Hydrolysis of pyrethroids by human and rat tissues: Examination of intestinal, liver and serum carboxylesterases

International Nuclear Information System (INIS)

Crow, J. Allen; Borazjani, Abdolsamad; Potter, Philip M.; Ross, Matthew K.

2007-01-01

Hydrolytic metabolism of pyrethroid insecticides in humans is one of the major catabolic pathways that clear these compounds from the body. Rodent models are often used to determine the disposition and clearance rates of these esterified compounds. In this study the distribution and activities of esterases that catalyze pyrethroid metabolism have been investigated in vitro using several human and rat tissues, including small intestine, liver and serum. The major esterase in human intestine is carboxylesterase 2 (hCE2). We found that the pyrethroid trans-permethrin is effectively hydrolyzed by a sample of pooled human intestinal microsomes (5 individuals), while deltamethrin and bioresmethrin are not. This result correlates well with the substrate specificity of recombinant hCE2 enzyme. In contrast, a sample of pooled rat intestinal microsomes (5 animals) hydrolyze trans-permethrin 4.5-fold slower than the sample of human intestinal microsomes. Furthermore, it is demonstrated that pooled samples of cytosol from human or rat liver are ∼ 2-fold less hydrolytically active (normalized per mg protein) than the corresponding microsomal fraction toward pyrethroid substrates; however, the cytosolic fractions do have significant amounts (∼ 40%) of the total esteratic activity. Moreover, a 6-fold interindividual variation in carboxylesterase 1 protein expression in human hepatic cytosols was observed. Human serum was shown to lack pyrethroid hydrolytic activity, but rat serum has hydrolytic activity that is attributed to a single CE isozyme. We purified the serum CE enzyme to homogeneity to determine its contribution to pyrethroid metabolism in the rat. Both trans-permethrin and bioresmethrin were effectively cleaved by this serum CE, but deltamethrin, esfenvalerate, alpha-cypermethrin and cis-permethrin were slowly hydrolyzed. Lastly, two model lipase enzymes were examined for their ability to hydrolyze pyrethroids. However, no hydrolysis products could be detected

Expression and kinetic properties of a recombinant 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase isoenzyme of human liver.

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Deyashiki, Y; Tamada, Y; Miyabe, Y; Nakanishi, M; Matsuura, K; Hara, A

1995-08-01

Human liver cytosol contains multiple forms of 3 alpha-hydroxysteroid dehydrogenase and dihydrodiol dehydrogenase with hydroxysteroid dehydrogenase activity, and multiple cDNAs for the enzymes have been cloned from human liver cDNA libraries. To understand the relationship of the multiple enzyme froms to the genes, a cDNA, which has been reported to code for an isoenzyme of human liver 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase, was expressed in Escherichia coli. The recombinant enzyme showed structural and functional properties almost identical to those of the isoenzyme purified from human liver. In addition, the recombinant isoenzyme efficiently reduced 5 alpha-dihydrotestosterone and 5 beta-dihydrocortisone, the known substrates of human liver 3 alpha-hydroxysteroid dehydrogenase and chlordecone reductase previously purified, which suggests that these human liver enzymes are identical. Furthermore, the steady-state kinetic data for NADP(+)-linked (S)-1-indanol oxidation by the recombinant isoenzyme were consistent with a sequential ordered mechanism in which NADP+ binds first. Phenolphthalein inhibited this isoenzyme much more potently than it did the other human liver dihydrodiol dehydrogenases, and was a competitive inhibitor (Ki = 20 nM) that bound to the enzyme-NADP+ complex.

Total and cytosolic concentrations of twenty metals/metalloids in the liver of brown trout Salmo trutta (Linnaeus, 1758) from the karstic Croatian river Krka.

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Dragun, Zrinka; Filipović Marijić, Vlatka; Krasnići, Nesrete; Ivanković, Dušica; Valić, Damir; Žunić, Jakov; Kapetanović, Damir; Smrzlić, Irena Vardić; Redžović, Zuzana; Grgić, Ivana; Erk, Marijana

2018-01-01

Total and cytosolic concentrations of twenty metals/metalloids in the liver of brown trout Salmo trutta (Linnaeus, 1758) were studied in the period from April 2015 to May 2016 at two sampling sites on Croatian river Krka, to establish if river water contamination with metals/metalloids downstream of Knin town has influenced metal bioaccumulation in S. trutta liver. Differences were observed between two sites, with higher concentrations of several elements (Ag, As, Ca, Co, Na, Se, Sr, V) found downstream of Knin town, whereas few others (Cd, Cs, Mo, Tl) were, unexpectedly, increased at the Krka River spring. However, total metal/metalloid concentrations in the liver of S. trutta from both sites of the Krka River were still mainly below previously reported levels for pristine freshwaters worldwide. The analysis of seasonal changes of metal/metalloid concentrations in S. trutta liver and their association with fish sex and size mostly indicated their independence of fish physiology, making them good indicators of water contamination and exposure level. Metal/metalloid concentrations in the metabolically available hepatic cytosolic fractions reported in this study are the first data of that kind for S. trutta liver, and the majority of analyzed elements were present in the cytosol in the quantity higher than 50% of their total concentrations, thus indicating their possible availability for toxic effects. However, the special attention should be directed to As, Cd, Cs, and Tl, which under the conditions of increased exposure tended to accumulate more within the cytosol. Although metal/metalloid concentrations in S. trutta liver were still rather low, monitoring of the Krka River water quality and of the health status of its biota is essential due to a trend of higher metal/metalloid bioaccumulation downstream of Knin town, especially taking into consideration the proximity of National Park Krka and the need for its conservation. Copyright © 2017 Elsevier Inc. All

Effect of starvation, diabetes and insulin on the casein kinase 2 from rat liver cytosol.

OpenAIRE

Martos, C; Plana, M; Guasch, M D; Itarte, E

1985-01-01

Starvation, diabetes and insulin did not alter the concentration of casein kinases in rat liver cytosol. However, the Km for casein of casein kinase 2 from diabetic rats was about 2-fold lower than that from control animals. Administration of insulin to control rats did not alter this parameter, but increased the Km for casein of casein kinase 2 in diabetic rats. Starvation did not affect the kinetic constants of casein kinases. The effect of diabetes on casein kinase 2 persisted after partia...

Dipeptidyl peptidase-4 greatly contributes to the hydrolysis of vildagliptin in human liver.

Science.gov (United States)

Asakura, Mitsutoshi; Fujii, Hideaki; Atsuda, Koichiro; Itoh, Tomoo; Fujiwara, Ryoichi

2015-04-01

The major metabolic pathway of vildagliptin in mice, rats, dogs, and humans is hydrolysis at the cyano group to produce a carboxylic acid metabolite M20.7 (LAY151), whereas the major metabolic enzyme of vildagliptin has not been identified. In the present study, we determined the contribution rate of dipeptidyl peptidase-4 (DPP-4) to the hydrolysis of vildagliptin in the liver. We performed hydrolysis assay of the cyano group of vildagliptin using mouse, rat, and human liver samples. Additionally, DPP-4 activities in each liver sample were assessed by DPP-4 activity assay using the synthetic substrate H-glycyl-prolyl-7-amino-4-methylcoumarin (Gly-Pro-AMC). M20.7 formation rates in liver microsomes were higher than those in liver cytosol. M20.7 formation rate was significantly positively correlated with the DPP-4 activity using Gly-Pro-AMC in liver samples (r = 0.917, P vildagliptin hydrolysis in the liver. Additionally, we established stable single expression systems of human DPP-4 and its R623Q mutant, which is the nonsynonymous single-nucleotide polymorphism of human DPP-4, in human embryonic kidney 293 (HEK293) cells to investigate the effect of R623Q mutant on vildagliptin-hydrolyzing activity. M20.7 formation rate in HEK293 cells expressing human DPP-4 was significantly higher than that in control HEK293 cells. Interestingly, R623Q mutation resulted in a decrease of the vildagliptin-hydrolyzing activity. Our findings might be useful for the prediction of interindividual variability in vildagliptin pharmacokinetics. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

Copper, Zinc Superoxide Dismutase is Primarily a Cytosolic Protein in Human Cells

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Crapo, James D.; Oury, Tim; Rabouille, Catherine; Slot, Jan W.; Chang, Ling-Yi

1992-11-01

The intracellular localization of human copper, zinc superoxide dismutase (Cu,Zn-SOD; superoxide:superoxide oxidoreductase, EC 1.15.1.1) was evaluated by using EM immunocytochemistry and both isolated human cell lines and human tissues. Eight monoclonal antibodies raised against either native or recombinant human Cu,Zn-SOD and two polyclonal antibodies raised against either native or recombinant human Cu,Zn-SOD were used. Fixation with 2% paraformaldehyde/0.2% glutaraldehyde was found necessary to preserve normal distribution of the protein. Monoclonal antibodies were less effective than polyclonal antibodies in recognizing the antigen after adequate fixation of tissue. Cu,Zn-SOD was found widely distributed in the cell cytosol and in the cell nucleus, consistent with it being a soluble cytosolic protein. Mitochondria and secretory compartments did not label for this protein. In human cells, peroxisomes showed a labeling density slightly less than that of cytoplasm.

Cytosolic and nuclear caspase-8 have opposite impact on survival after liver resection for hepatocellular carcinoma

International Nuclear Information System (INIS)

Koschny, Ronald; Schemmer, Peter; Schirmacher, Peter; Ganten, Tom M; Brost, Sylvia; Hinz, Ulf; Sykora, Jaromir; Batke, Emanuela M; Singer, Stephan; Breuhahn, Kai; Stremmel, Wolfgang; Walczak, Henning

2013-01-01

An imbalance between proliferation and apoptosis is one of the main features of carcinogenesis. TRAIL (TNF-related apoptosis-inducing ligand) induces apoptosis upon binding to the TRAIL death receptors, TRAIL receptor 1 (TRAIL-R1) and TRAIL-R2, whereas binding to TRAIL-R3 and TRAIL-R4 might promote cell survival and proliferation. The anti-tumor activity of TRAIL-R1 and TRAIL-R2 agonists is currently investigated in clinical trials. To gain further insight into the regulation of apoptosis in hepatocellular carcinoma (HCC), we investigated the TRAIL pathway and the regulators of apoptosis caspase-8, Bcl-xL and Mcl-1 in patients with HCC regarding patient survival. We analyzed 157 hepatocellular carcinoma patients who underwent partial liver resection or orthotopic liver transplantation and healthy control liver tissue using immunohistochemistry on tissue microarrays for the expression of TRAIL-R1 to TRAIL-R4, caspase-8, Bcl-xL and Mcl-1. Immunohistochemical data were evaluated for potential associations with clinico-pathological parameters and survival. Whereas TRAIL-R1 was downregulated in HCC in comparison to normal liver tissue, TRAIL-R2 and –R4 were upregulated in HCC, especially in G2 and G3 tumors. TRAIL-R1 downregulation and upregulation of TRAIL-R2 and TRAIL-R4 correlated with tumor dedifferentiation (G2/G3). TRAIL-R3, Bcl-xL and Mcl-1 showed no differential expression in tumor tissue compared to normal tissue. The expression levels of TRAIL receptors did not correlate with patient survival after partial hepatectomy. Interestingly, in tumor tissue, but not in normal hepatocytes, caspase-8 showed a strong nuclear staining. Low cytosolic and high nuclear staining intensity of caspase-8 significantly correlated with impaired survival after partial hepatectomy, which, for cytosolic caspase-8, was independent from tumor grade. Assessment of TRAIL-receptor expression patterns may have therapeutic implications for the use of TRAIL receptor agonists in HCC therapy

Effect of propylthiouracil on 125I-L-triiodothyronine binding to the nuclei and on malic enzyme activity in rat liver cytosol

International Nuclear Information System (INIS)

Knopp, J.

1980-01-01

The effects of propylthiouracil (PTU), triiodothyronine (T 3 ) and thyroxine (T 4 ) on the malic enzyme activity of rat liver cytosol and on the binding of 125 I-L-triiodothyronine to the nuclear fraction were examined. A significant decrease in in vitro binding of 125 I-T 3 to the liver nuclei was found in rats fed PTU for 12 h, 24 h and 120 h. The hepatic malic enzyme activity was unchanged after 12 h, but markedly decreased after 24 h and 120 h of PTU treatment. In 120 h PTU fed animals the effect of T 3 on the malic enzyme activity was five times higher in comparison with an equimolar dose of T 4 . Finally, the effect of T 4 on the malic enzyme activity was observed which might result from the intrinsic activity of T 4 under the conditions of a decreased T 4 to T 3 conversion due to the PTU feeding. The obtained results show that there is a certain correlation between the nuclear T 3 binding and the cytosol malic enzyme activity which is presumably induced directly through the m-RNA synthesis stimulated by thyroid hormones. (author)

An alpha-glucose-1-phosphate phosphodiesterase is present in rat liver cytosol

International Nuclear Information System (INIS)

Srisomsap, C.; Richardson, K.L.; Jay, J.C.; Marchase, R.B.

1989-01-01

UDP-glucose:glycoprotein glucose-1-phosphotransferase (Glc-phosphotransferase) catalyzes the transfer of alpha-Glc-1-P from UDP-Glc to mannose residues on acceptor glycoproteins. The predominant acceptor for this transfer in both mammalian cells and Paramecium is a cytoplasmic glycoprotein of 62-63 kDa. When cytoplasmic proteins from rat liver were fractionated by preparative isoelectric focusing following incubation of a liver homogenate with the 35S-labeled phosphorothioate analogue of UDP-Glc ([beta-35S]UDP-Glc), the acceptor was found to have a pI of about 6.0. This fraction, when not labeled prior to the focusing, became very heavily labeled when mixed with [beta-35S]. UDP-Glc and intact liver microsomes, a rich source of the Glc-phosphotransferase. In addition, it was observed that the isoelectric fractions of the cytosol having pI values of 2-3.2 contained a degradative activity, alpha-Glc-1-P phosphodiesterase, that was capable of removing alpha-Glc-1-P, monitored through radioactive labeling both in the sugar and the phosphate, as an intact unit from the 62-kDa acceptor. Identification of the product of this cleavage was substantiated by its partial transformation to UDP-Glc in the presence of UTP and UDP-Glc pyrophosphorylase. The alpha-Glc-1-P phosphodiesterase had a pH optimum of 7.5 and was not effectively inhibited by any of the potential biochemical inhibitors that were tested. Specificity for the Glc-alpha-1-P-6-Man diester was suggested by the diesterase's inability to degrade UDP-Glc or glucosylphosphoryldolichol. This enzyme may be important in the regulation of secretion since the alpha-Glc-1-P present on the 62-kDa phosphoglycoprotein appears to be removed and then rapidly replaced in response to secretagogue

Purification and properties of a 3 alpha-hydroxysteroid dehydrogenase of rat liver cytosol and its inhibition by anti-inflammatory drugs.

OpenAIRE

Penning, T M; Mukharji, I; Barrows, S; Talalay, P

1984-01-01

An NAD(P)-dependent 3 alpha-hydroxysteroid dehydrogenase (EC 1.1.1.50) was purified to homogeneity from rat liver cytosol, where it is responsible for most if not all of the capacity for the oxidation of androsterone, 1-acenaphthenol and benzenedihydrodiol (trans-1,2-dihydroxycyclohexa-3,5-diene). The dehydrogenase has many properties (substrate specificity, pI, Mr, amino acid composition) in common with the dihydrodiol dehydrogenase (EC 1.3.1.20) purified from the same source [Vogel, Bentley...

New insights into the posttranslational regulation of human cytosolic thioredoxin by S-palmitoylation

Energy Technology Data Exchange (ETDEWEB)

Xu, Zhiyu; Zhong, Liangwei, E-mail: liazho@ucas.ac.cn

2015-05-15

High level of palmitate is associated with metabolic disorders. We recently showed that enhanced level of S-palmitoylated cytosolic thioredoxin (Trx1) in mouse liver was new characteristic feature of insulin resistance. However, our understanding of the effect of S-palmitoylation on Trx1 is limited, and the tissue specificity of Trx1 S-palmitoylation is unclear. Here we show that S-palmitoylation also occurs at Cys73 of Trx1 in living endothelial cells, and the level of S-palmitoylated Trx1 undergoes regulation by insulin signaling. Trx1 prefers thiol-thioester exchange with palmitoyl-CoA to acetyl-CoA. S-palmitoylation alters conformation or secondary structure of Trx1, as well as decreases the ability of Trx1 to transfer electrons from thioredoxin reductase to S-nitrosylated protein–tyrosine phosphatase 1B and S-nitroso-glutathione. Our results demonstrate that S-palmitoylation is an important post-translational modification of human Trx1. - Highlights: • S-palmitoylation occurs at Cys73 of Trx1 in living endothelial cells. • Insulin signaling may regulate level of S-palmitoylated Trx1 in the cells. • S-palmitoylation plays significant effects on Trx1 structure and functions.

Studies of the activity of cytosol on the mixed disulfide bond formed by proteins and radioprotector mercaptoethylguanidine

Energy Technology Data Exchange (ETDEWEB)

Horvath, M [National Inst. of Oncology, Budapest (Hungary); Holland, J [Orszagos Onkologiai Intezet, Budapest (Hungary)

1979-01-01

The cytoplasm of normal and tumorous rat liver cells contains a heat-resistant compound with reducing ability to break the mixed disulfide bond of albumin-/sup 14/C-mercaptoethylguanidine. The reducing activity of cytosol is destoryed by 1000 krd /sup 60/Co-gamma-ray doses in diluted solution. In vivo supralethal of rats does not affect the activity of cytosol prepared from liver cells.

Unique hepatic cytosolic arginase evolved independently in ureogenic freshwater air-breathing teleost, Heteropneustes fossilis.

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Shilpee Srivastava

Full Text Available Hepatic cytosolic arginase (ARG I, an enzyme of the urea cycle operating in the liver of ureotelic animals, is reported to be present in an ammoniotelic freshwater air-breathing teleost, Heteropneustes fossilis which has ureogenic potential. Antibodies available against mammalian ARG I showed no cross reactivity with the H. fossilis ARG I. We purified unique ARG I from H. fossilis liver. Purified ARG I is a homotrimer with molecular mass 75 kDa and subunit molecular mass of 24 kDa. The pI value of the enzyme was 8.5. It showed maximum activity at pH 10.5 and 55°C. The Km of purified enzyme for L-arginine was 2.65±0.39 mM. L-ornithine and N(ω-hydroxy-L-arginine showed inhibition of the ARG I activity, with Ki values 0.52±0.02mM and 0.08±0.006mM, respectively. Antibody raised against the purified fish liver ARG I showed exclusive specificity, and has no cross reactivity against fish liver ARG II and mammalian liver ARG I and ARG II. We found another isoform of arginase bound to the outer membrane of the mitochondria which was released by 150-200 mM KCl in the extraction medium. This isoform was immunologically different from the soluble cytosolic and mitochondrial arginase. The results of present study support that hepatic cytosolic arginase evolved in this ureogenic freshwater teleost, H. fossilis. Phylogenetic analysis confirms an independent evolution event that occurred much after the evolution of the cytosolic arginase of ureotelic vertebrates.

A field survey of metal binding to metallothionein and other cytosolic ligands in liver of eels using an on-line isotope dilution method in combination with size exclusion (SE) high pressure liquid chromatography (HPLC) coupled to Inductively Coupled Plasma time-of-flight Mass Spectrometry (ICP-TOFMS).

Science.gov (United States)

Van Campenhout, Karen; Goenaga Infante, Heidi; Goemans, Geert; Belpaire, Claude; Adams, Freddy; Blust, Ronny; Bervoets, Lieven

2008-05-15

The effect of metal exposure on the accumulation and cytosolic speciation of metals in livers of wild populations of European eel with special emphasis on metallothioneins (MT) was studied. Four sampling sites in Flanders showing different degrees of heavy metal contamination were selected for this purpose. An on-line isotope dilution method in combination with size exclusion (SE) high pressure liquid chromatography (HPLC) coupled to Inductively Coupled Plasma time-of-flight Mass Spectrometry (ICP-TOFMS) was used to study the cytosolic speciation of the metals. The distribution of the metals Cd, Cu, Ni, Pb and Zn among cytosolic fractions displayed strong differences. The cytosolic concentration of Cd, Ni and Pb increased proportionally with the total liver levels. However, the cytosolic concentrations of Cu and Zn only increased above a certain liver tissue threshold level. Cd, Cu and Zn, but not Pb and Ni, were largely associated with the MT pool in correspondence with the environmental exposure and liver tissue concentrations. Most of the Pb and Ni and a considerable fraction of Cu and Zn, but not Cd, were associated to High Molecular Weight (HMW) fractions. The relative importance of the Cu and Zn in the HMW fraction decreased with increasing contamination levels while the MT pool became progressively more important. The close relationship between the cytosolic metal load and the total MT levels or the metals bound on the MT pool indicates that the metals, rather than other stress factors, are the major factor determining MT induction.

Cytosolic distributions of highly toxic metals Cd and Tl and several essential elements in the liver of brown trout (Salmo trutta L.) analyzed by size exclusion chromatography and inductively coupled plasma mass spectrometry.

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Dragun, Zrinka; Krasnići, Nesrete; Kolar, Nicol; Filipović Marijić, Vlatka; Ivanković, Dušica; Erk, Marijana

2018-05-15

Cytosolic distributions of nonessential metals Cd and Tl and seven essential elements among compounds of different molecular masses were studied in the liver of brown trout (Salmo trutta) from the karstic Krka River in Croatia. Analyses were done by size exclusion high performance liquid chromatography and high resolution inductively coupled plasma mass spectrometry. Common feature of Cd and Tl, as highly toxic elements, was their distribution within only two narrow peaks. The increase of cytosolic Cd concentrations was reflected in marked increase of Cd elution within low molecular mass peak (maximum at ∼15 kDa), presumably containing metallothioneins (MTs), which indicated successful Cd detoxification in brown trout liver under studied exposure conditions. Contrary, the increase of cytosolic Tl concentrations was reflected in marked increase of Tl elution within high molecular mass peak (maximum at 140 kDa), which probably indicated incomplete Tl detoxification. Common feature of the majority of studied essential elements was their distribution within more peaks, often broad and not well resolved, which is consistent with their numerous physiological functions. Among observed associations of essential metals/nonmetal to proteins, the following could be singled out: Cu and Zn association to MTs, Fe association to storage protein ferritin, and Se association to compounds of very low molecular masses (<5 kDa). The obtained results present the first step towards identification of metal-binding compounds in hepatic cytosol of brown trout, and thus a significant contribution to better understanding of metal fate in the liver of that important bioindicator species. Copyright © 2018 Elsevier Ltd. All rights reserved.

Circadian Rhythm of Hepatic Cytosolic and Nuclear Estrogen and Androgen Receptors

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FRANCAVILLA, ANTONIO; EAGON, PATRICIA K.; DiLEO, ALFREDO; VAN THIEL, DAVID H.; PANELLA, CARMINE; POLIMENO, LORENZO; AMORUSO, CINZIA; INGROSSO, MARCELLO; AQUILINO, A. MARIA; STARZL, THOMAS E.

2010-01-01

Mammalian liver is a sex steroid-responsive tissue. The effects of these hormones presumably are mediated by hepatic estrogen receptors (ER) and androgen receptors (AR). Serum levels of sex hormones display circadian rhythms. Further, estrogens and androgens are commonly administered; administration of these agents is associated frequently with liver disease. Therefore, we investigated whether the cytosolic and nuclear sex steroid receptors also display a similar circadian rhythm, and whether variations occurred in the distribution of receptors between cytosolic and nuclear compartments. Animals were killed every 4 h from midnight till the following midnight; cytosolic and nuclear levels of both ER and AR were measured. Cytosolic ER reached a maximum level at 4 AM, and a minimum at 8 PM and midnight of both days. Nuclear ER was highest at 8 AM and lowest at 4 PM and 8 PM, a pattern which parallels variations in serum estradiol levels. Cytosolic AR was highest at 8 PM and lowest at midnight and 4 AM. Nuclear AR was highest at 4 AM and lowest at 4 PM and 8 PM. The highest level of nuclear AR does not correspond to the maximum serum testosterone level, which occurred at 4 PM. The total hepatic content of both ER and AR was not constant over the 24-h period, but varied considerably with time of day. These studies suggest that both ER and AR show a distinct circadian rhythm in subcellular compartmentalization, and that total hepatic content of ER and AR varies significantly during a 24-h period. PMID:3710067

Ontogenic differences in human liver 4-hydroxynonenal detoxification are associated with in vitro injury to fetal hematopoietic stem cells

International Nuclear Information System (INIS)

Gardner, James L.; Doi, Adriana M.; Pham, Robert T.; Huisden, Christiaan M.; Gallagher, Evan P.

2003-01-01

4-hydroxynonenal (4HNE) is a highly mutagenic and cytotoxic α,β-unsaturated aldehyde that can be produced in utero during transplacental exposure to prooxidant compounds. Cellular protection against 4HNE injury is provided by alcohol dehydrogenases (ADH), aldehyde reductases (ALRD), aldehyde dehydrogenases (ALDH), and glutathione S-transferases (GST). In the present study, we examined the comparative detoxification of 4HNE by aldehyde-metabolizing enzymes in a panel of adult and second-trimester prenatal liver tissues and report the toxicological ramifications of ontogenic 4HNE detoxification in vitro. The initial rates of 4HNE oxidation and reduction were two- to fivefold lower in prenatal liver subcellular fractions as compared to adult liver, and the rates of GST conjugation of 4HNE were not detectable in either prenatal or adult cytosolic fractions. GSH-affinity purification of hepatic cytosol yielded detectable and roughly equivalent rates of GST-4HNE conjugation for the two age groups. Consistent with the inefficient oxidative and reductive metabolism of 4HNE in prenatal liver, cytosolic fractions prepared from prenatal liver exhibited a decreased ability to protect against 4HNE-protein adduct formation relative to adults. Prenatal liver hematopoietic stem cells (HSC), which constitute a significant percentage of prenatal liver cell populations, exhibited ALDH activities toward 4HNE, but little reductive or conjugative capacity toward 4HNE through ALRD, ADH, and GST. Cultured HSC exposed to 5 μM 4HNE exhibited a loss in viability and readily formed one or more high molecular weight 4HNE-protein adduct(s). Collectively, our results indicate that second trimester prenatal liver has a lower ability to detoxify 4HNE relative to adults, and that the inefficient detoxification of 4HNE underlies an increased susceptibility to 4HNE injury in sensitive prenatal hepatic cell targets

Sex-related difference in the inductions by perfluoro-octanoic acid of peroxisomal beta-oxidation, microsomal 1-acylglycerophosphocholine acyltransferase and cytosolic long-chain acyl-CoA hydrolase in rat liver.

Science.gov (United States)

Kawashima, Y; Uy-Yu, N; Kozuka, H

1989-01-01

Inductions by perfluoro-octanoic acid (PFOA) of hepatomegaly, peroxisomal beta-oxidation, microsomal 1-acylglycerophosphocholine acyltransferase and cytosolic long-chain acyl-CoA hydrolase were compared in liver between male and female rats. Marked inductions of these four parameters were seen concurrently in liver of male rats, whereas the inductions in liver of female rats were far less pronounced. The sex-related difference in the response of rat liver to PFOA was much more marked than that seen with p-chlorophenoxyisobutyric acid (clofibric acid) or 2,2'-(decamethylenedithio)diethanol (tiadenol). Hormonal manipulations revealed that this sex-related difference in the inductions is strongly dependent on sex hormones, namely that testosterone is necessary for the inductions, whereas oestradiol prevented the inductions by PFOA. PMID:2570571

in Human Liver Diseases

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Minoru Fujimoto

2010-01-01

Full Text Available Toll-like receptor (TLR signaling pathways are strictly coordinated by several mechanisms to regulate adequate innate immune responses. Recent lines of evidence indicate that the suppressor of cytokine signaling (SOCS family proteins, originally identified as negative-feedback regulators in cytokine signaling, are involved in the regulation of TLR-mediated immune responses. SOCS1, a member of SOCS family, is strongly induced upon TLR stimulation. Cells lacking SOCS1 are hyperresponsive to TLR stimulation. Thus, SOCS1 is an important regulator for both cytokine and TLR-induced responses. As an immune organ, the liver contains various types of immune cells such as T cells, NK cells, NKT cells, and Kupffer cells and is continuously challenged with gut-derived bacterial and dietary antigens. SOCS1 may be implicated in pathophysiology of the liver. The studies using SOCS1-deficient mice revealed that endogenous SOCS1 is critical for the prevention of liver diseases such as hepatitis, cirrhosis, and cancers. Recent studies on humans suggest that SOCS1 is involved in the development of various liver disorders in humans. Thus, SOCS1 and other SOCS proteins are potential targets for the therapy of human liver diseases.

Detection on immunoblot of new proteins from the soluble fraction of the cell recognized either by anti-liver-kidney microsome antibodies type 1 or by anti-liver cytosol antibodies type 1--relationship with hepatitis C virus infection.

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Ballot, E; Desbos, A; Monier, J C

1996-09-01

Antibodies directed against liver cytosol protein, called anti-liver cytosol type 1 (LC1 Ab), have been described by both immunofluorescence (IF) and immunodiffusion techniques in sera from patients with autoimmune hepatitis (AIH). They have never been found in association with antibodies directed against the hepatitis C virus (HCV), unlike the anti-liver-kidney microsome antibodies type 1 (LKM1 Ab), the serological marker of AIH type 2. This suggests that there are two subgroups of AIH type 2, i.e., HCV-related and non-HCV-related. In this study, immunoblotting experiments were performed using proteins from the soluble phase of the rat liver cell; 141 sera which tested positive for LKM1 Ab by IF, 24 identified as having LC1 Ab by IF, and 50 from blood donors as controls were analyzed. Three bands were stained by LC1 Ab sera more often than by the control sera, and with a statistically significant frequency. These 3 proteins were located at apparent Mr 50,000, 55,000, and 60,000. The LKM1 Ab-positive sera as defined by IF stained six bands with a statistically significant frequency compared to the controls. Their apparent Mr were 35,000, 39,000, 47,000, 50,000, 55,000, and 60,000. LKM1 Ab-positive sera which were anti-HCV negative recognized a 60,000 protein belonging to the soluble phase of the cell, with a statistically significant frequency compared to LKM1 Ab-positive sera which were anti-HCV positive. This 60,000 protein was also recognized by LC1 Ab-positive sera, which were almost always anti-HCV negative. The presence of antibodies against a 60,000 protein from the soluble phase of the cell is discussed in terms of the anti-HCV serological markers found in the sera from patients with AIH.

Subcellular Distribution of NAD+ between Cytosol and Mitochondria Determines the Metabolic Profile of Human Cells*

Science.gov (United States)

VanLinden, Magali R.; Dölle, Christian; Pettersen, Ina K. N.; Kulikova, Veronika A.; Niere, Marc; Agrimi, Gennaro; Dyrstad, Sissel E.; Palmieri, Ferdinando; Nikiforov, Andrey A.; Tronstad, Karl Johan; Ziegler, Mathias

2015-01-01

The mitochondrial NAD pool is particularly important for the maintenance of vital cellular functions. Although at least in some fungi and plants, mitochondrial NAD is imported from the cytosol by carrier proteins, in mammals, the mechanism of how this organellar pool is generated has remained obscure. A transporter mediating NAD import into mammalian mitochondria has not been identified. In contrast, human recombinant NMNAT3 localizes to the mitochondrial matrix and is able to catalyze NAD+ biosynthesis in vitro. However, whether the endogenous NMNAT3 protein is functionally effective at generating NAD+ in mitochondria of intact human cells still remains to be demonstrated. To modulate mitochondrial NAD+ content, we have expressed plant and yeast mitochondrial NAD+ carriers in human cells and observed a profound increase in mitochondrial NAD+. None of the closest human homologs of these carriers had any detectable effect on mitochondrial NAD+ content. Surprisingly, constitutive redistribution of NAD+ from the cytosol to the mitochondria by stable expression of the Arabidopsis thaliana mitochondrial NAD+ transporter NDT2 in HEK293 cells resulted in dramatic growth retardation and a metabolic shift from oxidative phosphorylation to glycolysis, despite the elevated mitochondrial NAD+ levels. These results suggest that a mitochondrial NAD+ transporter, similar to the known one from A. thaliana, is likely absent and could even be harmful in human cells. We provide further support for the alternative possibility, namely intramitochondrial NAD+ synthesis, by demonstrating the presence of endogenous NMNAT3 in the mitochondria of human cells. PMID:26432643

The tobacco carcinogen NNK is stereoselectively reduced by human pancreatic microsomes and cytosols.

Science.gov (United States)

Trushin, Neil; Leder, Gerhard; El-Bayoumy, Karam; Hoffmann, Dietrich; Beger, Hans G; Henne-Bruns, Doris; Ramadani, Marco; Prokopczyk, Bogdan

2008-07-01

Cigarette smoking increases the risk of cancer of the pancreas. The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is the only known environmental compound that induces pancreatic cancer in laboratory animals. Concentrations of NNK are significantly higher in the pancreatic juice of smokers than in that of nonsmokers. The chiral NNK metabolite, (R,S)-4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) is itself a potent pancreatic carcinogen in rats. The carcinogenicity of NNAL is related to its stereochemistry; (S)-NNAL is a more potent lung tumorigen in the A/J mouse than is (R)-NNAL. In this study, we determined the potential of the human pancreas to convert NNK into NNAL. Human pancreatic microsomes and cytosols were incubated with [5-(3)H]NNK, and the metabolic products were determined by high-performance liquid chromatography (HPLC). (S)-NNAL was the predominant isomer formed in all cytosolic incubations. In ten microsomal samples, NNAL was formed at an average rate of 3.8 +/- 1.6 pmol/mg/min; (R)-NNAL was the predominant isomer in this group. The average rate of NNAL formation in 18 other microsomal samples was significantly lower, 0.13 +/- 0.12 pmol/mg/min (p < 0.001); (S)-NNAL was the predominant isomer formed in this group. In human pancreatic tissues, there is intraindividual variability regarding the capacity for, and stereoselectivity of, carbonyl reduction of NNK.

In vitro metabolism of [14C]-toluene by human and rat liver microsomes and liver slices

International Nuclear Information System (INIS)

Chapman, D.E.; Moore, T.J.; Michener, S.R.; Powis, G.

1990-01-01

Toluene metabolites produced by liver microsomes from six human donors included benzylalcohol (Balc), benzaldehyde (Bald) and benzoic acid (Bacid). Microsomes from only one human donor metabolized toluene to p-cresol and o-cresol. Human liver microsomes also metabolized Balc to Bald. Balc metabolism required NADPH, was inhibited by carbon monoxide, and was decreased at a buffer pH of 10. Balc metabolism was not inhibited by ADP-ribose or sodium azide. These results suggest that cytochrome P450 is responsible for the in vitro metabolism of Balc by human liver microsomes. Toluene metabolites formed by human liver slices and released into the incubation media included hippuric acid, and Bacid. Cresols or cresol-conjugates were not detected in liver slice incubation media from any human donor. Toluene metabolism by human liver was compared to metabolism by comparable liver preparations from male Fischer F344 rats. Rates of toluene metabolism by human liver microsomes and liver slices were 9-fold and 1.3-fold greater than for rat liver, respectively. Covalent binding of toluene to human liver microsomes and liver slices was 21-fold and 4-fold greater than for comparable rat liver preparations. Covalent binding of toluene to human microsomes required NADPH, was significantly decreased by coincubation with 4 mM cysteine or 4 mM glutathione, and radioactivity associated with microsomes was decreased by subsequent digestion of microsomes with protease. These results suggest that toluene metabolism and covalent binding of toluene are underestimated if the male Fischer 344 rat is used as a model for human toluene metabolism

Comparative Study of Human Liver Ferritin and Chicken Liver by Moessbauer Spectroscopy. Preliminary Results

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Oshtrakh, M. I. [Ural State Technical University - UPI, Division of Applied Biophysics, Faculty of Physical Techniques and Devices for Quality Control (Russian Federation); Milder, O. B.; Semionkin, V. A. [Ural State Technical University - UPI, Faculty of Experimental Physics (Russian Federation); Prokopenko, P. G. [Russian State Medical University, Faculty of Biochemistry (Russian Federation); Malakheeva, L. I. [Simbio Holding, Science Consultation Department (Russian Federation)

2004-12-15

A comparative study of normal human liver ferritin and livers from normal chicken and chicken with Marek disease was made by Moessbauer spectroscopy. Small differences of quadrupole splitting and isomer shift were found for human liver ferritin and chicken liver. Moessbauer parameters for liver from normal chicken and chicken with Marek disease were the same.

Comparative Study of Human Liver Ferritin and Chicken Liver by Moessbauer Spectroscopy. Preliminary Results

International Nuclear Information System (INIS)

Oshtrakh, M. I.; Milder, O. B.; Semionkin, V. A.; Prokopenko, P. G.; Malakheeva, L. I.

2004-01-01

A comparative study of normal human liver ferritin and livers from normal chicken and chicken with Marek disease was made by Moessbauer spectroscopy. Small differences of quadrupole splitting and isomer shift were found for human liver ferritin and chicken liver. Moessbauer parameters for liver from normal chicken and chicken with Marek disease were the same.

Purification and characterization of rat liver minoxidil sulphotransferase.

Science.gov (United States)

Hirshey, S J; Falany, C N

1990-01-01

Minoxidil (Mx), a pyrimidine N-oxide, is used therapeutically as an antihypertensive agent and to induce hair growth in patients with male pattern baldness. Mx NO-sulphate has been implicated as the agent active in producing these effects. This paper describes the purification of a unique sulphotransferase (ST) from rat liver cytosol that is capable of catalysing the sulphation of Mx. By using DEAE-Sepharose CL-6B chromatography, hydroxyapatite chromatography and ATP-agarose affinity chromatography, Mx-ST activity was purified 240-fold compared with the activity in cytosol. The purified enzyme was also capable of sulphating p-nitrophenol (PNP) at low concentrations (less than 10 microM). Mx-ST was purified to homogeneity, as evaluated by SDS/PAGE and reverse-phase h.p.l.c. The active form of the enzyme had a molecular mass of 66,000-68,000 Da as estimated by gel exclusion chromatography and a subunit molecular mass of 35,000 Da. The apparent Km values for Mx, 3'-phosphoadenosine 5'-phosphosulphate and PNP were 625 microM, 5.0 microM and 0.5 microM respectively. However, PNP displayed potent substrate inhibition at concentrations above 1.2 microM. Antibodies raised in rabbits to the pure enzyme detected a single band in rat liver cytosol with a subunit molecular mass of 35,000 Da, as determined by immunoblotting. The anti-(rat Mx-ST) antibodies also reacted with the phenol-sulphating form of human liver phenol sulphotransferase, suggesting some structural similarity between these proteins. Images Fig. 5. Fig. 6. Fig. 7. PMID:2241904

HUMAN LIVER SLICES EXPRESS THE SAME LIDOCAINE BIOTRANSFORMATION RATE AS ISOLATED HUMAN HEPATOCYTES

NARCIS (Netherlands)

OLINGA, P; MEIJER, DKF; SLOOFF, MJH; GROOTHUIS, GMM; Merema, M.T.

1993-01-01

In order to investigate whether liver slices are a valuable tool for the assessment of drug metabolism in human liver, we compared the phase I metabolism of lidocaine in human liver slices and hepatocytes prepared from three human livers. Lidocaine is mainly metabolised to monoethylglycinexylidide

Subcellular Distribution of NAD+ between Cytosol and Mitochondria Determines the Metabolic Profile of Human Cells.

Science.gov (United States)

VanLinden, Magali R; Dölle, Christian; Pettersen, Ina K N; Kulikova, Veronika A; Niere, Marc; Agrimi, Gennaro; Dyrstad, Sissel E; Palmieri, Ferdinando; Nikiforov, Andrey A; Tronstad, Karl Johan; Ziegler, Mathias

2015-11-13

The mitochondrial NAD pool is particularly important for the maintenance of vital cellular functions. Although at least in some fungi and plants, mitochondrial NAD is imported from the cytosol by carrier proteins, in mammals, the mechanism of how this organellar pool is generated has remained obscure. A transporter mediating NAD import into mammalian mitochondria has not been identified. In contrast, human recombinant NMNAT3 localizes to the mitochondrial matrix and is able to catalyze NAD(+) biosynthesis in vitro. However, whether the endogenous NMNAT3 protein is functionally effective at generating NAD(+) in mitochondria of intact human cells still remains to be demonstrated. To modulate mitochondrial NAD(+) content, we have expressed plant and yeast mitochondrial NAD(+) carriers in human cells and observed a profound increase in mitochondrial NAD(+). None of the closest human homologs of these carriers had any detectable effect on mitochondrial NAD(+) content. Surprisingly, constitutive redistribution of NAD(+) from the cytosol to the mitochondria by stable expression of the Arabidopsis thaliana mitochondrial NAD(+) transporter NDT2 in HEK293 cells resulted in dramatic growth retardation and a metabolic shift from oxidative phosphorylation to glycolysis, despite the elevated mitochondrial NAD(+) levels. These results suggest that a mitochondrial NAD(+) transporter, similar to the known one from A. thaliana, is likely absent and could even be harmful in human cells. We provide further support for the alternative possibility, namely intramitochondrial NAD(+) synthesis, by demonstrating the presence of endogenous NMNAT3 in the mitochondria of human cells. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Cigarette smoke toxicants as substrates and inhibitors for human cytosolic SULTs

International Nuclear Information System (INIS)

Yasuda, Shin; Idell, Steven; Fu Jian; Carter, Glendora; Snow, Rhodora; Liu, M.-C.

2007-01-01

The current study was designed to examine the role of sulfation in the metabolism of cigarette smoke toxicants and clarify whether these toxicants, by serving as substrates for the cytosolic sulfotransferases (SULTs), may interfere with the sulfation of key endogenous compounds. By metabolic labeling, [ 35 S]sulfated species were found to be generated and released into the media of HepG2 human hepatoma cells and primary human lung endothelial cells labeled with [ 35 S]sulfate in the presence of cigarette smoke extract (CSE). Concomitantly, several [ 35 S]sulfated metabolites observed in the medium in the absence of CSE either decreased or disappeared. Eleven previously prepared human cytosolic SULTs were tested for sulfating activity with CSE and known cigarette smoke toxicants as substrates. Activity data revealed SULT1A1, SULT1A2, SULT1A3, and SULT1C2 as major enzymes responsible for their sulfation. To examine their inhibitory effects on the sulfation of 17β-estradiol by SULT1A1, enzymatic assays were performed in the presence of three representative toxicant compounds, namely N-hydroxy-4-aminobiphenyl (N-OH-4-ABP), 4-aminobiphenyl (4-ABP) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). IC 50 values determined for the sulfation of 17β-estradiol by SULT1A1 were 11.8 μM, 28.2 μM, and 500 μM, respectively, for N-OH-4-ABP, 4-ABP and PhIP. Kinetic analyses indicated that the mechanism underlying the inhibition of 17β-estradiol sulfation by these cigarette smoke toxicants is of a mixed competitive-noncompetitive type. Metabolic labeling experiments clearly showed inhibition of the production of [ 35 S]sulfated 17β-estradiol by N-OH-4-ABP in a concentration-dependent manner in HepG2 cells. Taken together, these results suggest that sulfation plays a significant role in the metabolism of cigarette smoke compounds. By serving as substrates for SULTs, cigarette smoke toxicants may interfere with the metabolism of 17β-estradiol and other endogenous

Glutathionylation regulates cytosolic NADP+-dependent isocitrate dehydrogenase activity.

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Shin, Seoung Woo; Oh, Chang Joo; Kil, In Sup; Park, Jeen-Woo

2009-04-01

Cytosolic NADP+-dependent isocitrate dehydrogenase (IDPc) is susceptible to inactivation by numerous thiol-modifying reagents. This study now reports that Cys269 of IDPc is a target for S-glutathionylation and that this modification is reversed by dithiothreitol as well as enzymatically by cytosolic glutaredoxin in the presence of GSH. Glutathionylated IDPc was significantly less susceptible than native protein to peptide fragmentation by reactive oxygen species and proteolytic digestion. Glutathionylation may play a protective role in the degradation of protein through the structural alterations of IDPc. HEK293 cells treated with diamide displayed decreased IDPc activity and accumulated glutathionylated enzyme. Using immunoprecipitation with an anti-IDPc IgG and immunoblotting with an anti-GSH IgG, we purified and positively identified glutathionylated IDPc from the kidneys of mice subjected to ischemia/reperfusion injury and from the livers of ethanol-administered rats. These results suggest that IDPc activity is modulated through enzymatic glutathionylation and deglutathionylation during oxidative stress.

Thyroid Hormone-Induced Cytosol-to-Nuclear Translocation of Rat Liver Nrf2 Is Dependent on Kupffer Cell Functioning

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Luis A. Videla

2012-01-01

Full Text Available L-3,3′,5-triiodothyronine (T3 administration upregulates nuclear factor-E2-related factor 2 (Nrf2 in rat liver, which is redox-sensitive transcription factor mediating cytoprotection. In this work, we studied the role of Kupffer cell respiratory burst activity, a process related to reactive oxygen species generation and liver homeostasis, in Nrf2 activation using the macrophage inactivator gadolinium chloride (GdCl3; 10 mg/kg i.v. 72 h before T3 [0.1 mg/kg i.p.] or NADPH oxidase inhibitor apocynin (1.5 mmol/L added to the drinking water for 7 days before T3, and determinations were performed 2 h after T3. T3 increased nuclear/cytosolic Nrf2 content ratio and levels of heme oxygenase 1 (HO-1, catalytic subunit of glutamate cysteine ligase, and thioredoxin (Western blot over control values, proteins whose gene transcription is induced by Nrf2. These changes were suppressed by GdCl3 treatment prior to T3, an agent-eliciting Kupffer-cell depletion, inhibition of colloidal carbon phagocytosis, and the associated respiratory burst activity, with enhancement in nuclear inhibitor of Nrf2 kelch-like ECH-associated protein 1 (Keap1/Nrf2 content ratios suggesting Nrf2 degradation. Under these conditions, T3-induced tumor necrosis factor-α (TNF-α response was eliminated by previous GdCl3 administration. Similar to GdCl3, apocynin given before T3 significantly reduced liver Nrf2 activation and HO-1 expression, a NADPH oxidase inhibitor eliciting abolishment of colloidal carbon-induced respiratory burst activity without altering carbon phagocytosis. It is concluded that Kupffer cell functioning is essential for upregulation of liver Nrf2-signaling pathway by T3. This contention is supported by suppression of the respiratory burst activity of Kupffer cells and the associated reactive oxygen species production by GdCl3 or apocynin given prior to T3, thus hindering Nrf2 activation.

Isolation of primary human hepatocytes from normal and diseased liver tissue: a one hundred liver experience.

Directory of Open Access Journals (Sweden)

Ricky H Bhogal

2011-03-01

Full Text Available Successful and consistent isolation of primary human hepatocytes remains a challenge for both cell-based therapeutics/transplantation and laboratory research. Several centres around the world have extensive experience in the isolation of human hepatocytes from non-diseased livers obtained from donor liver surplus to surgical requirement or at hepatic resection for tumours. These livers are an important but limited source of cells for therapy or research. The capacity to isolate cells from diseased liver tissue removed at transplantation would substantially increase availability of cells for research. However no studies comparing the outcome of human hepatocytes isolation from diseased and non-diseased livers presently exist. Here we report our experience isolating human hepatocytes from organ donors, non-diseased resected liver and cirrhotic tissue. We report the cell yields and functional qualities of cells isolated from the different types of liver and demonstrate that a single rigorous protocol allows the routine harvest of good quality primary hepatocytes from the most commonly accessible human liver tissue samples.

A novel “humanized mouse” model for autoimmune hepatitis and the association of gut microbiota with liver inflammation

Science.gov (United States)

Yuksel, Muhammed; Wang, Yipeng; Tai, Ningwen; Peng, Jian; Guo, Junhua; Beland, Kathie; Lapierre, Pascal; David, Chella; Alvarez, Fernando; Colle, Isabelle; Yan, Huiping; Mieli-Vergani, Giorgina; Vergani, Diego; Ma, Yun; Wen, Li

2016-01-01

Background Autoimmune hepatitis (AIH) in humans is a severe inflammatory liver disease, characterized by interface hepatitis, the presence of circulating autoantibodies and hyper-gammaglobulinemia. There are two types of AIH, type-1 (AIH-1) and type-2 (AIH-2) characterized by distinct autoimmune serology. Patients with AIH-1 are positive for anti-smooth muscle and/or anti-nuclear (SMA/ANA) autoantibodies whereas patients with AIH-2 have anti-liver kidney microsomal type 1 (anti-LKM1) and/or anti-liver cytosol type 1 (anti-LC1) autoantibodies. Cytochrome P4502D6 (CYP2D6) is the antigenic target of anti-LKM1 and formiminotransferase cyclodeaminase (FTCD) is the antigenic target of anti-LC1. It is known that AIH, both type-1 and type-2, is strongly linked to the Human Leukocyte Antigen (HLA) alleles -DR3, -DR4 and -DR7. However, the direct evidence of the association of HLA with AIH is lacking. Methods We developed a novel mouse model of AIH using the HLA-DR3 transgenic mouse on the non-obese diabetic (NOD) background (HLA-DR3 NOD) by immunization of HLA-DR3− and HLA-DR3+ NOD mice with a DNA plasmid, coding for human CYP2D6/FTCD fusion protein. Results Immunization with CYP2D6/FTCD leads to a sustained elevation of alanine aminotransferase (ALT), development of ANA and anti-LKM1/anti-LC1 autoantibodies, chronic immune cell infiltration and parenchymal fibrosis on liver histology in HLA-DR3+ mice. Immunized mice also showed an enhanced Th1 immune response and paucity of the frequency of regulatory T-cell (Treg) in the liver. Moreover, HLA-DR3+ mice with exacerbated AIH showed reduced diversity and total load of gut bacteria. Conclusion Our humanized animal model has provided a novel experimental tool to further elucidate the pathogenesis of AIH and to evaluate the efficacy and safety of immunoregulatory therapeutic interventions in vivo. PMID:26185095

Autoimmune hepatitis-specific antibodies against soluble liver antigen and liver cytosol type 1 in patients with chronic viral hepatitis.

Science.gov (United States)

Rigopoulou, Eirini I; Mytilinaiou, Maria; Romanidou, Ourania; Liaskos, Christos; Dalekos, George N

2007-02-04

Non-organ specific autoantibodies are highly prevalent in patients with chronic hepatitis C (HCV). Among them, anti-liver kidney microsomal type 1 (LKM1) antibody--the serological marker of type 2 autoimmune hepatitis (AIH-2)--is detected in up to 11% of the HCV-infected subjects. On the other hand, anti-liver cytosol type 1 antibodies (anti-LC1)--either in association with anti-LKM1, or in isolation--and anti-soluble liver antigen antibodies (anti-SLA) have been considered as useful and specific diagnostic markers for AIH. However, their specificity for AIH has been questioned by some recent studies, which have shown the detection of anti-LC1 and anti-SLA by immunoprecipitation assays in HCV patients irrespective of their anti-LKM1 status. The aim of the present study was to test the anti-LC1 and anti-SLA presence by specific enzyme linked immunosorbent assays (ELISAs), in a large group of Greek HCV-infected patients with or without anti-LKM1 reactivity as firstly, immunoprecipitation assays are limited to few specialized laboratories worldwide and cannot be used routinely and secondly, to assess whether application of such tests has any relevance in the context of patients with viral hepatitis since antibody detection based on such ELISAs has not been described in detail in large groups of HCV patients. One hundred and thirty eight consecutive HCV patients (120 anti-LKM1 negative and 18 anti-LKM1 positive) were investigated for the presence of anti-LC1 and anti-SLA by commercial ELISAs. A similar number (120) of chronic hepatitis B virus (HBV) infected patients seronegative for anti-LKM1 was also tested as pathological controls. Six out of 18 (33%) anti-LKM(pos)/HCV(pos) patients tested positive for anti-LC1 compared to 1/120 (0.83%) anti-LKM(neg)/HCV(pos) patients and 0/120 (0%) of the anti-LKM1(neg)/HBV(pos) patients (p LKM1) or HBV-infected patients. We showed that anti-LC1 and anti-SLA autoantibodies are not detected by conventional assays in a large group of

Enzymatic sulfation of tocopherols and tocopherol metabolites by human cytosolic sulfotransferases.

Science.gov (United States)

Hashiguchi, Takuyu; Kurogi, Katsuhisa; Sakakibara, Yoichi; Yamasaki, Masao; Nishiyama, Kazuo; Yasuda, Shin; Liu, Ming-Cheh; Suiko, Masahito

2011-01-01

Tocopherols are essential micronutrients for mammals widely known as potent lipid-soluble antioxidants that are present in cell membranes. Recent studies have demonstrated that most of the carboxychromanol (CEHC), a tocopherol metabolite, in the plasma exists primarily in sulfate- and glucuronide-conjugated forms. To gain insight into the enzymatic sulfation of tocopherols and their metabolites, a systematic investigation was performed using all 14 known human cytosolic sulfotransferases (SULTs). The results showed that the members of the SULT1 family displayed stronger sulfating activities toward tocopherols and their metabolites. These enzymes showed a substrate preference for γ-tocopherol over α-tocopherol and for γ-CEHC over other CEHCs. Using A549 human lung epithelial cells in a metabolic labeling study, a similar trend in the sulfation of tocopherols and CEHCs was observed. Collectively, the results obtained indicate that SULT-mediated enzymatic sulfation of tocopherols and their metabolites is a significant pathway for regulation of the homeostasis and physiological functions of these important compounds.

Comparative characterization of thyroid hormone receptors and binding proteins in rat liver nucleus, plasma membrane, and cytosol by photoaffinity labeling with L-thyroxine

International Nuclear Information System (INIS)

Dozin, B.; Cahnmann, H.J.; Nikodem, V.M.

1985-01-01

Photoaffinity labeling with underivatized thyroxine (T4) was used to identify and compare the T4 binding proteins in rat liver cytosol, nuclear extract, and purified plasma membrane. When these subcellular fractions were incubated with a tracer concentration of [125I]T4, irradiated with light above 300 nm, and individually analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the radioactivity profiles revealed the presence of T4 binding proteins of molecular masses of 70, 52, 43, 37, 30, and 26 kilodaltons (kDa) in cytosol, of 96, 56, 45, and 35 kDa in nuclear extract, and of 70, 44, and 30 kDa in plasma membrane. Competition experiments performed in the presence of a 1000-fold excess of unlabeled T4 demonstrated that these binding proteins display different hormone binding activities. The similar electrophoretic mobilities of some binding proteins present in the different subcellular fractions, i.e., the 70-, 43-45-, and 30-kDa proteins, suggested that these proteins might be identical. However, double-labeling experiments in which plasma membrane, nuclear extract, and cytosol were photolabeled with either [125I] or [131I]T4 and mixed, two at a time, in all possible combinations showed that from one cellular fraction to another, the radioactivity peaks corresponding to the approximately 70-, 43-45-, and 30-kDa proteins were not superimposed. Their relative positions on the gel differed by one or two slices, which indicated differences in molecular mass of 1.9-3.6 kDa. Moreover, enzymatic digestion with Staphylococcus aureus V8 protease of these three proteins, prepared from each subcellular fraction, yielded dissimilar peptide patterns

Radiological evaluation of a liver simulator in comparison to a human real liver

International Nuclear Information System (INIS)

Toledo, Janine M.; Campos, Tarcisio P.R. de

2009-01-01

The present study evaluates the radiological features of a human real healthy liver reproducing its characteristics on a developed liver simulator. The radiological evaluation will be performed through radiological methods such as CT and X-ray images, density and weight measurements, as well as representation of the coloration and texture. According to literature, the liver is the highest weight organ and gland of the body, weighing approximately 1,5 kg. On the liver, the nutrients are absorbed from the digestive tract and are prosecuted and stored for future use by other organs. Also the liver is responsible for the neutralization and elimination of various toxic substances. Thus, it is an interface between the digestive system and the blood. Besides, this organ is the principal source of plasmatic proteins like the albumin, transport of graxos oily acids. Due to its proprieties, the liver holds a large amount of radionuclides on any uptake from external source. The liver simulator was designed to have the same density, weight and corresponding shape. The radiographic image was produced by conventional X-rays machine, in which the radiographic applied parameters were the same applied to abdomen. The result of the radiographic and CT images demonstrates radiological equivalence between the simulator and human real liver. Hounsfield number of the synthetic liver tissue was found on the range of human livers. Therefore, due to its similar shape, chemical composition, radiological response, the liver simulator can be used to investigate ionizing radiation procedures during radiation therapy intervention. (author)

Dynamic movement of cytochrome c from mitochondria into cytosol and peripheral circulation in massive hepatic cell injury.

Science.gov (United States)

Kobayashi, Yoshinori; Mori, Masaaki; Naruto, Takuya; Kobayashi, Naoki; Sugai, Toshiyuki; Imagawa, Tomoyuki; Yokota, Shumpei

2004-12-01

In the process of apoptosis, it is known that the transition of cytochrome c from mitochondria into the cytosol occurs, and tumor necrosis factor (TNF)-alpha is one of the molecules responsible for this event. But in the state of hypercytokine induced by D-galactosamine (D-GaIN)/Lipopolysaccharide (LPS), the localization of cytochrome c is little known. Rats were administrated with D-GaIN(700 mg/kg)/LPS(200 microg/kg). Blood and tissue samples were collected and examined for levels of pro-inflammatory cytokines, the apoptosis of liver cells, and the localization of cytochrome c. Before administration of D-GaIN/LPS, cytochrome c was definitely localized in the mitochondria. At 2 h after simultaneous administration of D-GaIN/LPS, cytochrome c had accumulated in the cytosol following abrupt increases of plasma TNF-alpha. Massive cell destruction due to apoptosis proved by Terminal deoxynucleo-tidyl transferase-mediated dUTP nick end labeling staining was observed in liver tissue 4 h later and markedly increased levels of cytochrome c were detected in the plasma 12 h after D-GaIN/LPS administration. Liver injury induced by simultaneous administration of D-GaIN/LPS was closely associated with the production of TNF-alpha, and also with the dynamic movement of cytochrome c from the mitochondria into the cytosol, and then into the systemic circulation. The detection of plasma cytochrome c levels may be a useful clinical tool for the detection of apoptosis in vivo.

Metabolic fate of desomorphine elucidated using rat urine, pooled human liver preparations, and human hepatocyte cultures as well as its detectability using standard urine screening approaches.

Science.gov (United States)

Richter, Lilian H J; Kaminski, Yeda Rumi; Noor, Fozia; Meyer, Markus R; Maurer, Hans H

2016-09-01

Desomorphine is an opioid misused as "crocodile", a cheaper alternative to heroin. It is a crude synthesis product homemade from codeine with toxic byproducts. The aim of the present work was to investigate the metabolic fate of desomorphine in vivo using rat urine and in vitro using pooled human liver microsomes and cytosol as well as human liver cell lines (HepG2 and HepaRG) by Orbitrap-based liquid chromatography-high resolution-tandem mass spectrometry or hydrophilic interaction liquid chromatography. According to the identified metabolites, the following metabolic steps could be proposed: N-demethylation, hydroxylation at various positions, N-oxidation, glucuronidation, and sulfation. The cytochrome P450 (CYP) initial activity screening revealed CYP3A4 to be the only CYP involved in all phase I steps. UDP-glucuronyltransferase (UGT) initial activity screening showed that UGT1A1, UGT1A8, UGT1A9, UGT1A10, UGT2B4, UGT2B7, UGT2B15, and UGT2B17 formed desomorphine glucuronide. Among the tested in vitro models, HepaRG cells were identified to be the most suitable tool for prediction of human hepatic phase I and II metabolism of drugs of abuse. Finally, desomorphine (crocodile) consumption should be detectable by all standard urine screening approaches mainly via the parent compound and/or its glucuronide assuming similar kinetics in rats and humans.

Cellular processing of gold nanoparticles: CE-ICP-MS evidence for the speciation changes in human cytosol.

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Legat, Joanna; Matczuk, Magdalena; Timerbaev, Andrei R; Jarosz, Maciej

2018-01-01

The cellular uptake of gold nanoparticles (AuNPs) may (or may not) affect their speciation, but information on the chemical forms in which the particles exist in the cell remains obscure. An analytical method based on the use of capillary electrophoresis hyphenated with inductively coupled plasma mass spectrometry (ICP-MS) has been proposed to shed light on the intracellular processing of AuNPs. It was observed that when being introduced into normal cytosol, the conjugates of 10-50 nm AuNPs with albumin evolved in human serum stayed intact. On the contrary, under simulated cancer cytosol conditions, the nanoconjugates underwent decomposition, the rate of which and the resulting metal speciation patterns were strongly influenced by particle size. The new peaks that appeared in ICP-MS electropherograms could be ascribed to nanosized species, as upon ultracentrifugation, they quantitatively precipitated whereas the supernatant showed only trace Au signals. Our present study is the first step to unravel a mystery of the cellular chemistry for metal-based nanomedicines.

[Effects of cytosolic bacteria on cyclic GMP-AMP synthase expression in human gingival tissues and periodontal ligament cells].

Science.gov (United States)

Xiaojun, Yang; Yongmei, Tan; Zhihui, Tian; Ting, Zhou; Wanghong, Zhao; Jin, Hou

2017-04-01

This work aims to determine the effect of cytosolic bacteria on the expression of cyclic GMP-AMP synthase (cGAS) in human periodontal ligament cells (hPDLCs) and gingival tissues. The ability of Porphyromonas gingivalis (P. gingivalis) to invade hPDLCs was detected using laser scanning confocal microscope assay at a multiplicity of infection of 10. P. gingivalis-infected cells were sorted by fluorescence-activated cell sorting (FACS). Then, quantitative real time reverse transcription polymerase chain reaction (qRT-PCR) and Western blot were used to detect cGAS expression in infected cells. Finally, the location and expression of cGAS in inflammatory and normal gingival tissues were investigated by immunohistochemistry. P. gingivalis actively invaded hPDLCs. Moreover, cGAS expression significantly increased in P. gingivalis-infected cells. Although cGAS was expressed in the epithelial and subepithelial cells of both inflamed and normal gingival tissues, cGAS expression significantly increased in inflamed gingival tissues. Cytosolic bacteria can upregulate cGAS expression in infected cells. These data suggest that cGAS may act as pattern-recognition receptors and participate in recognizing cytosolic nucleic acid pathogen-associated molecular patterns.
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Purification and characterization of akr1b10 from human liver: role in carbonyl reduction of xenobiotics.

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Martin, Hans-Jörg; Breyer-Pfaff, Ursula; Wsol, Vladimir; Venz, Simone; Block, Simone; Maser, Edmund

2006-03-01

Members of the aldo-keto reductase (AKR) superfamily have a broad substrate specificity in catalyzing the reduction of carbonyl group-containing xenobiotics. In the present investigation, a member of the aldose reductase subfamily, AKR1B10, was purified from human liver cytosol. This is the first time AKR1B10 has been purified in its native form. AKR1B10 showed a molecular mass of 35 kDa upon gel filtration and SDS-polyacrylamide gel electrophoresis. Kinetic parameters for the NADPH-dependent reduction of the antiemetic 5-HT3 receptor antagonist dolasetron, the antitumor drugs daunorubicin and oracin, and the carcinogen 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK) to the corresponding alcohols have been determined by HPLC. Km values ranged between 0.06 mM for dolasetron and 1.1 mM for daunorubicin. Enzymatic efficiencies calculated as kcat/Km were more than 100 mM-1 min-1 for dolasetron and 1.3, 0.43, and 0.47 mM-1 min-1 for daunorubicin, oracin, and NNK, respectively. Thus, AKR1B10 is one of the most significant reductases in the activation of dolasetron. In addition to its reducing activity, AKR1B10 catalyzed the NADP+-dependent oxidation of the secondary alcohol (S)-1-indanol to 1-indanone with high enzymatic efficiency (kcat/Km=112 mM-1 min-1). The gene encoding AKR1B10 was cloned from a human liver cDNA library and the recombinant enzyme was purified. Kinetic studies revealed lower activity of the recombinant compared with the native form. Immunoblot studies indicated large interindividual variations in the expression of AKR1B10 in human liver. Since carbonyl reduction of xenobiotics often leads to their inactivation, AKR1B10 may play a role in the occurrence of chemoresistance of tumors toward carbonyl group-bearing cytostatic drugs.

The interaction of bacterial magnetosomes and human liver cancer cells in vitro

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Wang, Pingping, E-mail: wangpp@mail.iee.ac.cn [Beijing Key Laboratory of Bioelectromagnetism, Institute of Electrical Engineering, Chinese Academy of Sciences, Beijing 100190 (China); Chen, Chuanfang [Beijing Key Laboratory of Bioelectromagnetism, Institute of Electrical Engineering, Chinese Academy of Sciences, Beijing 100190 (China); Chen, Changyou [Beijing Key Laboratory of Bioelectromagnetism, Institute of Electrical Engineering, Chinese Academy of Sciences, Beijing 100190 (China); University of Chinese Academy of Sciences, Beijing 100049 (China); Li, Yue; Pan, Weidong; Song, Tao [Beijing Key Laboratory of Bioelectromagnetism, Institute of Electrical Engineering, Chinese Academy of Sciences, Beijing 100190 (China)

2017-04-01

As the biogenic magnetic nanomaterial, bacterial magnetic nanoparticles, namely magnetosomes, provide many advantages for potential biomedical applications. As such, interactions among magnetosomes and target cells should be elucidated to develop their bioapplications and evaluate their biocompatibilities. In this study, the interaction of magnetosomes and human liver cancer HepG2 cells was examined. Prussian blue staining revealed numerous stained particles in or on the cells. Intracellular iron concentrations, measured through inductively coupled plasma optical emission spectroscopy, increased with the increasing concentration of the magnetosomes. Transmission electron microscopy images showed that magnetosomes could be internalized in cells, mainly encapsulated in membrane vesicles, such as endosomes and lysosomes, and partly found as free particles in the cytosol. Some of the magnetosomes on cellular surfaces were encapsulated through cell membrane ruffling, which is the initiating process of endocytosis. Applying low temperature treatment and using specific endocytic inhibitors, we validated that macropinocytosis and clathrin-mediated endocytosis were involved in magnetosome uptake by HepG2 cells. Consequently, we revealed the interaction and intrinsic endocytic mechanisms of magnetosomes and HepG2 cells. This study provides a basis for the further research on bacterial magnetosome applications in liver diseases. - Highlights: • Bacterial magnetosomes interact with HepG2 cells in a dose-dependent manner. • Magnetosomes are wrapped by membrane ruffling on cell surface. • Internalized magnetosomes mainly localize in endosomes and lysosomes. • Macropinocytosis and CME are involved in the cellular uptake of magnetosomes.

A cytosolic protein factor from the naked mole-rat activates proteasomes of other species and protects these from inhibition

Science.gov (United States)

Rodriguez, Karl A.; Osmulski, Pawel A.; Pierce, Anson; Weintraub, Susan T.; Gaczynska, Maria; Buffenstein, Rochelle

2015-01-01

The naked mole-rat maintains robust proteostasis and high levels of proteasome-mediated proteolysis for most of its exceptional (~31y) life span. Here, we report that the highly active proteasome from the naked mole-rat liver resists attenuation by a diverse suite of proteasome-specific small molecule inhibitors. Moreover, mouse, human, and yeast proteasomes exposed to the proteasome-depleted, naked mole-rat cytosolic fractions, recapitulate the observed inhibition resistance, and mammalian proteasomes also show increased activity. Gel filtration coupled with mass spectrometry and atomic force microscopy indicates that these traits are supported by a protein factor that resides in the cytosol. This factor interacts with the proteasome and modulates its activity. Although HSP72 and HSP40 (Hdj1) are among the constituents of this factor, the observed phenomenon, such as increasing peptidase activity and protecting against inhibition cannot be reconciled with any known chaperone functions. This novel function may contribute to the exceptional protein homeostasis in the naked mole-rat and allow it to successfully defy aging. PMID:25018089

Extracellular Matrix Molecular Remodeling in Human Liver Fibrosis Evolution.

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Andrea Baiocchini

Full Text Available Chronic liver damage leads to pathological accumulation of ECM proteins (liver fibrosis. Comprehensive characterization of the human ECM molecular composition is essential for gaining insights into the mechanisms of liver disease. To date, studies of ECM remodeling in human liver diseases have been hampered by the unavailability of purified ECM. Here, we developed a decellularization method to purify ECM scaffolds from human liver tissues. Histological and electron microscopy analyses demonstrated that the ECM scaffolds, devoid of plasma and cellular components, preserved the three-dimensional ECM structure and zonal distribution of ECM components. This method has been then applied on 57 liver biopsies of HCV-infected patients at different stages of liver fibrosis according to METAVIR classification. Label-free nLC-MS/MS proteomics and computation biology were performed to analyze the ECM molecular composition in liver fibrosis progression, thus unveiling protein expression signatures specific for the HCV-related liver fibrotic stages. In particular, the ECM molecular composition of liver fibrosis was found to involve dynamic changes in matrix stiffness, flexibility and density related to the dysregulation of predominant collagen, elastic fibers and minor components with both structural and signaling properties. This study contributes to the understanding of the molecular bases underlying ECM remodeling in liver fibrosis and suggests new molecular targets for fibrolytic strategies.

Genome-Derived Cytosolic DNA Mediates Type I Interferon-Dependent Rejection of B Cell Lymphoma Cells

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Yu J. Shen

2015-04-01

Full Text Available The DNA damage response (DDR induces the expression of type I interferons (IFNs, but the underlying mechanisms are poorly understood. Here, we show the presence of cytosolic DNA in different mouse and human tumor cells. Treatment of cells with genotoxic agents increased the levels of cytosolic DNA in a DDR-dependent manner. Cloning of cytosolic DNA molecules from mouse lymphoma cells suggests that cytosolic DNA is derived from unique genomic loci and has the potential to form non-B DNA structures, including R-loops. Overexpression of Rnaseh1, which resolves R-loops, reduced the levels of cytosolic DNA, type I Ifn transcripts, and type I IFN-dependent rejection of lymphoma cells. Live-cell imaging showed a dynamic contact of cytosolic DNA with mitochondria, an important organelle for innate immune recognition of cytosolic nucleotides. In summary, we found that cytosolic DNA is present in many tumor cells and contributes to the immunogenicity of tumor cells.

Human immunodeficiency virus infection and the liver.

Science.gov (United States)

Crane, Megan; Iser, David; Lewin, Sharon R

2012-03-27

Liver disease in human immunodeficiency virus (HIV)-infected individuals encompasses the spectrum from abnormal liver function tests, liver decompensation, with and without evidence of cirrhosis on biopsy, to non-alcoholic liver disease and its more severe form, non-alcoholic steatohepatitis and hepatocellular cancer. HIV can infect multiple cells in the liver, leading to enhanced intrahepatic apoptosis, activation and fibrosis. HIV can also alter gastro-intestinal tract permeability, leading to increased levels of circulating lipopolysaccharide that may have an impact on liver function. This review focuses on recent changes in the epidemiology, pathogenesis and clinical presentation of liver disease in HIV-infected patients, in the absence of co-infection with hepatitis B virus or hepatitis C virus, with a specific focus on issues relevant to low and middle income countries.

Subcellular distribution of styrene oxide in rat liver

International Nuclear Information System (INIS)

Pacifici, G.M.; Cuoci, L.; Rane, A.

1984-01-01

The subcellular distribution of ( 3 H)-styrene-7,8-oxide was studied in the rat liver. The compound was added to liver homogenate to give a final concentration of 2 X 10(-5); 2 X 10(-4) and 2 X 10(-3) M. Subcellular fractions were obtained by differential centrifugation. Most of styrene oxide (59-88%) was associated with the cytosolic fraction. Less than 15 percent of the compound was retrieved in each of the nuclear, mitochondrial and microsomal fractions. A considerable percentage of radioactivity was found unextractable with the organic solvents, suggesting that styrene oxide reacted with the endogenous compounds. The intracellular distribution of this epoxide was also studied in the perfused rat liver. Comparable results with those previously described were obtained. The binding of styrene oxide to the cytosolic protein was investigated by equilibrium dialysis and ultrafiltration. Only a small percentage of the compound was bound to protein

ESX-1-mediated translocation to the cytosol controls virulence of mycobacteria

KAUST Repository

Houben, Diane; Demangel, Caroline; Van Ingen, Jakko; Perez, Jorge; Baldeó n, Lucy R.; Abdallah, Abdallah; Caleechurn, Laxmee; Bottai, Daria; Van Zon, Maaike; De Punder, Karin; Van Der Laan, Tridia; Kant, Arie; Bossers-De Vries, Ruth; Willemsen, Peter Th J; Bitter, Wilbert M.; Van Soolingen, Dick; Brosch, Roland; Van Der Wel, Nicole N.; Peters, Peter J.

2012-01-01

Mycobacterium species, including Mycobacterium tuberculosis and Mycobacterium leprae, are among the most potent human bacterial pathogens. The discovery of cytosolic mycobacteria challenged the paradigm that these pathogens exclusively localize within the phagosome of host cells. As yet the biological relevance of mycobacterial translocation to the cytosol remained unclear. In this current study we used electron microscopy techniques to establish a clear link between translocation and mycobacterial virulence. Pathogenic, patient-derived mycobacteria species were found to translocate to the cytosol, while non-pathogenic species did not. We were further able to link cytosolic translocation with pathogenicity by introducing the ESX-1 (type VII) secretion system into the non-virulent, exclusively phagolysosomal Mycobacterium bovis BCG. Furthermore, we show that translocation is dependent on the C-terminus of the early-secreted antigen ESAT-6. The C-terminal truncation of ESAT-6 was shown to result in attenuation in mice, again linking translocation to virulence. Together, these data demonstrate the molecular mechanism facilitating translocation of mycobacteria. The ability to translocate from the phagolysosome to the cytosol is with this study proven to be biologically significant as it determines mycobacterial virulence. © 2012 Blackwell Publishing Ltd.

ESX-1-mediated translocation to the cytosol controls virulence of mycobacteria

KAUST Repository

Houben, Diane

2012-05-08

Mycobacterium species, including Mycobacterium tuberculosis and Mycobacterium leprae, are among the most potent human bacterial pathogens. The discovery of cytosolic mycobacteria challenged the paradigm that these pathogens exclusively localize within the phagosome of host cells. As yet the biological relevance of mycobacterial translocation to the cytosol remained unclear. In this current study we used electron microscopy techniques to establish a clear link between translocation and mycobacterial virulence. Pathogenic, patient-derived mycobacteria species were found to translocate to the cytosol, while non-pathogenic species did not. We were further able to link cytosolic translocation with pathogenicity by introducing the ESX-1 (type VII) secretion system into the non-virulent, exclusively phagolysosomal Mycobacterium bovis BCG. Furthermore, we show that translocation is dependent on the C-terminus of the early-secreted antigen ESAT-6. The C-terminal truncation of ESAT-6 was shown to result in attenuation in mice, again linking translocation to virulence. Together, these data demonstrate the molecular mechanism facilitating translocation of mycobacteria. The ability to translocate from the phagolysosome to the cytosol is with this study proven to be biologically significant as it determines mycobacterial virulence. © 2012 Blackwell Publishing Ltd.

Dataset of protein species from human liver

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Stanislav Naryzhny

2017-06-01

Full Text Available This article contains data related to the research article entitled “Zipf׳s law in proteomics” (Naryzhny et al., 2017 [1]. The protein composition in the human liver or hepatocarcinoma (HepG2 cells extracts was estimated using a filter-aided sample preparation (FASP protocol. The protein species/proteoform composition in the human liver was determined by two-dimensional electrophoresis (2-DE followed by Electrospray Ionization Liquid Chromatography-Tandem Mass Spectrometry (ESI LC-MS/MS. In the case of two-dimensional electrophoresis (2-DE, the gel was stained with Coomassie Brilliant Blue R350, and image analysis was performed with ImageMaster 2D Platinum software (GE Healthcare. The 96 sections in the 2D gel were selected and cut for subsequent ESI LC-MS/MS and protein identification. If the same protein was detected in different sections, it was considered to exist as different protein species/proteoforms. A list of human liver proteoforms detected in this way is presented.

Long-term culture of human liver tissue with advanced hepatic functions.

Science.gov (United States)

Ng, Soon Seng; Xiong, Anming; Nguyen, Khanh; Masek, Marilyn; No, Da Yoon; Elazar, Menashe; Shteyer, Eyal; Winters, Mark A; Voedisch, Amy; Shaw, Kate; Rashid, Sheikh Tamir; Frank, Curtis W; Cho, Nam Joon; Glenn, Jeffrey S

2017-06-02

A major challenge for studying authentic liver cell function and cell replacement therapies is that primary human hepatocytes rapidly lose their advanced function in conventional, 2-dimensional culture platforms. Here, we describe the fabrication of 3-dimensional hexagonally arrayed lobular human liver tissues inspired by the liver's natural architecture. The engineered liver tissues exhibit key features of advanced differentiation, such as human-specific cytochrome P450-mediated drug metabolism and the ability to support efficient infection with patient-derived inoculums of hepatitis C virus. The tissues permit the assessment of antiviral agents and maintain their advanced functions for over 5 months in culture. This extended functionality enabled the prediction of a fatal human-specific hepatotoxicity caused by fialuridine (FIAU), which had escaped detection by preclinical models and short-term clinical studies. The results obtained with the engineered human liver tissue in this study provide proof-of-concept determination of human-specific drug metabolism, demonstrate the ability to support infection with human hepatitis virus derived from an infected patient and subsequent antiviral drug testing against said infection, and facilitate detection of human-specific drug hepatotoxicity associated with late-onset liver failure. Looking forward, the scalability and biocompatibility of the scaffold are also ideal for future cell replacement therapeutic strategies.

A study of human liver ferritin and chicken liver and spleen using Moessbauer spectroscopy with high velocity resolution

Energy Technology Data Exchange (ETDEWEB)

Oshtrakh, M. I., E-mail: oshtrakh@mail.utnet.ru [Ural State Technical University-UPI, Faculty of Physical Techniques and Devices for Quality Control (Russian Federation); Milder, O. B.; Semionkin, V. A. [Ural State Technical University-UPI, Faculty of Experimental Physics (Russian Federation)

2008-01-15

Lyophilized samples of human liver ferritin and chicken liver and spleen were measured at room temperature using Moessbauer spectroscopy with high velocity resolution. An increase in the velocity resolution of Moessbauer spectroscopy permitted us to increase accuracy and decrease experimental error in determining the hyperfine parameters of human liver ferritin and chicken liver and spleen. Moessbauer spectroscopy with high velocity resolution may be very useful for revealing small differences in hyperfine parameters during biomedical research.

Human cytosolic thymidine kinase: purification and physical characterization of the enzyme from HeLa cells

International Nuclear Information System (INIS)

Sherley, J.L.; Kelly, T.J.

1988-01-01

The mammalian cytosolic thymidine kinase is one of a number of enzymes involved in DNA replication whose activities increase dramatically during S phase of the cell cycle. As a first step in defining the mechanisms that control the S phase induction of thymidine kinase activity, the authors have purified the human enzyme from HeLa cells and raised a specific immune serum against the purified protein. The enzyme was isolated from cells arrested in S phase by treatment with methotrexate and purified to near homogeneity by ion-exchange and affinity chromatography. Stabilization of the purified enzyme was achieved by the addition of digitonin. An electrophoretic R/sub m/ of 0.2 in nondenaturing gels characterizes the purified enzyme activity as cytosolic thymidine kinase. The enzyme has a Stoke's radius of 40 A determined by gel filtration and a sedimentation coefficient of 5.5 S determined by glycerol gradient sedimentation. Based on these hydrodynamic values, a native molecular weight of 96,000 was calculated for the purified enzyme. When electrophoresed in denaturing sodium dodecyl sulfate-polyacrylamide gels under reducing conditions, the most purified enzyme fraction was found to contain one predominant polypeptide of M/sub r/ = 24,000. Several lines of evidence indicate that this polypeptide is responsible for thymidine kinase enzymatic activity

Subcellular distribution of curium in beagle liver

International Nuclear Information System (INIS)

Bruenger, F.W.; Grube, B.J.; Atherton, D.R.; Taylor, G.N.; Stevens, W.

1976-01-01

The subcellular distribution of curium ( 243 244 Cm) was studied in canine liver from 2 hr to 47 days after injection of 3 μCi 243 244 Cm/kg of body weight. The pattern of distribution for Cm was similar to other trivalent actinide elements studied previously (Am, Cf). Initially (2 hr), most of the nuclide was found in the cytosol and at least 90 percent was protein bound. About 70 percent of the Cm was bound to ferritin, approximately 5 percent was associated with a protein of MW approximately 200,000, and approximately 25 percent was found in the low-molecular-weight region (approximately 5000). The decrease in the Cm content of cytosol, nuclei, and microsomes coincided with an increase in the amount associated with mitochondria and lysosomes. The concentration of the Cm in the mitochondrial fraction was higher than it was in the lysosomal fraction at each time studied. In the mitochondrial fraction approximately 30 percent of the Cm was bound to membranous or granular material, and 70 percent was found in the soluble fraction. The Cm concentration initially associated with cell nuclei was high but had diminished to 20 percent of the 2 hr concentration by 20 days post injection (PI). The subcellular distribution of Cm in the liver of a dog which had received the same dose and was terminated because of severe liver damage was studied at 384 days PI. The liver weighed 130 g and contained approximately 30 percent of the injected Cm. In contrast, a normal liver weighs 280 g and at 2 hr PI contains approximately 40 percent of the injected dose. The subcellular distribution of Cm in this severely damaged liver differed from the pattern observed at earlier times after injection. The relative concentration of Cm in the cytosol was doubled; it was higher in the nuclei-debris fraction; and it was lower in the mitochondrial and lysosomal fractions when compared to earlier times

Hepatic cholesterol ester hydrolase in human liver disease.

Science.gov (United States)

Simon, J B; Poon, R W

1978-09-01

Human liver contains an acid cholesterol ester hydrolase (CEH) of presumed lysosomal origin, but its significance is unknown. We developed a modified CEH radioassay suitable for needle biopsy specimens and measured hepatic activity of this enzyme in 69 patients undergoing percutaneous liver biopsy. Histologically normal livers hydrolyzed 5.80 +/- 0.78 SEM mumoles of cholesterol ester per hr per g of liver protein (n, 10). Values were similar in alcoholic liver disease (n, 17), obstructive jaundice (n, 9), and miscellaneous hepatic disorders (n, 21). In contrast, mean hepatic CEH activity was more than 3-fold elevated in 12 patients with acute hepatitis, 21.05 +/- 2.45 SEM mumoles per hr per g of protein (P less than 0.01). In 2 patients studied serially, CEH returned to normal as hepatitis resolved. CEH activity in all patients paralleled SGOT levels (r, 0.84; P less than 0.01). There was no correlation with serum levels of free or esterified cholesterol nor with serum activity of lecithin-cholesterol acyltransferase, the enzyme responsible for cholesterol esterification in plasma. These studies confirm the presence of CEH activity in human liver and show markedly increased activity in acute hepatitis. The pathogenesis and clinical significance of altered hepatic CEH activity in liver disease require further study.

Three-dimensional reconstructions of intrahepatic bile duct tubulogenesis in human liver

DEFF Research Database (Denmark)

Vestentoft, Peter S; Jelnes, Peter; Hopkinson, Branden M

2011-01-01

BACKGROUND: During liver development, intrahepatic bile ducts are thought to arise by a unique asymmetric mode of cholangiocyte tubulogenesis characterized by a series of remodeling stages. Moreover, in liver diseases, cells lining the Canals of Hering can proliferate and generate new hepatic...... in normal liver and in the extensive ductular reactions originating from intrahepatic bile ducts and branching into the parenchyma of the acetaminophen intoxicated liver. In the developing human liver, three-dimensional reconstructions using multiple marker proteins confirmed that the human intrahepatic...

Effect of the Human Amniotic Membrane on Liver Regeneration in Rats

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Mesut Sipahi

2015-01-01

Full Text Available Introduction. Operations are performed for broader liver surgery indications for a better understanding of hepatic anatomy/physiology and developments in operation technology. Surgery can cure some patients with liver metastasis of some tumors. Nevertheless, postoperative liver failure is the most feared complication causing mortality in patients who have undergone excision of a large liver mass. The human amniotic membrane has regenerative effects. Thus, we investigated the effects of the human amniotic membrane on regeneration of the resected liver. Methods. Twenty female Wistar albino rats were divided into control and experimental groups and underwent a 70% hepatectomy. The human amniotic membrane was placed over the residual liver in the experimental group. Relative liver weight, histopathological features, and biochemical parameters were assessed on postoperative day 3. Results. Total protein and albumin levels were significantly lower in the experimental group than in the control group. No difference in relative liver weight was observed between the groups. Hepatocyte mitotic count was significantly higher in the experimental group than in the control group. Hepatic steatosis was detected in the experimental group. Conclusion. Applying the amniotic membrane to residual liver adversely affected liver regeneration. However, mesenchymal stem cell research has the potential to accelerate liver regeneration investigations.

Molecular Structure of Human-Liver Glycogen.

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Bin Deng

Full Text Available Glycogen is a highly branched glucose polymer which is involved in maintaining blood-sugar homeostasis. Liver glycogen contains large composite α particles made up of linked β particles. Previous studies have shown that the binding which links β particles into α particles is impaired in diabetic mice. The present study reports the first molecular structural characterization of human-liver glycogen from non-diabetic patients, using transmission electron microscopy for morphology and size-exclusion chromatography for the molecular size distribution; the latter is also studied as a function of time during acid hydrolysis in vitro, which is sensitive to certain structural features, particularly glycosidic vs. proteinaceous linkages. The results are compared with those seen in mice and pigs. The molecular structural change during acid hydrolysis is similar in each case, and indicates that the linkage of β into α particles is not glycosidic. This result, and the similar morphology in each case, together imply that human liver glycogen has similar molecular structure to those of mice and pigs. This knowledge will be useful for future diabetes drug targets.

Detection of Cytosolic Shigella flexneri via a C-Terminal Triple-Arginine Motif of GBP1 Inhibits Actin-Based Motility

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Anthony S. Piro

2017-12-01

Full Text Available Dynamin-like guanylate binding proteins (GBPs are gamma interferon (IFN-γ-inducible host defense proteins that can associate with cytosol-invading bacterial pathogens. Mouse GBPs promote the lytic destruction of targeted bacteria in the host cell cytosol, but the antimicrobial function of human GBPs and the mechanism by which these proteins associate with cytosolic bacteria are poorly understood. Here, we demonstrate that human GBP1 is unique among the seven human GBP paralogs in its ability to associate with at least two cytosolic Gram-negative bacteria, Burkholderia thailandensis and Shigella flexneri. Rough lipopolysaccharide (LPS mutants of S. flexneri colocalize with GBP1 less frequently than wild-type S. flexneri does, suggesting that host recognition of O antigen promotes GBP1 targeting to Gram-negative bacteria. The targeting of GBP1 to cytosolic bacteria, via a unique triple-arginine motif present in its C terminus, promotes the corecruitment of four additional GBP paralogs (GBP2, GBP3, GBP4, and GBP6. GBP1-decorated Shigella organisms replicate but fail to form actin tails, leading to their intracellular aggregation. Consequentially, the wild type but not the triple-arginine GBP1 mutant restricts S. flexneri cell-to-cell spread. Furthermore, human-adapted S. flexneri, through the action of one its secreted effectors, IpaH9.8, is more resistant to GBP1 targeting than the non-human-adapted bacillus B. thailandensis. These studies reveal that human GBP1 uniquely functions as an intracellular “glue trap,” inhibiting the cytosolic movement of normally actin-propelled Gram-negative bacteria. In response to this powerful human defense program, S. flexneri has evolved an effective counterdefense to restrict GBP1 recruitment.

Current status of prediction of drug disposition and toxicity in humans using chimeric mice with humanized liver.

Science.gov (United States)

Kitamura, Shigeyuki; Sugihara, Kazumi

2014-01-01

1. Human-chimeric mice with humanized liver have been constructed by transplantation of human hepatocytes into several types of mice having genetic modifications that injure endogenous liver cells. Here, we focus on liver urokinase-type plasminogen activator-transgenic severe combined immunodeficiency (uPA/SCID) mice, which are the most widely used human-chimeric mice. Studies so far indicate that drug metabolism, drug transport, pharmacological effects and toxicological action in these mice are broadly similar to those in humans. 2. Expression of various drug-metabolizing enzymes is known to be different between humans and rodents. However, the expression pattern of cytochrome P450, aldehyde oxidase and phase II enzymes in the liver of human-chimeric mice resembles that in humans, not that in the host mice. 3. Metabolism of various drugs, including S-warfarin, zaleplon, ibuprofen, naproxen, coumarin, troglitazone and midazolam, in human-chimeric mice is mediated by human drug-metabolizing enzymes, not by host mouse enzymes, and thus resembles that in humans. 4. Pharmacological and toxicological effects of various drugs in human-chimeric mice are also similar to those in humans. 5. The current consensus is that chimeric mice with humanized liver are useful to predict drug metabolism catalyzed by cytochrome P450, aldehyde oxidase and phase II enzymes in humans in vivo and in vitro. Some remaining issues are discussed in this review.

Inhibition of rat, mouse, and human glutathione S-transferase by eugenol and its oxidation products

NARCIS (Netherlands)

Rompelberg, C.J.M.; Ploemen, J.H.T.M.; Jespersen, S.; Greef, J. van der; Verhagen, H.; Bladeren, P.J. van

1996-01-01

The irreversible and reversible inhibition of glutathione S-transferases (GSTs) by eugenol was studied in rat, mouse and man. Using liver cytosol of human, rat and mouse, species differences were found in the rate of irreversible inhibition of GSTs by eugenol in the presence of the enzyme

A Roadmap for Human Liver Differentiation from Pluripotent Stem Cells

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Lay Teng Ang

2018-02-01

Full Text Available How are closely related lineages, including liver, pancreas, and intestines, diversified from a common endodermal origin? Here, we apply principles learned from developmental biology to rapidly reconstitute liver progenitors from human pluripotent stem cells (hPSCs. Mapping the formation of multiple endodermal lineages revealed how alternate endodermal fates (e.g., pancreas and intestines are restricted during liver commitment. Human liver fate was encoded by combinations of inductive and repressive extracellular signals at different doses. However, these signaling combinations were temporally re-interpreted: cellular competence to respond to retinoid, WNT, TGF-β, and other signals sharply changed within 24 hr. Consequently, temporally dynamic manipulation of extracellular signals was imperative to suppress the production of unwanted cell fates across six consecutive developmental junctures. This efficiently generated 94.1% ± 7.35% TBX3+HNF4A+ human liver bud progenitors and 81.5% ± 3.2% FAH+ hepatocyte-like cells by days 6 and 18 of hPSC differentiation, respectively; the latter improved short-term survival in the Fah−/−Rag2−/−Il2rg−/− mouse model of liver failure.

PVA matches human liver in needle-tissue interaction.

Science.gov (United States)

de Jong, Tonke L; Pluymen, Loes H; van Gerwen, Dennis J; Kleinrensink, Gert-Jan; Dankelman, Jenny; van den Dobbelsteen, John J

2017-05-01

Medical phantoms can be used to study needle-tissue interaction and to train medical residents. The purpose of this research is to study the suitability of polyvinyl alcohol (PVA) as a liver tissue mimicking material in terms of needle-tissue interaction. Insertions into ex-vivo human livers were used for reference. Six PVA samples were created by varying the mass percentage of PVA to water (4m% and 7m%) and the number of freeze-thaw cycles (1, 2 and 3 cycles, 16hours of freezing at -19°C, 8hours of thawing). The inner needle of an 18 Gauge trocar needle with triangular tip was inserted 13 times into each of the samples, using an insertion velocity of 5 mm/s. In addition, 39 insertions were performed in two ex-vivo human livers. Axial forces on the needle were captured during insertion and retraction and characterized by friction along the needle shaft, peak forces, and number of peak forces per unit length. The concentration of PVA and the number of freeze-thaw cycles both influenced the mechanical interaction between needle and specimen. Insertions into 4m% PVA phantoms with 2 freeze-thaw cycles were comparable to human liver in terms of estimated friction along the needle shaft and the number of peak forces. Therefore, these phantoms are considered to be suitable liver mimicking materials for image-guided needle interventions. The mechanical properties of PVA hydrogels can be influenced in a controlled manner by varying the concentration of PVA and the number of freeze-thaw cycles, to mimic liver tissue characteristics. Copyright © 2017 Elsevier Ltd. All rights reserved.

Cytosolic adenylate changes during exercise in prawn muscle

International Nuclear Information System (INIS)

Thebault, M.T.; Raffin, J.P.; Pichon, R.

1994-01-01

31 P NMR and biochemical analysis were used to assess the effect of heavy exercise on cytosolic adenylate levels in Palaemon serratus abdominal muscle. At rest, the MgATP level corresponded to 85.5% of the total ATP content. The cytosolic adenylate concentrations of the prawn muscle are considerably different from that of vertebrates. The percentage of ADP bound to myofilaments was lower in the prawn muscle. Consequently, the level of free cytosolic AMP was greatly higher (thirty fold higher) than in vertebrate muscle. During vigorous work, the concentration of MgATP dropped and the cytosolic AMP accumulated, while the cytosolic adenine nucleotide pool decreased significantly. The phosphorylation potential value and the ATP/ADP ratio, calculated from the cytosolic adenylate, dropped acutely during the whole period of muscular contractions. On the contrary, the adenylate energy charge calculated from the cytosolic adenylate decreased slightly. Therefore, even in muscle displaying no AMP deamination, the adenylate charge is stabilized during exercise by the dynamic changes between cytosolic and bound adenylate species. (author). 21 refs., 2 tabs

Metabolic profiles of pomalidomide in human plasma simulated with pharmacokinetic data in control and humanized-liver mice.

Science.gov (United States)

Shimizu, Makiko; Suemizu, Hiroshi; Mitsui, Marina; Shibata, Norio; Guengerich, F Peter; Yamazaki, Hiroshi

2017-10-01

1. Pomalidomide has been shown to be potentially teratogenic in thalidomide-sensitive animal species such as rabbits. Screening for thalidomide analogs devoid of teratogenicity/toxicity - attributable to metabolites formed by cytochrome P450 enzymes - but having immunomodulatory properties is a strategic pathway towards development of new anticancer drugs. 2. In this study, plasma concentrations of pomalidomide, its primary 5-hydroxylated metabolite, and its glucuronide conjugate(s) were investigated in control and humanized-liver mice. Following oral administration of pomalidomide (100 mg/kg), plasma concentrations of 7-hydroxypomalidomide and 5-hydroxypomalidomide glucuronide were slightly higher in humanized-liver mice than in control mice. 3. Simulations of human plasma concentrations of pomalidomide were achieved with simplified physiologically-based pharmacokinetic models in both groups of mice in accordance with reported pomalidomide concentrations after low dose administration in humans. 4. The results indicate that pharmacokinetic profiles of pomalidomide were roughly similar between control mice and humanized-liver mice and that control and humanized-liver mice mediated pomalidomide 5-hydroxylation in vivo. Introducing one aromatic amino group into thalidomide resulted in less species differences in in vivo pharmacokinetics in control and humanized-liver mice.

New psychoactive substances: Studies on the metabolism of XLR-11, AB-PINACA, FUB-PB-22, 4-methoxy-α-PVP, 25-I-NBOMe, and meclonazepam using human liver preparations in comparison to primary human hepatocytes, and human urine.

Science.gov (United States)

Richter, Lilian H J; Maurer, Hans H; Meyer, Markus R

2017-10-05

New psychoactive substances (NPS) are an increasing problem in clinical and forensic toxicology. The knowledge of their metabolism is important for toxicological risk assessment and for developing toxicological urine screenings. Considering the huge numbers of NPS annually appearing on the market, metabolism studies should be realized in a fast, simple, cost efficient, and reliable way. Primary human hepatocytes (PHH) were recommended to be the gold standard for in vitro metabolism studies as they are expected to contain natural enzyme clusters, co-substrates, and drug transporters. In addition, they were already successfully used for metabolism studies of NPS. However, they also have disadvantages such as high costs and limited applicability without special equipment. The aims of the present study were therefore first to investigate exemplarily the phase I and phase II metabolism of six NPS (XLR-11, AB-PINACA, FUB-PB-22, 4-methoxy-α-PVP, 25-I-NBOMe, and meclonazepam) from different drug classes using pooled human S9 fraction (pS9) or pooled human liver microsomes combined with cytosol (pHLM/pHLC) after addition of the co-substrates for the main metabolic phase I and II reactions. Second to compare results to published data generated using primary human hepatocytes and human urine samples. Results of the incubations with pS9 or pHLM/pHLC were comparable in number and abundance of metabolites. Formation of metabolites, particularly after multi-step reactions needed a longer incubation time. However, incubations using human liver preparations resulted in a lower number of total detected metabolites compared to PHH, but they were still able to allow the identification of the main human urinary excretion products. Human liver preparations and particularly the pooled S9 fraction could be shown to be a sufficient and more cost-efficient alternative in context of metabolism studies also for developing toxicological urine screenings. It might be recommended to use the

Cell sources for in vitro human liver cell culture models

Science.gov (United States)

Freyer, Nora; Damm, Georg; Seehofer, Daniel; Knöspel, Fanny

2016-01-01

In vitro liver cell culture models are gaining increasing importance in pharmacological and toxicological research. The source of cells used is critical for the relevance and the predictive value of such models. Primary human hepatocytes (PHH) are currently considered to be the gold standard for hepatic in vitro culture models, since they directly reflect the specific metabolism and functionality of the human liver; however, the scarcity and difficult logistics of PHH have driven researchers to explore alternative cell sources, including liver cell lines and pluripotent stem cells. Liver cell lines generated from hepatomas or by genetic manipulation are widely used due to their good availability, but they are generally altered in certain metabolic functions. For the past few years, adult and pluripotent stem cells have been attracting increasing attention, due their ability to proliferate and to differentiate into hepatocyte-like cells in vitro. However, controlling the differentiation of these cells is still a challenge. This review gives an overview of the major human cell sources under investigation for in vitro liver cell culture models, including primary human liver cells, liver cell lines, and stem cells. The promises and challenges of different cell types are discussed with a focus on the complex 2D and 3D culture approaches under investigation for improving liver cell functionality in vitro. Finally, the specific application options of individual cell sources in pharmacological research or disease modeling are described. PMID:27385595

Molecular Aging of Human Liver: An Epigenetic/Transcriptomic Signature.

Science.gov (United States)

Bacalini, Maria Giulia; Franceschi, Claudio; Gentilini, Davide; Ravaioli, Francesco; Zhou, Xiaoyuan; Remondini, Daniel; Pirazzini, Chiara; Giuliani, Cristina; Marasco, Elena; Gensous, Noémie; Di Blasio, Anna Maria; Ellis, Ewa; Gramignoli, Roberto; Castellani, Gastone; Capri, Miriam; Strom, Stephen; Nardini, Christine; Cescon, Matteo; Grazi, Gian Luca; Garagnani, Paolo

2018-03-15

The feasibility of liver transplantation from old healthy donors suggests that this organ is able to preserve its functionality during aging. To explore the biological basis of this phenomenon, we characterized the epigenetic profile of liver biopsies collected from 45 healthy liver donors ranging from 13 to 90 years old using the Infinium HumanMethylation450 BeadChip. The analysis indicates that a large remodeling in DNA methylation patterns occurs, with 8823 age-associated differentially methylated CpG probes. Notably, these age-associated changes tended to level off after the age of 60, as confirmed by Horvath's clock. Using stringent selection criteria we further identified a DNA methylation signature of aging liver including 75 genomic regions. We demonstrated that this signature is specific for liver compared to other tissues and that it is able to detect biological age-acceleration effects associated with obesity. Finally we combined DNA methylation measurements with available expression data. Although the intersection between the two omic characterizations was low, both approaches suggested a previously unappreciated role of epithelial-mesenchymal transition and Wnt signaling pathways in the aging of human liver.

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

International Nuclear Information System (INIS)

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

2004-01-01

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

Human plasma concentrations of tolbutamide and acetaminophen extrapolated from in vivo animal pharmacokinetics using in vitro human hepatic clearances and simple physiologically based pharmacokinetic modeling for radio-labeled microdose clinical studies

International Nuclear Information System (INIS)

Yamazaki, Hiroshi; Kunikane, Eriko; Nishiyama, Sayako; Murayama, Norie; Shimizu, Makiko; Sugiyama, Yuichi; Chiba, Koji; Ikeda, Toshihiko

2015-01-01

The aim of the current study was to extrapolate the pharmacokinetics of drug substances orally administered in humans from rat pharmacokinetic data using tolbutamide and acetaminophen as model compounds. Adjusted animal biomonitoring equivalents from rat studies based on reported plasma concentrations were scaled to human biomonitoring equivalents using known species allometric scaling factors. In this extrapolation, in vitro metabolic clearance data were obtained using liver preparations. Rates of tolbutamide elimination were roughly similar in rat and human liver microsome experiments, but acetaminophen elimination by rat liver microsomes and cytosolic preparations showed a tendency to be faster than those in humans. Using a simple physiologically based pharmacokinetic (PBPK) model, estimated human plasma concentrations of tolbutamide and acetaminophen were consistent with reported concentrations. Tolbutamide cleared in a roughly similar manner in humans and rats, but medical-dose levels of acetaminophen cleared (dependent on liver metabolism) more slowly from plasma in humans than it did in rats. The data presented here illustrate how pharmacokinetic data in combination with a simple PBPK model can be used to assist evaluations of the pharmacological/toxicological potential of new drug substances and for estimating human radiation exposures from radio-labeled drugs when planning human studies. (author)

Assessment of emerging biomarkers of liver injury in human subjects.

Science.gov (United States)

Schomaker, Shelli; Warner, Roscoe; Bock, Jeff; Johnson, Kent; Potter, David; Van Winkle, Joyce; Aubrecht, Jiri

2013-04-01

Hepatotoxicity remains a major challenge in drug development. Although alanine aminotransferase (ALT) remains the gold standard biomarker of liver injury, alternative biomarker strategies to better predict the potential for severe drug-induced liver injury (DILI) are essential. In this study, we evaluated the utility of glutamate dehydrogenase (GLDH), purine nucleoside phosphorylase (PNP), malate dehydrogenase (MDH), and paraxonase 1 (PON1) as indicators of liver injury in cohorts of human subjects, including healthy subjects across age and gender, subjects with a variety of liver impairments, and several cases of acetaminophen poisoning. In the healthy subjects, levels of GLDH and MDH were not affected by age or gender. Reference ranges for GLDH and MDH in healthy subjects were 1-10 and 79-176U/L, respectively. In contrast, the levels of PON1 and PNP were not consistent across cohorts of healthy subjects. Furthermore, GLDH and MDH had a strong correlation with elevated ALT levels and possessed a high predictive power for liver injury, as determined by ROC analysis. In contrast, PON1 and PNP did not detect liver injury in our study. Finally, evaluation of patients with acetaminophen-induced liver injury provided evidence that both GLDH and MDH might have utility as biomarkers of DILI in humans. This study is the first to evaluate GLDH, MDH, PON1, and PNP in a large number of human subjects and, and it provides an impetus for prospective clinical studies to fully evaluate the diagnostic value of GLDH and MDH for detection of liver injury.

Cranberry juice suppressed the diclofenac metabolism by human liver microsomes, but not in healthy human subjects

Science.gov (United States)

Ushijima, Kentarou; Tsuruoka, Shu-ichi; Tsuda, Hidetoshi; Hasegawa, Gohki; Obi, Yuri; Kaneda, Tae; Takahashi, Masaki; Maekawa, Tomohiro; Sasaki, Tomohiro; Koshimizu, Taka-aki; Fujimura, Akio

2009-01-01

AIM To investigate a potential interaction between cranberry juice and diclofenac, a substrate of CYP2C9. METHODS The inhibitory effect of cranberry juice on diclofenac metabolism was determined using human liver microsome assay. Subsequently, we performed a clinical trial in healthy human subjects to determine whether the repeated consumption of cranberry juice changed the diclofenac pharmacokinetics. RESULTS Cranberry juice significantly suppressed diclofenac metabolism by human liver microsomes. On the other hand, repeated consumption of cranberry juice did not influence the diclofenac pharmacokinetics in human subjects. CONCLUSIONS Cranberry juice inhibited diclofenac metabolism by human liver microsomes, but not in human subjects. Based on the present and previous findings, we think that although cranberry juice inhibits CYP2C9 activity in vitro, it does not change the pharmacokinetics of medications metabolized by CYP2C9 in clinical situations. PMID:19694738

Human precision-cut liver slices as a model to test antifibrotic drugs in the early onset of liver fibrosis

NARCIS (Netherlands)

Westra, Inge M.; Mutsaers, Henricus A. M.; Luangmonkong, Theerut; Hadi, Mackenzie; Oosterhuis, Dorenda; de Jong, Koert P.; Groothuis, Geny M. M.; Olinga, Peter

Liver fibrosis is the progressive accumulation of connective tissue ultimately resulting in loss of organ function. Currently, no effective antifibrotics are available due to a lack of reliable human models. Here we investigated the fibrotic process in human precision-cut liver slices (PCLS) and

Interaction of rocuronium with human liver cytochromes P450

OpenAIRE

Anzenbacherova, Eva; Spicakova, Alena; Jourova, Lenka; Ulrichova, Jitka; Adamus, Milan; Bachleda, Petr; Anzenbacher, Pavel

2015-01-01

Rocuronium is a neuromuscular blocking agent acting as a competitive antagonist of acetylcholine. Results of an inhibition of eight individual liver microsomal cytochromes P450 (CYP) are presented. As the patients are routinely premedicated with diazepam, possible interaction of diazepam with rocuronium has been also studied. Results indicated that rocuronium interacts with human liver microsomal CYPs by binding to the substrate site. Next, concentration dependent inhibition of liver micro...

Surface-Enhanced Raman Spectroscopy (SERS Tracking of Chelerythrine, a Na+/K+ Pump Inhibitor, into Cytosol and Plasma Membrane Fractions of Human Lens Epithelial Cell Cultures

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Kevin M. Dorney

2013-12-01

Full Text Available Background/Aims: The quaternary benzo-phenanthridine alkaloid (QBA chelerythrine (CET is a pro-apoptotic drug and Na+/K+ pump (NKP inhibitor in human lens epithelial cells (HLECs. In order to obtain further insight into the mechanism of NKP inhibition by CET, its sub-cellular distribution was quantified in cytosolic and membrane fractions of HLEC cultures by surface-enhanced Raman spectroscopy (SERS. Methods: Silver nanoparticles (AgNPs prepared by the Creighton method were concentrated, and size-selected using a one-step tangential flow filtration approach. HLECs cultures were exposed to 50 μM CET in 300 mOsM phosphate-buffered NaCl for 30 min. A variety of cytosolic extracts, crude and purified membranes, prepared in lysing solutions in the presence and absence of a non-ionic detergent, were incubated with AgNPs and subjected to SERS analysis. Determinations of CET were based on a linear calibration plot of the integrated CET SERS intensity at its 659 cm-1 marker band as a function of CET concentration. Results: SERS detected chemically unaltered CET in both cytosol and plasma membrane fractions. Normalized for protein, the CET content was some 100 fold higher in the crude and purified plasma membrane fraction than in the soluble cytosolic extract. The total free CET concentration in the cytosol, free of membranes or containing detergent-solubilized membrane material, approached that of the incubation medium of HLECs. Conclusion: Given a negative membrane potential of HLECs the data suggest, but do not prove, that CET may traverse the plasma membrane as a positively charged monomer (CET+ accumulating near or above passive equilibrium distribution. These findings may contribute to a recently proposed hypothesis that CET binds to and inhibits the NKP through its cytosolic aspect.

Surface-enhanced Raman spectroscopy (SERS) tracking of chelerythrine, a Na(+)/K(+) pump inhibitor, into cytosol and plasma membrane fractions of human lens epithelial cell cultures.

Science.gov (United States)

Dorney, Kevin M; Sizemore, Ioana E P; Alqahtani, Tariq; Adragna, Norma C; Lauf, Peter K

2013-01-01

The quaternary benzo-phenanthridine alkaloid (QBA) chelerythrine (CET) is a pro-apoptotic drug and Na(+)/K(+) pump (NKP) inhibitor in human lens epithelial cells (HLECs). In order to obtain further insight into the mechanism of NKP inhibition by CET, its sub-cellular distribution was quantified in cytosolic and membrane fractions of HLEC cultures by surface-enhanced Raman spectroscopy (SERS). Silver nanoparticles (AgNPs) prepared by the Creighton method were concentrated, and size-selected using a one-step tangential flow filtration approach. HLECs cultures were exposed to 50 μM CET in 300 mOsM phosphate-buffered NaCl for 30 min. A variety of cytosolic extracts, crude and purified membranes, prepared in lysing solutions in the presence and absence of a non-ionic detergent, were incubated with AgNPs and subjected to SERS analysis. Determinations of CET were based on a linear calibration plot of the integrated CET SERS intensity at its 659 cm(-1) marker band as a function of CET concentration. SERS detected chemically unaltered CET in both cytosol and plasma membrane fractions. Normalized for protein, the CET content was some 100 fold higher in the crude and purified plasma membrane fraction than in the soluble cytosolic extract. The total free CET concentration in the cytosol, free of membranes or containing detergent-solubilized membrane material, approached that of the incubation medium of HLECs. Given a negative membrane potential of HLECs the data suggest, but do not prove, that CET may traverse the plasma membrane as a positively charged monomer (CET(+)) accumulating near or above passive equilibrium distribution. These findings may contribute to a recently proposed hypothesis that CET binds to and inhibits the NKP through its cytosolic aspect. © 2014 S. Karger AG, Basel.

Augmenter of liver regeneration (ALR) protects human hepatocytes against apoptosis

Energy Technology Data Exchange (ETDEWEB)

Ilowski, Maren [Liver Regeneration Group, Department of Surgery, Grosshadern Hospital, Ludwig Maximilians University, Munich (Germany); Kleespies, Axel [Department of Surgery, Grosshadern Hospital, Ludwig Maximilians University, Munich (Germany); Toni, Enrico N. de [Department of Medicine II, Grosshadern Hospital, Ludwig Maximilians University, Munich (Germany); Donabauer, Barbara [Liver Regeneration Group, Department of Surgery, Grosshadern Hospital, Ludwig Maximilians University, Munich (Germany); Jauch, Karl-Walter [Department of Surgery, Grosshadern Hospital, Ludwig Maximilians University, Munich (Germany); Hengstler, Jan G. [Leibniz Research Centre for Working Environment and Human Factors, Technical University, Dortmund (Germany); Thasler, Wolfgang E., E-mail: wolfgang.thasler@med.uni-muenchen.de [Liver Regeneration Group, Department of Surgery, Grosshadern Hospital, Ludwig Maximilians University, Munich (Germany); Department of Surgery, Grosshadern Hospital, Ludwig Maximilians University, Munich (Germany)

2011-01-07

Research highlights: {yields} ALR decreases cytochrome c release from mitochondria. {yields} ALR protects hepatocytes against apoptosis induction by ethanol, TRAIL, anti-Apo, TGF-{beta} and actinomycin D. {yields} ALR exerts a liver-specific anti-apoptotic effect. {yields} A possible medical usage of ALR regarding protection of liver cells during apoptosis inducing therapies. -- Abstract: Augmenter of liver regeneration (ALR) is known to support liver regeneration and to stimulate proliferation of hepatocytes. However, it is not known if ALR exerts anti-apoptotic effects in human hepatocytes and whether this protective effect is cell type specific. This is relevant, because compounds that protect the liver against apoptosis without undesired effects, such as protection of metastatic tumour cells, would be appreciated in several clinical settings. Primary human hepatocytes (phH) and organotypic cancer cell lines were exposed to different concentrations of apoptosis inducers (ethanol, TRAIL, anti-Apo, TGF-{beta}, actinomycin D) and cultured with or without recombinant human ALR (rhALR). Apoptosis was evaluated by the release of cytochrome c from mitochondria and by FACS with propidium iodide (PI) staining. ALR significantly decreased apoptosis induced by ethanol, TRAIL, anti-Apo, TGF-{beta} and actinomycin D. Further, the anti-apoptotic effect of ALR was observed in primary human hepatocytes and in HepG2 cells but not in bronchial (BC1), colonic (SW480), gastric (GC1) and pancreatic (L3.6PL) cell lines. Therefore, the hepatotrophic growth factor ALR acts in a liver specific manner with regards to both its mitogenic and its anti-apoptotic effect. Unlike the growth factors HGF and EGF, rhALR acts in a liver specific manner. Therefore, ALR is a promising candidate for further evaluation as a possible hepatoprotective factor in clinical settings.

Augmenter of liver regeneration (ALR) protects human hepatocytes against apoptosis

International Nuclear Information System (INIS)

Ilowski, Maren; Kleespies, Axel; Toni, Enrico N. de; Donabauer, Barbara; Jauch, Karl-Walter; Hengstler, Jan G.; Thasler, Wolfgang E.

2011-01-01

Research highlights: → ALR decreases cytochrome c release from mitochondria. → ALR protects hepatocytes against apoptosis induction by ethanol, TRAIL, anti-Apo, TGF-β and actinomycin D. → ALR exerts a liver-specific anti-apoptotic effect. → A possible medical usage of ALR regarding protection of liver cells during apoptosis inducing therapies. -- Abstract: Augmenter of liver regeneration (ALR) is known to support liver regeneration and to stimulate proliferation of hepatocytes. However, it is not known if ALR exerts anti-apoptotic effects in human hepatocytes and whether this protective effect is cell type specific. This is relevant, because compounds that protect the liver against apoptosis without undesired effects, such as protection of metastatic tumour cells, would be appreciated in several clinical settings. Primary human hepatocytes (phH) and organotypic cancer cell lines were exposed to different concentrations of apoptosis inducers (ethanol, TRAIL, anti-Apo, TGF-β, actinomycin D) and cultured with or without recombinant human ALR (rhALR). Apoptosis was evaluated by the release of cytochrome c from mitochondria and by FACS with propidium iodide (PI) staining. ALR significantly decreased apoptosis induced by ethanol, TRAIL, anti-Apo, TGF-β and actinomycin D. Further, the anti-apoptotic effect of ALR was observed in primary human hepatocytes and in HepG2 cells but not in bronchial (BC1), colonic (SW480), gastric (GC1) and pancreatic (L3.6PL) cell lines. Therefore, the hepatotrophic growth factor ALR acts in a liver specific manner with regards to both its mitogenic and its anti-apoptotic effect. Unlike the growth factors HGF and EGF, rhALR acts in a liver specific manner. Therefore, ALR is a promising candidate for further evaluation as a possible hepatoprotective factor in clinical settings.

Analysis of iron storage proteins in chicken liver and spleen tissues in comparison with human liver ferritin by Moessbauer spectroscopy

International Nuclear Information System (INIS)

Oshtrakh, M.I.; Milder, O.B.; Semionkin, V.A.; Malakheeva, L.I.; Prokopenko, P.G.

2006-01-01

Characterization of iron storage proteins in liver and spleen from normal chicken and chicken with lymphoid leukemia in comparison with human liver ferritin were considered by Moessbauer spectroscopy (preliminary results). Small differences in Moessbauer hyperfine parameters for both normal and lymphoid leukemia chicken liver and spleen were observed. The value of quadrupole splitting for human liver ferritin was higher than those for chicken tissues. A decrease of iron content in lymphoid leukemia chicken tissues was also found, however, the reason of this fact (pathology or feeding) was not clear yet. (author)

AGE WISE HISTOMORPHOLOGICAL CHANGES IN HUMAN LIVER

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Tribeni

2015-11-01

Full Text Available CONTEXT: Hepato cellular carcinoma (HCC results in between 2.5 lakhs to 1million deaths globally per annum. Liver transplantation nowadays is a well accepted treatment option for end-stage liver disease and acute liver failure. AIMS: Keeping this concept in view, a study was conducted in the Guwahati Zone of Northeast India, to compare the histomorphological features of the human liver in different age groups. SETTING AND DESIGN: Apparently healthy livers were obtained from 21 subjects on whom medicolegal post-mortems had been performed. Their ages varied from newborn to 90 years. Subjects were divided into 3 groups. 7 specimens were taken from each group. (1 Pediatric (2 Adult (3 Old age. METHODS AND MATERIALS: In all the above age groups, immediately after removal of the livers, they were washed in normal saline, dried with blotting paper and weighed in an electronic weighing machine. Sections of liver were fixed, processed, cut and stained with Harris Haematoxylin and Eosin stain. RESULTS: The liver loses weight from 50 years onwards. There appears to be racial and environmental differences in the change in liver weight in old age. Autopsy studies show a diminution of nearly 46% in liver weight between the 3rd and 10th decades of life. The liver decreases in size with age. The hepatocytes are radially disposed in the liver lobule. They are piled up, forming a layer one cell thick (except in young children in a fashion similar to the bricks of a wall. These plates are directed from the periphery of the lobule to its centre and anastomose freely forming a complex labyrinthine and sponge-like structure. CONCLUSIONS: From the findings in the present study it can be concluded that: 1. Nowadays, the measurement of liver volume has gained practical use in relation to liver transplantation. 2. We have compared the histomorphology of adult liver with a child. The findings in both the groups are very similar. This feature is important, since in

The cytosolic exonuclease TREX1 inhibits the innate immune response to HIV-1

Science.gov (United States)

Yan, Nan; Regalado-Magdos, Ashton D.; Stiggelbout, Bart; Lee-Kirsch, Min Ae; Lieberman, Judy

2010-01-01

Viral infection triggers innate immune sensors to produce type I interferons (IFN). However, HIV infection of T cells and macrophages does not trip these alarms. How HIV avoids activating nucleic acid sensors is unknown. The cytosolic exonuclease TREX1 suppressed IFN triggered by HIV. In Trex1−/− mouse cells and human CD4+ T cells and macrophages in which TREX1 was inhibited by RNA interference, cytosolic HIV DNA accumulated, and HIV infection induced type I IFN that inhibited HIV replication and spreading. TREX1 bound to cytosolic HIV DNA and digested excess HIV DNA that would otherwise activate IFN expression via a TBK1, STING and IRF3 dependent pathway. HIV-stimulated IFN production in cells deficient in TREX1 did not involve known nucleic acid sensors. PMID:20871604

Attenuated lipotoxicity and apoptosis is linked to exogenous and endogenous augmenter of liver regeneration by different pathways.

Science.gov (United States)

Weiss, Thomas S; Lupke, Madeleine; Ibrahim, Sara; Buechler, Christa; Lorenz, Julia; Ruemmele, Petra; Hofmann, Ute; Melter, Michael; Dayoub, Rania

2017-01-01

Nonalcoholic fatty liver disease (NAFLD) covers a spectrum from simple steatosis to nonalcoholic steatohepatitis (NASH) and cirrhosis. Free fatty acids (FFA) induce steatosis and lipo-toxicity and correlate with severity of NAFLD. In this study we aimed to investigate the role of exogenous and endogenous ALR (augmenter of liver regeneration) for FFA induced ER (endoplasmatic reticulum) -stress and lipoapoptosis. Primary human hepatocytes or hepatoma cells either treated with recombinant human ALR (rhALR, 15kDa) or expressing short form ALR (sfALR, 15kDa) were incubated with palmitic acid (PA) and analyzed for lipo-toxicity, -apoptosis, activation of ER-stress response pathways, triacylglycerides (TAG), mRNA and protein expression of lipid metabolizing genes. Both, exogenous rhALR and cytosolic sfALR reduced PA induced caspase 3 activity and Bax protein expression and therefore lipotoxicity. Endogenous sfALR but not rhALR treatment lowered TAG levels, diminished activation of ER-stress mediators C-Jun N-terminal kinase (JNK), X-box binding protein-1 (XBP1) and proapoptotic transcription factor C/EBP-homologous protein (CHOP), and reduced death receptor 5 protein expression. Cellular ALR exerts its lipid lowering and anti-apoptotic actions by enhancing FABP1, which binds toxic FFA, increasing mitochondrial β-oxidation by elevating the mitochondrial FFA transporter CPT1α, and decreasing ELOVL6, which delivers toxic FFA metabolites. We found reduced hepatic mRNA levels of ALR in a high fat diet mouse model, and of ALR and FOXA2, a transcription factor inducing ALR expression, in human steatotic as well as NASH liver samples, which may explain increased lipid deposition and reduced β-oxidation in NASH patients. Present study shows that exogenous and endogenous ALR reduce PA induced lipoapoptosis. Furthermore, cytosolic sfALR changes mRNA and protein expression of genes regulating lipid metabolism, reduces ER-stress finally impeding progression of NASH.

Attenuated lipotoxicity and apoptosis is linked to exogenous and endogenous augmenter of liver regeneration by different pathways.

Directory of Open Access Journals (Sweden)

Thomas S Weiss

Full Text Available Nonalcoholic fatty liver disease (NAFLD covers a spectrum from simple steatosis to nonalcoholic steatohepatitis (NASH and cirrhosis. Free fatty acids (FFA induce steatosis and lipo-toxicity and correlate with severity of NAFLD. In this study we aimed to investigate the role of exogenous and endogenous ALR (augmenter of liver regeneration for FFA induced ER (endoplasmatic reticulum -stress and lipoapoptosis. Primary human hepatocytes or hepatoma cells either treated with recombinant human ALR (rhALR, 15kDa or expressing short form ALR (sfALR, 15kDa were incubated with palmitic acid (PA and analyzed for lipo-toxicity, -apoptosis, activation of ER-stress response pathways, triacylglycerides (TAG, mRNA and protein expression of lipid metabolizing genes. Both, exogenous rhALR and cytosolic sfALR reduced PA induced caspase 3 activity and Bax protein expression and therefore lipotoxicity. Endogenous sfALR but not rhALR treatment lowered TAG levels, diminished activation of ER-stress mediators C-Jun N-terminal kinase (JNK, X-box binding protein-1 (XBP1 and proapoptotic transcription factor C/EBP-homologous protein (CHOP, and reduced death receptor 5 protein expression. Cellular ALR exerts its lipid lowering and anti-apoptotic actions by enhancing FABP1, which binds toxic FFA, increasing mitochondrial β-oxidation by elevating the mitochondrial FFA transporter CPT1α, and decreasing ELOVL6, which delivers toxic FFA metabolites. We found reduced hepatic mRNA levels of ALR in a high fat diet mouse model, and of ALR and FOXA2, a transcription factor inducing ALR expression, in human steatotic as well as NASH liver samples, which may explain increased lipid deposition and reduced β-oxidation in NASH patients. Present study shows that exogenous and endogenous ALR reduce PA induced lipoapoptosis. Furthermore, cytosolic sfALR changes mRNA and protein expression of genes regulating lipid metabolism, reduces ER-stress finally impeding progression of NASH.

"Cyt/Nuc," a Customizable and Documenting ImageJ Macro for Evaluation of Protein Distributions Between Cytosol and Nucleus.

Science.gov (United States)

Grune, Tilman; Kehm, Richard; Höhn, Annika; Jung, Tobias

2018-05-01

Large amounts of data from multi-channel, high resolution, fluorescence microscopic images require tools that provide easy, customizable, and reproducible high-throughput analysis. The freeware "ImageJ" has become one of the standard tools for scientific image analysis. Since ImageJ offers recording of "macros," even a complex multi-step process can be easily applied fully automated to large numbers of images, saving both time and reducing human subjective evaluation. In this work, we present "Cyt/Nuc," an ImageJ macro, able to recognize and to compare the nuclear and cytosolic areas of tissue samples, in order to investigate distributions of immunostained proteins between both compartments, while it documents in detail the whole process of evaluation and pattern recognition. As practical example, the redistribution of the 20S proteasome, the main intracellular protease in mammalian cells, is investigated in NZO-mouse liver after feeding the animals different diets. A significant shift in proteasomal distribution between cytosol and nucleus in response to metabolic stress was revealed using "Cyt/Nuc" via automatized quantification of thousands of nuclei within minutes. "Cyt/Nuc" is easy to use and highly customizable, matches the precision of careful manual evaluation and bears the potential for quick detection of any shift in intracellular protein distribution. © 2018 The Authors. Biotechnology Journal Published by Wiley-VCH Verlag GmbH & Co. KGaA.

The Arabidopsis cytosolic proteome

DEFF Research Database (Denmark)

Ito, Jun; Parsons, Harriet Tempé; Heazlewood, Joshua L.

2014-01-01

compartments. However, a detailed study of enriched cytosolic fractions from Arabidopsis cell culture has been performed only recently, with over 1,000 proteins reproducibly identified by mass spectrometry. The number of proteins allocated to the cytosol nearly doubles to 1,802 if a series of targeted...

PEPCK-M expression in mouse liver potentiates, not replaces, PEPCK-C mediated gluconeogenesis

Science.gov (United States)

Méndez-Lucas, Andrés; Duarte, João; Sunny, Nishanth E.; Satapati, Santhosh; He, TianTeng; Fu, Xiaorong; Bermúdez, Jordi; Burgess, Shawn C.; Perales, Jose C.

2013-01-01

Background & Aims Hepatic gluconeogenesis helps maintain systemic energy homeostasis by compensating for discontinuities in nutrient supply. Liver specific deletion of cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C) abolishes gluconeogenesis from mitochondrial substrates, deregulates lipid metabolism and affects TCA cycle. While, mouse liver almost exclusively expresses PEPCK-C, humans equally present a mitochondrial isozyme (PEPCK-M). Despite clear relevance to human physiology, the role of PEPCK-M and its gluconeogenic potential remain unknown. Here, we test the significance of PEPCK-M in gluconeogenesis and TCA cycle function in liver-specific PEPCK-C knockout and WT mice. Methods The effects of the overexpression of PEPCK-M were examined by a combination of tracer studies and molecular biology techniques. Partial PEPCK-C re-expression was used as a positive control. Metabolic fluxes were evaluated in isolated livers by NMR using 2H and 13C tracers. Gluconeogenic potential, together with metabolic profiling, were investigated in vivo and in primary hepatocytes. Results PEPCK-M expression partially rescued defects in lipid metabolism, gluconeogenesis and TCA cycle function impaired by PEPCK-C deletion, while ~10% re-expression of PEPCK-C normalized most parameters. When PEPCK-M was expressed in the presence of PEPCK-C, the mitochondrial isozyme amplified total gluconeogenic capacity, suggesting autonomous regulation of oxaloacetate to phosphoenolpyruvate fluxes by the individual isoforms. Conclusions We conclude that PEPCK-M has gluconeogenic potential per se, and cooperates with PEPCK-C to adjust gluconeogenic/TCA flux to changes in substrate or energy availability, hinting at a role in the regulation of glucose and lipid metabolism in human liver. PMID:23466304

Molecular cloning and nucleotide sequence of cDNA for human liver arginase

International Nuclear Information System (INIS)

Haraguchi, Y.; Takiguchi, M.; Amaya, Y.; Kawamoto, S.; Matsuda, I.; Mori, M.

1987-01-01

Arginase (EC3.5.3.1) catalyzes the last step of the urea cycle in the liver of ureotelic animals. Inherited deficiency of the enzyme results in argininemia, an autosomal recessive disorder characterized by hyperammonemia. To facilitate investigation of the enzyme and gene structures and to elucidate the nature of the mutation in argininemia, the authors isolated cDNA clones for human liver arginase. Oligo(dT)-primed and random primer human liver cDNA libraries in λ gt11 were screened using isolated rat arginase cDNA as a probe. Two of the positive clones, designated λ hARG6 and λ hARG109, contained an overlapping cDNA sequence with an open reading frame encoding a polypeptide of 322 amino acid residues (predicted M/sub r/, 34,732), a 5'-untranslated sequence of 56 base pairs, a 3'-untranslated sequence of 423 base pairs, and a poly(A) segment. Arginase activity was detected in Escherichia coli cells transformed with the plasmid carrying λ hARG6 cDNA insert. RNA gel blot analysis of human liver RNA showed a single mRNA of 1.6 kilobases. The predicted amino acid sequence of human liver arginase is 87% and 41% identical with those of the rat liver and yeast enzymes, respectively. There are several highly conserved segments among the human, rat, and yeast enzymes

Effects of the hepatocarcinogen nafenopin, a peroxisome proliferator, on the activities of rat liver glutathione-requiring enzymes and catalase in comparison to the action of phenobarbital.

Science.gov (United States)

Furukawa, K; Numoto, S; Furuya, K; Furukawa, N T; Williams, G M

1985-10-01

The biochemical effects in the livers of male rats of prolonged administration of the experimental hepatocarcinogen nafenopin, a hypolipidemic agent and peroxisome proliferator, were compared to those of another experimental liver carcinogen, phenobarbital, which acts as a neoplasm promoter. Feeding of nafenopin, 0.03 mmol/kg basal diet for up to 24 weeks increased the numbers of hepatic peroxisomes, increased catalase activity, markedly decreased cytosolic glutathione transferase activities toward two substrates, decreased cytosolic glutathione peroxidase activities toward H2O2 and two organic peroxides, and suppressed the age-related increase in gamma-glutamyl transpeptidase activity. In contrast the livers of rats fed an equimolar concentration of phenobarbital displayed increases in cytosolic glutathione transferase activities and enhancement of gamma-glutamyl transpeptidase activity but no changes in glutathione peroxidase activities. There was also an enhancement of catalase activity without apparent increase in peroxisome number. Enzyme kinetic analyses revealed that the cytosolic glutathione transferase activities toward two halogenonitrobenzene substrates were inhibited in the rats fed nafenopin and displayed elevated Km and decreased Vmax. Kinetic studies of glutathione transferase activities in which nafenopin was mixed with normal rat liver cytosols in the assay system revealed competitive type inhibition toward 1-chloro-2,4-dinitrobenzene and a noncompetitive type of inhibition toward 3,4-dichloronitrobenzene. Likewise activities of glutathione peroxidases toward H2O2 and cumene hydroperoxide were suppressed by in vitro addition. Thus the effects of nafenopin and phenobarbital on liver biochemistry were very different. The inhibition of hepatic biotransformation and scavenger systems by nafenopin is suggested to be relevant to its hepatocarcinogenicity.

Acute cadmium intoxication induces alpha-class glutathione S-transferase protein synthesis and enzyme activity in rat liver

International Nuclear Information System (INIS)

Casalino, Elisabetta; Sblano, Cesare; Calzaretti, Giovanna; Landriscina, Clemente

2006-01-01

Acute cadmium intoxication affects glutathione S-transferase (GST) in rat liver. It has been found that 24 h after i.p. cadmium administration to rats, at a dose of 2.5 mg CdCl 2 kg -1 body weight, the activity of this enzyme in liver cytosol increased by 40%. A less stimulatory effect persisted till 48 h and thereafter the enzyme activity normalized. Since, GST isoenzymes belong to different classes in mammalian tissues, we used quantitative immunoassays to verify which family of GST isoenzymes is influenced by this intoxication. Only alpha-class glutathione S-transferase (α-GST) proteins were detected in rat liver cytosol and their level increased by about 25%, 24 h after cadmium treatment. No pi-GST isoforms were found in liver cytosol from either normal or cadmium-treated rats. Co-administration of actinomycin D with cadmium normalized both the protein level and the activity of α-GST, suggesting that some effect occurs on enzyme transcription of these isoenzymes by this metal. On the other hand, it seems unlikely that the stimulatory effect is due to the high level of peroxides caused by lipid peroxidation, since Vitamin E administration strongly reduced the TBARS level, but did not cause any GST activity decrease

Expression pattern of thymosin beta 4 in the adult human liver

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S. Nemolato

2011-09-01

Full Text Available Thymosin beta-4 (Tβ4 is a member of beta-thymosins, a family of small peptides involved in polymerization of G-actin, and in many critical biological processes including apoptosis, cell migration, angiogenesis, and fibrosis. Previous studies in the newborn liver did not reveal any significant reactivity for Tβ4 during the intrauterine life. The aim of the present study was to investigate by immunohistochemistry Tβ4 expression in the adult normal liver. Thirty-five human liver samples, including 11 needle liver biopsies and 24 liver specimens obtained at autopsy, in which no pathological change was detected at the histological examination, were immunostained utilizing an anti-Tβ4 commercial antibody. Tβ4 was detected in the hepatocytes of all adult normal livers examined. A zonation of Tβ4 expression was evident in the vast majority of cases. Immunostaining was preferentially detected in zone 3, while a minor degree of reactivity was detected in periportal hepatocytes (zone 1. At higher power, Tβ4-reactive granules appeared mainly localized at the biliary pole of hepatocytes. In cases with a strong immunostaining, even perinuclear areas and the sinusoidal pole of hepatocytes appeared interested by immunoreactivity for Tβ4. The current work first evidences a strong diffuse expression of Tβ4 in the adult human liver, and adds hepatocytes to the list of human cells able to synthesize large amounts of Tβ4 in adulthood. Moreover, Tβ4 should be added to the liver proteins characterized by a zonate expression pattern, in a descending gradient from the terminal vein to the periportal areas of the liver acinus. Identifying the intimate role played by this peptide intracellularly and extracellularly, in physiology and in different liver diseases, is a major challenge for future research focusing on Tβ4.

Recellularization of rat liver: An in vitro model for assessing human drug metabolism and liver biology.

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Matthew J Robertson

Full Text Available Liver-like organoids that recapitulate the complex functions of the whole liver by combining cells, scaffolds, and mechanical or chemical cues are becoming important models for studying liver biology and drug metabolism. The advantages of growing cells in three-dimensional constructs include enhanced cell-cell and cell-extracellular matrix interactions and preserved cellular phenotype including, prevention of de-differentiation. In the current study, biomimetic liver constructs were made via perfusion decellularization of rat liver, with the goal of maintaining the native composition and structure of the extracellular matrix. We optimized our decellularization process to produce liver scaffolds in which immunogenic residual DNA was removed but glycosaminoglycans were maintained. When the constructs were recellularized with rat or human liver cells, the cells remained viable, capable of proliferation, and functional for 28 days. Specifically, the cells continued to express cytochrome P450 genes and maintained their ability to metabolize a model drug, midazolam. Microarray analysis showed an upregulation of genes involved in liver regeneration and fibrosis. In conclusion, these liver constructs have the potential to be used as test beds for studying liver biology and drug metabolism.

Application of chimeric mice with humanized liver for study of human-specific drug metabolism.

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Bateman, Thomas J; Reddy, Vijay G B; Kakuni, Masakazu; Morikawa, Yoshio; Kumar, Sanjeev

2014-06-01

Human-specific or disproportionately abundant human metabolites of drug candidates that are not adequately formed and qualified in preclinical safety assessment species pose an important drug development challenge. Furthermore, the overall metabolic profile of drug candidates in humans is an important determinant of their drug-drug interaction susceptibility. These risks can be effectively assessed and/or mitigated if human metabolic profile of the drug candidate could reliably be determined in early development. However, currently available in vitro human models (e.g., liver microsomes, hepatocytes) are often inadequate in this regard. Furthermore, the conduct of definitive radiolabeled human ADME studies is an expensive and time-consuming endeavor that is more suited for later in development when the risk of failure has been reduced. We evaluated a recently developed chimeric mouse model with humanized liver on uPA/SCID background for its ability to predict human disposition of four model drugs (lamotrigine, diclofenac, MRK-A, and propafenone) that are known to exhibit human-specific metabolism. The results from these studies demonstrate that chimeric mice were able to reproduce the human-specific metabolite profile for lamotrigine, diclofenac, and MRK-A. In the case of propafenone, however, the human-specific metabolism was not detected as a predominant pathway, and the metabolite profiles in native and humanized mice were similar; this was attributed to the presence of residual highly active propafenone-metabolizing mouse enzymes in chimeric mice. Overall, the data indicate that the chimeric mice with humanized liver have the potential to be a useful tool for the prediction of human-specific metabolism of xenobiotics and warrant further investigation.

Phosphorus-31 spectroscopic imaging of the human liver

International Nuclear Information System (INIS)

Biran, M.; Raffard, G.; Canioni, P.; Kien, P.

1993-01-01

During the last decade, progresses in the field of nuclear magnetic resonance spectroscopy (M.R.S.), have allowed the metabolic studies of complex biological systems. Since the coming out of whole body magnets, clinical applications are possible; they utilize magnetic field gradients coupled with selective pulse sequences. Study of the phosphorylated metabolism of human liver can be performed with sequences as ISIS, FROGS or 1D-CSI. But they present some disadvantages (for instance contamination by phosphocreatine from muscle). In the present work, we have studied the human liver in vivo by 31 P spectroscopic imaging. Several spectra could be acquired with only one acquisition. This study has needed the building of radiofrequency coils (surface coils), specially designed for liver observation (15 cm diameter 31 P coil and 19 cm diameter proton coil, both transmitter and receiver coils). Preliminary studies have been done on a phantom followed by in vivo measurements on healthy subject livers. We have obtained localized 31 P N.M.R. spectra corresponding to different voxels within the hepatic tissue. The conditions of acquisition of spectra and the problems related to the saturation of phosphorylated metabolite signals (in particular phosphodiesters) are discussed. (author). 5 figs., 15 refs

Massive and Reproducible Production of Liver Buds Entirely from Human Pluripotent Stem Cells

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Takanori Takebe

2017-12-01

Full Text Available Summary: Organoid technology provides a revolutionary paradigm toward therapy but has yet to be applied in humans, mainly because of reproducibility and scalability challenges. Here, we overcome these limitations by evolving a scalable organ bud production platform entirely from human induced pluripotent stem cells (iPSC. By conducting massive “reverse” screen experiments, we identified three progenitor populations that can effectively generate liver buds in a highly reproducible manner: hepatic endoderm, endothelium, and septum mesenchyme. Furthermore, we achieved human scalability by developing an omni-well-array culture platform for mass producing homogeneous and miniaturized liver buds on a clinically relevant large scale (>108. Vascularized and functional liver tissues generated entirely from iPSCs significantly improved subsequent hepatic functionalization potentiated by stage-matched developmental progenitor interactions, enabling functional rescue against acute liver failure via transplantation. Overall, our study provides a stringent manufacturing platform for multicellular organoid supply, thus facilitating clinical and pharmaceutical applications especially for the treatment of liver diseases through multi-industrial collaborations. : With the goal of clinical translation of liver bud transplant therapy, Takebe et al. established a massive organoid production platform from endoderm, endothelial, and mesenchymal progenitor populations specified entirely from human iPSCs, reproducibly demonstrating functionality both in vitro and in vivo. Keywords: iPSC, liver bud, organoid, transplantation, self-organization, endothelial, mesenchymal, liver failure, clinical grade

Kinetic studies of the inhibition of a human liver 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase isozyme by bile acids and anti-inflammatory drugs.

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Miyabe, Y; Amano, T; Deyashiki, Y; Hara, A; Tsukada, F

1995-01-01

We have investigated the steady-state kinetics for a cytosolic 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase isozyme of human liver and its inhibition by several bile acids and anti-inflammatory drugs such as indomethacin, flufemanic acid and naproxen. Initial velocity and product inhibition studies performed in the NADP(+)-linked (S)-1-indanol oxidation at pH 7.4 were consistent with a sequential ordered mechanism in which NADP+ binds first and leaves last. The bile acids and drugs, competitive inhibitors with respect to the alcohol substrate, exhibited uncompetitive inhibition with respect to the coenzyme, with Ki values less than 1 microM, whereas indomethacin exhibited noncompetitive inhibition (Ki < 24 microM). The kinetics of the inhibition by a mixture of the two inhibitors suggests that bile acids and drugs, except indomethacin, bind to overlapping sites at the active center of the enzyme-coenzyme binary complex.

Effect of low dose radiation on thymocyte cytosol and nuclei protein synthesis in mice

International Nuclear Information System (INIS)

Meng Qingyong; Chen Shali; Liu Shuzheng

2003-01-01

Objective: To the effect of low dose radiation on thymocyte cytosol and nuclei protein synthesis in mice. Methods: The expression of proteins was analyzed by gel filtration with Sephadex G-100 and HPLC based on separation of proteins on thymocyte cytosol and nuclei after whole-body irradiation with 75 mGy X-rays and sham-irradiation, and their biological activity was examined by mouse splenocyte proliferation and chromosome aberration of human peripheral blood lymphocytes. Results: HPLC analysis showed that there was a marked increase in expression of 61.4 kD protein in the extract of thymocyte cytosol and 30.4 kD protein in the extract of thymocyte nuclei in comparison with the corresponding fractions from the sham-irradiated control mice. These protein fractions from the thymocyte cytosol and nuclei of the irradiated mice showed both stimulating effect on normal T cell proliferation and protective effect on chromosome damage induced by high dose radiation. Conclusion: These findings might have implications in study of mechanism of immunoenhancement and cytogenetic adaptive response induced by low dose radiation

Liver Effects of Clinical Drugs Differentiated in Human Liver Slices

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Alison E. M. Vickers

2017-03-01

Full Text Available Drugs with clinical adverse effects are compared in an ex vivo 3-dimensional multi-cellular human liver slice model. Functional markers of oxidative stress and mitochondrial function, glutathione GSH and ATP levels, were affected by acetaminophen (APAP, 1 mM, diclofenac (DCF, 1 mM and etomoxir (ETM, 100 μM. Drugs targeting mitochondria more than GSH were dantrolene (DTL, 10 μM and cyclosporin A (CSA, 10 μM, while GSH was affected more than ATP by methimazole (MMI, 500 μM, terbinafine (TBF, 100 μM, and carbamazepine (CBZ 100 μM. Oxidative stress genes were affected by TBF (18%, CBZ, APAP, and ETM (12%–11%, and mitochondrial genes were altered by CBZ, APAP, MMI, and ETM (8%–6%. Apoptosis genes were affected by DCF (14%, while apoptosis plus necrosis were altered by APAP and ETM (15%. Activation of oxidative stress, mitochondrial energy, heat shock, ER stress, apoptosis, necrosis, DNA damage, immune and inflammation genes ranked CSA (75%, ETM (66%, DCF, TBF, MMI (61%–60%, APAP, CBZ (57%–56%, and DTL (48%. Gene changes in fatty acid metabolism, cholestasis, immune and inflammation were affected by DTL (51%, CBZ and ETM (44%–43%, APAP and DCF (40%–38%, MMI, TBF and CSA (37%–35%. This model advances multiple dosing in a human ex vivo model, plus functional markers and gene profile markers of drug induced human liver side-effects.

LeftyA sensitive cytosolic pH regulation and glycolytic flux in Ishikawa human endometrial cancer cells

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Salker, Madhuri S.; Zhou, Yuetao; Singh, Yogesh [Department of Physiology, University of Tuebingen, 72076 Tuebingen (Germany); Brosens, Jan [Division of Reproductive Health, Warwick Medical School, Clinical Sciences Research Laboratories, University Hospital, Coventry CV2 2DX (United Kingdom); Lang, Florian, E-mail: florian.lang@uni-tuebingen.de [Department of Physiology, University of Tuebingen, 72076 Tuebingen (Germany)

2015-05-08

Objective: LeftyA, a powerful regulator of stemness, embryonic differentiation, and reprogramming of cancer cells, counteracts cell proliferation and tumor growth. Key properties of tumor cells include enhanced glycolytic flux, which is highly sensitive to cytosolic pH and thus requires export of H{sup +} and lactate. H{sup +} extrusion is in part accomplished by Na{sup +}/H{sup +} exchangers, such as NHE1. An effect of LeftyA on transport processes has, however, never been reported. The present study thus explored whether LeftyA modifies regulation of cytosolic pH (pHi) in Ishikawa cells, a well differentiated endometrial carcinoma cell model. Methods: NHE1 transcript levels were determined by qRT-PCR, NHE1 protein abundance quantified by Western blotting, pH{sub i} estimated utilizing (2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein [BCECF] fluorescence, Na{sup +}/H{sup +} exchanger activity from Na{sup +} dependent realkalinization after an ammonium pulse, and lactate concentration in the supernatant utilizing an enzymatic assay and subsequent colorimetry. Results: A 2 h treatment with LeftyA (8 ng/ml) significantly decreased NHE1 transcript levels (by 99.6%), NHE1 protein abundance (by 71%), Na{sup +}/H{sup +} exchanger activity (by 55%), pHi (from 7.22 ± 0.02 to 7.05 ± 0.02), and lactate release (by 41%). Conclusions: LeftyA markedly down-regulates NHE1 expression, Na{sup +}/H{sup +} exchanger activity, pHi, and lactate release in Ishikawa cells. Those effects presumably contribute to cellular reprogramming and growth inhibition. - Highlights: • LeftyA, an inhibitor of tumor growth, reduces Na{sup +}/H{sup +}-exchanger activity by 55%. • LeftyA decreases NHE1 transcripts by 99.6% and NHE1 protein by 71%. • LeftyA decreases cytosolic pH from 7.22 ± 0.02 to 7.05 ± 0.02. • Cytosolic acidification by Lefty A decreases glycolysis by 41%. • Cytosolic acidification by Lefty A compromises energy production of tumor cells.

Completion of hepatitis C virus replication cycle in heterokaryons excludes dominant restrictions in human non-liver and mouse liver cell lines.

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Anne Frentzen

2011-04-01

Full Text Available Hepatitis C virus (HCV is hepatotropic and only infects humans and chimpanzees. Consequently, an immunocompetent small animal model is lacking. The restricted tropism of HCV likely reflects specific host factor requirements. We investigated if dominant restriction factors expressed in non-liver or non-human cell lines inhibit HCV propagation thus rendering these cells non-permissive. To this end we explored if HCV completes its replication cycle in heterokaryons between human liver cell lines and non-permissive cell lines from human non-liver or mouse liver origin. Despite functional viral pattern recognition pathways and responsiveness to interferon, virus production was observed in all fused cells and was only ablated when cells were treated with exogenous interferon. These results exclude that constitutive or virus-induced expression of dominant restriction factors prevents propagation of HCV in these cell types, which has important implications for HCV tissue and species tropism. In turn, these data strongly advocate transgenic approaches of crucial human HCV cofactors to establish an immunocompetent small animal model.

Human plasma metabolic profiles of benzydamine, a flavin-containing monooxygenase probe substrate, simulated with pharmacokinetic data from control and humanized-liver mice.

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Yamazaki-Nishioka, Miho; Shimizu, Makiko; Suemizu, Hiroshi; Nishiwaki, Megumi; Mitsui, Marina; Yamazaki, Hiroshi

2018-02-01

1. Benzydamine is used clinically as a nonsteroidal anti-inflammatory drug in oral rinses and is employed in preclinical research as a flavin-containing monooxygenase (FMO) probe substrate. In this study, plasma concentrations of benzydamine and its primary N-oxide and N-demethylated metabolites were investigated in control TK-NOG mice, in humanized-liver mice, and in mice whose liver cells had been ablated with ganciclovir. 2. Following oral administration of benzydamine (10 mg/kg) in humanized-liver TK-NOG mice, plasma concentrations of benzydamine N-oxide were slightly higher than those of demethyl benzydamine. In contrast, in control and ganciclovir-treated TK-NOG mice, concentrations of demethyl benzydamine were slightly higher than those of benzydamine N-oxide. 3. Simulations of human plasma concentrations of benzydamine and its N-oxide were achieved using simplified physiologically based pharmacokinetic models based on data from control TK-NOG mice and from reported benzydamine concentrations after low-dose administration in humans. Estimated clearance rates based on data from humanized-liver and ganciclovir-treated TK-NOG mice were two orders magnitude high. 4. The pharmacokinetic profiles of benzydamine were different for control and humanized-liver TK-NOG mice. Humanized-liver mice are generally accepted human models; however, drug oxidation in mouse kidney might need to be considered when probe substrates undergo FMO-dependent drug oxidation in mouse liver and kidney.

Human germline hedgehog pathway mutations predispose to fatty liver.

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Guillen-Sacoto, Maria J; Martinez, Ariel F; Abe, Yu; Kruszka, Paul; Weiss, Karin; Everson, Joshua L; Bataller, Ramon; Kleiner, David E; Ward, Jerrold M; Sulik, Kathleen K; Lipinski, Robert J; Solomon, Benjamin D; Muenke, Maximilian

2017-10-01

Non-alcoholic fatty liver disease (NAFLD) is the most common form of liver disease. Activation of hedgehog (Hh) signaling has been implicated in the progression of NAFLD and proposed as a therapeutic target; however, the effects of Hh signaling inhibition have not been studied in humans with germline mutations that affect this pathway. Patients with holoprosencephaly (HPE), a disorder associated with germline mutations disrupting Sonic hedgehog (SHH) signaling, were clinically evaluated for NAFLD. A combined mouse model of Hh signaling attenuation (Gli2 heterozygous null: Gli2 +/- ) and diet-induced NAFLD was used to examine aspects of NAFLD and hepatic gene expression profiles, including molecular markers of hepatic fibrosis and inflammation. Patients with HPE had a higher prevalence of liver steatosis compared to the general population, independent of obesity. Exposure of Gli2 +/- mice to fatty liver-inducing diets resulted in increased liver steatosis compared to wild-type mice. Similar to humans, this effect was independent of obesity in the mutant mice and was associated with decreased expression of pro-fibrotic and pro-inflammatory genes, and increased expression of PPARγ, a potent anti-fibrogenic and anti-inflammatory regulator. Interestingly, tumor suppressors p53 and p16INK4 were found to be downregulated in the Gli2 +/- mice exposed to a high-fat diet. Our results indicate that germline mutations disrupting Hh signaling promotes liver steatosis, independent of obesity, with reduced fibrosis. While Hh signaling inhibition has been associated with a better NAFLD prognosis, further studies are required to evaluate the long-term effects of mutations affecting this pathway. Lay summary: Non-alcoholic fatty liver disease (NAFLD) is characterized by excess fat deposition in the liver predominantly due to high calorie intake and a sedentary lifestyle. NAFLD progression is usually accompanied by activation of the Sonic hedgehog (SHH) pathway leading to fibrous

Isolation and characterization of adult human liver progenitors from ischemic liver tissue derived from therapeutic hepatectomies.

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Stachelscheid, Harald; Urbaniak, Thomas; Ring, Alexander; Spengler, Berlind; Gerlach, Jörg C; Zeilinger, Katrin

2009-07-01

Recent evidence suggests that progenitor cells in adult tissues and embryonic stem cells share a high resistance to hypoxia and ischemic stress. To study the ischemic resistance of adult liver progenitors, we characterized remaining viable cells in human liver tissue after cold ischemic treatment for 24-168 h, applied to the tissue before cell isolation. In vitro cultures of isolated cells showed a rapid decline of the number of different cell types with increasing ischemia length. After all ischemic periods, liver progenitor-like cells could be observed. The comparably small cells exhibited a low cytoplasm-to-nucleus ratio, formed densely packed colonies, and showed a hepatobiliary marker profile. The cells expressed epithelial cell adhesion molecule, epithelial-specific (CK8/18) and biliary-specific (CK7/19) cytokeratins, albumin, alpha-1-antitrypsin, cytochrome-P450 enzymes, as well as weak levels of hepatocyte nuclear factor-4 and gamma-glutamyl transferase, but not alpha-fetoprotein or Thy-1. In vitro survival and expansion was facilitated by coculture with mouse embryonic fibroblasts. Hepatic progenitor-like cells exhibit a high resistance to ischemic stress and can be isolated from human liver tissue after up to 7 days of ischemia. Ischemic liver tissue from various sources, thought to be unsuitable for cell isolation, may be considered as a prospective source of hepatic progenitor cells.

Inherited human group IVA cytosolic phospholipase A2 deficiency abolishes platelet, endothelial, and leucocyte eicosanoid generation

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Kirkby, Nicholas S.; Reed, Daniel M.; Edin, Matthew L.; Rauzi, Francesca; Mataragka, Stefania; Vojnovic, Ivana; Bishop-Bailey, David; Milne, Ginger L.; Longhurst, Hilary; Zeldin, Darryl C.; Mitchell, Jane A.; Warner, Timothy D.

2016-01-01

Eicosanoids are important vascular regulators, but the phospholipase A2 (PLA2) isoforms supporting their production within the cardiovascular system are not fully understood. To address this, we have studied platelets, endothelial cells, and leukocytes from 2 siblings with a homozygous loss-of-function mutation in group IVA cytosolic phospholipase A2 (cPLA2α). Chromatography/mass spectrometry was used to determine levels of a broad range of eicosanoids produced by isolated vascular cells, and in plasma and urine. Eicosanoid release data were paired with studies of cellular function. Absence of cPLA2α almost abolished eicosanoid synthesis in platelets (e.g., thromboxane A2, control 20.5 ± 1.4 ng/ml vs. patient 0.1 ng/ml) and leukocytes [e.g., prostaglandin E2 (PGE2), control 21.9 ± 7.4 ng/ml vs. patient 1.9 ng/ml], and this was associated with impaired platelet activation and enhanced inflammatory responses. cPLA2α-deficient endothelial cells showed reduced, but not absent, formation of prostaglandin I2 (prostacyclin; control 956 ± 422 pg/ml vs. patient 196 pg/ml) and were primed for inflammation. In the urine, prostaglandin metabolites were selectively influenced by cPLA2α deficiency. For example, prostacyclin metabolites were strongly reduced (18.4% of control) in patients lacking cPLA2α, whereas PGE2 metabolites (77.8% of control) were similar to healthy volunteer levels. These studies constitute a definitive account, demonstrating the fundamental role of cPLA2α to eicosanoid formation and cellular responses within the human circulation.—Kirkby, N. S., Reed, D. M., Edin, M. L., Rauzi, F., Mataragka, S., Vojnovic, I., Bishop-Bailey, D., Milne, G. L., Longhurst, H., Zeldin, D. C., Mitchell, J. A., Warner, T. D. Inherited human group IVA cytosolic phospholipase A2 deficiency abolishes platelet, endothelial, and leucocyte eicosanoid generation. PMID:26183771

Studies on cellular distribution of elements in human hepatocellular carcinoma samples by molecular activation analysis

International Nuclear Information System (INIS)

Deng Guilong; Chen Chunying; Zhang Peiqun; Zhao Jiujiang; Chai Zhifang

2005-01-01

The distribution patterns of 17 elements in the subcellular fractions of nuclei, mitochondria, lysosome, microsome and cytosol of human hepatocellular carcinoma (HCC) and normal liver samples were investigated by using molecular activation analysis (MAA) and differential centrifugation. Their significant difference was checked by the Studient's t-test. These elements exhibit inhomogeneous distributions in each subcellular fraction. Some elements have no significant difference between hepatocellular carcinoma and normal liver samples. However, the concentrations of Br, Ca, Cd and Cs are significantly higher in each component of hepatocarcinoma than in normal liver. The content of Fe in microsome of HCC is significantly lower, almost half of normal liver samples, but higher in other subcellular fractions than in those of normal tissues. The rare earth elements of La and Ce have the patterns similar to Fe. The concentrations of Sb and Zn in nuclei of HCC are obviously lower (P<0.05, P<0.05). The contents of K and Na are higher in cytosol of HCC (P<0.05). The distributions of Ba and Rb show no significant difference between two groups. The relationships of Fe, Cd and K with HCC were also discussed. The levels of some elements in subcellular fractions of tumor were quite different from those of normal liver, which suggested that trace elements might play important roles in the occurrence and development of hepatocellular carcinoma. (authors)

Studies on cellular distribution of elements in human hepatocellular carcinoma samples by molecular activation analysis

Energy Technology Data Exchange (ETDEWEB)

Guilong, Deng [Chinese Academy of Sciences, Beijing (China). Inst. of High Energy Physics, Key Laboratory of Nuclear Analytical Techniques; Department of General Surgery, the Second Affiliated Hospital, School of Medicine, Zhejiang Univ., Hangzhou (China); Chunying, Chen; Peiqun, Zhang; Jiujiang, Zhao; Zhifang, Chai [Chinese Academy of Sciences, Beijing (China). Inst. of High Energy Physics, Key Laboratory of Nuclear Analytical Techniques; Yingbin, Liu; Jianwei, Wang; Bin, Xu; Shuyou, Peng [Department of General Surgery, the Second Affiliated Hospital, School of Medicine, Zhejiang Univ., Hangzhou (China)

2005-07-15

The distribution patterns of 17 elements in the subcellular fractions of nuclei, mitochondria, lysosome, microsome and cytosol of human hepatocellular carcinoma (HCC) and normal liver samples were investigated by using molecular activation analysis (MAA) and differential centrifugation. Their significant difference was checked by the Studient's t-test. These elements exhibit inhomogeneous distributions in each subcellular fraction. Some elements have no significant difference between hepatocellular carcinoma and normal liver samples. However, the concentrations of Br, Ca, Cd and Cs are significantly higher in each component of hepatocarcinoma than in normal liver. The content of Fe in microsome of HCC is significantly lower, almost half of normal liver samples, but higher in other subcellular fractions than in those of normal tissues. The rare earth elements of La and Ce have the patterns similar to Fe. The concentrations of Sb and Zn in nuclei of HCC are obviously lower (P<0.05, P<0.05). The contents of K and Na are higher in cytosol of HCC (P<0.05). The distributions of Ba and Rb show no significant difference between two groups. The relationships of Fe, Cd and K with HCC were also discussed. The levels of some elements in subcellular fractions of tumor were quite different from those of normal liver, which suggested that trace elements might play important roles in the occurrence and development of hepatocellular carcinoma. (authors)

Autoimmune liver disease 2007.

Science.gov (United States)

Muratori, Paolo; Granito, Alessandro; Pappas, Georgios; Muratori, Luigi; Lenzi, Marco; Bianchi, Francesco B

2008-01-01

Autoimmune liver disease (ALD) includes a spectrum of diseases which comprises both cholestatic and hepatitic forms: autoimmune hepatitis (AIH), primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC) and the so called "overlap" syndromes where hepatitic and cholestatic damage coexists. All these diseases are characterized by an extremely high heterogeneity of presentation, varying from asymptomatic, acute (as in a subset of AIH) or chronic (with aspecific symptoms such as fatigue and myalgia in AIH or fatigue and pruritus in PBC and PSC). The detection and characterization of non organ specific autoantibodies plays a major role in the diagnostic approach of autoimmune liver disease; anti nuclear reactivities (ANA) and anti smooth muscle antibodies (SMA) mark type 1 AIH, liver kidney microsomal antibody type 1 (LKM1) and liver cytosol type 1 (LC1) are the serological markers of type 2 AIH; antimitochondrial antibodies (AMA) are associated with PBC, while no specific marker is found in PSC, since anticytoplasmic neutrophil antibodies with perinuclear pattern (atypical p-ANCA or p-ANNA) are also detected in a substantial proportion of type 1 AIH cases. Treatment options rely on immunosoppressive therapy (steroids and azathioprine) in AIH and on ursodeoxycholic acid in cholestatic conditions; in all these diseases liver transplantation remains the only therapeutical approach for the end stage of liver disease.

Redox characteristics of the eukaryotic cytosol

DEFF Research Database (Denmark)

López-Mirabal, H Reynaldo; Winther, Jakob R

2007-01-01

The eukaryotic cytoplasm has long been regarded as a cellular compartment in which the reduced state of protein cysteines is largely favored. Under normal conditions, the cytosolic low-molecular weight redox buffer, comprising primarily of glutathione, is highly reducing and reactive oxygen species...... (ROS) and glutathionylated proteins are maintained at very low levels. In the present review, recent progress in the understanding of the cytosolic thiol-disulfide redox metabolism and novel analytical approaches to studying cytosolic redox properties are discussed. We will focus on the yeast model...... organism, Saccharomyces cerevisiae, where the combination of genetic and biochemical approaches has brought us furthest in understanding the mechanisms underlying cellular redox regulation. It has been shown in yeast that, in addition to the enzyme glutathione reductase, other mechanisms may exist...

Development of Murine Cyp3a Knockout Chimeric Mice with Humanized Liver.

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Kato, Kota; Ohbuchi, Masato; Hamamura, Satoko; Ohshita, Hiroki; Kazuki, Yasuhiro; Oshimura, Mitsuo; Sato, Koya; Nakada, Naoyuki; Kawamura, Akio; Usui, Takashi; Kamimura, Hidetaka; Tateno, Chise

2015-08-01

We developed murine CYP3A knockout ko chimeric mice with humanized liver expressing human P450S similar to those in humans and whose livers and small intestines do not express murine CYP3A this: approach may overcome effects of residual mouse metabolic enzymes like Cyp3a in conventional chimeric mice with humanized liver, such as PXB-mice [urokinase plasminogen activator/severe combined immunodeficiency (uPA/SCID) mice repopulated with over 70% human hepatocytes] to improve the prediction of drug metabolism and pharmacokinetics in humans. After human hepatocytes were transplanted into Cyp3a KO/uPA/SCID host mice, human albumin levels logarithmically increased until approximately 60 days after transplantation, findings similar to those in PXB-mice. Quantitative real-time-polymerase chain reaction analyses showed that hepatic human P450s, UGTs, SULTs, and transporters mRNA expression levels in Cyp3a KO chimeric mice were also similar to those in PXB-mice and confirmed the absence of Cyp3a11 mRNA expression in mouse liver and intestine. Findings for midazolam and triazolam metabolic activities in liver microsomes were comparable between Cyp3a KO chimeric mice and PXB-mice. In contrast, these activities in the intestine of Cyp3a KO chimeric mice were attenuated compared with PXB-mice. Owing to the knockout of murine Cyp3a, hepatic Cyp2b10 and 2c55 mRNA levels in Cyp3a KO/uPA/SCID mice (without hepatocyte transplants) were 8.4- and 61-fold upregulated compared with PXB-mice, respectively. However, human hepatocyte transplantation successfully restored Cyp2b10 level nearly fully and Cyp2c55 level partly (still 13-fold upregulated) compared with those in PXB-mice. Intestinal Cyp2b10 and 2c55 were also repressed by human hepatocyte transplantation in Cyp3a KO chimeric mice. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

Inhibitors of ORAI1 Prevent Cytosolic Calcium-Associated Injury of Human Pancreatic Acinar Cells and Acute Pancreatitis in 3 Mouse Models

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Wen, Li; Voronina, Svetlana; Javed, Muhammad A.; Awais, Muhammad; Szatmary, Peter; Latawiec, Diane; Chvanov, Michael; Collier, David; Huang, Wei; Barrett, John; Begg, Malcolm; Stauderman, Ken; Roos, Jack; Grigoryev, Sergey; Ramos, Stephanie; Rogers, Evan; Whitten, Jeff; Velicelebi, Gonul; Dunn, Michael; Tepikin, Alexei V.; Criddle, David N.; Sutton, Robert

2015-01-01

Background & Aims Sustained activation of the cytosolic calcium concentration induces injury to pancreatic acinar cells and necrosis. The calcium release–activated calcium modulator ORAI1 is the most abundant Ca2+ entry channel in pancreatic acinar cells; it sustains calcium overload in mice exposed to toxins that induce pancreatitis. We investigated the roles of ORAI1 in pancreatic acinar cell injury and the development of acute pancreatitis in mice. Methods Mouse and human acinar cells, as well as HEK 293 cells transfected to express human ORAI1 with human stromal interaction molecule 1, were hyperstimulated or incubated with human bile acid, thapsigargin, or cyclopiazonic acid to induce calcium entry. GSK-7975A or CM_128 were added to some cells, which were analyzed by confocal and video microscopy and patch clamp recordings. Acute pancreatitis was induced in C57BL/6J mice by ductal injection of taurolithocholic acid 3-sulfate or intravenous' administration of cerulein or ethanol and palmitoleic acid. Some mice then were given GSK-7975A or CM_128, which inhibit ORAI1, at different time points to assess local and systemic effects. Results GSK-7975A and CM_128 each separately inhibited toxin-induced activation of ORAI1 and/or activation of Ca2+ currents after Ca2+ release, in a concentration-dependent manner, in mouse and human pancreatic acinar cells (inhibition >90% of the levels observed in control cells). The ORAI1 inhibitors also prevented activation of the necrotic cell death pathway in mouse and human pancreatic acinar cells. GSK-7975A and CM_128 each inhibited all local and systemic features of acute pancreatitis in all 3 models, in dose- and time-dependent manners. The agents were significantly more effective, in a range of parameters, when given at 1 vs 6 hours after induction of pancreatitis. Conclusions Cytosolic calcium overload, mediated via ORAI1, contributes to the pathogenesis of acute pancreatitis. ORAI1 inhibitors might be developed

Cytosolic delivery of materials with endosome-disrupting colloids

Science.gov (United States)

Helms, Brett A.; Bayles, Andrea R.

2016-03-15

A facile procedure to deliver nanocrystals to the cytosol of live cells that is both rapid and general. The technique employs a unique cationic core-shell polymer colloid that directs nanocrystals to the cytosol of living cells within a few hours of incubation. The present methods and compositions enable a host of advanced applications arising from efficient cytosolic delivery of nanocrystal imaging probes: from single particle tracking experiments to monitoring protein-protein interactions in live cells for extended periods.

Targeted induction of interferon-λ in humanized chimeric mouse liver abrogates hepatotropic virus infection.

Science.gov (United States)

Nakagawa, Shin-ichiro; Hirata, Yuichi; Kameyama, Takeshi; Tokunaga, Yuko; Nishito, Yasumasa; Hirabayashi, Kazuko; Yano, Junichi; Ochiya, Takahiro; Tateno, Chise; Tanaka, Yasuhito; Mizokami, Masashi; Tsukiyama-Kohara, Kyoko; Inoue, Kazuaki; Yoshiba, Makoto; Takaoka, Akinori; Kohara, Michinori

2013-01-01

The interferon (IFN) system plays a critical role in innate antiviral response. We presume that targeted induction of IFN in human liver shows robust antiviral effects on hepatitis C virus (HCV) and hepatitis B virus (HBV). This study used chimeric mice harboring humanized livers and infected with HCV or HBV. This mouse model permitted simultaneous analysis of immune responses by human and mouse hepatocytes in the same liver and exploration of the mechanism of antiviral effect against these viruses. Targeted expression of IFN was induced by treating the animals with a complex comprising a hepatotropic cationic liposome and a synthetic double-stranded RNA analog, pIC (LIC-pIC). Viral replication, IFN gene expression, IFN protein production, and IFN antiviral activity were analyzed (for type I, II and III IFNs) in the livers and sera of these humanized chimeric mice. Following treatment with LIC-pIC, the humanized livers of chimeric mice exhibited increased expression (at the mRNA and protein level) of human IFN-λs, resulting in strong antiviral effect on HBV and HCV. Similar increases were not seen for human IFN-α or IFN-β in these animals. Strong induction of IFN-λs by LIC-pIC occurred only in human hepatocytes, and not in mouse hepatocytes nor in human cell lines derived from other (non-hepatic) tissues. LIC-pIC-induced IFN-λ production was mediated by the immune sensor adaptor molecules mitochondrial antiviral signaling protein (MAVS) and Toll/IL-1R domain-containing adaptor molecule-1 (TICAM-1), suggesting dual recognition of LIC-pIC by both sensor adaptor pathways. These findings demonstrate that the expression and function of various IFNs differ depending on the animal species and tissues under investigation. Chimeric mice harboring humanized livers demonstrate that IFN-λs play an important role in the defense against human hepatic virus infection.

Cytosolic PrP Can Participate in Prion-Mediated Toxicity

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Thackray, Alana M.; Zhang, Chang; Arndt, Tina

2014-01-01

ABSTRACT Prion diseases are characterized by a conformational change in the normal host protein PrPC. While the majority of mature PrPC is tethered to the plasma membrane by a glycosylphosphatidylinositol anchor, topological variants of this protein can arise during its biosynthesis. Here we have generated Drosophila transgenic for cytosolic ovine PrP in order to investigate its toxic potential in flies in the absence or presence of exogenous ovine prions. While cytosolic ovine PrP expressed in Drosophila was predominantly detergent insoluble and showed resistance to low concentrations of proteinase K, it was not overtly detrimental to the flies. However, Drosophila transgenic for cytosolic PrP expression exposed to classical or atypical scrapie prion inocula showed a faster decrease in locomotor activity than similar flies exposed to scrapie-free material. The susceptibility to classical scrapie inocula could be assessed in Drosophila transgenic for panneuronal expression of cytosolic PrP, whereas susceptibility to atypical scrapie required ubiquitous PrP expression. Significantly, the toxic phenotype induced by ovine scrapie in cytosolic PrP transgenic Drosophila was transmissible to recipient PrP transgenic flies. These data show that while cytosolic PrP expression does not adversely affect Drosophila, this topological PrP variant can participate in the generation of transmissible scrapie-induced toxicity. These observations also show that PrP transgenic Drosophila are susceptible to classical and atypical scrapie prion strains and highlight the utility of this invertebrate host as a model of mammalian prion disease. IMPORTANCE During prion diseases, the host protein PrPC converts into an abnormal conformer, PrPSc, a process coupled to the generation of transmissible prions and neurotoxicity. While PrPC is principally a glycosylphosphatidylinositol-anchored membrane protein, the role of topological variants, such as cytosolic PrP, in prion-mediated toxicity and

A rapid and simple method for cryopreservation of human liver slices

NARCIS (Netherlands)

de Kanter, R; Olinga, Peter; Hof, I.H; de Jager, M.H; Verwillegen, W.A; Slooff, M.JH; Meijer, D.K F; Groothuis, Geny; Koster, H

1. Precision-cut liver slices represent a suitable and convenient in vitro preparation for studying metabolism and toxicity mechanisms of drugs and toxic chemicals. Particularly in the case of human liver slices, cryopreservation would enable more efficient utilization of this scarce and irregularly

In Vitro Generation of Functional Liver Organoid-Like Structures Using Adult Human Cells.

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Ramachandran, Sarada Devi; Schirmer, Katharina; Münst, Bernhard; Heinz, Stefan; Ghafoory, Shahrouz; Wölfl, Stefan; Simon-Keller, Katja; Marx, Alexander; Øie, Cristina Ionica; Ebert, Matthias P; Walles, Heike; Braspenning, Joris; Breitkopf-Heinlein, Katja

2015-01-01

In this study we used differentiated adult human upcyte® cells for the in vitro generation of liver organoids. Upcyte® cells are genetically engineered cell strains derived from primary human cells by lenti-viral transduction of genes or gene combinations inducing transient proliferation capacity (upcyte® process). Proliferating upcyte® cells undergo a finite number of cell divisions, i.e., 20 to 40 population doublings, but upon withdrawal of proliferation stimulating factors, they regain most of the cell specific characteristics of primary cells. When a defined mixture of differentiated human upcyte® cells (hepatocytes, liver sinusoidal endothelial cells (LSECs) and mesenchymal stem cells (MSCs)) was cultured in vitro on a thick layer of Matrigel™, they self-organized to form liver organoid-like structures within 24 hours. When further cultured for 10 days in a bioreactor, these liver organoids show typical functional characteristics of liver parenchyma including activity of cytochromes P450, CYP3A4, CYP2B6 and CYP2C9 as well as mRNA expression of several marker genes and other enzymes. In summary, we hereby describe that 3D functional hepatic structures composed of primary human cell strains can be generated in vitro. They can be cultured for a prolonged period of time and are potentially useful ex vivo models to study liver functions.

Chip-based human liver-intestine and liver-skin co-cultures--A first step toward systemic repeated dose substance testing in vitro.

Science.gov (United States)

Maschmeyer, Ilka; Hasenberg, Tobias; Jaenicke, Annika; Lindner, Marcus; Lorenz, Alexandra Katharina; Zech, Julie; Garbe, Leif-Alexander; Sonntag, Frank; Hayden, Patrick; Ayehunie, Seyoum; Lauster, Roland; Marx, Uwe; Materne, Eva-Maria

2015-09-01

Systemic repeated dose safety assessment and systemic efficacy evaluation of substances are currently carried out on laboratory animals and in humans due to the lack of predictive alternatives. Relevant international regulations, such as OECD and ICH guidelines, demand long-term testing and oral, dermal, inhalation, and systemic exposure routes for such evaluations. So-called "human-on-a-chip" concepts are aiming to replace respective animals and humans in substance evaluation with miniaturized functional human organisms. The major technical hurdle toward success in this field is the life-like combination of human barrier organ models, such as intestine, lung or skin, with parenchymal organ equivalents, such as liver, at the smallest biologically acceptable scale. Here, we report on a reproducible homeostatic long-term co-culture of human liver equivalents with either a reconstructed human intestinal barrier model or a human skin biopsy applying a microphysiological system. We used a multi-organ chip (MOC) platform, which provides pulsatile fluid flow within physiological ranges at low media-to-tissue ratios. The MOC supports submerse cultivation of an intact intestinal barrier model and an air-liquid interface for the skin model during their co-culture with the liver equivalents respectively at (1)/100.000 the scale of their human counterparts in vivo. To increase the degree of organismal emulation, microfluidic channels of the liver-skin co-culture could be successfully covered with human endothelial cells, thus mimicking human vasculature, for the first time. Finally, exposure routes emulating oral and systemic administration in humans have been qualified by applying a repeated dose administration of a model substance - troglitazone - to the chip-based co-cultures. Copyright © 2015. Published by Elsevier B.V.

Detection of driver metabolites in the human liver metabolic network using structural controllability analysis

Science.gov (United States)

2014-01-01

Background Abnormal states in human liver metabolism are major causes of human liver diseases ranging from hepatitis to hepatic tumor. The accumulation in relevant data makes it feasible to derive a large-scale human liver metabolic network (HLMN) and to discover important biological principles or drug-targets based on network analysis. Some studies have shown that interesting biological phenomenon and drug-targets could be discovered by applying structural controllability analysis (which is a newly prevailed concept in networks) to biological networks. The exploration on the connections between structural controllability theory and the HLMN could be used to uncover valuable information on the human liver metabolism from a fresh perspective. Results We applied structural controllability analysis to the HLMN and detected driver metabolites. The driver metabolites tend to have strong ability to influence the states of other metabolites and weak susceptibility to be influenced by the states of others. In addition, the metabolites were classified into three classes: critical, high-frequency and low-frequency driver metabolites. Among the identified 36 critical driver metabolites, 27 metabolites were found to be essential; the high-frequency driver metabolites tend to participate in different metabolic pathways, which are important in regulating the whole metabolic systems. Moreover, we explored some other possible connections between the structural controllability theory and the HLMN, and find that transport reactions and the environment play important roles in the human liver metabolism. Conclusion There are interesting connections between the structural controllability theory and the human liver metabolism: driver metabolites have essential biological functions; the crucial role of extracellular metabolites and transport reactions in controlling the HLMN highlights the importance of the environment in the health of human liver metabolism. PMID:24885538

Cytosolic iron chaperones: Proteins delivering iron cofactors in the cytosol of mammalian cells.

Science.gov (United States)

Philpott, Caroline C; Ryu, Moon-Suhn; Frey, Avery; Patel, Sarju

2017-08-04

Eukaryotic cells contain hundreds of metalloproteins that are supported by intracellular systems coordinating the uptake and distribution of metal cofactors. Iron cofactors include heme, iron-sulfur clusters, and simple iron ions. Poly(rC)-binding proteins are multifunctional adaptors that serve as iron ion chaperones in the cytosolic/nuclear compartment, binding iron at import and delivering it to enzymes, for storage (ferritin) and export (ferroportin). Ferritin iron is mobilized by autophagy through the cargo receptor, nuclear co-activator 4. The monothiol glutaredoxin Glrx3 and BolA2 function as a [2Fe-2S] chaperone complex. These proteins form a core system of cytosolic iron cofactor chaperones in mammalian cells. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Effects of whole body x-ray irradiation on induction by phenobarbital of rat liver glucose-6-phosphate dehydrogenase and glutathione reductase

Energy Technology Data Exchange (ETDEWEB)

Bitny-Szlachto, S.; Szyszko, A. (Wojskowy Inst. Higieny i Epidemiologii, Warsaw (Poland))

1979-01-01

In rats treated with phenobarbital (3x100 mg/kg, i.p.), liver G-6-P dehydrogenase activity increased by 70% in the cytosol and in the 9.000xg supernatant, and only by 20% in microsomes. Moreover, the phenobarbital treatment increased rat liver GSSG reductase activity by 30%. On the other hand, activity of the liver microsomal G-6-P dehydrogenase was found to increase by some 20% in whole body irradiated, both control and phenobarbital treated rats. In rats irradiated with 600 R prior to the first dose of the inducer there was not noted any increase in G-6-P dehydrogenase of the 9.000xg supernatant, and increase in the cytosol activity dropped to 38%. Thus, induction of the soluble liver G-6-P dehydrogenase by phenobarbital has turned out to be radiosensitive, whereas phenobarbital induction of GSSG reductase was unaffected by irradiation.

Targeted induction of interferon-λ in humanized chimeric mouse liver abrogates hepatotropic virus infection.

Directory of Open Access Journals (Sweden)

Shin-ichiro Nakagawa

Full Text Available BACKGROUND & AIMS: The interferon (IFN system plays a critical role in innate antiviral response. We presume that targeted induction of IFN in human liver shows robust antiviral effects on hepatitis C virus (HCV and hepatitis B virus (HBV. METHODS: This study used chimeric mice harboring humanized livers and infected with HCV or HBV. This mouse model permitted simultaneous analysis of immune responses by human and mouse hepatocytes in the same liver and exploration of the mechanism of antiviral effect against these viruses. Targeted expression of IFN was induced by treating the animals with a complex comprising a hepatotropic cationic liposome and a synthetic double-stranded RNA analog, pIC (LIC-pIC. Viral replication, IFN gene expression, IFN protein production, and IFN antiviral activity were analyzed (for type I, II and III IFNs in the livers and sera of these humanized chimeric mice. RESULTS: Following treatment with LIC-pIC, the humanized livers of chimeric mice exhibited increased expression (at the mRNA and protein level of human IFN-λs, resulting in strong antiviral effect on HBV and HCV. Similar increases were not seen for human IFN-α or IFN-β in these animals. Strong induction of IFN-λs by LIC-pIC occurred only in human hepatocytes, and not in mouse hepatocytes nor in human cell lines derived from other (non-hepatic tissues. LIC-pIC-induced IFN-λ production was mediated by the immune sensor adaptor molecules mitochondrial antiviral signaling protein (MAVS and Toll/IL-1R domain-containing adaptor molecule-1 (TICAM-1, suggesting dual recognition of LIC-pIC by both sensor adaptor pathways. CONCLUSIONS: These findings demonstrate that the expression and function of various IFNs differ depending on the animal species and tissues under investigation. Chimeric mice harboring humanized livers demonstrate that IFN-λs play an important role in the defense against human hepatic virus infection.

Evaluation of the oxidative status in Oreochromis niloticus fed with tuna by-product meal: possible human health impact.

Science.gov (United States)

Saїdi, Saber Abdelkader; Azaza, Mohamed Salah; van Pelt, Jos; El Feki, Abdelfattah

2014-10-01

The influence of tuna by-product meal (TBM) diets on the activities of antioxidant enzymes in the muscles, gill and kidney of tilapia was evaluated. The control diet (A0) used fish meal (FM) as the sole source of animal protein and the other three diets (A10-A30), 33%-100% of FM was substituted by TBM at 10% increments. The cytotoxicity of liver and muscles cytosol extracts in human liver cell HepG2 was undertaken. The activities of glutathione S-transferase, superoxide dismutase, catalase and TBARS values in the kidney of fish fed with diet A30 were significantly different (p<0.05) and no alterations were observed in muscle and gill anti-oxidant activities. Our study has confirmed that the cytosol extract does not exhibit cytotoxic effects on the HepG2 cells. These results indicate that the use of TBM as a total replacement for FM into tilapia diets seems to have oxidative stress, thus generating effect on fish metabolism.

Calcium signaling in liver.

Science.gov (United States)

Gaspers, Lawrence D; Thomas, Andrew P

2005-01-01

In hepatocytes, hormones linked to the formation of the second messenger inositol 1,4,5-trisphosphate (InsP3) evoke transient increases or spikes in cytosolic free calcium ([Ca2+]i), that increase in frequency with the agonist concentration. These oscillatory Ca2+ signals are thought to transmit the information encoded in the extracellular stimulus to down-stream Ca2+-sensitive metabolic processes. We have utilized both confocal and wide field fluorescence microscopy techniques to study the InsP3-dependent signaling pathway at the cellular and subcellular levels in the intact perfused liver. Typically InsP3-dependent [Ca2+]i spikes manifest as Ca2+ waves that propagate throughout the entire cytoplasm and nucleus, and in the intact liver these [Ca2+]i increases are conveyed through gap junctions to encompass entire lobular units. The translobular movement of Ca2+ provides a means to coordinate the function of metabolic zones of the lobule and thus, liver function. In this article, we describe the characteristics of agonist-evoked [Ca2+]i signals in the liver and discuss possible mechanisms to explain the propagation of intercellular Ca2+ waves in the intact organ.

Human Liver Cells Expressing Albumin and Mesenchymal Characteristics Give Rise to Insulin-Producing Cells

Directory of Open Access Journals (Sweden)

Irit Meivar-Levy

2011-01-01

Full Text Available Activation of the pancreatic lineage in the liver has been suggested as a potential autologous cell replacement therapy for diabetic patients. Transcription factors-induced liver-to-pancreas reprogramming has been demonstrated in numerous species both in vivo and in vitro. However, human-derived liver cells capable of acquiring the alternate pancreatic repertoire have never been characterized. It is yet unknown whether hepatic-like stem cells or rather adult liver cells give rise to insulin-producing cells. Using an in vitro experimental system, we demonstrate that proliferating adherent human liver cells acquire mesenchymal-like characteristics and a considerable level of cellular plasticity. However, using a lineage-tracing approach, we demonstrate that insulin-producing cells are primarily generated in cells enriched for adult hepatic markers that coexpress both albumin and mesenchymal markers. Taken together, our data suggest that adult human hepatic tissue retains a substantial level of developmental plasticity, which could be exploited in regenerative medicine approaches.

Impaired methylation as a novel mechanism for proteasome suppression in liver cells

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Osna, Natalia A., E-mail: nosna@UNMC.edu [Liver Study Unit, The Omaha Veterans Affairs VA Medical Center, Omaha, NE 68105 (United States); Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE 68105 (United States); White, Ronda L.; Donohue, Terrence M. [Liver Study Unit, The Omaha Veterans Affairs VA Medical Center, Omaha, NE 68105 (United States); Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE 68105 (United States); Beard, Michael R. [Department of Molecular Biosciences, University of Adelaide (Australia); Tuma, Dean J.; Kharbanda, Kusum K. [Liver Study Unit, The Omaha Veterans Affairs VA Medical Center, Omaha, NE 68105 (United States); Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE 68105 (United States)

2010-01-08

The proteasome is a multi-catalytic protein degradation enzyme that is regulated by ethanol-induced oxidative stress; such suppression is attributed to CYP2E1-generated metabolites. However, under certain conditions, it appears that in addition to oxidative stress, other mechanisms are also involved in proteasome regulation. This study investigated whether impaired protein methylation that occurs during exposure of liver cells to ethanol, may contribute to suppression of proteasome activity. We measured the chymotrypsin-like proteasome activity in Huh7CYP cells, hepatocytes, liver cytosols and nuclear extracts or purified 20S proteasome under conditions that maintain or prevent protein methylation. Reduction of proteasome activity of hepatoma cell and hepatocytes by ethanol or tubercidin was prevented by simultaneous treatment with S-adenosylmethionine (SAM). Moreover, the tubercidin-induced decline in proteasome activity occurred in both nuclear and cytosolic fractions. In vitro exposure of cell cytosolic fractions or highly purified 20S proteasome to low SAM:S-adenosylhomocysteine (SAH) ratios in the buffer also suppressed proteasome function, indicating that one or more methyltransferase(s) may be associated with proteasomal subunits. Immunoblotting a purified 20S rabbit red cell proteasome preparation using methyl lysine-specific antibodies revealed a 25 kDa proteasome subunit that showed positive reactivity with anti-methyl lysine. This reactivity was modified when 20S proteasome was exposed to differential SAM:SAH ratios. We conclude that impaired methylation of proteasome subunits suppressed proteasome activity in liver cells indicating an additional, yet novel mechanism of proteasome activity regulation by ethanol.

Human Precision-Cut Liver Slices as an ex Vivo Model to Study Idiosyncratic Drug-Induced Liver Injury

NARCIS (Netherlands)

Hadi, Mackenzie; Westra, Inge M.; Starokozhko, Viktoriia; Dragovic, Sanja; Merema, M.T.; Groothuis, Geny M. M.

Idiosyncratic drug-induced liver injury (IDILI) is a major problem during drug development and has caused drug withdrawal and black-box warnings. Because of the low concordance of the hepatotoxicity of drugs in animals and humans, robust screening methods using human tissue are needed to predict

Alpha-tocopherol transfer factor (aTTF) from rat liver mediates the transfer of d-alpha-[3H]-tocopherol from liposomes to human erythrocyte ghosts and exhibits saturation kinetics

International Nuclear Information System (INIS)

Verdon, C.P.; Blumberg, J.B.

1986-01-01

aTTF was observed to transfer d-alpha-[ 3 H]-tocopherol ( 3 HaT) from egg lecithin liposomes to human erythrocyte ghosts (EG). aTTF may be associated with the 32,000-35,000 MW alpha-Tocopherol Binding Protein previously described to transfer 3 HaT from liposomes to rat liver microsomes and mitochondria prepared by ammonium sulfate precipitation of rat liver cytosol followed by dialysis against 50 mM TRIS-HCl/1 mM EDTA buffer, pH 7.4. Assay for aTTF activity consisted of incubating liposomal 3 HaT and EG in the presence of aTTF or buffer blank for various time periods at 37 0 C, then counting the resulting radioactivity in washed EG after pelleting by centrifugation. Liposomes were prelabeled-with non-exchangable glycerol-[ 14 C]-trioleate to correct for liposomes adhering to pelleted EG. Greater than 50% of the tritium found with the EG pellet was recovered by HPLC as 3 HaT. aTTF activity increased with increasing liposomal 3 HaT concentration before reaching a plateau. aTTF activity was similarly saturated by increasing EG concentrations. The same assay conditions with buffer blank along resulted in negligible transfer activity

Human precision-cut liver slices as an ex vivo model to study idiosyncratic drug-induced liver injury

NARCIS (Netherlands)

Hadi, Mackenzie; Westra, Inge; Starokozhko, Viktoriia; Dragovic, Sanja; Merema, Maja; Groothuis, Genoveva

2013-01-01

Idiosyncratic drug-induced liver injury (IDILI) is a major problem during drug development and has caused drug withdrawal and black-box warnings. Due to the low concordance of the hepatotoxicity of drugs in animals and humans, robust screening methods using human tissue are needed to predict and to

Malate-aspartate shuttle and exogenous NADH/cytochrome c electron transport pathway as two independent cytosolic reducing equivalent transfer systems.

Science.gov (United States)

Abbrescia, Daniela Isabel; La Piana, Gianluigi; Lofrumento, Nicola Elio

2012-02-15

In mammalian cells aerobic oxidation of glucose requires reducing equivalents produced in glycolytic phase to be channelled into the phosphorylating respiratory chain for the reduction of molecular oxygen. Data never presented before show that the oxidation rate of exogenous NADH supported by the malate-aspartate shuttle system (reconstituted in vitro with isolated liver mitochondria) is comparable to the rate obtained on activation of the cytosolic NADH/cytochrome c electron transport pathway. The activities of these two reducing equivalent transport systems are independent of each other and additive. NADH oxidation induced by the malate-aspartate shuttle is inhibited by aminooxyacetate and by rotenone and/or antimycin A, two inhibitors of the respiratory chain, while the NADH/cytochrome c system remains insensitive to all of them. The two systems may simultaneously or mutually operate in the transfer of reducing equivalents from the cytosol to inside the mitochondria. In previous reports we suggested that the NADH/cytochrome c system is expected to be functioning in apoptotic cells characterized by the presence of cytochrome c in the cytosol. As additional new finding the activity of reconstituted shuttle system is linked to the amount of α-ketoglutarate generated inside the mitochondria by glutamate dehydrogenase rather than by aspartate aminotransferase. Copyright © 2011 Elsevier Inc. All rights reserved.

Mice with humanized liver endothelium

NARCIS (Netherlands)

el Filali, E.

2014-01-01

The only curative treatment option for a large proportion of patients suffering from a liver disorder is liver transplantation. The use of ex vivo genetically modified autologous liver cells instead of whole liver transplantation could overcome the problem of donor scarcity. Even though clinical

Chimeric mice with humanized liver: Application in drug metabolism and pharmacokinetics studies for drug discovery.

Science.gov (United States)

Naritomi, Yoichi; Sanoh, Seigo; Ohta, Shigeru

2018-02-01

Predicting human drug metabolism and pharmacokinetics (PK) is key to drug discovery. In particular, it is important to predict human PK, metabolite profiles and drug-drug interactions (DDIs). Various methods have been used for such predictions, including in vitro metabolic studies using human biological samples, such as hepatic microsomes and hepatocytes, and in vivo studies using experimental animals. However, prediction studies using these methods are often inconclusive due to discrepancies between in vitro and in vivo results, and interspecies differences in drug metabolism. Further, the prediction methods have changed from qualitative to quantitative to solve these issues. Chimeric mice with humanized liver have been developed, in which mouse liver cells are mostly replaced with human hepatocytes. Since human drug metabolizing enzymes are expressed in the liver of these mice, they are regarded as suitable models for mimicking the drug metabolism and PK observed in humans; therefore, these mice are useful for predicting human drug metabolism and PK. In this review, we discuss the current state, issues, and future directions of predicting human drug metabolism and PK using chimeric mice with humanized liver in drug discovery. Copyright © 2017 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.

Cytosolic cholesterol ester hydrolase in adrenal cortex

OpenAIRE

Tocher, Douglas R.

1983-01-01

Cholesterol ester hydrolase (CEH) in adrenocortical cytosol was known to be phosphorylated and activated, in response to ACTH in a cAMPdependent protein kinase mediated process. The purification of CEH from bovine adrenocortical cytosol was attempted. The use of detergents to solubilise the enzyme from lipid-rich aggregates was investigated and sodium cholate was found to be effective. A purification procedure using cholate solubilised enzyme was developed. The detergent int...

The importance of cytosolic glutamine synthetase in nitrogen assimilation and recycling

Energy Technology Data Exchange (ETDEWEB)

Bernard, S.M.; Habash, D.Z.

2009-07-02

Glutamine synthetase assimilates ammonium into amino acids, thus it is a key enzyme for nitrogen metabolism. The cytosolic isoenzymes of glutamine synthetase assimilate ammonium derived from primary nitrogen uptake and from various internal nitrogen recycling pathways. In this way, cytosolic glutamine synthetase is crucial for the remobilization of protein-derived nitrogen. Cytosolic glutamine synthetase is encoded by a small family of genes that are well conserved across plant species. Members of the cytosolic glutamine synthetase gene family are regulated in response to plant nitrogen status, as well as to environmental cues, such as nitrogen availability and biotic/abiotic stresses. The complex regulation of cytosolic glutamine synthetase at the transcriptional to post-translational levels is key to the establishment of a specific physiological role for each isoenzyme. The diverse physiological roles of cytosolic glutamine synthetase isoenzymes are important in relation to current agricultural and ecological issues.

Proteomic profiling in incubation medium of mouse, rat and human precision-cut liver slices for biomarker detection regarding acute drug-induced liver injury

NARCIS (Netherlands)

van Swelm, Rachel P L; Hadi, Mackenzie; Laarakkers, Coby M M; Masereeuw, R.|info:eu-repo/dai/nl/155644033; Groothuis, Geny M M; Russel, Frans G M

Drug-induced liver injury is one of the leading causes of drug withdrawal from the market. In this study, we investigated the applicability of protein profiling of the incubation medium of human, mouse and rat precision-cut liver slices (PCLS) exposed to liver injury-inducing drugs for biomarker

Induction of biotransformation enzymes by the carcinogenic air-pollutant 3-nitrobenzanthrone in liver, kidney and lung, after intra-tracheal instillation in rats.

Science.gov (United States)

Mizerovská, Jana; Dračínská, Helena; Frei, Eva; Schmeiser, Heinz H; Arlt, Volker M; Stiborová, Marie

2011-02-28

3-Nitrobenzanthrone (3-NBA), a carcinogenic air pollutant, was investigated for its ability to induce cytochrome P450 (CYP) 1A1/2 and NAD(P)H:quinone oxidoreductase (NQO1) in liver, kidney and lung of rats treated by intra-tracheal instillation. The organs used were from a previous study performed to determine the persistence of 3-NBA-derived DNA adducts in target and non-target tissues (Bieler et al., Carcinogenesis 28 (2007) 1117-1121, [22]). NQO1 is the enzyme reducing 3-NBA to N-hydroxy-3-aminobenzanthrone (N-OH-3-ABA) and CYP1A enzymes oxidize a human metabolite of 3-NBA, 3-aminobenzanthrone (3-ABA), to yield the same reactive intermediate. 3-NBA and 3-ABA are both activated to species forming DNA adducts by cytosols and/or microsomes isolated from rat lung, the target organ for 3-NBA carcinogenicity, and from liver and kidney. Each compound generated the same five DNA adducts detectable by (32)P-postlabelling. When hepatic cytosols from rats treated with 0.2 or 2mg/kg body weight of 3-NBA were incubated with 3-NBA, DNA adduct formation was 3.2- and 8.6-fold higher, respectively, than in incubations with cytosols from control animals. Likewise, cytosols isolated from lungs and kidneys of rats exposed to 3-NBA more efficiently activated 3-NBA than those of control rats. This increase corresponded to an increase in protein levels and enzymatic activities of NQO1. Incubations of hepatic, pulmonary or renal microsomes of 3-NBA-treated rats with 3-ABA led to an 9.6-fold increase in DNA-adduct formation relative to controls. The highest induction in DNA-adduct levels was found in lung. The stimulation of DNA-adduct formation correlated with expression of CYP1A1/2 induced by the intra-tracheal instillation of 3-NBA. The results demonstrate that 3-NBA induces NQO1 and CYP1A1/2 in livers, lungs and kidneys of rats after intra-tracheal instillation, thereby enhancing its own genotoxic and carcinogenic potential. Copyright © 2010 Elsevier B.V. All rights reserved.

A murine model of type 2 autoimmune hepatitis: Xenoimmunization with human antigens.

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Lapierre, Pascal; Djilali-Saiah, Idriss; Vitozzi, Susana; Alvarez, Fernando

2004-04-01

Autoimmune hepatitis (AIH) is characterized by an immune-mediated injury of the hepatic parenchyma of unknown pathogenesis. Type 2 AIH is identified by the presence of anti-liver-kidney microsomes type 1 (anti-LKM1) and anti-liver cytosol type 1 (anti-LC1) autoantibodies. The current study shows that a murine model of AIH can be generated by DNA immunization against type 2 AIH self-antigens (P450 2D6 and formiminotransferase-cyclodeaminase). A pCMV plasmid containing the N-terminal region of mouse CTLA-4 and the antigenic region of human CYP2D6 (672-1,377 bp) and human formiminotransferase cyclodeaminase (FTCD; 1,232-1,668 bp) was used for DNA immunization of C57BL/6 female mice. Immunized mice showed elevated levels of alanine aminotransferase (ALT), with peaks at 4 and 7 months postinjection. Periportal, portal, and intralobular liver inflammatory infiltrates were observed at histology. Mainly CD4+ lymphocytes, but also CD8+ and B lymphocytes, were found in the liver. Cytotoxic-specific T cells were found in both the liver and spleen of these animals. Mice developed anti-LKM1 and anti-LC1 antibodies of immunoglobulin G2 (IgG2) subclass, against specific mouse autoantigens. The ALT levels correlated with both the presence of anti-LKM1/anti-LC1 antibodies and the presence of liver necroinflammation. In conclusion, in mice, DNA immunization against human autoantigens breaks tolerance and induces an autoimmune liver disease. Molecular mimicry between foreign and self-antigens explains the liver injury. This model of AIH resembles human type 2 AIH and will be helpful for the study of its pathogenesis.

Protocol for Isolation of Primary Human Hepatocytes and Corresponding Major Populations of Non-parenchymal Liver Cells

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Pfeiffer, Elisa; Zeilinger, Katrin; Seehofer, Daniel; Damm, Georg

2016-01-01

Beside parenchymal hepatocytes, the liver consists of non-parenchymal cells (NPC) namely Kupffer cells (KC), liver endothelial cells (LEC) and hepatic Stellate cells (HSC). Two-dimensional (2D) culture of primary human hepatocyte (PHH) is still considered as the "gold standard" for in vitro testing of drug metabolism and hepatotoxicity. It is well-known that the 2D monoculture of PHH suffers from dedifferentiation and loss of function. Recently it was shown that hepatic NPC play a central role in liver (patho-) physiology and the maintenance of PHH functions. Current research focuses on the reconstruction of in vivo tissue architecture by 3D- and co-culture models to overcome the limitations of 2D monocultures. Previously we published a method to isolate human liver cells and investigated the suitability of these cells for their use in cell cultures in Experimental Biology and Medicine1. Based on the broad interest in this technique the aim of this article was to provide a more detailed protocol for the liver cell isolation process including a video, which will allow an easy reproduction of this technique. Human liver cells were isolated from human liver tissue samples of surgical interventions by a two-step EGTA/collagenase P perfusion technique. PHH were separated from the NPC by an initial centrifugation at 50 x g. Density gradient centrifugation steps were used for removal of dead cells. Individual liver cell populations were isolated from the enriched NPC fraction using specific cell properties and cell sorting procedures. Beside the PHH isolation we were able to separate KC, LEC and HSC for further cultivation. Taken together, the presented protocol allows the isolation of PHH and NPC in high quality and quantity from one donor tissue sample. The access to purified liver cell populations could allow the creation of in vivo like human liver models. PMID:27077489

Structure of Human GIVD Cytosolic Phospholipase A2 Reveals Insights into Substrate Recognition

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Wang, Hui; Klein, Michael G.; Snell, Gyorgy; Lane, Weston; Zou, Hua; Levin, Irena; Li, Ke; Sang, Bi-Ching (Takeda Cali)

2016-07-01

Cytosolic phospholipases A2 (cPLA2s) consist of a family of calcium-sensitive enzymes that function to generate lipid second messengers through hydrolysis of membrane-associated glycerophospholipids. The GIVD cPLA2 (cPLA2δ) is a potential drug target for developing a selective therapeutic agent for the treatment of psoriasis. Here, we present two X-ray structures of human cPLA2δ, capturing an apo state, and in complex with a substrate-like inhibitor. Comparison of the apo and inhibitor-bound structures reveals conformational changes in a flexible cap that allows the substrate to access the relatively buried active site, providing new insight into the mechanism for substrate recognition. The cPLA2δ structure reveals an unexpected second C2 domain that was previously unrecognized from sequence alignments, placing cPLA2δ into the class of membrane-associated proteins that contain a tandem pair of C2 domains. Furthermore, our structures elucidate novel inter-domain interactions and define three potential calcium-binding sites that are likely important for regulation and activation of enzymatic activity. These findings provide novel insights into the molecular mechanisms governing cPLA2's function in signal transduction.

Fialuridine induces acute liver failure in chimeric TK-NOG mice: a model for detecting hepatic drug toxicity prior to human testing.

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Dan Xu

2014-04-01

Full Text Available Seven of 15 clinical trial participants treated with a nucleoside analogue (fialuridine [FIAU] developed acute liver failure. Five treated participants died, and two required a liver transplant. Preclinical toxicology studies in mice, rats, dogs, and primates did not provide any indication that FIAU would be hepatotoxic in humans. Therefore, we investigated whether FIAU-induced liver toxicity could be detected in chimeric TK-NOG mice with humanized livers.Control and chimeric TK-NOG mice with humanized livers were treated orally with FIAU 400, 100, 25, or 2.5 mg/kg/d. The response to drug treatment was evaluated by measuring plasma lactate and liver enzymes, by assessing liver histology, and by electron microscopy. After treatment with FIAU 400 mg/kg/d for 4 d, chimeric mice developed clinical and serologic evidence of liver failure and lactic acidosis. Analysis of liver tissue revealed steatosis in regions with human, but not mouse, hepatocytes. Electron micrographs revealed lipid and mitochondrial abnormalities in the human hepatocytes in FIAU-treated chimeric mice. Dose-dependent liver toxicity was detected in chimeric mice treated with FIAU 100, 25, or 2.5 mg/kg/d for 14 d. Liver toxicity did not develop in control mice that were treated with the same FIAU doses for 14 d. In contrast, treatment with another nucleotide analogue (sofosbuvir 440 or 44 mg/kg/d po for 14 d, which did not cause liver toxicity in human trial participants, did not cause liver toxicity in mice with humanized livers.FIAU-induced liver toxicity could be readily detected using chimeric TK-NOG mice with humanized livers, even when the mice were treated with a FIAU dose that was only 10-fold above the dose used in human participants. The clinical features, laboratory abnormalities, liver histology, and ultra-structural changes observed in FIAU-treated chimeric mice mirrored those of FIAU-treated human participants. The use of chimeric mice in preclinical toxicology

Fialuridine induces acute liver failure in chimeric TK-NOG mice: a model for detecting hepatic drug toxicity prior to human testing.

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Xu, Dan; Nishimura, Toshi; Nishimura, Sachiko; Zhang, Haili; Zheng, Ming; Guo, Ying-Ying; Masek, Marylin; Michie, Sara A; Glenn, Jeffrey; Peltz, Gary

2014-04-01

Seven of 15 clinical trial participants treated with a nucleoside analogue (fialuridine [FIAU]) developed acute liver failure. Five treated participants died, and two required a liver transplant. Preclinical toxicology studies in mice, rats, dogs, and primates did not provide any indication that FIAU would be hepatotoxic in humans. Therefore, we investigated whether FIAU-induced liver toxicity could be detected in chimeric TK-NOG mice with humanized livers. Control and chimeric TK-NOG mice with humanized livers were treated orally with FIAU 400, 100, 25, or 2.5 mg/kg/d. The response to drug treatment was evaluated by measuring plasma lactate and liver enzymes, by assessing liver histology, and by electron microscopy. After treatment with FIAU 400 mg/kg/d for 4 d, chimeric mice developed clinical and serologic evidence of liver failure and lactic acidosis. Analysis of liver tissue revealed steatosis in regions with human, but not mouse, hepatocytes. Electron micrographs revealed lipid and mitochondrial abnormalities in the human hepatocytes in FIAU-treated chimeric mice. Dose-dependent liver toxicity was detected in chimeric mice treated with FIAU 100, 25, or 2.5 mg/kg/d for 14 d. Liver toxicity did not develop in control mice that were treated with the same FIAU doses for 14 d. In contrast, treatment with another nucleotide analogue (sofosbuvir 440 or 44 mg/kg/d po) for 14 d, which did not cause liver toxicity in human trial participants, did not cause liver toxicity in mice with humanized livers. FIAU-induced liver toxicity could be readily detected using chimeric TK-NOG mice with humanized livers, even when the mice were treated with a FIAU dose that was only 10-fold above the dose used in human participants. The clinical features, laboratory abnormalities, liver histology, and ultra-structural changes observed in FIAU-treated chimeric mice mirrored those of FIAU-treated human participants. The use of chimeric mice in preclinical toxicology studies could improve

Cytosolic antibody delivery by lipid-sensitive endosomolytic peptide

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Akishiba, Misao; Takeuchi, Toshihide; Kawaguchi, Yoshimasa; Sakamoto, Kentarou; Yu, Hao-Hsin; Nakase, Ikuhiko; Takatani-Nakase, Tomoka; Madani, Fatemeh; Gräslund, Astrid; Futaki, Shiroh

2017-08-01

One of the major obstacles in intracellular targeting using antibodies is their limited release from endosomes into the cytosol. Here we report an approach to deliver proteins, which include antibodies, into cells by using endosomolytic peptides derived from the cationic and membrane-lytic spider venom peptide M-lycotoxin. The delivery peptides were developed by introducing one or two glutamic acid residues into the hydrophobic face. One peptide with the substitution of leucine by glutamic acid (L17E) was shown to enable a marked cytosolic liberation of antibodies (immunoglobulins G (IgGs)) from endosomes. The predominant membrane-perturbation mechanism of this peptide is the preferential disruption of negatively charged membranes (endosomal membranes) over neutral membranes (plasma membranes), and the endosomolytic peptide promotes the uptake by inducing macropinocytosis. The fidelity of this approach was confirmed through the intracellular delivery of a ribosome-inactivation protein (saporin), Cre recombinase and IgG delivery, which resulted in a specific labelling of the cytosolic proteins and subsequent suppression of the glucocorticoid receptor-mediated transcription. We also demonstrate the L17E-mediated cytosolic delivery of exosome-encapsulated proteins.

Polymorphic Human Sulfotransferase 2A1 Mediates the Formation of 25-Hydroxyvitamin D3-3-O-Sulfate, a Major Circulating Vitamin D Metabolite in Humans.

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Wong, Timothy; Wang, Zhican; Chapron, Brian D; Suzuki, Mizuki; Claw, Katrina G; Gao, Chunying; Foti, Robert S; Prasad, Bhagwat; Chapron, Alenka; Calamia, Justina; Chaudhry, Amarjit; Schuetz, Erin G; Horst, Ronald L; Mao, Qingcheng; de Boer, Ian H; Thornton, Timothy A; Thummel, Kenneth E

2018-04-01

Metabolism of 25-hydroxyvitamin D 3 (25OHD 3 ) plays a central role in regulating the biologic effects of vitamin D in the body. Although cytochrome P450-dependent hydroxylation of 25OHD 3 has been extensively investigated, limited information is available on the conjugation of 25OHD 3 In this study, we report that 25OHD 3 is selectively conjugated to 25OHD 3 -3- O -sulfate by human sulfotransferase 2A1 (SULT2A1) and that the liver is a primary site of metabolite formation. At a low (50 nM) concentration of 25OHD 3 , 25OHD 3 -3- O -sulfate was the most abundant metabolite, with an intrinsic clearance approximately 8-fold higher than the next most efficient metabolic route. In addition, 25OHD 3 sulfonation was not inducible by the potent human pregnane X receptor agonist, rifampicin. The 25OHD 3 sulfonation rates in a bank of 258 different human liver cytosols were highly variable but correlated with the rates of dehydroepiandrosterone sulfonation. Further analysis revealed a significant association between a common single nucleotide variant within intron 1 of SULT2A1 (rs296361; minor allele frequency = 15% in whites) and liver cytosolic SULT2A1 content as well as 25OHD 3 -3- O -sulfate formation rate, suggesting that variation in the SULT2A1 gene contributes importantly to interindividual differences in vitamin D homeostasis. Finally, 25OHD 3 -3- O -sulfate exhibited high affinity for the vitamin D binding protein and was detectable in human plasma and bile but not in urine samples. Thus, circulating concentrations of 25OHD 3 -3- O -sulfate appear to be protected from rapid renal elimination, raising the possibility that the sulfate metabolite may serve as a reservoir of 25OHD 3 in vivo, and contribute indirectly to the biologic effects of vitamin D. Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.

MicroRNA-mediated suppression of oncolytic adenovirus replication in human liver.

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Erkko Ylösmäki

Full Text Available MicroRNAs (miRNAs are important and ubiquitous regulators of gene expression that can suppress their target genes by translational inhibition as well as mRNA destruction. Cell type-specific miRNA expression patterns have been successfully exploited for targeting the expression of experimental and therapeutic gene constructs, for example to reduce pathogenic effects of cancer virotherapy in normal tissues. In order to avoid liver damage associated with systemic or intrahepatic delivery of oncolytic adenoviruses we have introduced the concept of suppressing adenovirus replication in hepatic cells by inserting target elements for the liver-specific miR122 into the viral genome. Here we show using ex vivo cultured tissue specimens that six perfectly complementary miR122 target sites in the 3' untranslated region of the viral E1A gene are sufficient in the absence of any other genetic modifications to prevent productive replication of serotype 5 adenovirus (Ad5 in normal human liver. This modification did not compromise the replicative capacity of the modified virus in cancer tissue derived from a colon carcinoma liver metastasis or its oncolytic potency in a human lung cancer xenograft mouse model. Unlike wild-type Ad5, the modified virus did not result in increased serum levels of liver enzymes in infected mice. These results provide a strong preclinical proof of concept for the use of miR122 target sites for reducing the risk of liver damage caused by oncolytic adenoviruses, and suggest that ectopic miR122 target elements should be considered as an additional safety measure included in any therapeutic virus or viral vector posing potential hazard to the liver.

Anti-liver-kidney microsome antibody type 1 recognizes human cytochrome P450 db1.

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Gueguen, M; Yamamoto, A M; Bernard, O; Alvarez, F

1989-03-15

Anti-liver-kidney microsome antibody type 1 (LKM1), present in the sera of a group of children with autoimmune hepatitis, was recently shown to recognize a 50 kDa protein identified as rat liver cytochromes P450 db1 and db2. High homology between these two members of the rat P450 IID subfamily and human P450 db1 suggested that anti-LKM1 antibody is directed against this human protein. To test this hypothesis, a human liver cDNA expression library in phage lambda GT-11 was screened using rat P450 db1 cDNA as a probe. Two human cDNA clones were found to be identical to human P450 db1 by restriction mapping. Immunoblot analysis using as antigen, the purified fusion protein from one of the human cDNA clones showed that only anti-LKM1 with anti-50 kDa reactivity recognized the fusion protein. This fusion protein was further used to develop an ELISA test that was shown to be specific for sera of children with this disease. These results: 1) identify the human liver antigen recognized by anti-LKM1 auto-antibodies as cytochrome P450 db1, 2) allow to speculate that mutation on the human P450 db1 gene could alter its expression in the hepatocyte and make it auto-antigenic, 3) provide a simple and specific diagnostic test for this disease.

A microscale human liver platform that supports the hepatic stages of Plasmodium falciparum and vivax.

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March, Sandra; Ng, Shengyong; Velmurugan, Soundarapandian; Galstian, Ani; Shan, Jing; Logan, David J; Carpenter, Anne E; Thomas, David; Sim, B Kim Lee; Mota, Maria M; Hoffman, Stephen L; Bhatia, Sangeeta N

2013-07-17

The Plasmodium liver stage is an attractive target for the development of antimalarial drugs and vaccines, as it provides an opportunity to interrupt the life cycle of the parasite at a critical early stage. However, targeting the liver stage has been difficult. Undoubtedly, a major barrier has been the lack of robust, reliable, and reproducible in vitro liver-stage cultures. Here, we establish the liver stages for both Plasmodium falciparum and Plasmodium vivax in a microscale human liver platform composed of cryopreserved, micropatterned human primary hepatocytes surrounded by supportive stromal cells. Using this system, we have successfully recapitulated the full liver stage of P. falciparum, including the release of infected merozoites and infection of overlaid erythrocytes, as well as the establishment of small forms in late liver stages of P. vivax. Finally, we validate the potential of this platform as a tool for medium-throughput antimalarial drug screening and vaccine development. Copyright © 2013 Elsevier Inc. All rights reserved.

Liver Progenitor Cell Line HepaRG Differentiated in a Bioartificial Liver Effectively Supplies Liver Support to Rats with Acute Liver Failure

NARCIS (Netherlands)

Nibourg, Geert A. A.; Chamuleau, Robert A. F. M.; van der Hoeven, Tessa V.; Maas, Martinus A. W.; Ruiter, An F. C.; Lamers, Wouter H.; Oude Elferink, Ronald P. J.; van Gulik, Thomas M.; Hoekstra, Ruurdtje

2012-01-01

A major roadblock to the application of bioartificial livers is the need for a human liver cell line that displays a high and broad level of hepatic functionality. The human bipotent liver progenitor cell line HepaRG is a promising candidate in this respect, for its potential to differentiate into

Production of factor VIII by human liver sinusoidal endothelial cells transplanted in immunodeficient uPA mice.

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Marina E Fomin

Full Text Available Liver sinusoidal endothelial cells (LSECs form a semi-permeable barrier between parenchymal hepatocytes and the blood. LSECs participate in liver metabolism, clearance of pathological agents, immunological responses, architectural maintenance of the liver and synthesis of growth factors and cytokines. LSECs also play an important role in coagulation through the synthesis of Factor VIII (FVIII. Herein, we phenotypically define human LSECs isolated from fetal liver using flow cytometry and immunofluorescence microscopy. Isolated LSECs were cultured and shown to express endothelial markers and markers specific for the LSEC lineage. LSECs were also shown to engraft the liver when human fetal liver cells were transplanted into immunodeficient mice with liver specific expression of the urokinase-type plasminogen activator (uPA transgene (uPA-NOG mice. Engrafted cells expressed human Factor VIII at levels approaching those found in human plasma. We also demonstrate engraftment of adult LSECs, as well as hepatocytes, transplanted into uPA-NOG mice. We propose that overexpression of uPA provides beneficial conditions for LSEC engraftment due to elevated expression of the angiogenic cytokine, vascular endothelial growth factor. This work provides a detailed characterization of human midgestation LSECs, thereby providing the means for their purification and culture based on their expression of CD14 and CD32 as well as a lack of CD45 expression. The uPA-NOG mouse is shown to be a permissive host for human LSECs and adult hepatocytes, but not fetal hepatoblasts. Thus, these mice provide a useful model system to study these cell types in vivo. Demonstration of human FVIII production by transplanted LSECs encourages further pursuit of LSEC transplantation as a cellular therapy for the treatment of hemophilia A.

Characterization of the cytosolic distribution of priority pollutant metals and metalloids in the digestive gland cytosol of marine mussels: seasonal and spatial variability.

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Strižak, Zeljka; Ivanković, Dušica; Pröfrock, Daniel; Helmholz, Heike; Cindrić, Ana-Marija; Erk, Marijana; Prange, Andreas

2014-02-01

Cytosolic profiles of several priority pollutant metals (Cu, Cd, Zn, Pb) and metalloid As were analyzed in the digestive gland of the mussel (Mytilus galloprovincialis) sampled at locations with different environmental pollution levels along the Croatian coast in the spring and summer season. Size-exclusion chromatography (SEC) connected to inductively coupled plasma mass spectrometry (ICP-MS) was used to determine selected elements bound to cytosolic biomolecules separated based on their molecular size. Copper, cadmium and zinc eluted mostly associated with high molecular weight (HMW) and medium molecular weight (MMW) biomolecules, but with a more prominent elution in the MMW peak at polluted locations which were probably associated with the 20 kDa metallothionein (MT). Elution of all three metals within this peak was also strongly correlated with cytosolic Cd as strong inducer of MT. Lead mostly eluted in HMW biomolecule range, but in elevated cytosolic Pb concentrations, significant amount eluted in low molecular weight (LMW) biomolecules. Arsenic, on the other hand eluted almost completely in LMW range, but we could not distinguish specific molecular weight biomolecules which would be predominant in detoxification mechanism. Seasonal variability in element abundance within specific peaks was present, although not in the same extent, for all elements and locations, especially for As. The results confirm the suitability of the distribution of selected metals/metalloids among different cytosolic ligands as potential indicator for metal exposure. Obtained findings can also serve as guidelines for further separation and characterization of specific cytosolic metal-binding biomolecules. © 2013.

Chromosomal instability drives metastasis through a cytosolic DNA response.

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Bakhoum, Samuel F; Ngo, Bryan; Laughney, Ashley M; Cavallo, Julie-Ann; Murphy, Charles J; Ly, Peter; Shah, Pragya; Sriram, Roshan K; Watkins, Thomas B K; Taunk, Neil K; Duran, Mercedes; Pauli, Chantal; Shaw, Christine; Chadalavada, Kalyani; Rajasekhar, Vinagolu K; Genovese, Giulio; Venkatesan, Subramanian; Birkbak, Nicolai J; McGranahan, Nicholas; Lundquist, Mark; LaPlant, Quincey; Healey, John H; Elemento, Olivier; Chung, Christine H; Lee, Nancy Y; Imielenski, Marcin; Nanjangud, Gouri; Pe'er, Dana; Cleveland, Don W; Powell, Simon N; Lammerding, Jan; Swanton, Charles; Cantley, Lewis C

2018-01-25

Chromosomal instability is a hallmark of cancer that results from ongoing errors in chromosome segregation during mitosis. Although chromosomal instability is a major driver of tumour evolution, its role in metastasis has not been established. Here we show that chromosomal instability promotes metastasis by sustaining a tumour cell-autonomous response to cytosolic DNA. Errors in chromosome segregation create a preponderance of micronuclei whose rupture spills genomic DNA into the cytosol. This leads to the activation of the cGAS-STING (cyclic GMP-AMP synthase-stimulator of interferon genes) cytosolic DNA-sensing pathway and downstream noncanonical NF-κB signalling. Genetic suppression of chromosomal instability markedly delays metastasis even in highly aneuploid tumour models, whereas continuous chromosome segregation errors promote cellular invasion and metastasis in a STING-dependent manner. By subverting lethal epithelial responses to cytosolic DNA, chromosomally unstable tumour cells co-opt chronic activation of innate immune pathways to spread to distant organs.

BAF is a cytosolic DNA sensor that leads to exogenous DNA avoiding autophagy.

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Kobayashi, Shouhei; Koujin, Takako; Kojidani, Tomoko; Osakada, Hiroko; Mori, Chie; Hiraoka, Yasushi; Haraguchi, Tokuko

2015-06-02

Knowledge of the mechanisms by which a cell detects exogenous DNA is important for controlling pathogen infection, because most pathogens entail the presence of exogenous DNA in the cytosol, as well as for understanding the cell's response to artificially transfected DNA. The cellular response to pathogen invasion has been well studied. However, spatiotemporal information of the cellular response immediately after exogenous double-stranded DNA (dsDNA) appears in the cytosol is lacking, in part because of difficulties in monitoring when exogenous dsDNA enters the cytosol of the cell. We have recently developed a method to monitor endosome breakdown around exogenous materials using transfection reagent-coated polystyrene beads incorporated into living human cells as the objective for microscopic observations. In the present study, using dsDNA-coated polystyrene beads (DNA-beads) incorporated into living cells, we show that barrier-to-autointegration factor (BAF) bound to exogenous dsDNA immediately after its appearance in the cytosol at endosome breakdown. The BAF(+) DNA-beads then assembled a nuclear envelope (NE)-like membrane and avoided autophagy that targeted the remnants of the endosome membranes. Knockdown of BAF caused a significant decrease in the assembly of NE-like membranes and increased the formation of autophagic membranes around the DNA-beads, suggesting that BAF-mediated assembly of NE-like membranes was required for the DNA-beads to evade autophagy. Importantly, BAF-bound beads without dsDNA also assembled NE-like membranes and avoided autophagy. We propose a new role for BAF: remodeling intracellular membranes upon detection of dsDNA in mammalian cells.

A bioenergetics systems evaluation of ketogenic diet liver effects.

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Hutfles, Lewis J; Wilkins, Heather M; Koppel, Scott J; Weidling, Ian W; Selfridge, J Eva; Tan, Eephie; Thyfault, John P; Slawson, Chad; Fenton, Aron W; Zhu, Hao; Swerdlow, Russell H

2017-09-01

Ketogenic diets induce hepatocyte fatty acid oxidation and ketone body production. To further evaluate how ketogenic diets affect hepatocyte bioenergetic infrastructure, we analyzed livers from C57Bl/6J male mice maintained for 1 month on a ketogenic or standard chow diet. Compared with the standard diet, the ketogenic diet increased cytosolic and mitochondrial protein acetylation and also altered protein succinylation patterns. SIRT3 protein decreased while SIRT5 protein increased, and gluconeogenesis, oxidative phosphorylation, and mitochondrial biogenesis pathway proteins were variably and likely strategically altered. The pattern of changes observed can be used to inform a broader systems overview of how ketogenic diets affect liver bioenergetics.

The draft genome of the carcinogenic human liver fluke Clonorchis sinensis

Science.gov (United States)

2011-01-01

Background Clonorchis sinensis is a carcinogenic human liver fluke that is widespread in Asian countries. Increasing infection rates of this neglected tropical disease are leading to negative economic and public health consequences in affected regions. Experimental and epidemiological studies have shown a strong association between the incidence of cholangiocarcinoma and the infection rate of C. sinensis. To aid research into this organism, we have sequenced its genome. Results We combined de novo sequencing with computational techniques to provide new information about the biology of this liver fluke. The assembled genome has a total size of 516 Mb with a scaffold N50 length of 42 kb. Approximately 16,000 reliable protein-coding gene models were predicted. Genes for the complete pathways for glycolysis, the Krebs cycle and fatty acid metabolism were found, but key genes involved in fatty acid biosynthesis are missing from the genome, reflecting the parasitic lifestyle of a liver fluke that receives lipids from the bile of its host. We also identified pathogenic molecules that may contribute to liver fluke-induced hepatobiliary diseases. Large proteins such as multifunctional secreted proteases and tegumental proteins were identified as potential targets for the development of drugs and vaccines. Conclusions This study provides valuable genomic information about the human liver fluke C. sinensis and adds to our knowledge on the biology of the parasite. The draft genome will serve as a platform to develop new strategies for parasite control. PMID:22023798

Comparative Metabolism Study of Five Protoberberine Alkaloids in Liver Microsomes from Rat, Rhesus Monkey, and Human.

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Li, Yan; Zhou, Yanyan; Si, Nan; Han, Lingyu; Ren, Wei; Xin, Shaokun; Wang, Hongjie; Zuo, Ran; Wei, Xiaolu; Yang, Jian; Zhao, Haiyu; Bian, Baolin

2017-11-01

Protoberberine alkaloids including berberine, palmatine, jatrorrhizine, coptisine, and epiberberine are major components in many medicinal plants. They have been widely used for the treatment of cancer, inflammation, diabetes, depression, hypertension, and various infectious areas. However, the metabolism of five protoberberine alkaloids among different species has not been clarified previously. In order to elaborate on the in vitro metabolism of them, a comparative analysis of their metabolic profile in rat, rhesus monkey, and human liver microsomes was carried out using ultrahigh-performance liquid chromatography coupled with a high-resolution linear trap quadrupole-Orbitrap mass spectrometer (UHPLC-electrospray ionization-Orbitrap MS) for the first time. Each metabolite was identified and semiquantified by its accurate mass data and peak area. Fifteen metabolites were characterized based on accurate MS/MS spectra and the proposed MS/MS fragmentation pathways including demethylation, hydroxylation, and methyl reduction. Among them, the content of berberine metabolites in human liver microsomes was similar with those in rhesus monkey liver microsomes, whereas berberine in rat liver microsomes showed no demethylation metabolites and the content of metabolites showed significant differences with that in human liver microsomes. On the contrary, the metabolism of palmatine in rat liver microsomes resembled that in human liver microsomes. The content of jatrorrhizine metabolites presented obvious differences in all species. The HR-ESI-MS/MS fragmentation behavior of protoberberine alkaloids and their metabolic profile in rat, rhesus monkey, and human liver microsomes were investigated for the first time. The results demonstrated that the biotransformation characteristics of protoberberine alkaloids among different species had similarities as well differences that would be beneficial for us to better understand the pharmacological activities of protoberberine alkaloids

Radionuclide imaging of the liver in human fascioliasis

International Nuclear Information System (INIS)

Rivera, J.V.; Bermudez, R.H.

1984-01-01

The clinical, laboratory, and scintigraphic findings in four cases of human fascioliasis are described. Acute onset of fever, abdominal pain, and weight loss in a person who has ingested watercress constitutes the clinical syndrome often seen. Eosinophilia and alteration in liver function tests, particularly alkaline phosphatase are frequent. Tc-99m sulfur colloid images showed hepatomegaly in four patients, focal defects in two, splenomegaly in three, and increased splenic uptake in two. Gallium citrate (Ga 67) images show increased uptake in the focal lesions in two of two. Sonographic imaging showed focal lucent abnormality in one of three. Liver biopsy findings were nonspecific. The differential diagnosis from other invasive parasitic diseases is discussed. A possible role of hepatic imaging in the evaluation of fascioliasis is suggested

Liver lipase and high-density lipoprotein. Lipoprotein changes after incubation of human serum with rat liver lipase.

Science.gov (United States)

Groot, P H; Scheek, L M; Jansen, H

1983-05-16

Human sera were incubated with rat liver lipase after inactivation of lecithin:cholesterol acyltransferase, and the changes in serum lipoprotein composition were measured. In the presence of liver lipase serum triacylglycerol and phosphatidylcholine were hydrolyzed. The main changes in the concentrations of these lipids were found in the high-density lipoprotein fraction. Subfractionation of high-density lipoprotein by rate-zonal ultracentrifugation showed a prominent decrease in all constituents of high-density lipoprotein2, a smaller decrease in the 'light' high-density lipoprotein3 and an increase in the 'heavy' high-density lipoprotein3. These data support a concept in which liver lipase is involved in high-density lipoprotein2 phospholipid and triacylglycerol catabolism and suggest that as a result of this action high-density lipoprotein2 is converted into high-density lipoprotein3.

Morphology and some biomechanical properties of human liver and spleen

Czech Academy of Sciences Publication Activity Database

Stingl, J.; Bača, V.; Čech, V.; Kovanda, J.; Kovandová, H.; Mandys, Václav; Rejmontová, J.; Sosna, B.

2002-01-01

Roč. 24, - (2002), s. 285-289 ISSN 0930-1038 Institutional research plan: CEZ:AV0Z5039906 Keywords : Human liver and spleen Subject RIV: FE - Other Internal Medicine Disciplines Impact factor: 0.252, year: 2002

Cyclic GMP-AMP Synthase Is the Cytosolic Sensor of Plasmodium falciparum Genomic DNA and Activates Type I IFN in Malaria.

Science.gov (United States)

Gallego-Marin, Carolina; Schrum, Jacob E; Andrade, Warrison A; Shaffer, Scott A; Giraldo, Lina F; Lasso, Alvaro M; Kurt-Jones, Evelyn A; Fitzgerald, Katherine A; Golenbock, Douglas T

2018-01-15

Innate immune receptors have a key role in the sensing of malaria and initiating immune responses. As a consequence of infection, systemic inflammation emerges and is directly related to signs and symptoms during acute disease. We have previously reported that plasmodial DNA is the primary driver of systemic inflammation in malaria, both within the phagolysosome and in the cytosol of effector cells. In this article, we demonstrate that Plasmodium falciparum genomic DNA delivered to the cytosol of human monocytes binds and activates cyclic GMP-AMP synthase (cGAS). Activated cGAS synthesizes 2'3'-cGAMP, which we subsequently can detect using liquid chromatography-tandem mass spectrometry. 2'3'-cGAMP acts as a second messenger for STING activation and triggers TBK1/IRF3 activation, resulting in type I IFN production in human cells. This induction of type I IFN was independent of IFI16. Access of DNA to the cytosolic compartment is mediated by hemozoin, because incubation of purified malaria pigment with DNase abrogated IFN-β induction. Collectively, these observations implicate cGAS as an important cytosolic sensor of P. falciparum genomic DNA and reveal the role of the cGAS/STING pathway in the induction of type I IFN in response to malaria parasites. Copyright © 2018 by The American Association of Immunologists, Inc.

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.

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

International Nuclear Information System (INIS)

Yannam, Govardhana Rao; Han, Bing; Setoyama, Kentaro; Yamamoto, Toshiyuki; Ito, Ryotaro; Brooks, Jenna M.; Guzman-Lepe, Jorge; Galambos, Csaba; Fong, Jason V.; Deutsch, Melvin; Quader, Mubina A.; Yamanouchi, Kosho; Kabarriti, Rafi; Mehta, Keyur; Soto-Gutierrez, Alejandro

2014-01-01

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

Humanizing π-class glutathione S-transferase regulation in a mouse model alters liver toxicity in response to acetaminophen overdose.

Directory of Open Access Journals (Sweden)

Matthew P Vaughn

Full Text Available Glutathione S-transferases (GSTs metabolize drugs and xenobiotics. Yet despite high protein sequence homology, expression of π-class GSTs, the most abundant of the enzymes, varies significantly between species. In mouse liver, hepatocytes exhibit high mGstp expression, while in human liver, hepatocytes contain little or no hGSTP1 mRNA or hGSTP1 protein. π-class GSTs are known to be critical determinants of liver responses to drugs and toxins: when treated with high doses of acetaminophen, mGstp1/2+/+ mice suffer marked liver damage, while mGstp1/2-/- mice escape liver injury.To more faithfully model the contribution of π-class GSTs to human liver toxicology, we introduced hGSTP1, with its exons, introns, and flanking sequences, into the germline of mice carrying disrupted mGstp genes. In the resultant hGSTP1+mGstp1/2-/- strain, π-class GSTs were regulated differently than in wild-type mice. In the liver, enzyme expression was restricted to bile duct cells, Kupffer cells, macrophages, and endothelial cells, reminiscent of human liver, while in the prostate, enzyme production was limited to basal epithelial cells, reminiscent of human prostate. The human patterns of hGSTP1 transgene regulation were accompanied by human patterns of DNA methylation, with bisulfite genomic sequencing revealing establishment of an unmethylated CpG island sequence encompassing the gene promoter. Unlike wild-type or mGstp1/2-/- mice, when hGSTP1+mGstp1/2-/- mice were overdosed with acetaminophen, liver tissues showed limited centrilobular necrosis, suggesting that π-class GSTs may be critical determinants of toxin-induced hepatocyte injury even when not expressed by hepatocytes.By recapitulating human π-class GST expression, hGSTP1+mGstp1/2-/- mice may better model human drug and xenobiotic toxicology.

Three-Dimensional Culture of Human Embryonic Stem Cell Derived Hepatic Endoderm and Its Role in Bioartificial Liver Construction

Directory of Open Access Journals (Sweden)

Ruchi Sharma

2010-01-01

Full Text Available The liver carries out a range of functions essential for bodily homeostasis. The impairment of liver functions has serious implications and is responsible for high rates of patient morbidity and mortality. Presently, liver transplantation remains the only effective treatment, but donor availability is a major limitation. Therefore, artificial and bioartificial liver devices have been developed to bridge patients to liver transplantation. Existing support devices improve hepatic encephalopathy to a certain extent; however their usage is associated with side effects. The major hindrance in the development of bioartificial liver devices and cellular therapies is the limited availability of human hepatocytes. Moreover, primary hepatocytes are difficult to maintain and lose hepatic identity and function over time even with sophisticated tissue culture media. To overcome this limitation, renewable cell sources are being explored. Human embryonic stem cells are one such cellular resource and have been shown to generate a reliable and reproducible supply of human hepatic endoderm. Therefore, the use of human embryonic stem cell-derived hepatic endoderm in combination with tissue engineering has the potential to pave the way for the development of novel bioartificial liver devices and predictive drug toxicity assays.

Mechanistic logic underlying the axonal transport of cytosolic proteins

Science.gov (United States)

Scott, David A.; Das, Utpal; Tang, Yong; Roy, Subhojit

2011-01-01

Proteins vital to presynaptic function are synthesized in the neuronal perikarya and delivered into synapses via two modes of axonal transport. While membrane-anchoring proteins are conveyed in fast axonal transport via motor-driven vesicles, cytosolic proteins travel in slow axonal transport; via mechanisms that are poorly understood. We found that in cultured axons, populations of cytosolic proteins tagged to photoactivable-GFP (PA-GFP) move with a slow motor-dependent anterograde bias; distinct from vesicular-trafficking or diffusion of untagged PA-GFP. The overall bias is likely generated by an intricate particle-kinetics involving transient assembly and short-range vectorial spurts. In-vivo biochemical studies reveal that cytosolic proteins are organized into higher-order structures within axon-enriched fractions that are largely segregated from vesicles. Data-driven biophysical modeling best predicts a scenario where soluble molecules dynamically assemble into mobile supra-molecular structures. We propose a model where cytosolic proteins are transported by dynamically assembling into multi-protein complexes that are directly/indirectly conveyed by motors. PMID:21555071

Gymnocypris przewalskii decreases cytosolic carbonic anhydrase expression to compensate for respiratory alkalosis and osmoregulation in the saline-alkaline lake Qinghai.

Science.gov (United States)

Yao, Zongli; Guo, Wenfei; Lai, Qifang; Shi, Jianquan; Zhou, Kai; Qi, Hongfang; Lin, Tingting; Li, Ziniu; Wang, Hui

2016-01-01

Naked carp (Gymnocypris przewalskii), endemic to the saline-alkaline Lake Qinghai, have the capacity to tolerate combined high salinity and alkalinity, but migrate to spawn in freshwater rivers each year. In this study, the full-length cDNA of the cytosolic carbonic anhydrase c isoform of G. przewalskii (GpCAc) was amplified and sequenced; mRNA levels and enzyme activity of GpCAc and blood chemistry were evaluated to understand the compensatory responses as the naked carp returned to the saline-alkaline lake after spawning. We found that GpCAc had a total length of 1400 bp and encodes a peptide of 260 amino acids. Comparison of the deduced amino acid sequences and phylogenetic analysis showed that GpCAc was a member of the cytosolic carbonic anhydrase II-like c family. Cytosolic-carbonic-anhydrase-c-specific primers were used to analyze the tissue distribution of GpCAc mRNA expression. Expression of GpCAc mRNA was found in brain, gill, liver, kidney, gut, and muscle tissues, but primarily in the gill and posterior kidney; however, none was evident in red blood cells. Transferring fish from river water to lake water resulted in a respiratory alkalosis, osmolality, and ion rise in the blood, as well as significant decreases in the expression and enzyme activity of GpCAc in both the gill and kidney within 96 h. These results indicate that GpCAc may play an important role in the acclimation to both high salinity and carbonate alkalinity. Specifically, G. przewalskii decreases cytosolic carbonic anhydrase c expression to compensate for a respiratory alkalosis and to aid in osmoregulation during the transition from river to saline-alkaline lake.

DMPD: Cytosolic DNA recognition for triggering innate immune responses. [Dynamic Macrophage Pathway CSML Database

Lifescience Database Archive (English)

Full Text Available 18280611 Cytosolic DNA recognition for triggering innate immune responses. Takaoka ...A, Taniguchi T. Adv Drug Deliv Rev. 2008 Apr 29;60(7):847-57. Epub 2007 Dec 31. (.png) (.svg) (.html) (.csml) Show Cytosol...ic DNA recognition for triggering innate immune responses. PubmedID 18280611 Title Cytosolic D

Metabolism of lysergic acid diethylamide (LSD) to 2-oxo-3-hydroxy LSD (O-H-LSD) in human liver microsomes and cryopreserved human hepatocytes.

Science.gov (United States)

Klette, K L; Anderson, C J; Poch, G K; Nimrod, A C; ElSohly, M A

2000-10-01

The metabolism of lysergic acid diethylamide (LSD) to 2-oxo-3-hydroxy lysergic acid diethylamide (O-H-LSD) was investigated in liver microsomes and cyropreserved hepatocytes from humans. Previous studies have demonstrated that O-H-LSD is present in human urine at concentrations 16-43 times greater than LSD, the parent compound. Additionally, these studies have determined that O-H-LSD is not generated during the specimen extraction and analytical processes or due to parent compound degradation in aqueous urine samples. However, these studies have not been conclusive in demonstrating that O-H-LSD is uniquely produced during in vivo metabolism. Phase I drug metabolism was investigated by incubating human liver microsomes and cryopreserved human hepatocytes with LSD. The reaction was quenched at various time points, and the aliquots were extracted using liquid partitioning and analyzed by liquid chromatography-mass spectrometry. O-H-LSD was positively identified in all human liver microsomal and human hepatocyte fractions incubated with LSD. In addition, O-H-LSD was not detected in any microsomal or hepatocyte fraction not treated with LSD nor in LSD specimens devoid of microsomes or hepatocytes. This study provides definitive evidence that O-H-LSD is produced as a metabolic product following incubation of human liver microsomes and hepatocytes with LSD.

Extended normothermic extracorporeal perfusion of isolated human liver after warm ischaemia: a preliminary report.

Science.gov (United States)

Bellomo, Rinaldo; Marino, Bruno; Starkey, Graeme; Fink, Michael; Wang, Bao Zhong; Eastwood, Glenn M; Peck, Leah; Young, Helen; Houston, Shane; Skene, Alison; Opdam, Helen; Jones, Robert

2014-09-01

Donation after circulatory death (DCD) livers are at markedly increased risk of primary graft dysfunction and biliary tract ischaemia. Normothermic extracorporeal liver perfusion (NELP) may increase the ability to transplant DCD livers and may allow their use for artificial extracorporeal liver support of patients with fulminant liver failure. We conducted two proof-of-concept experiments using human livers after DCD to assess the feasibility and functional efficacy of NELP over an extended period. We applied extracorporeal membrane oxygenation, parenteral nutrition, separate hepatic artery and portal vein perfusion and physiological perfusion pressures to two livers obtained after DCD. We achieved NELP and evidence of liver function (bile production, paracetamol removal and maintenance of normal lactate levels) in both livers; one for 24 hours and the other for 43 hours. Histological examination showed areas of patchy ischaemia but preserved biliary ducts and canaliculi. Our experiments justify further investigations of the feasibility and efficacy of extended DCD liver preservation by ex-vivo perfusion.

A physiologically based pharmacokinetic model for ethylene oxide in mouse, rat, and human.

Science.gov (United States)

Fennell, T R; Brown, C D

2001-06-15

Ethylene oxide (EO) is widely used as a gaseous sterilant and industrial intermediate and is a direct-acting mutagen and carcinogen. The objective of these studies was to develop physiologically based pharmacokinetic (PB-PK) models for EO to describe the exposure-tissue dose relationship in rodents and humans. We previously reported results describing in vitro and in vivo kinetics of EO metabolism in male and female F344 rats and B6C3F1 mice. These studies were extended by determining the kinetics of EO metabolism in human liver cytosol and microsomes. The results indicate enzymatically catalyzed GSH conjugation via cytosolic glutathione S-transferase (cGST) and hydrolysis via microsomal epoxide hydrolase (mEH) occur in both rodents and humans. The in vitro kinetic constants were scaled to account for cytosolic (cGST) and microsomal (mEH) protein content and incorporated into PB-PK descriptions for mouse, rat, and human. Flow-limited models adequately predicted blood and tissue EO levels, disposition, and elimination kinetics determined experimentally in rats and mice, with the exception of testis concentrations, which were overestimated. Incorporation of a diffusion-limited description for testis improved the ability of the model to describe testis concentrations. The model accounted for nonlinear increases in blood and tissue concentrations that occur in mice on exposure to EO concentrations greater than 200 ppm. Species differences are predicted in the metabolism and exposure-dose relationship, with a nonlinear relationship observed in the mouse as a result of GSH depletion. These models represent an essential step in developing a mechanistically based EO exposure-dose-response description for estimating human risk from exposure to EO. Copyright 2001 Academic Press.

Well-defined polypeptide-based systems as non-viral vectors for cytosolic delivery

OpenAIRE

Niño Pariente, Amaya

2017-01-01

A convenient cytosolic drug delivery constitutes a very powerful tool for the treatment and/or prevention of several relevant human diseases. Along with recent advances in therapeutic technologies based on biomacromolecules (e.g. oligonucleotides or proteins), we also require the development of technologies which improve the transport of therapeutic molecules to the cell of choice. This has led to the emergence of a variety of promising methods over the last 20 years. Despite significant prog...

Cytosol cathepsin-D content and proliferative activity of human breast cancer. The Comitato Italiano per il Controllo di Qualita del Laboratorio in Oncologia.

Science.gov (United States)

Paradiso, A; Mangia, A; Correale, M; Abbate, I; Ferri, G; Piffanelli, A; Catozzi, L; Amadori, D; Riccobon, A; De Lena, M

1992-01-01

Mitogenic properties have been demonstrated in vitro for the lysosomal acidic protease cathepsin-D (cath-D). We investigated possible relationships between cath-D cytosol cell content and tumor proliferative activity in a series of 129 operable breast cancer patients. For total cytosol cath-D evaluation, a solid phase two-site immunoradiometric assay was utilized on tumor cell cytosol obtained for hormone receptor assay (DCC method). The percentage of S-phase cells was analyzed by 3H-thymidine autoradiographic assay. Median 3H-thymidine Labeling Index (3H-Tdr-LI) of the series was 2.7%; median cath-D content resulted 57 pmol/mg of protein cytosol and was significantly higher in node-positive with respect to the node-negative subgroup (p < 0.03). When classified in low, intermediate or high tumor cath-D content and slow or fast proliferative activity (cut-off: median values of the series), no significant agreement was found between the two variables. Statistical analysis, however, showed that a significant inverse correlation existed in node positive tumors between cath-D and 3H-Tdr-LI values which was even more evident in N-positive high estrogen receptor-positive (ER+) cases (coefficient of correlation = 0.6828; p = 0.0001). Cytosol cath-D content cannot be generally proposed as a direct marker of proliferative activity for operable breast cancer.

Pentose pathway in human liver

International Nuclear Information System (INIS)

Magnusson, I.; Chandramouli, V.; Schumann, W.C.; Kumaran, K.; Wahren, J.; Landau, B.R.

1988-01-01

[1- 14 C]Ribose and [1- 14 C]glucose were given to normal subjects along with glucose loads (1 g per kg of body weight) after administration of diflunisal and acetaminophen, drugs that are excreted in urine as glucuronides. Distributions of 14 C were determined in the carbons of the excreted glucoronides and in the glucose from blood samples drawn from hepatic veins before and after glucagon administration. Eighty percent or more of the 14 C from [1- 14 C]ribose incorporated into the glucuronic acid moiety of the glucuronides was in carbons 1 and 3, with less than 8% in carbon 2. In glucuronic acid from glucuronide excreted when [2- 14 C]glucose was given, 3.5-8.1% of the 14 C was in carbon 1, 2.5-4.3% in carbon 3, and more than 70% in carbon 2. These distributions are in accord with the glucuronides sampling the glucose unit of the glucose 6-phosphate pool that is a component of the pentose pathway and is intermediate in glycogen formation. It is concluded that the glucuronic acid conjugates of the drugs can serve as a noninvasive means of sampling hepatic glucose 6-phosphate. In human liver, as in animal liver, the classical pentose pathway functions, not the L-type pathway, and only a small percentage of the glucose is metabolized via the pathway

Fatty acid translocase promoted hepatitis B virus replication by upregulating the levels of hepatic cytosolic calcium.

Science.gov (United States)

Huang, Jian; Zhao, Lei; Yang, Ping; Chen, Zhen; Ruan, Xiong Z; Huang, Ailong; Tang, Ni; Chen, Yaxi

2017-09-15

Hepatitis B virus (HBV) is designated a "metabolovirus" due to the intimate connection between the virus and host metabolism. The nutrition state of the host plays a relevant role in the severity of HBV infection. Metabolic syndrome (MS) is prone to increasing HBV DNA loads and accelerating the progression of liver disease in patients with chronic hepatitis B (CHB). Cluster of differentiation 36 (CD36), also named fatty acid translocase, is known to facilitate long-chain fatty acid uptake and contribute to the development of MS. We recently found that CD36 overexpression enhanced HBV replication. In this study, we further explored the mechanism by which CD36 overexpression promotes HBV replication. Our data showed that CD36 overexpression increased HBV replication, and CD36 knockdown inhibited HBV replication. RNA sequencing found some of the differentially expressed genes were involved in calcium ion homeostasis. CD36 overexpression elevated the cytosolic calcium level, and CD36 knockdown decreased the cytosolic calcium level. Calcium chelator BAPTA-AM could override the HBV replication increased by CD36 overexpression, and the calcium activator thapsigargin could improve the HBV replication reduced by CD36 knockdown. We further found that CD36 overexpression activated Src kinase, which plays an important role in the regulation of the store-operated Ca 2+ channel. An inhibitor of Src kinase (SU6656) significantly reduced the CD36-induced HBV replication. We identified a novel link between CD36 and HBV replication, which is associated with cytosolic calcium and the Src kinase pathway. CD36 may represent a potential therapeutic target for the treatment of CHB patients with MS. Copyright © 2017 Elsevier Inc. All rights reserved.

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

Directory of Open Access Journals (Sweden)

Donna N Douglas

2010-02-01

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

Rapid increase of bile salt secretion is associated with bile duct injury after human liver transplantation

NARCIS (Netherlands)

Geuken, Erwin; Visser, Dorien; Kuipers, Folkert; Blokzijl, Hans; Leuvenink, Henri G. D.; de Jong, Koert P.; Peeters, Paul M. J. G.; Jansen, Peter L. M.; Slooff, Maarten J. H.; Gouw, Annette S. H.; Porte, Robert J.

2004-01-01

BACKGROUND/AIMS: Biliary strictures are a serious cause of morbidity after liver transplantation. We have studied the role of altered bile composition as a mechanism of bile duct injury after human liver transplantation. METHODS: In 28 liver transplant recipients, bile samples were collected daily

Rapid increase of bile salt secretion is associated with bile duct injury after human liver transplantation

NARCIS (Netherlands)

Geuken, E; Visser, D; Kuipers, F; Blokzijl, H; Leuvenink, HGD; de Jong, KP; Peeters, PMJG; Jansen, PLM; Slooff, MJH; Gouw, ASH; Porte, RJ

2004-01-01

Background/Aims: Biliary strictures are a serious cause of morbidity after liver transplantation. We have studied the role of altered bile composition as a mechanism of bile duct injury after human liver transplantation. Methods: In 28 liver transplant recipients, bile samples were collected daily

In vivo time-harmonic multifrequency elastography of the human liver

International Nuclear Information System (INIS)

Tzschätzsch, Heiko; Guo, Jing; Streitberger, Kaspar-Josche; Fischer, Thomas; Sack, Ingolf; Ipek-Ugay, Selcan; Braun, Jürgen; Gentz, Enno; Klaua, Robert; Schultz, Michael

2014-01-01

Elastography is capable of noninvasively detecting hepatic fibrosis by imposing mechanical stress and measuring the viscoelastic response in the liver. Magnetic resonance elastography (MRE) relies on time-harmonic vibrations, while most dynamic ultrasound elastography methods employ transient stimulation methods. This study attempts to benefit from the advantages of time-harmonic tissue stimulation, i.e. relative insensitivity to obesity and ascites and mechanical approachability of the entire liver, and the advantages of ultrasound, i.e. time efficiency, low costs, and wide availability, by introducing in vivo time-harmonic elastography (THE) of the human liver using ultrasound and a broad range of harmonic stimulation frequencies. THE employs continuous harmonic shear vibrations at 7 frequencies from 30 to 60 Hz in a single examination and determines the elasticity and the viscosity of the liver from the dispersion of the shear wave speed within the applied frequency range. The feasibility of the method is demonstrated in the livers of eight healthy volunteers and a patient with cirrhosis. Multifrequency MRE at the same drive frequencies was used as elastographic reference method. Similar values of shear modulus and shear viscosity according the Kelvin–Voigt model were obtained by MRE and THE, indicating that the new method is suitable for in vivo quantification of the shear viscoelastic properties of the liver, however, in real-time and at a fraction of the costs of MRE. In conclusion, THE may provide a useful tool for fast assessment of the viscoelastic properties of the liver at low costs and without limitations in obesity, ascites or hemochromatosis. (paper)

Metabolism of styrene in the human liver in vitro: interindividual variation and enantioselectivity

NARCIS (Netherlands)

Wenker, M. A.; Kezić, S.; Monster, A. C.; de Wolff, F. A.

2001-01-01

1. The interindividual variation and enantioselectivity of the in vitro styrene oxidation by cytochrome P450 have been investigated in 20 human microsomal liver samples. Liver samples were genotyped for the CYP2E1*6 and CYP2E1*5B alleles. 2. Kinetic analysis indicated the presence of at least two

Potential genotoxic and cytotoxicity of emamectin benzoate in human normal liver cells.

Science.gov (United States)

Zhang, Zhijie; Zhao, Xinyu; Qin, Xiaosong

2017-10-10

Pesticide residue inducing cancer-related health problems draw people more attention recently. Emamectin benzoate (EMB) has been widely used in agriculture around the world based on its specificity targets. Although potential risk and the molecular mechanism of EMB toxicity to human liver has not been well-characterized. Unlike well-reported toxicity upon central nervous system, potential genotoxic and cytotoxicity of EMB in human liver cell was ignored and very limited. In this study, we identify genotoxicity and cytotoxicity of EMB to human normal liver cells (QSG7701 cell line) in vitro . We demonstrate that EMB inhibited the viability of QSG7701 cells and induced the DNA damage. Established assays of cytotoxicity were performed to characterize the mechanism of EMB toxicity on QSG7701 cells. Typical chromatin condensation and DNA fragmentation indicated the apoptosis of QSG7701 cells induced by EMB. And the intracellular biochemical results demonstrated that EMB-enhanced apoptosis of QSG7701 cells concurrent with generated ROS, a loss of mitochondrial membrane potential, the cytochrome-c release, up regulate the Bax/Bcl-2 and the activation of caspase-9/-3. Our results of EMB induces the death of QSG7701 cells maybe via mitochondrial-mediated intrinsic apoptotic pathways would contribute to promote the awareness of EMB as an extensive used pesticide to human being effects and reveal the underlying mechanisms of potential genotoxic.

Interaction of rocuronium with human liver cytochromes P450.

Science.gov (United States)

Anzenbacherova, Eva; Spicakova, Alena; Jourova, Lenka; Ulrichova, Jitka; Adamus, Milan; Bachleda, Petr; Anzenbacher, Pavel

2015-02-01

Rocuronium is a neuromuscular blocking agent acting as a competitive antagonist of acetylcholine. Results of an inhibition of eight individual liver microsomal cytochromes P450 (CYP) are presented. As the patients are routinely premedicated with diazepam, possible interaction of diazepam with rocuronium has been also studied. Results indicated that rocuronium interacts with human liver microsomal CYPs by binding to the substrate site. Next, concentration dependent inhibition of liver microsomal CYP3A4 down to 42% (at rocuronium concentration 189 μM) was found. This effect has been confirmed with two CYP3A4 substrates, testosterone (formation of 6β-hydroxytestosterone) and diazepam (temazepam formation). CYP2C9 and CYP2C19 activities were inhibited down to 75-80% (at the same rocuronium concentration). Activities of other microsomal CYPs have not been inhibited by rocuronium. To prove the possibility of rocuronium interaction with other drugs (diazepam), the effect of rocuronium on formation of main diazepam metabolites, temazepam (by CYP3A4) and desmethyldiazepam, (also known as nordiazepam; formed by CYP2C19) in primary culture of human hepatocytes has been examined. Rocuronium has caused inhibition of both reactions by 20 and 15%, respectively. The results open a possibility that interactions of rocuronium with drugs metabolized by CYP3A4 (and possibly also CYP2C19) may be observed. Copyright © 2014 Japanese Pharmacological Society. Production and hosting by Elsevier B.V. All rights reserved.

Decreased hepatotoxic bile acid composition and altered synthesis in progressive human nonalcoholic fatty liver disease

International Nuclear Information System (INIS)

Lake, April D.; Novak, Petr; Shipkova, Petia; Aranibar, Nelly; Robertson, Donald; Reily, Michael D.; Lu, Zhenqiang; Lehman-McKeeman, Lois D.; Cherrington, Nathan J.

2013-01-01

Bile acids (BAs) have many physiological roles and exhibit both toxic and protective influences within the liver. Alterations in the BA profile may be the result of disease induced liver injury. Nonalcoholic fatty liver disease (NAFLD) is a prevalent form of chronic liver disease characterized by the pathophysiological progression from simple steatosis to nonalcoholic steatohepatitis (NASH). The hypothesis of this study is that the ‘classical’ (neutral) and ‘alternative’ (acidic) BA synthesis pathways are altered together with hepatic BA composition during progression of human NAFLD. This study employed the use of transcriptomic and metabolomic assays to study the hepatic toxicologic BA profile in progressive human NAFLD. Individual human liver samples diagnosed as normal, steatosis, and NASH were utilized in the assays. The transcriptomic analysis of 70 BA genes revealed an enrichment of downregulated BA metabolism and transcription factor/receptor genes in livers diagnosed as NASH. Increased mRNA expression of BAAT and CYP7B1 was observed in contrast to decreased CYP8B1 expression in NASH samples. The BA metabolomic profile of NASH livers exhibited an increase in taurine together with elevated levels of conjugated BA species, taurocholic acid (TCA) and taurodeoxycholic acid (TDCA). Conversely, cholic acid (CA) and glycodeoxycholic acid (GDCA) were decreased in NASH liver. These findings reveal a potential shift toward the alternative pathway of BA synthesis during NASH, mediated by increased mRNA and protein expression of CYP7B1. Overall, the transcriptomic changes of BA synthesis pathway enzymes together with altered hepatic BA composition signify an attempt by the liver to reduce hepatotoxicity during disease progression to NASH. - Highlights: ► Altered hepatic bile acid composition is observed in progressive NAFLD. ► Bile acid synthesis enzymes are transcriptionally altered in NASH livers. ► Increased levels of taurine and conjugated bile acids

Decreased hepatotoxic bile acid composition and altered synthesis in progressive human nonalcoholic fatty liver disease

Energy Technology Data Exchange (ETDEWEB)

Lake, April D. [University of Arizona, Department of Pharmacology and Toxicology, Tucson, AZ 85721 (United States); Novak, Petr [Biology Centre ASCR, Institute of Plant Molecular Biology, Ceske Budejovice 37001 (Czech Republic); Shipkova, Petia; Aranibar, Nelly; Robertson, Donald; Reily, Michael D. [Pharmaceutical Candidate Optimization, Bristol-Myers Squibb Co., Princeton, NJ 08543 (United States); Lu, Zhenqiang [The Arizona Statistical Consulting Laboratory, University of Arizona, Tucson, AZ 85721 (United States); Lehman-McKeeman, Lois D. [Pharmaceutical Candidate Optimization, Bristol-Myers Squibb Co., Princeton, NJ 08543 (United States); Cherrington, Nathan J., E-mail: cherrington@pharmacy.arizona.edu [University of Arizona, Department of Pharmacology and Toxicology, Tucson, AZ 85721 (United States)

2013-04-15

Bile acids (BAs) have many physiological roles and exhibit both toxic and protective influences within the liver. Alterations in the BA profile may be the result of disease induced liver injury. Nonalcoholic fatty liver disease (NAFLD) is a prevalent form of chronic liver disease characterized by the pathophysiological progression from simple steatosis to nonalcoholic steatohepatitis (NASH). The hypothesis of this study is that the ‘classical’ (neutral) and ‘alternative’ (acidic) BA synthesis pathways are altered together with hepatic BA composition during progression of human NAFLD. This study employed the use of transcriptomic and metabolomic assays to study the hepatic toxicologic BA profile in progressive human NAFLD. Individual human liver samples diagnosed as normal, steatosis, and NASH were utilized in the assays. The transcriptomic analysis of 70 BA genes revealed an enrichment of downregulated BA metabolism and transcription factor/receptor genes in livers diagnosed as NASH. Increased mRNA expression of BAAT and CYP7B1 was observed in contrast to decreased CYP8B1 expression in NASH samples. The BA metabolomic profile of NASH livers exhibited an increase in taurine together with elevated levels of conjugated BA species, taurocholic acid (TCA) and taurodeoxycholic acid (TDCA). Conversely, cholic acid (CA) and glycodeoxycholic acid (GDCA) were decreased in NASH liver. These findings reveal a potential shift toward the alternative pathway of BA synthesis during NASH, mediated by increased mRNA and protein expression of CYP7B1. Overall, the transcriptomic changes of BA synthesis pathway enzymes together with altered hepatic BA composition signify an attempt by the liver to reduce hepatotoxicity during disease progression to NASH. - Highlights: ► Altered hepatic bile acid composition is observed in progressive NAFLD. ► Bile acid synthesis enzymes are transcriptionally altered in NASH livers. ► Increased levels of taurine and conjugated bile acids

Dynamin-Related Protein 1 Translocates from the Cytosol to Mitochondria during UV-Induced Apoptosis

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Zhang, Zhenzhen; Wu, Shengnan; Feng, Jie

2011-01-01

Mitochondria are dynamic structures that frequently divide and fuse with one another to form interconnecting network. This network disintegrates into punctiform organelles during apoptosis. However, the mechanisms involved in these processes are still not well characterized. In this study, we investigate the role of dynamin-related protein 1 (Drp1), a large GTPase that mediates outer mitochondrial membrane fission, in mitochondrial dynamics in response to UV irradiation in human lung adenocarcinoma cells (ASTC-α-1) and HeLa cells. Using time-lapse fluorescent imaging, we find that Drp1 primarily distributes in cytosol under physiological conditions. After UV treatment, Drp1 translocates from cytosol to mitochondria, indicating the enhancement of Drp1 mitochondrial accumulation. Our results suggest that Drp1 is involved in the regulation of transition from an interconnecting network to a punctiform mitochondrial phenotype during UV-induced apoptosis.

Distribution of sterol carrier protein2 (SCP2) in rat tissues and evidence for slow turnover in liver and adrenal cortex

International Nuclear Information System (INIS)

Kharroubi, A.; Chanderbhan, R.; Fiskum, G.; Noland, B.J.; Scallen, T.J.; Vahouny, G.V.

1986-01-01

Sterol carrier protein 2 (SCP 2 ) has been implicated in the regulation of the terminal stages of hepatic cholesterol biosynthesis, and in sterol utilization for adrenal steroid hormone and hepatic bile acid synthesis. In the present studies, a highly sensitive radioimmunoassay, using [ 125 I] SCP 2 , has been developed. Highest levels of SCP 2 were found in rat liver with progressively lower levels in intestinal mucosa, adrenal, kidney, lung and testis. SCP 2 levels were low or absent in heart, brain, skeletal muscle and serum. Liver SCP 2 was largely (44%) associated with the microsomal fraction, while in adrenal, 46% was associated with mitochondria, a distribution which is consistent with the proposed roles for SCP 2 in these tissues. Levels of SCP 2 in AS 30D hepatoma cells were only 5% of those in normal liver. In liver there was no indication of diurnal rhythm of SCP 2 in the cytosol and only slight variation of the microsomal SCP 2 levels. Fasting has only slight effects on SCP 2 concentration of rat liver microsomes and cytosol. Neither ACTH nor cycloheximide treatment of rats had a significant effect on SCP 2 distribution in the adrenal. In general, these findings indicate that SCP 2 has a low turn-over rate

Demonstration of Hepatitis C Virus RNA with In Situ Hybridization Employing a Locked Nucleic Acid Probe in Humanized Liver of Infected Chimeric Mice and in Needle-Biopsied Human Liver

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Kazuya Shiogama

2013-01-01

Full Text Available Background. In situ hybridization (ISH with high sensitivity has been requested to demonstrate hepatitis C virus (HCV RNA in formalin-fixed, paraffin-embedded (FFPE sections of the liver. Methods. ISH employing a locked-nucleic-acid- (LNA-modified oligonucleotide probe and biotin-free catalyzed signal amplification system (CSAII was applied to HCV-RNA detection in the liver tissue. Nested reverse-transcription polymerase chain reaction (RT-PCR was performed for HCV genotyping using total RNA extracted from FFPE sections. The target tissues included FFPE tissue sections of humanized livers in HCV-infected chimeric mice (HCV genotypes 1a, 1b, and 2a and noninfected and of needle-biopsied livers from HCV-infected patients. Results. HCV-RNA was demonstrated with the ISH technique in HCV-infected liver tissues from both chimeric mice and 9 (82% of 11 patients with HCV infection. The HCV signals were sensitive to RNase. Nested RT-PCR confirmed the genotype in 8 (73% of 11 livers (type 1b: 6 lesions and type 2a: 2 lesions. HCV-RNA was not identified in chronic hepatitis B lesions, fatty liver, autoimmune hepatitis, and hepatocellular carcinoma. Conclusion. ISH using the LNA-modified oligonucleotide probe and CSAII was applicable to detecting HCV-RNA in routinely prepared FFPE liver specimens.

Monochloramine produces reactive oxygen species in liver by converting xanthine dehydrogenase into xanthine oxidase.

Science.gov (United States)

Sakuma, Satoru; Miyoshi, Emi; Sadatoku, Namiko; Fujita, Junko; Negoro, Miki; Arakawa, Yukio; Fujimoto, Yohko

2009-09-15

In the present study, we assessed the influence of monochloramine (NH(2)Cl) on the conversion of xanthine dehydrogenase (XD) into xanthine oxidase (XO) in rat liver in vitro. When incubated with the partially purified cytosolic fraction from rat liver, NH(2)Cl (2.5-20 microM) dose-dependently enhanced XO activity concomitant with a decrease in XD activity, implying that NH(2)Cl can convert XD into the reactive oxygen species (ROS) producing form XO. The NH(2)Cl (5 microM)-induced XD/XO interconversion in the rat liver cytosol was completely inhibited when added in combination with an inhibitor of NH(2)Cl methionine (25 microM). A sulfhydryl reducing agent, dithiothreitol at concentrations of 0.1, 1 and 5 mM also dose-dependently reversed the NH(2)Cl (5 microM)-induced XD/XO interconversion. These imply that NH(2)Cl itself acts on the XD/XO interconversion, and that this conversion occurs at the cysteine residues in XD. Furthermore, using the fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate, it was found that NH(2)Cl could increase ROS generation in the cytoplasm of rat primary hepatocyte cultures, and that this increase might be reversed by an XO inhibitor, allopurinol. These results suggest that NH(2)Cl has the potential to convert XD into XO in the liver, which in turn may induce the ROS generation in this region.

Methods of measuring metabolism during surgery in humans: focus on the liver-brain relationship.

Science.gov (United States)

Battezzati, Alberto; Bertoli, Simona

2004-09-01

The purpose of this work is to review recent advances in setting methods and models for measuring metabolism during surgery in humans. Surgery, especially solid organ transplantation, may offer unique experimental models in which it is ethically acceptable to gain information on difficult problems of amino acid and protein metabolism. Two areas are reviewed: the metabolic study of the anhepatic phase during liver transplantation and brain microdialysis during cerebral surgery. The first model offers an innovative approach to understand the relative role of liver and extrahepatic organs in gluconeogenesis, and to evaluate whether other organs can perform functions believed to be exclusively or almost exclusively performed by the liver. The second model offers an insight to intracerebral metabolism that is closely bound to that of the liver. The recent advances in metabolic research during surgery provide knowledge immediately useful for perioperative patient management and for a better control of surgical stress. The studies during the anhepatic phase of liver transplantation have showed that gluconeogenesis and glutamine metabolism are very active processes outside the liver. One of the critical organs for extrahepatic glutamine metabolism is the brain. Microdialysis studies helped to prove that in humans there is an intense trafficking of glutamine, glutamate and alanine among neurons and astrocytes. This delicate network is influenced by systemic amino acid metabolism. The metabolic dialogue between the liver and the brain is beginning to be understood in this light in order to explain the metabolic events of brain damage during liver failure.

Human amnion epithelial cells expressing HLA-G as novel cell-based treatment for liver disease.

Science.gov (United States)

Strom, Stephen C; Gramignoli, Roberto

2016-09-01

Despite routine liver transplantation and supporting medical therapies, thousands of patients currently wait for an organ and there is an unmet need for more refined and widely available regenerative strategies to treat liver diseases. Cell transplants attempt to maximize the potential for repair and/or regeneration in liver and other organs. Over 40years of laboratory pre-clinical research and 25years of clinical procedures have shown that certain liver diseases can be treated by the infusion of isolated cells (hepatocyte transplant). However, like organ transplants, hepatocyte transplant suffers from a paucity of tissues useful for cell production. Alternative sources have been investigated, yet with limited success. The tumorigenic potential of pluripotent stem cells together with their primitive level of hepatic differentiation, have limited the use of stem cell populations. Stem cell sources from human placenta, and the amnion tissue in particular are receiving renewed interest in the field of regenerative medicine. Unlike pluripotent stem cells, human amnion epithelial (AE) cells are easily available without ethical or religious concerns; they do not express telomerase and are not immortal or tumorigenic when transplanted. In addition, AE cells have been reported to express genes normally expressed in mature liver, when transplanted into the liver. Moreover, because of the possibility of an immune-privileged status related to their expression of HLA-G, it might be possible to transplant human AE cells without immunosuppression of the recipient. Copyright © 2016. Published by Elsevier Inc.

Cytosolic Pellino-1-Mediated K63-Linked Ubiquitination of IRF5 in M1 Macrophages Regulates Glucose Intolerance in Obesity

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Donghyun Kim

2017-07-01

Full Text Available IRF5 is a signature transcription factor that induces M1 macrophage polarization. However, little is known regarding cytosolic proteins that induce IRF5 activation for M1 polarization. Here, we report the interaction between ubiquitin E3 ligase Pellino-1 and IRF5 in the cytoplasm, which increased nuclear translocation of IRF5 by K63-linked ubiquitination in human and mouse M1 macrophages. LPS and/or IFN-γ increased Pellino-1 expression, and M1 polarization was attenuated in Pellino-1-deficient macrophages in vitro and in vivo. Defective M1 polarization in Pellino-1-deficient macrophages improved glucose intolerance in mice fed a high-fat diet. Furthermore, macrophages in adipose tissues from obese humans exhibited increased Pellino-1 expression and IRF5 nuclear translocation compared with nonobese subjects, and these changes are associated with insulin resistance index. This study demonstrates that cytosolic Pellino-1-mediated K63-linked ubiquitination of IRF5 in M1 macrophages regulates glucose intolerance in obesity, suggesting a cytosolic mediator function of Pellino-1 in TLR4/IFN-γ receptor-IRF5 axis during M1 polarization.

Molecular cloning of cDNAs of human liver and placenta NADH-cytochrome b5 reductase

International Nuclear Information System (INIS)

Yubisui, T.; Naitoh, Y.; Zenno, S.; Tamura, M.; Takeshita, M.; Sakaki, Y.

1987-01-01

A cDNA coding for human liver NADH-cytochrome b 5 reductase was cloned from a human liver cDNA library constructed in phage λgt11. The library was screened by using an affinity-purified rabbit antibody against NADH-cytochrome b 5 reductase of human erythrocytes. A cDNA about 1.3 kilobase pairs long was isolated. By using the cDNA as a probe, another cDNA (pb 5 R141) of 1817 base pairs was isolated that hybridized with a synthetic oligonucleotide encoding Pro-Asp-Ile-Lys-Tyr-Pro, derived from the amino acid sequence at the amino-terminal region of the enzyme from human erythrocytes. Furthermore, by using the pb 5 R141 as a probe, cDNA clones having more 5' sequence were isolated from a human placenta cDNA library. The amino acid sequences deduced from the nucleotide sequences of these cDNA clones overlapped each other and consisted of a sequence that completely coincides with that of human erythrocytes and a sequence of 19 amino acid residues extended at the amino-terminal side. The latter sequence closely resembles that of the membrane-binding domain of steer liver microsomal enzyme

Endosomolytic Nano-Polyplex Platform Technology for Cytosolic Peptide Delivery To Inhibit Pathological Vasoconstriction.

Science.gov (United States)

Evans, Brian C; Hocking, Kyle M; Kilchrist, Kameron V; Wise, Eric S; Brophy, Colleen M; Duvall, Craig L

2015-06-23

A platform technology has been developed and tested for delivery of intracellular-acting peptides through electrostatically complexed nanoparticles, or nano-polyplexes, formulated from an anionic endosomolytic polymer and cationic therapeutic peptides. This delivery platform has been initially tested and optimized for delivery of two unique vasoactive peptides, a phosphomimetic of heat shock protein 20 and an inhibitor of MAPKAP kinase II, to prevent pathological vasoconstriction (i.e., vasospasm) in human vascular tissue. These peptides inhibit vasoconstriction and promote vasorelaxation by modulating actin dynamics in vascular smooth muscle cells. Formulating these peptides into nano-polyplexes significantly enhances peptide uptake and retention, facilitates cytosolic delivery through a pH-dependent endosomal escape mechanism, and enhances peptide bioactivity in vitro as measured by inhibition of F-actin stress fiber formation. In comparison to treatment with the free peptides, which were endowed with cell-penetrating sequences, the nano-polyplexes significantly increased vasorelaxation, inhibited vasoconstriction, and decreased F-actin formation in the human saphenous vein ex vivo. These results suggest that these formulations have significant potential for treatment of conditions such as cerebral vasospasm following subarachnoid hemorrhage. Furthermore, because many therapeutic peptides include cationic cell-penetrating segments, this simple and modular platform technology may have broad applicability as a cost-effective approach for enhancing the efficacy of cytosolically active peptides.

Bioartificial liver and liver transplantation: new modalities for the treatment of liver failure

Directory of Open Access Journals (Sweden)

DING Yitao

2017-09-01

Full Text Available The main features of liver failure are extensive necrosis of hepatocytes, rapid disease progression, and poor prognosis, and at present, there are no effective drugs and methods for the treatment of liver failure. This article summarizes four treatment methods for liver failure, i.e., medical treatment, cell transplantation, liver transplantation, and artificial liver support therapy, and elaborates on the existing treatment methods. The current medical treatment regimen should be optimized; cell transplantation has not been used in clinical practice; liver transplantation is the most effective method, but it is limited by donor liver shortage and high costs; artificial liver can effectively remove toxic substances in human body. Therefore, this article puts forward artificial liver as a transition for liver transplantation; artificial liver can buy time for liver regeneration or liver transplantation and prolong patients′ survival time and thus has a promising future. The new treatment modality of bioartificial liver combined with liver transplantation may bring good news to patients with liver failure.

Scaffold-free 3D bio-printed human liver tissue stably maintains metabolic functions useful for drug discovery.

Science.gov (United States)

Kizawa, Hideki; Nagao, Eri; Shimamura, Mitsuru; Zhang, Guangyuan; Torii, Hitoshi

2017-07-01

The liver plays a central role in metabolism. Although many studies have described in vitro liver models for drug discovery, to date, no model has been described that can stably maintain liver function. Here, we used a unique, scaffold-free 3D bio-printing technology to construct a small portion of liver tissue that could stably maintain drug, glucose, and lipid metabolism, in addition to bile acid secretion. This bio-printed normal human liver tissue maintained expression of several kinds of hepatic drug transporters and metabolic enzymes that functioned for several weeks. The bio-printed liver tissue displayed glucose production via cAMP/protein kinase A signaling, which could be suppressed with insulin. Bile acid secretion was also observed from the printed liver tissue, and it accumulated in the culture medium over time. We observed both bile duct and sinusoid-like structures in the bio-printed liver tissue, which suggested that bile acid secretion occurred via a sinusoid-hepatocyte-bile duct route. These results demonstrated that our bio-printed liver tissue was unique, because it exerted diverse liver metabolic functions for several weeks. In future, we expect our bio-printed liver tissue to be applied to developing new models that can be used to improve preclinical predictions of long-term toxicity in humans, generate novel targets for metabolic liver disease, and evaluate biliary excretion in drug development.

AMC-Bio-Artificial Liver culturing enhances mitochondrial biogenesis in human liver cell lines: The role of oxygen, medium perfusion and 3D configuration

NARCIS (Netherlands)

Adam, Aziza A. A.; van Wenum, Martien; van der Mark, Vincent A.; Jongejan, Aldo; Moerland, Perry D.; Houtkooper, Riekelt H.; Wanders, Ronald J. A.; Oude Elferink, Ronald P.; Chamuleau, Robert A. F. M.; Hoekstra, Ruurdtje

2017-01-01

Human liver cell lines, like HepaRG and C3A, acquire higher functionality when cultured in the AMC-Bio-Artificial Liver (AMC-BAL). The three main differences between BAL and monolayer culture are the oxygenation (40% vs 20%O2), dynamic vs absent medium perfusion and 3D vs 2D configuration. Here, we

Differential genomic effects of six different TiO2 nanomaterials on human liver HepG2 cells

Science.gov (United States)

Engineered nanoparticles are reported to cause liver toxicity in vivo. To better assess the mechanism of the in vivo liver toxicity, we used the human hepatocarcinoma cells (HepG2) as a model system. Human HepG2 cells were exposed to 6 TiO2 nanomaterials (with dry primary partic...

Increased diacylglycerols characterize hepatic lipid changes in progression of human nonalcoholic fatty liver disease; comparison to a murine model.

Science.gov (United States)

Gorden, D Lee; Ivanova, Pavlina T; Myers, David S; McIntyre, J Oliver; VanSaun, Michael N; Wright, J Kelly; Matrisian, Lynn M; Brown, H Alex

2011-01-01

The spectrum of nonalcoholic fatty liver disease (NAFLD) includes steatosis, nonalcoholic steatohepatitis (NASH), and progression to cirrhosis. While differences in liver lipids between disease states have been reported, precise composition of phospholipids and diacylglycerols (DAG) at a lipid species level has not been previously described. The goal of this study was to characterize changes in lipid species through progression of human NAFLD using advanced lipidomic technology and compare this with a murine model of early and advanced NAFLD. Utilizing mass spectrometry lipidomics, over 250 phospholipid and diacylglycerol species (DAGs) were identified in normal and diseased human and murine liver extracts. Significant differences between phospholipid composition of normal and diseased livers were demonstrated, notably among DAG species, consistent with previous reports that DAG transferases are involved in the progression of NAFLD and liver fibrosis. In addition, a novel phospholipid species (ether linked phosphatidylinositol) was identified in human cirrhotic liver extracts. Using parallel lipidomics analysis of murine and human liver tissues it was determined that mice maintained on a high-fat diet provide a reproducible model of NAFLD in regards to specificity of lipid species in the liver. These studies demonstrated that novel lipid species may serve as markers of advanced liver disease and importantly, marked increases in DAG species are a hallmark of NAFLD. Elevated DAGs may contribute to altered triglyceride, phosphatidylcholine (PC), and phosphatidylethanolamine (PE) levels characteristic of the disease and specific DAG species might be important lipid signaling molecules in the progression of NAFLD.

In vitro biotransformation of tris(2-butoxyethyl) phosphate (TBOEP) in human liver and serum

Energy Technology Data Exchange (ETDEWEB)

Van den Eede, Nele, E-mail: nele.vandeneede@uantwerpen.be [Toxicological Center, Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein 1, 2610 Wilrijk, Antwerp (Belgium); Erratico, Claudio [Toxicological Center, Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein 1, 2610 Wilrijk, Antwerp (Belgium); Exarchou, Vassiliki [Natural Products & Food Research and Analysis (NatuRA), Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein 1, 2610 Wilrijk, Antwerp (Belgium); Maho, Walid; Neels, Hugo [Toxicological Center, Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein 1, 2610 Wilrijk, Antwerp (Belgium); Covaci, Adrian, E-mail: adrian.covaci@uantwerpen.be [Toxicological Center, Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein 1, 2610 Wilrijk, Antwerp (Belgium)

2015-04-15

Tris(2-butoxyethyl) phosphate (TBOEP) is a plasticizer present in indoor dust, reaching levels of several micrograms per gram. Such levels could lead to significant daily exposure of adults and children. Currently, no toxicokinetic data are available to estimate TBOEP clearance in humans after uptake and therefore, one objective of this study was to investigate intrinsic clearance of TBOEP by human liver microsome (HLM) and serum enzymes. Another objective was to generate information to identify and prioritize several metabolites of TBOEP for investigation of human exposure by biomonitoring. 1D and 2D-NMR methodologies were successfully applied on a mixture of the metabolites to confirm the structure of 3-HO-TBOEP (bis(2-butoxyethyl) 3-hydroxyl-2-butoxyethyl phosphate) and to tentatively assign structures to 1-HO-TBOEP and 2-HO-TBOEP. HO-TBOEP isomers and bis(2-butoxyethyl) phosphate (BBOEP), bis(2-butoxyethyl) hydroxyethyl phosphate (BBOEHEP) were further monitored by liquid chromatography–tandem mass spectrometry. Rates of formation of BBOEHEP and HO-TBOEP metabolites by liver enzymes were best described by the Michaelis–Menten model. Apparent K{sub m} values for BBOEHEP, 3-HO-TBOEP, and sum of 1- and 2-HO-TBOEP isomer formation were 152, 197 and 148 μM, respectively. Apparent V{sub max} values for the formation of BBOEHEP, 3-HO-TBOEP, and the sum of 1- and 2-HO-TBOEP isomers were 2560, 643, and 254 pmol/min/mg protein, respectively. No detectable formation of BBOEP occurred with liver or serum enzymes. Our findings indicate that intrinsic clearance of TBOEP is mainly catalyzed by oxidative enzymes in the liver and that its major in vitro metabolite is BBOEHEP. These findings can be applied in human biomonitoring studies and risk assessment. - Highlights: • First steps in the elucidation of TBOEP toxicokinetics • Quantification of TBOEP metabolites in human serum and liver microsomes • No detectable formation of BBOEP occurred with liver or serum

Hsp70 facilitates trans-membrane transport of bacterial ADP-ribosylating toxins into the cytosol of mammalian cells.

Science.gov (United States)

Ernst, Katharina; Schmid, Johannes; Beck, Matthias; Hägele, Marlen; Hohwieler, Meike; Hauff, Patricia; Ückert, Anna Katharina; Anastasia, Anna; Fauler, Michael; Jank, Thomas; Aktories, Klaus; Popoff, Michel R; Schiene-Fischer, Cordelia; Kleger, Alexander; Müller, Martin; Frick, Manfred; Barth, Holger

2017-06-02

Binary enterotoxins Clostridium (C.) botulinum C2 toxin, C. perfringens iota toxin and C. difficile toxin CDT are composed of a transport (B) and a separate non-linked enzyme (A) component. Their B-components mediate endocytic uptake into mammalian cells and subsequently transport of the A-components from acidic endosomes into the cytosol, where the latter ADP-ribosylate G-actin resulting in cell rounding and cell death causing clinical symptoms. Protein folding enzymes, including Hsp90 and peptidyl-prolyl cis/trans isomerases facilitate transport of the A-components across endosomal membranes. Here, we identified Hsp70 as a novel host cell factor specifically interacting with A-components of C2, iota and CDT toxins to facilitate their transport into the cell cytosol. Pharmacological Hsp70-inhibition specifically prevented pH-dependent trans-membrane transport of A-components into the cytosol thereby protecting living cells and stem cell-derived human miniguts from intoxication. Thus, Hsp70-inhibition might lead to development of novel therapeutic strategies to treat diseases associated with bacterial ADP-ribosylating toxins.

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.

A membrane model for cytosolic calcium oscillations. A study using Xenopus oocytes.

OpenAIRE

Jafri, M S; Vajda, S; Pasik, P; Gillo, B

1992-01-01

Cytosolic calcium oscillations occur in a wide variety of cells and are involved in different cellular functions. We describe these calcium oscillations by a mathematical model based on the putative electrophysiological properties of the endoplasmic reticulum (ER) membrane. The salient features of our membrane model are calcium-dependent calcium channels and calcium pumps in the ER membrane, constant entry of calcium into the cytosol, calcium dependent removal from the cytosol, and buffering ...

Microsomal UDP-glucuronyltransferase-catalyzed bilirubin diglucuronide formation in human liver

NARCIS (Netherlands)

Peters, W. H.; Jansen, P. L.

1986-01-01

Human liver microsomal bilirubin UDP-glucuronyltransferase catalyzes formation of bilirubin mono- and diglucuronide. KmUDPGA and Vmax of the enzyme are 0.6 mM and 1.69 nmol/mg protein X min. In vitro, bilirubin readily dissolves in the microsomal lipid phase. Taking this into account a Kmbilirubin

Fluctuations in Cytosolic Calcium Regulate the Neuronal Malate-Aspartate NADH Shuttle

DEFF Research Database (Denmark)

Satrústegui, Jorgina; Bak, Lasse K

2015-01-01

that MAS is regulated by fluctuations in cytosolic Ca(2+) levels, and that this regulation is required to maintain a tight coupling between neuronal activity and mitochondrial respiration and oxidative phosphorylation. At cytosolic Ca(2+) fluctuations below the threshold of the mitochondrial calcium...

Human liver phosphatase 2A: cDNA and amino acid sequence of two catalytic subunit isotypes

International Nuclear Information System (INIS)

Arino, J.; Woon, Chee Wai; Brautigan, D.L.; Miller, T.B. Jr.; Johnson, G.L.

1988-01-01

Two cDNA clones were isolated from a human liver library that encode two phosphatase 2A catalytic subunits. The two cDNAs differed in eight amino acids (97% identity) with three nonconservative substitutions. All of the amino acid substitutions were clustered in the amino-terminal domain of the protein. Amino acid sequence of one human liver clone (HL-14) was identical to the rabbit skeletal muscle phosphatase 2A cDNA (with 97% nucleotide identity). The second human liver clone (HL-1) is encoded by a separate gene, and RNA gel blot analysis indicates that both mRNAs are expressed similarly in several human clonal cell lines. Sequence comparison with phosphatase 1 and 2A indicates highly divergent amino acid sequences at the amino and carboxyl termini of the proteins and identifies six highly conserved regions between the two proteins that are predicted to be important for phosphatase enzymatic activity

Proton MR spectroscopic features of the human liver: in-vivo application to the normal condition

International Nuclear Information System (INIS)

Cho, Soon Gu; Kim, Mi Young; Kim, Young Soo; Choi, Won; Shin, Seok Hwan; Ok, Chul Soo; Suh, Chang Hae

1999-01-01

To determine the feasibility of MR spectroscopy in the living human liver, and to evaluate the corresponding proton MR spectroscopic features. In fifteen normal volunteers with neither previous nor present liver disease, the proton MR spectroscopic findings were reviewed. Twelve subjects were male and three were female ; they were aged between 28 and 32 (mean, 30) years. MR spectroscopy involved the use of a 1.5T GE Signa Horizon system with body coil(GE Medical System, Milwaukee, U.S.A). We used STEAM (Stimulated Echo-Acquisition Mode) with 3000/30 msec of TR/TE for signal acquisition, and the prone position without respiratory interruption. Mean and standard deviation of the ratios of glutamate+glutamine/lipids, phosphomonoesters/lipids, and glycogen+glucose/lipids were calculated from the area of their peaks. The proton MR spectroscopic findings of normal human livers showed four distinctive peaks, i.e. lipids, glutamate and glutamine complex, phosphomonoesters, and glycogen and glucose complex. The mean and standard deviation of the ratios of glutamate+glutamine/lipids, phosphomonoesters/lipids, and glycogen+glucose/lipids were 0.02±0.01, 0.01±0.01, and 0.04±0.03, respectively. In living normal human livers, MR spectroscopy can be successfully applied. When applied to a liver whose condition is pathologic, the findings can be used as a standard

Overexpression of human virus surface glycoprotein precursors induces cytosolic unfolded protein response in Saccharomyces cerevisiae

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Sasnauskas Kęstutis

2011-05-01

Full Text Available Abstract Background The expression of human virus surface proteins, as well as other mammalian glycoproteins, is much more efficient in cells of higher eukaryotes rather than yeasts. The limitations to high-level expression of active viral surface glycoproteins in yeast are not well understood. To identify possible bottlenecks we performed a detailed study on overexpression of recombinant mumps hemagglutinin-neuraminidase (MuHN and measles hemagglutinin (MeH in yeast Saccharomyces cerevisiae, combining the analysis of recombinant proteins with a proteomic approach. Results Overexpressed recombinant MuHN and MeH proteins were present in large aggregates, were inactive and totally insoluble under native conditions. Moreover, the majority of recombinant protein was found in immature form of non-glycosylated precursors. Fractionation of yeast lysates revealed that the core of viral surface protein aggregates consists of MuHN or MeH disulfide-linked multimers involving eukaryotic translation elongation factor 1A (eEF1A and is closely associated with small heat shock proteins (sHsps that can be removed only under denaturing conditions. Complexes of large Hsps seem to be bound to aggregate core peripherally as they can be easily removed at high salt concentrations. Proteomic analysis revealed that the accumulation of unglycosylated viral protein precursors results in specific cytosolic unfolded protein response (UPR-Cyto in yeast cells, characterized by different action and regulation of small Hsps versus large chaperones of Hsp70, Hsp90 and Hsp110 families. In contrast to most environmental stresses, in the response to synthesis of recombinant MuHN and MeH, only the large Hsps were upregulated whereas sHsps were not. Interestingly, the amount of eEF1A was also increased during this stress response. Conclusions Inefficient translocation of MuHN and MeH precursors through ER membrane is a bottleneck for high-level expression in yeast. Overexpression of

A human cytochrome P-450 is recognized by anti-liver/kidney microsome antibodies in autoimmune chronic hepatitis.

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Kiffel, L; Loeper, J; Homberg, J C; Leroux, J P

1989-02-28

1- Anti-liver/kidney microsome autoantibodies type 1 (anti-LKM1), observed in some children with chronic active hepatitis, were used to isolate their antigen in human liver microsomes. A protein, called P-LKM1 was thus purified. This protein was recognized by a rabbit antiserum directed against the related human cytochromes P-450 bufI and P-450 bufII. 2- A human liver microsomal protein immunoprecipitated with anti-LKM1 sera was also recognized by anti cytochromes P-450 bufI/II antibodies. 3- Anti-LKM1 antibodies potently inhibited microsomal bufuralol 1'-hydroxylation. These results displayed the possible identity between cytochrome P-450 bufI/II and LKM1 antigen.

Dynamic PET of human liver inflammation: impact of kinetic modeling with optimization-derived dual-blood input function.

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Wang, Guobao; Corwin, Michael T; Olson, Kristin A; Badawi, Ramsey D; Sarkar, Souvik

2018-05-30

The hallmark of nonalcoholic steatohepatitis is hepatocellular inflammation and injury in the setting of hepatic steatosis. Recent work has indicated that dynamic 18F-FDG PET with kinetic modeling has the potential to assess hepatic inflammation noninvasively, while static FDG-PET did not show a promise. Because the liver has dual blood supplies, kinetic modeling of dynamic liver PET data is challenging in human studies. The objective of this study is to evaluate and identify a dual-input kinetic modeling approach for dynamic FDG-PET of human liver inflammation. Fourteen human patients with nonalcoholic fatty liver disease were included in the study. Each patient underwent one-hour dynamic FDG-PET/CT scan and had liver biopsy within six weeks. Three models were tested for kinetic analysis: traditional two-tissue compartmental model with an image-derived single-blood input function (SBIF), model with population-based dual-blood input function (DBIF), and modified model with optimization-derived DBIF through a joint estimation framework. The three models were compared using Akaike information criterion (AIC), F test and histopathologic inflammation reference. The results showed that the optimization-derived DBIF model improved the fitting of liver time activity curves and achieved lower AIC values and higher F values than the SBIF and population-based DBIF models in all patients. The optimization-derived model significantly increased FDG K1 estimates by 101% and 27% as compared with traditional SBIF and population-based DBIF. K1 by the optimization-derived model was significantly associated with histopathologic grades of liver inflammation while the other two models did not provide a statistical significance. In conclusion, modeling of DBIF is critical for kinetic analysis of dynamic liver FDG-PET data in human studies. The optimization-derived DBIF model is more appropriate than SBIF and population-based DBIF for dynamic FDG-PET of liver inflammation. © 2018

Purification and characterization of glutaryl-CoA dehydrogenase from porcine and human liver

International Nuclear Information System (INIS)

Lenich, A.C.

1985-01-01

Glutaryl-CoA dehydrogenase (GCDH) was purified from porcine liver mitochondria by pH and ammonium sulfate fractionations followed by a series of column chromatographies. The purified porcine enzyme was found by sodium dodecyl-sulfate polyacrylamide gel electrophoresis to have a subunit molecular weight of 47,800 and by gradient polyacrylamide gel electrophoresis (PAGE) to have a native molecular weight of approximately 186,000. The product of the GCDH reaction with its primary substrate, glutaryl-CoA, was investigated by radio-gas chromatography and found to be crotonyl-CoA. Alternate substrates as well as crotonyl-CoA, the glutaryl-CoA reaction end product, demonstrated competitive inhibition when incubated with (1,5- 14 C)-glutaryl-CoA in the presence of porcine GCDH. Kinetic parameters for the interaction of both ETF and glutaryl-CoA with porcine GCDH were determined. Purified porcine GCDH was used to produce an antiserum which cross-reacted with human liver GCDH with a reaction of partial identity, but proved too insensitive to detect GCDH in control human fibroblasts. As a result of these negative findings, GCDH was purified by a series of column chromatographies from human liver. The purified human enzyme was found by SDS-PAGE and gel filtration to have subunit and native molecular weights of 58,800 and 256,000 respectively

[Detection of human parvovirus B19, human bocavirus and human parvovirus 4 infections in blood samples among 95 patients with liver disease in Nanjing by nested PCR].

Science.gov (United States)

Tong, Rui; Zhou, Wei-Min; Liu, Xi-Jun; Wang, Yue; Lou, Yong-Liang; Tan, Wen-Jie

2013-04-01

To analyze the infection of human parvovirus B19, human bocavirus (HBoV) and human parvovirus 4 (PARV4) in blood samples among patients with liver disease in Nanjing by molecular detection. Nested PCR assays were designed and validated to detect B19, HBoV and PARV4, respectively. The assays were used to screen three parvoviruses in blood samples from 95 patients with different liver disease in Nanjing. The parvovirus infection was analyzed statistically. The detection limits were 10 copies of genomic DNA equivalents per reaction for each assays and the good specificity were observed. The frequency of B19 and HBoV were 2/95 (2.1%) and 9/95 (9.5%) in blood samples respectively. No PARV4 was detected. HBoV was detected in 3/5 patients with drug-induced hepatitis. Both B19 and HBoV infection were detected in blood from patients with liver disease.

Automation of metabolic stability studies in microsomes, cytosol and plasma using a 215 Gilson liquid handler.

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Linget, J M; du Vignaud, P

1999-05-01

A 215 Gilson liquid handler was used to automate enzymatic incubations using microsomes, cytosol and plasma. The design of automated protocols are described. They were based on the use of 96 deep well plates and on HPLC-based methods for assaying the substrate. The assessment of those protocols was made with comparison between manual and automated incubations, reliability and reproducibility of automated incubations in microsomes and cytosol. Examples of the use of those programs in metabolic studies in drug research, i.e. metabolic screening in microsomes and plasma were shown. Even rapid processes (with disappearance half lives as low as 1 min) can be analysed. This work demonstrates how stability studies can be automated to save time, render experiments involving human biological media less hazardous and may be improve inter-laboratory reproducibility.

Identification and characterization of metabolites of ASP015K, a novel oral Janus kinase inhibitor, in rats, chimeric mice with humanized liver, and humans.

Science.gov (United States)

Nakada, Naoyuki; Oda, Kazuo

2015-01-01

1. Here, we elucidated the structure of metabolites of novel oral Janus kinase inhibitor ASP015K in rats and humans and evaluated the predictability of human metabolites using chimeric mice with humanized liver (PXB mice). 2. Rat biological samples collected after oral dosing of (14)C-labelled ASP015K were examined using a liquid chromatography-radiometric detector and mass spectrometer (LC-RAD/MS). The molecular weight of metabolites in human and the liver chimeric mouse biological samples collected after oral dosing of non-labelled ASP015K was also investigated via LC-MS. Metabolites were also isolated from rat bile samples and analyzed using nuclear magnetic resonance. 3. Metabolic pathways of ASP015K in rats and humans were found to be glucuronide conjugation, methyl conjugation, sulfate conjugation, glutathione conjugation, hydroxylation of the adamantane ring and N-oxidation of the 1H-pyrrolo[2,3-b]pyridine ring. The main metabolite of ASP015K in rats was the glucuronide conjugate, while the main metabolite in humans was the sulfate conjugate. Given that human metabolites were produced by human hepatocytes in chimeric mice with humanized liver, this human model mouse was believed to be useful in predicting the human metabolic profile of various drug candidates.

Control of oxygen tension recapitulates zone-specific functions in human liver microphysiology systems.

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Lee-Montiel, Felipe T; George, Subin M; Gough, Albert H; Sharma, Anup D; Wu, Juanfang; DeBiasio, Richard; Vernetti, Lawrence A; Taylor, D Lansing

2017-10-01

This article describes our next generation human Liver Acinus MicroPhysiology System (LAMPS). The key demonstration of this study was that Zone 1 and Zone 3 microenvironments can be established by controlling the oxygen tension in individual devices over the range of ca. 3 to 13%. The oxygen tension was computationally modeled using input on the microfluidic device dimensions, numbers of cells, oxygen consumption rates of hepatocytes, the diffusion coefficients of oxygen in different materials and the flow rate of media in the MicroPhysiology System (MPS). In addition, the oxygen tension was measured using a ratiometric imaging method with the oxygen sensitive dye, Tris(2,2'-bipyridyl) dichlororuthenium(II) hexahydrate (RTDP) and the oxygen insensitive dye, Alexa 488. The Zone 1 biased functions of oxidative phosphorylation, albumin and urea secretion and Zone 3 biased functions of glycolysis, α1AT secretion, Cyp2E1 expression and acetaminophen toxicity were demonstrated in the respective Zone 1 and Zone 3 MicroPhysiology System. Further improvements in the Liver Acinus MicroPhysiology System included improved performance of selected nonparenchymal cells, the inclusion of a porcine liver extracellular matrix to model the Space of Disse, as well as an improved media to support both hepatocytes and non-parenchymal cells. In its current form, the Liver Acinus MicroPhysiology System is most amenable to low to medium throughput, acute through chronic studies, including liver disease models, prioritizing compounds for preclinical studies, optimizing chemistry in structure activity relationship (SAR) projects, as well as in rising dose studies for initial dose ranging. Impact statement Oxygen zonation is a critical aspect of liver functions. A human microphysiology system is needed to investigate the impact of zonation on a wide range of liver functions that can be experimentally manipulated. Because oxygen zonation has such diverse physiological effects in the liver, we

Cytosolic Phosphoenolpyruvate Carboxykinase Does Not Solely Control the Rate of Hepatic Gluconeogenesis in the Intact Mouse Liver

OpenAIRE

Burgess, Shawn C.; He, TianTeng; Yan, Zheng; Lindner, Jill; Sherry, A. Dean; Malloy, Craig R.; Browning, Jeffrey D.; Magnuson, Mark A.

2007-01-01

When dietary carbohydrate is unavailable, glucose required to support metabolism in vital tissues is generated via gluconeogenesis in the liver. Expression of phosphoenolpyruvate carboxykinase (PEPCK), commonly considered the control point for liver gluconeogenesis, is normally regulated by circulating hormones to match systemic glucose demand. However, this regulation fails in diabetes. Because other molecular and metabolic factors can also influence gluconeogenesis, the explicit role of PEP...

Human Adipose Tissue Derived Stem Cells Promote Liver Regeneration in a Rat Model of Toxic Injury

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Eva Koellensperger

2013-01-01

Full Text Available In the light of the persisting lack of donor organs and the risks of allotransplantations, the possibility of liver regeneration with autologous stem cells from adipose tissue (ADSC is an intriguing alternative. Using a model of a toxic liver damage in Sprague Dawley rats, generated by repetitive intraperitoneal application of retrorsine and allyl alcohol, the ability of human ADSC to support the restoration of liver function was investigated. A two-thirds hepatectomy was performed, and human ADSC were injected into one remaining liver lobe in group 1 (n = 20. Injection of cell culture medium performed in group 2 (n = 20 served as control. Cyclosporine was applied to achieve immunotolerance. Blood samples were drawn weekly after surgery to determine liver-correlated blood values. Six and twelve weeks after surgery, animals were sacrificed and histological sections were analyzed. ADSC significantly raised postoperative albumin (P < 0.017, total protein (P < 0.031, glutamic oxaloacetic transaminase (P < 0.001, and lactate dehydrogenase (P < 0.04 levels compared to injection of cell culture medium alone. Transplanted cells could be found up to twelve weeks after surgery in histological sections. This study points towards ADSC being a promising alternative to hepatocyte or liver organ transplantation in patients with severe liver failure.

Alloxan-Induced Diabetes Causes Morphological and Ultrastructural Changes in Rat Liver that Resemble the Natural History of Chronic Fatty Liver Disease in Humans

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Amanda Natália Lucchesi

2015-01-01

Full Text Available Purpose. This study evaluated the long-term effects of alloxan-induced diabetes in rat liver. Methods. Thirty nondiabetic control rats (NC and 30 untreated diabetic (UD rats were divided into three subgroups sacrificed after 6, 14, or 26 weeks. Clinical and laboratory parameters were assessed. Fresh liver weight and its relationship with body weight were obtained, and liver tissue was analyzed. Results. UD rats showed sustained hyperglycemia, high glycosylated hemoglobin, and low plasma insulin. High serum levels of AST and ALT were observed in UD rats after 2 weeks, but only ALT remained elevated throughout the experiment. Fresh liver weight was equal between NC and UD rats, but the fresh liver weight/body weight ratio was significantly higher in UD rats after 14 and 26 weeks. UD rats showed liver morphological changes characterized by hepatic sinusoidal enlargement and micro- and macrovesicular hepatocyte fatty degeneration with progressive liver structure loss, steatohepatitis, and periportal fibrosis. Ultrastructural changes of hepatocytes, such as a decrease in the number of intracytoplasmic organelles and degeneration of mitochondria, rough endoplasmic reticulum, and nuclei, were also observed. Conclusion. Alloxan-induced diabetes triggered liver morphological and ultrastructural changes that closely resembled human disease, ranging from steatosis to steatohepatitis and liver fibrosis.

A computer-simulated liver phantom (virtual liver phantom) for multidetector computed tomography evaluation

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Funama, Yoshinori [Kumamoto University, Department of Radiological Sciences, School of Health Sciences, Kumamoto (Japan); Awai, Kazuo; Nakayama, Yoshiharu; Liu, Da; Yamashita, Yasuyuki [Kumamoto University, Department of Diagnostic Radiology, Graduate School of Medical Sciences, Kumamoto (Japan); Miyazaki, Osamu; Goto, Taiga [Hitachi Medical Corporation, Tokyo (Japan); Hori, Shinichi [Gate Tower Institute of Image Guided Therapy, Osaka (Japan)

2006-04-15

The purpose of study was to develop a computer-simulated liver phantom for hepatic CT studies. A computer-simulated liver phantom was mathematically constructed on a computer workstation. The computer-simulated phantom was calibrated using real CT images acquired by an actual four-detector CT. We added an inhomogeneous texture to the simulated liver by referring to CT images of chronically damaged human livers. The mean CT number of the simulated liver was 60 HU and we added numerous 5-to 10-mm structures with 60{+-}10 HU/mm. To mimic liver tumors we added nodules measuring 8, 10, and 12 mm in diameter with CT numbers of 60{+-}10, 60{+-}15, and 60{+-}20 HU. Five radiologists visually evaluated similarity of the texture of the computer-simulated liver phantom and a real human liver to confirm the appropriateness of the virtual liver images using a five-point scale. The total score was 44 in two radiologists, and 42, 41, and 39 in one radiologist each. They evaluated that the textures of virtual liver were comparable to those of human liver. Our computer-simulated liver phantom is a promising tool for the evaluation of the image quality and diagnostic performance of hepatic CT imaging. (orig.)

The participation of human hepatic P450 isoforms, flavin-containing monooxygenases and aldehyde oxidase in the biotransformation of the insecticide fenthion

International Nuclear Information System (INIS)

Leoni, Claudia; Buratti, Franca M.; Testai, Emanuela

2008-01-01

Although fenthion (FEN) is widely used as a broad spectrum insecticide on various crops in many countries, very scant data are available on its biotransformation in humans. In this study the in vitro human hepatic FEN biotransformation was characterized, identifying the relative contributions of cytochrome P450 (CYPs) and/or flavin-containing monooxygenase (FMOs) by using single c-DNA expressed human enzymes, human liver microsomes and cytosol and CYP/FMO-specific inhibitors. Two major metabolites, FEN-sulfoxide and FEN-oxon (FOX), are formed by some CYPs although at very different levels, depending on the relative CYP hepatic content. Formation of further oxidation products and the reduction of FEN-sulfoxide back to FEN by the cytosolic aldehyde oxidase enzyme were ruled out. Comparing intrinsic clearance values, FOX formation seemed to be favored and at low FEN concentrations CYP2B6 and 1A2 are mainly involved in its formation. At higher levels, a more widespread CYP involvement was evident, as in the case of FEN-sulfoxide, although a higher efficiency of CYP2C family was suggested. Hepatic FMOs were able to catalyze only sulfoxide formation, but at low FEN concentrations hepatic FEN sulfoxidation is predominantly P450-driven. Indeed, the contribution of the hepatic isoforms FMO 3 and FMO 5 was generally negligible, although at high FEN concentrations FMO's showed activities comparable to the active CYPs, accounting for up to 30% of total sulfoxidation. Recombinant FMO 1 showed the highest efficiency with respect to CYPs and the other FMOs, but it is not expressed in the adult human liver. This suggests that FMO 1 -catalysed sulfoxidation may represent the major extra-hepatic pathway of FEN biotransformation

Determination of cobalt in human liver by atomic absorption spectrometry with electrothermal atomization

International Nuclear Information System (INIS)

Caldas, E.D.; Gine-Rosias, M.F.; Dorea, J.G.

1991-01-01

A detailed study of the use of electrothermal atomic absorption spectrometry for the determination of cobalt in human liver is described. Comparisons of sample digestion using nitric acid or nitric acid plus perchloric acid, atomization procedures and the application of palladium and magnesium nitrate chemical modifiers were studied using NBS SRM 1577a Bovine Liver. The best results were achieved with sample decomposition in nitric acid, atomization from the tube wall and no chemical modifier. Cobalt was determined in 90 samples of livers from foetuses and deceased newborns using the standard addition method with an average recovery of 99.8%. (author). 30 refs.; 4 figs.; 2 tabs

Liver-related deaths in persons infected with the human immunodeficiency virus: the D:A:D study

NARCIS (Netherlands)

Weber, Rainer; Sabin, Caroline A.; Friis-Møller, Nina; Reiss, Peter; El-Sadr, Wafaa M.; Kirk, Ole; Dabis, Francois; Law, Matthew G.; Pradier, Christian; de Wit, Stephane; Akerlund, Börje; Calvo, Gonzalo; D'Arminio Monforte, Antonella; Rickenbach, Martin; Ledergerber, Bruno; Phillips, Andrew N.; Lundgren, Jens D.

2006-01-01

BACKGROUND: An increasing proportion of deaths among human immunodeficiency virus (HIV)-infected persons with access to combination antiretroviral therapy (cART) are due to complications of liver diseases. METHODS: We investigated the frequency of and risk factors associated with liver-related

Accumulation of free oligosaccharides and tissue damage in cytosolic α-mannosidase (Man2c1)-deficient mice.

Science.gov (United States)

Paciotti, Silvia; Persichetti, Emanuele; Klein, Katharina; Tasegian, Anna; Duvet, Sandrine; Hartmann, Dieter; Gieselmann, Volkmar; Beccari, Tommaso

2014-04-04

Free Man(7-9)GlcNAc2 is released during the biosynthesis pathway of N-linked glycans or from misfolded glycoproteins during the endoplasmic reticulum-associated degradation process and are reduced to Man5GlcNAc in the cytosol. In this form, free oligosaccharides can be transferred into the lysosomes to be degraded completely. α-Mannosidase (MAN2C1) is the enzyme responsible for the partial demannosylation occurring in the cytosol. It has been demonstrated that the inhibition of MAN2C1 expression induces accumulation of Man(8-9)GlcNAc oligosaccharides and apoptosis in vitro. We investigated the consequences caused by the lack of cytosolic α-mannosidase activity in vivo by the generation of Man2c1-deficient mice. Increased amounts of Man(8-9)GlcNAc oligosaccharides were recognized in all analyzed KO tissues. Histological analysis of the CNS revealed neuronal and glial degeneration with formation of multiple vacuoles in deep neocortical layers and major telencephalic white matter tracts. Enterocytes of the small intestine accumulate mannose-containing saccharides and glycogen particles in their apical cytoplasm as well as large clear vacuoles in retronuclear position. Liver tissue is characterized by groups of hepatocytes with increased content of mannosyl compounds and glycogen, some of them undergoing degeneration by hydropic swelling. In addition, lectin screening showed the presence of mannose-containing saccharides in the epithelium of proximal kidney tubules, whereas scattered glomeruli appeared collapsed or featured signs of fibrosis along Bowman's capsule. Except for a moderate enrichment of mannosyl compounds and glycogen, heterozygous mice were normal, arguing against possible toxic effects of truncated Man2c1. These findings confirm the key role played by Man2c1 in the catabolism of free oligosaccharides.

Paraoxonase activity in liver of Pacu, Piaractus mesopotamicus Holmberg (Characidae

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Vera Lucia F. Cunha Bastos

1998-01-01

Full Text Available Enzymatic production of p-nitrophenol in liver of Piaractus mesopotamicus Holmberg, 1887 was consistently assayed at pH 8.5 using 7.5 mM paraoxon as substrate. This pacu liver paraoxonase activity was activated by NaCl. Apparent values of K M were 2.42 x 10-3 M in the presence of 0.5 M NaCl and 8.99 x 10-3 M without NaCl. Apparent maximum velocity values calculated in the absence and presence of 0.5 M NaCl were 1.09 x 10-3 µmoles/min/mg of proteins and 1.29 x 100-3 µmoles/min/mg of proteins, respectively. These Vmax values are fifty-fold the value described for trout (Salmo trutta Linnaeus, 1758 liver paraoxonase. Paraoxonase activity of pacu liver homogenates was recovered as much in cytosolic as in particulate cellular subtractions, but the particulate subtractions showed higher specific activities. The data presented here indicate that hepatic hydrolysis of organophosphorous pesticides may not be an important detoxification process in pacu.

Sinusoidal obstruction syndrome (SOS): A light and electron microscopy study in human liver.

Science.gov (United States)

Vreuls, C P H; Driessen, A; Olde Damink, S W M; Koek, G H; Duimel, H; van den Broek, M A J; Dejong, C H C; Braet, F; Wisse, E

2016-05-01

Oxaliplatin is an important chemotherapeutic agent, used in the treatment of hepatic colorectal metastases, and known to induce the sinusoidal obstruction syndrome (SOS). Pathophysiological knowledge concerning SOS is based on a rat model. Therefore, the aim was to perform a comprehensive study of the features of human SOS, using both light microscopy (LM) and electron microscopy (EM). Included were all patients of whom wedge liver biopsies were collected during a partial hepatectomy for colorectal liver metastases, in a 4-year period. The wedge biopsy were perfusion fixated and processed for LM and EM. The SOS lesions were selected by LM and details were studied using EM. Material was available of 30 patients, of whom 28 patients received neo-adjuvant oxaliplatin. Eighteen (64%) of the 28 patients showed SOS lesions, based on microscopy. The lesions consisted of sinusoidal endothelial cell detachment from the space of Disse on EM. In the enlarged space of Disse a variable amount of erythrocytes were located. Sinusoidal endothelial cell detachment was present in human SOS, accompanied by enlargement of the space of Disse and erythrocytes in this area. These findings, originally described in a rat model, were now for the first time confirmed in human livers under clinically relevant settings. Copyright © 2016 Elsevier Ltd. All rights reserved.

CHARACTERIZATION OF HUMAN LIVER MICROSOMAL UDP-GLYCOSYLTRANSFERASES USING PHOTOAFFINITY ANALOGS

NARCIS (Netherlands)

LITTLE, JM; DRAKE, RR; VONK, R; KUIPERS, F; LESTER, R; RADOMINSKA, A

The photoaffinity analogs [beta-P-32]5-azido-UDP-glucuronic acid ([P-32]5N3UDP-GlcUA) and [beta-P-32]5-azido-UDP-glucose ([P-32]5N(3)UDP-Glc) were used to characterize UDP-glycosyl-transferases of microsomes prepared from human liver. Photoincorporation of both probes into proteins in the 50- to

From the Cover: Cell-replacement therapy for diabetes: Generating functional insulin-producing tissue from adult human liver cells

Science.gov (United States)

Sapir, Tamar; Shternhall, Keren; Meivar-Levy, Irit; Blumenfeld, Tamar; Cohen, Hamutal; Skutelsky, Ehud; Eventov-Friedman, Smadar; Barshack, Iris; Goldberg, Iris; Pri-Chen, Sarah; Ben-Dor, Lya; Polak-Charcon, Sylvie; Karasik, Avraham; Shimon, Ilan; Mor, Eytan; Ferber, Sarah

2005-05-01

Shortage in tissue availability from cadaver donors and the need for life-long immunosuppression severely restrict the large-scale application of cell-replacement therapy for diabetic patients. This study suggests the potential use of adult human liver as alternate tissue for autologous beta-cell-replacement therapy. By using pancreatic and duodenal homeobox gene 1 (PDX-1) and soluble factors, we induced a comprehensive developmental shift of adult human liver cells into functional insulin-producing cells. PDX-1-treated human liver cells express insulin, store it in defined granules, and secrete the hormone in a glucose-regulated manner. When transplanted under the renal capsule of diabetic, immunodeficient mice, the cells ameliorated hyperglycemia for prolonged periods of time. Inducing developmental redirection of adult liver offers the potential of a cell-replacement therapy for diabetics by allowing the patient to be the donor of his own insulin-producing tissue. pancreas | transdifferentiation

The Use of an Acellular Oxygen Carrier in a Human Liver Model of Normothermic Machine Perfusion.

Science.gov (United States)

Laing, Richard W; Bhogal, Ricky H; Wallace, Lorraine; Boteon, Yuri; Neil, Desley A H; Smith, Amanda; Stephenson, Barney T F; Schlegel, Andrea; Hübscher, Stefan G; Mirza, Darius F; Afford, Simon C; Mergental, Hynek

2017-11-01

Normothermic machine perfusion of the liver (NMP-L) is a novel technique that preserves liver grafts under near-physiological conditions while maintaining their normal metabolic activity. This process requires an adequate oxygen supply, typically delivered by packed red blood cells (RBC). We present the first experience using an acellular hemoglobin-based oxygen carrier (HBOC) Hemopure in a human model of NMP-L. Five discarded high-risk human livers were perfused with HBOC-based perfusion fluid and matched to 5 RBC-perfused livers. Perfusion parameters, oxygen extraction, metabolic activity, and histological features were compared during 6 hours of NMP-L. The cytotoxicity of Hemopure was also tested on human hepatic primary cell line cultures using an in vitro model of ischemia reperfusion injury. The vascular flow parameters and the perfusate lactate clearance were similar in both groups. The HBOC-perfused livers extracted more oxygen than those perfused with RBCs (O2 extraction ratio 13.75 vs 9.43 % ×10 per gram of tissue, P = 0.001). In vitro exposure to Hemopure did not alter intracellular levels of reactive oxygen species, and there was no increase in apoptosis or necrosis observed in any of the tested cell lines. Histological findings were comparable between groups. There was no evidence of histological damage caused by Hemopure. Hemopure can be used as an alternative oxygen carrier to packed red cells in NMP-L perfusion fluid.

Specific binding of [alpha-32P]GTP to cytosolic and membrane-bound proteins of human platelets correlates with the activation of phospholipase C

International Nuclear Information System (INIS)

Lapetina, E.G.; Reep, B.R.

1987-01-01

We have assessed the binding of [alpha- 32 P]GTP to platelet proteins from cytosolic and membrane fractions. Proteins were separated by NaDodSO 4 /PAGE and electrophoretically transferred to nitrocellulose. Incubation of the nitrocellulose blots with [alpha- 32 P]GTP indicated the presence of specific and distinct GTP-binding proteins in cytosol and membranes. Binding was prevented by 10-100 nM GTP and by 100 nM guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S]) or GDP; binding was unaffected by 1 nM-1 microM ATP. One main GTP-binding protein (29.5 kDa) was detected in the membrane fraction, while three others (29, 27, and 21 kDa) were detected in the soluble fraction. Two cytosolic GTP-binding proteins (29 and 27 kDa) were degraded by trypsin; another cytosolic protein (21 kDa) and the membrane-bound protein (29.5 kDa) were resistant to the action of trypsin. Treatment of intact platelets with trypsin or thrombin, followed by lysis and fractionation, did not affect the binding of [alpha- 32 P]GTP to the membrane-bound protein. GTP[gamma S] still stimulated phospholipase C in permeabilized platelets already preincubated with trypsin. This suggests that trypsin-resistant GTP-binding proteins might regulate phospholipase C stimulated by GTP[gamma S

Induction of phosphatidylcholine biosynthesis via CDPcholine pathway in lung and liver of rats following intratracheal administration of DDT and endosulfan

International Nuclear Information System (INIS)

Narayan, S.; Dani, H.M.; Misra, U.K.

1989-01-01

The induction of phosphatidylcholine (PC) biosynthesis via the CDPcholine pathway in lung and liver of rats has been shown following the intratracheal administration of 1,1,1-trichloro-2m2-bis(p-chlorophenyl) ethane (DDT) (5 mg/100 g body weight) and endosulfan (1 mg/100 g body weight) for 3 days. Controls received only the vehicle solution (groundnut oil, 0.1 m1/100 g body weight). The treatment of DDT and endosulfan significantly increased the PC contents and the incorporation of radioactive [methyl-3H]choline into PC of lung and liver microsomes. The incorporation of radioactive [methyl-14C]methionine into microsomal PC of lung and liver was not affected significantly by treatment with either of the insecticides. 1,4,5,6,7-hexachloro-5-norbornene-2,3-dimethano cyclic sulfite (endosulfan) administration significantly increased the activity of choline kinase and phosphocholine cytidylyltransferase (both cytosolic and microsomal) of lung, whereas DDT increased the activity of only latter. In liver, both DDT and endosulfan administration significantly increased the activity of choline kinase and phosphocholine cytidylyltransferase (both cytosolic and microsomal). However, the activity of phosphocholinetransferase was not affected in both lung and liver microsomes of rats treated with these insecticides. The PC precursor pool sizes, choline and phosphorylcholine, of lung and liver tissues were not altered by DDT and endosulfan treatments. The present results suggest that the increased level of PC and incorporation of radioactive [methyl-3H]choline into microsomal PC could be the result of increased activity of choline kinase and phosphocholine cytidylyltransferase of lung and liver of rats following intratracheal administration of DDT and endosulfan

Trapping of cis-2-butene-1,4-dial to measure furan metabolism in human liver microsomes by cytochrome P450 enzymes.

Science.gov (United States)

Gates, Leah A; Lu, Ding; Peterson, Lisa A

2012-03-01

Furan is a liver toxicant and carcinogen in rodents. It is classified as a possible human carcinogen, but the human health effects of furan exposure remain unknown. The oxidation of furan by cytochrome P450 (P450) enzymes is necessary for furan toxicity. The product of this reaction is the reactive α,β-unsaturated dialdehyde, cis-2-butene-1,4-dial (BDA). To determine whether human liver microsomes metabolize furan to BDA, a liquid chromatography/tandem mass spectrometry method was developed to detect and quantify BDA by trapping this reactive metabolite with N-acetyl-l-cysteine (NAC) and N-acetyl-l-lysine (NAL). Reaction of NAC and NAL with BDA generates N-acetyl-S-[1-(5-acetylamino-5-carboxypentyl)-1H-pyrrol-3-yl]-l-cysteine (NAC-BDA-NAL). Formation of NAC-BDA-NAL was quantified in 21 different human liver microsomal preparations. The levels of metabolism were comparable to that observed in F-344 rat and B6C3F1 mouse liver microsomes, two species known to be sensitive to furan-induced toxicity. Studies with recombinant human liver P450s indicated that CYP2E1 is the most active human liver furan oxidase. The activity of CYP2E1 as measured by p-nitrophenol hydroxylase activity was correlated to the extent of NAC-BDA-NAL formation in human liver microsomes. The formation of NAC-BDA-NAL was blocked by CYP2E1 inhibitors but not other P450 inhibitors. These results suggest that humans are capable of oxidizing furan to its toxic metabolite, BDA, at rates comparable to those of species sensitive to furan exposure. Therefore, humans may be susceptible to furan's toxic effects.

Preoperative assessment of congestive liver dysfunction using technetium-99m galactosyl human Serum albumin liver scintigraphy in patients with severe valvular heart disease

International Nuclear Information System (INIS)

Nishi, Hiroyuki; Matsumiya, Goro; Takano, Hiroshi; Ichikawa, Hajime; Miyagawa, Shigeru; Sawa, Yoshiki; Takahashi, Toshiki

2007-01-01

Severe valvular heart disease is often complicated by congestive liver dysfunction, which greatly compromises the operative results. We evaluated congestive liver dysfunction by a novel approach using technetium-99m galactosyl human serum albumin ( 99m Tc-GSA) with liver scintigraphy. Between 1998 and 2004, we performed scintigraphy accompanied by 99m Tc-GSA in 28 patients who had valvular heart disease with moderate-to-severe tricuspid regurgitation and who showed symptoms of right heart failure. Based on the results, we calculated a receptor index (LHL15) and an index of blood clearance (HH15) and assessed the correlation between these factors and postoperative liver dysfunction, defined as the maximum serum total bilirubin level (max T-bil) as >2.0 mg/dl. Nineteen patients, including four who died in hospital, had postoperative liver dysfunction. The level of HH15 was significantly higher and the level of cholinesterase was significantly lower (P 99m Tc-GSA is a clinically useful predictor of postoperative liver dysfunction in patients with severe valvular disease. (author)

PEDIATRIC LIVER TRANSPLANTATION WITH EX-SITU LIVER TRANSECTION AND THE APPLICATION OF THE HUMAN FIBRINOGEN AND THROMBIN SPONGE IN THE WOUND AREA.

Science.gov (United States)

Vicentine, Fernando Pompeu Piza; Gonzalez, Adriano Miziara; Azevedo, Ramiro Anthero de; Benini, Barbara Burza; Linhares, Marcelo Moura; Lopes-Filho, Gaspar de Jesus; Martins, Jose Luiz; Salzedas-Netto, Alcides Augusto

2016-01-01

Surgical strategy to increase the number of liver transplants in the pediatric population is the ex-situ liver transection (reduction or split). However, it is associated with complications such as hemorrhage and leaks. The human fibrinogen and thrombin sponge is useful for improving hemostasis in liver surgery. Compare pediatric liver transplants with ex-situ liver transection (reduction or split) with or without the human fibrinogen and thrombin sponge. Was performed a prospective analysis of 21 patients submitted to liver transplantation with ex-situ liver transection with the application of the human fibrinogen and thrombin sponge in the wound area (group A) and retrospective analysis of 59 patients without the sponge (group B). The characteristics of recipients and donors were similar. There were fewer reoperations due to bleeding in the wound area in group A (14.2%) compared to group B (41.7%, p=0.029). There was no difference in relation to the biliary leak (group A: 17.6%, group B: 5.1%, p=0.14). There was a lower number of reoperations due to bleeding of the wound area of ​​the hepatic graft when the human fibrinogen and thrombin sponge were used. Estratégia cirúrgica para aumentar o número de transplantes hepáticos na população pediátrica é a transecção hepática ex-situ (redução ou split). No entanto, ela está associada com complicações, tais como hemorragia e fístulas. A esponja de fibrinogênio e trombina humana é útil para melhorar a hemostasia nas operações hepáticas. Comparar transplantes hepáticos pediátricos com transecção hepática ex-situ (redução ou split) com ou sem a esponja de fibrinogênio e trombina humana. Foi realizada análise prospectiva de 21 pacientes submetidos ao transplante de fígado com transecção hepática ex-situ com a aplicação da esponja de fibrinogênio e trombina humana na área cruenta (grupo A) e análise retrospectiva de 59 pacientes sem a esponja (grupo B). As características dos

Liver X receptor alpha mediated genistein induction of human dehydroepiandrosterone sulfotransferase (hSULT2A1) in Hep G2 cells

Energy Technology Data Exchange (ETDEWEB)

Chen, Yue; Zhang, Shunfen [Department of Physiological Sciences, Center for Veterinary Health Sciences, Oklahoma State University, Stillwater, OK 74078 (United States); Zhou, Tianyan [Department of Pharmaceutics, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100083 (China); Huang, Chaoqun; McLaughlin, Alicia [Department of Physiological Sciences, Center for Veterinary Health Sciences, Oklahoma State University, Stillwater, OK 74078 (United States); Chen, Guangping, E-mail: guangping.chen@okstate.edu [Department of Physiological Sciences, Center for Veterinary Health Sciences, Oklahoma State University, Stillwater, OK 74078 (United States)

2013-04-15

Cytosolic sulfotransferases are one of the major families of phase II drug metabolizing enzymes. Sulfotransferase-catalyzed sulfonation regulates hormone activities, metabolizes drugs, detoxifies xenobiotics, and bioactivates carcinogens. Human dehydroepiandrosterone sulfotransferase (hSULT2A1) plays important biological roles by sulfating endogenous hydroxysteroids and exogenous xenobiotics. Genistein, mainly existing in soy food products, is a naturally occurring phytoestrogen with both chemopreventive and chemotherapeutic potential. Our previous studies have shown that genistein significantly induces hSULT2A1 in Hep G2 and Caco-2 cells. In this study, we investigated the roles of liver X receptor (LXRα) in the genistein induction of hSULT2A1. LXRs have been shown to induce expression of mouse Sult2a9 and hSULT2A1 gene. Our results demonstrate that LXRα mediates the genistein induction of hSULT2A1, supported by Western blot analysis results, hSULT2A1 promoter driven luciferase reporter gene assay results, and mRNA interference results. Chromatin immunoprecipitation (ChIP) assay results demonstrate that genistein increase the recruitment of hLXRα binding to the hSULT2A1 promoter. These results suggest that hLXRα plays an important role in the hSULT2A1 gene regulation. The biological functions of phytoestrogens may partially relate to their induction activity toward hydroxysteroid SULT. - Highlights: ► Liver X receptor α mediated genistein induction of hSULT2A1 in Hep G2 cells. ► LXRα and RXRα dimerization further activated this induction. ► Western blot results agreed well with luciferase reporter gene assay results. ► LXRs gene silencing significantly decreased hSULT2A1 expression. ► ChIP analysis suggested that genistein enhances hLXRα binding to the hSULT2A1 promoter.

Suppression of Human Liver Cancer Cell Migration and Invasion via the GABAA Receptor

International Nuclear Information System (INIS)

Chen, Zhi-ao; Bao, Mei-yan; Xu, Yong-fen; Zha, Ruo-peng; Shi, Hai-bing; Chen, Tao-yang; He, Xiang-huo

2012-01-01

To investigate the roles of the γ-aminobutyric acid (GABA) in the metastasis of hepatocellular carcinoma (HCC) and to explore the potential of a novel therapeutic approach for the treatment of HCC. The expression levels of GABA receptor subunit genes in various HCC cell lines and patients‘ tissues were detected by quantitative real-time polymerase chain reaction and Western blot analysis. Transwell cell migration and invasion assays were carried out for functional analysis. The effects of GABA on liver cancer cell cytoskeletal were determined by immunofluorescence staining. And the effects of GABA on HCC metastasis in nude mice were evaluated using an in vivo orthotopic model of liver cancer. The mRNA level of GABA receptor subunits varied between the primary hepatocellular carcinoma tissue and the adjacent non-tumor liver tissue. GABA inhibited human liver cancer cell migration and invasion via the ionotropic GABA A receptor as a result of the induction of liver cancer cell cytoskeletal reorganization. Pretreatment with GABA also significantly reduced intrahepatic liver metastasis and primary tumor formation in vivo. These findings introduce a potential and novel therapeutic approach for the treatment of cancer patients based on the modulation of the GABAergic system

Retargeting the Clostridium botulinum C2 toxin to the neuronal cytosol.

Science.gov (United States)

Pavlik, Benjamin J; Hruska, Elizabeth J; Van Cott, Kevin E; Blum, Paul H

2016-03-30

Many biological toxins are known to attack specific cell types, delivering their enzymatic payloads to the cytosol. This process can be manipulated by molecular engineering of chimeric toxins. Using toxins with naturally unlinked components as a starting point is advantageous because it allows for the development of payloads separately from the binding/translocation components. Here the Clostridium botulinum C2 binding/translocation domain was retargeted to neural cell populations by deleting its non-specific binding domain and replacing it with a C. botulinum neurotoxin binding domain. This fusion protein was used to deliver fluorescently labeled payloads to Neuro-2a cells. Intracellular delivery was quantified by flow cytometry and found to be dependent on artificial enrichment of cells with the polysialoganglioside receptor GT1b. Visualization by confocal microscopy showed a dissociation of payloads from the early endosome indicating translocation of the chimeric toxin. The natural Clostridium botulinum C2 toxin was then delivered to human glioblastoma A172 and synchronized HeLa cells. In the presence of the fusion protein, native cytosolic enzymatic activity of the enzyme was observed and found to be GT1b-dependent. This retargeted toxin may enable delivery of therapeutics to peripheral neurons and be of use in addressing experimental questions about neural physiology.

Fatty acid-binding protein in liver and small intestine of the preruminant calf

International Nuclear Information System (INIS)

Jenkins, K.J.

1986-01-01

Cytosol obtained from differential centrifugation of homogenates from liver and small intestine mucosa was incubated with 1-[ 14 C] oleic acid or 1-[ 14 C] palmitic acid and filtered through Sephadex G-75. Elution profiles for both tissues showed radioactivity in two main peaks, the first corresponding to binding of fatty acid to high molecular weight proteins and the second to a protein fraction with a molecular weight of approximately 12,000 daltons. The low molecular weight fraction had high fatty acid-binding activity, which was greater for oleic than palmitic acid. The findings demonstrate the presence of fatty acid-binding protein in liver and intestinal mucosa of the preruminant calf

Rapid production of human liver scaffolds for functional tissue engineering by high shear stress oscillation-decellularization

NARCIS (Netherlands)

Mazza, G. (Giuseppe); Al-Akkad, W. (Walid); Telese, A. (Andrea); Longato, L. (Lisa); Urbani, L. (Luca); Robinson, B. (Benjamin); Hall, A. (Andrew); Kong, K. (Kenny); Frenguelli, L. (Luca); Marrone, G. (Giusi); Willacy, O. (Oliver); Shaeri, M. (Mohsen); A.J. Burns (Alan); Malago, M. (Massimo); Gilbertson, J. (Janet); Rendell, N. (Nigel); Moore, K. (Kevin); Hughes, D. (David); Notingher, I. (Ioan); Jell, G. (Gavin); Del Rio Hernandez, A. (Armando); P. de Coppi (Paolo); Rombouts, K. (Krista); Pinzani, M. (Massimo)

2017-01-01

textabstractThe development of human liver scaffolds retaining their 3-dimensional structure and extra-cellular matrix (ECM) composition is essential for the advancement of liver tissue engineering. We report the design and validation of a new methodology for the rapid and accurate production of

An Experimental Study to Measure the Mechanical Properties of the Human Liver.

Science.gov (United States)

Karimi, Alireza; Shojaei, Ahmad

2018-01-01

Since the liver is one of the most important organs of the body that can be injured during trauma, that is, during accidents like car crashes, understanding its mechanical properties is of great interest. Experimental data is needed to address the mechanical properties of the liver to be used for a variety of applications, such as the numerical simulations for medical purposes, including the virtual reality simulators, trauma research, diagnosis objectives, as well as injury biomechanics. However, the data on the mechanical properties of the liver capsule is limited to the animal models or confined to the tensile/compressive loading under single direction. Therefore, this study was aimed at experimentally measuring the axial and transversal mechanical properties of the human liver capsule under both the tensile and compressive loadings. To do that, 20 human cadavers were autopsied and their liver capsules were excised and histologically analyzed to extract the mean angle of a large fibers population (bundle of the fine collagen fibers). Thereafter, the samples were cut and subjected to a series of axial and transversal tensile/compressive loadings. The results revealed the tensile elastic modulus of 12.16 ± 1.20 (mean ± SD) and 7.17 ± 0.85 kPa under the axial and transversal loadings respectively. Correspondingly, the compressive elastic modulus of 196.54 ± 13.15 and 112.41 ± 8.98 kPa were observed under the axial and transversal loadings respectively. The compressive axial and transversal maximum/failure stress of the capsule were 32.54 and 37.30 times higher than that of the tensile ones respectively. The capsule showed a stiffer behavior under the compressive load compared to the tensile one. In addition, the axial elastic modulus of the capsule was found to be higher than that of the transversal one. The findings of the current study have implications not only for understanding the mechanical properties of the human capsule tissue under tensile

A new view of the bacterial cytosol environment.

Directory of Open Access Journals (Sweden)

Benjamin P Cossins

2011-06-01

Full Text Available The cytosol is the major environment in all bacterial cells. The true physical and dynamical nature of the cytosol solution is not fully understood and here a modeling approach is applied. Using recent and detailed data on metabolite concentrations, we have created a molecular mechanical model of the prokaryotic cytosol environment of Escherichia coli, containing proteins, metabolites and monatomic ions. We use 200 ns molecular dynamics simulations to compute diffusion rates, the extent of contact between molecules and dielectric constants. Large metabolites spend ∼80% of their time in contact with other molecules while small metabolites vary with some only spending 20% of time in contact. Large non-covalently interacting metabolite structures mediated by hydrogen-bonds, ionic and π stacking interactions are common and often associate with proteins. Mg(2+ ions were prominent in NIMS and almost absent free in solution. Κ(+ is generally not involved in NIMSs and populates the solvent fairly uniformly, hence its important role as an osmolyte. In simulations containing ubiquitin, to represent a protein component, metabolite diffusion was reduced owing to long lasting protein-metabolite interactions. Hence, it is likely that with larger proteins metabolites would diffuse even more slowly. The dielectric constant of these simulations was found to differ from that of pure water only through a large contribution from ubiquitin as metabolite and monatomic ion effects cancel. These findings suggest regions of influence specific to particular proteins affecting metabolite diffusion and electrostatics. Also some proteins may have a higher propensity for associations with metabolites owing to their larger electrostatic fields. We hope that future studies may be able to accurately predict how binding interactions differ in the cytosol relative to dilute aqueous solution.

Volumetric Growth of the Liver in the Human Fetus: An Anatomical, Hydrostatic, and Statistical Study

Directory of Open Access Journals (Sweden)

Michał Szpinda

2015-01-01

Full Text Available Using anatomical, hydrostatic, and statistical methods, liver volumes were assessed in 69 human fetuses of both sexes aged 18–30 weeks. No sex differences were found. The median of liver volume achieved by hydrostatic measurements increased from 6.57 cm3 at 18–21 weeks through 14.36 cm3 at 22–25 weeks to 20.77 cm3 at 26–30 weeks, according to the following regression: y = −26.95 + 1.74 × age ± Z  × (−3.15 + 0.27 × age. The median of liver volume calculated indirectly according to the formula liver volume = 0.55 × liver length × liver transverse diameter × liver sagittal diameter increased from 12.41 cm3 at 18–21 weeks through 28.21 cm3 at 22–25 weeks to 49.69 cm3 at 26–30 weeks. There was a strong relationship (r=0.91, p<0.001 between the liver volumes achieved by hydrostatic (x and indirect (y methods, expressed by y = −0.05 + 2.16x  ± 7.26. The liver volume should be calculated as follows liver volume = 0.26 × liver length × liver transverse diameter × liver sagittal diameter. The age-specific liver volumes are of great relevance in the evaluation of the normal hepatic growth and the early diagnosis of fetal micro- and macrosomias.

Postneonatal Mortality and Liver Changes in Cloned Pigs Associated with Human Tumor Necrosis Factor Receptor I-Fc and Human Heme Oxygenase-1 Overexpression.

Science.gov (United States)

Kim, Geon A; Jin, Jun-Xue; Lee, Sanghoon; Taweechaipaisankul, Anukul; Oh, Hyun Ju; Hwang, Joing-Ik; Ahn, Curie; Saadeldin, Islam M; Lee, Byeong Chun

2017-01-01

Soluble human tumor necrosis factor (shTNFRI-Fc) and human heme oxygenase 1 (hHO-1) are key regulators for protection against oxidative and inflammatory injury for xenotransplantation. Somatic cells with more than 10 copy numbers of shTNFRI-Fc and hHO-1 were employed in somatic cell nuclear transfer to generate cloned pigs, thereby resulting in seven cloned piglets. However, produced piglets were all dead within 24 hours after birth. Obviously, postnatal death with liver apoptosis was reported in the higher copy number of shTNFRI-Fc and hHO-1 piglets. In liver, the transcript levels of ferritin heavy chain, light chain, transferrin, and inducible nitric oxide synthase were significantly highly expressed compared to those of lower copy number of shTNFRI-Fc and hHO-1 piglets ( P hHO-1 piglets ( P hHO-1 overexpression may apparently induce free iron in the liver and exert oxidative stress by enhancing reactive oxygen species production and block normal postneonatal liver metabolism.

Extracellular Vesicles from Human Liver Stem Cells Reduce Injury in an Ex Vivo Normothermic Hypoxic Rat Liver Perfusion Model.

Science.gov (United States)

Rigo, Federica; De Stefano, Nicola; Navarro-Tableros, Victor; David, Ezio; Rizza, Giorgia; Catalano, Giorgia; Gilbo, Nicholas; Maione, Francesca; Gonella, Federica; Roggio, Dorotea; Martini, Silvia; Patrono, Damiano; Salizzoni, Mauro; Camussi, Giovanni; Romagnoli, Renato

2018-05-01

The gold standard for organ preservation before transplantation is static cold storage, which is unable to fully protect suboptimal livers from ischemia/reperfusion injury. An emerging alternative is normothermic machine perfusion (NMP), which permits organ reconditioning. Here, we aimed to explore the feasibility of a pharmacological intervention on isolated rat livers by using a combination of NMP and human liver stem cells-derived extracellular vesicles (HLSC-EV). We established an ex vivo murine model of NMP capable to maintain liver function despite an ongoing hypoxic injury induced by hemodilution. Livers were perfused for 4 hours without (control group, n = 10) or with HLSC-EV (treated group, n = 9). Bile production was quantified; perfusate samples were collected hourly to measure metabolic (pH, pO2, pCO2) and cytolysis parameters (AST, alanine aminotransferase, lactate dehydrogenase). At the end of perfusion, we assessed HLSC-EV engraftment by immunofluorescence, tissue injury by histology, apoptosis by terminal deoxynucleotidyl transferase dUTP nick-end labeling assay, tissue hypoxia-inducible factor 1-α, and transforming growth factor-beta 1 RNA expression by quantitative reverse transcription-polymerase chain reaction. During hypoxic NMP, livers were able to maintain homeostasis and produce bile. In the treated group, AST (P = 0.018) and lactate dehydrogenase (P = 0.032) levels were significantly lower than those of the control group at 3 hours of perfusion, and AST levels persisted lower at 4 hours (P = 0.003). By the end of NMP, HLSC-EV had been uptaken by hepatocytes, and EV treatment significantly reduced histological damage (P = 0.030), apoptosis (P = 0.049), and RNA overexpression of hypoxia-inducible factor 1-α (P < 0.0001) and transforming growth factor-beta 1 (P = 0.014). HLSC-EV treatment, even in a short-duration model, was feasible and effectively reduced liver injury during hypoxic NMP.

Mice with chimeric livers are an improved model for human lipoprotein metabolism.

Science.gov (United States)

Ellis, Ewa C S; Naugler, Willscott Edward; Nauglers, Scott; Parini, Paolo; Mörk, Lisa-Mari; Jorns, Carl; Zemack, Helen; Sandblom, Anita Lövgren; Björkhem, Ingemar; Ericzon, Bo-Göran; Wilson, Elizabeth M; Strom, Stephen C; Grompe, Markus

2013-01-01

Rodents are poor model for human hyperlipidemias because total cholesterol and low density lipoprotein levels are very low on a normal diet. Lipoprotein metabolism is primarily regulated by hepatocytes and we therefore assessed whether chimeric mice extensively repopulated with human cells can model human lipid and bile acid metabolism. FRG [ F ah(-/-) R ag2(-/-)Il2r g (-/-)]) mice were repopulated with primary human hepatocytes. Serum lipoprotein lipid composition and distribution (VLDL, LDL, and HDL) was analyzed by size exclusion chromatography. Bile was analyzed by LC-MS or by GC-MS. RNA expression levels were measured by quantitative RT-PCR. Chimeric mice displayed increased LDL and VLDL fractions and a lower HDL fraction compared to wild type, thus significantly shifting the ratio of LDL/HDL towards a human profile. Bile acid analysis revealed a human-like pattern with high amounts of cholic acid and deoxycholic acid (DCA). Control mice had only taurine-conjugated bile acids as expcted, but highly repopulated mice had glycine-conjugated cholic acid as found in human bile. RNA levels of human genes involved in bile acid synthesis including CYP7A1, and CYP27A1 were significantly upregulated as compared to human control liver. However, administration of recombinant hFGF19 restored human CYP7A1 levels to normal. Humanized-liver mice showed a typical human lipoprotein profile with LDL as the predominant lipoprotein fraction even on a normal diet. The bile acid profile confirmed presence of an intact enterohepatic circulation. Although bile acid synthesis was deregulated in this model, this could be fully normalized by FGF19 administration. Taken together these data indicate that chimeric FRG-mice are a useful new model for human lipoprotein and bile-acid metabolism.

Metabolism of ethylbenzene by human liver microsomes and recombinant human cytochrome P450s (CYP).

Science.gov (United States)

Sams, Craig; Loizou, George D; Cocker, John; Lennard, Martin S

2004-03-07

The enzyme kinetics of the initial hydroxylation of ethylbenzene to form 1-phenylethanol were determined in human liver microsomes. The individual cytochrome P450 (CYP) forms catalysing this reaction were identified using selective inhibitors and recombinant preparations of hepatic CYPs. Production of 1-phenylethanol in hepatic microsomes exhibited biphasic kinetics with a high affinity, low Km, component (mean Km = 8 microM; V(max) = 689 pmol/min/mg protein; n = 6 livers) and a low affinity, high Km, component (Km = 391 microM; V(max) = 3039 pmol/min/mg protein; n = 6). The high-affinity component was inhibited 79%-95% (mean 86%) by diethyldithiocarbamate, and recombinant CYP2E1 was shown to metabolise ethylbenzene with low Km (35 microM), but also low (max) (7 pmol/min/pmol P450), indicating that this isoform catalysed the high-affinity component. Recombinant CYP1A2 and CYP2B6 exhibited high V(max) (88 and 71 pmol/min/pmol P450, respectively) and high Km (502 and 219 microM, respectively), suggesting their involvement in catalysing the low-affinity component. This study has demonstrated that CYP2E1 is the major enzyme responsible for high-affinity side chain hydroxylation of ethylbenzene in human liver microsomes. Activity of this enzyme in the population is highly variable due to induction or inhibition by physiological factors, chemicals in the diet or some pharmaceuticals. This variability can be incorporated into the risk assessment process to improve the setting of occupational exposure limits and guidance values for biological monitoring.

Volumetric Growth of the Liver in the Human Fetus: An Anatomical, Hydrostatic, and Statistical Study

Science.gov (United States)

Szpinda, Michał; Paruszewska-Achtel, Monika; Mila-Kierzenkowska, Celestyna; Elminowska-Wenda, Gabriela; Dombek, Małgorzata; Szpinda, Anna; Badura, Mateusz

2015-01-01

Using anatomical, hydrostatic, and statistical methods, liver volumes were assessed in 69 human fetuses of both sexes aged 18–30 weeks. No sex differences were found. The median of liver volume achieved by hydrostatic measurements increased from 6.57 cm3 at 18–21 weeks through 14.36 cm3 at 22–25 weeks to 20.77 cm3 at 26–30 weeks, according to the following regression: y = −26.95 + 1.74 × age ± Z  × (−3.15 + 0.27 × age). The median of liver volume calculated indirectly according to the formula liver volume = 0.55 × liver length × liver transverse diameter × liver sagittal diameter increased from 12.41 cm3 at 18–21 weeks through 28.21 cm3 at 22–25 weeks to 49.69 cm3 at 26–30 weeks. There was a strong relationship (r = 0.91, p hydrostatic (x) and indirect (y) methods, expressed by y = −0.05 + 2.16x  ± 7.26. The liver volume should be calculated as follows liver volume = 0.26 × liver length × liver transverse diameter × liver sagittal diameter. The age-specific liver volumes are of great relevance in the evaluation of the normal hepatic growth and the early diagnosis of fetal micro- and macrosomias. PMID:26413551

Anti-soluble liver antigen/liver-pancreas (SLA/LP) antibodies in pediatric patients with autoimmune hepatitis.

Science.gov (United States)

Vitozzi, Susana; Djilali-Saiah, Idriss; Lapierre, Pascal; Alvarez, Fernando

2002-12-01

Antibodies against soluble liver antigen/liver-pancreas (SLA/LP) have been associated with severe autoimmune hepatitis (AIH) and poor outcome, but most of these reports have focused on adult patients. The aim of this study was to assess the prevalence and clinical significance of anti-SLA/LP antibodies in a pediatric population with AIH. We developed a quantitative enzyme-linked immunoassay (ELISA), a Western blot (WB) and an immunoprecipitation assay (IPA) based on recombinant cDNA from activated Jurkat cells. The specificity of these tests was validated by testing 200 serum samples from healthy subjects, and from patients with liver and non-liver diseases. Anti-SLA/LP antibodies were found in patients with type 1 and type 2 AIH. The prevalence of these antibodies in patients with type 1 AIH was: 42% when tested by ELISA, 15% by WB and 50% by IPA. In patients with type 2 AIH, the prevalence rates were 42% by ELISA, 18% by WB and 44% by IPA. The mean titer values for anti-SLA/LP antibodies was significantly higher in type 2 AIH (1:1,300 +/- 339) than in type 1 AIH (1:600 +/- 71; p LKM1) and anti-liver cytosol type 1 (LC1) antibodies in sera. The presence of anti-SLA/LP showed a significant female preponderance in type 1 and 2 AIH patients (p = 0.0003 and p = 0.003, respectively), and was significantly correlated with a lower age at diagnosis (p = 0.05) in type 1 AIH patients. In conclusion, anti-SLA/LP antibodies in pediatric patients are associated with both type 1 and 2 AIH.

Diagnosis and Management of Paediatric Autoimmune Liver Disease: ESPGHAN Hepatology Committee Position Statement.

Science.gov (United States)

Mieli-Vergani, Giorgina; Vergani, Diego; Baumann, Ulrich; Czubkowski, Piotr; Debray, Dominique; Dezsofi, Antal; Fischler, Björn; Gupte, Girish; Hierro, Loreto; Indolfi, Giuseppe; Jahnel, Jörg; Smets, Françoise; Verkade, Henkjan J; Hadzic, Nedim

2017-11-03

Paediatric autoimmune liver disease is characterised by inflammatory liver histology, circulating autoantibodies and increased levels of IgG, in the absence of a known etiology. Three conditions have a likely autoimmune pathogenesis: autoimmune hepatitis (AIH), autoimmune sclerosing cholangitis (ASC), and de novo AIH after liver transplantation. Two types of paediatric AIH are recognized according to seropositivity for smooth muscle and/or antinuclear antibody (SMA/ANA, AIH-1) or liver kidney microsomal type 1 and/or anti-liver cytosol type 1 antibodies (anti-LKM-1/anti-LC-1; AIH-2).Pertinent issues addressing the diagnosis, treatment and long term follow up were formulated by a core group of ESPGHAN members. They have commissioned the first authors with execution of this project. Initially, they have performed a systematic literature search on MEDLINE, ResearchGate and Mendeley databases over the last 30 years and produced a document focusing on prospective and retrospective studies in children. The ESPGHAN core group and ESPGHAN Hepatology Committee members voted on each recommendation, using a formal voting technique.

Limits to anaerobic energy and cytosolic concentration in the living cell

Science.gov (United States)

Paglietti, A.

2015-11-01

For many physical systems at any given temperature, the set of all states where the system's free energy reaches its largest value can be determined from the system's constitutive equations of internal energy and entropy, once a state of that set is known. Such an approach is fraught with complications when applied to a living cell, because the cell's cytosol contains thousands of solutes, and thus thousands of state variables, which makes determination of its state impractical. We show here that, when looking for the maximum energy that the cytosol can store and release, detailed information on cytosol composition is redundant. Compatibility with cell's life requires that a single variable that represents the overall concentration of cytosol solutes must fall between defined limits, which can be determined by dehydrating and overhydrating the cell to its maximum capacity. The same limits are shown to determine, in particular, the maximum amount of free energy that a cell can supply in fast anaerobic processes, starting from any given initial state. For a typical skeletal muscle in normal physiological conditions this energy, i.e., the maximum anaerobic capacity to do work, is calculated to be about 960 J per kg of muscular mass. Such energy decreases as the overall concentration of solutes in the cytosol is increased. Similar results apply to any kind of cell. They provide an essential tool to understand and control the macroscopic response of single cells and multicellular cellular tissues alike. The applications include sport physiology, cell aging, disease produced cell damage, drug absorption capacity, to mention the most obvious ones.

Effect of C-terminal of human cytosolic thymidine kinase (TK1) on in vitro stability and enzymatic properties

DEFF Research Database (Denmark)

Munch-Petersen, Birgitte; Munch-Petersen, Sune; Berenstein, Dvora

2006-01-01

Thymidine kinase (TK1) is a key enzyme in the salvage pathway of nucleotide metabolism and catalyzes the first rate-limiting step in the synthesis of dTTP, transfer of a gamma-phosphate group from a nucleoside triphosphate to the 5′-hydroxyl group of thymidine, thus forming dTMP. TK1 is cytosolic...

Developmental changes in cytosolic coupling between epidermis cells as visualized by photoactivation of fluorescein

DEFF Research Database (Denmark)

Liu, Xiangdong; Martens, Helle; Schulz, Alexander

Developmental changes in cytosolic coupling between epidermis cells as visualized by photoactivation of fluorescein.......Developmental changes in cytosolic coupling between epidermis cells as visualized by photoactivation of fluorescein....

Laser Capture and Deep Sequencing Reveals the Transcriptomic Programmes Regulating the Onset of Pancreas and Liver Differentiation in Human Embryos

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Rachel E. Jennings

2017-11-01

Full Text Available To interrogate the alternative fates of pancreas and liver in the earliest stages of human organogenesis, we developed laser capture, RNA amplification, and computational analysis of deep sequencing. Pancreas-enriched gene expression was less conserved between human and mouse than for liver. The dorsal pancreatic bud was enriched for components of Notch, Wnt, BMP, and FGF signaling, almost all genes known to cause pancreatic agenesis or hypoplasia, and over 30 unexplored transcription factors. SOX9 and RORA were imputed as key regulators in pancreas compared with EP300, HNF4A, and FOXA family members in liver. Analyses implied that current in vitro human stem cell differentiation follows a dorsal rather than a ventral pancreatic program and pointed to additional factors for hepatic differentiation. In summary, we provide the transcriptional codes regulating the start of human liver and pancreas development to facilitate stem cell research and clinical interpretation without inter-species extrapolation.

Developmental anatomy of the liver from computerized three-dimensional reconstructions of four human embryos (from Carnegie stage 14 to 23).

Science.gov (United States)

Lhuaire, Martin; Tonnelet, Romain; Renard, Yohann; Piardi, Tullio; Sommacale, Daniele; Duparc, Fabrice; Braun, Marc; Labrousse, Marc

2015-07-01

Some aspects of human embryogenesis and organogenesis remain unclear, especially concerning the development of the liver and its vasculature. The purpose of this study was to investigate, from a descriptive standpoint, the evolutionary morphogenesis of the human liver and its vasculature by computerized three-dimensional reconstructions of human embryos. Serial histological sections of four human embryos at successive stages of development belonging to three prestigious French historical collections were digitized and reconstructed in 3D using software commonly used in medical radiology. Manual segmentation of the hepatic anatomical regions of interest was performed section by section. In this study, human liver organogenesis was examined at Carnegie stages 14, 18, 21 and 23. Using a descriptive and an analytical method, we showed that these stages correspond to the implementation of the large hepatic vascular patterns (the portal system, the hepatic artery and the hepatic venous system) and the biliary system. To our knowledge, our work is the first descriptive morphological study using 3D computerized reconstructions from serial histological sections of the embryonic development of the human liver between Carnegie stages 14 and 23. Copyright © 2015 Elsevier GmbH. All rights reserved.

All-In-One: Advanced preparation of Human Parenchymal and Non-Parenchymal Liver Cells.

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Melanie Werner

Full Text Available Liver cells are key players in innate immunity. Thus, studying primary isolated liver cells is necessary for determining their role in liver physiology and pathophysiology. In particular, the quantity and quality of isolated cells are crucial to their function. Our aim was to isolate a large quantity of high-quality human parenchymal and non-parenchymal cells from a single liver specimen.Hepatocytes, Kupffer cells, liver sinusoidal endothelial cells, and stellate cells were isolated from liver tissues by collagenase perfusion in combination with low-speed centrifugation, density gradient centrifugation, and magnetic-activated cell sorting. The purity and functionality of cultured cell populations were controlled by determining their morphology, discriminative cell marker expression, and functional activity.Cell preparation yielded the following cell counts per gram of liver tissue: 2.0 ± 0.4 × 10(7 hepatocytes, 1.8 ± 0.5 × 10(6 Kupffer cells, 4.3 ± 1.9 × 10(5 liver sinusoidal endothelial cells, and 3.2 ± 0.5 × 10(5 stellate cells. Hepatocytes were identified by albumin (95.5 ± 1.7% and exhibited time-dependent activity of cytochrome P450 enzymes. Kupffer cells expressed CD68 (94.5 ± 1.2% and exhibited phagocytic activity, as determined with 1 μm latex beads. Endothelial cells were CD146(+ (97.8 ± 1.1% and exhibited efficient uptake of acetylated low-density lipoprotein. Hepatic stellate cells were identified by the expression of α-smooth muscle actin (97.1 ± 1.5%. These cells further exhibited retinol (vitamin A-mediated autofluorescence.Our isolation procedure for primary parenchymal and non-parenchymal liver cells resulted in cell populations of high purity and quality, with retained physiological functionality in vitro. Thus, this system may provide a valuable tool for determining liver function and disease.

Differentiation of human-induced pluripotent stem cell under flow conditions to mature hepatocytes for liver tissue engineering

DEFF Research Database (Denmark)

Starokozhko, Viktoriia; Hemmingsen, Mette; Larsen, Layla

2018-01-01

Hepatic differentiation of human-induced pluripotent stem cells (hiPSCs) under flow conditions in a 3D scaffold is expected to be a major step forward for construction of bioartificial livers. The aims of this study were to induce hepatic differentiation of hiPSCs under perfusion conditions...... and to perform functional comparisons with fresh human precision-cut liver slices (hPCLS), an excellent benchmark for the human liver in vivo. The majority of the mRNA expression of CYP isoenzymes and transporters and the tested CYP activities, Phase II metabolism, and albumin, urea, and bile acid synthesis...... in the hiPSC-derived cells reached values that overlap those of hPCLS, which indicates a higher degree of hepatic differentiation than observed until now. Differentiation under flow compared with static conditions had a strong inducing effect on Phase II metabolism and suppressed AFP expression but resulted...

Cytosolic NADP+-dependent isocitrate dehydrogenase plays a key role in lipid metabolism.

Science.gov (United States)

Koh, Ho-Jin; Lee, Su-Min; Son, Byung-Gap; Lee, Soh-Hyun; Ryoo, Zae Young; Chang, Kyu-Tae; Park, Jeen-Woo; Park, Dong-Chan; Song, Byoung J; Veech, Richard L; Song, Hebok; Huh, Tae-Lin

2004-09-17

NADPH is an essential cofactor for many enzymatic reactions including glutathione metabolism and fat and cholesterol biosynthesis. We have reported recently an important role for mitochondrial NADP(+)-dependent isocitrate dehydrogenase in cellular defense against oxidative damage by providing NADPH needed for the regeneration of reduced glutathione. However, the role of cytosolic NADP(+)-dependent isocitrate dehydrogenase (IDPc) is still unclear. We report here for the first time that IDPc plays a critical role in fat and cholesterol biosynthesis. During differentiation of 3T3-L1 adipocytes, both IDPc enzyme activity and its protein content were increased in parallel in a time-dependent manner. Increased expression of IDPc by stable transfection of IDPc cDNA positively correlated with adipogenesis of 3T3-L1 cells, whereas decreased IDPc expression by an antisense IDPc vector retarded adipogenesis. Furthermore, transgenic mice with overexpressed IDPc exhibited fatty liver, hyperlipidemia, and obesity. In the epididymal fat pads of the transgenic mice, the expressions of adipocyte-specific genes including peroxisome proliferator-activated receptor gamma were markedly elevated. The hepatic and epididymal fat pad contents of acetyl-CoA and malonyl-CoA in the transgenic mice were significantly lower, whereas the total triglyceride and cholesterol contents were markedly higher in the liver and serum of transgenic mice compared with those measured in wild type mice, suggesting that the consumption rate of those lipogenic precursors needed for fat biosynthesis must be increased by elevated IDPc activity. Taken together, our findings strongly indicate that IDPc would be a major NADPH producer required for fat and cholesterol synthesis.

In vitro Inhibitory Effects of Andrographis paniculata, Gynura procumbens, Ficus deltoidea, and Curcuma xanthorrhiza Extracts and Constituents on Human Liver Glucuronidation Activity.

Science.gov (United States)

Husni, Zulhilmi; Ismail, Sabariah; Zulkiffli, Mohd Halimhilmi; Afandi, Atiqah; Haron, Munirah

2017-07-01

Andrographis paniculata , Gynura procumbens , Ficus deltoidea and Curcuma xanthorrhiza are commonly consumed as herbal medicines. However their effects on human liver glucuronidation activity are not yet evaluated. In this study, we evaluate the inhibitory Effects of Andrographis paniculata, Gynura procumbens, Ficus deltoidea and Curcuma xanthorrhiza extracts and their constituents on human liver glucuronidation activity. Herbal extracts (aqueous, methanolic and ethanolic extracts) and their constituents were incubated with human liver microsomes with the addition of UDPGA to initiate the reaction. Working concentrations of herbal extracts and their constituents ranged from 10 μg/mL to 1000 μg/mL and 10 μM to 300 μM respectively. IC50 was determined by monitoring the decrement of glucuronidation activity with the increment of herbal extracts or phytochemical constituent's concentrations. All herbal extracts inhibited human liver glucuronidation activity in range of 34.69 μg/mL to 398.10 μg/mL whereas for the constituents, only xanthorrhizol and curcumin (constituents of Curcuma xanthorrhiza ) inhibited human liver glucuronidation activity with IC50 of 538.50 and 32.26 μM respectively. In the present study, we have proved the capabilities of Andrographis paniculata , Gynura procumbens , Ficus deltoidea and Curcuma xanthorrhiza to interfere with in vitro glucuronidation process in human liver microsomes. This study documented the capabilities of Andrographis paniculata , Gynura procumbens , Ficus deltoidea and Curcuma xanthorrhiza to inhibit human liver glucuronidation activity which may affect the metabolism of therapeutic drugs or hazardous toxicants that follow the same glucuronidation pathway. Abbreviations used: UGT: Uridine 5'-diphospho-glucuronosyltransferase; 4-MU: 4-methylumbelliferone; IC50: Half Maximal Inhibitory Concentration; Km: Michaelis constant; Vmax: Maximum velocity.

Ammodytoxin, a neurotoxic secreted phospholipase A2, can act in the cytosol of the nerve cell

International Nuclear Information System (INIS)

Petrovic, Uros; Sribar, Jernej; Paris, Alenka; Rupnik, Marjan; Krzan, Mojca; Vardjan, Nina; Gubensek, Franc; Zorec, Robert; Krizaj, Igor

2004-01-01

Recent identification of intracellular proteins that bind ammodytoxin (calmodulin, 14-3-3 proteins, and R25) suggests that this snake venom presynaptically active phospholipase A 2 acts intracellularly. As these ammodytoxin acceptors are cytosolic and mitochondrial proteins, the toxin should be able to enter the cytosol of a target cell and remain stable there to interact with them. Using laser scanning confocal microscopy we show here that Alexa-labelled ammodytoxin entered the cytoplasm of the rat hippocampal neuron and subsequently also its nucleus. The transport of proteins into the nucleus proceeds via the cytosol of a cell, therefore, ammodytoxin passed the cytosol of the neuron on its way to the nucleus. Although it is not yet clear how ammodytoxin is translocated into the cytosol of the neuron, our results demonstrate that its stability in the cytosol is not in question, providing the evidence that the toxin can act in this cellular compartment

Radiation-induced liver damage

International Nuclear Information System (INIS)

Marcial, V.A.; Santiago-Delpin, E.A.; Lanaro, A.E.; Castro-Vita, H.; Arroyo, G.; Moscol, J.A.; Gomez, C.; Velazquez, J.; Prado, K.

1977-01-01

Due to the recent increase in the use of radiation therapy in the treatment of cancer with or without chemotherapy, the risk of liver radiation damage has become a significant concern for the radiotherapist when the treated tumour is located in the upper abdomen or lower thorax. Clinically evident radiation liver damage may result in significant mortality, but at times patients recover without sequelae. The dose of 3000 rads in 3 weeks to the entire liver with 5 fractions per week of 200 rads each, seems to be tolerated well clinically by adult humans. Lower doses may lead to damage when used in children, when chemotherapy is added, as in recent hepatectomy cases, and in the presence of pre-existent liver damage. Reduced fractionation may lead to increased damage. Increased fractionation, limitation of the dose delivered to the entire liver, and restriction of the high dose irradiation volume may afford protection. With the aim of studying the problems of hepatic radiation injury in humans, a project of liver irradiation in the dog is being conducted. Mongrel dogs are being conditioned, submitted to pre-irradiation studies (haemogram, blood chemistry, liver scan and biopsy), irradiated under conditions resembling human cancer therapy, and submitted to post-irradiation evaluation of the liver. Twenty-two dogs have been entered in the study but only four qualify for the evaluation of all the study parameters. It has been found that dogs are susceptible to liver irradiation damage similar to humans. The initial mortality has been high mainly due to non-radiation factors which are being kept under control at the present phase of the study. After the initial experiences, the study will involve variations in total dose and fractionation, and the addition of anticoagulant therapy for possible prevention of radiation liver injury. (author)

Effect of ultrasound frequency on the Nakagami statistics of human liver tissues.

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Po-Hsiang Tsui

Full Text Available The analysis of the backscattered statistics using the Nakagami parameter is an emerging ultrasound technique for assessing hepatic steatosis and fibrosis. Previous studies indicated that the echo amplitude distribution of a normal liver follows the Rayleigh distribution (the Nakagami parameter m is close to 1. However, using different frequencies may change the backscattered statistics of normal livers. This study explored the frequency dependence of the backscattered statistics in human livers and then discussed the sources of ultrasound scattering in the liver. A total of 30 healthy participants were enrolled to undergo a standard care ultrasound examination on the liver, which is a natural model containing diffuse and coherent scatterers. The liver of each volunteer was scanned from the right intercostal view to obtain image raw data at different central frequencies ranging from 2 to 3.5 MHz. Phantoms with diffuse scatterers only were also made to perform ultrasound scanning using the same protocol for comparisons with clinical data. The Nakagami parameter-frequency correlation was evaluated using Pearson correlation analysis. The median and interquartile range of the Nakagami parameter obtained from livers was 1.00 (0.98-1.05 for 2 MHz, 0.93 (0.89-0.98 for 2.3 MHz, 0.87 (0.84-0.92 for 2.5 MHz, 0.82 (0.77-0.88 for 3.3 MHz, and 0.81 (0.76-0.88 for 3.5 MHz. The Nakagami parameter decreased with the increasing central frequency (r = -0.67, p < 0.0001. However, the effect of ultrasound frequency on the statistical distribution of the backscattered envelopes was not found in the phantom results (r = -0.147, p = 0.0727. The current results demonstrated that the backscattered statistics of normal livers is frequency-dependent. Moreover, the coherent scatterers may be the primary factor to dominate the frequency dependence of the backscattered statistics in a liver.

Prenatal iron deficiency causes sex-dependent mitochondrial dysfunction and oxidative stress in fetal rat kidneys and liver.

Science.gov (United States)

Woodman, Andrew G; Mah, Richard; Keddie, Danae; Noble, Ronan M N; Panahi, Sareh; Gragasin, Ferrante S; Lemieux, Hélène; Bourque, Stephane L

2018-06-01

Prenatal iron deficiency alters fetal developmental trajectories, which results in persistent changes in organ function. Here, we studied the effects of prenatal iron deficiency on fetal kidney and liver mitochondrial function. Pregnant Sprague-Dawley rats were fed partially or fully iron-restricted diets to induce a state of moderate or severe iron deficiency alongside iron-replete control rats. We assessed mitochondrial function via high-resolution respirometry and reactive oxygen species generation via fluorescence microscopy on gestational d 21. Hemoglobin levels were reduced in dams in the moderate (-31%) and severe groups (-54%) compared with controls, which was accompanied by 55% reductions in fetal hemoglobin levels in both moderate and severe groups versus controls. Male iron-deficient kidneys exhibited globally reduced mitochondrial content and respiration, as well as increased cytosolic superoxide and decreased NO. Female iron-deficient kidneys exhibited complex II down-regulation and increased mitochondrial oxidative stress. Male iron-deficient livers exhibited reduced complex IV respiration and increased cytosolic superoxide, whereas female liver tissues exhibited no alteration in oxidant levels or mitochondrial function. These findings indicate that prenatal iron deficiency causes changes in mitochondrial content and function as well as oxidant status in a sex- and organ-dependent manner, which may be an important mechanism that underlies the programming of cardiovascular disease.-Woodman, A. G., Mah, R., Keddie, D., Noble, R. M. N., Panahi, S., Gragasin, F. S., Lemieux, H., Bourque, S. L. Prenatal iron deficiency causes sex-dependent mitochondrial dysfunction and oxidative stress in fetal rat kidneys and liver.

Organizers and activators: Cytosolic Nox proteins impacting on vascular function.

Science.gov (United States)

Schröder, Katrin; Weissmann, Norbert; Brandes, Ralf P

2017-08-01

NADPH oxidases of the Nox family are important enzymatic sources of reactive oxygen species (ROS) in the cardiovascular system. Of the 7 members of the Nox family, at least three depend for their activation on specific cytosolic proteins. These are p47phox and its homologue NoxO1 and p67phox and its homologue NoxA1. Also the Rho-GTPase Rac is important but as this protein has many additional functions, it will not be covered here. The Nox1 enzyme is preferentially activated by the combination of NoxO1 with NoxA1, whereas Nox2 gains highest activity with p47phox together with p67phox. As p47phox, different to NoxO1 contains an auto inhibitory region it has to be phosphorylated prior to complex formation. In the cardio-vascular system, all cytosolic Nox proteins are expressed but the evidence for their contribution to ROS production is not well established. Most data have been collected for p47phox, whereas NoxA1 has basically not yet been studied. In this article the specific aspects of cytosolic Nox proteins in the cardiovascular system with respect to Nox activation, their expression and their importance will be reviewed. Finally, it will be discussed whether cytosolic Nox proteins are suitable pharmacological targets to tamper with vascular ROS production. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Nicotine induces fibrogenic changes in human liver via nicotinic acetylcholine receptors expressed on hepatic stellate cells

Energy Technology Data Exchange (ETDEWEB)

Soeda, Junpei; Morgan, Maelle; McKee, Chad; Mouralidarane, Angelina; Lin, ChingI [University College London, Centre for Hepatology, Royal Free Hospital, London NW3 2PF (United Kingdom); Roskams, Tania [Department of Morphology and Molecular Pathology, University of Leuven (Belgium); Oben, Jude A., E-mail: j.oben@ucl.ac.uk [University College London, Centre for Hepatology, Royal Free Hospital, London NW3 2PF (United Kingdom); Department of Gastroenterology and Hepatology, Guy' s and St Thomas' Hospital, London SE1 7EH (United Kingdom)

2012-01-06

Highlights: Black-Right-Pointing-Pointer Cigarette smoke may induce liver fibrosis via nicotine receptors. Black-Right-Pointing-Pointer Nicotine induces proliferation of hepatic stellate cells (HSCs). Black-Right-Pointing-Pointer Nicotine activates hepatic fibrogenic pathways. Black-Right-Pointing-Pointer Nicotine receptor antagonists attenuate HSC proliferation. Black-Right-Pointing-Pointer Nicotinic receptor antagonists may have utility as novel anti-fibrotic agents. -- Abstract: Background and aims: Cigarette smoke (CS) may cause liver fibrosis but possible involved mechanisms are unclear. Among the many chemicals in CS is nicotine - which affects cells through nicotinic acetylcholine receptors (nAChR). We studied the effects of nicotine, and involved pathways, on human primary hepatic stellate cells (hHSCs), the principal fibrogenic cells in the liver. We then determined possible disease relevance by assaying nAChR in liver samples from human non-alcoholic steatohepatitis (NASH). Methods: hHSC were isolated from healthy human livers and nAChR expression analyzed - RT-PCR and Western blotting. Nicotine induction of hHSC proliferation, upregulation of collagen1-{alpha}2 and the pro-fibrogenic cytokine transforming growth factor beta 1 (TGF-{beta}1) was determined along with involved intracellular signaling pathways. nAChR mRNA expression was finally analyzed in whole liver biopsies obtained from patients diagnosed with non-alcoholic steatohepatitis (NASH). Results: hHSCs express muscle type ({alpha}1, {beta}1, delta and epsilon) and neuronal type ({alpha}3, {alpha}6, {alpha}7, {beta}2 and {beta}4) nAChR subunits at the mRNA level. Among these subunits, {alpha}3, {alpha}7, {beta}1 and {epsilon} were predominantly expressed as confirmed by Western blotting. Nicotine induced hHSC proliferation was attenuated by mecamylamine (p < 0.05). Additionally, collagen1-{alpha}2 and TGF-{beta}1 mRNA expression were significantly upregulated by nicotine and inhibited by

Nicotine induces fibrogenic changes in human liver via nicotinic acetylcholine receptors expressed on hepatic stellate cells

International Nuclear Information System (INIS)

Soeda, Junpei; Morgan, Maelle; McKee, Chad; Mouralidarane, Angelina; Lin, ChingI; Roskams, Tania; Oben, Jude A.

2012-01-01

Highlights: ► Cigarette smoke may induce liver fibrosis via nicotine receptors. ► Nicotine induces proliferation of hepatic stellate cells (HSCs). ► Nicotine activates hepatic fibrogenic pathways. ► Nicotine receptor antagonists attenuate HSC proliferation. ► Nicotinic receptor antagonists may have utility as novel anti-fibrotic agents. -- Abstract: Background and aims: Cigarette smoke (CS) may cause liver fibrosis but possible involved mechanisms are unclear. Among the many chemicals in CS is nicotine – which affects cells through nicotinic acetylcholine receptors (nAChR). We studied the effects of nicotine, and involved pathways, on human primary hepatic stellate cells (hHSCs), the principal fibrogenic cells in the liver. We then determined possible disease relevance by assaying nAChR in liver samples from human non-alcoholic steatohepatitis (NASH). Methods: hHSC were isolated from healthy human livers and nAChR expression analyzed – RT-PCR and Western blotting. Nicotine induction of hHSC proliferation, upregulation of collagen1-α2 and the pro-fibrogenic cytokine transforming growth factor beta 1 (TGF-β1) was determined along with involved intracellular signaling pathways. nAChR mRNA expression was finally analyzed in whole liver biopsies obtained from patients diagnosed with non-alcoholic steatohepatitis (NASH). Results: hHSCs express muscle type (α1, β1, delta and epsilon) and neuronal type (α3, α6, α7, β2 and β4) nAChR subunits at the mRNA level. Among these subunits, α3, α7, β1 and ε were predominantly expressed as confirmed by Western blotting. Nicotine induced hHSC proliferation was attenuated by mecamylamine (p < 0.05). Additionally, collagen1-α2 and TGF-β1 mRNA expression were significantly upregulated by nicotine and inhibited by mecamylamine. α1 and α3-nAChR mRNA expression was significantly upregulated in NASH fibrosis compared to normal livers. Conclusion: Nicotine at levels in smokers’ blood is pro-fibrogenic, through

Human intrahepatic ILC2 are IL-13positive amphiregulinpositive and their frequency correlates with model of end stage liver disease score.

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Hannah C Jeffery

Full Text Available Innate lymphoid cells (ILC have been implicated in the initiation of inflammation and fibrosis in mice. However, ILC have not been characterized in inflamed human liver tissue.Human intrahepatic lymphocytes were isolated by mechanical digestion and phenotyped by flow cytometry. Conditioned medium from cultures of primary human biliary epithelial cells, stellate cells, fibroblasts and inflamed human liver tissue was used to model the effects of the inflammatory liver environment of ILC phenotype and function.All three ILC subsets were present in the human liver, with the ILC1 (CRTH2negCD117neg subset constituting around 70% of intrahepatic ILCs. Both NCRpos (NKp44+ and NCRneg ILC3 (CRTH2negCD117pos subsets were also detected. ILC2 (CRTH2pos frequency correlated with disease severity measured by model of end stage liver disease (MELD scoring leading us to study this subset in more detail. ILC2 displayed a tissue resident CD69+ CD161++ phenotype and expressed chemokine receptor CCR6 allowing them to respond to CCL20 secreted by cholangiocytes and stellate cells. ILC2 expressed integrins VLA-5 and VLA-6 and the IL-2 and IL-7 cytokine receptors CD25 and CD127 although IL-2 and IL-7 were barely detectable in inflamed liver tissue. Although biliary epithelial cells secrete IL-33, intrahepatic ILC2 had low expression of the ST2 receptor. Intrahepatic ILC2 secreted the immunoregulatory and repair cytokines IL-13 and amphiregulin.Intrahepatic ILC2 express receptors allowing them to be recruited to bile ducts in inflamed portal tracts. Their frequencies increased with worsening liver function. Their secretion of IL-13 and amphiregulin suggests they may be recruited to promote resolution and repair and thereby they may contribute to ongoing fibrogenesis in liver disease.

Rearrangement of a common cellular DNA domain on chromosome 4 in human primary liver tumors

International Nuclear Information System (INIS)

Pasquinelli, C.; Garreau, F.; Bougueleret, L.; Cariani, E.; Thiers, V.; Croissant, O.; Hadchouel, M.; Tiollais, P.; Brechot, C.; Grzeschik, K.H.

1988-01-01

Hepatitis B virus (HBV) DNA integration has been shown to occur frequently in human hepatocellular carcinomas. The authors have investigated whether common cellular DNA domains might be rearranged, possibly by HBV integration, in human primary liver tumors. Unique cellular DNA sequences adjacent to an HBV integration site were isolated from a patient with hepatitis B surface antigen-positive hepatocellular carcinoma. These probes detected rearrangement of this cellular region of chromosomal DNA in 3 of 50 additional primary liver tumors studied. Of these three tumor samples, two contained HBV DNA, without an apparent link between the viral DNA and the rearranged allele; HBV DNA sequences were not detected in the third tumor sample. By use of a panel of somatic cell hybrids, these unique cellular DNA sequences were shown to be located on chromosome 4. Therefore, this region of chromosomal DNA might be implicated in the formation of different tumors at one step of liver cell transformation, possible related to HBV integration

The sinusoidal lining cells in "normal" human liver. A scanning electron microscopic investigation

DEFF Research Database (Denmark)

Horn, T; Henriksen, Jens Henrik Sahl; Christoffersen, P

1986-01-01

The scanning electron microscopic was used to study the fenestrations of human liver sinusoids. Thirteen biopsies, where light microscopy and transmission electron microscopy revealed normal sinusoidal architecture, were investigated. The number of fenestrae was calculated in acinar zone 3...

Gene expression profiling and secretome analysis differentiate adult-derived human liver stem/progenitor cells and human hepatic stellate cells.

Directory of Open Access Journals (Sweden)

Silvia Berardis

Full Text Available Adult-derived human liver stem/progenitor cells (ADHLSC are obtained after primary culture of the liver parenchymal fraction. The cells are of fibroblastic morphology and exhibit a hepato-mesenchymal phenotype. Hepatic stellate cells (HSC derived from the liver non-parenchymal fraction, present a comparable morphology as ADHLSC. Because both ADHLSC and HSC are described as liver stem/progenitor cells, we strived to extensively compare both cell populations at different levels and to propose tools demonstrating their singularity. ADHLSC and HSC were isolated from the liver of four different donors, expanded in vitro and followed from passage 5 until passage 11. Cell characterization was performed using immunocytochemistry, western blotting, flow cytometry, and gene microarray analyses. The secretion profile of the cells was evaluated using Elisa and multiplex Luminex assays. Both cell types expressed α-smooth muscle actin, vimentin, fibronectin, CD73 and CD90 in accordance with their mesenchymal origin. Microarray analysis revealed significant differences in gene expression profiles. HSC present high expression levels of neuronal markers as well as cytokeratins. Such differences were confirmed using immunocytochemistry and western blotting assays. Furthermore, both cell types displayed distinct secretion profiles as ADHLSC highly secreted cytokines of therapeutic and immuno-modulatory importance, like HGF, interferon-γ and IL-10. Our study demonstrates that ADHLSC and HSC are distinct liver fibroblastic cell populations exhibiting significant different expression and secretion profiles.

DMPD: Regulation of arachidonic acid release and cytosolic phospholipase A2activation. [Dynamic Macrophage Pathway CSML Database

Lifescience Database Archive (English)

Full Text Available 10080535 Regulation of arachidonic acid release and cytosolic phospholipase A2activ...on of arachidonic acid release and cytosolic phospholipase A2activation. PubmedID 10080535 Title Regulation ...of arachidonic acid release and cytosolic phospholipase A2activation. Authors Gij

Quantitative assessment of cellular uptake and cytosolic access of antibody in living cells by an enhanced split GFP complementation assay

Energy Technology Data Exchange (ETDEWEB)

Kim, Ji-sun; Choi, Dong-Ki; Park, Seong-wook; Shin, Seung-Min; Bae, Jeomil [Department of Molecular Science and Technology, Ajou University, Suwon 443-749 (Korea, Republic of); Kim, Dong-Myung [Department of Chemical Engineering and Applied Chemistry, Chungnam National University, Daejeon 305-764 (Korea, Republic of); Yoo, Tae Hyeon [Department of Molecular Science and Technology, Ajou University, Suwon 443-749 (Korea, Republic of); Kim, Yong-Sung, E-mail: kimys@ajou.ac.kr [Department of Molecular Science and Technology, Ajou University, Suwon 443-749 (Korea, Republic of)

2015-11-27

Considering the number of cytosolic proteins associated with many diseases, development of cytosol-penetrating molecules from outside of living cells is highly in demand. To gain access to the cytosol after cellular uptake, cell-penetrating molecules should be released from intermediate endosomes prior to the lysosomal degradation. However, it is very challenging to distinguish the pool of cytosolic-released molecules from those trapped in the endocytic vesicles. Here we describe a method to directly demonstrate the cytosolic localization and quantification of cytosolic amount of a cytosol-penetrating IgG antibody, TMab4, based on enhanced split GFP complementation system. We generated TMab4 genetically fused with one GFP fragment and separately established HeLa cells expressing the other GFP fragment in the cytosol such that the complemented GFP fluorescence is observed only when extracellular-treated TMab4 reaches the cytosol after cellular internalization. The high affinity interactions between streptavidin-binding peptide 2 and streptavidin was employed as respective fusion partners of GFP fragments to enhance the sensitivity of GFP complementation. With this method, cytosolic concentration of TMab4 was estimated to be about 170 nM after extracellular treatment of HeLa cells with 1 μM TMab4 for 6 h. We also found that after cellular internalization into living cells, nearly 1.3–4.3% of the internalized TMab4 molecules escaped into the cytosol from the endocytic vesicles. Our enhanced split GFP complementation assay provides a useful tool to directly quantify cytosolic amount of cytosol-penetrating agents and allows cell-based high-throughput screening for cytosol-penetrating agents with increased endosomal-escaping activity.

Gene expression analysis of precision-cut human liver slices indicates stable expression of ADME-Tox related genes

NARCIS (Netherlands)

Elferink, M. G. L.; Olinga, P.; van Leeuwen, E. M.; Bauerschmidt, S.; Polman, J.; Schoonen, W. G.; Heisterkamp, S. H.; Groothuis, G. M. M.

2011-01-01

In the process of drug development it is of high importance to test the safety of new drugs with predictive value for human toxicity. A promising approach of toxicity testing is based on shifts in gene expression profiling of the liver. Toxicity screening based on animal liver cells cannot be

Ca2+-mobilizing agonists increase mitochondrial ATP production to accelerate cytosolic Ca2+ removal: aberrations in human complex I deficiency.

NARCIS (Netherlands)

Visch, H.J.; Koopman, W.J.H.; Zeegers, D.; Emst-de Vries, S.E. van; Kuppeveld, F.J.M. van; Heuvel, L.W. van den; Smeitink, J.A.M.; Willems, P.H.G.M.

2006-01-01

Previously, we reported that both the bradykinin (Bk)-induced increase in mitochondrial ATP concentration ([ATP]M) and the rate of cytosolic Ca2+ removal are significantly decreased in skin fibroblasts from a patient with an isolated complex I deficiency. Here we demonstrate that the mitochondrial

ADAM12 in human liver cancers: TGF-beta-regulated expression in stellate cells is associated with matrix remodeling

DEFF Research Database (Denmark)

Le Pabic, Hélène; Bonnier, Dominique; Wewer, Ulla M

2003-01-01

"A disintegrin and metalloproteinases" (ADAMs) form a family of cell-surface glycoproteins with potential protease and cell-adhesion activities. We have investigated ADAM expression in human liver cancers and their regulation by several cytokines involved in liver injury. Using degenerative RT...... carcinomas (up to 3- and 6-fold, respectively) and liver metastases from colonic carcinomas (up to 40- and 60-fold, respectively). The up-regulation of both ADAM9 and ADAM12 was correlated with an increase in matrix metalloproteinase 2 expression and activity. In conclusion, in liver cancers ADAM9 and ADAM12......-PCR, cDNA encoding sequences for ADAM9 and ADAM12 were identified in human activated hepatic stellate cells (HSCs). Northern blot analyses showed that HSCs, but not hepatocytes, expressed transcripts for ADAM9 messenger RNA (mRNA) and both the long and short forms of ADAM12. This expression...

Tc-99 m-GSA liver scintigraphy in alcoholic liver cirrhosis

International Nuclear Information System (INIS)

Itano, Satoshi; Harada, Masaru; Nagamatsu, Hiroaki

2003-01-01

We compared 15 alcoholic liver cirrhosis patients with 10 viral liver cirrhosis patients using technetium-99 m-galactosyl human serum albumin (Tc-99 m-GSA) liver scintigraphy and could clinically reveal the disorder of metabolism of asialoglycoprotein in alcoholic liver cirrhosis. Receptor index (LHL 15 = liver count divided by the sum of liver and heart counts at 15 minutes) was significantly (p <0.01) lower in patients with alcoholic cirrhosis (median: 0.821), compared with patients with viral cirrhosis (0.915). Grading score, which was an index showed by the difference in the isotope uptake patterns between liver and heart, was significantly (p <0.01) worse in patients with alcoholic cirrhosis, compared with patients with viral cirrhosis. These results suggested that alcoholic liver cirrhosis had a specific disorder of a metabolic function for asialoglycoprotein. (author)

Amarogentin Induces Apoptosis of Liver Cancer Cells via Upregulation of p53 and Downregulation of Human Telomerase Reverse Transcriptase in Mice

Science.gov (United States)

Li, Runqin; Zhang, Yinglin

2016-01-01

Background and Objective: Amarogentin has been reported to have a preventive effect on liver cancer via inducing cancer cell apoptosis. We attempted to elucidate the roles of p53-associated apoptosis pathways in the chemopreventive mechanism of amarogentin. The findings of this study will facilitate the development of a novel supplementary strategy for the treatment of liver cancer. Materials and Methods: The purity of amarogentin was assessed by high-performance liquid chromatography. The inhibitory ratios of the liver cell lines were determined using a Cell Counting Kit-8 following treatment with a gradient concentration of amarogentin. Cell apoptosis was detected by flow cytometry using annexin V-fluorescein isothiocyanate/propidium iodide kits. The gene and protein expression of p53-associated molecules, such as Akt, human telomerase reverse transcriptase, RelA, and p38, was detected by real-time quantitative polymerase chain reaction, Western blotting, and immunohistochemical staining in liver cancer cells and mouse tumor tissues after treatment with amarogentin. Results: The inhibitory effect of amarogentin on cell proliferation was more obvious in liver cancer cells, and amarogentin was more likely to induce the apoptosis of liver cancer cells than that of normal liver cells. The gene and protein expression levels of Akt, RelA, and human telomerase reverse transcriptase were markedly higher in the control group than in the preventive group and treatment groups. Only the expression of human telomerase reverse transcriptase was downregulated, accompanied by the upregulation of p53. Conclusion: The results of our study suggest that amarogentin promotes apoptosis of liver cancer cells by the upregulation of p53 and downregulation of human telomerase reverse transcriptase and prevents the malignant transformation of these cells. PMID:27402632

Liver lipid molecules induce PEPCK-C gene transcription and attenuate insulin action

International Nuclear Information System (INIS)

Chen Guoxun

2007-01-01

Cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C) plays key roles in gluconeogenesis, glyceroneogenesis, and cataplerosis. Experiments were designed to examine the effects of endogenous lipid molecules from rat livers on the expression of PEPCK-C gene in primary rat hepatocytes. The lipid extracts prepared from livers of Zucker fatty, lean, and Wistar rats induced the expression levels of PEPCK-C transcripts. Insulin-mediated reduction of PEPCK-C gene expression was attenuated by the same treatment. The lipid extracts induced the relative luciferase activity of reporter gene constructs that contain a 2.2-kb 5' promoter fragment of PEPCK-C gene, but not the construct that contains only the 3' untranslated region (UTR) of its mRNA. The estimated half life of PEPCK-C transcripts in the presence of the lipid extract is the same as that in the absence of it. My results demonstrate for the first time that endogenous lipid molecules induce PEPCK-C gene transcription and attenuate insulin action in liver

Mechanisms of acetaminophen-induced cell death in primary human hepatocytes

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

2014-09-15

Acetaminophen (APAP) overdose is the most prevalent cause of drug-induced liver injury in western countries. Numerous studies have been conducted to investigate the mechanisms of injury after APAP overdose in various animal models; however, the importance of these mechanisms for humans remains unclear. Here we investigated APAP hepatotoxicity using freshly isolated primary human hepatocytes (PHH) from either donor livers or liver resections. PHH were exposed to 5 mM, 10 mM or 20 mM APAP over a period of 48 h and multiple parameters were assessed. APAP dose-dependently induced significant hepatocyte necrosis starting from 24 h, which correlated with the clinical onset of human liver injury after APAP overdose. Interestingly, cellular glutathione was depleted rapidly during the first 3 h. APAP also resulted in early formation of APAP-protein adducts (measured in whole cell lysate and in mitochondria) and mitochondrial dysfunction, indicated by the loss of mitochondrial membrane potential after 12 h. Furthermore, APAP time-dependently triggered c-Jun N-terminal kinase (JNK) activation in the cytosol and translocation of phospho-JNK to the mitochondria. Both co-treatment and post-treatment (3 h) with the JNK inhibitor SP600125 reduced JNK activation and significantly attenuated cell death at 24 h and 48 h after APAP. The clinical antidote N-acetylcysteine offered almost complete protection even if administered 6 h after APAP and a partial protection when given at 15 h. Conclusion: These data highlight important mechanistic events in APAP toxicity in PHH and indicate a critical role of JNK in the progression of injury after APAP in humans. The JNK pathway may represent a therapeutic target in the clinic. - Highlights: • APAP reproducibly causes cell death in freshly isolated primary human hepatocytes. • APAP induces adduct formation, JNK activation and mitochondrial dysfunction in PHH. • Mitochondrial adducts and JNK translocation are delayed in PHH compared to

Spatial modeling of the membrane-cytosolic interface in protein kinase signal transduction.

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Wolfgang Giese

2018-04-01

Full Text Available The spatial architecture of signaling pathways and the interaction with cell size and morphology are complex, but little understood. With the advances of single cell imaging and single cell biology, it becomes crucial to understand intracellular processes in time and space. Activation of cell surface receptors often triggers a signaling cascade including the activation of membrane-attached and cytosolic signaling components, which eventually transmit the signal to the cell nucleus. Signaling proteins can form steep gradients in the cytosol, which cause strong cell size dependence. We show that the kinetics at the membrane-cytosolic interface and the ratio of cell membrane area to the enclosed cytosolic volume change the behavior of signaling cascades significantly. We suggest an estimate of average concentration for arbitrary cell shapes depending on the cell volume and cell surface area. The normalized variance, known from image analysis, is suggested as an alternative measure to quantify the deviation from the average concentration. A mathematical analysis of signal transduction in time and space is presented, providing analytical solutions for different spatial arrangements of linear signaling cascades. Quantification of signaling time scales reveals that signal propagation is faster at the membrane than at the nucleus, while this time difference decreases with the number of signaling components in the cytosol. Our investigations are complemented by numerical simulations of non-linear cascades with feedback and asymmetric cell shapes. We conclude that intracellular signal propagation is highly dependent on cell geometry and, thereby, conveys information on cell size and shape to the nucleus.

Effect of alcohol consumption on the liver detoxication capacity as measured by [13C2]aminopyrine and L-[1-13C]phenylalanine breath tests.

Science.gov (United States)

Wutzke, Klaus D; Wigger, Marianne

2009-09-01

The aim of this study was to investigate the hepatic microsomal and cytosolic functions by using the 13CO2 breath test in healthy subjects either before or after consumption of red wine. Twelve adults received [13C2]aminopyrine and L-[1-13C]phenylalanine together with a standardised dinner. Expired air samples were taken over 6 h. After a wash-out period, the subjects consumed 0.4 ml ethanol per kg per day together with dinner over a 7.5-day period on average. Thereafter, 13C-tracer administration was repeated under identical conditions. The 13CO2 enrichments were measured by isotope ratio mass spectrometry. The mean cumulative percentage 13C-dose recovery after administration of [13C2]aminopyrine and L-[1-13C]phenylalanine either without or with red wine consumption amounted to 17.0+/-4.4 vs. 14.7+/-3.1% (p=0.170) and 14.0+/-2.8 vs. 11.5+/-3.9% (p=0.084), respectively. Moderate alcohol consumption does not induce significant short-term changes of the microsomal and the cytosolic function of the human liver in healthy subjects.

Pressurized liquid extraction-assisted mussel cytosol preparation for the determination of metals bound to metallothionein-like proteins

International Nuclear Information System (INIS)

Santiago-Rivas, Sandra; Moreda-Pineiro, Antonio; Bermejo-Barrera, Pilar; Moreda-Pineiro, Jorge; Alonso-Rodriguez, Elia; Muniategui-Lorenzo, Soledad; Lopez-Mahia, Purificacion; Prada-Rodriguez, Dario

2007-01-01

The possibilities of pressurized liquid extraction (PLE) have been novelty tested to assist the cytosol preparation from wet mussel soft tissue before the determination of metals bound to metallothionein-like proteins (MLPs). Results obtained after PLE were compared with those obtained after a classical blending procedure for mussel cytosolic preparation. Isoforms MLP-1 (retention time of 4.1 min) and MLP-2 (retention time of 7.4 min) were separated by anion exchange high-performance liquid chromatography (HPLC) and the concentrations of Ba, Cu, Mn, Sr and Zn bound to MLP isoforms were directly measured by inductively coupled plasma-atomic emission spectrometry (ICP-OES) as a multi-element detector. The optimized PLE-assisted mussel cytosol preparation has consisted of one extraction cycle at room temperature and 1500 psi for 2 min. Since separation between the solid mussel residue and the extract (cytosol) is performed by the PLE system, the cytosol preparation method is faster than conventional cytosol preparation methods by cutting/blending using Ultraturrax or Stomacher devices

Arsenic induces apoptosis in mouse liver is mitochondria dependent and is abrogated by N-acetylcysteine

International Nuclear Information System (INIS)

Santra, Amal; Chowdhury, Abhijit; Ghatak, Subhadip; Biswas, Ayan; Dhali, Gopal Krishna

2007-01-01

Arsenicosis, caused by arsenic contamination of drinking water supplies, is a major public health problem in India and Bangladesh. Chronic liver disease, often with portal hypertension occurs in chronic arsenicosis, contributes to the morbidity and mortality. The early cellular events that initiate liver cell injury due to arsenicosis have not been studied. Our aim was to identify the possible mechanisms related to arsenic-induced liver injury in mice. Liver injury was induced in mice by arsenic treatment. The liver was used for mitochondrial oxidative stress, mitochondrial permeability transition (MPT). Evidence of apoptosis was sought by TUNEL test, caspase assay and histology. Pretreatment with N-acetyl-L-cysteine (NAC) was done to modulate hepatic GSH level. Arsenic treatment in mice caused liver injury associated with increased oxidative stress in liver mitochondria and alteration of MPT. Altered MPT facilitated cytochrome c release in the cytosol, activation of caspase 9 and caspase 3 activities and apoptotic cell death. Pretreatment of NAC to arsenic-treated mice abrogated all these alteration suggesting a glutathione (GSH)-dependent mechanism. Oxidative stress in mitochondria and inappropriate MPT are important in the pathogenesis of arsenic induced apoptotic liver cell injury. The phenomenon is GSH dependent and supplementation of NAC might have beneficial effects

Regulation of coagulation factor XI expression by microRNAs in the human liver.

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Salam Salloum-Asfar

Full Text Available High levels of factor XI (FXI increase the risk of thromboembolic disease. However, the genetic and environmental factors regulating FXI expression are still largely unknown. The aim of our study was to evaluate the regulation of FXI by microRNAs (miRNAs in the human liver. In silico prediction yielded four miRNA candidates that might regulate FXI expression. HepG2 cells were transfected with miR-181a-5p, miR-23a-3p, miR-16-5p and miR-195-5p. We used mir-494, which was not predicted to bind to F11, as a negative control. Only miR-181a-5p caused a significant decrease both in FXI protein and F11 mRNA levels. In addition, transfection with a miR-181a-5p inhibitor in PLC/PRF/5 hepatic cells increased both the levels of F11 mRNA and extracellular FXI. Luciferase assays in human colon cancer cells deficient for Dicer (HCT-DK demonstrated a direct interaction between miR-181a-5p and 3'untranslated region of F11. Additionally, F11 mRNA levels were inversely and significantly correlated with miR-181a-5p levels in 114 healthy livers, but not with miR-494. This study demonstrates that FXI expression is directly regulated by a specific miRNA, miR-181a-5p, in the human liver. Future studies are necessary to further investigate the potential consequences of miRNA dysregulation in pathologies involving FXI.

Functional contribution of elevated circulating and hepatic non-classical CD14CD16 monocytes to inflammation and human liver fibrosis.

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Henning W Zimmermann

Full Text Available BACKGROUND: Monocyte-derived macrophages critically perpetuate inflammatory responses after liver injury as a prerequisite for organ fibrosis. Experimental murine models identified an essential role for the CCR2-dependent infiltration of classical Gr1/Ly6C(+ monocytes in hepatic fibrosis. Moreover, the monocyte-related chemokine receptors CCR1 and CCR5 were recently recognized as important fibrosis modulators in mice. In humans, monocytes consist of classical CD14(+CD16(- and non-classical CD14(+CD16(+ cells. We aimed at investigating the relevance of monocyte subpopulations for human liver fibrosis, and hypothesized that 'non-classical' monocytes critically exert inflammatory as well as profibrogenic functions in patients during liver disease progression. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed circulating monocyte subsets from freshly drawn blood samples of 226 patients with chronic liver disease (CLD and 184 healthy controls by FACS analysis. Circulating monocytes were significantly expanded in CLD-patients compared to controls with a marked increase of the non-classical CD14(+CD16(+ subset that showed an activated phenotype in patients and correlated with proinflammatory cytokines and clinical progression. Correspondingly, CD14(+CD16(+ macrophages massively accumulated in fibrotic/cirrhotic livers, as evidenced by immunofluorescence and FACS. Ligands of monocyte-related chemokine receptors CCR2, CCR1 and CCR5 were expressed at higher levels in fibrotic and cirrhotic livers, while CCL3 and CCL4 were also systemically elevated in CLD-patients. Isolated monocyte/macrophage subpopulations were functionally characterized regarding cytokine/chemokine expression and interactions with primary human hepatic stellate cells (HSC in vitro. CD14(+CD16(+ monocytes released abundant proinflammatory cytokines. Furthermore, CD14(+CD16(+, but not CD14(+CD16(- monocytes could directly activate collagen-producing HSC. CONCLUSIONS/SIGNIFICANCE: Our data

Gene expression profiling in human precision cut liver slices in response to the FXR agonist obeticholic acid.

Science.gov (United States)

Ijssennagger, Noortje; Janssen, Aafke W F; Milona, Alexandra; Ramos Pittol, José M; Hollman, Danielle A A; Mokry, Michal; Betzel, Bark; Berends, Frits J; Janssen, Ignace M; van Mil, Saskia W C; Kersten, Sander

2016-05-01

The bile acid-activated farnesoid X receptor (FXR) is a nuclear receptor regulating bile acid, glucose and cholesterol homeostasis. Obeticholic acid (OCA), a promising drug for the treatment of non-alcoholic steatohepatitis (NASH) and type 2 diabetes, activates FXR. Mouse studies demonstrated that FXR activation by OCA alters hepatic expression of many genes. However, no data are available on the effects of OCA in the human liver. Here we generated gene expression profiles in human precision cut liver slices (hPCLS) after treatment with OCA. hPCLS were incubated with OCA for 24 h. Wild-type or FXR(-/-) mice received OCA or vehicle by oral gavage for 7 days. Transcriptomic analysis showed that well-known FXR target genes, including NR0B2 (SHP), ABCB11 (BSEP), SLC51A (OSTα) and SLC51B (OSTβ), and ABCB4 (MDR3) are regulated by OCA in hPCLS. Ingenuity pathway analysis confirmed that 'FXR/RXR activation' is the most significantly changed pathway upon OCA treatment. Comparison of gene expression profiles in hPCLS and mouse livers identified 18 common potential FXR targets. ChIP-sequencing in mouse liver confirmed FXR binding to IR1 sequences of Akap13, Cgnl1, Dyrk3, Pdia5, Ppp1r3b and Tbx6. Our study shows that hPCLS respond to OCA treatment by upregulating well-known FXR target genes, demonstrating its suitability to study FXR-mediated gene regulation. We identified six novel bona-fide FXR target genes in both mouse and human liver. Finally, we discuss a possible explanation for changes in high or low density lipoprotein observed in NASH and primary biliary cholangitis patients treated with OCA based on the genomic expression profile in hPCLS. Copyright © 2016 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Phenobarbital induces cell cycle transcriptional responses in mouse liver humanized for constitutive androstane and pregnane x receptors.

Science.gov (United States)

Luisier, Raphaëlle; Lempiäinen, Harri; Scherbichler, Nina; Braeuning, Albert; Geissler, Miriam; Dubost, Valerie; Müller, Arne; Scheer, Nico; Chibout, Salah-Dine; Hara, Hisanori; Picard, Frank; Theil, Diethilde; Couttet, Philippe; Vitobello, Antonio; Grenet, Olivier; Grasl-Kraupp, Bettina; Ellinger-Ziegelbauer, Heidrun; Thomson, John P; Meehan, Richard R; Elcombe, Clifford R; Henderson, Colin J; Wolf, C Roland; Schwarz, Michael; Moulin, Pierre; Terranova, Rémi; Moggs, Jonathan G

2014-06-01

The constitutive androstane receptor (CAR) and the pregnane X receptor (PXR) are closely related nuclear receptors involved in drug metabolism and play important roles in the mechanism of phenobarbital (PB)-induced rodent nongenotoxic hepatocarcinogenesis. Here, we have used a humanized CAR/PXR mouse model to examine potential species differences in receptor-dependent mechanisms underlying liver tissue molecular responses to PB. Early and late transcriptomic responses to sustained PB exposure were investigated in liver tissue from double knock-out CAR and PXR (CAR(KO)-PXR(KO)), double humanized CAR and PXR (CAR(h)-PXR(h)), and wild-type C57BL/6 mice. Wild-type and CAR(h)-PXR(h) mouse livers exhibited temporally and quantitatively similar transcriptional responses during 91 days of PB exposure including the sustained induction of the xenobiotic response gene Cyp2b10, the Wnt signaling inhibitor Wisp1, and noncoding RNA biomarkers from the Dlk1-Dio3 locus. Transient induction of DNA replication (Hells, Mcm6, and Esco2) and mitotic genes (Ccnb2, Cdc20, and Cdk1) and the proliferation-related nuclear antigen Mki67 were observed with peak expression occurring between 1 and 7 days PB exposure. All these transcriptional responses were absent in CAR(KO)-PXR(KO) mouse livers and largely reversible in wild-type and CAR(h)-PXR(h) mouse livers following 91 days of PB exposure and a subsequent 4-week recovery period. Furthermore, PB-mediated upregulation of the noncoding RNA Meg3, which has recently been associated with cellular pluripotency, exhibited a similar dose response and perivenous hepatocyte-specific localization in both wild-type and CAR(h)-PXR(h) mice. Thus, mouse livers coexpressing human CAR and PXR support both the xenobiotic metabolizing and the proliferative transcriptional responses following exposure to PB.

The liver taxis of receptor mediated lactosaminated human growth hormone

International Nuclear Information System (INIS)

Chen Zelian; Shi Lin; Li Tongling; Pang Qijie; He Juying; Guan Changtian

2002-01-01

Radiography imaging is used to assess liver taxis mechanism of anti-dwarfism drug lactosaminated human growth hormone (L-rhGH). Both L-rhGH and rhGH labelled with 131 I are used to study their biodistribution in animals (including rabbits, cocks and rats). The results show that L-rhGH is of specific hepatic targeting property, and the maximum hepatic concentration rate is 76.8%, which is two times of rhGH. Its hepatic binding is receptor mediated

Characterization of the methotrexate transport pathway in murine L1210 leukemia cells: Involvement of a membrane receptor and a cytosolic protein

International Nuclear Information System (INIS)

Price, E.M.; Ratnam, M.; Rodeman, K.M.; Freisheim, J.H.

1988-01-01

A radioiodinated photoaffinity analogue of methotrexate, N α -(4-amino-4-deoxy-10-methyl-pteroyl)-N ε -(4-azidosalicylyl)-L-lysine (APA-ASA-Lys), was recently used to identify the plasma membrane derived binding protein involved in the transport of this folate antagonist into murine L1210 cells. The labeled protein has an apparent molecular weight of 46K-48K when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but no such labeling occurs in a methotrexate transport-defective cell line (L1210/R81). Labeling of the total cytosolic protein from disrupted cells, followed by electrophoresis and autoradiography, showed, among other proteins, a 21K band, corresponding to dihydrofolate reductase (DHFR), in both the parent and R81 cells and a 38K band only in the parent cells. However, when whole cells were UV irradiated at various times at 37 degree C following addition of radiolabeled APA-ASA-Lys, the 38K protein and DHFR were the only cytosolic proteins labeled in the parent cells, while the intact R81 cells showed no labeled cytosolic protein, since the photoprobe is not transported. Further, when the parent cells were treated with a pulse of radiolabeled photoprobe, followed by UV irradiation at different times at 37 degree C, the probe appeared sequentially on the 48K membrane protein and both the 38K cytosolic protein and dihydrofolate reductase. A 48K protein could be detected in both parent L1210 cells and the R81 cells on Western blots using antisera to a membrane folate binding protein from human placenta. These results suggest a vectorial transport of APA-ASA-Lys or methotrexate and reduced folate coenzymes into murine L1210 cells mediated by a 48K integral membrane protein and a 38K cytosolic or peripheral membrane protein. The 38K protein may help in the trafficking of reduced folate coenzymes, shuttling them to various cytosolic targets

Establishment of a general NAFLD scoring system for rodent models and comparison to human liver pathology.

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Wen Liang

Full Text Available The recently developed histological scoring system for non-alcoholic fatty liver disease (NAFLD by the NASH Clinical Research Network (NASH-CRN has been widely used in clinical settings, but is increasingly employed in preclinical research as well. However, it has not been systematically analyzed whether the human scoring system can directly be converted to preclinical rodent models. To analyze this, we systematically compared human NAFLD liver pathology, using human liver biopsies, with liver pathology of several NAFLD mouse models. Based upon the features pertaining to mouse NAFLD, we aimed at establishing a modified generic scoring system that is applicable to broad spectrum of rodent models.The histopathology of NAFLD was analyzed in several different mouse models of NAFLD to define generic criteria for histological assessment (preclinical scoring system. For validation of this scoring system, 36 slides of mouse livers, covering the whole spectrum of NAFLD, were blindly analyzed by ten observers. Additionally, the livers were blindly scored by one observer during two separate assessments longer than 3 months apart.The criteria macrovesicular steatosis, microvesicular steatosis, hepatocellular hypertrophy, inflammation and fibrosis were generally applicable to rodent NAFLD. The inter-observer reproducibility (evaluated using the Intraclass Correlation Coefficient between the ten observers was high for the analysis of macrovesicular steatosis and microvesicular steatosis (ICC = 0.784 and 0.776, all p<0.001, respectively and moderate for the analysis of hypertrophy and inflammation (ICC = 0.685 and 0.650, all p<0.001, respectively. The intra-observer reproducibility between the different observations of one observer was high for the analysis of macrovesicular steatosis, microvesicular steatosis and hypertrophy (ICC = 0.871, 0.871 and 0.896, all p<0.001, respectively and very high for the analysis of inflammation (ICC = 0.931, p

Subcellular partitioning of non-essential trace metals (Ag, As, Cd, Ni, Pb, and Tl) in livers of American (Anguilla rostrata) and European (Anguilla anguilla) yellow eels

Energy Technology Data Exchange (ETDEWEB)

Rosabal, Maikel [Institut national de la recherche scientifique, Centre Eau Terre et Environnement (INRS–ETE), 490 de la Couronne, Québec (Québec) G1K 9A9 (Canada); Pierron, Fabien [Université de Bordeaux, UMR EPOC CNRS 5805, F-33400 Talence (France); CNRS, EPOC, UMR 5805, F-33400 Talence (France); Couture, Patrice [Institut national de la recherche scientifique, Centre Eau Terre et Environnement (INRS–ETE), 490 de la Couronne, Québec (Québec) G1K 9A9 (Canada); Baudrimont, Magalie [Université de Bordeaux, UMR EPOC CNRS 5805, F-33400 Talence (France); CNRS, EPOC, UMR 5805, F-33400 Talence (France); Hare, Landis [Institut national de la recherche scientifique, Centre Eau Terre et Environnement (INRS–ETE), 490 de la Couronne, Québec (Québec) G1K 9A9 (Canada); Campbell, Peter G.C., E-mail: peter.campbell@ete.inrs.ca [Institut national de la recherche scientifique, Centre Eau Terre et Environnement (INRS–ETE), 490 de la Couronne, Québec (Québec) G1K 9A9 (Canada)

2015-03-15

Highlights: • Handling of hepatic metals consistently involved cytosolic, thermostable ligands. • Granule-like fractions are also involved in the detoxification of Ni, Pb, and Tl. • Despite these sequestration mechanisms, metal detoxification is incomplete. • Along the metal gradient, concentrations increase in metal-sensitive fractions. • This increase could represent a toxicological risk for the yellow eels. - Abstract: We determined the intracellular compartmentalization of the trace metals Ag, As, Cd, Ni, Pb, and Tl in the livers of yellow eels collected from the Saint Lawrence River system in Canada (Anguilla rostrata) and in the area of the Gironde estuary in France (Anguilla anguilla). Differential centrifugation, NaOH digestion and thermal shock were used to separate eel livers into putative “sensitive” fractions (heat-denatured proteins, mitochondria and microsomes + lysosomes) and detoxified metal fractions (heat-stable peptides/proteins and granules). The cytosolic heat-stable fraction (HSP) was consistently involved in the detoxification of all trace metals. In addition, granule-like structures played a complementary role in the detoxification of Ni, Pb, and Tl in both eel species. However, these detoxification mechanisms were not completely effective because increasing trace metal concentrations in whole livers were accompanied by significant increases in the concentrations of most trace metals in “sensitive” subcellular fractions, that is, mitochondria, heat-denatured cytosolic proteins and microsomes + lysosomes. Among these “sensitive” fractions, mitochondria were the major binding sites for As, Cd, Pb, and Tl. This accumulation of non-essential metals in “sensitive” fractions likely represents a health risk for eels inhabiting the Saint Lawrence and Gironde environments.

VE-cadherin expression allows identification of a new class of hematopoietic stem cells within human embryonic liver.

Science.gov (United States)

Oberlin, Estelle; Fleury, Maud; Clay, Denis; Petit-Cocault, Laurence; Candelier, Jean-Jacques; Mennesson, Benoît; Jaffredo, Thierry; Souyri, Michèle

2010-11-25

Edification of the human hematopoietic system during development is characterized by the production of waves of hematopoietic cells separated in time, formed in distinct embryonic sites (ie, yolk sac, truncal arteries including the aorta, and placenta). The embryonic liver is a major hematopoietic organ wherein hematopoietic stem cells (HSCs) expand, and the future, adult-type, hematopoietic cell hierarchy becomes established. We report herein the identification of a new, transient, and rare cell population in the human embryonic liver, which coexpresses VE-cadherin, an endothelial marker, CD45, a pan-hematopoietic marker, and CD34, a common endothelial and hematopoietic marker. This population displays an outstanding self-renewal, proliferation, and differentiation potential, as detected by in vitro and in vivo hematopoietic assays compared with its VE-cadherin negative counterpart. Based on VE-cadherin expression, our data demonstrate the existence of 2 phenotypically and functionally separable populations of multipotent HSCs in the human embryo, the VE-cadherin(+) one being more primitive than the VE-cadherin(-) one, and shed a new light on the hierarchical organization of the embryonic liver HSC compartment.

Insulin Induces an Increase in Cytosolic Glucose Levels in 3T3-L1 Cells with Inhibited Glycogen Synthase Activation

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Helena H. Chowdhury

2014-10-01

Full Text Available Glucose is an important source of energy for mammalian cells and enters the cytosol via glucose transporters. It has been thought for a long time that glucose entering the cytosol is swiftly phosphorylated in most cell types; hence the levels of free glucose are very low, beyond the detection level. However, the introduction of new fluorescence resonance energy transfer-based glucose nanosensors has made it possible to measure intracellular glucose more accurately. Here, we used the fluorescent indicator protein (FLIPglu-600µ to monitor cytosolic glucose dynamics in mouse 3T3-L1 cells in which glucose utilization for glycogen synthesis was inhibited. The results show that cells exhibit a low resting cytosolic glucose concentration. However, in cells with inhibited glycogen synthase activation, insulin induced a robust increase in cytosolic free glucose. The insulin-induced increase in cytosolic glucose in these cells is due to an imbalance between the glucose transported into the cytosol and the use of glucose in the cytosol. In untreated cells with sensitive glycogen synthase activation, insulin stimulation did not result in a change in the cytosolic glucose level. This is the first report of dynamic measurements of cytosolic glucose levels in cells devoid of the glycogen synthesis pathway.

Mass spectrometry-based proteomic analysis of human liver cytochrome(s) P450

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Shrivas, Kamlesh; Mindaye, Samuel T.; Getie-Kebtie, Melkamu; Alterman, Michail A., E-mail: Michail.Alterman@fda.hhs.gov

2013-02-15

The major objective of personalized medicine is to select optimized drug therapies and to a large degree such mission is determined by the expression profiles of cytochrome(s) P450 (CYP). Accordingly, a proteomic case study in personalized medicine is provided by the superfamily of cytochromes P450. Our knowledge about CYP isozyme expression on a protein level is very limited and based exclusively on DNA/mRNA derived data. Such information is not sufficient because transcription and translation events do not lead to correlated levels of expressed proteins. Here we report expression profiles of CYPs in human liver obtained by mass spectrometry (MS)-based proteomic approach. We analyzed 32 samples of human liver microsomes (HLM) of different sexes, ages and ethnicity along with samples of recombinant human CYPs. We have experimentally confirmed that each CYP isozyme can be effectively differentiated by their unique isozyme-specific tryptic peptide(s). Trypsin digestion patterns for almost 30 human CYP isozymes were established. Those findings should assist in selecting tryptic peptides suitable for MS-based quantitation. The data obtained demonstrate remarkable differences in CYP expression profiles. CYP2E1, CYP2C8 and CYP4A11 were the only isozymes found in all HLM samples. Female and pediatric HLM samples revealed much more diverse spectrum of expressed CYPs isozymes compared to male HLM. We have confirmed expression of a number of “rare” CYP (CYP2J2, CYP4B1, CYP4V2, CYP4F3, CYP4F11, CYP8B1, CYP19A1, CYP24A1 and CYP27A1) and obtained first direct experimental data showing expression of such CYPs as CYP2F1, CYP2S1, CYP2W1, CYP4A22, CYP4X1, and CYP26A1 on a protein level. - Highlights: ► First detailed proteomic analysis of CYP isozymes expression in human liver ► Trypsin digestion patterns for almost 30 human CYP isozymes established ► The data obtained demonstrate remarkable differences in CYP expression profiles. ► Female HLM samples revealed more

Mass spectrometry-based proteomic analysis of human liver cytochrome(s) P450

International Nuclear Information System (INIS)

Shrivas, Kamlesh; Mindaye, Samuel T.; Getie-Kebtie, Melkamu; Alterman, Michail A.

2013-01-01

The major objective of personalized medicine is to select optimized drug therapies and to a large degree such mission is determined by the expression profiles of cytochrome(s) P450 (CYP). Accordingly, a proteomic case study in personalized medicine is provided by the superfamily of cytochromes P450. Our knowledge about CYP isozyme expression on a protein level is very limited and based exclusively on DNA/mRNA derived data. Such information is not sufficient because transcription and translation events do not lead to correlated levels of expressed proteins. Here we report expression profiles of CYPs in human liver obtained by mass spectrometry (MS)-based proteomic approach. We analyzed 32 samples of human liver microsomes (HLM) of different sexes, ages and ethnicity along with samples of recombinant human CYPs. We have experimentally confirmed that each CYP isozyme can be effectively differentiated by their unique isozyme-specific tryptic peptide(s). Trypsin digestion patterns for almost 30 human CYP isozymes were established. Those findings should assist in selecting tryptic peptides suitable for MS-based quantitation. The data obtained demonstrate remarkable differences in CYP expression profiles. CYP2E1, CYP2C8 and CYP4A11 were the only isozymes found in all HLM samples. Female and pediatric HLM samples revealed much more diverse spectrum of expressed CYPs isozymes compared to male HLM. We have confirmed expression of a number of “rare” CYP (CYP2J2, CYP4B1, CYP4V2, CYP4F3, CYP4F11, CYP8B1, CYP19A1, CYP24A1 and CYP27A1) and obtained first direct experimental data showing expression of such CYPs as CYP2F1, CYP2S1, CYP2W1, CYP4A22, CYP4X1, and CYP26A1 on a protein level. - Highlights: ► First detailed proteomic analysis of CYP isozymes expression in human liver ► Trypsin digestion patterns for almost 30 human CYP isozymes established ► The data obtained demonstrate remarkable differences in CYP expression profiles. ► Female HLM samples revealed more

Long-term outcomes of liver transplant patients with human immunodeficiency virus infection and end-stage-liver-disease: single center experience

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Vernadakis S

2011-08-01

Full Text Available Abstract Objective Orthotopic-liver-transplantation (OLT in patients with Human-Immunodeficiency-Virus infection (HIV and end-stage-liver-disease (ESDL is rarely reported. The purpose of this study is to describe our institutional experience on OLT for HIV positive patients. Material and methods This is a retrospective study of all HIV-infected patients who underwent OLT at the University Hospital of Essen, from January 1996 to December 2009. Age, sex, HIV transmission-way, CDC-stage, etiology of ESDL, concomitant liver disease, last CD4cell count and HIV-viral load prior to OLT were collected and analysed. Standard calcineurin-inhibitors-based immunosuppression was applied. All patients received anti-fungal and anti-pneumocystis carinii pneumonia prophylaxis post-OLT. Results Eight transplanted HIV-infected patients with a median age of 46 years (range 35-61 years were included. OLT indications were HCV (n = 5, HBV (n = 2, HCV/HBV/HDV-related cirrhosis (n = 1 and acute liver-failure (n = 1. At OLT, CD4 cell-counts ranged from 113-621 cells/μl, and HIV viral-loads from Conclusions OLT in HIV-infected patients and ESLD is an acceptable therapeutic option in selected patients. Long-term survival can be achieved without HIV disease-progression under antiretroviral therapy and management of the viral hepatitis co-infection.

CULTIVATION OF HUMAN LIVER CELLS AND ADIPOSE-DERIVED MESENCHYMAL STROMAL CELLS IN PERFUSION BIOREACTOR

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Yu. В. Basok

2018-01-01

Full Text Available Aim: to show the progress of the experiment of cultivation of human liver cells and adipose-derived mesenchymal stromal cells in perfusion bioreactor.Materials and methods. The cultivation of a cell-engineered construct, consisting of a biopolymer microstructured collagen-containing hydrogel, human liver cells, adipose-derived mesenchymal stromal cells, and William’s E Medium, was performed in a perfusion bioreactor.Results. On the 7th day large cells with hepatocyte morphology – of a polygonal shape and a centrally located round nucleus, – were present in the culture chambers of the bioreactor. The metabolic activity of hepatocytes in cell-engineered constructs was confi rmed by the presence of urea in the culture medium on the seventh day of cultivation in the bioreactor and by the resorption of a biopolymer microstructured collagen-containing hydrogel.

The nutritional geometry of liver disease including non-alcoholic fatty liver disease.

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Simpson, Stephen J; Raubenheimer, David; Cogger, Victoria C; Macia, Laurence; Solon-Biet, Samantha M; Le Couteur, David G; George, Jacob

2018-02-01

Nutrition has a profound effect on chronic liver disease, especially non-alcoholic fatty liver disease (NAFLD). Most observational studies and clinical trials have focussed on the effects of total energy intake, or the intake of individual macronutrients and certain micronutrients, such as vitamin D, on liver disease. Although these studies have shown the importance of nutrition on hepatic outcomes, there is not yet any unifying framework for understanding the relationship between diet and liver disease. The Geometric Framework for Nutrition (GFN) is an innovative model for designing nutritional experiments or interpreting nutritional data that can determine the effects of nutrients and their interactions on animal behaviour and phenotypes. Recently the GFN has provided insights into the relationship between dietary energy and macronutrients on obesity and ageing in mammals including humans. Mouse studies using the GFN have disentangled the effects of macronutrients on fatty liver and the gut microbiome. The GFN is likely to play a significant role in disentangling the effects of nutrients on liver disease, especially NAFLD, in humans. Copyright © 2017 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Cytosolic 5'-nucleotidase II interacts with the leucin rich repeat of NLR family member Ipaf.

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Federico Cividini

Full Text Available IMP/GMP preferring cytosolic 5'-nucleotidase II (cN-II is a bifunctional enzyme whose activities and expression play crucial roles in nucleotide pool maintenance, nucleotide-dependent pathways and programmed cell death. Alignment of primary amino acid sequences of cN-II from human and other organisms show a strong conservation throughout the entire vertebrata taxon suggesting a fundamental role in eukaryotic cells. With the aim to investigate the potential role of this homology in protein-protein interactions, a two hybrid system screening of cN-II interactors was performed in S. cerevisiae. Among the X positive hits, the Leucin Rich Repeat (LRR domain of Ipaf was found to interact with cN-II. Recombinant Ipaf isoform B (lacking the Nucleotide Binding Domain was used in an in vitro affinity chromatography assay confirming the interaction obtained in the screening. Moreover, co-immunoprecipitation with proteins from wild type Human Embryonic Kidney 293 T cells demonstrated that endogenous cN-II co-immunoprecipitated both with wild type Ipaf and its LRR domain after transfection with corresponding expression vectors, but not with Ipaf lacking the LRR domain. These results suggest that the interaction takes place through the LRR domain of Ipaf. In addition, a proximity ligation assay was performed in A549 lung carcinoma cells and in MDA-MB-231 breast cancer cells and showed a positive cytosolic signal, confirming that this interaction occurs in human cells. This is the first report of a protein-protein interaction involving cN-II, suggesting either novel functions or an additional level of regulation of this complex enzyme.

Liver Immunology

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Bogdanos, Dimitrios P.; Gao, Bin; Gershwin, M. Eric

2014-01-01

The liver is the largest organ in the body and is generally regarded by non-immunologists as not having lymphoid function. However, such is far from accurate. This review highlights the importance of the liver as a lymphoid organ. Firstly, we discuss experimental data surrounding the role of liver as a lymphoid organ. The liver facilitates a tolerance rather than immunoreactivity, which protects the host from antigenic overload of dietary components and drugs derived from the gut and is also instrumental to fetal immune tolerance. Loss of liver tolerance leads to autoaggressive phenomena which if are not controlled by regulatory lymphoid populations may lead to the induction of autoimmune liver diseases. Liver-related lymphoid subpopulations also act as critical antigen-presenting cells. The study of the immunological properties of liver and delineation of the microenvironment of the intrahepatic milieu in normal and diseased livers provides a platform to understand the hierarchy of a series of detrimental events which lead to immune-mediated destruction of the liver and the rejection of liver allografts. The majority of emphasis within this review will be on the normal mononuclear cell composition of the liver. However, within this context, we will discus select, but not all, immune mediated liver disease and attempt to place these data in the context of human autoimmunity. PMID:23720323

Detection of titanium particles in human liver and spleen and possible health implications

NARCIS (Netherlands)

Heringa, M.B.; Peters, R.J.B.; Bleys, R.L.A.W.; Lee, van der M.K.; Tromp, P.C.; Kesteren, van P.C.E.; Eijkeren, van J.C.H.; Undas, A.K.; Oomen, A.G.; Bouwmeester, H.

2018-01-01

Background: Titanium dioxide (TiO2) is produced at high volumes and applied in many consumer and food products. Recent toxicokinetic modelling indicated the potential of TiO2 to accumulate in human liver and spleen upon daily oral exposure, which is not routinely investigated in chronic animal

Gene expression data from acetaminophen-induced toxicity in human hepatic in vitro systems and clinical liver samples

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Robim M. Rodrigues

2016-06-01

Full Text Available This data set is composed of transcriptomics analyses of (i liver samples from patients suffering from acetaminophen-induced acute liver failure (ALF and (ii hepatic cell systems exposed to acetaminophen and their respective controls. The in vitro systems include widely employed cell lines i.e. HepaRG and HepG2 cells as well as a novel stem cell-derived model i.e. human skin-precursors-derived hepatocyte-like cells (hSKP-HPC. Data from primary human hepatocytes was also added to the data set “Open TG-GATEs: a large-scale toxicogenomics database” (Igarashi et al., 2015 [1]. Changes in gene expression due to acetaminophen intoxication as well as comparative information between human in vivo and in vitro samples are provided. The microarray data have been deposited in NCBI׳s Gene Expression Omnibus and are accessible through GEO Series accession number GEO: GSE74000. The provided data is used to evaluate the predictive capacity of each hepatic in vitro system and can be directly compared with large-scale publically available toxicogenomics databases. Further interpretation and discussion of these data feature in the corresponding research article “Toxicogenomics-based prediction of acetaminophen-induced liver injury using human hepatic cell systems” (Rodrigues et al., 2016 [2].

Apoptotic DNA Degradation into Oligonucleosomal Fragments, but Not Apoptotic Nuclear Morphology, Relies on a Cytosolic Pool of DFF40/CAD Endonuclease*

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Iglesias-Guimarais, Victoria; Gil-Guiñon, Estel; Gabernet, Gisela; García-Belinchón, Mercè; Sánchez-Osuna, María; Casanelles, Elisenda; Comella, Joan X.; Yuste, Victor J.

2012-01-01

Apoptotic cell death is characterized by nuclear fragmentation and oligonucleosomal DNA degradation, mediated by the caspase-dependent specific activation of DFF40/CAD endonuclease. Here, we describe how, upon apoptotic stimuli, SK-N-AS human neuroblastoma-derived cells show apoptotic nuclear morphology without displaying concomitant internucleosomal DNA fragmentation. Cytotoxicity afforded after staurosporine treatment is comparable with that obtained in SH-SY5Y cells, which exhibit a complete apoptotic phenotype. SK-N-AS cell death is a caspase-dependent process that can be impaired by the pan-caspase inhibitor q-VD-OPh. The endogenous inhibitor of DFF40/CAD, ICAD, is correctly processed, and dff40/cad cDNA sequence does not reveal mutations altering its amino acid composition. Biochemical approaches show that both SH-SY5Y and SK-N-AS resting cells express comparable levels of DFF40/CAD. However, the endonuclease is poorly expressed in the cytosolic fraction of healthy SK-N-AS cells. Despite this differential subcellular distribution of DFF40/CAD, we find no differences in the subcellular localization of both pro-caspase-3 and ICAD between the analyzed cell lines. After staurosporine treatment, the preferential processing of ICAD in the cytosolic fraction allows the translocation of DFF40/CAD from this fraction to a chromatin-enriched one. Therefore, the low levels of cytosolic DFF40/CAD detected in SK-N-AS cells determine the absence of DNA laddering after staurosporine treatment. In these cells DFF40/CAD cytosolic levels can be restored by the overexpression of their own endonuclease, which is sufficient to make them proficient at degrading their chromatin into oligonucleosome-size fragments after staurosporine treatment. Altogether, the cytosolic levels of DFF40/CAD are determinants in achieving a complete apoptotic phenotype, including oligonucleosomal DNA degradation. PMID:22253444

Cytosolic Cl- Affects the Anticancer Activity of Paclitaxel in the Gastric Cancer Cell Line, MKN28 Cell

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Sachie Tanaka

2017-05-01

Full Text Available Background/Aims: Our previous study revealed that cytosolic Cl- affected neurite elongation promoted via assembly of microtubule in rat pheochromocytoma PC12D cells and Cl-–induced blockade of intrinsic GTPase enhanced tubulin polymerization in vitro. Paclitaxel (PTX is a microtubule-targeted chemotherapeutic drug and stabilizes microtubules resulting in mainly blockade of mitosis at the metaphase-anaphase transition and induction of apoptosis. In the present study, we tried to clarify whether the cytosolic Cl- affected PTX ability to inhibit cell growth in the gastric cancer cell line, MKN28. Methods: To clarify the cytosolic Cl- action on PTX-induced cell death and metaphase-anaphase transition in the gastric cancer cell line, MKN28 cell, and PTX-induced tubulin polymerization, we performed cell proliferation assay, cytosolic Cl- concentration measurement, immunofluorescence microscopy, and in vitro tubulin polymerization assay. Results: The decline of cytosolic Cl- weakened the cytotoxic effect of PTX on cell proliferation of MKN28 cells, which could pass through the metaphase-anaphase transition. Moreover, in vitro PTX-induced tubulin polymerization was diminished under the low Cl- condition. Conclusions: Our results strongly suggest that the upregulation of cytosolic Cl- concentration would enhance the antitumor effect of PTX, and that the cytosolic Cl- would be one of the key targets for anti-cancer therapy.

Postneonatal Mortality and Liver Changes in Cloned Pigs Associated with Human Tumor Necrosis Factor Receptor I-Fc and Human Heme Oxygenase-1 Overexpression

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Geon A. Kim

2017-01-01

Full Text Available Soluble human tumor necrosis factor (shTNFRI-Fc and human heme oxygenase 1 (hHO-1 are key regulators for protection against oxidative and inflammatory injury for xenotransplantation. Somatic cells with more than 10 copy numbers of shTNFRI-Fc and hHO-1 were employed in somatic cell nuclear transfer to generate cloned pigs, thereby resulting in seven cloned piglets. However, produced piglets were all dead within 24 hours after birth. Obviously, postnatal death with liver apoptosis was reported in the higher copy number of shTNFRI-Fc and hHO-1 piglets. In liver, the transcript levels of ferritin heavy chain, light chain, transferrin, and inducible nitric oxide synthase were significantly highly expressed compared to those of lower copy number of shTNFRI-Fc and hHO-1 piglets (P<0.05. Also, H2O2 contents were increased, and superoxide dismutase was significantly lower in the higher copy number of shTNFRI-Fc and hHO-1 piglets (P<0.05. These results indicate that TNFRI-Fc and hHO-1 overexpression may apparently induce free iron in the liver and exert oxidative stress by enhancing reactive oxygen species production and block normal postneonatal liver metabolism.

Differentiation of human-induced pluripotent stem cell under flow conditions to mature hepatocytes for liver tissue engineering

DEFF Research Database (Denmark)

Starokozhko, Viktoriia; Hemmingsen, Mette; Larsen, Layla

2018-01-01

and to perform functional comparisons with fresh human precision-cut liver slices (hPCLS), an excellent benchmark for the human liver in vivo. The majority of the mRNA expression of CYP isoenzymes and transporters and the tested CYP activities, Phase II metabolism, and albumin, urea, and bile acid synthesis...... in the hiPSC-derived cells reached values that overlap those of hPCLS, which indicates a higher degree of hepatic differentiation than observed until now. Differentiation under flow compared with static conditions had a strong inducing effect on Phase II metabolism and suppressed AFP expression but resulted...... in slightly lower activity of some of the Phase I metabolism enzymes. Gene expression data indicate that hiPSCs differentiated into both hepatic and biliary directions. In conclusion, the hiPSC differentiated under flow conditions towards hepatocytes express a wide spectrum of liver functions at levels...

Potent inhibition of cytochrome P450 2B6 by sibutramine in human liver microsomes.

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Bae, Soo Hyeon; Kwon, Min Jo; Choi, Eu Jin; Zheng, Yu Fen; Yoon, Kee Dong; Liu, Kwang-Hyeon; Bae, Soo Kyung

2013-09-05

The present study was performed to evaluate the potency and specificity of sibutramine as an inhibitor of the activities of nine human CYP isoforms in liver microsomes. Using a cocktail assay, the effects of sibutramine on specific marker reactions of the nine CYP isoforms were measured in human liver microsomes. Sibutramine showed potent inhibition of CYP2B6-mediated bupropion 6-hydroxylation with an IC50 value of 1.61μM and Ki value of 0.466μM in a competitive manner at microsomal protein concentrations of 0.25mg/ml; this was 3.49-fold more potent than the typical CYP2B6 inhibitor thio-TEPA (Ki=1.59μM). In addition, sibutramine slightly inhibited CYP2C19 activity (Ki=16.6μM, noncompetitive inhibition) and CYP2D6 activity (Ki=15.7μM, noncompetitive inhibition). These observations indicated 35.6- and 33.7-fold decreases in inhibition potency, respectively, compared with that of CYP2B6 by sibutramine. However, no inhibition of CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2D6, or CYP2E1 activities was observed. In addition, the CYP2B6 inhibitory potential of sibutramine was enhanced at a lower microsomal protein concentration of 0.05mg/ml. After 30min preincubation of human liver microsomes with sibutramine in the presence of NADPH, no shift in IC50 was observed in terms of inhibition of the activities of the nine CYPs, suggesting that sibutramine is not a time-dependent inactivator. These observations suggest that sibutramine is a selective and potent inhibitor of CYP2B6 in vitro, whereas inhibition of other CYPs is substantially lower. These in vitro data support the use of sibutramine as a well-known inhibitor of CYP2B6 for routine screening of P450 reversible inhibition when human liver microsomes are used as the enzyme source. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

TRIM56-mediated monoubiquitination of cGAS for cytosolic DNA sensing.

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Seo, Gil Ju; Kim, Charlotte; Shin, Woo-Jin; Sklan, Ella H; Eoh, Hyungjin; Jung, Jae U

2018-02-09

Intracellular nucleic acid sensors often undergo sophisticated modifications that are critical for the regulation of antimicrobial responses. Upon recognition of DNA, the cytosolic sensor cyclic GMP-AMP (cGAMP) synthase (cGAS) produces the second messenger cGAMP, which subsequently initiates downstream signaling to induce interferon-αβ (IFNαβ) production. Here we report that TRIM56 E3 ligase-induced monoubiquitination of cGAS is important for cytosolic DNA sensing and IFNαβ production to induce anti-DNA viral immunity. TRIM56 induces the Lys335 monoubiquitination of cGAS, resulting in a marked increase of its dimerization, DNA-binding activity, and cGAMP production. Consequently, TRIM56-deficient cells are defective in cGAS-mediated IFNαβ production upon herpes simplex virus-1 (HSV-1) infection. Furthermore, TRIM56-deficient mice show impaired IFNαβ production and high susceptibility to lethal HSV-1 infection but not to influenza A virus infection. This adds TRIM56 as a crucial component of the cytosolic DNA sensing pathway that induces anti-DNA viral innate immunity.

Induction of Cytosolic Acetyl-Coenzyme A Carboxylase in Pea Leaves by Ultraviolet-B Irradiation

OpenAIRE

Tomokazu, Konishi; Takahiro, Kamoi; Ryuichi, Matsuno; Yukiko, Sasaki; Department of Food Science and Technology, Faculty of Agriculture, Kyoto University:(Present)Laboratory of Molecular Genetics, Biotechnology Institute, Akita Prefectural College of Agriculture; Department of Food Science and Technology, Faculty of Agriculture, Kyoto University; Department of Food Science and Technology, Faculty of Agriculture, Kyoto University; Department of Food Science and Technology, Faculty of Agriculture, Kyoto University:(Present)Laboratory of Plant Molecular Biology, School of Agricultural Sciences, Nagoya University

1996-01-01

Levels of subunits of two acetyl-coenzyme A carboxylases were high in small leaves of Pisum sativum, decreased with growth, and remained constant in fully expanded leaves. Irradiation of fully expanded leaves induced the cytosolic isozyme only. This result suggests a key role for the cytosolic enzyme in protection against UV-B.

Aryl hydrocarbon receptor nuclear translocator in human liver is regulated by miR-24

Energy Technology Data Exchange (ETDEWEB)

Oda, Yuki; Nakajima, Miki; Mohri, Takuya [Drug Metabolism and Toxicology, Division of Pharmaceutical Sciences, Graduate School of Medical Science, Kanazawa University, Kakuma-machi, Kanazawa 920-1192 (Japan); Takamiya, Masataka; Aoki, Yasuhiro [Department of Legal Medicine, Iwate Medical University School of Medicine, 19-1 Uchimaru, Morioka 020-8505 (Japan); Fukami, Tatsuki [Drug Metabolism and Toxicology, Division of Pharmaceutical Sciences, Graduate School of Medical Science, Kanazawa University, Kakuma-machi, Kanazawa 920-1192 (Japan); Yokoi, Tsuyoshi, E-mail: tyokoi@kenroku.kanazawa-u.ac.jp [Drug Metabolism and Toxicology, Division of Pharmaceutical Sciences, Graduate School of Medical Science, Kanazawa University, Kakuma-machi, Kanazawa 920-1192 (Japan)

2012-05-01

Aryl hydrocarbon receptor nuclear translocator (ARNT) forms a heterodimer with aryl hydrocarbon receptor or hypoxia inducible factor 1α to mediate biological responses to xenobiotic exposure and hypoxia. Although the regulation mechanism of the ARNT expression is largely unknown, earlier studies reported that the human ARNT protein level was decreased by hydrogen peroxide or reactive oxygen species. These stimuli increase the miR-24 level in various human cell lines. In silico analysis predicts that some microRNAs including miR-16 and miR-23b may bind to ARNT mRNA. This background prompted us to investigate whether human ARNT is regulated by microRNAs. Overexpression of miR-24 into HuH-7 and HepG2 cells significantly decreased the ARNT protein level, but not the ARNT mRNA level, indicating translational repression. However, overexpression of miR-16 or miR-23b caused no change in the ARNT expression. The miR-24-dependent down-regulation of ARNT decreased the expression of its downstream genes such as CYP1A1 and carbonic anhydrase IX. Luciferase assay was performed to determine the element on the ARNT mRNA to which miR-24 binds. Finally, it was demonstrated that the miR-24 levels in a panel of 26 human livers were inversely correlated with the protein levels or the translational efficiency of ARNT. Taken together, we found that miR-24 negatively regulates ARNT expression in human liver, affecting the expression of its downstream genes. miR-24 would be one of the factors underlying the mechanisms by which ARNT protein is decreased by reactive oxygen species. -- Highlights: ► Overexpression of miR-24 into human cell lines decreased the ARNT protein level. ► miR-24-dependent down-regulation of ARNT affected the expression of CYP1A1 and CA IX. ► Luciferase assay was performed to identify functional MREs for miR-24 in ARNT mRNA. ► The miR-24 levels inversely correlated with the ARNT protein levels in human liver.

Stiffness of hyaluronic acid gels containing liver extracellular matrix supports human hepatocyte function and alters cell morphology.

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Deegan, Daniel B; Zimmerman, Cynthia; Skardal, Aleksander; Atala, Anthony; Shupe, Thomas D

2015-03-01

Tissue engineering and cell based liver therapies have utilized primary hepatocytes with limited success due to the failure of hepatocytes to maintain their phenotype in vitro. In order to overcome this challenge, hyaluronic acid (HA) cell culture substrates were formulated to closely mimic the composition and stiffness of the normal liver cellular microenvironment. The stiffness of the substrate was modulated by adjusting HA hydrogel crosslinking. Additionally, the repertoire of bioactive molecules within the HA substrate was bolstered by supplementation with normal liver extracellular matrix (ECM). Primary human hepatocyte viability and phenotype were determined over a narrow physiologically relevant range of substrate stiffnesses from 600 to 4600Pa in both the presence and absence of liver ECM. Cell attachment, viability, and organization of the actin cytoskeleton improved with increased stiffness up to 4600Pa. These differences were not evident in earlier time points or substrates containing only HA. However, gene expression for the hepatocyte markers hepatocyte nuclear factor 4 alpha (HNF4α) and albumin significantly decreased on the 4600Pa stiffness at day 7 indicating that cells may not have maintained their phenotype long-term at this stiffness. Function, as measured by albumin secretion, varied with both stiffness and time in culture and peaked at day 7 at the 1200Pa stiffness, slightly below the stiffness of normal liver ECM at 3000Pa. Overall, gel stiffness affected primary human hepatocyte cell adhesion, functional marker expression, and morphological characteristics dependent on both the presence of liver ECM in gel substrates and time in culture. Copyright © 2015 Elsevier Ltd. All rights reserved.

Blood cell oxidative stress precedes hemolysis in whole blood-liver slice co-cultures of rat, dog, and human tissues

International Nuclear Information System (INIS)

Vickers, Alison E.M.; Sinclair, John R.; Fisher, Robyn L.; Morris, Stephen R.; Way, William

2010-01-01

A novel in vitro model to investigate time-dependent and concentration-dependent responses in blood cells and hemolytic events is studied for rat, dog, and human tissues. Whole blood is co-cultured with a precision-cut liver slice. Methimazole (MMI) was selected as a reference compound, since metabolism of its imidazole thione moiety is linked with hematologic disorders and hepatotoxicity. An oxidative stress response occurred in all three species, marked by a decline in blood GSH levels by 24 h that progressed, and preceded hemolysis, which occurred at high MMI concentrations in the presence of a liver slice with rat (≥ 1000 μM at 48 h) and human tissues (≥ 1000 μM at 48 h, ≥ 750 μM at 72 h) but not dog. Human blood-only cultures exhibited a decline of GSH levels but minimal to no hemolysis. The up-regulation of liver genes for heme degradation (Hmox1 and Prdx1), iron cellular transport (Slc40a1), and GSH synthesis and utilization (mGST1 and Gclc) were early markers of the oxidative stress response. The up-regulation of the Kupffer cell lectin Lgals3 gene expression indicated a response to damaged red blood cells, and Hp (haptoglobin) up-regulation is indicative of increased hemoglobin uptake. Up-regulation of liver IL-6 and IL-8 gene expression suggested an activation of an inflammatory response by liver endothelial cells. In summary, MMI exposure led to an oxidative stress response in blood cells, and an up-regulation of liver genes involved with oxidative stress and heme homeostasis, which was clearly separate and preceded frank hemolysis.

PHYSICAL CONTACT BETWEEN HUMAN VASCULAR ENDOTHELIAL AND SMOOTH MUSCLE CELLS MODULATES CYTOSOLIC AND NUCLEAR CALCIUM HOMEOSTASIS.

Science.gov (United States)

Hassan, Ghada S; Jacques, Danielle; D'Orleans-Juste, Pedro; Magder, Sheldon; Bkaily, Ghassan

2018-05-14

The interaction between vascular endothelial cells (VECs) and vascular smooth muscle cells (VSMCs) plays an important role in the modulation of vascular tone. There is however no information on whether direct physical communication regulates the intracellular calcium levels of human VECs (hVECs) and/or hVSMCs . Thus, the objective of the study is to verify whether co-culture of hVECs and hVSMCs modulates cytosolic ([Ca2+]c) and nuclear calcium ([Ca2+]n) levels via physical contact and/or factors released by both cell types. Quantitative 3D confocal microscopy for [Ca2+]c and [Ca2+]n measurement was performed in cultured hVECs or hVSMCs or in co-culture of hVECs-hVSMCs. Our results show that: 1) physical contact between hVECs-hVECs or hVSMCs-hVSMCs does not affect [Ca2+]c and [Ca2+]n in these two cell types; 2) physical contact between hVECs and hVSMCs induces a significant increase only of [Ca2+]n of hVECs without affecting the level of [Ca2+]c and [Ca2+]n of hVSMCs; and 3) preconditioned culture medium of hVECs or hVSMCs does not affect [Ca2+]c and [Ca2+]n of both types of cells. We concluded that physical contact between hVECs and hVSMCs only modulates [Ca2+]n in hVECs. The increase of [Ca2+]n in hVECs may modulate nuclear functions that are calcium dependent.

The effect of mitochondrial dysfunction on cytosolic nucleotide metabolism

DEFF Research Database (Denmark)

Madsen, Claus Desler; Lykke, Anne; Rasmussen, Lene Juel

2010-01-01

Several enzymes of the metabolic pathways responsible for metabolism of cytosolic ribonucleotides and deoxyribonucleotides are located in mitochondria. Studies described in this paper suggest dysfunction of the mitochondria to affect these metabolic pathways and limit the available levels...

Detection and characterization of Ah receptor in tissue and cells from human tonsils

International Nuclear Information System (INIS)

Lorenzen, A.; Okey, A.B.

1991-01-01

Ah receptor was identified and characterized in cytosol and nuclear extracts from human tonsils obtained at surgery from children 2 to 6 years of age. Ah receptor was found in cytosol prepared from whole-tonsil homogenates as well as in cytosol and nuclear fractions prepared from tonsil lymphocytes or tonsil fibroblasts grown in primary culture. Cytosolic Ah receptor was detectable in tonsillar tissue with either halogenated (2,3,7,8-[3H]tetrachlorodibenzo-p-dioxin (TCDD)) or nonhalogenated (3-[3H]methylcholanthrene and [3H]benzo[a]pyrene) aromatic hydrocarbons and sedimented at approximately 9 S after velocity sedimentation on sucrose gradients. The apparent binding affinity (Kd) of [3H]TCDD for Ah receptor ranged from 3 to 12 nM in cytosols from seven different donors. The same analyses indicated a concentration of Ah receptor in human tonsils of approximately 100-300 fmol/mg cytosolic protein. Incubation of either tonsil lymphocytes or tonsil fibroblasts with [3H]TCDD resulted in transformation of cytosolic Ah receptor to a nuclear binding form which could be detected as a specifically labeled peak sedimenting at approximately 6 S on sucrose gradients. These data demonstrate the existence of Ah receptor in human tonsils and suggest that this immune organ may be an appropriate model for further studies on the mechanism and manifestation of aromatic hydrocarbon-induced immunotoxicity in man

Autoimmune liver serology: current diagnostic and clinical challenges.

Science.gov (United States)

Bogdanos, Dimitrios-P; Invernizzi, Pietro; Mackay, Ian-R; Vergani, Diego

2008-06-07

Liver-related autoantibodies are crucial for the correct diagnosis and classification of autoimmune liver diseases (AiLD), namely autoimmune hepatitis types 1 and 2 (AIH-1 and 2), primary biliary cirrhosis (PBC), and the sclerosing cholangitis variants in adults and children. AIH-1 is specified by anti-nuclear antibody (ANA) and smooth muscle antibody (SMA). AIH-2 is specified by antibody to liver kidney microsomal antigen type-1 (anti-LKM1) and anti-liver cytosol type 1 (anti-LC1). SMA, ANA and anti-LKM antibodies can be present in de-novo AIH following liver transplantation. PBC is specified by antimitochondrial antibodies (AMA) reacting with enzymes of the 2-oxo-acid dehydrogenase complexes (chiefly pyruvate dehydrogenase complex E2 subunit) and disease-specific ANA mainly reacting with nuclear pore gp210 and nuclear body sp100. Sclerosing cholangitis presents as at least two variants, first the classical primary sclerosing cholangitis (PSC) mostly affecting adult men wherein the only (and non-specific) reactivity is an atypical perinuclear antineutrophil cytoplasmic antibody (p-ANCA), also termed perinuclear anti-neutrophil nuclear antibodies (p-ANNA) and second the childhood disease called autoimmune sclerosing cholangitis (ASC) with serological features resembling those of type 1 AIH. Liver diagnostic serology is a fast-expanding area of investigation as new purified and recombinant autoantigens, and automated technologies such as ELISAs and bead assays, become available to complement (or even compete with) traditional immunofluorescence procedures. We survey for the first time global trends in quality assurance impacting as it does on (1) manufacturers/purveyors of kits and reagents, (2) diagnostic service laboratories that fulfill clinicians' requirements, and (3) the end-user, the physician providing patient care, who must properly interpret test results in the overall clinical context.

The proteome of human liver peroxisomes: identification of five new peroxisomal constituents by a label-free quantitative proteomics survey.

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Thomas Gronemeyer

Full Text Available The peroxisome is a key organelle of low abundance that fulfils various functions essential for human cell metabolism. Severe genetic diseases in humans are caused by defects in peroxisome biogenesis or deficiencies in the function of single peroxisomal proteins. To improve our knowledge of this important cellular structure, we studied for the first time human liver peroxisomes by quantitative proteomics. Peroxisomes were isolated by differential and Nycodenz density gradient centrifugation. A label-free quantitative study of 314 proteins across the density gradient was accomplished using high resolution mass spectrometry. By pairing statistical data evaluation, cDNA cloning and in vivo colocalization studies, we report the association of five new proteins with human liver peroxisomes. Among these, isochorismatase domain containing 1 protein points to the existence of a new metabolic pathway and hydroxysteroid dehydrogenase like 2 protein is likely involved in the transport or β-oxidation of fatty acids in human peroxisomes. The detection of alcohol dehydrogenase 1A suggests the presence of an alternative alcohol-oxidizing system in hepatic peroxisomes. In addition, lactate dehydrogenase A and malate dehydrogenase 1 partially associate with human liver peroxisomes and enzyme activity profiles support the idea that NAD(+ becomes regenerated during fatty acid β-oxidation by alternative shuttling processes in human peroxisomes involving lactate dehydrogenase and/or malate dehydrogenase. Taken together, our data represent a valuable resource for future studies of peroxisome biochemistry that will advance research of human peroxisomes in health and disease.

In vivo hepatic differentiation potential of human umbilical cord-derived mesenchymal stem cells: Therapeutic effect on liver fibrosis/cirrhosis

OpenAIRE

Zhang, Guo-Zun; Sun, Hui-Cong; Zheng, Li-Bo; Guo, Jin-Bo; Zhang, Xiao-Lan

2017-01-01

AIM To investigate the hepatic differentiation potential of human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) and to evaluate their therapeutic effect on liver fibrosis/cirrhosis. METHODS A CCl4-induced liver fibrotic/cirrhotic rat model was used to assess the effect of hUC-MSCs. Histopathology was assessed by hematoxylin and eosin (H&E), Masson trichrome and Sirius red staining. The liver biochemical profile was measured using a Beckman Coulter analyzer. Expression analysis was ...

Roles of different forms of cytochrome P450 in the activation of the promutagen 6-aminochrysene to genotoxic metabolites in human liver microsomes.

Science.gov (United States)

Yamazaki, H; Mimura, M; Oda, Y; Inui, Y; Shiraga, T; Iwasaki, K; Guengerich, F P; Shimada, T

1993-07-01

We reported previously that the potent mutagen 6-aminochrysene is catalyzed principally by rat liver microsomal P4501A and P4502B enzymes to reactive metabolites that induce umu gene expression in O-acetyltransferase-over-expressing strain Salmonella typhimurium NM2009; the proposal was made that there are different mechanisms in the formation of reactive N-hydroxylated and diolepoxide metabolites by P450 enzymes (Yamazaki, H. and Shimada, T., Biochem. Pharmacol., 44, 913-920, 1992). Here we further examined the roles of human liver P450 enzymes and the mechanism of activation of 6-aminochrysene by rat and human P450 enzymes in the Salmonella tester strains. Liver microsomes from 18 different human samples catalyzed activation of 6-aminochrysene more efficiently in S. typhimurium NM2009 than in the original strain of S. typhimurium TA1535/pSK1002. The rates of 6-aminochrysene activation in 18 human liver samples showed good correlation to the contents of P4502B6 as well as contents of P4503A4 and the respective mono-oxygenase activities catalyzed by P4503A4. Among purified P450 enzymes examined, P4501A2 as well as P4503A4 were highly active in transforming 6-amino-chrysene to reactive metabolites, suggesting the involvement of different human P450 enzymes in the reaction. Four human samples that contained relatively high levels of particular P450 enzymes in their microsomes were selected and used for further characterization. Liver microsomes from human samples HL-13 and HL-4 that contained the highest levels of P4502B6 and P4503A4 respectively, were sensitive to the respective antibodies raised against monkey P4502B and human P4503A4; the activity in sample HL-16 having the highest level of P4501A2 was inhibited by anti-P4501A2 IgG. alpha-Naphthoflavone enhanced the activation of 6-aminochrysene very significantly in human liver microsomes enriched in P4503A4 and P4502B6 enzymes. Pentachlorophenol, an inhibitor of acetyltransferase activity, suppressed the

Extracellular vesicles from human liver stem cells restore argininosuccinate synthase deficiency.

Science.gov (United States)

Herrera Sanchez, Maria Beatriz; Previdi, Sara; Bruno, Stefania; Fonsato, Valentina; Deregibus, Maria Chiara; Kholia, Sharad; Petrillo, Sara; Tolosano, Emanuela; Critelli, Rossana; Spada, Marco; Romagnoli, Renato; Salizzoni, Mauro; Tetta, Ciro; Camussi, Giovanni

2017-07-27

Argininosuccinate synthase (ASS)1 is a urea cycle enzyme that catalyzes the conversion of citrulline and aspartate to argininosuccinate. Mutations in the ASS1 gene cause citrullinemia type I, a rare autosomal recessive disorder characterized by neonatal hyperammonemia, elevated citrulline levels, and early neonatal death. Treatment for this disease is currently restricted to liver transplantation; however, due to limited organ availability, substitute therapies are required. Recently, extracellular vesicles (EVs) have been reported to act as intercellular transporters carrying genetic information responsible for cell reprogramming. In previous studies, we isolated a population of stem cell-like cells known as human liver stem cells (HLSCs) from healthy liver tissue. Moreover, EVs derived from HLSCs were reported to exhibit regenerative effects on the liver parenchyma in models of acute liver injury. The aim of this study was to evaluate whether EVs derived from normal HLSCs restored ASS1 enzymatic activity and urea production in hepatocytes differentiated from HLSCs derived from a patient with type I citrullinemia. HLSCs were isolated from the liver of a patient with type I citrullinemia (ASS1-HLSCs) and characterized by fluorescence-activated cell sorting (FACS), immunofluorescence, and DNA sequencing analysis. Furthermore, their differentiation capabilities in vitro were also assessed. Hepatocytes differentiated from ASS1-HLSCs were evaluated by the production of urea and ASS enzymatic activity. EVs derived from normal HLSCs were purified by differential ultracentrifugation followed by floating density gradient. The EV content was analyzed to identify the presence of ASS1 protein, mRNA, and ASS1 gene. In order to obtain ASS1-depleted EVs, a knockdown of the ASS1 gene in HLSCs was performed followed by EV isolation from these cells. Treating ASS1-HLSCs with EVs from HLSCs restored both ASS1 activity and urea production mainly through the transfer of ASS1 enzyme

Bisphenol A sulfonation is impaired in metabolic and liver disease

International Nuclear Information System (INIS)

Yalcin, Emine B.; Kulkarni, Supriya R.; Slitt, Angela L.; King, Roberta

2016-01-01

Background: Bisphenol A (BPA) is a widely used industrial chemical and suspected endocrine disruptor to which humans are ubiquitously exposed. The liver metabolizes and facilitates BPA excretion through glucuronidation and sulfonation. The sulfotransferase enzymes contributing to BPA sulfonation (detected in human and rodents) is poorly understood. Objectives: To determine the impact of metabolic and liver disease on BPA sulfonation in human and mouse livers. Methods: The capacity for BPA sulfonation was determined in human liver samples that were categorized into different stages of metabolic and liver disease (including obesity, diabetes, steatosis, and cirrhosis) and in livers from ob/ob mice. Results: In human liver tissues, BPA sulfonation was substantially lower in livers from subjects with steatosis (23%), diabetes cirrhosis (16%), and cirrhosis (18%), relative to healthy individuals with non-fatty livers (100%). In livers of obese mice (ob/ob), BPA sulfonation was lower (23%) than in livers from lean wild-type controls (100%). In addition to BPA sulfonation activity, Sult1a1 protein expression decreased by 97% in obese mouse livers. Conclusion: Taken together these findings establish a profoundly reduced capacity of BPA elimination via sulfonation in obese or diabetic individuals and in those with fatty or cirrhotic livers versus individuals with healthy livers. - Highlights: • Present study demonstrates that hepatic SULT 1A1/1A3 are primarily sulfonate BPA in mouse and human. • Hepatic BPA sulfonation is profoundly reduced steatosis, diabetes and cirrhosis. • With BPA-S detectable in urine under low or common exposures, these findings are novel and important.

Bisphenol A sulfonation is impaired in metabolic and liver disease

Energy Technology Data Exchange (ETDEWEB)

Yalcin, Emine B.; Kulkarni, Supriya R.; Slitt, Angela L., E-mail: angela_slitt@uri.edu; King, Roberta, E-mail: rking@uri.edu

2016-02-01

Background: Bisphenol A (BPA) is a widely used industrial chemical and suspected endocrine disruptor to which humans are ubiquitously exposed. The liver metabolizes and facilitates BPA excretion through glucuronidation and sulfonation. The sulfotransferase enzymes contributing to BPA sulfonation (detected in human and rodents) is poorly understood. Objectives: To determine the impact of metabolic and liver disease on BPA sulfonation in human and mouse livers. Methods: The capacity for BPA sulfonation was determined in human liver samples that were categorized into different stages of metabolic and liver disease (including obesity, diabetes, steatosis, and cirrhosis) and in livers from ob/ob mice. Results: In human liver tissues, BPA sulfonation was substantially lower in livers from subjects with steatosis (23%), diabetes cirrhosis (16%), and cirrhosis (18%), relative to healthy individuals with non-fatty livers (100%). In livers of obese mice (ob/ob), BPA sulfonation was lower (23%) than in livers from lean wild-type controls (100%). In addition to BPA sulfonation activity, Sult1a1 protein expression decreased by 97% in obese mouse livers. Conclusion: Taken together these findings establish a profoundly reduced capacity of BPA elimination via sulfonation in obese or diabetic individuals and in those with fatty or cirrhotic livers versus individuals with healthy livers. - Highlights: • Present study demonstrates that hepatic SULT 1A1/1A3 are primarily sulfonate BPA in mouse and human. • Hepatic BPA sulfonation is profoundly reduced steatosis, diabetes and cirrhosis. • With BPA-S detectable in urine under low or common exposures, these findings are novel and important.

Expression and Regulation of the Bile Acid Transporter, OST alpha-OST beta in Rat and Human Intestine and Liver

NARCIS (Netherlands)

Khan, Ansar A.; Chow, Edwin C. Y.; Porte, Robert J.; Pang, K. Sandy; Groothuis, Geny M. M.

The regulation of the OST alpha and OST beta expression was studied in the rat jejunum, ileum, colon and liver and in human ileum and liver by ligands for the farnesoid X receptor (FXR), pregnane X receptor (PXR), vitamin D receptor (VDR) and glucocorticoid receptor (GR) using precision cut tissue

Metabolism of 5-fluorouracil in human liver: an in vivo 19F NMR study

International Nuclear Information System (INIS)

Mohankrishnan, P.; Sprigg, J.; Cardwell, D.; Komoroski, R.A.; Hutchins, L.; Nauke, S.; Williamson, M.R.; Jagannathan, N.R.

1999-01-01

In vivo fluorine-19 nuclear magnetic resonance ( 19 F NMR) spectroscopy was used to study the metabolism and pharmacokinetics of 5-fluorouracil (5-FU) in human liver. Nine patients received 5-FU, and additional chemotherapeutic agents (methotrexate, leucovorin, or levamisole) either prophylactically after breast cancer surgery or for colorectal cancer. The time constant for the disappearance of 5-FU from the liver in vivo varied from 5 to 17 min, while the time constant for the appearance of α-fluoro-β-alanine (the major catabolite of 5 FU) varied from 7 to 86 min. The modulators of 5-FU metabolism did not appear to affect the time constant for the disappearance of 5-FU from the liver or for the appearance of α-fluoro-β-alanine. Results obtained indicate that the pharmacokinetics of 5-FU and α-fluoro-β-alanine may vary substantially at different times in a given individual. (author)

Negative Effect of Ellagic Acid on Cytosolic pH Regulation and Glycolytic Flux in Human Endometrial Cancer Cells

OpenAIRE

Khalid N. M. Abdelazeem; Yogesh Singh; Florian Lang; Madhuri S. Salker

2017-01-01

Background/Aims: Key properties of tumor cells include enhanced glycolytic flux with excessive consumption of glucose and formation of lactate. As glycolysis is highly sensitive to cytosolic pH, maintenance of glycolysis requires export of H+ ions, which is in part accomplished by Na+/H+ exchangers, such as NHE1. The carrier is sensitive to oxidative stress. Growth of tumor cells could be suppressed by the polyphenol Ellagic acid, which is found in various fruits and vegetables. An effect of ...

UPLC/MS MS data of testosterone metabolites in human and zebrafish liver microsomes and whole zebrafish larval microsomes

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Moayad Saad

2018-02-01

Full Text Available This article represents data regarding a study published in Toxicology in vitro entitled “ in vitro CYP-mediated drug metabolism in the zebrafish (embryo using human reference compounds” (Saad et al., 2017 [1]. Data were acquired with ultra-performance liquid chromatography – accurate mass mass spectrometry (UPLC-amMS. A full spectrum scan was conducted for the testosterone (TST metabolites from the microsomal stability assay in zebrafish and humans. The microsomal proteins were extracted from adult zebrafish male (MLM and female (FLM livers, whole body homogenates of 96 h post fertilization larvae (EM and a pool of human liver microsomes from 50 donors (HLM. Data are expressed as the abundance from the extracted ion chromatogram of the metabolites.

Comparison of monoclonal antibodies and tritiated ligands for estrogen receptor assays in 241 breast cancer cytosols

International Nuclear Information System (INIS)

Goussard, J.; Lechevrel, C.; Martin, P.M.; Roussel, G.

1986-01-01

Estrogen receptor determinations have been performed on 241 cytosols from 160 breast cancer tumors using both radioactive ligands ([ 3 H]-estradiol, [3H]R2858) and monoclonal antibodies (Abbott ER-EIA Kit) to compare the two methods and to evaluate the clinical usefulness of the new immunological, simplified assay. Intra- and interassay reproducibility of the enzyme immunoassay (EIA) method was studied during a 6-month period on 35 standard curves with 4 different batches of monoclonal antibodies. Intraassay coefficients of variation studied on duplicates were smaller than 5% in most cases and reproducibility of the curves showed coefficients of variation lower than 10% except for standard 0 and 5 fmol/ml. Pooled cytosols used as control for the dextran coated charcoal method had interassay variation coefficients between 3.8 and 11.4%. Reproducibility has been studied on clinical specimens assayed twice at two different periods with either EIA or dextran coated charcoal methods. Slopes obtained were 1.05 and 0.96, respectively. A good stability of EIA results was obtained with protein concentrations in the range 4-0.15 mg/ml cytosol. No significant effects of dithiothreitol or monothioglycerol (1 mM) on EIA and dextran coated charcoal assay were observed. Eighty breast cancer cytosols were assayed with both EIA and Scatchard analysis. The slope of the regression curve obtained was 1.04 (r = 0.963). Cytosols were assayed by EIA and by a saturating concentration of tritiated ligand (5 nM). With 153 cytosols the EIA/5 nM slope was 1.34 (r = 0.978). This slope can be compared with the slope Scatchard/5 nM obtained with 90 cytosols: 1.29 (r = 0.985). Absence of cross-reactivity of monoclonal ER antibodies with progesterone receptor was observed

Alterations in cytosol free calcium in horseradish roots simultaneously exposed to lanthanum(III) and acid rain.

Science.gov (United States)

Zhang, Xuanbo; Wang, Lihong; Zhou, Anhua; Zhou, Qing; Huang, Xiaohua

2016-04-01

The extensive use of rare earth elements (REEs) has increased their environmental levels. REE pollution concomitant with acid rain in many agricultural regions can affect crop growth. Cytosol free calcium ions (Ca(2+)) play an important role in almost all cellular activities. However, no data have been reported regarding the role of cytosol free Ca(2+) in plant roots simultaneously exposed to REE and acid rain. In this study, the effects of exposures to lanthanum(III) and acid rain, independently and in combination, on cytosol free Ca(2+) levels, root activity, metal contents, biomass, cytosol pH and La contents in horseradish roots were investigated. The simultaneous exposures to La(III) and acid rain increased or decreased the cytosol free Ca(2+) levels, depending on the concentration of La(III), and these effects were more evident than independent exposure to La(III) or acid rain. In combined exposures, cytosol free Ca(2+) played an important role in the regulation of root activity, metal contents and biomass. These roles were closely related to La(III) dose, acid rain strength and treatment mode (independent exposure or simultaneous exposure). A low concentration of La(III) (20 mg L(-1)) could alleviate the adverse effects on the roots caused by acid rain, and the combined exposures at higher concentrations of La(III) and acid rain had synergic effects on the roots. Copyright © 2015 Elsevier Inc. All rights reserved.

Initial prevalence of anal human papilloma virus infection in liver transplant recipients.

Science.gov (United States)

Grąt, Michał; Grąt, Karolina; Hołówko, Wacław; Malejczyk, Magdalena; Walter de Walthoffen, Szymon; Lewandowski, Zbigniew; Kobryń, Konrad; Patkowski, Waldemar; Majewski, Sławomir; Młynarczyk, Grażyna; Krawczyk, Marek

2014-08-01

Although liver transplant recipients are at increased risk of human papilloma virus (HPV)-related anal cancer, limited data are available regarding the initial prevalence of anal HPV infection in this population. Anal swabs collected from 50 liver transplant recipients within the first three postoperative weeks were subjected to real-time polymerase chain reaction for detection of the four HPV genotypes: 6, 11, 16, and 18. Predictors of any, low-risk, and high-risk anal HPV infection were evaluated. Overall, the prevalence of any anal HPV infection was 18.0%, with the corresponding rates for high- and low-risk HPV genotypes being 8.0% and 10.0%, respectively. Infection with any type of anal HPV was higher in patients with hepatitis B virus (HBV) infection (P = 0.027), ≥3 sexual partners (P = 0.031), and alcoholic liver disease (P = 0.063). HBV infection was the only factor significantly associated with high-risk HPV infection (P = 0.038). Male sex (P = 0.050), age ≥52 years (P = 0.016), ≥30 sexual partners (P = 0.003), age at first intercourse ≤18 years (P = 0.045), and time since first intercourse ≥38 years (P = 0.012) were identified as predictors of low-risk HPV infection. These results indicate that HPV vaccination of liver transplant candidates and screening for anal HPV infection in high-risk groups should be considered. © 2014 Steunstichting ESOT.

A high level of liver-specific expression of oncogenic KrasV12 drives robust liver tumorigenesis in transgenic zebrafish

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Anh Tuan Nguyen

2011-11-01

Human liver cancer is one of the deadliest cancers worldwide, with hepatocellular carcinoma (HCC being the most common type. Aberrant Ras signaling has been implicated in the development and progression of human HCC, but a complete understanding of the molecular mechanisms of this protein in hepatocarcinogenesis remains elusive. In this study, a stable in vivo liver cancer model using transgenic zebrafish was generated to elucidate Ras-driven tumorigenesis in HCC. Using the liver-specific fabp10 (fatty acid binding protein 10 promoter, we overexpressed oncogenic krasV12 specifically in the transgenic zebrafish liver. Only a high level of krasV12 expression initiated liver tumorigenesis, which progressed from hyperplasia to benign and malignant tumors with activation of the Ras-Raf-MEK-ERK and Wnt–β-catenin pathways. Histological diagnosis of zebrafish tumors identified HCC as the main lesion. The tumors were invasive and transplantable, indicating malignancy of these HCC cells. Oncogenic krasV12 was also found to trigger p53-dependent senescence as a tumor suppressive barrier in the pre-neoplastic stage. Microarray analysis of zebrafish liver hyperplasia and HCC uncovered the deregulation of several stage-specific and common biological processes and signaling pathways responsible for krasV12-driven liver tumorigenesis that recapitulated the molecular hallmarks of human liver cancer. Cross-species comparisons of cancer transcriptomes further defined a HCC-specific gene signature as well as a liver cancer progression gene signature that are evolutionarily conserved between human and zebrafish. Collectively, our study presents a comprehensive portrait of molecular mechanisms during progressive Ras-induced HCC. These observations indicate the validity of our transgenic zebrafish to model human liver cancer, and this model might act as a useful platform for drug screening and identifying new therapeutic targets.

Test systems in drug discovery for hazard identification and risk assessment of human drug-induced liver injury.

Science.gov (United States)

Weaver, Richard J; Betts, Catherine; Blomme, Eric A G; Gerets, Helga H J; Gjervig Jensen, Klaus; Hewitt, Philip G; Juhila, Satu; Labbe, Gilles; Liguori, Michael J; Mesens, Natalie; Ogese, Monday O; Persson, Mikael; Snoeys, Jan; Stevens, James L; Walker, Tracy; Park, B Kevin

2017-07-01

The liver is an important target for drug-induced toxicities. Early detection of hepatotoxic drugs requires use of well-characterized test systems, yet current knowledge, gaps and limitations of tests employed remains an important issue for drug development. Areas Covered: The current state of the science, understanding and application of test systems in use for the detection of drug-induced cytotoxicity, mitochondrial toxicity, cholestasis and inflammation is summarized. The test systems highlighted herein cover mostly in vitro and some in vivo models and endpoint measurements used in the assessment of small molecule toxic liabilities. Opportunities for research efforts in areas necessitating the development of specific tests and improved mechanistic understanding are highlighted. Expert Opinion: Use of in vitro test systems for safety optimization will remain a core activity in drug discovery. Substantial inroads have been made with a number of assays established for human Drug-induced Liver Injury. There nevertheless remain significant gaps with a need for improved in vitro tools and novel tests to address specific mechanisms of human Drug-Induced Liver Injury. Progress in these areas will necessitate not only models fit for application, but also mechanistic understanding of how chemical insult on the liver occurs in order to identify translational and quantifiable readouts for decision-making.

Exchange of cytosolic content between T cells and tumor cells activates CD4 T cells and impedes cancer growth.

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Matthias Hardtke-Wolenski

Full Text Available BACKGROUND: T cells are known to participate in the response to tumor cells and react with cytotoxicity and cytokine release. At the same time tumors established versatile mechanisms for silencing the immune responses. The interplay is far from being completely understood. In this study we show contacts between tumor cells and lymphocytes revealing novel characteristics in the interaction of T cells and cancer cells in a way not previously described. METHODS/ FINDINGS: Experiments are based on the usage of a hydrophilic fluorescent dye that occurs free in the cytosol and thus transfer of fluorescent cytosol from one cell to the other can be observed using flow cytometry. Tumor cells from cell lines of different origin or primary hepatocellular carcinoma (HCC cells were incubated with lymphocytes from human and mice. This exposure provoked a contact dependent uptake of tumor derived cytosol by lymphocytes--even in CD4⁺ T cells and murine B cells--which could not be detected after incubation of lymphocytes with healthy cells. The interaction was a direct one, not requiring the presence of accessory cells, but independent of cytotoxicity and TCR engagement. Electron microscopy disclosed 100-200 nm large gaps in the cell membranes of connected cells which separated viable and revealed astonishing outcome. While the lymphocytes were induced to proliferate in a long term fashion, the tumor cells underwent a temporary break in cell division. The in vitro results were confirmed in vivo using a murine acute lymphoblastic leukemia (ALL model. The arrest of tumor proliferation resulted in a significant prolonged survival of challenged mice. CONCLUSIONS: The reported cell-cell contacts reveal new characteristics i.e. the enabling of cytosol flow between the cells including biological active proteins that influence the cell cycle and biological behaviour of the recipient cells. This adds a completely new aspect in tumor induced immunology.

Influence of nanoparticles accumulation on optical properties of human normal and cancerous liver tissue in vitro estimated by OCT

International Nuclear Information System (INIS)

Zhou, Fang; Wei, Huajiang; Guo, Zhouyi; Ye, Xiangping; Hu, Kun; Wu, Guoyong; Yang, Hongqin; Xie, Shusen; He, Yonghong

2015-01-01

In this work, the potential use of nanoparticles as contrast agents by using spectral domain optical coherence tomography (SD-OCT) in liver tissue was demonstrated. Gold nanoparticles (average size of 25 and 70 nm), were studied in human normal and cancerous liver tissues in vitro, respectively. Each sample was monitored with SD-OCT functional imaging for 240 min. Continuous OCT monitoring showed that, after application of gold nanoparticles, the OCT signal intensities of normal liver and cancerous liver tissue both increase with time, and the larger nanoparticles tend to produce a greater signal enhancement in the same type of tissue. The results show that the values of attenuation coefficients have significant differences between normal liver tissue and cancerous liver tissue. In addition, 25 nm gold nanoparticles allow higher penetration depth than 70 nm gold nanoparticles in liver tissues. (paper)

High-affinity binding of [3H]estradiol-17 beta by an estrogen receptor in the liver of the turtle

International Nuclear Information System (INIS)

Ho, S.M.; Fehrer, S.; Yu, M.; Liang, L.C.; Press, D.

1988-01-01

Specific [3H]estradiol-17 beta ([3H]E2) binding activity (EBA) with characteristics of an estrogen receptor (ER) was demonstrated in cytosols and nuclear extracts of the female turtle, Chrysemys picta. Three different receptor assays (dextran-coated charcoal assay, hydroxylapatite batch procedure, and DNA-cellulose chromatography) were evaluated in terms of their applicability in analyzing large numbers of samples. For the measurement of cytosolic EBA, the hydroxylapatite batch procedure was found to be the most reliable assay. On the other hand, the dextran-coated charcoal assay was found to be the most appropriate method for the measurement of nuclear EBA. Turtle hepatic EBA binds [3H]E2 with high affinity (cytosolic, 17.4 +/- 2.8 X 10(9) M-1; nuclear, 17.7 +/- 1.9 X 10(9) M-1), limited capacity (cytosolic, 133.7 +/- 4.6 fmol/g tissue; nuclear, 81.1 +/- 9.0 fmol/g tissue), and strict steroid specificity. The EBA bound natural estrogens (E2, estrone, estriol) as well as the nonsteroidal estrogen, diethylstilbestrol, but exhibited little affinity for androgens, progesterone, or corticosterone. The turtle hepatic EBA resembled mammalian and avian ERs in terms of binding characteristics; however, unlike mammalian and avian ERs it was shown to be heat-labile. Incubation at 30 degrees caused rapid loss of [3H]E2 binding activity in both cytosolic and nuclear fractions. The exchange between [3H]E2 and the endogenously bound estrogen was slow at 4 and 15 degrees, but the exchange process was facilitated in the presence of the chaotropic salt, NaSCN. Establishment of quantitation methods for both cytosolic and nuclear forms of EBA will enable future investigation of the mechanism and regulation of estrogen action in the liver of this turtle species

Peroxisome proliferator-activated receptor α (PPARα mRNA expression in human hepatocellular carcinoma tissue and non-cancerous liver tissue

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Kurokawa Tsuyoshi

2011-12-01

Full Text Available Abstract Background Peroxisome proliferator-activated receptor α (PPARα regulates lipid metabolism in the liver. It is unclear, however, how this receptor changes in liver cancer tissue. On the other hand, mouse carcinogenicity studies showed that PPARα is necessary for the development of liver cancer induced by peroxisome proliferators, and the relationship between PPARα and the development of liver cancer have been the focus of considerable attention. There have been no reports, however, demonstrating that PPARα is involved in the development of human liver cancer. Methods The subjects were 10 patients who underwent hepatectomy for hepatocellular carcinoma. We assessed the expression of PPARα mRNA in human hepatocellular carcinoma tissue and non-cancerous tissue, as well as the expression of target genes of PPARα, carnitine palmitoyltransferase 1A and cyclin D1 mRNAs. We also evaluated glyceraldehyde 3-phosphate dehydrogenase, a key enzyme in the glycolytic system. Results The amounts of PPARα, carnitine palmitoyltransferase 1A and glyceraldehyde 3-phosphate dehydrogenase mRNA in cancerous sections were significantly increased compared to those in non-cancerous sections. The level of cyclin D1 mRNA tends to be higher in cancerous than non-cancerous sections. Although there was a significant correlation between the levels of PPARα mRNA and cyclin D1 mRNA in both sections, however the correlation was higher in cancerous sections. Conclusion The present investigation indicated increased expression of PPARα mRNA and mRNAs for PPARα target genes in human hepatocellular carcinoma. These results might be associated with its carcinogenesis and characteristic features of energy production.

Liver afferents contribute to water drinking-induced sympathetic activation in human subjects: a clinical trial.

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Marcus May

Full Text Available Water drinking acutely increases sympathetic activity in human subjects. In animals, the response appears to be mediated through transient receptor potential channel TRPV4 activation on osmosensitive hepatic spinal afferents, described as osmopressor response. We hypothesized that hepatic denervation attenuates water drinking-induced sympathetic activation. We studied 20 liver transplant recipients (44±2.6 years, 1.2±0.1 years post transplant as model of hepatic denervation and 20 kidney transplant recipients (43±2.6 years, 0.8±0.1 years post transplant as immunosuppressive drug matched control group. Before and after 500 ml water ingestion, we obtained venous blood samples for catecholamine analysis. We also monitored brachial and finger blood pressure, ECG, and thoracic bioimpedance. Plasma norepinephrine concentration had changed by 0.01±0.07 nmol/l in liver and by 0.21±0.07 nmol/l in kidney transplant recipients (p<0.05 between groups after 30-40 minutes of water drinking. While blood pressure and systemic vascular resistance increased in both groups, the responses tended to be attenuated in liver transplant recipients. Our findings support the idea that osmosensitive hepatic afferents are involved in water drinking-induced sympathetic activation in human subjects.ClinicalTrials.gov NCT01237431.

Liver Afferents Contribute to Water Drinking-Induced Sympathetic Activation in Human Subjects: A Clinical Trial

Science.gov (United States)

May, Marcus; Gueler, Faikah; Barg-Hock, Hannelore; Heiringhoff, Karl-Heinz; Engeli, Stefan; Heusser, Karsten; Diedrich, André; Brandt, André; Strassburg, Christian P.; Tank, Jens; Sweep, Fred C. G. J.; Jordan, Jens

2011-01-01

Water drinking acutely increases sympathetic activity in human subjects. In animals, the response appears to be mediated through transient receptor potential channel TRPV4 activation on osmosensitive hepatic spinal afferents, described as osmopressor response. We hypothesized that hepatic denervation attenuates water drinking-induced sympathetic activation. We studied 20 liver transplant recipients (44±2.6 years, 1.2±0.1 years post transplant) as model of hepatic denervation and 20 kidney transplant recipients (43±2.6 years, 0.8±0.1 years post transplant) as immunosuppressive drug matched control group. Before and after 500 ml water ingestion, we obtained venous blood samples for catecholamine analysis. We also monitored brachial and finger blood pressure, ECG, and thoracic bioimpedance. Plasma norepinephrine concentration had changed by 0.01±0.07 nmol/l in liver and by 0.21±0.07 nmol/l in kidney transplant recipients (pwater drinking. While blood pressure and systemic vascular resistance increased in both groups, the responses tended to be attenuated in liver transplant recipients. Our findings support the idea that osmosensitive hepatic afferents are involved in water drinking-induced sympathetic activation in human subjects. Trial Registration ClinicalTrials.gov NCT01237431 PMID:22016786

Human fetal liver stromal cells that overexpress bFGF support growth and maintenance of human embryonic stem cells.

Directory of Open Access Journals (Sweden)

Jiafei Xi

Full Text Available In guiding hES cell technology toward the clinic, one key issue to be addressed is to culture and maintain hES cells much more safely and economically in large scale. In order to avoid using mouse embryonic fibroblasts (MEFs we isolated human fetal liver stromal cells (hFLSCs from 14 weeks human fetal liver as new human feeder cells. hFLSCs feeders could maintain hES cells for 15 passages (about 100 days. Basic fibroblast growth factor (bFGF is known to play an important role in promoting self-renewal of human embryonic stem (hES cells. So, we established transgenic hFLSCs that stably express bFGF by lentiviral vectors. These transgenic human feeder cells--bFGF-hFLSCs maintained the properties of H9 hES cells without supplementing with any exogenous growth factors. H9 hES cells culturing under these conditions maintained all hES cell features after prolonged culture, including the developmental potential to differentiate into representative tissues of all three embryonic germ layers, unlimited and undifferentiated proliferative ability, and maintenance of normal karyotype. Our results demonstrated that bFGF-hFLSCs feeder cells were central to establishing the signaling network among bFGF, insulin-like growth factor 2 (IGF-2, and transforming growth factor β (TGF-β, thereby providing the framework in which hES cells were instructed to self-renew or to differentiate. We also found that the conditioned medium of bFGF-hFLSCs could maintain the H9 hES cells under feeder-free conditions without supplementing with bFGF. Taken together, bFGF-hFLSCs had great potential as feeders for maintaining pluripotent hES cell lines more safely and economically.

Influence of starvation, triton WR-1339 and [131I]-human serum albumin on rat liver lysosomes

International Nuclear Information System (INIS)

Harikumar, P.; Ninjoor, V.

1986-01-01

The response of rat liver lysosomes to starvation and administration of lysosomotropic agents viz. Triton WR-1339 and [ 131 I]-human serum albumin, was assessed in terms of their distribution pattern after isopycnic sucrose density gradient centrifugation. Starvation induced changes in lysosomes appeared to be similar to that produced by the detergent uptake. Both the treatments caused a distinct decline in the equilibration densities of the organelles. On the other hand, injected labelled protein failed to comigrate with the lysosomal markers in starved as well as Triton treated rats and conspicuously remained in a region of high specific gravity in the gradient. These findings indicate retarded fusion between secondary lysosomes and [ 131 I]-human serum albumin containing phagosomes in the livers of rats subjected to starvation or detergent treatment. (author)

Immune mediated liver failure.

Science.gov (United States)

Wang, Xiaojing; Ning, Qin

2014-01-01

Liver failure is a clinical syndrome of various etiologies, manifesting as jaundice, encephalopathy, coagulopathy and circulatory dysfunction, which result in subsequent multiorgan failure. Clinically, liver failure is classified into four categories: acute, subacute, acute-on-chronic and chronic liver failure. Massive hepatocyte death is considered to be the core event in the development of liver failure, which occurs when the extent of hepatocyte death is beyond the liver regenerative capacity. Direct damage and immune-mediated liver injury are two major factors involved in this process. Increasing evidence has suggested the essential role of immune-mediated liver injury in the pathogenesis of liver failure. Here, we review the evolved concepts concerning the mechanisms of immune-mediated liver injury in liver failure from human and animal studies. Both innate and adaptive immunity, especially the interaction of various immune cells and molecules as well as death receptor signaling system are discussed. In addition, we highlight the concept of "immune coagulation", which has been shown to be related to the disease progression and liver injury exacerbation in HBV related acute-on-chronic liver failure.

Ecto- and cytosolic 5'-nucleotidases in normal and AMP deaminase-deficient human skeletal muscle

DEFF Research Database (Denmark)

Hanisch, Frank; Hellsten, Ylva; Zierz, Stephan

2006-01-01

homogenate 5'-nucleotidase and ectoN, or in cN-I expression on Western blots. No correlation for age, fibre type distribution and AMPD1 genotype was found for whole homogenate nucleotidase, total cN and cN-I using multiple linear regression analysis. There was no gender-specific difference in the activities...... with a homozygous C34T mutation, cN-I might be a more important pathway for AMP removal. We determined activities of AMP deaminase, cN-I, total cytosolic 5'-nucleotidase (total cN), ecto-5'-nucleotidase (ectoN) and whole homogenate 5'-nucleotidase activity in skeletal muscle biopsies from patients with different...... AMPD1 genotypes [homozygotes for C34T mutation (TT); heterozygotes for C34T mutation (CT); and homozygotes for wild type (CC): diseased controls CC; and normal controls CC]. AMP deaminase activity showed genotype-dependent differences. Total cN activity in normal controls accounted for 57...

Processing highly porous calcium phosphate ceramics for use in bioreactor cores for culturing human liver cells in-vitro

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Finoli, Anthony

Chronic liver disease is the 11th highest cause of death in the United States claiming over 30,000 lives in 2009. The current treatment for chronic liver failure is liver transplantation but the availability of tissue is far less than the number of patients in need. To develop human liver tissue in the lab a 3D culturing environment must be created to support the growth of a complex tissue. Hydroxyapatite (HAp) has been chosen as a scaffold material because of its biocompatibility in the body and the ability to create a bioresorbable scaffold. By using a ceramic material, it is possible to create a three dimensional, protective environment in which tissue can grow. The first part of this study is to examine the behavior of adult human liver cells grown on composites of HAp and different biocompatible hydrogels. Porous HAp has been created using an emulsion foaming technique and cells are injected into the structure after being suspended in a hydrogel and are kept in culture for up to 28 days. Functional assays, gene expression and fluorescent microscopy will be used to examine these cultures. The second part of this study will be to develop a processing technique to create a resorbable scaffold that incorporates a vascular system template. Previous experiments have shown the high temperature decomposition of HAp into resorbable calcium phosphates will be used to create a multiphase material. By controlling the amount of transformation product formed, it is proposed that the resorption of the scaffold can be tailored. To introduce a pore network to guide the growth of a vascular system, a positive-negative casting technique has also been developed. A positive polymer copy can be made of a natural vascular system and ceramic is foamed around the copy. During sintering, the polymer is pyrolyzed leaving a multiscale pore network in the ceramic. By combining these techniques, it is proposed that a calcium phosphate bioreactor core can be processed that is suitable for

[Molecular detection of hepatitis E virus in pig livers destined for human consumption in the state of Nuevo Leon, Mexico].

Science.gov (United States)

Cantú-Martínez, Marco Antonio; Roig-Sagués, Artur Xavier; Cedillo-Rosales, Sibilina; Zamora-Ávila, Diana Elisa; Avalos-Ramírez, Ramiro

2013-04-01

Molecular detection of HEV in pig livers destined for human consumption in Nuevo Leon, Mexico. 87 livers were collected from pigs slaughtered in TIF and 40 livers from butchers. A 212 pb fragment of HEV ORF2 gene was amplified by semi-nested RT-PCR. 19.54% (17) of tif's and 22.5% (9) of butcher's livers were positive for HEV. Sequencing of the amplified products showed a 94%-95% homology with the sequences reported for genotype 3. Our results indicate that HEV is circulating in swine herds in the state, constituting a probable source of contamination of pig meat products.

Enhancement of UVB radiation-mediated apoptosis by knockdown of cytosolic NADP+-dependent isocitrate dehydrogenase in HaCaT cells

OpenAIRE

Lee, Su Jeong; Park, Jeen-Woo

2014-01-01

Ultraviolet B (UVB) radiation induces the production of reactive oxygen species (ROS) that promote apoptotic cell death. We showed that cytosolic NADP+-dependent isocitrate dehydrogenase (IDPc) plays an essential role in the control of cellular redox balance and defense against oxidative damage, by supplying NADPH for antioxidant systems. In this study, we demonstrated that knockdown of IDPc expression by RNA interference enhances UVB-induced apoptosis of immortalized human HaCaT keratinocyte...

Reversed electrogenic sodium bicarbonate cotransporter 1 is the major acid loader during recovery from cytosolic alkalosis in mouse cortical astrocytes.

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Theparambil, Shefeeq M; Naoshin, Zinnia; Thyssen, Anne; Deitmer, Joachim W

2015-08-15

The regulation of H(+) i from cytosolic alkalosis has generally been attributed to the activity of Cl(-) -coupled acid loaders/base extruders in most cell types, including brain cells. The present study demonstrates that outwardly-directed sodium bicarbonate cotransport via electrogenic sodium bicarbonate cotransporter 1 (NBCe1) mediates the major fraction of H(+) i regulation from cytosolic alkalosis in mouse cortical astrocytes. Cl(-) -coupled acid-loading transporters play only a minor role in the regulation of H(+) i from alkalosis in mouse cortical astrocytes. NBCe1-mediated H(+) i regulation from alkalosis was dominant, with the support of intracellular carbonic anhydrase II, even when the intra- and extracellular [HCO3 (-) ] was very low (sodium bicarbonate cotransporter 1 (NBCe1) and for carbonic anhydrase (CA) isoform II. An acute cytosolic alkalosis was induced by the removal of either CO2 /HCO3 (-) or butyric acid, and the subsequent acid loading was analysed by monitoring changes in cytosolic H(+) or Na(+) using ion-sensitive fluorescent dyes. We have identified that NBCe1 reverses during alkalosis and contributes more than 70% to the rate of recovery from alkalosis by extruding Na(+) and HCO3 (-) . After CA inhibition or in CAII-knockout (KO) cells, the rate of recovery was reduced by 40%, and even by 70% in the nominal absence of CO2 /HCO3 (-) . Increasing the extracellular K(+) concentration modulated the rate of acid loading in wild-type cells, but not in NBCe1-KO cells. Removing chloride had only a minor effect on the recovery from alkalosis. Reversal of NBCe1 by reducing pH/[HCO3 (-) ] was demonstrated in astrocytes and in Xenopus oocytes, in which human NBCe1 was heterologously expressed. The results obtained suggest that reversed NBCe1, supported by CAII activity, plays a major role in acid-loading cortical astrocytes to support recovery from cytosolic alkalosis. © 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.

Identification of cytosolic peroxisome proliferator binding protein as a member of the heat shock protein HSP70 family.

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Alvares, K; Carrillo, A; Yuan, P M; Kawano, H; Morimoto, R I; Reddy, J K

1990-01-01

Clofibrate and many of its structural analogues induce proliferation of peroxisomes in the hepatic parenchymal cells of rodents and certain nonrodent species including primates. This induction is tissue specific, occurring mainly in the liver parenchymal cells and to a lesser extent in the kidney cortical epithelium. The induction of peroxisomes is associated with a predictable pleiotropic response, characterized by hepatomegaly, and increased activities and mRNA levels of certain peroxisomal enzymes. Using affinity chromatography, we had previously isolated a protein that binds to clofibric acid. We now show that this protein is homologous with the heat shock protein HSP70 family by analysis of amino acid sequences of isolated peptides from trypsin-treated clofibric acid binding protein and by cross-reactivity with a monoclonal antibody raised against the conserved region of the 70-kDa heat shock proteins. The clofibric acid-Sepharose column could bind HSP70 proteins isolated from various species, which could then be eluted with either clofibric acid or ATP. Conversely, when a rat liver cytosol containing multiple members of the HSP70 family was passed through an ATP-agarose column, and eluted with clofibric acid, only P72 (HSC70) was eluted. These results suggest that clofibric acid, a peroxisome proliferator, preferentially interacts with P72 at or near the ATP binding site. Images PMID:2371272

Quantification of human hepatic binding protein (HBP) via 99mTc-galactosyl-neoglycoalbumin (NGA) liver scintigraphy

International Nuclear Information System (INIS)

Virgolini, I.; Hoebart, J.; Bergmann, H.; Sinzinger, H.; Mueller, C.; Angelberger, P.

1991-01-01

99m Tc-galactosyl-neoglycoalbumin ( 99m Tc-NGA) was synthesized by covalent coupling of 2-imino-2-methoxyethyl-1-thio-β-D-galactopyranoside to the primary amino groups of human serum albumin. Injections of 99m Tc-NGA (150 MBq; 3.5 mg (=50 nmol)/ml) demonstrated the liver to be the exclusive site of tracer-uptake. Simulation of 99m Tc-NGA-kinetics allowed quantification of binding to the hepatic binding protein (HBP). Using this model we studied 250 patients with various liver disease. In alcoholic liver cirrhosis such patients with Child B and Child C stage cirrhosis had a lower HBP-concentration in the liver compared to control individuals (0.85-1.2 μmol/l). The group with the most advanced cirrhosis (Child C stage) had a significantly lower HBP-concentration (0.20-0.45 μmol/l) than Child A patients (0.60-0.85 μmol/l; p 99m Tc-galactosyl-neoglycoalbumin (NGA) scanning of the liver during the course of the disease. Return of liver function tests to normal values was associated with an increased hepatic imaging size as well as increase in HBP-concentration (up to a 3-fold of initial concentration). In patients exhibiting a prolonged course of the disease changes in NGA-kinetic data were borderline and the hepatic image size unchanged. The values obtained for HBP-concentration in the liver amounted to 0.30-0.50 μmol/l liver for patients with hepatoma, to 0.40-0.60 μmol/l in patients with liver metastasis and to 0.90-1.20 μmol/l in cancer patients without liver malignancy. It is concluded that scintigraphic evaluation of functional hepatic cell mass using the new receptor-tracer 99m Tc-NGA provides an in vivo diagnostic mean allowing quantitative data on liver function beside assessment of liver morphology. (Authors)

Systemic administration of a novel human umbilical cord mesenchymal stem cells population accelerates the resolution of acute liver injury

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Burra Patrizia

2012-07-01

Full Text Available Abstract Background Hepatocytes and stem cells transplantation may be an alternative to liver transplantation in acute or chronic liver disease. We aimed to evaluate the therapeutic potential of mesenchymal stem cells from human umbilical cord (UCMSCs, a readily available source of mesenchymal stem cells, in the CCl4-induced acute liver injury model. Methods Mesenchymal stem cells profile was analyzed by flow cytometry. In order to evaluate the capability of our UCMSCs to differentiate in hepatocytes, cells were seeded on three different supports, untreated plastic support, MatrigelTM and human liver acellular matrix. Cells were analyzed by immunocitochemistry for alpha-fetoprotein and albumin expression, qPCR for hepatocyte markers gene expression, Periodic Acid-Schiff staining for glycogen storage, ELISA for albumin detection and colorimetric assay for urea secretion. To assess the effects of undifferentiated UCMSCs in hepatic regeneration after an acute liver injury, we transplanted them via tail vein in mice injected intraperitoneally with a single dose of CCl4. Livers were analyzed by histological evaluation for damage quantification, immunostaining for Kupffer and stellate cells/liver myofibroblasts activation and for UCMSCs homing. Pro- and anti-inflammatory cytokines gene expression was evaluated by qPCR analysis and antioxidant enzyme activity was measured by catalase quantification. Data were analyzed by Mann–Whitney U-test, Kruskal-Wallis test and Cuzick’s test followed by Bonferroni correction for multiple comparisons. Results We have standardized the isolation procedure to obtain a cell population with hepatogenic properties prior to in vivo transplantation. When subjected to hepatogenic differentiation on untreated plastic support, UCMSCs differentiated in hepatocyte-like cells as demonstrated by their morphology, progressive up-regulation of mature hepatocyte markers, glycogen storage, albumin and urea secretion. However

Nanocapsule-mediated cytosolic siRNA delivery for anti-inflammatory treatment.

Science.gov (United States)

Jiang, Ying; Hardie, Joseph; Liu, Yuanchang; Ray, Moumita; Luo, Xiang; Das, Riddha; Landis, Ryan F; Farkas, Michelle E; Rotello, Vincent M

2018-06-05

The use of nanoparticle-stabilized nanocapsules for cytosolic siRNA delivery for immunomodulation in vitro and in vivo is reported. These NPSCs deliver siRNA directly to the cytosol of macrophages in vitro with concomitant knockdown of gene expression. In vivo studies showed directed delivery of NPSCs to the spleen, enabling gene silencing of macrophages, with preliminary studies showing 70% gene knockdown at a siRNA dose of 0.28 mg/kg. Significantly, the delivery of siRNA targeting tumor necrosis factor-α efficiently silenced TNF-α expression in LPS-challenged mice, demonstrating efficacy in modulating immune response in an organ-selective manner. This research highlights the potential of the NPSC platform for targeted immunotherapy and further manipulation of the immune system. Copyright © 2018 Elsevier B.V. All rights reserved.

Bioengineered Liver Models for Drug Testing and Cell Differentiation Studies

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Gregory H. Underhill

2018-01-01

Full Text Available In vitro models of the human liver are important for the following: (1 mitigating the risk of drug-induced liver injury to human beings, (2 modeling human liver diseases, (3 elucidating the role of single and combinatorial microenvironmental cues on liver cell function, and (4 enabling cell-based therapies in the clinic. Methods to isolate and culture primary human hepatocytes (PHHs, the gold standard for building human liver models, were developed several decades ago; however, PHHs show a precipitous decline in phenotypic functions in 2-dimensional extracellular matrix–coated conventional culture formats, which does not allow chronic treatment with drugs and other stimuli. The development of several engineering tools, such as cellular microarrays, protein micropatterning, microfluidics, biomaterial scaffolds, and bioprinting, now allow precise control over the cellular microenvironment for enhancing the function of both PHHs and induced pluripotent stem cell–derived human hepatocyte-like cells; long-term (4+ weeks stabilization of hepatocellular function typically requires co-cultivation with liver-derived or non–liver-derived nonparenchymal cell types. In addition, the recent development of liver organoid culture systems can provide a strategy for the enhanced expansion of therapeutically relevant cell types. Here, we discuss advances in engineering approaches for constructing in vitro human liver models that have utility in drug screening and for determining microenvironmental determinants of liver cell differentiation/function. Design features and validation data of representative models are presented to highlight major trends followed by the discussion of pending issues that need to be addressed. Overall, bioengineered liver models have significantly advanced our understanding of liver function and injury, which will prove useful for drug development and ultimately cell-based therapies.

Cytosolic labile zinc: a marker for apoptosis in the developing rat brain.

Science.gov (United States)

Lee, Joo-Yong; Hwang, Jung Jin; Park, Mi-Ha; Koh, Jae-Young

2006-01-01

Cytosolic zinc accumulation was thought to occur specifically in neuronal death (necrosis) following acute injury. However, a recent study demonstrated that zinc accumulation also occurs in adult rat neurons undergoing apoptosis following target ablation, and in vitro experiments have shown that zinc accumulation may play a causal role in various forms of apoptosis. Here, we examined whether intraneuronal zinc accumulation occurs in central neurons undergoing apoptosis during development. Embryonic and newborn Sprague-Dawley rat brains were double-stained for terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling (TUNEL) detection of apoptosis and immunohistochemical detection of stage-specific neuronal markers, such as nestin, proliferating cell nuclear antigen (PCNA), TuJ1 and neuronal nuclear specific protein (NeuN). The results revealed that apoptotic cell death occurred in neurons of diverse stages (neural stem cells, and dividing, young and adult neurons) throughout the brain during the embryonic and early postnatal periods. Further staining of brain sections with acid fuchsin or zinc-specific fluorescent dyes showed that all of the apoptotic neurons were acidophilic and contained labile zinc in their cell bodies. Cytosolic zinc accumulation was also observed in cultured cortical neurons undergoing staurosporine- or sodium nitroprusside (SNP)-induced apoptosis. In contrast, zinc chelation with CaEDTA or N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) reduced SNP-induced apoptosis but not staurosporine-induced apoptosis, indicating that cytosolic zinc accumulation does not play a causal role in all forms of apoptosis. Finally, the specific cytosolic zinc accumulation may have a practical application as a relatively simple marker for neurons undergoing developmental apoptosis.

Purification of nonspecific lipid transfer protein (sterol carrier protein 2) from human liver and its deficiency in livers from patients with cerebro-hepato-renal (Zellweger) syndrome

NARCIS (Netherlands)

Amerongen, A. van; Helms, J.B.; Krift, T.P. van der; Schutgens, R.B.H.; Wirtz, K.W.A.

1987-01-01

The nonspecific lipid transfer protein (i.e., sterol carrier protein 2) from human liver was purified to homogeneity using ammonium sulfate precipitation, CM-cellulose chromatography, molecular sieve chromatography and fast protein liquid chromatography. Its amino acid composition was determined and

Cytosolic NADP(+)-dependent isocitrate dehydrogenase regulates cadmium-induced apoptosis.

Science.gov (United States)

Shin, Seoung Woo; Kil, In Sup; Park, Jeen-Woo

2010-04-01

Cadmium ions have a high affinity for thiol groups. Therefore, they may disturb many cellular functions. We recently reported that cytosolic NADP(+)-dependent isocitrate dehydrogenase (IDPc) functions as an antioxidant enzyme to supply NADPH, a major source of reducing equivalents to the cytosol. Cadmium decreased the activity of IDPc both as a purified enzyme and in cultured cells. In the present study, we demonstrate that the knockdown of IDPc expression in HEK293 cells greatly enhances apoptosis induced by cadmium. Transfection of HEK293 cells with an IDPc small interfering RNA significantly decreased the activity of IDPc and enhanced cellular susceptibility to cadmium-induced apoptosis as indicated by the morphological evidence of apoptosis, DNA fragmentation and condensation, cellular redox status, mitochondria redox status and function, and the modulation of apoptotic marker proteins. Taken together, our results suggest that suppressing the expression of IDPc enhances cadmium-induced apoptosis of HEK293 cells by increasing disruption of the cellular redox status. Copyright 2009 Elsevier Inc. All rights reserved.

Interplay of Mg2+, ADP, and ATP in the cytosol and mitochondria: unravelling the role of Mg2+ in cell respiration.

Science.gov (United States)

Gout, Elisabeth; Rébeillé, Fabrice; Douce, Roland; Bligny, Richard

2014-10-28

In animal and plant cells, the ATP/ADP ratio and/or energy charge are generally considered key parameters regulating metabolism and respiration. The major alternative issue of whether the cytosolic and mitochondrial concentrations of ADP and ATP directly mediate cell respiration remains unclear, however. In addition, because only free nucleotides are exchanged by the mitochondrial ADP/ATP carrier, whereas MgADP is the substrate of ATP synthase (EC 3.6.3.14), the cytosolic and mitochondrial Mg(2+) concentrations must be considered as well. Here we developed in vivo/in vitro techniques using (31)P-NMR spectroscopy to simultaneously measure these key components in subcellular compartments. We show that heterotrophic sycamore (Acer pseudoplatanus L.) cells incubated in various nutrient media contain low, stable cytosolic ADP and Mg(2+) concentrations, unlike ATP. ADP is mainly free in the cytosol, but complexed by Mg(2+) in the mitochondrial matrix, where [Mg(2+)] is tenfold higher. In contrast, owing to a much higher affinity for Mg(2+), ATP is mostly complexed by Mg(2+) in both compartments. Mg(2+) starvation used to alter cytosolic and mitochondrial [Mg(2+)] reversibly increases free nucleotide concentration in the cytosol and matrix, enhances ADP at the expense of ATP, decreases coupled respiration, and stops cell growth. We conclude that the cytosolic ADP concentration, and not ATP, ATP/ADP ratio, or energy charge, controls the respiration of plant cells. The Mg(2+) concentration, remarkably constant and low in the cytosol and tenfold higher in the matrix, mediates ADP/ATP exchange between the cytosol and matrix, [MgADP]-dependent mitochondrial ATP synthase activity, and cytosolic free ADP homeostasis.

PEPCK-C reexpression in the liver counters neonatal hypoglycemia in Pck1 del/del mice, unmasking role in non-gluconeogenic tissues.

Science.gov (United States)

Semakova, Jana; Hyroššová, Petra; Méndez-Lucas, Andrés; Cutz, Ernest; Bermudez, Jordi; Burgess, Shawn; Alcántara, Soledad; Perales, José C

2017-02-01

Whole body cytosolic phosphoenolpyruvate carboxykinase knockout (PEPCK-C KO) mice die early after birth with profound hypoglycemia therefore masking the role of PEPCK-C in adult, non-gluconeogenic tissues where it is expressed. To investigate whether PEPCK-C deletion in the liver was critically responsible for the hypoglycemic phenotype, we reexpress this enzyme in the liver of PEPCK-C KO pups by early postnatal administration of PEPCK-C-expressing adenovirus. This maneuver was sufficient to partially rescue hypoglycemia and allow the pups to survive and identifies the liver as a critical organ, and hypoglycemia as the critical pathomechanism, leading to early postnatal death in the whole-body PEPCK-C knockout mice. Pathology assessment of survivors also suggest a possible role for PEPCK-C in lung maturation and muscle metabolism.

Hepatic Differentiation of Human Induced Pluripotent Stem Cells in a Perfused 3D Porous Polymer Scaffold for Liver Tissue Engineering

DEFF Research Database (Denmark)

Hemmingsen, Mette; Muhammad, Haseena Bashir; Mohanty, Soumyaranjan

A huge shortage of liver organs for transplantation has motivated the research field of tissue engineering to develop bioartificial liver tissue and even a whole liver. The goal of NanoBio4Trans is to create a vascularized bioartificial liver tissue, initially as a liver-support system. Due...... to limitations of primary hepatocytes regarding availability and maintenance of functionality, stem cells and especially human induced pluripotent stem cells (hIPS cells) are an attractive cell source for liver tissue engineering. The aim of this part of NanoBio4Trans is to optimize culture and hepatic...... differentiation of hIPS-derived definitive endoderm (DE) cells in a 3D porous polymer scaffold built-in a perfusable bioreactor. The use of a microfluidic bioreactor array enables the culture of 16 independent tissues in one experimental run and thereby an optimization study to be performed....

Featured Article: Isolation, characterization, and cultivation of human hepatocytes and non-parenchymal liver cells

Science.gov (United States)

Pfeiffer, Elisa; Kegel, Victoria; Zeilinger, Katrin; Hengstler, Jan G; Nüssler, Andreas K; Seehofer, Daniel

2015-01-01

Primary human hepatocytes (PHH) are considered to be the gold standard for in vitro testing of xenobiotic metabolism and hepatotoxicity. However, PHH cultivation in 2D mono-cultures leads to dedifferentiation and a loss of function. It is well known that hepatic non-parenchymal cells (NPC), such as Kupffer cells (KC), liver endothelial cells (LEC), and hepatic stellate cells (HSC), play a central role in the maintenance of PHH functions. The aims of the present study were to establish a protocol for the simultaneous isolation of human PHH and NPC from the same tissue specimen and to test their suitability for in vitro co-culture. Human PHH and NPC were isolated from tissue obtained by partial liver resection by a two-step EDTA/collagenase perfusion technique. The obtained cell fractions were purified by Percoll density gradient centrifugation. KC, LEC, and HSC contained in the NPC fraction were separated using specific adherence properties and magnetic activated cell sorting (MACS®). Identified NPC revealed a yield of 1.9 × 106 KC, 2.7 × 105 LEC and 4.7 × 105 HSC per gram liver tissue, showing viabilities >90%. Characterization of these NPC showed that all populations went through an activation process, which influenced the cell fate. The activation of KC strongly depended on the tissue quality and donor anamnesis. KC became activated in culture in association with a loss of viability within 4–5 days. LEC lost specific features during culture, while HSC went through a transformation process into myofibroblasts. The testing of different culture conditions for HSC demonstrated that they can attenuate, but not prevent dedifferentiation in vitro. In conclusion, the method described allows the isolation and separation of PHH and NPC in high quality and quantity from the same donor. PMID:25394621

Prognostic significance of cytosolic pS2 content in ovarian tumors

International Nuclear Information System (INIS)

Raigoso, P.; Allende, T.; Zeidan, N.; Llana, B.; Bernardo, L.; Roiz, C.; Tejuca, S.; Vazquez, J.; Lamelas, M.L.

2002-01-01

Aim: pS2 is an estrogen regulated peptide which has been associated with a good prognosis an with a more favorable response to treatment in breast cancer patients. In ovarian tumors, the expression of pS2 was demonstrated at both mRNA and protein levels. In addition, it has been showed significant association of pS2 with mucinous differentiation or well differentiation grade of the tumors. However, it is little know about the prognostic significance of the pS2 content in ovarian carcinomas. The aims of the present work were to analyze the cytosolic pS2 content in benign and malignant ovarian tumors, its relationship with clinico-pathologic parameters, steroid receptor status, and prognostic significance. Material and Methods: We analysed the cytosolic concentrations of pS2 in 91 specimen ovarian tissues by an immunoradiometric assay (ELSA-pS2, CIS, France). The tissues were 8 normal ovaries, 43 benign tumors and 40 malignant ovarian tumors. The same ovarian tissues processed to pS2 were analyzed to Estrogen (ER) and Progesterone (PgR) Receptor status. These steroid receptors were quantified biochemically following commercial ELISA method (ABBOTT Diagnostics, Germany). The relationship between cytosolic content and clinico-pathologic factors was examined by the Mann-Whitney or Kruskall-Wallis test. Correlation between steroid receptors and pS2 content was calculated with the Spearman test. Survival curves were calculated using the Kaplan-Meier method and compared by the log-rank test. Differences were considered significant at 5% probability level. Results: pS2 could be detected in 30 cases (32.9%) with values ranged from 0.04 to 89 ng/mg prt. Only one normal ovary showed detectable levels of pS2 and there were not differences in cytosolic content between benign and malignant ovarian tumors. The pS2 levels were only associated to mucinous differentiation in both benign and malignant ovarian tumors (p=0.029 and p=0.015, respectively). Significantly higher

Mediator-assisted Simultaneous probing of Cytosolic and Mitochondrial Redox activity in living cells

DEFF Research Database (Denmark)

Heiskanen, Arto; Spegel, Christer; Kostesha, Natalie

2009-01-01

the ferricyanide-menadione double mediator system to study the effect of dicoumarol, an inhibitor of cytosolic and mitochondrial oxidoreductases and an uncoupler of the electron transport chain. Evaluation of the role of NAD(P)H-producing pathways in mediating biological effects is facilitated by introducing...... either fructose or glucose as the carbon source, yielding either NADH or NADPH through the glycolytic or pen-rose phosphate pathway, respectively. Respiratory noncompetent cells show greater inhibition of cytosolic menadione-reducing enzymes when NADH rather than NADPH is produced. Spectrophotometric...

Plasmodium falciparum full life cycle and Plasmodium ovale liver stages in humanized mice.

Science.gov (United States)

Soulard, Valérie; Bosson-Vanga, Henriette; Lorthiois, Audrey; Roucher, Clémentine; Franetich, Jean-François; Zanghi, Gigliola; Bordessoulles, Mallaury; Tefit, Maurel; Thellier, Marc; Morosan, Serban; Le Naour, Gilles; Capron, Frédérique; Suemizu, Hiroshi; Snounou, Georges; Moreno-Sabater, Alicia; Mazier, Dominique

2015-07-24

Experimental studies of Plasmodium parasites that infect humans are restricted by their host specificity. Humanized mice offer a means to overcome this and further provide the opportunity to observe the parasites in vivo. Here we improve on previous protocols to achieve efficient double engraftment of TK-NOG mice by human primary hepatocytes and red blood cells. Thus, we obtain the complete hepatic development of P. falciparum, the transition to the erythrocytic stages, their subsequent multiplication, and the appearance of mature gametocytes over an extended period of observation. Furthermore, using sporozoites derived from two P. ovale-infected patients, we show that human hepatocytes engrafted in TK-NOG mice sustain maturation of the liver stages, and the presence of late-developing schizonts indicate the eventual activation of quiescent parasites. Thus, TK-NOG mice are highly suited for in vivo observations on the Plasmodium species of humans.

Detection of human leukocyte antigen compatibility and antibodies in liver transplantation in China

Institute of Scientific and Technical Information of China (English)

Xue-Qin Meng; Xuan Zhang; Jun Fan; Lin Zhou; Bing Hao; Xiao-Ming Chen; Wei-Hang Ma; Shu-Sen Zheng

2009-01-01

BACKGROUND: The exact roles of human leukocyte antigen (HLA) compatibility, HLA antibodies and underlying diseases in acute rejection of liver transplants are not clear. Moreover, cytomegalovirus (CMV) infection, one of the most common infections after transplantation, is related to HLA genotype and the incidence of acute rejection. METHODS: Since there are controversial reports, we analyzed the impact of HLA matching, HLA antibodies and underlying diseases in 38 liver transplant recipients in China, and assessed the association of CMV infection and HLA compatibility. RESULTS: The frequency of no HLA compatibility was high in patients without antigenemia (P=0.019). All 17 patients with HLA-A matching developed antigenemia (P0.05). In patients with acute rejection, no differences were found in the incidence of acute rejection in transplants for hepatitis B, tumors, or combined hepatitis B and tumors (P>0.05).CONCLUSIONS: There are fewer acute rejections in transplants with more HLA compatibilities. Speciifc investigations of underlying diseases and HLA typing may be necessary in liver transplantation. The mechanisms of CMV infection and HLA matching should be further studied. HLA before transplantation should be examined for the prevention of acute rejection and CMV infection.

The chaperone BAG6 captures dislocated glycoproteins in the cytosol.

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Jasper H L Claessen

Full Text Available Secretory and membrane (glycoproteins are subject to quality control in the endoplasmic reticulum (ER to ensure that only functional proteins reach their destination. Proteins deemed terminally misfolded and hence functionally defective may be dislocated to the cytosol, where the proteasome degrades them. What we know about this process stems mostly from overexpression of tagged misfolded proteins, or from situations where viruses have hijacked the quality control machinery to their advantage. We know of only very few endogenous substrates of ER quality control, most of which are degraded as part of a signaling pathway, such as Insig-1, but such examples do not necessarily represent terminally misfolded proteins. Here we show that endogenous dislocation clients are captured specifically in association with the cytosolic chaperone BAG6, or retrieved en masse via their glycan handle.

Characterization of HBV integration patterns and timing in liver cancer and HBV-infected livers.

Science.gov (United States)

Furuta, Mayuko; Tanaka, Hiroko; Shiraishi, Yuichi; Unida, Takuro; Imamura, Michio; Fujimoto, Akihiro; Fujita, Masahi; Sasaki-Oku, Aya; Maejima, Kazuhiro; Nakano, Kaoru; Kawakami, Yoshiiku; Arihiro, Koji; Aikata, Hiroshi; Ueno, Masaki; Hayami, Shinya; Ariizumi, Shun-Ichi; Yamamoto, Masakazu; Gotoh, Kunihito; Ohdan, Hideki; Yamaue, Hiroki; Miyano, Satoru; Chayama, Kazuaki; Nakagawa, Hidewaki

2018-05-18

Integration of Hepatitis B virus (HBV) into the human genome can cause genetic instability, leading to selective advantages for HBV-induced liver cancer. Despite the large number of studies for HBV integration into liver cancer, little is known about the mechanism of initial HBV integration events owing to the limitations of materials and detection methods. We conducted an HBV sequence capture, followed by ultra-deep sequencing, to screen for HBV integrations in 111 liver samples from human-hepatocyte chimeric mice with HBV infection and human clinical samples containing 42 paired samples from non-tumorous and tumorous liver tissues. The HBV infection model using chimeric mice verified the efficiency of our HBV-capture analysis and demonstrated that HBV integration could occur 23 to 49 days after HBV infection via microhomology-mediated end joining and predominantly in mitochondrial DNA. Overall HBV integration sites in clinical samples were significantly enriched in regions annotated as exhibiting open chromatin, a high level of gene expression, and early replication timing in liver cells. These data indicate that HBV integration in liver tissue was biased according to chromatin accessibility, with additional selection pressures in the gene promoters of tumor samples. Moreover, an integrative analysis using paired non-tumorous and tumorous samples and HBV-related transcriptional change revealed the involvement of TERT and MLL4 in clonal selection. We also found frequent and non-tumorous liver-specific HBV integrations in FN1 and HBV-FN1 fusion transcript. Extensive survey of HBV integrations facilitates and improves the understanding of the timing and biology of HBV integration during infection and HBV-related hepatocarcinogenesis.

(99m) Tc-labelled human serum albumin cannot replace (125) I-labelled human serum albumin to determine plasma volume in patients with liver disease

DEFF Research Database (Denmark)

Henriksen, Ulrik Lütken; Henriksen, Jens H; Bendtsen, Flemming

2013-01-01

Summary Background and aims Determination of plasma volume (PV) is important in several clinical situations. Thus, patients with liver disease often have augmented PV as part of their sodium–water retention. This study was undertaken to compare PV determination by two indicators: technetium......-labelled human serum albumin (99mTc-HSA) and iodine-labelled human serum albumin (125I-HSA), as the former may have advantages at repeated measurements and the latter is the classical gold standard. Study population and methods In 88 patients, (64 with liver disease, mainly cirrhosis, and 24 patients without...... In all patients, a close correlation was present between PV determined by the two indicators (r = 0·89, Pdetermined with 99mTc-HSA exceeded PV determined with 125I-HSA by 367 ml (5·2 ml kg...

Glutathione delays varies as-tocopherol oxidation and subsequent lipid peroxidation in rat liver microsomes

International Nuclear Information System (INIS)

Robey, S.; Mavis, R.

1986-01-01

A method has been developed for in vitro trace radiolabeling of rat liver microsomes with 3 H-α-tocopherol (αT*) which allows virtually complete oxidation of the αT* under oxidizing conditions. The supernatant of a 16,000 xg centrifugation of homogenized rat liver, containing the cytosolic rat liver vitamin E (VE) transfer protein, was incubated with an ethanolic solution of αT* for 10 minutes at 37 0 C. Labeled microsomes were collected in the washed 100,000 xg pellet. Microsomes were then incubated with 30 μM Fe 2+ in an NADPH-generating system, and both production of malondialdehyde (MDA) (a product of lipid peroxidation) and oxidation of αT* were monitored over a time course in the presence and absence of glutathione (GSH). The results indicate virtually complete oxidation of αT* precedes significant membrane lipid peroxidation, and that addition of 5 mM GSH delays both αT* oxidation and subsequent MDA production. This suggests that the previously observed VE-dependent heat labile inhibition of microsomal lipid peroxidation by GSH involves maintaining membrane levels of α-tocopherol

Transcriptional profiling of human liver identifies sex-biased genes associated with polygenic dyslipidemia and coronary artery disease.

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Yijing Zhang

Full Text Available Sex-differences in human liver gene expression were characterized on a genome-wide scale using a large liver sample collection, allowing for detection of small expression differences with high statistical power. 1,249 sex-biased genes were identified, 70% showing higher expression in females. Chromosomal bias was apparent, with female-biased genes enriched on chrX and male-biased genes enriched on chrY and chr19, where 11 male-biased zinc-finger KRAB-repressor domain genes are distributed in six clusters. Top biological functions and diseases significantly enriched in sex-biased genes include transcription, chromatin organization and modification, sexual reproduction, lipid metabolism and cardiovascular disease. Notably, sex-biased genes are enriched at loci associated with polygenic dyslipidemia and coronary artery disease in genome-wide association studies. Moreover, of the 8 sex-biased genes at these loci, 4 have been directly linked to monogenic disorders of lipid metabolism and show an expression profile in females (elevated expression of ABCA1, APOA5 and LDLR; reduced expression of LIPC that is consistent with the lower female risk of coronary artery disease. Female-biased expression was also observed for CYP7A1, which is activated by drugs used to treat hypercholesterolemia. Several sex-biased drug-metabolizing enzyme genes were identified, including members of the CYP, UGT, GPX and ALDH families. Half of 879 mouse orthologs, including many genes of lipid metabolism and homeostasis, show growth hormone-regulated sex-biased expression in mouse liver, suggesting growth hormone might play a similar regulatory role in human liver. Finally, the evolutionary rate of protein coding regions for human-mouse orthologs, revealed by dN/dS ratio, is significantly higher for genes showing the same sex-bias in both species than for non-sex-biased genes. These findings establish that human hepatic sex differences are widespread and affect diverse cell

Low dose perfluorooctanoate exposure promotes cell proliferation in a human non-tumor liver cell line

Energy Technology Data Exchange (ETDEWEB)

Zhang, Hongxia; Cui, Ruina [Key Laboratory of Animal Ecology and Conservation Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101 (China); Guo, Xuejiang [State Key Laboratory of Reproductive Medicine, Nanjing Medical University, Nanjing 210029 (China); Hu, Jiayue [Key Laboratory of Animal Ecology and Conservation Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101 (China); Dai, Jiayin, E-mail: daijy@ioz.ac.cn [Key Laboratory of Animal Ecology and Conservation Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101 (China)

2016-08-05

Highlights: • Differential expression of proteins induced by PFOA in HL-7702 was identified. • Most of the differentially expressed proteins are related to cell proliferation. • A low dose of PFOA stimulates HL-7702 cell proliferation. • A high dose of PFOA inhibits HL-7702 cell proliferation. - Abstract: Perfluorooctanoate (PFOA) is a well-known persistent organic pollutant widely found in the environment, wildlife and humans. Medical surveillance and experimental studies have investigated the potential effects of PFOA on human livers, but the hepatotoxicity of PFOA on humans and its underlying mechanism remain to be clarified. We exposed a human liver cell line (HL-7702) to 50 μM PFOA for 48 h and 96 h, and identified 111 significantly differentially expressed proteins by iTRAQ analysis. A total of 46 proteins were related to cell proliferation and apoptosis. Through further analysis of the cell cycle, apoptosis and their related proteins, we found that low doses of PFOA (50–100 μM) promoted cell proliferation and numbers by promoting cells from the G1 to S phases, whereas high doses of PFOA (200–400 μM) led to reduced HL-7702 cell numbers compared with that of the control mainly due to cell cycle arrest in the G0/G1 phase. To our knowledge, this is the first report on the promotion of cell cycle progression in human cells following PFOA exposure.

HBsAg-redirected T cells exhibit antiviral activity in HBV-infected human liver chimeric mice.

Science.gov (United States)

Kruse, Robert L; Shum, Thomas; Tashiro, Haruko; Barzi, Mercedes; Yi, Zhongzhen; Whitten-Bauer, Christina; Legras, Xavier; Bissig-Choisat, Beatrice; Garaigorta, Urtzi; Gottschalk, Stephen; Bissig, Karl-Dimiter

2018-04-06

Chronic hepatitis B virus (HBV) infection remains incurable. Although HBsAg-specific chimeric antigen receptor (HBsAg-CAR) T cells have been generated, they have not been tested in animal models with authentic HBV infection. We generated a novel CAR targeting HBsAg and evaluated its ability to recognize HBV+ cell lines and HBsAg particles in vitro. In vivo, we tested whether human HBsAg-CAR T cells would have efficacy against HBV-infected hepatocytes in human liver chimeric mice. HBsAg-CAR T cells recognized HBV-positive cell lines and HBsAg particles in vitro as judged by cytokine production. However, HBsAg-CAR T cells did not kill HBV-positive cell lines in cytotoxicity assays. Adoptive transfer of HBsAg-CAR T cells into HBV-infected humanized mice resulted in accumulation within the liver and a significant decrease in plasma HBsAg and HBV-DNA levels compared with control mice. Notably, the fraction of HBV core-positive hepatocytes among total human hepatocytes was greatly reduced after HBsAg-CAR T cell treatment, pointing to noncytopathic viral clearance. In agreement, changes in surrogate human plasma albumin levels were not significantly different between treatment and control groups. HBsAg-CAR T cells have anti-HBV activity in an authentic preclinical HBV infection model. Our results warrant further preclinical exploration of HBsAg-CAR T cells as immunotherapy for HBV. Copyright © 2018 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Low dose perfluorooctanoate exposure promotes cell proliferation in a human non-tumor liver cell line

International Nuclear Information System (INIS)

Zhang, Hongxia; Cui, Ruina; Guo, Xuejiang; Hu, Jiayue; Dai, Jiayin

2016-01-01

Highlights: • Differential expression of proteins induced by PFOA in HL-7702 was identified. • Most of the differentially expressed proteins are related to cell proliferation. • A low dose of PFOA stimulates HL-7702 cell proliferation. • A high dose of PFOA inhibits HL-7702 cell proliferation. - Abstract: Perfluorooctanoate (PFOA) is a well-known persistent organic pollutant widely found in the environment, wildlife and humans. Medical surveillance and experimental studies have investigated the potential effects of PFOA on human livers, but the hepatotoxicity of PFOA on humans and its underlying mechanism remain to be clarified. We exposed a human liver cell line (HL-7702) to 50 μM PFOA for 48 h and 96 h, and identified 111 significantly differentially expressed proteins by iTRAQ analysis. A total of 46 proteins were related to cell proliferation and apoptosis. Through further analysis of the cell cycle, apoptosis and their related proteins, we found that low doses of PFOA (50–100 μM) promoted cell proliferation and numbers by promoting cells from the G1 to S phases, whereas high doses of PFOA (200–400 μM) led to reduced HL-7702 cell numbers compared with that of the control mainly due to cell cycle arrest in the G0/G1 phase. To our knowledge, this is the first report on the promotion of cell cycle progression in human cells following PFOA exposure.

Peroxisomal abnormalities in the immortalized human hepatocyte (IHH) cell line.

Science.gov (United States)

Klouwer, Femke C C; Koster, Janet; Ferdinandusse, Sacha; Waterham, Hans R

2017-04-01

The immortalized human hepatocyte (IHH) cell line is increasingly used for studies related to liver metabolism, including hepatic glucose, lipid, lipoprotein and triglyceride metabolism, and the effect of therapeutic interventions. To determine whether the IHH cell line is a good model to investigate hepatic peroxisomal metabolism, we measured several peroxisomal parameters in IHH cells and, for comparison, HepG2 cells and primary skin fibroblasts. This revealed a marked plasmalogen deficiency and a deficient fatty acid α-oxidation in the IHH cells, due to a defect of PEX7, a cytosolic receptor protein required for peroxisomal import of a subset of peroxisomal proteins. These abnormalities have consequences for the lipid homeostasis of these cells and thus should be taken into account for the interpretation of data previously generated by using this cell line and when considering using this cell line for future research.

Defective i6A37 modification of mitochondrial and cytosolic tRNAs results from pathogenic mutations in TRIT1 and its substrate tRNA.

Directory of Open Access Journals (Sweden)

John W Yarham

2014-06-01

Full Text Available Identifying the genetic basis for mitochondrial diseases is technically challenging given the size of the mitochondrial proteome and the heterogeneity of disease presentations. Using next-generation exome sequencing, we identified in a patient with severe combined mitochondrial respiratory chain defects and corresponding perturbation in mitochondrial protein synthesis, a homozygous p.Arg323Gln mutation in TRIT1. This gene encodes human tRNA isopentenyltransferase, which is responsible for i6A37 modification of the anticodon loops of a small subset of cytosolic and mitochondrial tRNAs. Deficiency of i6A37 was previously shown in yeast to decrease translational efficiency and fidelity in a codon-specific manner. Modelling of the p.Arg323Gln mutation on the co-crystal structure of the homologous yeast isopentenyltransferase bound to a substrate tRNA, indicates that it is one of a series of adjacent basic side chains that interact with the tRNA backbone of the anticodon stem, somewhat removed from the catalytic center. We show that patient cells bearing the p.Arg323Gln TRIT1 mutation are severely deficient in i6A37 in both cytosolic and mitochondrial tRNAs. Complete complementation of the i6A37 deficiency of both cytosolic and mitochondrial tRNAs was achieved by transduction of patient fibroblasts with wild-type TRIT1. Moreover, we show that a previously-reported pathogenic m.7480A>G mt-tRNASer(UCN mutation in the anticodon loop sequence A36A37A38 recognised by TRIT1 causes a loss of i6A37 modification. These data demonstrate that deficiencies of i6A37 tRNA modification should be considered a potential mechanism of human disease caused by both nuclear gene and mitochondrial DNA mutations while providing insight into the structure and function of TRIT1 in the modification of cytosolic and mitochondrial tRNAs.

Merlin, the product of NF2 gene, is associated with aromatase expression and estrogen formation in human liver tissues and liver cancer cells.

Science.gov (United States)

Cocciadiferro, Letizia; Miceli, Vitale; Granata, Orazia M; Carruba, Giuseppe

2017-09-01

The product of neurofibromatosis type 2 (NF2) gene, also known as Merlin/neurofibromin 2, homeostatically regulates liver stem cells by controlling abundance and signaling of epidermal growth factor receptor (EGFR), with a mechanism independent of the Hippo pathway. We have reported that locally elevated estrogen formation, driven by abnormally high expression and function of aromatase, may be implicated in development and progression of human hepatocellular carcinoma (HCC) through activation of a rapid signaling pathway mediated by amphiregulin (AREG) and EGFR. We have recently presented a model by which the aromatase-estrogen-amphiregulin-EGFR axis is activated in response to tissue injury and/or inflammatory disease, with its alteration eventually leading to development of major human tumors (liver, breast, prostate) and other chronic diseases (diabetes, obesity, Alzheimer's and heart disease). In this study, we investigated NF2 expression in liver cancer cells and tissues in relation to aromatase expression/function, estrogen receptor (ER) status and amphiregulin. Our data indicate that NF2 expression is associated with aromatase and AREG expression, being elevated in HCC tissues and HepG2 cells, intermediate in cirrhotic tissues and Huh7 cells, and lower in nontumoral liver and HA22T cells. In addition, NF2 expression is inversely related to wild type hERα66 and proportional to the expression of the membrane-associated hERα36 splice variant, as measured by exon-specific RT-PCR analysis, both in vivo and in vitro. Furthermore, incubation with estradiol induced a significant decrease of NF2 expression in both HA22T and Huh7 cells (over 54% and 22%, respectively), while no change could be observed in HepG2 cells, this effect being inversely related to aromatase expression and activity in HCC cell lines. Based on the above combined evidence, we hypothesize that NF2 behaves as a protein sensing tissue damage and aromatase-driven local estrogen formation

Cytosol-dependent membrane fusion in ER, nuclear envelope and nuclear pore assembly: biological implications.

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Rafikova, Elvira R; Melikov, Kamran; Chernomordik, Leonid V

2010-01-01

Endoplasmic reticulum and nuclear envelope rearrangements after mitosis are often studied in the reconstitution system based on Xenopus egg extract. In our recent work we partially replaced the membrane vesicles in the reconstitution mix with protein-free liposomes to explore the relative contributions of cytosolic and transmembrane proteins. Here we discuss our finding that cytosolic proteins mediate fusion between membranes lacking functional transmembrane proteins and the role of membrane fusion in endoplasmic reticulum and nuclear envelope reorganization. Cytosol-dependent liposome fusion has allowed us to restore, without adding transmembrane nucleoporins, functionality of nuclear pores, their spatial distribution and chromatin decondensation in nuclei formed at insufficient amounts of membrane material and characterized by only partial decondensation of chromatin and lack of nuclear transport. Both the mechanisms and the biological implications of the discovered coupling between spatial distribution of nuclear pores, chromatin decondensation and nuclear transport are discussed.

Glucose acutely reduces cytosolic and mitochondrial H2O2 in rat pancreatic beta-cells.

Science.gov (United States)

Deglasse, Jean-Philippe; Roma, Leticia Prates; Pastor-Flores, Daniel; Gilon, Patrick; Dick, Tobias P; Jonas, Jean-Christophe

2018-05-14

Whether H2O2 contributes to the glucose-dependent stimulation of insulin secretion by pancreatic β-cells is highly controversial. We used two H2O2-sensitive probes, roGFP2-Orp1 and HyPer with its pH-control SypHer, to test the acute effects of glucose, monomethylsuccinate, leucine with glutamine, and α-ketoisocaproate, on β-cell cytosolic and mitochondrial H2O2 concentrations. We then tested the effects of low H2O2 and menadione concentrations on insulin secretion. RoGFP2-Orp1 was more sensitive than HyPer to H2O2 (response at 2-5 vs. 10µM) and less pH-sensitive. Under control conditions, stimulation with glucose reduced mitochondrial roGFP2-Orp1 oxidation without affecting cytosolic roGFP2-Orp1 and HyPer fluorescence ratios, except for the pH-dependent effects on HyPer. However, stimulation with glucose decreased the oxidation of both cytosolic probes by 15µM exogenous H2O2. The glucose effects were not affected by overexpression of catalase, mitochondrial catalase or superoxide dismutase 1 and 2. They followed the increase in NAD(P)H autofluorescence, were maximal at 5mM glucose in the cytosol and 10mM glucose in the mitochondria, and were partly mimicked by the other nutrients. Exogenous H2O2 (1-15µM) did not affect insulin secretion. By contrast, menadione (1-5µM) did not increase basal insulin secretion but reduced the stimulation of insulin secretion by 20mM glucose. Subcellular changes in β-cell H2O2 levels are better monitored with roGFP2-Orp1 than HyPer/SypHer. Nutrients acutely lower mitochondrial H2O2 levels in β-cells and promote degradation of exogenously supplied H2O2 in both cytosolic and mitochondrial compartments. The glucose-dependent stimulation of insulin secretion occurs independently of a detectable increase in β-cell cytosolic or mitochondrial H2O2 levels.

Lack of cilia and differentiation defects in the liver of human foetuses with the Meckel syndrome.

Science.gov (United States)

Clotman, Frédéric; Libbrecht, Louis; Killingsworth, Murray C; Loo, Christine C K; Roskams, Tania; Lemaigre, Frédéric P

2008-03-01

Meckel syndrome is an autosomal-recessive disease characterized by a combination of renal cysts, anomalies of the central nervous system, polydactyly and ductal plate malformations (DPM), which are hepatic anomalies consisting of excessive and abnormal foetal biliary structures. Among the genomic loci associated with Meckel syndrome, mutations in four genes were recently identified. These genes code for proteins associated with primary cilia and are possibly involved in cell differentiation. The aim of the present work was to investigate the formation of the primary cilia and the differentiation of the hepatic cells in foetuses with Meckel syndrome. Sections of livers from human foetuses with Meckel syndrome were analysed by immunofluorescence, immunohistochemistry and electron microscopy. The primary cilia of the biliary cells were absent in some Meckel foetuses, but were present in others. In addition, defects in hepatic differentiation were observed in Meckel livers, as evidenced by the presence of hybrid cells co-expressing hepatocytic and biliary markers. Defects in cilia formation occur in some Meckel livers, and most cases show DPM associated with abnormal hepatic cell differentiation. Because differentiation precedes the formation of the cilia during liver development, we propose that defective differentiation may constitute the initial defect in the liver of Meckel syndrome foetuses.

RPA and Rad51 constitute a cell intrinsic mechanism to protect the cytosol from self DNA.

Science.gov (United States)

Wolf, Christine; Rapp, Alexander; Berndt, Nicole; Staroske, Wolfgang; Schuster, Max; Dobrick-Mattheuer, Manuela; Kretschmer, Stefanie; König, Nadja; Kurth, Thomas; Wieczorek, Dagmar; Kast, Karin; Cardoso, M Cristina; Günther, Claudia; Lee-Kirsch, Min Ae

2016-05-27

Immune recognition of cytosolic DNA represents a central antiviral defence mechanism. Within the host, short single-stranded DNA (ssDNA) continuously arises during the repair of DNA damage induced by endogenous and environmental genotoxic stress. Here we show that short ssDNA traverses the nuclear membrane, but is drawn into the nucleus by binding to the DNA replication and repair factors RPA and Rad51. Knockdown of RPA and Rad51 enhances cytosolic leakage of ssDNA resulting in cGAS-dependent type I IFN activation. Mutations in the exonuclease TREX1 cause type I IFN-dependent autoinflammation and autoimmunity. We demonstrate that TREX1 is anchored within the outer nuclear membrane to ensure immediate degradation of ssDNA leaking into the cytosol. In TREX1-deficient fibroblasts, accumulating ssDNA causes exhaustion of RPA and Rad51 resulting in replication stress and activation of p53 and type I IFN. Thus, the ssDNA-binding capacity of RPA and Rad51 constitutes a cell intrinsic mechanism to protect the cytosol from self DNA.

Determination of trace elements in human liver biopsy samples by ICP-MS and TXRF: hepatic steatosis and nickel accumulation

Energy Technology Data Exchange (ETDEWEB)

Varga, Imre; Szoboszlai, Norbert [L. Eoetvoes University, Department of Inorganic and Analytical Chemistry, P.O. Box 32, Budapest (Hungary); Szebeni, Agnes [MI Central Hospital, Ultrasound Laboratory, Budapest (Hungary); Kovacs, Bela [Debrecen University - Centre of Agricultural Sciences, Department of Food Science and Quality Assurance, P.O. Box 36, Debrecen (Hungary)

2005-10-01

Human liver biopsy samples, collected from 52 individuals, were analysed by inductively coupled plasma-mass spectrometry (ICP-MS) and total reflection X-ray fluorescence (TXRF) spectrometry in a retrospective study (i.e. patient selection and liver biopsy were not for the purpose of element analysis). The freeze-dried samples (typically 0.5-2 mg dry weight) were digested in a laboratory microwave digestion system and solutions with a final volume of 1 mL were prepared. The concentrations of Cr, Mn, Fe, Ni, Cu, Zn, Rb, and Pb were determined by use of a Thermo Elemental X7 ICP-MS spectrometer. TXRF measurements were performed with an Atomika Extra IIA spectrometer. Yttrium was employed as an internal standard, prepared by dissolution of 5N-purity yttria (Y{sub 2}O{sub 3}) in our laboratory. The accuracy was tested by analysis of NIST 1577a Bovine Liver certified reference material. The concentrations of Fe, Cu, Zn, and Rb determined in human liver biopsy samples were in good agreement with data published by other authors. The distribution of nickel in the samples was surprisingly uneven - nickel concentrations ranged from 0.7 to 12 {mu}g g{sup -1} (dry weight) in 38 samples and in several samples were extremely high, 36-693 {mu}g g{sup -1}. Analysis of replicate procedural blanks and control measurements were performed to prevent misinterpretation of the data. For patients with steatosis (n=14) Ni concentrations were consistently high except for two who had levels close to those measured for the normal group. As far as we are aware no previous literature data are available on the association of steatosis with high concentration of nickel in human liver biopsies taken from living patients. (orig.)

Dexamethasone-induced haptoglobin release by calf liver parenchymal cells.

Science.gov (United States)

Higuchi, H; Katoh, N; Miyamoto, T; Uchida, E; Yuasa, A; Takahashi, K

1994-08-01

Parenchymal cells were isolated from the liver of male calves, and monolayer cultures formed were treated with glucocorticoids to examine whether haptoglobin, appearance of which is associated with hepatic lipidosis (fatty liver) in cattle, is induced by steroid hormones. Without addition of dexamethasone, only trace amounts of haptoglobin were detected in culture medium. With addition of dexamethasone (10(-12) to 10(-4) M), considerable amounts of haptoglobin were released into the medium. Maximal release was observed at concentrations of 10(-8) to 10(-6) M dexamethasone. Haptoglobin release was similarly induced by cortisol, although the effect was less potent than that of dexamethasone. Actinomycin D (a known protein synthesis inhibitor) dose-dependently reduced amounts of haptoglobin released in response to 10(-8) M dexamethasone. Dexamethasone also induced annexin I, which is known to be synthesized in response to glucocorticoids. Dexamethasone treatment resulted in reduced protein kinase C activity in the cell cytosol, which has been shown to be an early event in dexamethasone-treated cells. Other than glucocorticoids, estradiol induced haptoglobin release, whereas progesterone was less effective. The association of haptoglobin with hepatic lipidosis can be reasonably explained by the fact that haptoglobin production by the liver is induced by glucocorticoids and estradiol, and these steroid hormones are triggers for development of hepatic lipidosis in cattle.

Human hepatocytes loaded in 3D bioprinting generate mini-liver.

Science.gov (United States)

Zhong, Cheng; Xie, Hai-Yang; Zhou, Lin; Xu, Xiao; Zheng, Shu-Sen

2016-10-01

Because of an increasing discrepancy between the number of potential liver graft recipients and the number of organs available, scientists are trying to create artificial liver to mimic normal liver function and therefore, to support the patient's liver when in dysfunction. 3D printing technique meets this purpose. The present study was to test the feasibility of 3D hydrogel scaffolds for liver engineering. We fabricated 3D hydrogel scaffolds with a bioprinter. The biocompatibility of 3D hydrogel scaffolds was tested. Sixty nude mice were randomly divided into four groups, with 15 mice in each group: control, hydrogel, hydrogel with L02 (cell line HL-7702), and hydrogel with hepatocyte growth factor (HGF). Cells were cultured and deposited in scaffolds which were subsequently engrafted into livers after partial hepatectomy and radiation-induced liver damage (RILD). The engrafted tissues were examined after two weeks. The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), albumin, total bilirubin, CYP1A2, CYP2C9, glutathione S-transferase (a-GST), and UDP-glucuronosyl transferase (UGT-2) were compared among the groups. Hematoxylin-eosin (HE) staining and immunohistochemistry of cKit and cytokeratin 18 (CK18) of engrafted tissues were evaluated. The survival time of the mice was also compared among the four groups. 3D hydrogel scaffolds did not impact the viability of cells. The levels of ALT, AST, albumin, total bilirubin, CYP1A2, CYP2C9, a-GST and UGT-2 were significantly improved in mice engrafted with 3D scaffold loaded with L02 compared with those in control and scaffold only (P<0.05). HE staining showed clear liver tissue and immunohistochemistry of cKit and CK18 were positive in the engrafted tissue. Mice treated with 3D scaffold+L02 cells had longer survival time compared with those in control and scaffold only (P<0.05). 3D scaffold has the potential of recreating liver tissue and partial liver functions and can be used in the

Targeting Cytosolic Nucleic Acid-Sensing Pathways for Cancer Immunotherapies.

Science.gov (United States)

Iurescia, Sandra; Fioretti, Daniela; Rinaldi, Monica

2018-01-01

The innate immune system provides the first line of defense against pathogen infection though also influences pathways involved in cancer immunosurveillance. The innate immune system relies on a limited set of germ line-encoded sensors termed pattern recognition receptors (PRRs), signaling proteins and immune response factors. Cytosolic receptors mediate recognition of danger damage-associated molecular patterns (DAMPs) signals. Once activated, these sensors trigger multiple signaling cascades, converging on the production of type I interferons and proinflammatory cytokines. Recent studies revealed that PRRs respond to nucleic acids (NA) released by dying, damaged, cancer cells, as danger DAMPs signals, and presence of signaling proteins across cancer types suggests that these signaling mechanisms may be involved in cancer biology. DAMPs play important roles in shaping adaptive immune responses through the activation of innate immune cells and immunological response to danger DAMPs signals is crucial for the host response to cancer and tumor rejection. Furthermore, PRRs mediate the response to NA in several vaccination strategies, including DNA immunization. As route of double-strand DNA intracellular entry, DNA immunization leads to expression of key components of cytosolic NA-sensing pathways. The involvement of NA-sensing mechanisms in the antitumor response makes these pathways attractive drug targets. Natural and synthetic agonists of NA-sensing pathways can trigger cell death in malignant cells, recruit immune cells, such as DCs, CD8 + T cells, and NK cells, into the tumor microenvironment and are being explored as promising adjuvants in cancer immunotherapies. In this minireview, we discuss how cGAS-STING and RIG-I-MAVS pathways have been targeted for cancer treatment in preclinical translational researches. In addition, we present a targeted selection of recent clinical trials employing agonists of cytosolic NA-sensing pathways showing how these pathways

Case study: an evaluation of the human relevance of the synthetic pyrethroid metofluthrin-induced liver tumors in rats based on mode of action.

Science.gov (United States)

Yamada, Tomoya; Uwagawa, Satoshi; Okuno, Yasuyoshi; Cohen, Samuel M; Kaneko, Hideo

2009-03-01

In recent years, mode of action (MOA) frameworks have been developed through the International Life Sciences Institute Risk Science Institute and the International Programme on Chemical Safety, including an evaluation of the human relevance of the animal MOA data. In the present paper, the MOA for rat liver tumors induced by Metofluthrin is first analyzed through this framework based on data from studies on Metofluthrin and information on related chemicals from the literature. The human relevance of the rat liver carcinogenic response is then discussed based upon the human relevance framework. Two-year treatment with high dose of Metofluthrin produced hepatocellular tumors in both sexes of the Wistar rats. Metofluthrin induced CYP2B (increased smooth endoplasmic reticulum), resulted in increased liver weights which were associated with centrilobular hepatocyte hypertrophy, and induction of increased hepatocellular DNA replications. The above parameters related to the key events in Metofluthrin-induced liver tumors were observed at or below tumorigenic dose levels. Furthermore, CYP2B induction by Metofluthrin was shown to involve activation of the constitutive androstane receptor in rat hepatocytes. Based on the evidence, including a comparison with the results with another chemical, phenobarbital, acting by a similar MOA, it is reasonable to conclude that Metofluthrin will not have any hepatocarcinogenic activity in humans.

Protective Effect of Exogenous Dehydro-epiandrosterone Sulfate (DHEAS) on Liver Cell Organs of Whole Body y-Irradiated Rats

International Nuclear Information System (INIS)

Abdel-Fattah, K.I.; El-Gawish, M.A.; Abou-Safi, H.M.

2005-01-01

Dehydroepiandrosterone (DHEA) and its sulfate (DHES) are adrenal hormones. They are powerful endogenous antioxidants and are important in protecting the cells from damage. The present work aimed to evaluate the exogenous DHEAS as a protector against the whole body exposure to gamma radiation damages on DNA and RNA content of the nuclear fraction, calcium and acid phosphatase in the mitochondria fraction and glutathione (GSH) and malonaldehyde (MDA) in the cytosol fraction in the liver of male rats. Fifty male albino rats weighing 130-150 g were categorized into the following groups: 1-Control untreated. 2-Exposed to whole body gamma irradiation (6.5 Gy). 3-Received a single oral administration of DHEAS at a dose level of 200 mg/kg b.wt. 4-Administered with DHEAS (200 mg/kg) two h pre-exposure to whole body gamma irradiation (6.5 Gy). Three time intervals were determined for tissue sampling: after one day, one week and two weeks post irradiation (groups 2 and 4) and post administration of DHEAS (group 3). The results showed that: 1- DHEAS has a radioprotective effect on DNA and RNA content decreases in the liver nuclear fraction. 2- It significantly ameliorated the changes in mitochondria Ca21 content and acid phosphatase activity. 3- It improved both GSH and MDA contents in the cytosolic fraction. It could be concluded that, DHEAS showed an obvious protective role against the hazard of gamma radiation on liver cells. Several mechanisms were discussed about its effects. Therefore, more investigations are needed to understand well the role of DHEAS in protecting the animal tissues against ionizing radiation hazard

Phytanic acid alpha-oxidation: decarboxylation of 2-hydroxyphytanoyl-CoA to pristanic acid in human liver

NARCIS (Netherlands)

Verhoeven, N. M.; Wanders, R. J.; Schor, D. S.; Jansen, G. A.; Jakobs, C.

1997-01-01

The degradation of the first intermediate in the alpha-oxidation of phytanic acid, 2-hydroxyphytanoyl-CoA, was investigated. Human liver homogenates were incubated with 2-hydroxyphytanoyl-CoA or 2-hydroxyphytanic acid, after which formation of 2-ketophytanic acid and pristanic acid were studied.

A new factor from enteric bacteria of rats amplifying induction of liver enzyme by glucocorticoid. Pt. 2

International Nuclear Information System (INIS)

Kido, Hiroshi; Higashi, Takao; Katanuma, Nobuhiko

1977-01-01

1) An amplifier of the action of glucocorticoid was purified from Proteus mirabilis as described previously. It was found that it amplified the induction of liver tyrosine aminotransferase by dexamethasone markedly with doses of dexamethasone that caused minimal enzyme induction, but had little effect with doses that caused maximal induction. Thus the amplification may represent a saving of glucocorticoid. The amplification of enzyme activity was brought about by increase in amount of enzyme. 2) The amplification was observed when the amplifier was administered before or with dexamethasone, but not when it was given 2 h after dexamethasone. These results and the finding that actinomycin D inhibited the amplification indicate that the amplifier does not act on the translational level of enzyme induction. 3) It was found that the amplifier increased both incorporation of [ 3 H]dexamethasone into the cytosol and binding of [ 3 H]dexamethasone to cytosol protein and that it decreased decay of the [ 3 H]dexamethasone protein complex. (orig.) [de

The induction of autoimmune hepatitis in the human leucocyte antigen-DR4 non-obese diabetic mice autoimmune hepatitis mouse model.

Science.gov (United States)

Yuksel, M; Xiao, X; Tai, N; Vijay, G M; Gülden, E; Beland, K; Lapierre, P; Alvarez, F; Hu, Z; Colle, I; Ma, Y; Wen, L

2016-11-01

Autoimmune hepatitis (AIH) is a chronic liver disease characterized by progressive inflammation, female preponderance and seropositivity for autoantibodies such as anti-smooth muscle actin and/or anti-nuclear, anti-liver kidney microsomal type 1 (anti-LKM1) and anti-liver cytosol type 1 (anti-LC1) in more than 80% of cases. AIH is linked strongly to several major histocompatibility complex (MHC) alleles, including human leucocyte antigen (HLA)-DR3, -DR7 and -DR13. HLA-DR4 has the second strongest association with adult AIH, after HLA-DR3. We investigated the role of HLA-DR4 in the development of AIH by immunization of HLA-DR4 (DR4) transgenic non-obese diabetic (NOD) mice with DNA coding for human CYP2D6/FTCD fusion autoantigen. Immunization of DR4 mice leads to sustained mild liver injury, as assessed biochemically by elevated alanine aminotransferase, histologically by interface hepatitis, plasma cell infiltration and mild fibrosis and immunologically by the development of anti-LKM1/anti-LC1 antibodies. In addition, livers from DR4 mice had fewer regulatory T cells (T regs ), which had decreased programmed death (PD)-1 expression. Splenic T regs from these mice also showed impaired inhibitory capacity. Furthermore, DR4 expression enhanced the activation status of CD8 + T cells, macrophages and dendritic cells in naive DR4 mice compared to naive wild-type (WT) NOD mice. Our results demonstrate that HLA-DR4 is a susceptibility factor for the development of AIH. Impaired suppressive function of T regs and reduced PD-1 expression may result in spontaneous activation of key immune cell subsets, such as antigen-presenting cells and CD8 + T effectors, facilitating the induction of AIH and persistent liver damage. © 2016 British Society for Immunology.

The induction of autoimmune hepatitis in the human leucocyte antigen‐DR4 non‐obese diabetic mice autoimmune hepatitis mouse model

Science.gov (United States)

Yuksel, M.; Xiao, X.; Tai, N.; Vijay, G. M.; Gülden, E.; Beland, K.; Lapierre, P.; Alvarez, F.; Hu, Z.; Colle, I.; Ma, Y.

2016-01-01

Summary Autoimmune hepatitis (AIH) is a chronic liver disease characterized by progressive inflammation, female preponderance and seropositivity for autoantibodies such as anti‐smooth muscle actin and/or anti‐nuclear, anti‐liver kidney microsomal type 1 (anti‐LKM1) and anti‐liver cytosol type 1 (anti‐LC1) in more than 80% of cases. AIH is linked strongly to several major histocompatibility complex (MHC) alleles, including human leucocyte antigen (HLA)‐DR3, ‐DR7 and ‐DR13. HLA‐DR4 has the second strongest association with adult AIH, after HLA‐DR3. We investigated the role of HLA‐DR4 in the development of AIH by immunization of HLA‐DR4 (DR4) transgenic non‐obese diabetic (NOD) mice with DNA coding for human CYP2D6/FTCD fusion autoantigen. Immunization of DR4 mice leads to sustained mild liver injury, as assessed biochemically by elevated alanine aminotransferase, histologically by interface hepatitis, plasma cell infiltration and mild fibrosis and immunologically by the development of anti‐LKM1/anti‐LC1 antibodies. In addition, livers from DR4 mice had fewer regulatory T cells (Tregs), which had decreased programmed death (PD)‐1 expression. Splenic Tregs from these mice also showed impaired inhibitory capacity. Furthermore, DR4 expression enhanced the activation status of CD8+ T cells, macrophages and dendritic cells in naive DR4 mice compared to naive wild‐type (WT) NOD mice. Our results demonstrate that HLA‐DR4 is a susceptibility factor for the development of AIH. Impaired suppressive function of Tregs and reduced PD‐1 expression may result in spontaneous activation of key immune cell subsets, such as antigen‐presenting cells and CD8+ T effectors, facilitating the induction of AIH and persistent liver damage. PMID:27414259