Biomedical subjects

Nazzareno Ballatori

Publications and source records attributed to Nazzareno Ballatori.

At least 19 recordsLinked to original sources

PubMed2006

4-Hydroxyacetophenone-induced choleresis in rats is mediated by the Mrp2-dependent biliary secretion of its glucuronide conjugate.

PURPOSE: The present study examined the underlying mechanism by which 4-hydroxyacetophenone (4-HA), a bioactive compound found in several medicinal herbs, exerts its potent stimulatory effects on hepatic bile secretion. METHODS: Bile flow, and biliary excretion of 4-HA, its metabolites, and inorganic electrolytes was examined in both normal Wistar rats and in TR(-) Wistar rats that have a congenital defect in the multidrug resistance-associated protein-2, Mrp2/Abcc2. The effects of 4-HA were also examined in animals treated with buthionine sulfoximine to decrease hepatic glutathione (GSH) levels. RESULTS: In normal rats, 4-HA dramatically increased bile flow rate, whereas it failed to exert a choleretic effect in TR(-) rats. This choleresis was not explained by increased biliary output of Na(+), K(+), Cl(-) or HCO(3) (-), or by increased biliary GSH excretion. Depletion of hepatic GSH with buthionine sulfoximine had no effect on the 4-HA-induced choleresis. HPLC analysis revealed that a single major compound was present in bile, namely.4-hydroxyacetophenone-4-O-beta-glucuronide, and that the parent compound was not detected in bile. Biliary excretion of the glucuronide was directly correlated with the increases in bile flow. In contrast to normal rats, this 4-HA metabolite was not present in bile of TR(-) rats. CONCLUSIONS: These results demonstrate that the major biliary metabolite of 4-HA in rats is the 4-O-beta-glucuronide, a compound that is secreted into bile at high concentrations, and may thus account in large part for the choleretic effects of 4-HA. Transport of this metabolite across the canalicular membrane into bile requires expression of the Mrp2 transport protein.

Acetophenones↗

PubMed2006

Retention of structural and functional polarity in cultured skate hepatocytes undergoing in vitro morphogenesis.

The present study characterized a primary culture model of hepatocytes isolated from the little skate, Leucoraja erinacea, that maintain remarkable structural and functional polarity over 7 days in culture. Skate hepatocytes were isolated as clusters of 3-20 hepatocytes surrounding a bile canaliculus, rather than as single cells. Trypan blue and propidium iodide exclusion was found to be >98%, and the cells maintained high intracellular concentrations of K+, ATP, and reduced glutathione (GSH), and high ratios of ATP/ADP and GSH/GSSG. Glutathione S-transferase activity remained constant, whereas cytochrome P450 activity declined to 16% of initial levels after 7 days. Quantitative RT-PCR analysis revealed that the mRNA levels of several genes remained constant over the 7-day period, whereas Bsep, the canalicular bile salt export pump, levels declined slowly to 30% of initial values. In the presence of dexamethasone, the cells underwent a morphogenesis in which the clusters reannealed into a three-dimensional network of chords. During this morphogenesis, skate hepatocytes clusters maintained a polarized distribution of actin filaments and microtubules, as well as apical and basolateral membrane domains. Polarity of membrane transport systems was confirmed both morphologically, using antibodies raised against Bsep and Mrp2, the canalicular multispecific organic anion transporter, and functionally, by monitoring secretion of the fluorescent organic anions NBD-taurocholate, a Bsep substrate, and fluorescein-methotrexate, an Mrp2 substrate, into the bile canalicular spaces. Overall, the results indicate that in contrast with mammalian hepatocytes, isolated skate hepatocyte clusters retain polarity in culture, and provide an excellent system for investigating long-term effects of drugs and xenobiotics on hepatobiliary functions, and for studying in vitro morphogenesis.

Animals↗

PubMed2006

Upregulation of a basolateral FXR-dependent bile acid efflux transporter OSTalpha-OSTbeta in cholestasis in humans and rodents.

Organic solute transporter (OSTalpha-OSTbeta) is a novel heteromeric bile acid and sterol transporter expressed at the basolateral membranes of epithelium in the ileum, kidney, and liver. To determine whether OSTalpha-OSTbeta undergoes farnesoid X receptor (FXR)-dependent adaptive regulation following cholestatic liver injury, mRNA and protein expression levels were analyzed in patients with primary biliary cirrhosis (PBC) and following common bile duct ligation (CBDL) in rats and Fxr null and wild-type mice. Hepatic OSTalpha and OSTbeta mRNA increased 3- and 32-fold, respectively, in patients with PBC compared with controls, whereas expression of Ostalpha and Ostbeta also increased in the liver of rats and mice following CBDL. In contrast, expression of Ostalpha and Ostbeta mRNA was generally lower in Fxr null mice, and CBDL failed to enhance expression of Ostalpha and Ostbeta compared with wild-type mice. HepG2 cells treated for 24 h with chenodeoxycholic acid, a selective FXR ligand, had higher levels of OSTalpha and OSTbeta mRNA and protein. Increases in OST protein were visualized by confocal microscopy at the plasma membrane. These results indicate that expression of Ostalpha and Ostbeta are highly regulated in response to cholestasis and that this response is dependent on the FXR bile acid receptor.

Animals↗

PubMed2006

Bile acids induce ileal damage during experimental necrotizing enterocolitis.

BACKGROUND & AIMS: Necrotizing enterocolitis (NEC) is the most common gastrointestinal emergency of premature infants. While the effect of bile acids (BAs) on intestinal mucosal injury is known, we investigated the contribution of BAs during the development of NEC in neonatal rats. METHODS: Premature rats were fed with cow's milk-based formula and subjected to asphyxia and cold stress to develop NEC. Jejunal and ileal luminal BAs, portal blood BAs, and messenger RNA and protein for the apical sodium-dependent bile acid transporter, the ileal bile acid binding protein, and the heteromeric organic solute transporter (Ostalpha/Ostbeta)were evaluated. RESULTS: Ileal luminal BAs levels were increased significantly during disease development and the removal of ileal BAs significantly decreased the incidence and severity of disease. Furthermore, when NEC was reduced via treatment with epidermal growth factor (EGF), BA levels were reduced significantly. Jejunal luminal BA levels were similar between animals with NEC and controls, but portal/ileal luminal BA ratios were decreased significantly in animals with NEC. The apical sodium-dependent bile acid transporter was up-regulated at the site of injury in animals with NEC and decreased after EGF treatment; however, the ileal bile acid binding protein was up-regulated only in the NEC and EGF group. Ostalpha/Ostbeta expression was low in all groups, and only slightly increased in the NEC group. CONCLUSIONS: These data strongly suggest that BAs play a role in the development of ileal damage in experimental NEC and that alterations in BA transport in the neonatal ileum may contribute to disease development.

Animals↗

PubMed2005

Molecular mechanisms of reduced glutathione transport: role of the MRP/CFTR/ABCC and OATP/SLC21A families of membrane proteins.

The initial step in reduced glutathione (GSH) turnover in all mammalian cells is its transport across the plasma membrane into the extracellular space; however, the mechanisms of GSH transport are not clearly defined. GSH export is required for the delivery of its constituent amino acids to other tissues, detoxification of drugs, metals, and other reactive compounds of both endogenous and exogenous origin, protection against oxidant stress, and secretion of hepatic bile. Recent studies indicate that some members of the multidrug resistance-associated protein (MRP/CFTR or ABCC) family of ATP-binding cassette (ABC) proteins, as well as some members of the organic anion transporting polypeptide (OATP or SLC21A) family of transporters contribute to this process. In particular, five of the 12 members of the MRP/CFTR family appear to mediate GSH export from cells namely, MRP1, MRP2, MRP4, MRP5, and CFTR. Additionally, two members of the OATP family, rat Oatp1 and Oatp2, have been identified as GSH transporters. For the Oatp1 transporter, efflux of GSH may provide the driving force for the uptake of extracellular substrates. In humans, OATP-B and OATP8 do not appear to transport GSH; however, other members of this family have yet to be characterized in regards to GSH transport. In yeast, the ABC proteins Ycf1p and Bpt1p transport GSH from the cytosol into the vacuole, whereas Hgt1p mediates GSH uptake across the plasma membrane. Because transport is a key step in GSH homeostasis and is intimately linked to its biological functions, GSH export proteins are likely to modulate essential cellular functions.

ATP Binding Cassette Transporter, Subfamily B↗

PubMed2005

OSTalpha-OSTbeta: a major basolateral bile acid and steroid transporter in human intestinal, renal, and biliary epithelia.

The cellular and subcellular localization and mechanism of transport of the heteromeric organic solute transporter (OST) OSTalpha-OSTbeta was examined in human and rodent epithelia. The two subunits of the transporter were expressed together in human small intestine, kidney, and liver, tissues that also express the apical sodium-dependent bile acid uptake transporter ASBT (SLC10A2). Indirect immunofluorescence microscopy localized OSTalpha and OSTbeta to the basolateral membrane of mouse, rat, and human ileal enterocytes, renal proximal tubular cells, and cholangiocytes. Transport in OSTalpha-OSTbeta-expressing Xenopus laevis oocytes was unaffected by depletion of intracellular adenosine triphosphate, or by changes in transmembrane Na(+), K(+), H(+), or Cl(-) concentration gradients. However, the oocytes demonstrated robust substrate efflux and trans-stimulation, indicating that transport occurs by facilitated diffusion. Madin Darby canine kidney cells coexpressing mouse Ostalpha and Ostbeta exhibited enhanced apical to basolateral transport of the major glycine and taurine conjugated bile acid species. In conclusion, the selective localization of OSTalpha and OSTbeta to the basolateral plasma membrane of epithelial cells responsible for bile acid and sterol reabsorption, the substrate selectivity of the transporter, and the facilitated diffusion transport mode collectively indicate that OSTalpha-OSTbeta is a key basolateral transporter for the reabsorption of these important steroid-derived molecules.

Amino Acid Sequence↗

PubMed2005

Biology of a novel organic solute and steroid transporter, OSTalpha-OSTbeta.

Using a comparative approach, recent studies have identified and functionally characterized a new type of organic solute and steroid transporter (OST) from skate, mouse, rat, and human genomes. In contrast to all other organic anion transporters identified to date, transport activity requires the coexpression of two distinct gene products, a predicted 340-amino acid, seven-transmembrane (TM) domain protein (OSTalpha) and a putative 128-amino acid, single-TM domain ancillary polypeptide (OSTbeta). When OSTalpha and OSTbeta are coexpressed in Xenopus oocytes, they are able to mediate transport of estrone 3-sulfate, dehydroepiandrosterone 3- sulfate, taurocholate, digoxin, and prostaglandin E2, indicating a role in the disposition of key cellular metabolites or signaling molecules. OSTalpha and OSTbeta are expressed at relatively high levels in intestine, kidney, and liver, but they are also expressed at lower levels in many human tissues. Indirect immunofluorescence microscopy revealed that intestinal OSTalpha and OSTbeta proteins are localized to the baso-lateral membrane of mouse enterocytes. In MDCK cells, mouse Ostalpha-Ostbeta mediated the vectorial movement of taurocholate from the apical to the basolateral membrane, but not in the opposite direction, indicating basolateral efflux of bile acids. Overall, these findings indicate that OSTalpha-OSTbeta is a heteromeric transporter that is localized to the basolateral membrane of specific epithelial tissues and serves to regulate the export and disposition of bile acids and structurally related compounds from the cell. If confirmed, this model would have important implications for the body's handling of various steroid-derived molecules and may provide a new pharmacologic target for altering sterol homeostasis.

Amino Acid Motifs↗

PubMed2004

The heteromeric organic solute transporter alpha-beta, Ostalpha-Ostbeta, is an ileal basolateral bile acid transporter.

Bile acids are transported across the ileal enterocyte brush border membrane by the well characterized apical sodium-dependent bile acid transporter (Asbt) Slc10a2; however, the carrier(s) responsible for transporting bile acids across the ileocyte basolateral membrane into the portal circulation have not been fully identified. Transcriptional profiling of wild type and Slc10a2 null mice was employed to identify a new candidate basolateral bile acid carrier, the heteromeric organic solute transporter (Ost)alpha-Ostbeta. By Northern blot analysis, Ostalpha and Ostbeta mRNA was detected only in mouse kidney and intestine, mirroring the horizontal gradient of expression of Asbt in the gastrointestinal tract. Analysis of Ostalpha and Ostbeta protein expression by immunohistochemistry localized both subunits to the basolateral surface of the mouse ileal enterocyte. The transport properties of Ostalpha-Ostbeta were analyzed in stably transfected Madin-Darby canine kidney cells. Co-expression of mouse Ostalpha-Ostbeta, but not the individual subunits, stimulated Na(+)-independent bile acid uptake and the apical-to-basolateral transport of taurocholate. In contrast, basolateral-to-apical transport was not affected by Ostalpha-Ostbeta expression. Co-expression of Ostalpha and Ostbeta was required to convert the Ostalpha subunit to a mature glycosylated endoglycosidase H-resistant form, suggesting that co-expression facilitates the trafficking of Ostalpha through the Golgi apparatus. Immunolocalization studies showed that co-expression was necessary for plasma membrane expression of both Ostalpha and Ostbeta. These results demonstrate that the mouse Ostalpha-Ostbeta heteromeric transporter is a basolateral bile acid carrier and may be responsible for bile acid efflux in ileum and other ASBT-expressing tissues.

Animals↗

PubMed2004

Activation of plasma membrane reduced glutathione transport in death receptor apoptosis of HepG2 cells.

Cells undergoing apoptosis release reduced glutathione (GSH) into the extracellular space; however, the physiological significance and the mechanism behind the GSH export remain unclear. The present study demonstrates that GSH is released by HepG2 cells undergoing Fas, tumor necrosis factor alpha (TNFalpha), or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-stimulated cell death. GSH release was observed at times when extracellular lactate dehydrogenase (LDH) activity and propidium iodide (PI) incorporation were low, suggesting that the GSH release does not occur because of nonspecific cell damage, but is occurring through a specific transport system. Caspase 3-like proteases were activated before GSH was released, indicating that protease may be involved in signaling GSH release. To investigate the mechanism of GSH release, studies were performed in the presence of GSH transport inhibitors, as well as 25 mM GSH in the media. Two organic anion transporter inhibitors, probenecid and dibromosulfophthalein (DBSP), were effective in inhibiting Fas-stimulated GSH release. The addition of 25 mM GSH to the extracellular media also prevented the loss of intracellular GSH and delayed cell death. These findings suggest that an organic anion transporter is involved in GSH release during apoptosis, and that maintenance of intracellular GSH levels during apoptosis provides protection for the cell.

Apoptosis↗

PubMed2003

Lack of biliary lipid excretion in the little skate, Raja erinacea, indicates the absence of functional Mdr2, Abcg5, and Abcg8 transporters.

The ABC transporters bile salt export pump (BSEP; encoded by the ABCB11 gene), MDR3 P-glycoprotein (ABCB4), and sterolin 1 and 2 (ABCG5 and ABCG8) are crucial for the excretion of bile salt, phospholipid, and cholesterol, respectively, into the bile of mammals. The current paradigm is that phospholipid excretion mainly serves to protect membranes of the biliary tree against bile salt micelles. Bile salt composition and cytotoxicity, however, differ greatly between species. We investigated whether biliary phospholipid and cholesterol excretion occurs in a primitive species, the little skate, which almost exclusively excretes the sulphated bile alcohol scymnolsulphate. We observed no phospholipid and very little cholesterol excretion into bile of these animals. Conversely, when scymnolsulphate was added to the perfusate of isolated mouse liver perfusions, it was very well capable of driving biliary phospholipid and cholesterol excretion. Furthermore, in an erythrocyte cytolysis assay, scymnolsulphate was found to be at least as cytotoxic as taurocholate. These results demonstrate that the little skate does not have a system for the excretion of phospholipid and cholesterol and that both the MDR3 and the two half-transporter genes, ABCG5 and ABCG8, have evolved relatively late in evolution to mediate biliary lipid excretion. Little skate plasma membranes may be protected against bile salt micelles mainly by their high sphingomyelin content.

ATP Binding Cassette Transporter, Subfamily B↗

PubMed2003

Functional complementation between a novel mammalian polygenic transport complex and an evolutionarily ancient organic solute transporter, OSTalpha-OSTbeta.

These studies identify an organic solute transporter (OST) that is generated when two novel gene products are co-expressed, namely human OSTalpha and OSTbeta or mouse OSTalpha and OSTbeta. The results also demonstrate that the mammalian proteins are functionally complemented by evolutionarily divergent Ostalpha-Ostbeta proteins recently identified in the little skate, Raja erinacea, even though the latter exhibit only 25-41% predicted amino acid identity with the mammalian proteins. Human, mouse, and skate OSTalpha proteins are predicted to contain seven transmembrane helices, whereas the OSTbeta sequences are predicted to have a single transmembrane helix. Human OSTalpha-OSTbeta and mouse Ostalpha-Ostbeta cDNAs were cloned from liver mRNA, sequenced, expressed in Xenopus laevis oocytes, and tested for their ability to functionally complement the corresponding skate proteins by measuring transport of [3H]estrone 3-sulfate. None of the proteins elicited a transport signal when expressed individually in oocytes; however, all nine OSTalpha-OSTbeta combinations (i.e. OSTalpha-OSTbeta pairs from human, mouse, or skate) generated robust estrone 3-sulfate transport activity. Transport was sodium-independent, saturable, and inhibited by other steroids and anionic drugs. Human and mouse OSTalpha-OSTbeta also were able to mediate transport of taurocholate, digoxin, and prostaglandin E2 but not of estradiol 17beta-d-glucuronide or p-aminohippurate. OSTalpha and OSTbeta were able to reach the oocyte plasma membrane when expressed either individually or in pairs, indicating that co-expression is not required for proper membrane targeting. Interestingly, OSTalpha and OSTbeta mRNAs were highly expressed and widely distributed in human tissues, with the highest levels occurring in the testis, colon, liver, small intestine, kidney, ovary, and adrenal gland.

Amino Acid Sequence↗

PubMed2003

N-glycosylation controls functional activity of Oatp1, an organic anion transporter.

Rat Oatp1 (Slc21a1) is an organic anion-transporting polypeptide believed to be an anion exchanger. To characterize its mechanism of transport, Oatp1 was expressed in Saccharomyces cerevisiae under control of the GAL1 promoter. Protein was present at high levels in isolated S. cerevisiae secretory vesicles but had minimal posttranslational modifications and failed to exhibit taurocholate transport activity. Apparent molecular mass (M) of Oatp1 in yeast was similar to that of unmodified protein, approximately 62 kDa, whereas in liver plasma membranes Oatp1 has an M of approximately 85 kDa. To assess whether underglycosylation of Oatp1 in yeast suppressed functional activity, Oatp1 was expressed in Xenopus laevis oocytes with and without tunicamycin, a glycosylation inhibitor. With tunicamycin, M of Oatp1 decreased from approximately 72 to approximately 62 kDa and transport activity was nearly abolished. Mutations to four predicted N-glycosylation sites on Oatp1 (Asn to Asp at positions 62, 124, 135, and 492) revealed a cumulative effect on function of Oatp1, leading to total loss of taurocholate transport activity when all glycosylation sites were removed. M of the quadruple mutant was approximately 62 kDa, confirming that these asparagine residues are sites of glycosylation in Oatp1. Relatively little of the quadruple mutant was able to reach the plasma membrane, and most remained in unidentified intracellular compartments. In contrast, two of the triple mutants tested (N62/124/135D and N124/135/492D) were present in the plasma membrane fraction yet exhibited minimal transport activity. These results demonstrate that both membrane targeting and functional activity of Oatp1 are controlled by the extent of N-glycosylation.

Animals↗

PubMed2003

Increased synaptosomal dopamine content and brain concentration of paraquat produced by selective dithiocarbamates.

Exposure to pesticides may be a risk factor for Parkinson's disease based on epidemiologic data in humans, animal models and in vitro studies. Different dithiocarbamate pesticides potentiate the toxicity of both 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and paraquat in mouse models of Parkinsonism by an unknown mechanism. This study examined the effects of commercially used dithiocarbamates on [3H]dopamine transport in striatal synaptosomal vesicles and on the concentration of [14C]paraquat in vivo in mice. Different ethylenebis-dithiocarbamates and diethyl-dithiocarbamate increased dopamine accumulation in synaptosomes, whereas dimethyl-dithiocarbamate and methyl-dithiocarbamate did not. Increased dopamine accumulation in synaptosomes was dose dependent and was related to the carbon backbone of these molecules. The dithiocarbamates that increased accumulation of dopamine did not alter the influx of dopamine, but rather delayed the efflux out of synaptosomes. These same dithiocarbamates also increased the tissue content of [14C]paraquat in vivo by a mechanism that appeared to be distinct from the dopamine transporter. There was a consistent relationship between the dithiocarbamates that increased synaptosomal accumulation of dopamine and tissue content of paraquat, with those previously demonstrated to enhance paraquat toxicity in vivo. These results suggest that selective dithiocarbamates may alter the kinetics of different endogenous and exogenous compounds to enhance their neurotoxicity.

Animals↗

PubMed2003

Exploiting genome data to understand the function, regulation, and evolutionary origins of toxicologically relevant genes.

The wealth of new information coming from the many genome sequencing projects is providing unprecedented opportunities for major advances in all areas of biology, including the environmental health sciences. To facilitate this discovery process, experts in the fields of functional genomics and informatics and the emerging field of toxicogenomics recently gathered at the Mount Desert Island Biological Laboratory in Salisbury Cove, Maine, site of a National Institute of Environmental Health Sciences Marine and Freshwater Biomedical Science Center, to share their ideas and latest research findings. The goal of the symposium was to highlight approaches that may be used to identify and characterize toxicologically relevant genes being discovered in the genome sequencing projects. Many of the approaches rely heavily on comparative models as a way of identifying gene homology, ontology, and physiologic function, and on the availability of databases that facilitate storage, analysis, interpretation, and widespread dissemination of relevant data.

Animals↗

PubMed2002

Molecular characterization of a multidrug resistance-associated protein, Mrp2, from the little skate.

Multidrug resistance protein Mrp2 (symbol Abcc2) in liver plays a significant role in the biliary excretion of organic anionic conjugates. Mutations in human MRP2 result in defects in excretion of conjugated bilirubin and other cholephiles known as the Dubin-Johnson syndrome. Previous studies indicate that transporters with Mrp2-like functions are present in ancient vertebrates. We have now characterized an Mrp2 ortholog at the molecular level from the liver of the small skate, Raja erinacea, a marine vertebrate that evolved approximately 200 million years ago. The full-length skate Mrp2 cDNA is 6 kb and encodes for a 1,564-amino acid peptide with 56% identity to human Mrp2. Northern blot analysis demonstrated that skate Mrp2 is abundantly expressed in skate liver, intestine, and kidney. Immunoblots reveal a 180-kDa protein in skate liver. Immunofluorescence studies locate skate Mrp2 to the apical membrane of hepatocytes, renal tubules, and intestine. A PDZ-interacting motif is also found at its COOH terminus. Further sequence analysis indicates that transmembrane domains 1, 9, 11, 16, and 17 are the most highly conserved transmembrane domains between skate Mrp2 and mammalian MRP2/Mrp2s. This analysis indicates that Mrp2 orthologs evolved early in vertebrate evolution and that conserved domains may be important determinants of Mrp2 substrate specificity.

Amino Acid Sequence↗

PubMed2002

Transport of a neurotoxicant by molecular mimicry: the methylmercury-L-cysteine complex is a substrate for human L-type large neutral amino acid transporter (LAT) 1 and LAT2.

Methylmercury (MeHg) readily crosses cell membrane barriers to reach its target tissue, the brain. Although it is generally assumed that this rapid transport is due to simple diffusion, recent studies have demonstrated that MeHg is transported as a hydrophilic complex, and possibly as an L-cysteine complex on the ubiquitous L-type large neutral amino acid transporters (LATs). To test this hypothesis, studies were carried out in Xenopus laevis oocytes expressing two of the major L-type carriers in humans, LAT1-4F2 heavy chain (4F2hc) and LAT2-4F2hc. Oocytes expressing LAT1-4F2hc or LAT2-4F2hc demonstrated enhanced uptake of [(14)C]MeHg when administered as the L-cysteine or D,L-homocysteine complexes, but not when administered as the D-cysteine, N -acetyl-L-cysteine, penicillamine or GSH complexes. Kinetic analysis of transport indicated that the apparent affinities ( K (m)) of MeHg-L-cysteine uptake by LAT1 and LAT2 (98+/-8 and 64+/-8 microM respectively) were comparable with those for methionine (99+/-9 and 161+/-11 microM), whereas the V (max) values were higher for MeHg-L-cysteine, indicating that it may be a better substrate than the endogenous amino acid. Uptake and efflux of [(3)H]methionine and [(14)C]MeHg-L-cysteine were trans -stimulated by leucine and phenylalanine, but not by glutamate, indicating that MeHg-L-cysteine is both a cis - and trans -substrate. In addition, [(3)H]methionine efflux was trans -stimulated by leucine and phenylalanine even in the presence of an inwardly directed methionine gradient, demonstrating concentrative transport by both LAT1 and LAT2. The present results describe a major molecular mechanism by which MeHg is transported across cell membranes and indicate that metal complexes may form a novel class of substrates for amino acid carriers. These transport proteins may therefore participate in metal ion homoeostasis and toxicity.

Amino Acid Transport System y+↗

PubMed2002

Defining the molecular and cellular basis of toxicity using comparative models.

A critical element of any experimental design is the selection of the model that will be used to test the hypothesis. As Claude Bernard proposed over 100 years ago "the solution of a physiological or pathological problem often depends solely on the appropriate choice of the animal for the experiment so as to make the result clear and searching." Likewise, the Danish physiologist August Krogh in 1929 wrote that "For a large number of problems there will be some animal of choice, or a few such animals, on which it can be most conveniently studied." This scientific principle has been validated repeatedly in the intervening years as investigators have described unique models that exploit natural differences in chemical and molecular structure, biochemical function, or physiological response between different cells, tissues, and organisms to address specific hypotheses. Despite the power of this comparative approach, investigators have generally been reluctant to utilize nonmammalian or nonclassical experimental models to address questions of human biology. The perception has been that studies in relatively simple or evolutionarily ancient organisms would provide little insight into "complex" human biology. This perception, although always somewhat misguided, is now even less tenable given the results of the genome sequencing projects, which demonstrate that the human genome is remarkably similar to that of evolutionarily ancient organisms. Thus, the various life forms on Earth share much more in common then anyone had previously envisioned. This realization provides additional rationale for the use of nonclassical experimental models and provides perhaps the strongest validation of Bernard's and Krogh's assertions. This overview emphasizes some of the special attributes of alternative animal models that may be exploited to define the molecular and cellular basis of toxicity. For each attribute, selected examples of animal models and experimental approaches are presented. It focuses on the areas of neurotoxicology, reproductive and developmental toxicology, organ systems toxicology, carcinogenesis, and functional genomics/toxicogenomics and highlights the use of fish, avian, Drosophila, Caenorhabditis elegans, and yeast models in such studies.

Animals↗

PubMed2002

The editosome for cytidine to uridine mRNA editing has a native complexity of 27S: identification of intracellular domains containing active and inactive editing factors.

Apolipoprotein B mRNA cytidine to uridine editing requires the assembly of a multiprotein editosome comprised minimally of the catalytic subunit, apolipoprotein B mRNA editing catalytic subunit 1 (APOBEC-1), and an RNA-binding protein, APOBEC-1 complementation factor (ACF). A rat homolog has been cloned with 93.5% identity to human ACF (huACF). Peptide-specific antibodies prepared against huACF immunoprecipitated a rat protein of similar mass as huACF bound to apolipoprotein B (apoB) RNA in UV cross-linking reactions, thereby providing evidence that the p66, mooring sequence-selective, RNA-binding protein identified previously in rat liver by UV cross-linking and implicated in editosome assembly is a functional homolog of huACF. The rat protein (p66/ACF) was distributed in both the nucleus and cytoplasm of rat primary hepatocytes. Within a thin section, a significant amount of total cellular p66/ACF was cytoplasmic, with a concentration at the outer surface of the endoplasmic reticulum. Native APOBEC-1 co-fractionated with p66/ACF in the cytoplasm as 60S complexes. In the nucleus, the biological site of apoB mRNA editing, native p66/ACF, was localized to heterochromatin and fractionated with APOBEC-1 as 27S editosomes. When apoB mRNA editing was stimulated in rat primary hepatocytes with ethanol or insulin, the abundance of p66/ACF in the nucleus markedly increased. It is proposed that the heterogeneity in size of complexes containing editing factors is functionally significant and reflects functionally engaged editosomes in the nucleus and an inactive cytoplasmic pool of factors.

APOBEC-1 Deaminase↗