Hepatocidol toxicity of Listeria species

A novel rat hepatocidal test, based on morphological changes in monolayer culture and the percentage of lactate dehydrogenase (LDH) released into the medium after exposure to culture filtrates of Listeria spp. was used to determine listerial toxicity and pathogenicity. Primary cultures of rat hepatocytes exposed to brain heart infusion (BHI) culture filtrates from ATCC strains of Listeria monocytogenes and L. ivanovii, released 91-92% and 95% of LDH after 3 h and 18.5 h, respectively. Cultured monolayers changed from normal hepatocytes into nonviable round forms. Brain heart infusion broth and BHI culture filtrates of other Listeria spp. were nontoxic to hepatocytes. The rat hepatocidal test is a quantitative and rapid system for studying listerial toxicity and pathogenicity.     

Lactosylceramide-induced stimulation of liposome uptake by Kupffer cells in vivo

Incorporation of 8 mol percent lactosylceramide into small unilamellar vesicles consisting of cholesterol and sphingomyelin in an equimolar ratio and containing [³H]inulin as a marker resulted in an increase in total liver uptake and a drastic change in intrahepatic distribution of the liposomes after intravenous injection into rats. The control vesicles without glycolipid accumulated predominantly in the hepatocytes, but incorporation of the glycolipid resulted in a larger stimulation of Kupffer-cell uptake (3.2-fold) than of hepatocyte uptake (1.2-fold). Liposome preparations both with and without lactosylceramide in which part of the sphingomyelin was replaced by phosphatidylserine, resulting in a net negative charge of the vesicles, were cleared much more rapidly from the blood and taken up by the liver to higher extents. The negative charge had, however, no influence on the intrahepatic distributions. The fast hepatic uptake of the negatively charged liposomes allowed competition experiments with substrates for the galactose receptors on liver cells. Inhibition of blood clearance and liver uptake of lactosylceramide-containing liposomes by $\text{N-}\text{acetyl-}\text{d}\text{-galactosamine}$ indicated the involvement of specific recognition sites for the liposomal galactose residues. This inhibitory effect of $\text{N-}\text{acetyl-}\text{d}\text{-galactosamine}$ was shown to be mainly the result of a decreased liposome uptake by the Kupffer cells, compatible with the reported presence of a galactose specific receptor on this cell type (Kolb-Bachofen et al. (1982) Cell 29, 859–866). The difference between the results on sphingomyelin-based liposomes as described in this paper and those on phosphatidylcholine-based liposomes as published previously (Spanjer and Scherphof (1983) Biochim. Biophys. Acta 734, 40–47) are discussed.     

ZRT/IRT-like Protein 14 (ZIP14) Promotes the Cellular Assimilation of Iron from Transferrin

ZIP14 is a transmembrane metal ion transporter that is abundantly expressed in the liver, heart, and pancreas. Previous studies of HEK 293 cells and the hepatocyte cell lines AML12 and HepG2 established that ZIP14 mediates the uptake of non-transferrin-bound iron, a form of iron that appears in the plasma during pathologic iron overload. In this study we investigated the role of ZIP14 in the cellular assimilation of iron from transferrin, the circulating plasma protein that normally delivers iron to cells by receptor-mediated endocytosis. We also determined the subcellular localization of ZIP14 in HepG2 cells. We found that overexpression of ZIP14 in HEK 293T cells increased the assimilation of iron from transferrin without increasing levels of transferrin receptor 1 or the uptake of transferrin. To allow for highly specific and sensitive detection of endogenous ZIP14 in HepG2 cells, we used a targeted knock-in approach to generate a cell line expressing a FLAG-tagged ZIP14 allele. Confocal microscopic analysis of these cells detected ZIP14 at the plasma membrane and in endosomes containing internalized transferrin. HepG2 cells in which endogenous ZIP14 was suppressed by siRNA assimilated 50% less iron from transferrin compared with controls. The uptake of transferrin, however, was unaffected. We also found that ZIP14 can mediate the transport of iron at pH 6.5, the pH at which iron dissociates from transferrin within the endosome. These results suggest that endosomal ZIP14 participates in the cellular assimilation of iron from transferrin, thus identifying a potentially new role for ZIP14 in iron metabolism.     

The Aryl Hydrocarbon Receptor-interacting Protein (AIP) Is Required for Dioxin-induced Hepatotoxicity but Not for the Induction of the Cyp1a1 and Cyp1a2 Genes

The aryl hydrocarbon receptor (AHR) plays an essential role in the toxic response to environmental pollutants such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin), in the adaptive up-regulation of xenobiotic metabolizing enzymes, and in hepatic vascular development. In our model of AHR signaling, the receptor is found in a cytosolic complex with a number of molecular chaperones, including Hsp90, p23, and the aryl hydrocarbon receptor-interacting protein (AIP), also known as ARA9 and XAP2. To understand the role of AIP in adaptive and toxic aspects of AHR signaling, we generated a conditional mouse model where the Aip locus can be deleted in hepatocytes. Using this model, we demonstrate two important roles for the AIP protein in AHR biology. (i) The expression of AIP in hepatocytes is essential to maintain high levels of functional cytosolic AHR protein in the mammalian liver. (ii) Expression of the AIP protein is essential for dioxin-induced hepatotoxicity. Interestingly, classical AHR-driven genes show differential dependence on AIP expression. The Cyp1b1 and Ahrr genes require AIP expression for normal up-regulation by dioxin, whereas Cyp1a1 and Cyp1a2 do not. This differential dependence on AIP provides evidence that the mammalian genome contains more than one class of AHR-responsive genes and suggests that a search for AIP-dependent, AHR-responsive genes may guide us to the targets of the dioxin-induced hepatotoxicity.     

The adenylate cyclase activity of isolated hepatocytes actions of glucagon and sodium fluoride

Isolated hepatocytes converted exogenous [α-³²P]ATP to cyclic [³²P]AMP at high rates. This system was used for kinetic studies of the effects of glucagon, fluoride, free magnesium and free ATP^4? on adenylate cyclase. In the absence or presence of glucagon, free Mg²⁺ activated adenylate cyclase by decreasing the K_m for MgATP^2? without changing V. Free ATP^4? was not a potent inhibitor of adenylate cyclase and the only effect of glucagon was to increase V.Fluoride also increased the V of adenylate cyclase, but, in contrast to the results obtained with glucagon, the effect increased as the concentration of free Mg²⁺ increased. One explanation of the effect of fluoride, consistent with the idea that free Mg²⁺ activates adenylate cyclase and free ATP is not an inhibitor, is that fluoride increases the affinity of the enzyme for Mg²⁺. Weak inhibition of adenylate cyclase by ATP^4? in the presence of fluoride cannot be excluded.     

Inhibition of autophagy by benzyl alcohol

Benzyl alcohol caused a rather complete and selective inhibition of the methylamine sensitive (i.e., the putative lysosomal) pathway of protein degradation in isolated rat hepatocytes. The effect was found to be entirely reversible within 30 min of removing the agent. A morphometric examination of electron micrographs revealed that the inhibition of lysosomal protein degradation coincided with a block in the formation of autophagic vacuoles. The number of acidic vacuoles (i.e., vacuoles induced to swell by adding methylamine) was not drastically reduced.     

Stability and optimization of canalicular function in hepatocyte couplets

An enriched preparation of rat hepatocyte couplets was obtaied by collagense perfusion and subsequent elutriation (> 85 per cent couplets and triplets; viability of over 95 per cent). Canalicular secretory activity (the ability to accumulate cholyl-lysyl-fluorescein, CLF) was first apparent after 2 h of culture at 37°C and was present in over 80 per cent of the total population after 5–6 h. This remained almost constant for at least 4 h in both elutriated and directly plated cells. Initial storage of freshly prepared couplets at 4°C for up to 6 h prior to incubation had no adverse effect upon secretory function. Reduction of canalicular secretory activity occurred at a concentration of the hepatotoxic agent menadione (IC₅₀ 17 μM) that was lower than that required to induce mild plasma-membrane blebbing (IC₅₀ 43 μM). This study has optimized and characterized the canalicular secretory effectiveness and stability of an enriched preparation of hepatocyte couplets, and established the feasibility of studies of toxic agents on hepatobiliary function in a heterogeneous population of hepatocytes. In this preparation other biochemical parameters can be assessed, thus complementing previous techniques using individual couplets.     

Complex regulation of nucleoside transporter expression in epithelial and immune system cells

Nucleoside transporters have a variety of functions in the cell, such as the provision of substrates for nucleic acid synthesis and the modulation of purine receptors by determining agonist availability. They also transport a wide range of nucleoside-derived antiviral and anticancer drugs. Most mammalian cells coexpress several nucleoside transporter isoforms at the plasma membrane, which are differentially regulated. This paper reviews studies on nucleoside transporter regulation, which has been extensively characterized in the laboratory in several model systems: the hepatocyte, an epithelial cell type, and immune system cells, in particular B cells, which are non-polarized and highly specialized. The hepatocyte co-expresses at least two Na+-dependent nucleoside transporters, CNT1 and CNT2, which are up-regulated during cell proliferation but may undergo selective loss in certain experimental models of hepatocarcinomas. This feature is consistent with evidence that CNT expression also depends on the differentiation status of the hepatocyte. Moreover, substrate availability also modulates CNT expression in epithelial cells, as reported for hepatocytes and jejunum epithelia from rats fed nucleotide-deprived diets. In human B cell lines, CNT and ENT transporters are co-expressed but differentially regulated after B cell activation triggered by cytokines or phorbol esters, as described for murine bone marrow macrophages induced either to activate or to proliferate. The complex regulation of the expression and activity of nucleoside transporters hints at their relevance in cell physiology.     

Immunoregulatory effect of mesenchymal stem cell via mitochondria signaling pathways in allergic asthma

Asthma is a complicated lung disease, which has increased morbidity and mortality rates in worldwide. There is an overlap between asthma pathophysiology and mitochondrial dysfunction and MSCs may have regulatory effect on mitochondrial dysfunction and treats asthma. Therefore, immune-modulatory effect of MSCs and mitochondrial signaling pathways in asthma was studied.After culturing of MSCs and producing asthma animal model, the mice were treated with MSCs via IV via IT. BALf's eosinophil Counting, The levels of IL-4, −5, −13, −25, –33, INF-γ, Cys-LT, LTB4, LTC4, mitochondria genes expression of COX-1, COX-2, ND1, Nrf2, Cytb were measured and lung histopathological study were done.BALf's eosinophils, the levels of IL-4, −5, −13, −25, –33, LTB4, LTC4, Cys-LT, the mitochondria genes expression (COX-1, COX-2, Cytb and ND-1), perivascular and peribronchial inflammation, mucus hyper-production and hyperplasia of the goblet cell in pathological study were significantly decreased in MSCs-treated asthma mice and reverse trend was found about Nrf-2 gene expression, IFN-γ level and ratio of the INF-γ/IL-4.MSC therapy can control inflammation, immune-inflammatory factors in asthma and mitochondrial related genes, and prevent asthma immune-pathology.