The role of thiols in ATP-dependent transport of S-(2,4-dinitrophenyl) glutathione by rat liver plasma membrane vesicles

AbstractThe effect of thiol/disulfide exchange on ATP-dependent S-(2,4-dinitrophenyl)glutathione (GS-DNP) transport was studied in sodium nitrate treated rat liver plasma membrane vesicles. Transport followed Michaelis-Menten kinetics with an apparent Km of 9.6 μM for GS-DNP and 124 μM for ATP. 5,5′-Dithiobis(2-nitrobenzoate) (DTNB) and N-ethylmaleimide (NEM) efficiently inactivated GS-DNP transport activity in a dose- and time-dependent manner. Half-maximal inactivation occurred in 10 min at 40 μM for DTNB and 550 μM for NEM. Inactivation by DTNB was reversed by dithiothreitol. S-(N-Ethyl)maleimyl glutathione and/or ATP-Mg2+, but neither S-(N-ethyl)maleimyl cysteinylglycine nor oxidized glutathione could protect transport activity from inactivation by NEM or cystamine. These results suggest that reactive thiols are located near the active site of the transporter and that S-alkyl and the γ-glutamyl residues of glutathione are important for protection. Biological disulfides which were tested included cystine, oxidized glutathione, oxidized Coenzyme-A, oxidized lipoic acid, and oxidized lipoamide; cystamine was the most potent reversible inactivator. Molecular oxygen also inactivated transport activity, which was recovered on addition of dithiothreitol, suggesting intramolecular disulfide formation by vicinal thiols. We interpret these results to indicate that the ATP-dependent GS-DNP transporter contains two or more thiols which are necessary for the maintenance of transport activity. The reversible inactivation of the activity by biological disulfides suggests that the transporter may be regulated by thiol/disulfide exchange in vivo

Is the multidrug resistance an ATP channel?

The multidrug resistance ( mdr1 ) gene product, P-glycoprotein, is responsible for the ATP-dependent extrusion of a variety of compounds, including chemotherapeutic drugs, from cells. The data presented here show that cells with increaed levels of the P-glycoprotein release ATP to the medium in proportion to the concentration of the protein in their plasma membrane. Furthermore, measurements of whole-cell and single-channel currents with patch-clamp electrodes indicate that the P-glycoprotein serves as an ATP-conducting channel in the plasma membrane. These findings suggest an unusual role for the P-glycoprotein.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/38396/1/1840180131_ftp.pd

LKB1/AMPK and PKA Control ABCB11 Trafficking and Polarization in Hepatocytes.

Polarization of hepatocytes is manifested by bile canalicular network formation and activation of LKB1 and AMPK, which control cellular energy metabolism. The bile acid, taurocholate, also regulates development of the canalicular network through activation of AMPK. In the present study, we used collagen sandwich hepatocyte cultures from control and liver-specific LKB1 knockout mice to examine the role of LKB1 in trafficking of ABCB11, the canalicular bile acid transporter. In polarized hepatocytes, ABCB11 traffics from Golgi to the apical plasma membrane and endogenously cycles through the rab 11a-myosin Vb recycling endosomal system. LKB1 knockout mice were jaundiced, lost weight and manifested impaired bile canalicular formation and intracellular trafficking of ABCB11, and died within three weeks. Using live cell imaging, fluorescence recovery after photobleaching (FRAP), particle tracking, and biochemistry, we found that LKB1 activity is required for microtubule-dependent trafficking of ABCB11 to the canalicular membrane. In control hepatocytes, ABCB11 trafficking was accelerated by taurocholate and cAMP; however, in LKB1 knockout hepatocytes, ABCB11 trafficking to the apical membrane was greatly reduced and restored only by cAMP, but not taurocholate. cAMP acted through a PKA-mediated pathway which did not activate AMPK. Our studies establish a regulatory role for LKB1 in ABCB11 trafficking to the canalicular membrane, hepatocyte polarization, and canalicular network formation

Tyrosine Kinase Syk Non-Enzymatic Inhibitors and Potential Anti-Allergic Drug-Like Compounds Discovered by Virtual and In Vitro Screening

In the past decade, the spleen tyrosine kinase (Syk) has shown a high potential for the discovery of new treatments for inflammatory and autoimmune disorders. Pharmacological inhibitors of Syk catalytic site bearing therapeutic potential have been developed, with however limited specificity towards Syk. To address this topic, we opted for the design of drug-like compounds that could impede the interaction of Syk with its cellular partners while maintaining an active kinase protein. To achieve this challenging task, we used the powerful potential of intracellular antibodies for the modulation of cellular functions in vivo, combined to structure-based in silico screening. In our previous studies, we reported the anti-allergic properties of the intracellular antibody G4G11. With the aim of finding functional mimics of G4G11, we developed an Antibody Displacement Assay and we isolated the drug-like compound C-13, with promising in vivo anti-allergic activity. The likely binding cavity of this compound is located at the close vicinity of G4G11 epitope, far away from the catalytic site of Syk. Here we report the virtual screen of a collection of 500,000 molecules against this new cavity, which led to the isolation of 1000 compounds subsequently evaluated for their in vitro inhibitory effects using the Antibody Displacement Assay. Eighty five compounds were selected and evaluated for their ability to inhibit the liberation of allergic mediators from mast cells. Among them, 10 compounds inhibited degranulation with IC50 values ≤10 µM. The most bioactive compounds combine biological activity, significant inhibition of antibody binding and strong affinity for Syk. Moreover, these molecules show a good potential for oral bioavailability and are not kinase catalytic site inhibitors. These bioactive compounds could be used as starting points for the development of new classes of non-enzymatic inhibitors of Syk and for drug discovery endeavour in the field of inflammation related disorders

Reconstructing the Indian Origin and Dispersal of the European Roma: A Maternal Genetic Perspective

Previous genetic, anthropological and linguistic studies have shown that Roma (Gypsies) constitute a founder population dispersed throughout Europe whose origins might be traced to the Indian subcontinent. Linguistic and anthropological evidence point to Indo-Aryan ethnic groups from North-western India as the ancestral parental population of Roma. Recently, a strong genetic hint supporting this theory came from a study of a private mutation causing primary congenital glaucoma. In the present study, complete mitochondrial control sequences of Iberian Roma and previously published maternal lineages of other European Roma were analyzed in order to establish the genetic affinities among Roma groups, determine the degree of admixture with neighbouring populations, infer the migration routes followed since the first arrival to Europe, and survey the origin of Roma within the Indian subcontinent. Our results show that the maternal lineage composition in the Roma groups follows a pattern of different migration routes, with several founder effects, and low effective population sizes along their dispersal. Our data allowed the confirmation of a North/West migration route shared by Polish, Lithuanian and Iberian Roma. Additionally, eleven Roma founder lineages were identified and degrees of admixture with host populations were estimated. Finally, the comparison with an extensive database of Indian sequences allowed us to identify the Punjab state, in North-western India, as the putative ancestral homeland of the European Roma, in agreement with previous linguistic and anthropological studies

Genomic research, publics and experts in Latin America: Nation, race and body

The articles in this issue highlight contributions that studies of Latin America can make to wider debates about the effects of genomic science on public ideas about race and nation. We argue that current ideas about the power of genomics to transfigure and transform existing ways of thinking about human diversity are often overstated. If a range of social contexts are examined, the effects are uneven. Our data show that genomic knowledge can unsettle and reinforce ideas of nation and race; it can be both banal and highly politicized. In this introduction, we outline concepts of genetic knowledge in society; theories of genetics, nation and race; approaches to public understandings of science; and the Latin American contexts of transnational ideas of nation and race