Long term culture of MDCK strains alters chromosome content.

International audienceBACKGROUND: The Madin-Darby Canine Kidney (MDCK) cell line and its different strains are widely used as models for studying epithelial simple polarity. Recently Dukes et al. [BMC Cell Biology 12:43, 2011] provided a useful guide to the different MDCK strains, with a directory of where to buy them. The present work focused on chromosome content of MDCK cells, a parameter often disregarded by researchers working with these cells. FINDINGS: Using a general and reliable method for obtaining high yield of metaphasic preparations, the chromosome content of MDCK, MDCK I, MDCK II obtained from reliable sources was determined after maintaining them in culture for various periods of time. Within two months significant changes were observed in the range and the mean number of chromosomes of MDCK I and MDCK II cells. MDCK II cells routinely cultured in six different laboratories were also examined. In some of these cultures the cells have considerably drifted as shown by a high scattering in their number of chromosomes. CONCLUSION: These results entitle me to encourage researchers using MDCK cells obtained from reliable sources, to determine their chromosomal content upon receipt, to check this content after several passages, to use this feature to follow the possible drift of these cells, and finally to avoid working with cells maintained for more than two months in culture

Effect of L-methioninyl adenylate on the level of aminoacylation in vivo of tRNA(Met) from Escherichia coli K12

In cells of E.coli K12 grown exponentially in minimal medium, tRNA(met), tRNA(leu) and tRNA(ile) are aminoacylated at 100%, 80% and 64%, respectively. On addition of L-methioninyl adenylate to the growth medium, one observes a specific deacylation of tRNA(met). When more than 35% of tRNA(met) is deacylated, growth rate is reduced and becomes proportional to the amount of methionyl-tRNA formed

Expression, localization, and inducibility by bile acids of hepatobiliary transporters in the new polarized rat hepatic cell lines, Can 3-1 and Can 10.

International audienceSinusoidal and apical transporters are responsible for the uptake and biliary elimination of many compounds by hepatocytes. Few in vitro models are however available for analyzing such functions. The expression and bile-acid inducibility of 13 transporters and two nuclear receptors were investigated in the new rat polarized lines, Can 3-1 and Can 10, and in their unpolarized parent, Fao. The relative abundance of mRNA, the protein level, and their localization were examined by real-time quantitative PCR, Western blotting, immunofluorescence, and confocal microscopy. Compared with rat liver, mRNA levels of Fao cells were: negligible for Bsep/Abcb11; lower for the uptake transporters Ntcp and Oatps; similar for SHP, FXR, and Bcrp/Abcg2; and higher (four-fold to 160-fold) for the efflux pumps Mdr1b/Abcb1b, Mdr2/Abcb4, Mrp1/Abcc1, Mrp2/Abcc2, Mrp3/Abcc3, Abcg5, and Abcg8. This profile was mostly maintained (and improved for Bsep) in Can 10. Some transporters were less well expressed in Can 3-1. In both lines, sinusoidal (Ntcp, Mrp3) and canalicular transporters (Mdr-P-glycoproteins detected with C219 antibody, Mrp2) were localized at their correct poles. Bile-acid effects on polarity and mRNA levels of transporters were analyzed after a 6-day treatment with 50 microM taurocholic, chenodeoxycholic (CDCA), or ursodeoxycholic acid (UDCA). No polarization of Fao cells was induced; Can 10 and Can 3-1 polarity was maintained. CDCA and UDCA induced marked enhancement of the volume of Can 10 bile canaliculi. CDCA upregulated Bsep, Mdr2, SHP, Mdr1b, and Oatp2/1a4 in Can 10 (two- to seven-fold) and in Fao cells. Thus, Can 10 constitutes an attractive polarized model for studying vectorial hepatobiliary transport of endogenous and xenobiotic cholephilic compounds

ATP7B copper-regulated traffic and association with the tight junctions: copper excretion into the bile.: ATP7B and copper excretion by liver

International audienceBACKGROUND & AIMS: The copper transporter ATP7B plays a central role in the elimination of excess copper by the liver into the bile, yet the site of its action remains controversial. The studies reported here examine the correspondence between the site of ATP7B action and distribution and the pathways of copper disposal by the liver. METHODS: Microscopy and cell fractionation studies of polarized Can 10 cells forming long-branched bile canaliculi have been used to study the cellular distribution of ATP7B. Copper excretion into the bile was studied in perfused rat liver. RESULTS: Copper excess provokes a massive download of the ATP7B retained in the trans-Golgi network into the bile canalicular membrane. Furthermore, a stable ATP7B pool is localized to the tight junctions that seal the bile canaliculi. The profile of Cu(64) excretion into the bile by isolated rat livers perfused under one-pass conditions provides evidence of copper excretion by 2 separate mechanisms, transcytosis across the hepatocyte and paracellular transport throughout the tight junctions. CONCLUSIONS: Whereas the ATP7B retained in the trans-Golgi-network is massively translocated to the bile canalicular membrane in response to increased copper levels, a pool of ATP7B associated with the tight junctions remains stable. In situ studies indicate that copper is excreted into the bile by 2 separate pathways. The results are discussed in the frame of the normal and impeded excretion of copper into the bile

Modulation of hepatic copper-ATPase activity by insulin and glucagon involves protein kinase A (PKA) signaling pathway

International audienceDifferent studies have revealed copper imbalance in individuals suffering from diabetes and obesity, suggesting that regulation of glucose and/or fat metabolism could modulate cellular copper homeostasis. To test this hypothesis we investigated whether the key hormones of energy metabolism, insulin and glucagon, regulate the functional properties of the major hepatic copper-transporter, ATP7B (i.e., copper-dependent ATPase activity). We demonstrated that insulin reverses the effect of copper and stimulates retrograde trafficking of ATP7B from the canalicular membranes, consistent with the enhanced ability of ATP7B to sequester copper away from the cytosol. Physiological concentrations of insulin increase endogenous ATP7B activity in cultured hepatic cells and in tissues by 40%, whereas glucagon inhibits this activity by 70%. These effects were cancelled out when insulin and glucagon were combined. We also demonstrated that the opposite effects of the hormones on ATP7B activity involve receptor-mediated signaling pathways and membrane-bound kinases (PKA and PKB/Akt), which are reciprocally regulated by insulin and glucagon. Inhibiting insulin signaling at the level of its Tyr-kinase receptor, PI3K or PKB/Akt restored the basal activity of ATP7B. Insulin reduced endogenous PKA activity, whereas glucagon promoted PKA stimulation by approximately 100%. These findings demonstrate that the physiological modulation of ATP7B activity is linked to energy metabolism via insulin and glucagon, and could help to understand the mechanisms involved in the disruption of copper homeostasis in diabetic and obese patients

A Sulfur Tripod Glycoconjugate that Releases a High-Affinity Copper Chelator in Hepatocytes

International audienceReleased in the cell: Three N-acetylgalactosamine units, which recognize the asialoglycoprotein receptor, were tethered through disulfide bonds to the three coordinating thiol functions of a sulfur tripod ligand that has a high affinity for CuI (see scheme). The resulting glycoconjugate can be considered as a prodrug, because after uptake by hepatic cells the intracellular reducing glutathione (GSH) releases the high-affinity intracellular CuI chelator