Biomedical subjects

Nazzareno Ballatori

Publications and source records attributed to Nazzareno Ballatori.

24 records · Page 2Linked to original sources

PubMed2002

Inhibition of Mrp2- and Ycf1p-mediated transport by reducing agents: evidence for GSH transport on rat Mrp2.

Mammalian Mrp2 and its yeast orthologue, Ycf1p, mediate the ATP-dependent cellular export of a variety of organic anions. Ycf1p also appears to transport the endogenous tripeptide glutathione (GSH), whereas no ATP-dependent GSH transport has been detected in Mrp2-containing mammalian plasma membrane vesicles. Because GSH uptake measurements in isolated membrane vesicles are normally carried out in the presence of 5-10 mM dithiothreitol (DTT) to maintain the tripeptide in the reduced form, the present study examined the effects of DTT and other sulfhydryl-reducing agents on Ycf1p- and Mrp2-mediated transport activity. Uptake of S-dinitrophenyl glutathione (DNP-SG), a prototypic substrate of both proteins, was measured in Ycf1p-containing Saccharomyces cerevisiae vacuolar membrane vesicles and in Mrp2-containing rat liver canalicular plasma membrane vesicles. Uptake was inhibited in both vesicle systems in a concentration-dependent manner by DTT, dithioerythritol, and beta-mercaptoethanol, with concentrations of 10 mM inhibiting by approximately 40%. DTT's inhibition of DNP-SG transport was noncompetitive. In contrast, ATP-dependent transport of [(3)H]taurocholate, a substrate for yeast Bat1p and mammalian Bsep bile acid transporters, was not significantly affected by DTT. DTT also inhibited the ATP-dependent uptake of GSH by Ycf1p. As the DTT concentration in incubation solutions containing rat liver canalicular plasma membrane vesicles was gradually decreased, ATP-dependent GSH transport was now detected. These results demonstrate that Ycf1p and Mrp2 are inhibited by concentrations of reducing agents that are normally employed in studies of GSH transport. When this inhibition was partially relieved, ATP-dependent GSH transport was detected in rat liver canalicular plasma membranes, indicating that both Mrp2 and Ycf1p are able to transport GSH by an ATP-dependent mechanism.

ATP-Binding Cassette Transporters↗

PubMed2002

Apolipoprotein B mRNA editing and the reduction in synthesis and secretion of the atherogenic risk factor, apolipoprotein B100 can be effectively targeted through TAT-mediated protein transduction.

Hepatic very-low-density lipoprotein particles (VLDL) containing full-length apolipoprotein B100 are metabolized in the blood stream to low-density lipoprotein (LDL) particles, whose elevated levels increase the risk of atherosclerosis. Statins and bile-acid sequestrants are effective LDL-lowering therapies for many patients. Development of alternative therapies remains important for patients with adverse reactions to conventional therapy, with defects in the LDL receptor-dependent lipoprotein uptake pathway and for intervention in children. Editing of apoB mRNA by the enzyme APOBEC-1 changes a glutamine codon to a stop codon, leading to the synthesis and secretion of apoB48-containing VLDL, which are rapidly cleared before they can be metabolized to LDL. Human liver does not edit apoB mRNA because it does not express APOBEC-1. Although initially promising, enthusiasm for apobec-1 gene therapy for hypercholesterolemia was blunted by the finding that uncontrolled transgenic expression of APOBEC-1 led to nonspecific editing of mRNAs and pathology. We demonstrate that APOBEC-1 fused to TAT entered primary hepatocytes, where it induced a transient increase in mRNA editing activity and enhanced synthesis and secretion of VLDL containing apoB48. Protein transduction of APOBEC-1 transiently stimulated high levels of apoB mRNA editing in a dose-dependent manner without loss of fidelity. These results suggested that apoB mRNA editing should be re-evaluated as a LDL-lowering therapeutic target in the new context of protein transduction therapy.

APOBEC-1 Deaminase↗

PubMed2002

Identification of a mechanism by which the methylmercury antidotes N-acetylcysteine and dimercaptopropanesulfonate enhance urinary metal excretion: transport by the renal organic anion transporter-1.

N-Acetylcysteine (NAC) and dimercaptopropanesulfonate (DMPS) are sulfhydryl-containing compounds that produce a dramatic acceleration of urinary methylmercury (MeHg) excretion in poisoned animals, but the molecular mechanism for this effect is unknown. NAC and DMPS are themselves excreted in urine in high concentrations. The present study tested the hypothesis that the complexes formed between MeHg and these anionic chelating agents are transported from blood into proximal tubule cells by the basolateral membrane organic anion transporters (Oat) 1 and Oat3. Xenopus laevis oocytes expressing rat Oat1 showed increased uptake of [(14)C]MeHg when complexed with either NAC or DMPS but not when complexed with L-cysteine, glutathione, dimercaptosuccinate, penicillamine, or gamma-glutamylcysteine. In contrast, none of these MeHg complexes were transported by Oat3-expressing oocytes. The apparent K(m) values for Oat1-mediated transport were 31 +/- 2 microM for MeHg-NAC and 9 +/- 2 microM for MeHg-DMPS, indicating that these are relatively high-affinity substrates. Oat1-mediated uptake of [(14)C]MeHg-NAC and [(14)C]MeHg-DMPS was inhibited by prototypical substrates for Oat1, including p-aminohippurate (PAH), and was trans-stimulated when oocytes were preloaded with 2 mM glutarate but not glutamate. Conversely, efflux of [(3)H]PAH from Oat1-expressing oocytes was trans-stimulated by glutarate, PAH, NAC, DMPS, MeHg-NAC, MeHg-DMPS, and a mercapturic acid, indicating that these are transported solutes. [(3)H]PAH uptake was competitively inhibited by NAC (K(i) of 2.0 +/- 0.3 mM) and DMPS (K(i) of 0.10 +/- 0.02 mM), providing further evidence that these chelating agents are substrates for Oat1. These results indicate that the MeHg antidotes NAC and DMPS and their mercaptide complexes are transported by Oat1 but are comparatively poor substrates for Oat3. This is the first molecular identification of a transport mechanism by which these antidotes may enhance urinary excretion of toxic metals.

Acetylcysteine↗

PubMed2002

An evolutionarily ancient Oatp: insights into conserved functional domains of these proteins.

Cellular uptake of organic solutes is mediated in large part by a gene family of membrane transporters called OATPs (SLC21A). To study the structural determinants and evolutionary development of the SLC21A family, we have cloned and functionally characterized a highly expressed evolutionarily primitive Oatp from the liver of the small skate, Raja erinacea. A full-length cDNA (2.3 kb) was obtained that encodes a protein of 689 amino acids. The characteristics of this novel skate Oatp, including tissue expression, subcellular localization, substrate selectivity, Na(+) dependence, and inhibitor selectivity were generally similar to liver-specific human OATP-C and rat Oatp4. However, sequence comparisons with other OATPs indicate that this skate Oatp shares only approximately 40-50% amino acid identity with the liver-specific OATPs/Oatps and with human OATP-F. Further computer analysis revealed that the highest amino acid identities reside in the first external (78%) and internal loops (75%) and transmembrane domains 2 (76%), 3 (62%), 4 (70%), and 11 (64%). We propose that the conserved regions of the SLC21A transporter family may be critical structural determinants of substrate specificity and function.

Algorithms↗

PubMed2002

Glutathione secretion into rat milk and its subsequent gamma-glutamyltranspeptidase-mediated catabolism.

Reduced glutathione (GSH) is a critical cellular antioxidant and a cofactor for enzymes that detoxify carcinogens. GSH is present at high concentrations in all mammalian tissues, including mammary gland tissue (1- 2 mM), and recent studies indicate that the tripeptide may also be present in milk. The present study examined the hypothesis that GSH is secreted into rat milk, and that some of the secreted GSH is degraded by the ectoenzyme gamma-glutamyltranspeptidase (GGT) within the milk space. To test this possibility, milk was collected over a 4-hour period in anesthetized, 9- to 11-day postpartum Wistar rats, as well as in similar rats treated with the GGT inhibitor acivicin. GGT enzymatic activity was relatively high in rat mammary tissue, and acivicin was able to inhibit most ( approximately 80%), but not all of this activity. In control animals, GSH was present in milk, although at a relatively low concentration of approximately 20-25 microM. However, after treatment with acivicin, milk GSH concentration increased 3- to 4-fold. Most of the glutathione was in the reduced form, as no GSSG was detected (<2 microM) in any of the milk samples. These results demonstrate that GSH is secreted into rat milk, but that a large fraction of the secreted GSH is degraded by the ectoenzyme GGT. Taken together with previous studies indicating the presence of GSH synthetic and catabolic enzymes in mammary tissue, these data suggest that the mammary gland has a functional gamma-glutamyl cycle. This cycle may contribute to the secretion of cysteine into milk, which is a rate-limiting amino acid for neonatal growth and development.

Animals↗

PubMed2002

Transport of toxic metals by molecular mimicry.

Intracellular concentrations of essential metals are normally maintained within a narrow range, whereas the nonessential metals generally lack homeostatic controls. Some of the factors that contribute to metal homeostasis have recently been identified at the molecular level and include proteins that mediate import of essential metals from the extracellular environment, those that regulate delivery to specific intracellular proteins or compartments, and those that mediate metal export from the cell. Some of these proteins appear highly selective for a given essential metal; however, others are less specific and interact with multiple metals, including toxic metals. For example, DCT1 (divalent cation transporter-1; also known as NRAMP2 or DMT1) is considered to be a major cellular uptake mechanism for Fe(2+) and other essential divalent metals, but this protein also mediates uptake of Cd(2+), Pb(2+), and possibly of other toxic divalent metals. The ability of nonessential metals to interact with binding sites for essential metals is critical for their ability to gain access to specific cellular compartments and for their ability to disrupt normal biochemical or physiological functions. Another major mechanism by which metals traverse cell membranes and produce cell injury is by forming complexes whose overall structures mimic those of endogenous molecules. For example, it has long been known that arsenate and vanadate can compete with phosphate for transport and metabolism, thereby disrupting normal cellular functions. Similarly, cromate and molybdate can mimic sulfate in biological systems. Studies in our laboratory have focused on the transport and toxicity of methylmercury (MeHg) and inorganic mercury. Mercury has a high affinity for reduced sulfhydryl groups, including those of cysteine and glutathione (GSH). MeHg-l-cysteine is structurally similar to the amino acid methionine, and this complex is a substrate for transport systems that carry methionine across cell membranes. Once MeHg has entered the cell, some of it binds to GSH, and the resulting MeHg-glutathione complex appears to be a substrate for proteins that mediate cellular export of glutathione S-conjugates, including the apically located MRP2 (multidrug resistance-associated protein 2) transporter, a member of the adenosine triphosphate-binding cassette protein superfamily. Because other toxic metals also form complexes with endogenous molecules, comparable mechanisms may be involved in their membrane transport and disposition.

Animals↗