill training (BWSTT) and robotic-assisted gait training with overground gait training and other forms of physiotherapy in people with traumatic SCI. Systematic review conducted by researchers from Germany and Australia. An extensive search was conducted for randomised controlled trials involving people with traumatic SCI that compared either BWSTT or robotic-assisted gait training with overground gait training and other forms of physiotherapy. The two outcomes of interest were walking speed (m s(-1)) and walking distance (m). BWSTT and robotic-assisted gait training were analysed separately, and data were pooled across trials to derive mean between-group differences using a random-effects model. Thirteen randomised controlled trials involving 586 people were identified. Ten trials involving 462 participants compared BWSTT to overground gait training and other forms of physiotherapy, but only nine trials provided useable data. The pooled mean (95% confidence interval (CI)) between-group differences for walking speed and walking distance were -0.03 m s(-1) (-0.10 to 0.04) and -7 m (-45 to 31), respectively, favouring overground gait training. Five trials involving 344 participants compared robotic-assisted gait training to overground gait training and other forms of physiotherapy but only three provided useable data. The pooled mean (95% CI) between-group differences for walking speed and walking distance were -0.04 m s(-1) (95% CI -0.21 to 0.13) and -6 m (95% CI -86 to 74), respectively, favouring overground gait training. BWSTT and robotic-assisted gait training do not increase walking speed more than overground gait training and other forms of physiotherapy do, but their effects on walking distance are not clear.

  4. Body Image

    Science.gov (United States)

    ... About Us Contact Us Text size | Print | Body Image Developing a positive body image and a healthy mental attitude is crucial to ... on for tips to have a healthy body image. Topics About body image When you look in ...

  5. A sandwich-cultured rat hepatocyte system with increased metabolic competence evaluated by gene expression profiling

    NARCIS (Netherlands)

    Kienhuis, A.S.; Wortelboer, H.M.; Maas, W.J.; Herwijnen, M. van; Kleinjans, J.C.S.; Delft, J.H.M. van; Stierum, R.H.

    2007-01-01

    A rapid decline of cytochrome P450 (CYP450) enzyme activities remains a drawback of rat hepatocyte-based in vitro cultures. Consequently, judgment of the toxic potential of compounds that need bioactivation by CYP450s may not be adequate using this model. In the present study, an improved hepatocyte

  6. A dual-functional fibrous scaffold enhances P450 activity of cultured primary rat hepatocytes.

    Science.gov (United States)

    Chua, Kian-Ngiap; Tang, Yen-Ni; Quek, Chai-Hoon; Ramakrishna, Seeram; Leong, Kam W; Mao, Hai-Quan

    2007-09-01

    We have designed a novel dual-functional electrospun fibrous scaffold comprising two fiber mesh layers that were modified differently to induce two separate biological responses from hepatocytes. The first fiber layer was galactosylated on the surface to mediate hepatocyte attachment, while the second layer was loaded with 3-methylcholanthrene (3-Mc) to enhance cytochrome P450 activity of hepatocytes. Primary rat hepatocytes cultured on the galactosylated fibrous scaffolds loaded with different concentrations of 3-Mc were compared for their cell attachment efficiency, albumin secretion activity and cytochrome P450-dependent 7-ethoxycoumarin O-deethylase activity. This hybrid fibrous scaffold mediated hepatocyte attachment with slightly lower efficiency (76+/-2.3%) than a single-layer galactosylated fibrous scaffold (84+/-3.5%). More importantly, the cytochrome P450 activity of the hepatocytes cultured on the hybrid scaffold correlated well with the 3-Mc loading level. The results also showed that transfer of 3-Mc to hepatocytes through direct cell-fiber contact was the dominant transport route, with the induced cytochrome P450 activity being 1.9- to 4.8-fold higher than that of transfer of 3-Mc to hepatocytes via dissolution from fibers to medium. This study demonstrates the feasibility of creating multi-functional fibrous scaffolds that serve both as an adhesive substrate and as a delivery vehicle for bioactive molecules.

  7. Interactions between macrophage/Kupffer cells and hepatocytes in surgical sepsis

    Energy Technology Data Exchange (ETDEWEB)

    West, M.A.

    1988-01-01

    Experiments were performed to investigate the role of Kupffer cell/macrophage interactions with hepatocytes in modulating liver function during infections using direct in vitro cocultivation of rat macrophages or Kupffer cells with rat hepatocytes. Protein synthesis was assayed as a sensitive indicator of integrated hepatocellular function by measuring {sup 3}H-leucine incorporation into hepatocyte protein. Septic stimuli such as lipoploysaccharide and killed bacteria were added to cocultures of hepatocytes and macrophages or Kupffer cells and the responses compared to hepatocytes alone. Information about the types of proteins synthesized by hepatocytes under various culture conditions was determined using polyacrylamide gel electrophoresis and autoradiography. These experiments showed that septic stimuli alter the amount and type of protein synthesized by hepatocytes and had no direct effect on hepatocytes in the absence of macrophages or Kupffer cells. The mediator(s) appears to be a heat labile, soluble monokine(s) which is distinct from interleukin-1 or tumor necrosis factor. The important role of Kupffer cells/macrophages in mediating alterations in hepatocellular function in sepsis may ultimately improve patient care.

  8. Effects of nuclear translocation of tissue transglutaminase and the release of cytochrome C on hepatocyte apoptosis

    Institute of Scientific and Technical Information of China (English)

    宋良文; 马宪梅; 李扬; 崔雪梅; 王晓民

    2003-01-01

    Objective To assess the effects of nuclear translocation of tissue transglutaminase (TTG) and the release of cytochrome C on hepatocyte apoptosis and to reveal the mechanism of signal transduction of early apoptosis in injured hepatocytes. Methods Hepatocytes isolated from tissue transglutaminase gene knock-out rats and mice were stimulated with ethanol. Proteins from whole cell, cytoplasm and nuclei were extracted for determination of TTG activity by 14 C-putrescine incorporation. Distribution of TTG throughout the entire cell, as well as just nucleus was observed under a confocal scanning microscope. The amount of cytochrome C released from mitochondria was determined by ELISA. Cell apoptosis was observed by fluorescent cytochemistry.Results TTG activity in whole cells and nuclei was significantly increased after the hepatocytes were treated with ethanol. Cytochrome C release was remarkably increased in the cells isolated from rat and wild-type mouse after treatment with ethanol but not in TTG gene knock-out mice. Cellular apoptosis appeared in hepatocytes isolated from rats and wild-type mice but not in the hepatocytes from TTG gene knock-out mice after stimulation with ethanol.Conclusions Increased TTG in hepatocytes can be translocated into the nucleus and promote release of mitochondrial cytochrome C into the cytoplasm. Passing through a series of signal pathways, hepatocyte apoptosis is induced eventually.

  9. HepatoDyn: A Dynamic Model of Hepatocyte Metabolism That Integrates 13C Isotopomer Data.

    Directory of Open Access Journals (Sweden)

    Carles Foguet

    2016-04-01

    Full Text Available The liver performs many essential metabolic functions, which can be studied using computational models of hepatocytes. Here we present HepatoDyn, a highly detailed dynamic model of hepatocyte metabolism. HepatoDyn includes a large metabolic network, highly detailed kinetic laws, and is capable of dynamically simulating the redox and energy metabolism of hepatocytes. Furthermore, the model was coupled to the module for isotopic label propagation of the software package IsoDyn, allowing HepatoDyn to integrate data derived from 13C based experiments. As an example of dynamical simulations applied to hepatocytes, we studied the effects of high fructose concentrations on hepatocyte metabolism by integrating data from experiments in which rat hepatocytes were incubated with 20 mM glucose supplemented with either 3 mM or 20 mM fructose. These experiments showed that glycogen accumulation was significantly lower in hepatocytes incubated with medium supplemented with 20 mM fructose than in hepatocytes incubated with medium supplemented with 3 mM fructose. Through the integration of extracellular fluxes and 13C enrichment measurements, HepatoDyn predicted that this phenomenon can be attributed to a depletion of cytosolic ATP and phosphate induced by high fructose concentrations in the medium.

  10. Measurement of acetyl-CoA carboxylase activity in isolated hepatocytes

    NARCIS (Netherlands)

    Bijleveld, C.; Geelen, M.J.H.

    1987-01-01

    An assay is described for acetyl-CoA carboxylase activity in isolated hepatocytes. The assay is based on two principles: (a) The hepatocytes are made permeable by digitonin. 64 μg of digitonin per mg of cellular protein were most effective in exposing enzyme activity without a significant effect on

  11. Partial hepatectomy induces delayed hepatocyte proliferation and normal liver regeneration in ovariectomized mice

    Directory of Open Access Journals (Sweden)

    Umeda M

    2015-07-01

    Full Text Available Makoto Umeda,1 Masaki Hiramoto,1,2 Takeshi Imai1 1Department of Aging Intervention, National Center for Geriatrics and Gerontology, Obu, Aichi, Japan; 2Department of Biochemistry, Tokyo Medical University, Tokyo, Japan Abstract: Estrogens play central roles in sexual development, reproduction, and hepatocyte proliferation. The ovaries are one of the main organs for estradiol (E2 production. Ovariectomies (OVXs were performed on the female mice, and hepatocyte proliferation was analyzed. The ovariectomized mice exhibited delayed hepatocyte proliferation after partial hepatectomy (PH and also exhibited delayed and reduced E2 induction. Both E2 administration and PH induced the gene expression of estrogen receptor α (ERα. The transcripts of ERα were detected specifically in periportal hepatocytes after E2 administration and PH. Moreover, the E2 concentrations and hepatocyte proliferation rates were highest in the proestrus period of the estrous cycle. Taken together, these findings indicate that E2 accelerated ERα expression in periportal hepatocytes and hepatocyte proliferation in the female mice.Keywords: estrogen, ER, estrous cycle, hepatocyte proliferation, liver regeneration

  12. Prolonged Survival of Mice with Acute Liver Failure with Transplantation of Monkey Hepatocytes Cultured with an Antiapoptotic Pentapeptide V5

    OpenAIRE

    田中, 公章

    2007-01-01

    BACKGROUND: Because hepatocyte transplantation has been considered to be an attractive method to treat acute liver failure (ALF), efficient recovery of hepatocytes and maintenance of differentiated hepatocyte functions is of extreme importance. We here report the usefulness of an antiapoptotic pentapeptide V5, composed of Val-Pro-Met-Leu-Lys, in the monkey hepatocyte cultures. METHODS: We evaluated albumin production, metabolizing abilities of ammonia, lidocaine, and diazepam of monkey hepato...

  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. Discovering networks of perturbed biological processes in hepatocyte cultures.

    Directory of Open Access Journals (Sweden)

    Christopher D Lasher

    Full Text Available The liver plays a vital role in glucose homeostasis, the synthesis of bile acids and the detoxification of foreign substances. Liver culture systems are widely used to test adverse effects of drugs and environmental toxicants. The two most prevalent liver culture systems are hepatocyte monolayers (HMs and collagen sandwiches (CS. Despite their wide use, comprehensive transcriptional programs and interaction networks in these culture systems have not been systematically investigated. We integrated an existing temporal transcriptional dataset for HM and CS cultures of rat hepatocytes with a functional interaction network of rat genes. We aimed to exploit the functional interactions to identify statistically significant linkages between perturbed biological processes. To this end, we developed a novel approach to compute Contextual Biological Process Linkage Networks (CBPLNs. CBPLNs revealed numerous meaningful connections between different biological processes and gene sets, which we were successful in interpreting within the context of liver metabolism. Multiple phenomena captured by CBPLNs at the process level such as regulation, downstream effects, and feedback loops have well described counterparts at the gene and protein level. CBPLNs reveal high-level linkages between pathways and processes, making the identification of important biological trends more tractable than through interactions between individual genes and molecules alone. Our approach may provide a new route to explore, analyze, and understand cellular responses to internal and external cues within the context of the intricate networks of molecular interactions that control cellular behavior.

  15. 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 hepatocytes using either the bile acid glycochenodeoxycholic acid (GCDCA or TNFα in combination with actinomycin D (actD. AMPK, mTOR and phosphoinositide-3 kinase (PI3K/Akt were inhibited using pharmacological inhibitors. Apoptosis and necrosis were quantified by caspase activation, acridine orange staining and Sytox green staining respectively. RESULTS: Metformin dose-dependently reduces GCDCA-induced apoptosis, even when added 2 hours after GCDCA, without increasing necrotic cell death. Metformin does not protect against TNFα/ActD-induced apoptosis. The protective effect of metformin is dependent on an intact PI3-kinase/Akt pathway, but does not require AMPK/mTOR-signaling. Metformin does not inhibit NF-κB activation. CONCLUSION: Metformin protects against bile acid-induced apoptosis and could be considered in the treatment of chronic liver diseases accompanied by inflammation.

  16. Cytotoxicity of ortho-phenylphenol in isolated rat hepatocytes.

    Science.gov (United States)

    Nakagawa, Y; Moldéus, P; Moore, G A

    1992-01-22

    The effects of ortho-phenylphenol (OPP) and its metabolites, phenyl-hydroquinol (PHQ) and phenyl-benzoquinone (PBQ), on isolated rat hepatocytes were investigated. Addition of OPP (0.5-1.0 mM) to cells caused a dose-dependent cell death accompanied by the depletion of intracellular levels of ATP, glutathione (GSH) and protein thiols. GSH loss correlated with the formation of oxidized GSH. In addition, PHQ and especially PBQ (both at 0.5 mM) resulted in acute cell death with rapid depletion of ATP, GSH and protein thiols, and further low doses of PBQ (10-50 microM) elicited serious impairment of mitochondrial functions related to oxidative phosphorylation and Ca fluxes in isolated liver mitochondria. These results indicate that mitochondria are a target for these compounds and that OPP is itself toxic to hepatocytes even when metabolism is inhibited. The loss of cellular GSH and protein thiols accompanied by the impairment of mitochondrial function may be the main mechanisms of cytotoxicity induced by OPP and its metabolites.

  17. 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.

  18. The mechanism of methyl mercury toxicity in isolated rat hepatocytes.

    Science.gov (United States)

    Ashour, H; Abdel-Rahman, M; Khodair, A

    1993-07-01

    Mercury is the major component of dental amalgam restorative material, which typically has 50% pure elemental mercury. It is also used in some skin creams, and in the manufacturing of plastic, drugs and fungicides. The present study was designed to investigate the toxicity of methyl mercury (MeHg+) on isolated rat hepatocytes using several toxicity parameters. The hepatocytes were isolated by a collagenase perfusion technique and were incubated with different concentrations of MeHg+ (0.1-100 ppm) for 2 h. Through the incubation period the viability was determined by Trypan blue exclusion. Reduced glutathione (GSH) content and its enzymes, glutathione peroxidase (GSH-PX) and glutathione reductase (GSH-RX) were measured. Leakage of enzymes such as aspartate transaminase (AST), and alanine transaminase (ALT) were determined. The cell viability was reduced significantly after 1 h incubation when 0.1 and 1 ppm MeHg+ were applied. The decrease in the cell viability was dose- and time-dependent. A depletion of GSH content was observed with 100 ppm MeHg+ after 30 min of incubation. A significant decrease in GSH-RX was observed with 100 ppm during 15 and 30 min of incubation, while 10 ppm of MeHg+ significantly increased ALT leakage after 60 min. However, there was a significant increase in AST leakage with 100 ppm only. The present investigation indicates that the toxic effect of MeHg+ is most likely cytosolic enzyme related.

  19. Differentiation of human embryonic stem cells along a hepatocyte lineage and its application in liver regeneration

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Hepatocyte transplantation and bioartificial liver(BAL)as alternatives to liver transplantation offer the possibility of effective treatment for many inherited and acquired hepatic disorders.Unfortunately,the limited availability of donated livers and the variability of their derived hepatocytes make it difficult to obtain enough viable human hepatocytes for the hepatocyte-based therapies.Embryonic stem cells (ESCs),which could be isolated directly from the blastocyst inner cell mass,have permanent self-renewal capability and developmental pluripotency and therefore might be an ideal cell source in the treatment of hepatic discords.However,differentiation of hESCS into hepatocytes with significant numbers remains a challenge.This review updates our current understanding of differentiation of ESCs into hepatic lineage cells,their future therapeutic uses and problems in liver regeneration.

  20. In vivo N-acetyl cysteine reduce hepatocyte death by induced acetaminophen

    Science.gov (United States)

    Lin, Chih-Ju; Li, Feng-Chieh; Wang, Sheng-Shun; Lee, Hsuan-Shu; Dong, Chen-Yuan

    2011-07-01

    Acetaminophen (APAP) is the famous drug in global, and taking overdose Acetaminophen will intake hepatic cell injure. Desptie substantial progress in our understanding of the mechanism of hepatocellular injury during the last 40 years, many aspects of the pathophysiology are still unknown or controversial.1 In this study, mice are injected APAP overdose to damage hepatocyte. APAP deplete glutathione and ATP of cell, N-Acetyl Cysteine (NAC) plays an important role to protect hepatocytes be injury. N-Acetyl Cysteine provides mitochondrial to produce glutathione to release drug effect hepatocyte. By 6-carboxyfluorescein diacetate (6-CFDA) metabolism in vivo, glutathione keep depleting to observe the hepatocyte morphology in time. Without NAC, cell necrosis increase to plasma membrane damage to release 6-CFDA, that's rupture. After 6-CFDA injection, fluorescence will be retained in hepatocyte. For cell retain with NAC and without NAC are almost the same. With NAC, the number of cell rupture decreases about 75%.

  1. Depot-dependent effects of adipose tissue explants on co-cultured hepatocytes

    DEFF Research Database (Denmark)

    Du, Zhen-Yu; Ma, Tao; Lock, Erik-Jan;

    2011-01-01

    We have developed an in vitro hepatocyte-adipose tissue explant (ATE) co-culture model enabling examination of the effect of visceral and subcutaneous adipose tissues on primary rat hepatocytes. Initial analyses of inflammatory marker genes were performed in fractionated epididymal or inguinal...... levels of IL-6, TNF-a and PGE(2) in the media from inguinal ATEs co-cultured with primary rat hepatocytes were higher than that in the media from epididymal ATEs co-cultured with hepatocytes, although the significant difference was only seen in PGE(2). Lipolysis, measured as glycerol release, was similar...... in the ATEs isolated from inguinal and epididymal adipose tissues when cultured alone, but the glycerol release was higher in the ATEs isolated from epididymal than from inguinal adipose tissue when co-cultured with hepatocytes. Compared to epididymal ATEs, the ATEs from inguinal adipose tissue elicited...

  2. Measurement of Blood Coagulation Factor Synthesis in Cultures of Human Hepatocytes.

    Science.gov (United States)

    Heinz, Stefan; Braspenning, Joris

    2015-01-01

    An important function of the liver is the synthesis and secretion of blood coagulation factors. Within the liver, hepatocytes are involved in the synthesis of most blood coagulation factors, such as fibrinogen, prothrombin, factor V, VII, IX, X, XI, XII, as well as protein C and S, and antithrombin, whereas liver sinusoidal endothelial cells produce factor VIII and von Willebrand factor. Here, we describe methods for the detection and quantification of most blood coagulation factors in hepatocytes in vitro. Hepatocyte cultures indeed provide a valuable tool to study blood coagulation factors. In addition, the generation and expansion of hepatocytes or hepatocyte-like cells may be used in future for cell-based therapies of liver diseases, including blood coagulation factor deficiencies.

  3. Lineage tracing reveals conversion of liver sinusoidal endothelial cells into hepatocytes.

    Science.gov (United States)

    Tan, Zhaoli; Chen, Keyan; Shao, Yong; Gao, Lihua; Wang, Yan; Xu, Jianming; Jin, Yang; Hu, Xianwen; Wang, Youliang

    2016-09-01

    Although liver sinusoidal endothelial cells (LSECs) have long been known to contribute to liver regeneration following injury, the exact role of these cells in liver regeneration remains poorly understood. In this work, we performed lineage tracing of LSECs in mice carrying Tie2-Cre or VE-cadherin-Cre constructs to facilitate fate-mapping of LSECs in liver regeneration. Some YFP-positive LSECs were observed to convert into hepatocytes following a two-thirds partial hepatectomy (PH). Furthermore, human umbilical vein endothelial cells (HUVECs) could be triggered to convert into cells that closely resembled hepatocytes when cultured with serum from mice that underwent an extended PH. These findings suggest that mature non-hepatocyte LSECs play an essential role in mammalian liver regeneration by converting to hepatocytes. The conversion of LSECs to hepatocyte-like (iHep) cells may provide a new approach to tissue engineering.

  4. Omeprazole and lansoprazole enantiomers induce CYP3A4 in human hepatocytes and cell lines via glucocorticoid receptor and pregnane X receptor axis.

    Science.gov (United States)

    Novotna, Aneta; Dvorak, Zdenek

    2014-01-01

    Benzimidazole drugs lansoprazole and omeprazole are used for treatment of various gastrointestinal pathologies. Both compounds cause drug-drug interactions because they activate aryl hydrocarbon receptor and induce CYP1A genes. In the current paper, we examined the effects of lansoprazole and omeprazole enantiomers on the expression of key drug-metabolizing enzyme CYP3A4 in human hepatocytes and human cancer cell lines. Lansoprazole enantiomers, but not omeprazole, were equipotent inducers of CYP3A4 mRNA in HepG2 cells. All forms (S-, R-, rac-) of lansoprazole and omeprazole induced CYP3A4 mRNA and protein in human hepatocytes. The quantitative profiles of CYP3A4 induction by individual forms of lansoprazole and omeprazole exerted enantiospecific patterns. Lansoprazole dose-dependently activated pregnane X receptor PXR in gene reporter assays, and slightly modulated rifampicin-inducible PXR activity, with similar potency for each enantiomer. Omeprazole dose-dependently activated PXR and inhibited rifampicin-inducible PXR activity. The effects of S-omeprazole were much stronger as compared to those of R-omeprazole. All forms of lansoprazole, but not omeprazole, slightly activated glucocorticoid receptor and augmented dexamethasone-induced GR transcriptional activity. Omeprazole and lansoprazole influenced basal and ligand inducible expression of tyrosine aminotransferase, a GR-target gene, in HepG2 cells and human hepatocytes. Overall, we demonstrate here that omeprazole and lansoprazole enantiomers induce CYP3A4 in HepG2 cells and human hepatocytes. The induction comprises differential interactions of omeprazole and lansoprazole with transcriptional regulators PXR and GR, and some of the effects were enantiospecific. The data presented here might be of toxicological and clinical importance, since the effects occurred in therapeutically relevant concentrations.

  5. Fipronil induces CYP isoforms and cytotoxicity in human hepatocytes.

    Science.gov (United States)

    Das, Parikshit C; Cao, Yan; Cherrington, Nathan; Hodgson, Ernest; Rose, Randy L

    2006-12-15

    Recent studies have demonstrated the potential of pesticides to either inhibit or induce xenobiotic metabolizing enzymes in humans. Exposure of human hepatocytes to doses of fipronil (5-amino-1-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4-[(trifluoromethyl) sulfinyl]-1H-pyrazole-3-carbonitrile) ranging from 0.1 to 25 microM resulted in a dose dependent increase in CYP1A1 mRNA expression (3.5 to approximately 55-fold) as measured by the branched DNA assay. In a similar manner, CYP3A4 mRNA expression was also induced (10-30-fold), although at the higher doses induction returned to near control levels. CYP2B6 and 3A5 were also induced by fipronil, although at lower levels (2-3-fold). Confirmation of bDNA results were sought through western blotting and/or enzyme activity assays. Western blots using CYP3A4 antibody demonstrated a dose responsive increase from 0.5 to 1 microM followed by decreasing responses at higher concentrations. Similar increases and decreases were observed in CYP3A4-specific activity levels as measured using 6beta-hydroxytestosterone formation following incubation with testosterone. Likewise, activity levels for a CYP1A1-specific substrate, luciferin CEE, demonstrated that CYP1A1 enzyme activities were maximally induced by 1 microM fipronil followed by dramatically declining activity measurements at 10 and 25 microM. Cytotoxic effects of fipronil and fipronil sulfone were examined using the adenylate kinase and the trypan blue exclusion assays in HepG2 cells and human hepatocytes. The results indicate both that HepG2 cells and primary human hepatocytes are sensitive to the cytotoxic effects of fipronil. The maximum induction of adenylate kinase was ca. 3-fold greater than the respective controls in HepG2 and 6-10-fold in the case of primary hepatocytes. A significant time- and dose-dependent induction of adenylate kinase activity in HepG2 cells was noted from 0.1 to 12.5 microM fipronil followed by decreasing activities at 25 and 50 microM. For

  6. Macrophage secretory products induce an inflammatory phenotype in hepatocytes

    Institute of Scientific and Technical Information of China (English)

    Michelle Melino; Gethin P Thomas; Andrew D Clouston; Julie R Jonsson; Elizabeth E Powell; Victoria L Gadd; Gene V Walker; Richard Skoien; Helen D Barrie; Dinesh Jothimani; Leigh Horsfall; Alun Jones; Matthew J Sweet

    2012-01-01

    AIM:To investigate the influence of macrophages on hepatocyte phenotype and function.METHODS:Macrophages were differentiated from THP-1 monocytes via phorbol myristate acetate stimulation and the effects of monocyte or macrophageconditioned medium on HepG2 mRNA and protein expression determined.The in vivo relevance of these findings was confirmed using liver biopsies from 147patients with hepatitis C virus (HCV) infection.RESULTS:Conditioned media from macrophages,but not monocytes,induced a transient morphological change in hepatocytes associated with upregulation of vimentin (7.8 ± 2.5-fold,P =0.045) and transforming growth factor (TGF)-β1 (2.6 ± 0.2-fold,P < 0.001) and downregulation of epithelial cadherin (1.7 ± 0.02-fold,P =0.017) mRNA expression.Microarray analysis revealed significant upregulation of lipocalin-2 (17-fold,P < 0.001) and pathways associated with inflammation,and substantial downregulation of pathways related to hepatocyte function.In patients with chronic HCV,realtime polymerase chain reaction and immunohistochemistry confirmed an increase in lipocalin-2 mRNA (F0 1.0± 0.3,F1 2.2 ± 0.2,F2 3.0 ± 9.3,F3/4 4.0 ± 0.8,P =0.003) and protein expression (F1 1.0 ± 0.5,F2 1.3 ±0.4,F3/4 3.6 ± 0.4,P =0.014) with increasing liver injury.High performance liquid chromatography-tandem mass spectrometry analysis identified elevated levels of matrix metalloproteinase (MMP)-9 in macrophageconditioned medium,and a chemical inhibitor of MMP-9attenuated the change in morphology and mRNA expression of TGF-β1 (2.9 ± 0.2 vs 1.04 ± 0.1,P < 0.001)in macrophage-conditioned media treated HepG2 cells.In patients with chronic HCV infection,hepatic mRNA expression of CD163 (F0 1.0 ± 0.2,F1/2 2.8 ± 0.3,F3/4 5.3 ± 1.0,P =0.001) and MMP-9 (F0 1.0 ± 0.4,F1/2 2.8 ± 0.3,F3/4 4.1 ± 0.8,P =0.011) was significantly associated with increasing stage of fibrosis.CONCLUSION:Secreted macrophage products alter the phenotype and function of hepatocytes

  7. Metabolic Consequences of TGFb Stimulation in CulturedPrimary Mouse Hepatocytes Screened from Transcript Data with ModeScore 

    Directory of Open Access Journals (Sweden)

    Andreas Hoppe

    2012-11-01

    Full Text Available TGFb signaling plays a major role in the reorganization of liver tissue upon injury and is an important driver of chronic liver disease. This is achieved by a deep impact on a cohort of cellular functions. To comprehensively assess the full range of affected metabolic functions, transcript changes of cultured mouse hepatocytes were analyzed with a novel method (ModeScore, which predicts the activity of metabolic functions by scoring transcript expression changes with 987 reference flux distributions, which yielded the following hypotheses. TGFb multiplies down-regulation of most metabolic functions occurring in culture stressed controls. This is especially pronounced for tyrosine degradation, urea synthesis, glucuronization capacity, and cholesterol synthesis. Ethanol degradation and creatine synthesis are down-regulated only in TGFb treated hepatocytes, but not in the control. Among the few TGFb dependently up-regulated functions, synthesis of various collagens is most pronounced. Further interesting findings include: down-regulation of glucose export is postponed by TGFb, TGFb up-regulates the synthesis capacity of ketone bodies only as an early response, TGFb suppresses the strong up-regulation of Vanin, and TGFb induces re-formation of ceramides and sphingomyelin.

  8. Influence of 48 hours of cold storage in University of Wisconsin organ preservation solution on metabolic capacity of rat hepatocytes

    NARCIS (Netherlands)

    Olinga, Peter; Slooff, M.JH; Meijer, D.K F; Groothuis, Geny; Merema, M.T.

    1997-01-01

    Background/Aims: Suspensions of isolated hepatocytes are a valuable tool to study liver functions, For optimal use of the isolated hepatocytes, methods are needed to preserve the hepatocytes while maintaining their viability, metabolic and transport functions, Until now little has been known about t

  9. Endotoxin-stimulated Rat Hepatic Stellate Cells Induce Autophagy in Hepatocytes as a Survival Mechanism.

    Science.gov (United States)

    Dangi, Anil; Huang, Chao; Tandon, Ashish; Stolz, Donna; Wu, Tong; Gandhi, Chandrashekhar R

    2016-01-01

    Bacterial lipopolysaccharide (LPS)-stimulated hepatic stellate cells (HSCs) produce many cytokines including IFNβ, TNFα, and IL6, strongly inhibit DNA synthesis, but induce apoptosis of a small number of hepatocytes. In vivo administration of LPS (up to 10 mg/mL) causes modest inflammation and weight loss in rats but not mortality. We determined whether LPS-stimulated HSCs instigate mechanisms of hepatocyte survival. Rats received 10 mg/kg LPS (i.p.) and determinations were made at 6 h. In vitro, HSCs were treated with 100 ng/mL LPS till 24 h. The medium was transferred to hepatocytes, and determinations were made at 0-12 h. Controls were HSC-conditioned medium or medium-containing LPS. LPS treatment of rats caused autophagy in hepatocytes, a physiological process for clearance of undesirable material including injured or damaged organelles. This was accompanied by activation of c-Jun NH2 terminal kinase (JNK) and apoptosis of ~4-5% of hepatocytes. In vitro, LPS-conditioned HSC medium (LPS/HSC) induced autophagy in hepatocytes but apoptosis of only ~10% of hepatocytes. While LPS/HSC stimulated activation of JNK (associated with cell death), it also activated NFkB and ERK1/2 (associated with cell survival). LPS-stimulated HSCs produced IFNβ, and LPS/HSC-induced autophagy in hepatocytes and their apoptosis were significantly inhibited by anti-IFNβ antibody. Blockade of autophagy, on the other hand, strongly augmented hepatocyte apoptosis. While LPS-stimulated HSCs cause apoptosis of a subpopulation of hepatocytes by producing IFNβ, they also induce cell survival mechanisms, which may be of critical importance in resistance to liver injury during endotoxemia.

  10. Body contact and body language

    DEFF Research Database (Denmark)

    Winther, Helle Dagmar

    2008-01-01

    and the boundaries between self and world. In western societies, the modern premises for contact are in some ways developing from close contact to virtual communication. With this breadth of perspective in mind, the ques­tion is whether conscious and experimental work with body contact and body language in move......­ment psychology and education provide potential for intense personal develop­ment as well as for social and cultural learning processes. This performative research project originates from the research project entitled, Movement Psy­chol­ogy: The Language of the Body and the Psy­chol­ogy of Movement based...... on the Dance Therapy Form Dansergia. The author, who is a practi­tioner-researcher, is methodologically inspir­ed by phenomenology, performative methods and a narrative and auto-ethnographic approach. The project will be presented in an organic, cre­at­ive and performative way. Through a moving dia...

  11. CdSe/ZnS quantum dots induce hepatocyte pyroptosis and liver inflammation via NLRP3 inflammasome activation.

    Science.gov (United States)

    Lu, Yonghui; Xu, Shangcheng; Chen, Haiyan; He, Mindi; Deng, Youcai; Cao, Zhengwang; Pi, Huifeng; Chen, Chunhai; Li, Min; Ma, Qinlong; Gao, Peng; Ji, Yan; Zhang, Lei; Yu, Zhengping; Zhou, Zhou

    2016-06-01

    Increased biomedical applications of quantum dots (QDs) have raised considerable concern regarding their toxicological impact. However, the toxicity of QDs is largely unknown and the underlying mechanism is still undefined. This study was conducted to examine the hepatotoxicity of CdSe/ZnS core/shell QDs and the underlying mechanism. In hepatic L02 cells, the QDs caused cytotoxicity in a dose-dependent manner. The QDs were then shown to activate the NLR pyrin domain containing 3 (NLRP3) inflammasome in hepatocytes, leading to a novel pro-inflammatory form of cell death named pyroptosis. Further experiments demonstrated that the QDs induced mitochondrial reactive oxygen species (mtROS) production, and that both a mtROS and a total ROS scavenger attenuated QDs-induced NLRP3 activation and pyroptosis. In addition, QDs increased cytoplasmic calcium (Ca(2+)) levels, while a Ca(2+) release antagonist and chelator alleviated QDs-induced mtROS, NLRP3 activation and subsequent pyroptosis in hepatocytes. In vivo, QDs administration induced liver inflammation and dysfunction. Moreover, the QDs also resulted in NLRP3 activation in liver tissue. However, QDs-induced liver inflammation and dysfunction were abolished in NLRP3 knockout mice. Also, an elevation in mtROS was observed in liver after QDs administration, and the mtROS scavenger suppressed liver NLRP3 activation, inflammation and dysfunction induced by QDs. Our data suggest that QDs induced hepatocyte pyroptosis, liver inflammation and dysfunction via NLRP3 activation, which was caused by QDs-triggered mtROS production and Ca(2+) mobilization. Our results provide novel insights into QDs-induced hepatotoxicity and the underlying mechanism, facilitating control of the side effects of QDs.

  12. The neuronal nitric oxide synthase inhibitor NANT blocks acetaminophen toxicity and protein nitration in freshly isolated hepatocytes.

    Science.gov (United States)

    Banerjee, Sudip; Melnyk, Stepan B; Krager, Kimberly J; Aykin-Burns, Nukhet; Letzig, Lynda G; James, Laura P; Hinson, Jack A

    2015-12-01

    3-Nitrotyrosine (3NT) in liver proteins of mice treated with hepatotoxic doses of acetaminophen (APAP) has been postulated to be causative in toxicity. Nitration is by a reactive nitrogen species formed from nitric oxide (NO). The source of the NO is unclear. iNOS knockout mice were previously found to be equally susceptible to APAP toxicity as wildtype mice and iNOS inhibitors did not decrease toxicity in mice or in hepatocytes. In this work we examined the potential role of nNOS in APAP toxicity in hepatocytes using the specific nNOS inhibitor NANT (10 µM)(N-[(4S)-4-amino-5-[(2-aminoethyl)amino]pentyl]-N'-nitroguanidinetris (trifluoroacetate)). Primary hepatocytes (1 million/ml) from male B6C3F1 mice were incubated with APAP (1mM). Cells were removed and assayed spectrofluorometrically for reactive nitrogen and oxygen species using diaminofluorescein (DAF) and Mitosox red, respectively. Cytotoxicity was determined by LDH release into media. Glutathione (GSH, GSSG), 3NT, GSNO, acetaminophen-cysteine adducts, NAD, and NADH were measured by HPLC. APAP significantly increased cytotoxicity at 1.5-3.0 h. The increase was blocked by NANT. NANT did not alter APAP mediated GSH depletion or acetaminophen-cysteine adducts in proteins which indicated that NANT did not inhibit metabolism. APAP significantly increased spectroflurometric evidence of reactive nitrogen and oxygen formation at 0.5 and 1.0 h, respectively, and increased 3NT and GSNO at 1.5-3.0 h. These increases were blocked by NANT. APAP dramatically increased NADH from 0.5-3.0 h and this increase was blocked by NANT. Also, APAP decreased the Oxygen Consumption Rate (OCR), decreased ATP production, and caused a loss of mitochondrial membrane potential, which were all blocked by NANT.

  13. Reinforced Airfoil Shaped Body

    DEFF Research Database (Denmark)

    2011-01-01

    The present invention relates to an airfoil shaped body with a leading edge and a trailing edge extending along the longitudinal extension of the body and defining a profile chord, the airfoil shaped body comprising an airfoil shaped facing that forms the outer surface of the airfoil shaped body...... and surrounds an internal volume of the body, a distance member that is connected to the facing inside the body and extends from the facing and into the internal volume of the body, and at least one reinforcing member that operates in tension for reinforcing the facing against inward deflections...... and that is connected to the facing inside the internal volume of the body at the same side of the profile chord as the connection of the distance member to the facing and to the distance member at a distance from the facing....

  14. Differences in lysine adduction by acrolein and methyl vinyl ketone: implications for cytotoxicity in cultured hepatocytes.

    Science.gov (United States)

    Kaminskas, Lisa M; Pyke, Simon M; Burcham, Philip C

    2005-11-01

    Acrolein is a highly toxic environmental pollutant that readily alkylates the epsilon-amino group of lysine residues in proteins. In model systems, such chemistry involves sequential addition of two acrolein molecules to a given nitrogen, forming bis-Michael-adducted species that undergo aldol condensation and dehydration to form Nepsilon-(3-formyl-3,4-dehydropiperidino)lysine. Whether this ability to form cyclic adducts participates in the toxicity of acrolein is unknown. To address this issue, we compared the chemistry of protein adduction by acrolein to that of its close structural analogue methyl vinyl ketone, expecting that the alpha-methyl group would hinder the intramolecular cyclization of any bis-adducted species formed by methyl vinyl ketone. Both acrolein and methyl vinyl ketone displayed comparable protein carbonylating activity during in vitro studies with the model protein bovine serum albumin, confirming the alpha,beta,-unsaturated bond of both compounds is an efficient Michael acceptor for protein nucleophiles. However, differences in adduction chemistry became apparent during the use of electrospray ionization-MS to monitor reaction products in a lysine-containing peptide after modification by each compound. For example, although a Schiff base adduct was detected following reaction of the peptide with acrolein, an analogous species was not formed by methyl vinyl ketone. Furthermore, while ions corresponding to mono- and bis-Michael adducts were detected at the N-terminus and lysine residues following peptide modification by both carbonyls, only acrolein modification generated ions attributable to cyclic adducts. Despite these differences in adduction chemistry, in mouse hepatocytes, the two compounds exhibited very comparable abilities to induce rapid, concentration-dependent cell death as well as protein carbonylation. These findings suggest that the acute toxicity of short-chain alpha,beta-unsaturated carbonyl compounds involves their ability to

  15. Intracellular maturation of apolipoprotein[a] and assembly of lipoprotein[a] in primary baboon hepatocytes.

    Science.gov (United States)

    White, A L; Rainwater, D L; Lanford, R E

    1993-03-01

    The glycoprotein apolipoprotein[a] (apo[a]) is present in plasma at highly variable concentrations and appears as a number of genetically determined size isoforms (400-800 kDa), disulfide linked to apoB-100 in low density lipoprotein to produce lipoprotein [a](Lp[a]). Apo[a] is synthesized by the liver, but the site of association of apo[a] and apoB and factors that regulate its production are unknown. To examine the morphogenesis of the Lp[a] particle, baboon hepatocytes expressing a single, low molecular weight isoform of apo[a] were labeled with [35S]cysteine and methionine, and apo[a] was analyzed by immunoprecipitation and SDS-PAGE. Steady-state labeling revealed two molecular weight forms of apo[a] inside the cell. Only the large form was recovered from the culture medium. Pulse-chase studies and endoglycosidase treatment revealed that the lower molecular weight form of apo[a] represented a precursor with a prolonged residence time in the endoplasmic reticulum or an early Golgi compartment, after which it was processed to the mature form. A proportion of the mature form of apo[a] was rapidly secreted after synthesis, whereas the remainder had a prolonged residence time in a late Golgi compartment. In all experiments, apoB co-precipitated with apo[a] from the culture medium, but not from cell lysates. Density gradient ultracentrifugation and immunoblot analysis revealed that the majority of apo[a] was secreted into the medium in a free form, suggesting that the association between apo[a] and apoB occurred after secretion. Regulation of the movement of apo[a] between intracellular compartments may be one mechanism by which the plasma levels of Lp[a] are influenced.

  16. Beyond packing of hard spheres: The effects of core softness, non-additivity, intermediate-range repulsion, and many-body interactions on the glass-forming ability of bulk metallic glasses

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Kai; Fan, Meng; Liu, Yanhui; Schroers, Jan [Department of Mechanical Engineering and Materials Science, Yale University, New Haven, Connecticut 06520 (United States); Center for Research on Interface Structures and Phenomena, Yale University, New Haven, Connecticut 06520 (United States); Shattuck, Mark D. [Department of Mechanical Engineering and Materials Science, Yale University, New Haven, Connecticut 06520 (United States); Department of Physics and Benjamin Levich Institute, The City College of the City University of New York, New York, New York 10031 (United States); O’Hern, Corey S. [Department of Mechanical Engineering and Materials Science, Yale University, New Haven, Connecticut 06520 (United States); Center for Research on Interface Structures and Phenomena, Yale University, New Haven, Connecticut 06520 (United States); Department of Physics, Yale University, New Haven, Connecticut 06520 (United States); Department of Applied Physics, Yale University, New Haven, Connecticut 06520 (United States)

    2015-11-14

    When a liquid is cooled well below its melting temperature at a rate that exceeds the critical cooling rate R{sub c}, the crystalline state is bypassed and a metastable, amorphous glassy state forms instead. R{sub c} (or the corresponding critical casting thickness d{sub c}) characterizes the glass-forming ability (GFA) of each material. While silica is an excellent glass-former with small R{sub c} < 10{sup −2} K/s, pure metals and most alloys are typically poor glass-formers with large R{sub c} > 10{sup 10} K/s. Only in the past thirty years have bulk metallic glasses (BMGs) been identified with R{sub c} approaching that for silica. Recent simulations have shown that simple, hard-sphere models are able to identify the atomic size ratio and number fraction regime where BMGs exist with critical cooling rates more than 13 orders of magnitude smaller than those for pure metals. However, there are a number of other features of interatomic potentials beyond hard-core interactions. How do these other features affect the glass-forming ability of BMGs? In this manuscript, we perform molecular dynamics simulations to determine how variations in the softness and non-additivity of the repulsive core and form of the interatomic pair potential at intermediate distances affect the GFA of binary alloys. These variations in the interatomic pair potential allow us to introduce geometric frustration and change the crystal phases that compete with glass formation. We also investigate the effect of tuning the strength of the many-body interactions from zero to the full embedded atom model on the GFA for pure metals. We then employ the full embedded atom model for binary BMGs and show that hard-core interactions play the dominant role in setting the GFA of alloys, while other features of the interatomic potential only change the GFA by one to two orders of magnitude. Despite their perturbative effect, understanding the detailed form of the intermetallic potential is important for

  17. ‘ SILENT’ LARYNGEAL FOREIGN BODY

    Directory of Open Access Journals (Sweden)

    Chandrasekhar

    2015-06-01

    Full Text Available Laryngeal foreign bodies in adults are rare. The foreign bodies accidentally entering the larynx are symptomatic in the form of choking , stridor or even death. We are presenting a rare case of foreign body in the larynx in a 42 year old male who was symptom free except for dysphonia. The foreign body was removed successfully under local anesthesia.

  18. CYP2E1-dependent hepatotoxicity and oxidative damage after ethanol administration in human primary hepatocytes

    Institute of Scientific and Technical Information of China (English)

    Lie-Gang Liu; Hong Yan; Ping Yao; Wen Zhang; Li-Jun Zou; Fang-Fang Song; Ke Li; Xiu-Fa Sun

    2005-01-01

    AIM: To observe the relationship between ethanol-induced oxidative damage in human primary cultured hepatocytes and cytochrome P450 2E1 (CYP2E1) activity, in order to address if inhibition of CYP2E1 could attenuate ethanol-induced cellular damage.METHODS: The dose-dependent (25-100 mmol/L) and time-dependent (0-24 h) exposures of primary human cultured hepatocytes to ethanol were carried out. CYP2E1 activity and protein expression were detected by spectrophotometer and Western blot analysis respectively.Hepatotoxicity was investigated by determination of lactate dehydrogenase (LDH) and aspartate transaminase (AST) level in hepatocyte culture supernatants, as well as the intracellular formation of malondialdehyde (MDA).RESULTS: A dose-and time-dependent response between ethanol exposure and CYP2E1 activity in human hepatocytes was demonstrated. Moreover, there was a time-dependent increase of CYP2E1 protein after 100 mmol/L ethanol exposure. Meanwhile, ethanol exposure of hepatocytes caused a time-dependent increase of ceilular MDA level, LDH, and AST activities in supernatants.Furthermore, the inhibitor of CYP2E1, diallyl sulfide (DAS) could partly attenuate the increases of MDA, LDH, and AST in human hepatocytes.CONCLUSION: A positive relationship between ethanol-induced oxidative aamage in human primary cultured hepatocytes and CYP2E1 activity was exhibited, and the inhibition of CYP2E1 could partly attenuate ethanol-induced oxidative damage.

  19. Nrf2 is involved in maintaining hepatocyte identity during liver regeneration.

    Directory of Open Access Journals (Sweden)

    Yuhong Zou

    Full Text Available Nrf2, a central regulator of the cellular defense against oxidative stress and inflammation, participates in modulating hepatocyte proliferation during liver regeneration. It is not clear, however, whether Nrf2 regulates hepatocyte growth, an important cellular mechanism to regain the lost liver mass after partial hepatectomy (PH. To determine this, various analyses were performed in wild-type and Nrf2-null mice following PH. We found that, at 60 h post-PH, the vast majority of hepatocytes lacking Nrf2 reduced their sizes, activated hepatic progenitor markers (CD133, TWEAK receptor, and trefoil factor family 3, depleted HNF4α protein, and downregulated the expression of a group of genes critical for their functions. Thus, the identity of hepatocytes deficient in Nrf2 was transiently but massively impaired in response to liver mass loss. This event was associated with the coupling of protein depletion of hepatic HNF4α, a master regulator of hepatocyte differentiation, and concomitant inactivation of hepatic Akt1 and p70S6K, critical hepatocyte growth signaling molecules. We conclude that Nrf2 participates in maintaining newly regenerated hepatocytes in a fully differentiated state by ensuring proper regulation of HNF4α, Akt1, and p70S6K during liver regeneration.

  20. Improved isolation of murine hepatocytes for in vitro malaria liver stage studies

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    Penha-Gonçalves Carlos

    2007-12-01

    Full Text Available Abstract Background Primary hepatocyte cultures are a valuable tool for the understanding of cellular and molecular phenomena occurring during malaria liver stage. This paper describes an improved perfusion/dissociation procedure to isolate hepatocytes from mouse liver that is suitable for malaria studies and allows reproducible preparation of primary hepatocytes with consistent cell yields and controlled purity. Results This protocol is a detailed description of a technique to isolate and culture mouse hepatocytes and represents an improvement over previous descriptions of hepatocyte isolation for malaria studies, regarding three technical aspects: (1 dissociation reagents choice; (2 cell separation gradient and (3 cell purity control. Cell dissociation was optimized for a specific collagenase digestion media. The cell dissociation step was improved by using a three-layer discontinuous gradient. A cell purity check was introduced to monitor the expression of CD95 on hepatocytes using flow cytometry methods. Conclusion The procedure described allows reproducible recovery of one to three million hepatocytes per preparation with cell purity of about 90% as determined by FACS analysis. Completion of the protocol is usually achieved in about four hours per preparation and pooling is suggested for multiple preparations of larger number of cells.

  1. Mycelial culture of Phellinus linteus protects primary cultured rat hepatocytes against hepatotoxins.

    Science.gov (United States)

    Kim, S H; Lee, H S; Lee, S; Cho, J; Ze, K; Sung, J; Kim, Y C

    2004-12-01

    Hepatoprotective activity of Phellinus linteus was studied using H(2)O(2)- or galactosamine-injured primary cultures of rat hepatocytes as screening systems. The methanolic extract of the mycelial culture of Phellinus linteus significantly protected against hepatotoxins-induced toxicity in primary cultured rat hepatocytes as seen from the decreased level of glutamic pyruvic transaminase released from the injured hepatocytes. The methanolic extract of the mycelial culture of Phellinus linteus was subsequently fractionated with n-hexane, ethyl acetate, n-butanol and water. Among these fractions, 100 microg/mL of the ethyl acetate fraction was the most active one. The relative protections were 68.9 +/- 5.3% in H(2)O(2)-injured hepatocytes and 46.8 +/- 3.9% in galactosamine-injured hepatocytes, respectively. The ethyl acetate fraction appeared to maintain the glutathione level which was decreased by the treatment of H(2)O(2) or galactosamine and restored the level of RNA synthesis more than two times compared to galactosamine-injured hepatocytes. These results suggest that the ethyl acetate fraction of the mycelial culture of Phellinus linteus protects hepatocytes from H(2)O(2)- or galactosamine-induced injury by maintaining hepatic glutathione level and RNA synthesis as well.

  2. Sodium-independent, bicuculline-sensitive (/sup 3/H)GABA binding to isolated rat hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Minuk, G.Y.; Bear, C.E.; Sarjeant, E.J.

    1987-05-01

    To determine whether hepatocytes possess specific receptor sites for gamma-aminobutyric acid (GABA), a potent amino acid neurotransmitter, (/sup 3/H)GABA, was added to sodium-free suspensions of Percoll-purified hepatocytes derived from collagenase-perfused rat livers under various experimental conditions and in the presence or absence of specific GABA receptor agonists (muscimol) and antagonists (bicuculline). The effects of GABA, muscimol, and bicuculline on hepatocyte resting membrane potentials were also determined. Specific binding of (/sup 3/H)GABA to hepatocytes was a consistent finding. GABA-hepatocyte interactions were reversible and temperature dependent. Muscimol and bicuculline inhibited binding in a dose-dependent manner (IC50, 30 nM and 50 microM, respectively), whereas strychnine (1.0-100 microM), a nonspecific central nervous system stimulant, had no appreciable effect. Both GABA and muscimol (100 microM) caused significant hyperpolarization of hepatocyte resting membrane potential (delta PD 5.4 +/- 3.1 and 22.2 +/- 16.2 mV, respectively, means +/- SD, P less than 0.0005). Bicuculline (100 microM) inhibited the effect of muscimol (P less than 0.05). The results of this study suggest that specific GABA receptor sites exist on the surface of isolated rat hepatocytes. The presence of such sites raises the possibility that, in addition to adrenergic and cholinergic innervation, hepatic function may be influenced by GABA-ergic neurotransmitter mechanisms.

  3. Auxiliary liver organ formation by implantation of spleen-encapsulated hepatocytes.

    Science.gov (United States)

    Nishio, Reiji; Nakayama, Miyuki; Ikekita, Masahiko; Watanabe, Yoshifumi

    2006-09-01

    Hepatocyte transplantation is an attractive alternative to orthotopic liver transplantation. However, its application has been limited because of its short-term success only. Here we report a new approach to hepatocyte transplantation resulting in the generation of an auxiliary liver in vivo. Isolated primary hepatocytes were encapsulated in isolated spleens and then transplanted by attaching the spleens to the livers of recipient animals (mice or rats) using biodegradable adhesive. A vascular network was rapidly established, and protein molecules circulated freely between the transplanted spleen and the liver, to which they adhered. In contrast, the spleen, which did not adhere to the liver or adhered elsewhere (adipose tissue or peritoneum), did not become vascularized but shrank and died. Encapsulation of hepatocytes in an isolated spleen enhanced their survival significantly, and co-encapsulation of Engelbreth- Holm-Swarm gel together with the hepatocytes further enhanced it. The encapsulated hepatocytes expressed liver-specific differentiation genes for more than 3 weeks. Plasma albumin concentrations in Nagase analbuminemic rats began to increase 3 days after transplantation. The transplanted hepatic cells migrated into the liver parenchyma, whereas the spleen was absorbed. Thus, we have developed a novel, simple approach for the rapid and efficient formation of functional auxiliary liver using a modified hepatocyte transplantation method.

  4. 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.

  5. Restoration of cell polarity and bile excretion function of hepatocytes in sandwich-culture

    Institute of Scientific and Technical Information of China (English)

    ZHANG Xian-jie; WANG Ying; SUN Jia-bang; SONG Mao-min; QIAO Xin

    2007-01-01

    Objective:To investigate the nature of the restoration of cell polarity and bile excretion function in Sandwich-cultured hepatocytes.Methods:Freshly isolated hepatocytes from male Sprague-Dawley rats were cultured in a double layer collagen gel Sandwich configuration.Morphological changes were observed under a inverted microscope.The domain specific membrane associated protein DPP Ⅳ was tested by immunofluorescenee,and the bile excretion function was determined by using fluorescein diacetate.Hepatocytes cultured on a single layer of collagen gel were taken as control.Results:Adult rat hepatocytes cultured in a double layer collagen gel sandwich configuration regained its morphological and functional polarity and maintained polygonal morphology for at least 4 weeks.Immunofluorescence studies USing antibodies against DPP Ⅳ showed polarity restoration as early as 48 h.After cultured in the double layer collagen gel Sandwich configuration for 96 h the hepatocytes began to excrete bile;while hepatocytes cultured on a single layer collagen gel had no bile excretion.Conclusion:Hepatocytes cultured in a double layer collagen gel Sandwich configuration are able to regain their morphological and functional polarity givan certain conditions.Hepaotcyte culture is a useful tool for the study of polarity restoration.

  6. Inclusion bodies and hepatopathies in psittacines.

    Science.gov (United States)

    Pass, D A

    1987-01-01

    Intranuclear inclusion bodies in hepatic cells of seven Peach-faced Lovebirds (Agapornis roseicollis), two Black-masked Love birds (A. personata) and two galahs (Cacatua roseicapilla) were examined electron microscopically. Thin sections were prepared from formalin-fixed Epon-embedded tissue or from haematoxylin and eosin stained sections that were subsequently embedded in Epon. Discrete and diffuse eosinophilic inclusions in hepatocytes of eight birds were composed of filaments and virus particles were not demonstrated within them. Virus-like particles of at least three types were found in diffuse, lightly basophilic inclusions of similar appearance in hepatocytes and Kupffer cells or endothelial cells in four birds. Particles, 66 nm in diameter which were probably adenovirus were demonstrated in hepatocytes in one A. roseicollis. Particles 26 nm to 39 nm in diameter which resembled papovaviruses were seen in one A. personata and one C. roseicapilla. Particles of 40 nm to 50 nm in diameter were seen in one A. personata but because of the method of preparation it was not possible to state whether they were adenovirus or papovavirus.

  7. Hepatocyte growth factor reduces CXCL10 expression in keratinocytes.

    Science.gov (United States)

    Hisadome, Mitsuhiro; Ohnishi, Tomokazu; Kakimoto, Kyoko; Kusuyama, Joji; Bandow, Kenjiro; Kanekura, Takuro; Matsuguchi, Tetsuya

    2016-10-01

    Keratinocytes secrete vascular endothelial growth factor (VEGF) and angioregulatory chemokines during cutaneous wound healing. Hepatocyte growth factor (HGF) promotes skin re-epithelialization by increasing VEGF expression in keratinocytes. Here, we investigated the regulatory roles of HGF in the expression of genes encoding angiogenic and angiostatic chemokines in keratinocytes and found that HGF specifically inhibits mRNA expression of the angiostatic chemokine CXCL10 in both mouse primary keratinocytes and in the human keratinocyte cell line HaCaT through the MEK/ERK cascade. Furthermore, HGF inhibited tumor necrosis factor-α-induced CXCL10 expression at both mRNA and protein levels in HaCaT cells. Thus, HGF may orchestrate angiogenesis in wounded skin by modulating both VEGF and CXCL10 expression in keratinocytes. © 2016 Federation of European Biochemical Societies.

  8. Hepatocyte growth factor in lung repair and pulmonary fibrosis

    Institute of Scientific and Technical Information of China (English)

    Ronald Allan M PANGANIBAN; Regina M DAY

    2011-01-01

    Pulmonary remodeling is characterized by the permanent and progressive loss of the normal alveolar architecture, especially the loss of alveolar epithelial and endothelial cells, persistent proliferation of activated fibroblasts, or myoflbroblasts, and alteration of extracellular matrix. Hepatocyte growth factor (HGF) is a pleiotropic factor, which induces cellular motility, survival, proliferation, and morphogenesis, depending upon the cell type. In the adult, HGF has been demonstrated to play a critical role in tissue repair, including in the lung. Administration of HGF protein or ectopic expression of HGF has been demonstrated in animal models of pulmonary fibrosis to induce normal tissue repair and to prevent fibrotic remodeling. HGF-induced inhibition of fibrotic remodeling may occur via multiple direct and indirect mechanisms including the induction of cell survival and proliferation of pulmonary epithelial and endothelial cells, and the reduction of myofibroblast accumulation.

  9. 3D hepatic cultures simultaneously maintain primary hepatocyte and liver sinusoidal endothelial cell phenotypes.

    Directory of Open Access Journals (Sweden)

    Yeonhee Kim

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

  10. Development of mouse hepatocyte lines permissive for hepatitis C virus (HCV.

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    Hussein Hassan Aly

    Full Text Available The lack of a suitable small animal model for the analysis of hepatitis C virus (HCV infection has hampered elucidation of the HCV life cycle and the development of both protective and therapeutic strategies against HCV infection. Human and mouse harbor a comparable system for antiviral type I interferon (IFN induction and amplification, which regulates viral infection and replication. Using hepatocytes from knockout (ko mice, we determined the critical step of the IFN-inducing/amplification pathways regulating HCV replication in mouse. The results infer that interferon-beta promoter stimulator (IPS-1 or interferon A receptor (IFNAR were a crucial barrier to HCV replication in mouse hepatocytes. Although both IFNARko and IPS-1ko hepatocytes showed a reduced induction of type I interferons in response to viral infection, only IPS-1-/- cells circumvented cell death from HCV cytopathic effect and significantly improved J6JFH1 replication, suggesting IPS-1 to be a key player regulating HCV replication in mouse hepatocytes. We then established mouse hepatocyte lines lacking IPS-1 or IFNAR through immortalization with SV40T antigen. Expression of human (hCD81 on these hepatocyte lines rendered both lines HCVcc-permissive. We also found that the chimeric J6JFH1 construct, having the structure region from J6 isolate enhanced HCV replication in mouse hepatocytes rather than the full length original JFH1 construct, a new finding that suggests the possible role of the HCV structural region in HCV replication. This is the first report on the entry and replication of HCV infectious particles in mouse hepatocytes. These mouse hepatocyte lines will facilitate establishing a mouse HCV infection model with multifarious applications.

  11. Proliferation of L02 human hepatocytes in tolerized geneticall yimmunocompetent rats

    Institute of Scientific and Technical Information of China (English)

    Hu Lin; Qing Mao; Yu-Ming Wang; Li Jiang

    2008-01-01

    AIM: To investigate whether human hepatocytes could proliferate after transplantation to normal immunocompetent rats treated with 2-acetaminofluorene or Retrorsine and partial hepatectomy.METHODS: L02 hepatocyte-tolerant Sprague-Dawley rats were injected with Retrorsine, 2-acetaminofluorene or normal saline. L02 hepatocytes were then transplanted via the spleen. Human albumin and its mRNA, specific proliferating cell nuclear antigen (PCNA), L02 hepatocyte dynamic distribution, number density and area density of PCNA-positive cells in the liver were determined.RESULTS: All the examined indicators were not significantly different between the rats treated with 2-acetaminofluorene and normal saline, which was not the case with rats treated with Retrorsine. A dynamic distribution of L02 hepatocytes in the rat liver was detected from wk 1 to mo 6 after transplantation in the Retrorsine group and from wk 1 to 10 in the 2-acetaminofluorene group. Human albumin and its mRNA were detected from wk 2 to mo 6 in the Retrorsine group and from wk 1 to 8 in the 2-acetaminofiuorene group.Specific human PCNA was detected in the rat liver from wk 2 to mo 6 in the Retrorsine group and from wk 2 to 6 in the 2-acetaminofluorene group. Human albumin and its mRNA contents as well as the number of PCNA positive cells reached a peak at wk 4.CONCLUSION: L02 human hepatocytes could not proliferate significiantly after transplantation to the normal,immunocompetent rats treated with 2-acetaminofluorene.L02 human hepatocytes can survive for 10 wk after transplantation and express human albumin for 8 wk. L02human hepatocytes can proliferate and express human albumin for 6 mo after transplantation to the rats treated with Retrorsine. The chimeric L02 human hepatocytes,which then underwent transplantation into tolerant rats,were normal in morphogenesis, biochemistry and function.

  12. Response of porcine hepatocytes in primary culture to plasma from severe viral hepatitis patients

    Institute of Scientific and Technical Information of China (English)

    Yong-Bo Cheng; Ying-Jie Wang; Shi-Chang Zhang; Jun Liu; Zhi Chen; Jia-Jia Li

    2005-01-01

    AIM: To observe the effects of plasma from patients with severe viral hepatitis (SVHP) on the growth and metabolism of porcine hepatocytes and the clinical efficiency of bioartificial liver device.METHODS: Hepatocytes were isolated from male porcines by collagenase perfusion. The synthesis of DNA and total protein, leakages of AST and LDH, changes in glutathione (GSH), catalase and morphology of porcine hepatocytes exposed to SVHP were investigated to indicate the effect of plasma from patients with severe hepatitis on the growth, injury, detoxification, and morphology of porcine hepatocytes.RESULTS: The synthesis of DNA and protein was inhibited in the medium containing 100% SVHP compared to the controls. The leakages of LDH and AST increased in porcine hepatocytes following exposure to 100% SVHP for 5 h. The difference between 100% SVHP and 10% newborn calf serum (NCS) was significant in t-test (LDH: t = 24.552, P = 0.001; AST: t = 4.169, P =0.014). After exposure to SVHP for 24 h, alterations in GSH status were significant (F = 2.746, P<0.05) between porcine hepatocytes in 100% SVHP and 10% NCS, but no alteration occurred in the culture medium after 48 h (F = 4.378, P<0.05). A similar profile was observed in catalase activity. Many round vacuoles were observed in porcine hepatocytes cultured in SVHP. The membranes of these cells became indistinct and almost all the cells died on d 5.CONCLUSION: Plasma from patients with severe hepatitis inhibits the growth, injures membrane, disturbs GSH homeostasis and induces morphological changes of porcine hepatocytes. It is suggested that SVHP should be pretreated to reduce the toxin load and improve the performance of porcine hepatocytes in extracorporeal liver-support devices.

  13. 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

  14. Interaction of propionate and carnitine metabolism in isolated rat hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Brass, E.P.; Beyerinck, R.A.

    1987-05-01

    Propionate (P) and its metabolic products P-CoA and methylmalonyl-CoA can disrupt normal hepatic metabolism. Carnitine (Cn) has been shown to partially restore cellular function in the presence of P. This effect of Cn may result from removal of propionyl groups as propionylcarnitine (P-Cn). The present study examined the kinetics of P-Cn formation in rat hepatocytes, and the consequence of P-Cn formation on P and Cn metabolism. /sup 14/C-P was converted to CO/sub 2/, glucose and P-Cn in the hepatocyte system. Increasing concentrations of Cn up to 10.0 mM increased P-Cn formation from P without affecting CO/sub 2/ or glucose formation. Thus, 10.0 mM Cn increased total P metabolism by 40%. Metabolism of P was associated with a decrease in Cn concentration and an increase in short chain acylcarnitines (SCCn). In the absence of added Cn, 60 min incubation with P decreased Cn from 6.8 to 2.5 ..mu..M with a corresponding increase in SCCn. This effect of P to deplete free Cn was not seen to the same degree with butyrate in place of P. Similar increases in the formation of SCCn in the presence of P at the expense of free Cn were seen when the incubation Cn concentration was increased to 50 ..mu..M or 150 ..mu..M. HPLC methodologies to study specific acylcarnitines demonstrated the accumulation of large amounts of P-Cn in the incubations containing P, accounting for the depletion of free Cn.

  15. Evidence against a stem cell origin of new hepatocytes in a common mouse model of chronic liver injury.

    Science.gov (United States)

    Schaub, Johanna R; Malato, Yann; Gormond, Coralie; Willenbring, Holger

    2014-08-21

    Hepatocytes provide most liver functions, but they can also proliferate and regenerate the liver after injury. However, under some liver injury conditions, particularly chronic liver injury where hepatocyte proliferation is impaired, liver stem cells (LSCs) are thought to replenish lost hepatocytes. Conflicting results have been reported about the identity of LSCs and their contribution to liver regeneration. To address this uncertainty, we followed candidate LSC populations by genetic fate tracing in adult mice with chronic liver injury due to a choline-deficient, ethionine-supplemented diet. In contrast to previous studies, we failed to detect hepatocytes derived from biliary epithelial cells or mesenchymal liver cells beyond a negligible frequency. In fact, we failed to detect hepatocytes that were not derived from pre-existing hepatocytes. In conclusion, our findings argue against LSCs, or other nonhepatocyte cell types, providing a backup system for hepatocyte regeneration in this common mouse model of chronic liver injury.

  16. Evidence against a Stem Cell Origin of New Hepatocytes in a Common Mouse Model of Chronic Liver Injury

    Directory of Open Access Journals (Sweden)

    Johanna R. Schaub

    2014-08-01

    Full Text Available Hepatocytes provide most liver functions, but they can also proliferate and regenerate the liver after injury. However, under some liver injury conditions, particularly chronic liver injury where hepatocyte proliferation is impaired, liver stem cells (LSCs are thought to replenish lost hepatocytes. Conflicting results have been reported about the identity of LSCs and their contribution to liver regeneration. To address this uncertainty, we followed candidate LSC populations by genetic fate tracing in adult mice with chronic liver injury due to a choline-deficient, ethionine-supplemented diet. In contrast to previous studies, we failed to detect hepatocytes derived from biliary epithelial cells or mesenchymal liver cells beyond a negligible frequency. In fact, we failed to detect hepatocytes that were not derived from pre-existing hepatocytes. In conclusion, our findings argue against LSCs, or other nonhepatocyte cell types, providing a backup system for hepatocyte regeneration in this common mouse model of chronic liver injury.

  17. Signifying Bodies

    DEFF Research Database (Denmark)

     In our everyday lives we strive to stay healthy and happy, while we live as our selves, engage with each other, and discover an infinite world of possibilities. Health arises and diminishes as human beings draw on a vibrant ecology of actions, interactions and coactions. Intricate processes...... and health care. Eschewing all forms of dualism, the authors emphasise the interdependency of how we act, think, feel and function. They advocate a relational turn in health care, in which bodies live and learn from suffering and care. In this view, health is inseparable from both living beings...... of, for example, how rheumatoid arthritis sufferers view their treatment, how decisions are made in simulated emergencies, and how therapists and homeopaths use distributed language and cognition with their clients....

  18. In vitro drug metabolism of green tea catechins in human, monkey, dog, rat and mouse hepatocytes.

    Science.gov (United States)

    Chen, Wendy W; Qin, Geng-Yao; Zhang, Ting; Feng, Wan-Yong

    2012-06-01

    The metabolic fate of green tea catechins [(-)-epicatechin ((-)-EC), (-)-epicatechin-3-gallate (ECG) (-)- epigallocatechin (EGC) and (-)-epigallocatechin-3-gallate (EGCG)] in cryopreserved human, monkey, dog, rat and mouse hepatocytes was studied. Methylation, glucuronidation, sulfation and isomerization pathways of (-)-EC in all five species were found. Methylation, glucuronidation, sulfation, hydrolysis, isomerization and glucosidation pathways of ECG were found. Species differences in metabolism of (-)-EC or ECG were observed. Surprisingly, no metabolites of EGC or EGCG were detected, but chemical oxidation and polymerization were observed under these experimental conditions. It appeared that enzymatic reactions and chemical reactions were differentiated by an additional hydroxyl group on the B-ring between (-)-EC/ECG and EGC/EGCG. For (-)-EC, thirty-five metabolites including isomerized (M6. M10 and M25), glucuronidated (M3, M5 and M11), sulfated (M7, M15, M16, M18, M20, M23, M26), methylated (M2, M9, M12, M17, M19, M21, M27, M30, M32), glucuronated/methylated (M4, M8, M13, M14), sulfated/methylated (M22, M24, M28, M29, M31, M33, M34, M35) and diglucuronidate (M1), were detected and characterized. M11, M18, M19 and M23 were major metabolites in human hepatocytes; M11, M26 and M31 were major metabolites in monkey hepatocytes; M10, M20, M22, M26 and M31 were major metabolites in dog hepatocytes; M5, M6 and M10 were major metabolites in rat hepatocytes; and M5, M6 and M13 were major metabolites in mouse hepatocytes. For ECG, twelve metabolites including isomerized (M1), hydrolyzed (M3), glucosidated (M2), glucuronidated (M4 and M6), sulfated (M9, M11 and M12), methylated (M7), sulfated/glucuronidated/methylated (M8 and M10) and diglucuronidated (M5), were detected and characterized. M4, M11 and M12 were major metabolites in human hepatocytes; M11 and M12 were major metabolites in monkey hepatocytes; M3 and M11 were major metabolites in dog hepatocytes; M4, M6 and

  19. Foreign Body Retrieval

    Medline Plus

    Full Text Available ... are a form of radiation like light or radio waves. X-rays pass through most objects, including ... Many foreign bodies, like coins and batteries, are radio-opaque, meaning that x-rays will not pass ...

  20. 计及成形因素影响的车身结构疲劳分析%A Fatigue Analysis on Auto-body Structure with the Consideration of Forming Effects

    Institute of Scientific and Technical Information of China (English)

    廖代辉; 成艾国; 谢慧超

    2009-01-01

    Based on random vibration theory, the formulas for the random vibration response spectra of au-to-body structure are deduced. The alternative loading is converted to the loading with equivalent damage by using Goodman curve, and a relationship between stress amplitude and mean stress is established. Taking the fatigue limit of materials as the indicator of fatigue resistance, the residual stress is equivalent to mean stress for studying the effects of forming factors on fatigue performance. With the cross member at tail end of vehicle taken as an example, the dynamic stress calculation, fatigue life prediction and modification design of auto-body structure are performed by adopting fatigue analysis method based on the results of FEA for considering the effects of forming factors. The results show that the fatigue performance predicted well agrees with the results of vehicle road test.%基于随机振动理论,推导了车身结构随机振动响应谱的计算公式;采用Goodman曲线进行交变载荷的等损伤转换,建立了应力幅和平均应力之间的关系式;以材料的疲劳极限作为疲劳抗力指标,将残余应力等效为平均应力,以研究成形因素对疲劳性能的影响;以某汽车尾端横梁为例,采用基于有限元分析结果的疲劳分析法,考虑冲压成形因素的影响,进行了车身结构的动态应力计算、疲劳寿命预测和改进设计.结果表明,所预测的疲劳性能与整车道路试验结果吻合.

  1. Nitric oxide and redox regulation in the liver: part II. Redox biology in pathologic hepatocytes and implications for intervention.

    Science.gov (United States)

    Diesen, Diana L; Kuo, Paul C

    2011-05-01

    Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are created in normal hepatocytes and are critical for normal physiologic processes, including oxidative respiration, growth, regeneration, apoptosis, and microsomal defense. When the levels of oxidation products exceed the capacity of normal antioxidant systems, oxidative stress occurs. This type of stress, in the form of ROS and RNS, can be damaging to all liver cells, including hepatocytes, Kupffer cells, stellate cells, and endothelial cells, through induction of inflammation, ischemia, fibrosis, necrosis, apoptosis, or through malignant transformation by damaging lipids, proteins, and/or DNA. In Part I of this review, we will discuss basic redox biology in the liver, including a review of ROS, RNS, and antioxidants, with a focus on nitric oxide as a common source of RNS. We will then review the evidence for oxidative stress as a mechanism of liver injury in hepatitis (alcoholic, viral, nonalcoholic). In Part II of this review, we will review oxidative stress in common pathophysiologic conditions, including ischemia/reperfusion injury, fibrosis, hepatocellular carcinoma, iron overload, Wilson's disease, sepsis, and acetaminophen overdose. Finally, biomarkers, proteomic, and antioxidant therapies will be discussed as areas for future therapeutic interventions.

  2. Embryonic mouse STO cell-derived xenografts express hepatocytic functions in the livers of nonimmunosuppressed adult rats.

    Science.gov (United States)

    Zhang, Mingjun; Joseph, Brigid; Gupta, Sanjeev; Guest, I; Xu, Meng; Sell, Stewart; Son, Kyung-Hwa; Koch, Katherine S; Leffert, Hyam L

    2005-02-01

    Cells derived from embryonic mouse STO cell lines differentiate into hepatocytes when transplanted into the livers of nonimmunosuppressed dipeptidylpeptidase IV (DPPIV)-negative F344 rats. Within 1 day after intrasplenic injection, donor cells moved rapidly into the liver and were found in intravascular and perivascular sites; by 1 month, they were intrasinusoidal and also integrated into hepatic plates with approximately 2% efficiency and formed conjoint bile canaliculi. Neither donor cell proliferation nor host inflammatory responses were observed during this time. Detection of intrahepatic mouse COX1 mitochondrial DNA and mouse albumin mRNA in recipient rats indicated survival and differentiation of donor cells for at least 3 months. Mouse COX1 targets were also detected intrahepatically 4-9 weeks after STO cell injection into nonimmunosuppressed wild-type rats. In contrast to STO-transplanted rats, mouse DNA or RNA was not detectable in untreated or mock-transplanted rats or in rats injected with donor cell DNA. In cultured STO donor cells, DPPIV and glucose-6-phosphatase activities were observed in small clusters; in contrast, mouse major histocompatibility complex class I H-2Kq, H-2Dq, and H-2Lq and class II I-Aq markers were undetectable in vitro before or after interferon gamma treatment. Together with H-2K allele typing, which confirmed the Swiss mouse origin of the donor cells, these observations indicate that mouse-derived STO cell lines can differentiate along hepatocytic lineage and engraft into rat liver across major histocompatibility barriers.

  3. First characterization of AKB-48 metabolism, a novel synthetic cannabinoid, using human hepatocytes and high-resolution mass spectrometry.

    Science.gov (United States)

    Gandhi, Adarsh S; Zhu, Mingshe; Pang, Shaokun; Wohlfarth, Ariane; Scheidweiler, Karl B; Liu, Hua-Fen; Huestis, Marilyn A

    2013-10-01

    Since the federal authorities scheduled the first synthetic cannabinoids, JWH-018 and JWH-073, new synthetic cannabinoids were robustly marketed. N-(1-Adamantyl)-1-pentylindazole-3-carboxamide (AKB-48), also known as APINACA, was recently observed in Japanese herbal smoking blends. The National Forensic Laboratory Information System registered 443 reports of AKB-48 cases in the USA from March 2010 to January 2013. In May 2013, the Drug Enforcement Administration listed AKB-48 as a Schedule I drug. Recently, AKB-48 was shown to have twice the CB1 receptor binding affinity than CB2. These pharmacological effects and the difficulty in detecting the parent compound in urine highlight the importance of metabolite identification for developing analytical methods for clinical and forensic investigations. Using human hepatocytes and TripleTOF mass spectrometry, we identified 17 novel phase I and II AKB-48 metabolites, products of monohydroxylation, dihydroxylation, or trihydroxylation on the aliphatic adamantane ring or N-pentyl side chain. Glucuronide conjugation of some mono- and dihydroxylated metabolites also occurred. Oxidation and dihydroxylation on the adamantane ring and N-pentyl side chain formed a ketone. More metabolites were identified after 3 h of incubation than at 1 h. For the first time, we present a AKB-48 metabolic scheme obtained from human hepatocytes and high-resolution mass spectrometry. These data are needed to develop analytical methods to identify AKB-48 consumption in clinical and forensic testing.

  4. Photopatterning of hydrogel scaffolds coupled to filter materials using stereolithography for perfused 3D culture of hepatocytes.

    Science.gov (United States)

    Neiman, Jaclyn A Shepard; Raman, Ritu; Chan, Vincent; Rhoads, Mary G; Raredon, Micha Sam B; Velazquez, Jeremy J; Dyer, Rachel L; Bashir, Rashid; Hammond, Paula T; Griffith, Linda G

    2015-04-01

    In vitro models that recapitulate the liver's structural and functional complexity could prolong hepatocellular viability and function to improve platforms for drug toxicity studies and understanding liver pathophysiology. Here, stereolithography (SLA) was employed to fabricate hydrogel scaffolds with open channels designed for post-seeding and perfused culture of primary hepatocytes that form 3D structures in a bioreactor. Photopolymerizable polyethylene glycol-based hydrogels were fabricated coupled to chemically activated, commercially available filters (polycarbonate and polyvinylidene fluoride) using a chemistry that permitted cell viability, and was robust enough to withstand perfused culture of up to 1 µL/s for at least 7 days. SLA energy dose, photoinitiator concentrations, and pretreatment conditions were screened to determine conditions that maximized cell viability and hydrogel bonding to the filter. Multiple open channel geometries were readily achieved, and included ellipses and rectangles. Rectangular open channels employed for subsequent studies had final dimensions on the order of 350 µm by 850 µm. Cell seeding densities and flow rates that promoted cell viability were determined. Perfused culture of primary hepatocytes in hydrogel scaffolds in the presence of soluble epidermal growth factor (EGF) prolonged the maintenance of albumin production throughout the 7-day culture relative to 2D controls. This technique of bonding hydrogel scaffolds can be employed to fabricate soft scaffolds for a number of bioreactor configurations and applications.

  5. FY 2000 report on the results of the regional consortium R and D project - Regional new technology creation R and D. First year report. R and D of medicine toxicity assessment use hepatocyte chip; 2000 nendo chiiki consortium kenkyu kaihatsu jigyo. Chiiki consortium kenkyu kaihatsu / chiiki shingijutsu soshutsu kenkyu kaihatsu. Dokubutsu dokusei hyokayo kansaibo chip no kenkyu kaihatsu (dai 1 nendo) seika hokokusho

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    2001-03-01

    Judging that in the beginning of the toxicity test, the necessary information/knowledge are acquired not by using animal itself but only by using liver system (hepatocyte), the development was proceeded with of the hepatocyte chip substituting for the animal experiment. Studies were made in the following five fields: 1) design of functional matrices; 2) development of the pharmacological assessment technology; 3) development of cell sensing technology/biosensor fabrication; 4) study of a possibility and the limit of cell patterning technology; 5) survey of the market scale. In 1), studies were made of the establishment of the 3D culture method for hepatocyte and the construction of hepatocyte models for medicine assessment using pieces of liver. In the latter, by including capsule using the sugar introduced with galactose residue which is special sugar of hepatocyte to alginic acid and chitosan which are natural polymer and also hepatocyte isolated into sponge, aggregate was formed, and its use as a medicine assessment use liver model was tried to be made. (NEDO)

  6. Bursting bodies of water

    DEFF Research Database (Denmark)

    Rasmussen, Mattias Borg

    2014-01-01

    A silent threat is growing below receding glaciers: lakes are formed as the tongues of the glaciers draw back up the mountain, and huge and growing bodies of water beneath them are contained only be weak moraine walls.......A silent threat is growing below receding glaciers: lakes are formed as the tongues of the glaciers draw back up the mountain, and huge and growing bodies of water beneath them are contained only be weak moraine walls....

  7. Bursting bodies of water

    DEFF Research Database (Denmark)

    Rasmussen, Mattias Borg

    2014-01-01

    A silent threat is growing below receding glaciers: lakes are formed as the tongues of the glaciers draw back up the mountain, and huge and growing bodies of water beneath them are contained only be weak moraine walls.......A silent threat is growing below receding glaciers: lakes are formed as the tongues of the glaciers draw back up the mountain, and huge and growing bodies of water beneath them are contained only be weak moraine walls....

  8. Hepatic stellate cells on poly(DL-lactic acid surfaces control the formation of 3D hepatocyte co-culture aggregates in vitro

    Directory of Open Access Journals (Sweden)

    R J Thomas

    2006-01-01

    Full Text Available Evidence for the functional superiority of cells cultured as 3D aggregates or on 3D scaffolds over conventional 2D monolayer cultures has created interest in material and cell based methods that influence the formation and structure of multicellular aggregates in vitro. We have created a co-culture of primary rat hepatocytes and hepatic stellate cells on a poly(DL-lactic acid surface, a poor substrate for rat hepatocyte adhesion, to study the dynamics of multicellular spheroid formation and the resultant cell arrangement. The poly(DL-lactic acid surface allows dynamic and rapid interaction of hepatocytes and stellate cells to form co-culture spheroids in a complex multistage process (shown by time lapse microscopy. This spheroid morphology supports enhanced cell viability relative to a mono-culture mono-layer system (measured by lactate dehydrogenase leakage. The distribution of the aggregating cell type in the final structure is related to the mechanics of formation i.e. mainly central and peripheral. This study provides a unique and generically applicable insight into the dynamics of multicellular spheroid formation where aggregation is induced by one cell type and imposed on another. This has implications for 3D cell culture models and a wide number of currently used stromal co-culture systems.

  9. Detection of free radicals formed by in vitro metabolism of fluoride using EPR spectroscopy.

    Science.gov (United States)

    Pawłowska-Góral, Katarzyna; Pilawa, Barbara

    2011-10-01

    In many parts of the globe, where water contains large amount of fluoride, fluorosis is a serious public health problem. It is accompanied by many changes, not only in the bones, but practically in all organs of the body. Since it was discovered that oxidation stress, together with the peroxidation of lipids which accompanies it, results in many diseases, research has been carried out on this aspect of fluorosis. The findings, however, are incomplete and divergent. The aim of our study was to determine the presence of free radicals in hepatocytes exposed to fluoride in concentrations which do not lead to changes in the concentrations of calcium and magnesium ions. Free radical properties of hepatocytes incubated with fluoride were studied by an X-band electron paramagnetic resonance (EPR) spectroscopy. Hepatocytes are paramagnetic and broad unsymmetrical EPR spectra were obtained for them. Oxygen free radicals with g-factor of 2.0032 exist in hepatocytes. The effect of fluoride concentration and the time of incubation on free radicals amount in cells were examined. The amount of free radicals in hepatocytes increases with the increase of fluoride concentration for all the incubation times (10, 30, and 60 min). The amount of free radicals in hepatocytes decreases with the increase of time of incubation for all the used fluoride concentrations (0.002, 0.082, and 0.164 mmol/l). EPR spectra of the studied cells are homogeneously broadened. Continuous microwave saturation of EPR lines indicates that slow spin-lattice relaxation processes exist in the studied cells. Strong dipolar interactions responsible for the broadening (ΔB(pp): 1.45-1.87 mT) of the EPR spectra exist in the hepatocytes.

  10. In vitro glucuronidation of 2,2-bis(bromomethyl)-1,3-propanediol by microsomes and hepatocytes from rats and humans.

    Science.gov (United States)

    Rad, Golriz; Hoehle, Simone I; Kuester, Robert K; Sipes, I Glenn

    2010-06-01

    2,2-Bis(bromomethyl)-1,3-propanediol (BMP) is a brominated flame retardant used in unsaturated polyester resins. In a 2-year bioassay BMP was shown to be a multisite carcinogen in rats and mice. Because glucuronidation is the key metabolic transformation of BMP by rats, in this study the in vitro hepatic glucuronidation of BMP was compared across several species. In addition, the glucuronidation activities of human intestinal microsomes and specific human hepatic UDP-glucuronosyltransferase (UGT) enzymes for BMP were determined. To explore other possible routes of metabolism for BMP, studies were conducted with rat and human hepatocytes. Incubation of hepatic microsomes with BMP in the presence of UDP-glucuronic acid resulted in the formation of a BMP monoglucuronide. The order of hepatic microsomal glucuronidation activity of BMP was rats, mice > hamsters > monkeys > humans. The rate of glucuronidation by rat hepatic microsomes was 90-fold greater than that of human hepatic microsomes. Human intestinal microsomes converted BMP to BMP glucuronide at a rate even lower than that of human hepatic microsomes. Among the human UGT enzymes tested, only UGT2B7 had detectable glucuronidation activity for BMP. BMP monoglucuronide was the only metabolite formed when BMP was incubated with suspensions of freshly isolated hepatocytes from male F-344 rats or with cryopreserved human hepatocytes. Glucuronidation of BMP in human hepatocytes was extremely low. Overall, the results support in vivo studies in rats in which BMP glucuronide was the only metabolite found. The poor glucuronidation capacity of humans for BMP suggests that the pharmacokinetic profile of BMP in humans will be dramatically different from that of rodents.

  11. Effects of natural and synthetic estrogens and various environmental contaminants on vitellogenesis in fish primary hepatocytes: comparison of bream (Abramis brama) and carp (Cyprinus carpio).

    Science.gov (United States)

    Rankouhi, T Rouhani; Sanderson, J T; van Holsteijn, I; van Leeuwen, C; Vethaak, A D; van den Berg, M

    2004-09-01

    Interaction of environmental estrogens with the estrogen receptor (ER) has been shown in various fish species. Our objective was to compare the sensitivity of bream (Abramis brama) to (xeno-)estrogens with that of the carp (Cyprinus carpio), by measuring the effects of 17beta-estradiol (E2), estrone (E1), ethynylestradiol (EE2), bisphenol A (BPA), nonylphenol (NP), methoxychlor (MXCL), and halogenated aromatic hydrocarbons (HAHs) such as polychlorinated biphenyls (PCB126, PCB118), 2,3,7,8-tetrachlorodibenzo-dioxin (TCDD), and 2,3,4,7,8-pentachlorodibenzofuran (PCDF) on vitellogenesis in primary hepatocytes. Comparing the EC50 values in bream hepatocytes: EE2 (0.1-0.2 microM) estrogenic potency of E2 and E1, indicating interspecies differences. Exposure to BPA, NP, MXCL, and HAHs did not or only weakly induce vitellogenesis. Bream hepatocytes coexposed to E2 and TCDD, PCB126 or PCDF showed a concentration-dependent inhibition of E2-induced vitellogenesis. IC50 (concentration of a compound that elicits 50% inhibition of E2-induced vitellogenesis) values determined in bream were: TCDD (0.02-0.09 nM) estrogenic response. IC50 values and benchmark-concentration for TCDD and PCB126 in bream and carp hepatocytes were in the same range, indicating similar sensitivity to these compounds. Due to their anti-estrogenic capacity with benchmark-concentrations in the pM range TCDD, PCDF, and PCB126 may form a potential hazard for the reproductive success of fish species by inhibition of vitellogenesis.

  12. Short-term exercise reduces markers of hepatocyte apoptosis in nonalcoholic fatty liver disease

    DEFF Research Database (Denmark)

    Fealy, Ciaran E; Haus, Jacob M; Solomon, Thomas

    2012-01-01

    Increased hepatocyte apoptosis is a hallmark of nonalcoholic fatty liver disease (NAFLD) and contributes to the profibrogenic state responsible for the progression to nonalcoholic steatohepatitis (NASH). Strategies aimed at reducing apoptosis may result in better outcomes for individuals with NAF...

  13. Asialoglycoprotein receptor and liposome synergistically mediate the gene transfer into primary rat hepatocytes

    Institute of Scientific and Technical Information of China (English)

    李崇辉; 温守明; 翟海峰; 孙曼霁

    1999-01-01

    Gene transfer into primary rat hepatocytes was performed by employing cationic liposome as DNA carrier and the specific ligand of hepatic asialoglycoprotein receptor (ASGPR), asialofetuin, as liver-targeting ligand. The resuits showed that asialofetuin, when added to the gene transfer complexes, could significantly increase the hepatocyte transfeetion efficiency, and alleviate the cellular toxicity of Lipofectin. Several synthetic ligands of ASGPR (galactosyl albumin) could also increase the transfection efficiency of hepatocyte like asialofetuin. It was proved that ASGPR and cationic liposome could synergistically mediate the gene transfer into primary rat hepatoeytes. This novel gene delivery system provided a safer, more simple and efficient gene transfer method for primary hepatocytes, and showed prospecting application in hepatic gene therapy.

  14. Preparation and property analysis of a hepatocyte targeting pH-sensitive liposome

    Institute of Scientific and Technical Information of China (English)

    Si-Yuan Wen; Xiao-Hong Wang; Li Lin; Wei Guan; Sheng-Qi Wang

    2004-01-01

    AIM: To develop a hepatocyte targeting pH-sensitive liposome for drug delivery based on active targeting technology mediated by asialoglycoprotein receptors.METHODS: Four types of targeting molecules with galactose residue were synthesized and mixed with pH-sensitive lipids DC-chol/DOPE to prepare liposome with integrated property of hepatocyte specificity and pH sensitivity. Liposome 18-gal was selected with the best transfection activity through cellular uptake experiment. Property analysis was made through experiments of competitive inhibition of receptors,red blood cell hemolysis,in vitro cytotoxicity test by MTS assay and mediation of inhibitory effects of antisense phosphorothioate ODN on gene expression, etc.RESULTS: Liposome L8-gal had the desired properties of hepatocyte specificity, pH sensitivity, low cytotoxicity, and high transfection efficiency.CONCLUSION: Liposome 18-gal can be further developed as a potential hepatocyte- targeting delivery system.

  15. Recombinant Laminins Drive the Differentiation and Self-Organization of hESC-Derived Hepatocytes

    Directory of Open Access Journals (Sweden)

    Kate Cameron

    2015-12-01

    Full Text Available Stem cell-derived somatic cells represent an unlimited resource for basic and translational science. Although promising, there are significant hurdles that must be overcome. Our focus is on the generation of the major cell type of the human liver, the hepatocyte. Current protocols produce variable populations of hepatocytes that are the product of using undefined components in the differentiation process. This serves as a significant barrier to scale-up and application. To tackle this issue, we designed a defined differentiation process using recombinant laminin substrates to provide instruction. We demonstrate efficient hepatocyte specification, cell organization, and significant improvements in cell function and phenotype. This is driven in part by the suppression of unfavorable gene regulatory networks that control cell proliferation and migration, pluripotent stem cell self-renewal, and fibroblast and colon specification. We believe that this represents a significant advance, moving stem cell-based hepatocytes closer toward biomedical application.

  16. Recombinant Laminins Drive the Differentiation and Self-Organization of hESC-Derived Hepatocytes.

    Science.gov (United States)

    Cameron, Kate; Tan, Rosanne; Schmidt-Heck, Wolfgang; Campos, Gisela; Lyall, Marcus J; Wang, Yu; Lucendo-Villarin, Baltasar; Szkolnicka, Dagmara; Bates, Nicola; Kimber, Susan J; Hengstler, Jan G; Godoy, Patricio; Forbes, Stuart J; Hay, David C

    2015-12-08

    Stem cell-derived somatic cells represent an unlimited resource for basic and translational science. Although promising, there are significant hurdles that must be overcome. Our focus is on the generation of the major cell type of the human liver, the hepatocyte. Current protocols produce variable populations of hepatocytes that are the product of using undefined components in the differentiation process. This serves as a significant barrier to scale-up and application. To tackle this issue, we designed a defined differentiation process using recombinant laminin substrates to provide instruction. We demonstrate efficient hepatocyte specification, cell organization, and significant improvements in cell function and phenotype. This is driven in part by the suppression of unfavorable gene regulatory networks that control cell proliferation and migration, pluripotent stem cell self-renewal, and fibroblast and colon specification. We believe that this represents a significant advance, moving stem cell-based hepatocytes closer toward biomedical application.

  17. A fat option for the pig: Hepatocytic differentiated mesenchymal stem cells for translational research

    Energy Technology Data Exchange (ETDEWEB)

    Brückner, Sandra, E-mail: sandra.brueckner@medizin.uni-leipzig.de [University Hospital Leipzig, Department of Visceral, Transplantation, Thoracic and Vascular Surgery, Liebigstraße 21, Leipzig D-04103 (Germany); Tautenhahn, Hans-Michael, E-mail: hans-michael.tautenhahn@medizin.uni-leipzig.de [University Hospital Leipzig, Department of Visceral, Transplantation, Thoracic and Vascular Surgery, Liebigstraße 21, Leipzig D-04103 (Germany); TRM, Translational Centre for Regenerative Medicine, Philipp-Rosenthal-Str. 55, Leipzig D-04103 (Germany); Winkler, Sandra, E-mail: sandra.pelz@medizin.uni-leipzig.de [University Hospital Leipzig, Department of Visceral, Transplantation, Thoracic and Vascular Surgery, Liebigstraße 21, Leipzig D-04103 (Germany); Stock, Peggy, E-mail: peggy.stock@medizin.uni-leipzig.de [University Hospital Leipzig, Department of Visceral, Transplantation, Thoracic and Vascular Surgery, Liebigstraße 21, Leipzig D-04103 (Germany); Dollinger, Matthias, E-mail: matthias.dollinger@uniklinik-ulm.de [University Hospital Ulm, First Department of Medicine, Albert-Einstein-Allee 23, Ulm D-89081 (Germany); Christ, Bruno, E-mail: bruno.christ@medizin.uni-leipzig.de [University Hospital Leipzig, Department of Visceral, Transplantation, Thoracic and Vascular Surgery, Liebigstraße 21, Leipzig D-04103 (Germany); TRM, Translational Centre for Regenerative Medicine, Philipp-Rosenthal-Str. 55, Leipzig D-04103 (Germany)

    2014-02-15

    Study background: Extended liver resection is the only curative treatment option of liver cancer. Yet, the residual liver may not accomplish the high metabolic and regenerative capacity needed, which frequently leads to acute liver failure. Because of their anti-inflammatory and -apoptotic as well as pro-proliferative features, mesenchymal stem cells differentiated into hepatocyte-like cells might provide functional and regenerative compensation. Clinical translation of basic research requires pre-clinical approval in large animals. Therefore, we characterized porcine mesenchymal stem cells (MSC) from adipose tissue and bone marrow and their hepatocyte differentiation potential for future assessment of functional liver support after surgical intervention in the pig model. Methods: Mesenchymal surface antigens and multi-lineage differentiation potential of porcine MSC isolated by collagenase digestion either from bone marrow or adipose tissue (subcutaneous/visceral) were assessed by flow cytometry. Morphology and functional properties (urea-, glycogen synthesis and cytochrome P450 activity) were determined during culture under differentiation conditions and compared with primary porcine hepatocytes. Results: MSC from porcine adipose tissue and from bone marrow express the typical mesenchymal markers CD44, CD29, CD90 and CD105 but not haematopoietic markers. MSC from both sources displayed differentiation into the osteogenic as well as adipogenic lineage. After hepatocyte differentiation, expression of CD105 decreased significantly and cells adopted the typical polygonal morphology of hepatocytes. Glycogen storage was comparable in adipose tissue- and bone marrow-derived cells. Urea synthesis was about 35% lower in visceral than in subcutaneous adipose tissue-derived MSC. Cytochrome P450 activity increased significantly during differentiation and was twice as high in hepatocyte-like cells generated from bone marrow as from adipose tissue. Conclusion: The hepatocyte

  18. Hepatocyte isolation from resected benign tissues: Results of a 5-year experience

    Science.gov (United States)

    Meng, Fan-Ying; Liu, Li; Liu, Jun; Li, Chun-You; Wang, Jian-Ping; Yang, Feng-Hui; Chen, Zhi-Shui; Zhou, Ping

    2016-01-01

    AIM To analyze retrospectively a 5-year experience of human hepatocyte isolation from resected liver tissues with benign disease. METHODS We established a method of modified four-step retrograde perfusion to isolate primary human hepatocytes. Samples were collected from the resected livers of patients with intrahepatic duct calculi (n = 7) and liver hemangioma (n = 17). Only the samples weighing ≥ 15 g were considered suitable for hepatocyte isolation. By using the standard trypan blue exclusion technique, hepatocyte viability and yield were immediately determined after isolation. RESULTS Twenty-four liver specimens, weighing 15-42 g, were immediately taken from the margin of the removed samples and transferred to the laboratory for hepatocyte isolation. Warm ischemia time was 5-35 min and cold ischemia time was 15-45 min. For the 7 samples of intrahepatic duct calculi, the method resulted in a hepatocyte yield of 3.49 ± 2.31 × 106 hepatocytes/g liver, with 76.4% ± 10.7% viability. The 17 samples of liver hemangioma had significantly higher yield of cells (5.4 ± 1.71 × 106 cells/g vs 3.49 ± 2.31 × 106 cells/g, P 0.05). We obtained a cell yield of 5.31 ± 1.87 × 106 hepatocytes/g liver when the samples weighed > 20 g. However, for the tissues weighing ≤ 20 g, a reduction in yield was found (3.08 ± 1.86 × 106 cells/g vs 5.31 ± 1.87 × 106 cells/g, P < 0.05). CONCLUSION Benign diseased livers are valuable sources for large-number hepatocyte isolation. Our study represents the largest number of primary human hepatocytes isolated from resected specimens from patients with benign liver disease. We evaluated the effect of donor liver characteristics on cell isolation, and we found that samples of liver hemangioma can provide better results than intrahepatic duct calculi, in terms of cell yield. Furthermore, the size of the tissues can affect the outcome of hepatocyte isolation. PMID:27688659

  19. 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

  20. The Influence of Human Body's Waist Form on the Pattern Structure of Circular Skirt%人体腰部形态对斜裙样板结构的影响研究

    Institute of Scientific and Technical Information of China (English)

    张姝; 陆鑫; 马莲莹

    2012-01-01

    选用160/84A的人体模台,将腰围分别补正为64cm、68cm、72cm,通过样板叠加分析方法,考察样板中各工艺点的变量规律;并辅助着装观察方法,研究人体的腰部形态对斜裙造型的影响。研究表明:当臀围不变,腰围呈等差数列增加,斜裙样板结构中的腰口曲线横深比增大,腰口曲线弧度与底摆曲线弧度越平滑,下摆围度越小,这些变量都呈等差数列;而斜裙着装后底摆波浪个数减少,且起伏变小,平均起浪点越低。%This paper aims to explore the influence of human body' s waist form on the pattern structure of oblique skirt. By choosing the phantom of 160/84A, the waistlines are respectively corrected to 64cm, 68cm and 72cm. Through the analytic method of sample stacking, the variable law at different waist forms is studied. Together with the observation method of oblique skirt outfit modeling, the affect of human' s waist form on oblique skirt's shape is analysed. Research shows that: when the hip circumference length is changeless, the waist circumference length takes on arithmetic sequence increase ; oblique skirt model structure of waist mouth curve cross depth ratio increases and the smoother the waist entry curve radian and bottom pendulum curve radian, the smaller the skirt hem circumference degrees. The waist circumference is bigger, the waist thick-width rate is smaller. The number of skirt flare is less, the skirt wave is smaller and average wave point is lower.

  1. Differentiation and selection of hepatocyte precursors in suspension spheroid culture of transgenic murine embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Elke Gabriel

    Full Text Available Embryonic stem cell-derived hepatocyte precursor cells represent a promising model for clinical transplantations to diseased livers, as well as for establishment of in vitro systems for drug metabolism and toxicology investigations. This study aimed to establish an in vitro culture system for scalable generation of hepatic progenitor cells. We used stable transgenic clones of murine embryonic stem cells possessing a reporter/selection vector, in which the enhanced green fluorescent protein- and puromycin N-acetyltransferase-coding genes are driven by a common alpha-fetoprotein gene promoter. This allowed for "live" monitoring and puromycin selection of the desired differentiating cell type possessing the activated alpha-fetoprotein gene. A rotary culture system was established, sequentially yielding initially partially selected hepatocyte lineage-committed cells, and finally, a highly purified cell population maintained as a dynamic suspension spheroid culture, which progressively developed the hepatic gene expression phenotype. The latter was confirmed by quantitative RT-PCR analysis, which showed a progressive up-regulation of hepatic genes during spheroid culture, indicating development of a mixed hepatocyte precursor-/fetal hepatocyte-like cell population. Adherent spheroids gave rise to advanced differentiated hepatocyte-like cells expressing hepatic proteins such as albumin, alpha-1-antitrypsin, cytokeratin 18, E-cadherin, and liver-specific organic anion transporter 1, as demonstrated by fluorescent immunostaining. A fraction of adherent cells was capable of glycogen storage and of reversible up-take of indocyanine green, demonstrating their hepatocyte-like functionality. Moreover, after transplantation of spheroids into the mouse liver, the spheroid-derived cells integrated into recipient. These results demonstrate that large-scale hepatocyte precursor-/hepatocyte-like cultures can be established for use in clinical trials, as well as in

  2. Selection of medium for serum-free primary culture of adult rat hepatocytes.

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    Miyazaki,Masahiro

    1990-02-01

    Full Text Available To select a suitable medium for serum-free primary culture of adult rat hepatocytes, ten commercially-available synthetic media were compared for their ability to maintain the cells under serum-free and serum-supplemented conditions with special reference to attachment, survival and albumin secretion. It was found that Williams' medium E and DM-160 medium were the best among the ten media for maintaining hepatocytes under serum-free conditions in primary culture.

  3. Ultrastructure of Kupffer cells and hepatocytes in the Dubin-Johnson syndrome: A case report

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    Sobaniec-Lotowska, Maria Elzbieta; Lebensztejn, Dariusz Marek

    2006-01-01

    Ultrastructure of Kupffer cells and hepatocytes in liver bioptate was evaluated in a 17-year-old boy with Dubin–Johnson syndrome (DJS). The liver tissue obtained by needle biopsy was fixed in glutaraldehyde and paraformaldehyde and routinely processed for electron microscopic analysis. The ultrastructural examinations of liver bioptate revealed the accumulation of membrane-bound, electron-dense lysosomal granules within the cytoplasm of hepatocytes, characteristic of DJS. They were located ma...

  4. Urea production in long-term cultures of adult rat hepatocytes.

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    Sierra-Santoyo, A; López, M L; Hernández, A; Mendoza-Figueroa, T

    1994-04-01

    To study the functionality of the urea cycle in long-term cultures of adult rat hepatocytes, urea production and the activity of two urea cycle enzymes were measured in hepatocytes cultured on 3T3 cells for 15 days. Urea production was also measured in cultures maintained with medium containing either 0.4 mm arginine or 0.4 mm ornithine and in cultures exposed to different concentrations of NH(4)Cl, an in vivo inducer of urea production. In hepatocytes seeded on 3T3 cells, urea production decreased gradually to 50% of the initial value after 15 days. Urea production was similar in 3T3-hepatocyte cultures maintained for 11 days with medium containing ornithine or arginine. Hepatocytes exposed for 24 hr to 1, 3 and 5 mm NH(4)Cl showed an average increase in urea production of 25, 50 and 69%, respectively, above that of unexposed cultures over 15 days. Ornithine transcarbamylase (OTC) activity decreased by 84% after 5 days in culture and remained constant thereafter, while arginase activity remained constant over 15 days. In contrast, in hepatocytes seeded on plastic substratum, urea production decreased to 24% of the initial value after 8 days in culture. OTC and arginase activities also decreased to 13 and 10% of their initial values after 8 days in culture. These results show that 3T3-hepatocyte cultures from adult rats produce urea from ornithine and/or arginine for at least 15 days and respond to an inducer of urea production as in vivo. They also show that these cultures have decreasing and constant levels of OTC and arginase activities, respectively, owing probably to an adaptative response dependent on substrate concentrations and hormonal regulation. These findings also suggest that 3T3-hepatocyte cultures are a suitable in vitro system to study urea production, its regulation by substrates and hormones and its alteration by drugs and toxic chemicals.

  5. Effect of nonsteroidal anti-inflammatory drugs on glycogenolysis in isolated hepatocytes.

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    Brass, E. P.; Garrity, M. J.

    1985-01-01

    E-series prostaglandins have previously been demonstrated to inhibit hormone-stimulated glycogenolysis when added to isolated hepatocytes of the rat. In the present study, the effect of nonsteroidal anti-inflammatory drugs, which inhibit cyclo-oxygenase activity, on glycogenolysis was examined in the hepatocyte model. Ibuprofen (80 microM), indomethacin (50 microM) and meclofenamate (60 microM) all increased rates of glycogenolysis when added under basal conditions. In contrast, piroxicam (50...

  6. Bile acid-induced necrosis in primary human hepatocytes and in patients with obstructive cholestasis

    Energy Technology Data Exchange (ETDEWEB)

    Woolbright, Benjamin L.; Dorko, Kenneth [Department of Pharmacology, Toxicology & Therapeutics, University of Kansas Medical Center, Kansas City, KS (United States); Antoine, Daniel J.; Clarke, Joanna I. [MRC Centre for Drug Safety Science, Department of Molecular and Clinical Pharmacology, Institute of Translational Medicine, University of Liverpool, Liverpool (United Kingdom); Gholami, Parviz [Department of Internal Medicine, University of Kansas Medical Center, Kansas City, KS (United States); Li, Feng [Department of Pharmacology, Toxicology & Therapeutics, University of Kansas Medical Center, Kansas City, KS (United States); Kumer, Sean C.; Schmitt, Timothy M.; Forster, Jameson [Department of Surgery, University of Kansas Medical Center, Kansas City, KS (United States); Fan, Fang [Department of Pathology, University of Kansas Medical Center, Kansas City, KS (United States); Jenkins, Rosalind E.; Park, B. Kevin [MRC Centre for Drug Safety Science, Department of Molecular and Clinical Pharmacology, Institute of Translational Medicine, University of Liverpool, Liverpool (United Kingdom); Hagenbuch, Bruno [Department of Pharmacology, Toxicology & Therapeutics, University of Kansas Medical Center, Kansas City, KS (United States); Olyaee, Mojtaba [Department of Internal Medicine, University of Kansas Medical Center, Kansas City, KS (United States); Jaeschke, Hartmut, E-mail: hjaeschke@kumc.edu [Department of Pharmacology, Toxicology & Therapeutics, University of Kansas Medical Center, Kansas City, KS (United States)

    2015-03-15

    Accumulation of bile acids is a major mediator of cholestatic liver injury. Recent studies indicate bile acid composition between humans and rodents is dramatically different, as humans have a higher percent of glycine conjugated bile acids and increased chenodeoxycholate content, which increases the hydrophobicity index of bile acids. This increase may lead to direct toxicity that kills hepatocytes, and promotes inflammation. To address this issue, this study assessed how pathophysiological concentrations of bile acids measured in cholestatic patients affected primary human hepatocytes. Individual bile acid levels were determined in serum and bile by UPLC/QTOFMS in patients with extrahepatic cholestasis with, or without, concurrent increases in serum transaminases. Bile acid levels increased in serum of patients with liver injury, while biliary levels decreased, implicating infarction of the biliary tracts. To assess bile acid-induced toxicity in man, primary human hepatocytes were treated with relevant concentrations, derived from patient data, of the model bile acid glycochenodeoxycholic acid (GCDC). Treatment with GCDC resulted in necrosis with no increase in apoptotic parameters. This was recapitulated by treatment with biliary bile acid concentrations, but not serum concentrations. Marked elevations in serum full-length cytokeratin-18, high mobility group box 1 protein (HMGB1), and acetylated HMGB1 confirmed inflammatory necrosis in injured patients; only modest elevations in caspase-cleaved cytokeratin-18 were observed. These data suggest human hepatocytes are more resistant to human-relevant bile acids than rodent hepatocytes, and die through necrosis when exposed to bile acids. These mechanisms of cholestasis in humans are fundamentally different to mechanisms observed in rodent models. - Highlights: • Cholestatic liver injury is due to cytoplasmic bile acid accumulation in hepatocytes. • Primary human hepatocytes are resistant to BA-induced injury

  7. Human Upcyte Hepatocytes: Characterization of the Hepatic Phenotype and Evaluation for Acute and Long-Term Hepatotoxicity Routine Testing.

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    Tolosa, Laia; Gómez-Lechón, M José; López, Silvia; Guzmán, Carla; Castell, José V; Donato, M Teresa; Jover, Ramiro

    2016-07-01

    The capacity of human hepatic cell-based models to predict hepatotoxicity depends on the functional performance of cells. The major limitations of human hepatocytes include the scarce availability and rapid loss of the hepatic phenotype. Hepatoma cells are readily available and easy to handle, but are metabolically poor compared with hepatocytes. Recently developed human upcyte hepatocytes offer the advantage of combining many features of primary hepatocytes with the unlimited availability of hepatoma cells. We analyzed the phenotype of upcyte hepatocytes comparatively with HepG2 cells and adult primary human hepatocytes to characterize their functional features as a differentiated hepatic cell model. The transcriptomic analysis of liver characteristic genes confirmed that the upcyte hepatocytes expression profile comes closer to human hepatocytes than HepG2 cells. CYP activities were measurable and showed a similar response to prototypical CYP inducers than primary human hepatocytes. Upcyte hepatocytes also retained conjugating activities and key hepatic functions, e.g. albumin, urea, lipid and glycogen synthesis, at levels close to hepatocytes. We also investigated the suitability of this cell model for preclinical hepatotoxicity risk assessments using multiparametric high-content screening, as well as transcriptomics and targeted metabolomic analysis. Compounds with well-documented in vivo hepatotoxicity were screened after acute and repeated doses up to 1 week. The evaluation of complex mechanisms of cell toxicity, drug-induced steatosis and oxidative stress biomarkers demonstrated that, by combining the phenotype of primary human hepatocytes and the ease of handling of HepG2 cells, upcyte hepatocytes offer suitable properties to be potentially used for toxicological assessments during drug development.

  8. Detection of genotoxic carcinogens in the in vivo-in vitro hepatocyte DNA repair assay.

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    Mirsalis, J C; Tyson, C K; Butterworth, B E

    1982-01-01

    The in vivo-in vitro hepatocyte DNA repair assay has been shown to be useful in the evaluation of the carcinogenic potential of chemicals. The purpose of this study was to apply this assay to determining the genotoxicity of the compounds from a wide variety of structural classes. Male Fischer-334 rats were treated by gavage or ip injection with compounds dissolved in either corn oil, water, or dimethyl sulfoxide (DMSO). At selected times after treatment, hepatocytes were isolated by liver perfusion and cultured with 3H-thymidine, Unscheduled DNA synthesis (UDS) was measured by quantitative autoradiography as net grains/nucleus (NG); greater than or equal to 5 NG was considered positive. Water, corn oil, or DMSO controls produced -3 to -6 NG with less than or equal to 6% of the cells in repair. All genotoxic hepatocarcinogens tested produced strong positive responses of greater than 15 NG including dimethylnitrosamine, 2-acetylaminofluorene (2-AAF), azoxymethane, 1,2-dimethylhydrazine, benzidine, aflatoxin B1, 2,6-dinitrotoluene, and 2,4-diaminotoluene. The noncarcinogen, 2,6-diaminotoluene, was negative. The mutagen and rat brain carcinogen methyl methanesulfonate (MMS) and the rat pancreatic carcinogen azaserine were also positive. The carcinogens benzo(a)pyrene and 7,12-dimethylbenz(a)anthracene yielded from -2 to -4 NG. This negative response is consistent with their lack of carcinogenic activity in rat liver. MMS produced the greatest amount of UDS 2 hr after treatment whereas 2-AAF did not induce its maximum response until 12 hr post-treatment. The potent hepatotoxin carbon tetrachloride induced a 40-fold elevation in DNA replication 48 hr after a 400 mg/kg dose, but no UDS was observed at 2, 12, 24, or 48 hr post-treatment. The weak hepatocarcinogen safrole induced no UDS suggesting that it is either nongenotoxic or is metabolized to an active form at an extremely slow rate following a single administration. These results demonstrate that this assay is

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

    Directory of Open Access Journals (Sweden)

    Julien Fitamant

    2015-03-01

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

  10. [Rna content of gerbil hepatocytes after the flight aboard space platform Foton-M3].

    Science.gov (United States)

    Atiashkin, D A; Bykov, E G; Il'in, E A; Pashkov, A N

    2010-01-01

    The paper compares and contrasts the results of measuring the hepatocyte cytoplasm area and RNA content in 35 gerbils in three series of experiments, i.e. the vivarium control, modeled space flight (synchronous control) and exposure to the factors of 12-d Foton-M3 orbital flight. Central, intermediate and peripheral zones of hepatic lobes were subjected to histological and histochemical analyses to measure the hepatocyte cytoplasm area; the RNA content was determined from the level of cytoplasm basophilia after azure staining. Cytometric and cytophotometric investigations were performed using image analyzer Video-7-Test-Morpho. In the vivarium animals, hepatocytes with the largest cytoplasm localized predominantly in the intermediate and central zones of the lobes. Judging from the results of microdensitometry, the RNA content was particularly high in binucleate hepatocytes of the intermediate zone. In the synchronous control, hepatocytes tended to grow in size, in the peripheral zone specifically, whereas RNA content was largely equal no matter hepatocyte topography. After space flight, cytoplasm enlargement transcended this process in the vivarium animals. The cytoplasm RNA content along the entire liver parenchyma made a significant decrease equally as compared with the vivarium and synchronous control animals.

  11. Tethered spheroids as an in vitro hepatocyte model for drug safety screening.

    Science.gov (United States)

    Xia, Lei; Sakban, Rashidah Binte; Qu, Yinghua; Hong, Xin; Zhang, Wenxia; Nugraha, Bramasta; Tong, Wen Hao; Ananthanarayanan, Abhishek; Zheng, Baixue; Chau, Ian Yin-Yan; Jia, Ruirui; McMillian, Michael; Silva, Jose; Dallas, Shannon; Yu, Hanry

    2012-03-01

    Hepatocyte spheroids mimic many in vivo liver-tissue phenotypes but increase in size during extended culture which limits their application in drug testing applications. We have developed an improved hepatocyte 3D spheroid model, namely tethered spheroids, on RGD and galactose-conjugated membranes using an optimized hybrid ratio of the two bioactive ligands. Cells in the spheroid configuration maintained 3D morphology and uncompromised differentiated hepatocyte functions (urea and albumin production), while the spheroid bottom was firmly tethered to the substratum maintaining the spheroid size in multi-well plates. The oblate shape of the tethered spheroids, with an average height of 32 μm, ensured efficient nutrient, oxygen and drug access to all the cells within the spheroid structure. Cytochrome P450 induction by prototypical inducers was demonstrated in the tethered spheroids and was comparable or better than that observed with hepatocyte sandwich cultures. These data suggested that tethered 3D hepatocyte spheroids may be an excellent alternative to 2D hepatocyte culture models for drug safety applications.

  12. p38α regulates actin cytoskeleton and cytokinesis in hepatocytes during development and aging.

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    Tormos, Ana M; Rius-Pérez, Sergio; Jorques, María; Rada, Patricia; Ramirez, Lorena; Valverde, Ángela M; Nebreda, Ángel R; Sastre, Juan; Taléns-Visconti, Raquel

    2017-01-01

    Hepatocyte poliploidization is an age-dependent process, being cytokinesis failure the main mechanism of polyploid hepatocyte formation. Our aim was to study the role of p38α MAPK in the regulation of actin cytoskeleton and cytokinesis in hepatocytes during development and aging. Wild type and p38α liver-specific knock out mice at different ages (after weaning, adults and old) were used. We show that p38α MAPK deficiency induces actin disassembly upon aging and also cytokinesis failure leading to enhanced binucleation. Although the steady state levels of cyclin D1 in wild type and p38α knock out old livers remained unaffected, cyclin B1- a marker for G2/M transition- was significantly overexpressed in p38α knock out mice. Our findings suggest that hepatocytes do enter into S phase but they do not complete cell division upon p38α deficiency leading to cytokinesis failure and binucleation. Moreover, old liver-specific p38α MAPK knock out mice exhibited reduced F-actin polymerization and a dramatic loss of actin cytoskeleton. This was associated with abnormal hyperactivation of RhoA and Cdc42 GTPases. Long-term p38α deficiency drives to inactivation of HSP27, which seems to account for the impairment in actin cytoskeleton as Hsp27-silencing decreased the number and length of actin filaments in isolated hepatocytes. p38α MAPK is essential for actin dynamics with age in hepatocytes.

  13. Low-Density Lipoprotein Uptake Demonstrates a Hepatocyte Phenotype in the Dog, but Is Nonspecific.

    Science.gov (United States)

    Gow, Adam G; Muirhead, Rhona; Hay, David C; Argyle, David J

    2016-01-01

    Low-density lipoprotein (LDL) uptake is one of a number of tests used to demonstrate hepatocyte-like function after stem cell differentiation. Use of two compounds, LDL and acetylated LDL (AcLDL), has been described despite each having different mechanisms of uptake. Three primary hepatocyte cultures and three sets of mesenchymal stromal cell (MSC) cultures, derived from both adipose tissue and bone marrow, were harvested from dogs. Those cells were compared to commercially available human and mouse bone marrow-derived MSCs. LDL receptor expression was demonstrated by gene expression and immunofluorescence in all primary hepatocyte cultures, undifferentiated canine bone marrow MSCs and canine adipose MSCs. Undifferentiated human and mouse bone marrow MSCs also expressed the LDL receptor. In vitro, canine hepatocytes took up labeled LDL, but not AcLDL. All undifferentiated MSCs took up LDL, but not AcLDL. In conclusion, LDL and not AcLDL is a test of canine hepatocyte-like phenotype, but this is not tissue or species specific and, therefore, is not informative assay when testing proof of MSC to hepatocyte differentiation.

  14. Differential effect of fructose on fat metabolism and clock gene expression in hepatocytes vs. myotubes.

    Science.gov (United States)

    Chapnik, Nava; Rozenblit-Susan, Sigal; Genzer, Yoni; Froy, Oren

    2016-08-01

    In the liver, fructose bypasses the main rate-limiting step of glycolysis at the level of phosphofructokinase, allowing it to act as an unregulated substrate for de novo lipogenesis. It has been reported that consumption of large amounts of fructose increases de novo lipogenesis in the liver. However, the effect of fructose on ectopic deposition of muscle fat has been under dispute. Our aim was to study the effect of fructose on levels of genes and proteins involved in fatty acid oxidation and synthesis in hepatocytes vs. muscle cells. In addition, as fat accumulation leads to disruption of daily rhythms, we tested the effect of fructose treatment on clock gene expression. AML-12 hepatocytes and C2C12 myotubes were treated with fructose or glucose for 2 consecutive 24-h cycles and harvested every 6h. In contrast to glucose, fructose disrupted clock gene rhythms in hepatocytes, but in myotubes, it led to more robust rhythms. Fructose led to low levels of phosphorylated AMP-activated protein kinase (pAMPK) and high levels of LIPIN1 in hepatocytes compared with glucose. In contrast, fructose led to high pAMPK and low LIPIN1 and microsomal triacylglycerol transfer protein (MTTP) levels in myotubes compared with glucose. Analysis of fat content revealed that fructose led to less fat accumulation in myotubes compared to hepatocytes. In summary, fructose shifts metabolism towards fatty acid synthesis and clock disruption in hepatocytes, but not in myotubes. Copyright © 2016 Elsevier Ltd. All rights reserved.

  15. p38α regulates actin cytoskeleton and cytokinesis in hepatocytes during development and aging

    Science.gov (United States)

    Jorques, María; Rada, Patricia; Ramirez, Lorena; Valverde, Ángela M.; Nebreda, Ángel R.; Sastre, Juan

    2017-01-01

    Background Hepatocyte poliploidization is an age-dependent process, being cytokinesis failure the main mechanism of polyploid hepatocyte formation. Our aim was to study the role of p38α MAPK in the regulation of actin cytoskeleton and cytokinesis in hepatocytes during development and aging. Methods Wild type and p38α liver-specific knock out mice at different ages (after weaning, adults and old) were used. Results We show that p38α MAPK deficiency induces actin disassembly upon aging and also cytokinesis failure leading to enhanced binucleation. Although the steady state levels of cyclin D1 in wild type and p38α knock out old livers remained unaffected, cyclin B1- a marker for G2/M transition- was significantly overexpressed in p38α knock out mice. Our findings suggest that hepatocytes do enter into S phase but they do not complete cell division upon p38α deficiency leading to cytokinesis failure and binucleation. Moreover, old liver-specific p38α MAPK knock out mice exhibited reduced F-actin polymerization and a dramatic loss of actin cytoskeleton. This was associated with abnormal hyperactivation of RhoA and Cdc42 GTPases. Long-term p38α deficiency drives to inactivation of HSP27, which seems to account for the impairment in actin cytoskeleton as Hsp27-silencing decreased the number and length of actin filaments in isolated hepatocytes. Conclusions p38α MAPK is essential for actin dynamics with age in hepatocytes. PMID:28166285

  16. Hepatocyte-like cells from directed differentiation of mouse bone marrow cells in vitro

    Institute of Scientific and Technical Information of China (English)

    Xiao-lei SHI; Yu-dong QIU; Qiang LI; Ting XIE; Zhang-hua ZHU; Lei-lei CHEN; Lei LI; Yi-tao DING

    2005-01-01

    Aim: To design the effective directed differentiation medium to differentiate bone marrow cells into hepatocyte-like cells. Methods: Bone marrow cells were cultured in the directed differentiation media including fibroblast growth factor-4 (FGF-4) and oncostatin M (OSM). Hepatocyte-like cells from directed differentia tion of bone marrow cells were identified through cell morphology, RNA expressions by reverse transcriptase-polymerase chain reaction (RT-PCR), protein expressions by Western blot, and hepatocellular synthesis and metabolism functions by albumin ELISA, Periodic acid-Shiff staining and urea assay. Results:Some epithelial-like cells or polygonal cells appeared and increased in the course of the cell directed differentiation. Hepatocyte nucleur factor-3β (HNF-3β), albumin (ALB), cytokeratin 18 (CK18), transthyretin (TFR), glucose-6-phosphate (G6-Pase), and tyrosine aminotransferase (TAT) mRNA were expressed in the course of the directed differentiation. The directed differentiated cells on d 21 expressed HNF-3β, ALB, and CK18 proteins. The directed differentiated cells produced albumin and synthesized urea in a time-dependent manner. They could also synthesize glycogen. Conclusion: Our differentiation media, including FGF-4 and OSM, are effective to differentiate bone marrow cells into hepatocyte-like cells, which could be used for hepatocyte resources for bioartificial liver or hepatocyte transplantation.

  17. Palm kernel cake extract exerts hepatoprotective activity in heat-induced oxidative stress in chicken hepatocytes.

    Science.gov (United States)

    Oskoueian, Ehsan; Abdullah, Norhani; Idrus, Zulkifli; Ebrahimi, Mahdi; Goh, Yong Meng; Shakeri, Majid; Oskoueian, Armin

    2014-10-02

    Palm kernel cake (PKC), the most abundant by-product of oil palm industry is believed to contain bioactive compounds with hepatoprotective potential. These compounds may serve as hepatoprotective agents which could help the poultry industry to alleviate adverse effects of heat stress on liver function in chickens. This study was performed to evaluate the hepatoprotective potential of PKC extract in heat-induced oxidative stress in chicken hepatocytes. The nature of the active metabolites and elucidation of the possible mechanism involved were also investigated. The PKC extract possessed free radical scavenging activity with values significantly (p Heat-induced oxidative stress in chicken hepatocyte impaired the total protein, lipid peroxidation and antioxidant enzymes activity significantly (p heat-induced hepatocytes with PKC extract (125 μg/ml) and silymarin as positive control increased these values significantly (p stress biomarkers including TNF-like, IFN-γ and IL-1β genes; NF-κB, COX-2, iNOS and Hsp70 proteins expression upon heat stress in chicken hepatocytes. The PKC extract and silymarin were able to alleviate the expression of all of these biomarkers in heat-induced chicken hepatocytes. The gas chromatography-mass spectrometry analysis of PKC extract showed the presence of fatty acids, phenolic compounds, sugar derivatives and other organic compounds such as furfural which could be responsible for the observed hepatoprotective activity. Palm kernel cake extract could be a potential agent to protect hepatocytes function under heat induced oxidative stress.

  18. Protection of hepatocytes from cytotoxic T cell mediated killing by interferon-alpha.

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    Christian B Willberg

    Full Text Available BACKGROUND: Cellular immunity plays a key role in determining the outcome of hepatitis C virus (HCV infection, although the majority of infections become persistent. The mechanisms behind persistence are still not clear; however, the primary site of infection, the liver, may be critical. We investigated the ability of CD8+ T-cells (CTL to recognise and kill hepatocytes under cytokine stimulation. METHODS/PRINCIPLE FINDINGS: Resting hepatocytes cell lines expressed low levels of MHC Class I, but remained susceptible to CTL cytotoxicity. IFN-alpha treatment, in vitro, markedly increased hepatocyte MHC Class I expression, however, reduced sensitivity to CTL cytotoxicity. IFN-alpha stimulated hepatocyte lines were still able to present antigen and induce IFN-gamma expression in interacting CTL. Resistance to killing was not due to the inhibition of the FASL/FAS- pathway, as stimulated hepatocytes were still susceptible to FAS-mediated apoptosis. In vitro stimulation with IFN-alpha, or the introduction of a subgenomic HCV replicon into the HepG2 line, upregulated the expression of the granzyme-B inhibitor-proteinase inhibitor 9 (PI-9. PI-9 expression was also observed in liver tissue biopsies from patients with chronic HCV infection. CONCLUSION/SIGNIFICANCE: IFN-alpha induces resistance in hepatocytes to perforin/granzyme mediate CTL killing pathways. One possible mechanism could be through the expression of the PI-9. Hindrance of CTL cytotoxicity could contribute to the chronicity of hepatic viral infections.

  19. Biosafety in Ex Vivo Gene Therapy and Conditional Ablation of Lentivirally Transduced Hepatocytes in Nonhuman Primates

    Science.gov (United States)

    Menzel, Olivier; Birraux, Jacques; Wildhaber, Barbara E; Jond, Caty; Lasne, Françoise; Habre, Walid; Trono, Didier; Nguyen, Tuan H; Chardot, Christophe

    2009-01-01

    Ex vivo gene therapy is an interesting alternative to orthotopic liver transplantation (OLT) for treating metabolic liver diseases. In this study, we investigated its efficacy and biosafety in nonhuman primates. Hepatocytes isolated from liver lobectomy were transduced in suspension with a bicistronic liver-specific lentiviral vector and immediately autotransplanted (SLIT) into three cynomolgus monkeys. The vector encoded cynomolgus erythropoietin (EPO) and the conditional suicide gene herpes simplex virus-thymidine kinase (HSV-TK). Survival of transduced hepatocytes and vector dissemination were evaluated by detecting transgene expression and vector DNA. SLIT was safely performed within a day in all three subjects. Serum EPO and hematocrit rapidly increased post-SLIT and their values returned to baseline within about 1 month. Isoforms of EPO detected in monkeys' sera differed from the physiological renal EPO. In liver biopsies at months 8 and 15, we detected EPO protein, vector mRNA and DNA, demonstrating long-term survival and functionality of transplanted lentivirally transduced hepatocytes. Valganciclovir administration resulted in complete ablation of the transduced hepatocytes. We demonstrated the feasibility and biosafety of SLIT, and the long term (>1 year) functionality of lentivirally transduced hepatocytes in nonhuman primates. The HSV-TK/valganciclovir suicide strategy can increase the biosafety of liver gene therapy protocols by safely and completely ablating transduced hepatocytes on demand. PMID:19568222

  20. Significant improvement of survival by intrasplenic hepatocyte transplantation in totally hepatectomized rats.

    Science.gov (United States)

    Vogels, B A; Maas, M A; Bosma, A; Chamuleau, R A

    1996-01-01

    The effect of intrasplenic hepatocyte transplantation (HTX) was studied in an experimental model of acute liver failure in rats with chronic liver atrophy. Rats underwent a portacaval shunt operation on Day -14 to induce liver atrophy, and underwent total hepatectomy on Day 0 as a start of acute liver failure. Intrasplenic hepatocyte or sham transplantation was performed on Day -7,-3, or -1 (n = 4 to 6 per group). During the period following hepatectomy, mean arterial blood pressure was maintained above 80 mm Hg and hypoglycaemia was prevented. Severity of hepatic encephalopathy was assessed by clinical grading and EEG spectral analysis, together with determination of blood ammonia and plasma amino acid concentrations, and "survival" time. Histological examination of the spleen and lungs was performed after sacrifice. Intrasplenic hepatocyte transplantation resulted in a significant improvement in clinical grading in all transplanted groups (p 0.05), HTX at Day -3: 19.7 +/- 3.7 h vs. 6.5 +/- 0.3 h (p ammonia concentrations after total hepatectomy (p spleens after sacrifice showed clusters of hepatocytes in the red pulp. Hepatocytes present in the spleen for 3 and 7 days showed bile accumulation and spots of beginning necrosis. The present data show that in a hard model of complete liver failure in portacaval shunted rats, intrasplenic hepatocyte transplantation is able to prolong "survival" time significantly 2- to 3-fold. The relevance of this observation for human application is discussed.

  1. The Effect and Mechanism of Tamoxifen-Induced Hepatocyte Steatosis in Vitro

    Directory of Open Access Journals (Sweden)

    Fei Zhao

    2014-03-01

    Full Text Available The aim of this study was to determine the effect and mechanism of tamoxifen (TAM-induced steatosis in vitro. HepG 2 (Human hepatocellular liver carcinoma cell line cells were treated with different concentrations of TAM for 72 h. Steatosis of hepatocytes was determined after Oil Red O staining and measurement of triglyceride (TG concentration. The expressions of genes in the TG homeostasis pathway, including sterol regulatory element-binding protein-1c (SREBP-1c, peroxisome proliferator-activated receptor γ (PPARγ, CCAAT/enhancer-binding protein α (C/EBPα, fatty acid synthase (FAS, acetyl-CoA carboxylase (ACC, stearoyl-CoA desaturase (SCD, carnitine palmitoyltransferase 1 (CPT1 and microsomal triglyceride transfer protein (MTP, were examined using quantitative real-time PCR and Western blot analysis. Cell proliferation was examined using the cell counting kit-8 (CCK-8 assay. We found that hepatocytes treated with TAM had: (1 induced hepatocyte steatosis and increased hepatocyte TG; (2 upregulation of SREBP-1c, FAS, ACC, SCD and MTP mRNA expressions (300%, 600%, 70%, 130% and 160%, respectively; (3 corresponding upregulation of protein expression; and (4 no difference in HepG 2 cell proliferation. Our results suggest that TAM can induce hepatocyte steatosis in vitro and that the enhancement of fatty acid synthesis through the upregulations of SREBP-1c and its downstream target genes (FAS, ACC and SCD may be the key mechanism of TAM-induced hepatocyte steatosis.

  2. CM2 antigen, a potential novel molecule participating in glucuronide transport on rat hepatocyte canalicular membrane

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    L. Wang

    2012-06-01

    Full Text Available The polarized molecules predominately distributing at hepatocyte canalicular surface play a vital role in disclosing the process of bile formation and etiopathogenisis of cholestatic live diseases. Therefore, it is important to find novel polarized molecules on hepatocyte canalicular membrane. In the present study, canalicular membrane vesicles (CMVs isolated from rat hepatocyte by density gradient centrifugation were used as immunogens to produce hybridoma and 46 strains of monoclonal antibodies (mAb against CMVs were obtained. With a series of morphological assay methods, including immunohistochemistry, immunofluorescence and immuno-electron microscope, the antigens recognized by canalicular mAb1 (CM1 and canalicular mAb2 (CM2 were confirmed to predominately distribute at hepatocyte canalicular membrane. Transport activity assay revealed that CM2 could inhibit ATP-dependent E217βG uptake of rat hepatocyte CMVs. Meanwhile, Western blotting analysis showed that the molecular mass of CM2 antigen was approximately 110kDa, which was much less than Mr 180kDa of multidrug resistance-associated protein 2 (MRP2 involved in glucuronide transport. These data indicated that CM2 antigen might be a potential novel molecule participating in glucuronide transport on the hepatocyte canalicular membrane.

  3. Treatment of chronical myocardial ischemia by adenovirus-mediated hepatocyte growth factor gene transfer in minipigs

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Growth factor gene transfer-induced therapeutic angiogenesis has become a novel approach for the treatment of myocardial ischemia. In order to provide a basis for the clinical application of an adeno- virus with hepatocyte growth factor gene (Ad-HGF) in the treatment of myocardial ischemia, we estab- lished a minipig model of chronically ischemic myocardium in which an Ameroid constrictor was placed around the left circumflex branch of the coronary artery (LCX). A total of 18 minipigs were ran- domly divided into 3 groups: a surgery control group, a model group and an Ad-HGF treatment group implanted with Ameroid constrictor. Ad-HGF or the control agent was injected directly into the ischemic myocardium, and an improvement in heart function and blood supply were evaluated. The results showed that myocardial perfusion remarkably improved in the Ad-HGF group compared with that in both the control and model groups. Four weeks after the treatment, the density of newly formed blood vessels was higher and the number of collateral blood vessels was greater in the Ad-HGF group than in the model group. The area of myocardial ischemia reduced evidently and the left ventricular ejection fraction improved significantly in the Ad-HGF group. These results suggest that HGF gene therapy may become a novel approach in the treatment of chronically ischemic myocardium.

  4. Preconditioning of the liver for efficient repopulation by primary hepatocyte transplants.

    Science.gov (United States)

    Krause, Petra; Rave-Frank, Margret; Christiansen, Hans; Koenig, Sarah

    2014-01-01

    The therapeutic potential of liver cell transplantation has been demonstrated in multiple clinical studies to correct hereditary metabolic or chronic liver diseases. However, there are several outstanding issues, which need to be investigated: most notably donor cell engraftment and the subsequent selective expansion of transplanted cells. This protocol describes the preconditioning of the liver in a dipeptidyl peptidase-IV (DPPIV(-))-deficient rat model of efficient repopulation utilizing a selective external beam irradiation technique combined with regional transient portal ischemia (RTPI). Irradiation of the host liver impairs endogenous cell division, and the subsequent RTPI constitutes a strongly proliferative stimulus. Transplanted cells benefit from this stimulus, whereas endogenous cells have no ability to respond, due to a reduction in the mitotic capacity of the host liver. As described here, an effective preparative regime for liver repopulation is external beam liver irradiation in the form of a single dose of 25 Gy applied to the whole organ followed (4 days later) by RTPI of the right liver lobes lasting 90 min. After 1 h of reperfusion, the donor hepatocytes may be transplanted directly into the spleen as implantation site for further redistribution into the portal system and liver. This preparative regime certainly has the potential to be implemented in the clinic, since neither toxins nor highly potent carcinogens are used.

  5. Microstructured multi-well plate for three-dimensional packed cell seeding and hepatocyte cell culture.

    Science.gov (United States)

    Goral, Vasiliy N; Au, Sam H; Faris, Ronald A; Yuen, Po Ki

    2014-07-01

    In this article, we present a microstructured multi-well plate for enabling three-dimensional (3D) high density seeding and culture of cells through the use of a standard laboratory centrifuge to promote and maintain 3D tissue-like cellular morphology and cell-specific functionality in vitro without the addition of animal derived or synthetic matrices or coagulants. Each well has microfeatures on the bottom that are comprised of a series of ditches/open microchannels. The dimensions of the microchannels promote and maintain 3D tissue-like cellular morphology and cell-specific functionality in vitro. After cell seeding with a standard pipette, the microstructured multi-well plates were centrifuged to tightly pack cells inside the ditches in order to enhance cell-cell interactions and induce formation of 3D cellular structures during cell culture. Cell-cell interactions were optimized based on cell packing by considering dimensions of the ditches/open microchannels, orientation of the microstructured multi-well plate during centrifugation, cell seeding density, and the centrifugal force and time. With the optimized cell packing conditions, we demonstrated that after 7 days of cell culture, primary human hepatocytes adhered tightly together to form cord-like structures that resembled 3D tissue-like cellular architecture. Importantly, cell membrane polarity was restored without the addition of animal derived or synthetic matrices or coagulants.

  6. Protective effect of mild endoplasmic reticulum stress on radiation-induced bystander effects in hepatocyte cells

    Science.gov (United States)

    Xie, Yuexia; Ye, Shuang; Zhang, Jianghong; He, Mingyuan; Dong, Chen; Tu, Wenzhi; Liu, Peifeng; Shao, Chunlin

    2016-01-01

    Radiation-induced bystander effect (RIBE) has important implications for secondary cancer risk assessment during cancer radiotherapy, but the defense and self-protective mechanisms of bystander normal cells are still largely unclear. The present study found that micronuclei (MN) formation could be induced in the non-irradiated HL-7702 hepatocyte cells after being treated with the conditioned medium from irradiated hepatoma HepG2 cells under either normoxia or hypoxia, where the ratio of the yield of bystander MN induction to the yield of radiation-induced MN formation under hypoxia was much higher than that of normoxia. Nonetheless, thapsigargin induced endoplasmic reticulum (ER) stress and dramatically suppressed this bystander response manifested as the decrease of MN and apoptosis inductions. Meanwhile, the interference of BiP gene, a major ER chaperone, amplified the detrimental RIBE. More precisely, thapsigargin provoked ER sensor of PERK to initiate an instantaneous and moderate ER stress thus defensed the hazard form RIBE, while BiP depletion lead to persistently destroyed homeostasis of ER and exacerbated cell injury. These findings provide new insights that the mild ER stress through BiP-PERK-p-eIF2α signaling pathway has a profound role in protecting cellular damage from RIBE and hence may decrease the potential secondary cancer risk after cancer radiotherapy. PMID:27958308

  7. Effects of carbon tetrachloride on oxidative stress, inflammatory response and hepatocyte apoptosis in common carp (Cyprinus carpio)

    Energy Technology Data Exchange (ETDEWEB)

    Jia, Rui [Wuxi Fisheries College, Nanjing Agricultural University, Wuxi 214081 (China); Key Laboratory of Freshwater Fisheries and Germplasm Resources Utilization, Ministry of Agriculture, Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences, Wuxi 214081 (China); Cao, Li-Ping; Du, Jin-Liang [Key Laboratory of Freshwater Fisheries and Germplasm Resources Utilization, Ministry of Agriculture, Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences, Wuxi 214081 (China); International Joint Research Laboratory for Fish Immunopharmacology, Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences, Wuxi 214081 (China); Wang, Jia-Hao; Liu, Ying-Juan [Wuxi Fisheries College, Nanjing Agricultural University, Wuxi 214081 (China); Jeney, Galina [National Agricultural Research Center, Research Institute for Fisherie and, Aquaculture, Anna Light 8, Szarvas 5440 (Hungary); Xu, Pao, E-mail: xup@ffrc.cn [Wuxi Fisheries College, Nanjing Agricultural University, Wuxi 214081 (China); Key Laboratory of Freshwater Fisheries and Germplasm Resources Utilization, Ministry of Agriculture, Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences, Wuxi 214081 (China); Yin, Guo-Jun, E-mail: yingj@ffrc.cn [Wuxi Fisheries College, Nanjing Agricultural University, Wuxi 214081 (China); Key Laboratory of Freshwater Fisheries and Germplasm Resources Utilization, Ministry of Agriculture, Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences, Wuxi 214081 (China); International Joint Research Laboratory for Fish Immunopharmacology, Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences, Wuxi 214081 (China)

    2014-07-01

    Highlights: • We explored the underlying toxicology of CCl{sub 4} at the cellular and molecular levels. • QRT-PCR detected the gene expression of NF-κB and inflammatory cytokines. • The apoptosis and necrosis occurred simultaneously in carp liver damage. • CCl{sub 4} activated the TNF-α/NF-κB and TRL4/NF-κB signaling pathways. - Abstract: In the present study, the cellular and molecular mechanism of carbon tetrachloride (CCl{sub 4})-induced hepatotoxicity in fish was investigated by studying the effects of CCl{sub 4} on the oxidative stress, inflammatory response and hepatocyte apoptosis. Common carp were given an intraperitoneal injection of 30% CCl{sub 4} in arachis oil (0.5 ml/kg body weight). At 72 h post-injection, blood were collected to measure glutamate pyruvate transaminase (GPT), glutamate oxalate transaminase (GOT), superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT), glutathione (GSH), total antioxidant capacity (T-AOC) and malondialdehyde (MDA), liver samples were taken to analyze toll-like receptor 4 (TLR4), cytochrome P450 2E1 (CYP2E1) and gene expressions of inflammatory cytokines and nuclear factor-κB (NF-κB/cREL). Cell viability and apoptosis were analyzed after treatment of the primary hepatocytes with CCl{sub 4} at 8 mM. The results showed that CCl{sub 4} significantly increased the levels of GPT, GOT, MDA, TLR4 and CYP2E1, reduced the levels of SOD, GPx, CAT, GSH and T-AOC, and up-regulated the gene expressions of NF-κB/cREL and inflammatory cytokines including tumor necrosis factor-α (TNF-α), inducible nitric oxide synthase (iNOS), IL-1β, IL-6 and IL-12. In vitro, CCl{sub 4} caused a dramatic loss in cell viability and induced hepatocyte apoptosis. Overall results suggest that oxidative stress lipid peroxidation, and TNF-α/NF-κB and TRL4/NF-κB signaling pathways play important roles in CCl{sub 4}-induced hepatotoxicity in fish.

  8. Leflunomide attenuates hepatocyte injury by inhibiting Kupffer cells

    Institute of Scientific and Technical Information of China (English)

    Hong-Wei Yao; Jun Li; Ji-Qiang Chen; Shu-Yun Xu

    2004-01-01

    AIM: To investigate the importance of direct contact between Kupffer cells (KCs) and hepatocytes (HCs) during hepatic inflammatory responses, and the effect of leflunomide′s active metabolite, A771726, on cytokines in KCs, HCs and KC cocultures (DC cocultures).METHODS: KCs and HCs in liver were isolated by digestion with pronase and collagenase. Lipopolysaccharide (LPS)-induced inflammatory response in monocultures of rat HCs and KCs was compared with that in DC cocultures. Tumor necrosis factor-α (TNF-α) and interleukin-1 (IL-1)concentrations in different culture supernatants were measured with ELISA. TNF-α mRNA in KCs of inflammatory liver injury was analyzed with reverse transcriptase polymerase chain reaction (RT-PCR).RESULTS: DC cocultures strongly exhibited the production of TNF-α and IL-1 compared with other cultures, and these cytokines were mainly produced by KCs, especially by activated KCs. Time course studies revealed an increased production of TNF-α preceding the IL-1 production,suggesting that increased TNF-α levels could be involved in the increase of IL-1 production. Leflunomide′s active metabolite, A771726, had significantly inhibitory effect on TNF-αand IL-1 at protein and transcription levels, and the reduced production of IL-1 by A771726 was associated with the inhibitory action of A771726 on TNF-α.

  9. Pathogenesis of selective insulin resistance in isolated hepatocytes.

    Science.gov (United States)

    Cook, Joshua R; Langlet, Fanny; Kido, Yoshiaki; Accili, Domenico

    2015-05-29

    The development of insulin resistance (IR) in the liver is a key pathophysiologic event in the development of type 2 diabetes. Although insulin loses its ability to suppress glucose production, it largely retains its capacity to drive lipogenesis. This selective IR results in the characteristic hyperglycemia and dyslipidemia of type 2 diabetes. The delineation of two branched pathways of insulin receptor (InsR) signaling to glucose versus triglyceride production, one through FoxO and the other through SREBP-1c, provides a mechanism to account for this pathophysiological abnormality. We tested the complementary hypothesis that selective IR arises due to different intrinsic sensitivities of glucose production versus de novo lipogenesis to insulin as a result of cell-autonomous down-regulation of InsR number in response to chronic hyperinsulinemia. We demonstrate in mouse primary hepatocytes that chronic hyperinsulinemia abrogates insulin's inhibition of glucose production, but not its stimulation of de novo lipogenesis. Using a competitive inhibitor of InsR, we show that there is a 4-fold difference between levels of InsR inhibition required to cause resistance of glucose production versus lipogenesis to the actions of insulin. Our data support a parsimonious model in which differential InsR activation underlies the selective IR of glucose production relative to lipogenesis, but both processes require signaling through Akt1/2. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. 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.

  11. Amidine-bearing lipoplex targeting to hepatocyte cells

    Institute of Scientific and Technical Information of China (English)

    Yasuya Kudo; Kazunori Koiwai; Kazuhiro Shimizu; Shota Kusuki; Mina Sakuragi; Naohiko Shimada; Yoichi Takeda; Kazuo Sakurai

    2008-01-01

    A lipoplex (i.e., pDNA#1/lipid complex and transfection reagent for pDNA delivery) containing galactosylceramide (GalCer) and an amidine-bearing lipid (TRX) was examined whether the bound pDNA was specifically ingested by hepatocyte via asialoglycoprotein receptor (ASGPR) and then expressed protein. Gel electrophoresis and small-angle X-ray scattering (SAXS) confirmed that the TRX-GalCer liposome#2 complexed with pDNA and the resultant lipoplex took a hexagonally packed inverted cylinder structure when the GalCer composition was less than 20 wt.% of the total lipid. When the lipoplex carrying pGL3 (luciferase-cording pDNA) was administrated to HepG2, the luciferase activity was increased with increasing the GalCer composition until it reached 3 wt.% and then decreased upon further addition of GalCer. When we added galactose itself as a competitor, the luciferase activity was decreased, while glucose did not show such decrease, suggesting that HepG2 ingested the lipoplex via ASGPR-mediated endocytosis. This paper indicated that the hexagonally packed inverted cylinder structures of lipoplex may not always provide excellent transfection and presented a possibility that the TRX lipoplex#3 can obtain a cellular-targeting ability through the receptors for oligosaccharide.

  12. Protein phosphorylation in isolated hepatocytes of septic and endotoxemic rats

    Energy Technology Data Exchange (ETDEWEB)

    Deaciuc, I.V.; Spitzer, J.A. (Louisiana State Univ. Medical Center, New Orleans (USA))

    1989-11-01

    The purpose of this study was to investigate possible alterations induced by sepsis and endotoxicosis in the late phase of Ca2+-dependent signaling in rat liver. Hepatocytes isolated from septic or chronically endotoxin (ET)-treated rats were labeled with (32P)H3PO4 and stimulated with various agents. Proteins were resolved by one-dimensional polyacrylamide gel electrophoresis and autoradiographed. Vasopressin (VP)- and phenylephrine (PE)-induced responses were attenuated in both septic and ET-treated rats for cytosolic and membrane proteins compared with their respective controls. Glucagon and 12-O-myristate phorbol-13-acetate (TPA) affected only the phosphorylation of membrane proteins. Glucagon-induced changes in the phosphorylation of membrane proteins were affected by both sepsis and endotoxicosis, whereas TPA-stimulated phosphorylation was lowered only in endotoxicosis. Response to the Ca2+ ionophore A23187 was depressed in septic rats for cytosolic proteins. The phosphorylation of two cytosolic proteins, i.e., 93 and 61 kDa (previously identified as glycogen phosphorylase and pyruvate kinase, respectively), in response to VP, PE, and A23187 was severely impaired by endotoxicosis and sepsis. TPA did not affect the phosphorylation state of these two proteins. The results show that sepsis and endotoxicosis produce perturbations of the phosphorylation step in Ca2+ transmembrane signaling. Such changes can explain alterations of glycogenolysis and gluconeogenesis associated with sepsis and endotoxicosis.

  13. Hepatocyte growth factor, a biomarker of macroangiopathy in diabetes mellitus

    Institute of Scientific and Technical Information of China (English)

    Hiroyuki; Konya; Masayuki; Miuchi; Kahori; Satani; Satoshi; Matsutani; Taku; Tsunoda; Yuzo; Yano; Tomoyuki; Katsuno; Tomoya; Hamaguchi; Jun-Ichiro; Miyagawa; Mitsuyoshi; Namba

    2014-01-01

    Atherosclerotic involvements are an essential causal element of prospect in diabetes mellitus(DM), with carotid atherosclerosis(CA) being a common risk-factor for prospective crisis of coronary artery diseases(CAD) and/or cerebral infarction(CI) in DM subjects. From another point of view, several reports have supplied augmenting proof that hepatocyte growth factor(HGF) has a physiopathological part in DM involvements. HGF has been a mesenchymal-derived polyphenic factor which modulates development, motion, and morphosis of diverse cells, and has been regarded as a humor intermediator of epithelial-mesenchymal interplays. The serum concentrations of HGF have been elevated in subjects with CAD and CI, especially during the acute phase of both disturbances. In our study with 89 type 2 DM patients, the association between serum concentrationsof HGF and risk-factors for macrovascular complicationsinclusive of CA were examined. The average of serumHGF levels in the subjects was more elevated than thereference interval. The serum HGF concentrations associated positively with both intimal-media thickness(IMT)(r = 0.24, P = 0.0248) and plaque score(r = 0.27, P =0.0126), indicating a relationship between the elevatedHGF concentrations and advancement of CA involvements. Multivariate statistical analysis accentuated thatserum concentrations of HGF would be associated inde-pendently with IMT(standardized = 0.28, P = 0.0499).The review indicates what is presently known regardingserum HGF might be a new and meaningful biomarkerof macroangiopathy in DM subjects.

  14. Nuclear lactate dehydrogenase modulates histone modification in human hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Castonguay, Zachary; Auger, Christopher; Thomas, Sean C.; Chahma, M’hamed; Appanna, Vasu D., E-mail: vappanna@laurentian.ca

    2014-11-07

    Highlights: • Nuclear LDH is up-regulated under oxidative stress. • SIRT1 is co-immunoprecipitated bound to nuclear LDH. • Nuclear LDH is involved in histone deacetylation and epigenetics. - Abstract: It is becoming increasingly apparent that the nucleus harbors metabolic enzymes that affect genetic transforming events. Here, we describe a nuclear isoform of lactate dehydrogenase (nLDH) and its ability to orchestrate histone deacetylation by controlling the availability of nicotinamide adenine dinucleotide (NAD{sup +}), a key ingredient of the sirtuin-1 (SIRT1) deacetylase system. There was an increase in the expression of nLDH concomitant with the presence of hydrogen peroxide (H{sub 2}O{sub 2}) in the culture medium. Under oxidative stress, the NAD{sup +} generated by nLDH resulted in the enhanced deacetylation of histones compared to the control hepatocytes despite no discernable change in the levels of SIRT1. There appeared to be an intimate association between nLDH and SIRT1 as these two enzymes co-immunoprecipitated. The ability of nLDH to regulate epigenetic modifications by manipulating NAD{sup +} reveals an intricate link between metabolism and the processing of genetic information.

  15. La expresión corporal en la formación de maestros The Body Expression in Teachers Forming: Beginning Programs Study at Universities in Bogotá

    Directory of Open Access Journals (Sweden)

    Blanco Vega María De Jesús

    2011-01-01

    Full Text Available Esta investigación tuvo como propósito indagar las orientaciones, enfoques y perspectivas de la expresión corporal en los programas de Educación Inicial en universidades de Bogotá. El método desarrollado fue el cualitativo y el tipo de estudio documental. Se diseñó y aplicó una matriz como instrumento de análisis y descripción basado en la categorización de la información teniendo en cuenta los documentos fuente: Proyecto Educativo Institucional (PEI, Proyecto Educativo del Programa (PEP y Planes de Estudio. A manera de conclusiones se puede decir que las orientaciones teórico-conceptuales están fundamentadas en el eje pedagógico, las perspectivas de formación se centran en el campo disciplinar, profesionalización específica y complementaria; y los enfoques pedagógicos y metodológicos están basados en el cuerpo y la expresión. This investigation inquires guidance, approaches and perspectives about corporal expression in beginning education at universities of Bogota. The qualitative method and documentary study type was put into practice. Information setting up from source documents: PEI, PEP and Study Plans provided a Matrix designed and applied to analyze and describe the pedagogical line. Forming perspectives are placed in the discipline field and in the specific and complementary professional activity. The pedagogic and method approaches are based on body and expression.

  16. Body Clock

    Institute of Scientific and Technical Information of China (English)

    刘洪毓

    2000-01-01

    Body clocks” are biological methods of controling body activities.Every living thing has one. In humans, a body clock controls normal periods of sleeping and waking. It controls the time swhen you are most likely to feel pain.Eating, sleeping and exercising at about the same time each day will help keep body activities normal. But changes in your life, a new job, for example, destroy the balance and thus cause health problems.

  17. Uptake of albumin nanoparticle surface modified with glycyrrhizin by primary cultured rat hepatocytes

    Institute of Scientific and Technical Information of China (English)

    Sheng-Jun Mao; Shi-Xiang Hou; Ru He; Liang-Ke Zhang; Da-Peng Wei; Yue-Qi Bi; Hui Jin

    2005-01-01

    AIM: To investigate the uptake difference between bovine serum albumin nanoparticle (BSA-NP) and bovine serum albumin nanoparticles with their surface modified byglycyrrhizin (BSA-NP-GL) and to develop a novel hepatocyte targeting BSA-NP-GL based on active targeting technology mediated by specific binding site of GL on rat cellular membrane. METHODS: Calcein loaded bovine serum albumin nanoparticles (Cal-BSA-NP) were prepared by desolvation process. Glycyrrhizin was conjugated to the surface reactive amino groups (SRAG) of Cal-BSA-NP by sodium periodate oxidization, which resulted in calcein-loaded bovine serum albumin nanoparticles with their surface modified by glycyrrhizin (Cal-BSA-NP-GL). The morphology of the two types of prepared nanoparticles (NP) was observed by transmission electron microscopy. The diameter of NP was measured with a laser particle size analyzer. The interaction between Cal-BSA-NP-GL and primary cultured hepatocytes was studied through cellular uptake experiments. The uptake amount of Cal-BSA-NPGL and Cal-BSA-NP by rat hepatocytes was determinedby fluorospectrophotometry. Uptake characteristics were investigated through experiments of competitive inhibition of specific binding site of GL. RESULTS: Both Cal-BSA-NP-GL and Cal-BSA-NP had regular spherical surfaces. The average diameter of CalBSA-NP-GL and Cal-BSA-NP was 77 and 79 nm respectively. The uptake amount of the two NP by hepatocytes reached its maximum at 2 h after incubation. The uptake amount of Cal-BSA-NP-GL by rat hepatocytes was 4.43-fold higher than that of Cal-BSA-NP. There was a significant difference in the uptake of Cal-BSA-NP-GL and Cal-BSA-NP by hepatocytes (P<0.01). The uptake of Cal-BSA-NP-GL was inhibited when GL was added previously to isolated rat hepatocytes, and the uptake of Cal-BSA-NP was not affected by GL.CONCLUSION: A binding site of GL is present on the surface of rat hepatocytes, BSA-NP-GL may be internalized via this site by hepatocytes and can be used as

  18. Foxa1 reduces lipid accumulation in human hepatocytes and is down-regulated in nonalcoholic fatty liver.

    Directory of Open Access Journals (Sweden)

    Marta Moya

    Full Text Available Triglyceride accumulation in nonalcoholic fatty liver (NAFL results from unbalanced lipid metabolism which, in the liver, is controlled by several transcription factors. The Foxa subfamily of winged helix/forkhead box (Fox transcription factors comprises three members which play important roles in controlling both metabolism and homeostasis through the regulation of multiple target genes in the liver, pancreas and adipose tissue. In the mouse liver, Foxa2 is repressed by insulin and mediates fasting responses. Unlike Foxa2 however, the role of Foxa1 in the liver has not yet been investigated in detail. In this study, we evaluate the role of Foxa1 in two human liver cell models, primary cultured hepatocytes and HepG2 cells, by adenoviral infection. Moreover, human and rat livers were analyzed to determine Foxa1 regulation in NAFL. Results demonstrate that Foxa1 is a potent inhibitor of hepatic triglyceride synthesis, accumulation and secretion by repressing the expression of multiple target genes of these pathways (e.g., GPAM, DGAT2, MTP, APOB. Moreover, Foxa1 represses the fatty acid transporter protein FATP2 and lowers fatty acid uptake. Foxa1 also increases the breakdown of fatty acids by inducing peroxisomal fatty acid β-oxidation and ketone body synthesis. Finally, Foxa1 is able to largely up-regulate UCP1, thereby dissipating energy and consistently decreasing the mitochondria membrane potential. We also report that human and rat NAFL have a reduced Foxa1 expression, possibly through a protein kinase C-dependent pathway. We conclude that Foxa1 is an antisteatotic factor that coordinately tunes several lipid metabolic pathways to block triglyceride accumulation in hepatocytes. However, Foxa1 is down-regulated in human and rat NAFL and, therefore, increasing Foxa1 levels could protect from steatosis. Altogether, we suggest that Foxa1 could be a novel therapeutic target for NAFL disease and insulin resistance.

  19. [Differences in dynamics of insulin and insulin-like growth I (IGF-I) receptors internalization in isolated rat hepatocytes].

    Science.gov (United States)

    2013-01-01

    Insulin and IGF-I are two related peptides performing in the mammalian body functionally different roles of the metabolic and growth hormones, respectively. Internalization of the insulin-receptor complex (IRC) is the most important chain of mechanism of the action of hormone. To elucidate differences in the main stages of internalization of the two related hormones, the internalization dynamics of 125I-insulin and 125I-IGF-I was traced in isolated rat hepatocytes at 37 and 12 degrees C. There were established marked differences in the process of internalization of labeled hormones, which is stimulated by insulin and IGF-I. At 37 degrees C the insulin-stimulated internalization, unlike the process initiated by IGF-I, did not reach the maximal level for 1 h of incubation. However, essential differences in the internalization course of these two related peptide were obvious at the temperature of 12 degrees C. The internalization level of insulin receptors at 12 degrees C decreased by one third in spite of a significant increase of the insulin receptor binding on the hepatocytes plasma membrane. At 12 degrees C a slight decrease of the proportion of intracellular 125I-IGF-I correlated with a decrease in the 125I-IGF-I binding to receptors on the cell membrane. Internalization of IGF-I receptors was not affected by low temperature, as neither its level, nor the rate changed at 12 degrees C. The paradoxical decrease of the insulin-stimulated internalization at low temperature seems to represent a peculiar "inhibition mechanism" of immersion of IRC into the cell, which leads to accumulation of the complexes on the cell surface and possibly to a readjustment of the insulin biological activity. The resistance of internalization of the IGF-I receptor to cold seems to be related to the more ancient origin of this mechanism in the poikilothermal vertebrates.

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

    Directory of Open Access Journals (Sweden)

    Hua-Qing Wu

    2017-07-01

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

  1. Foxa1 Reduces Lipid Accumulation in Human Hepatocytes and Is Down-Regulated in Nonalcoholic Fatty Liver

    Science.gov (United States)

    Moya, Marta; Benet, Marta; Guzmán, Carla; Tolosa, Laia; García-Monzón, Carmelo; Pareja, Eugenia; Castell, José Vicente; Jover, Ramiro

    2012-01-01

    Triglyceride accumulation in nonalcoholic fatty liver (NAFL) results from unbalanced lipid metabolism which, in the liver, is controlled by several transcription factors. The Foxa subfamily of winged helix/forkhead box (Fox) transcription factors comprises three members which play important roles in controlling both metabolism and homeostasis through the regulation of multiple target genes in the liver, pancreas and adipose tissue. In the mouse liver, Foxa2 is repressed by insulin and mediates fasting responses. Unlike Foxa2 however, the role of Foxa1 in the liver has not yet been investigated in detail. In this study, we evaluate the role of Foxa1 in two human liver cell models, primary cultured hepatocytes and HepG2 cells, by adenoviral infection. Moreover, human and rat livers were analyzed to determine Foxa1 regulation in NAFL. Results demonstrate that Foxa1 is a potent inhibitor of hepatic triglyceride synthesis, accumulation and secretion by repressing the expression of multiple target genes of these pathways (e.g., GPAM, DGAT2, MTP, APOB). Moreover, Foxa1 represses the fatty acid transporter protein FATP2 and lowers fatty acid uptake. Foxa1 also increases the breakdown of fatty acids by inducing peroxisomal fatty acid β-oxidation and ketone body synthesis. Finally, Foxa1 is able to largely up-regulate UCP1, thereby dissipating energy and consistently decreasing the mitochondria membrane potential. We also report that human and rat NAFL have a reduced Foxa1 expression, possibly through a protein kinase C-dependent pathway. We conclude that Foxa1 is an antisteatotic factor that coordinately tunes several lipid metabolic pathways to block triglyceride accumulation in hepatocytes. However, Foxa1 is down-regulated in human and rat NAFL and, therefore, increasing Foxa1 levels could protect from steatosis. Altogether, we suggest that Foxa1 could be a novel therapeutic target for NAFL disease and insulin resistance. PMID:22238690

  2. Toxicity of different forms of graphene in a chicken embryo model.

    Science.gov (United States)

    Szmidt, Maciej; Sawosz, Ewa; Urbańska, Kaja; Jaworski, Sławomir; Kutwin, Marta; Hotowy, Anna; Wierzbicki, Mateusz; Grodzik, Marta; Lipińska, Ludwika; Chwalibog, André

    2016-10-01

    In the present work, the toxicity of three forms of graphene: pristine graphene (pG), graphene oxide (GO), and reduced graphene oxide (rGO) was investigated using a chicken embryo model. Fertilized chicken eggs were divided into the control group and groups administered with pG, GO, and rGO, in concentrations of 50, 500, and 5000 μg/ml. The experimental solutions were injected in ovo into the eggs, and at day 18 of incubation, the embryo survival, body and organ weights, the ultrastructure of liver samples, and the concentration of 8-hydroxy-2'-deoxyguanosine (8-OHdG) in the livers were measured. Survival of embryos decreased significantly after treatment with all types of graphene, but not in a dose-dependent manner. The body weights were only slightly affected by the highest doses of graphene, while the organ weights were not different among treatment groups. In all experimental groups, atypical hepatocyte ultrastructure and mitochondrial damage were observed. The concentration of the marker of DNA damage 8-OHdG in the liver significantly decreased after pG and rGO treatments.