Resorption of amino acids in kidney and intestine is mediated by transporters, which prefer groups of amino acids with similar physico-chemical properties. It is generally assumed that most neutral amino acids are transported across the apical membrane of epithelial cells by system B 0 . Here we have characterized a novel member of the Na ؉ -dependent neurotransmitter transporter family (B 0 AT1) isolated from mouse kidney, which shows all properties of system B 0 . Flux experiments showed that the transporter is Na ؉ -dependent, electrogenic, and actively transports most neutral amino acids but not anionic or cationic amino acids. Superfusion of mB 0 AT1-expressing oocytes with neutral amino acids generated inward currents, which were proportional to the fluxes observed with labeled amino acids. In situ hybridization showed strong expression in intestinal microvilli and in the proximal tubule of the kidney. Expression of mouse B 0 AT1 was restricted to kidney, intestine, and skin. It is generally assumed that mutations of the system B 0 transporter underlie autosomal recessive Hartnup disorder. In support of this notion mB 0 AT1 is located on mouse chromosome 13 in a region syntenic to human chromosome 5p15, the locus of Hartnup disorder. Thus, the human homologue of this transporter is an excellent functional and positional candidate for Hartnup disorder.

Hartnup disorder (OMIM 234500) is an autosomal recessive abnormality of renal and gastrointestinal neutral amino acid transport noted for its clinical variability. We localized a gene causing Hartnup disorder to chromosome 5p15.33 and cloned a new gene, SLC6A19, in this region. SLC6A19 is a sodiumdependent and chloride-independent neutral amino acid transporter, expressed predominately in kidney and intestine, with properties of system B 0. We identified six mutations in SLC6A19 that cosegregated with disease in the predicted recessive manner, with most affected individuals being compound heterozygotes. The disease-causing mutations that we tested reduced neutral amino acid transport function in vitro. Population frequencies for the most common mutated SLC6A19 alleles are 0.007 for 517G-A and 0.001 for 718C-T. Our findings indicate that SLC6A19 is the long-sought gene that is mutated in Hartnup disorder; its identification provides the opportunity to examine the inconsistent multisystemic features of this disorder. 14 C-Amino acid uptake 14 C-Leucine uptake Figure 1 Ion and voltage dependence and substrate specificity of SLC6A19 confirm that it has the predicted profile for system B 0. (a) Leucine uptake (pmol per 15 min) in oocytes transfected with SLC6A19 cRNA in which buffer containing NaCl (Na) was modified. The transport activity of oocytes injected with water was subtracted (n ¼ 3 experiments). Expression of SLC6A19 resulted in uptake of 100 mM 14 C-leucine at 2073 pmol per 15 min, which was more than twice that detected in oocytes injected with water (net activity 871.8 pmol per 15 min). Uptake of 14 C-leucine was sodium-dependent, as replacement of sodium ions by the impermeant N-methyl-D-glucamine (NMDG) or LiCl (Li) reduced the transport activity by 92% or 85%, respectively. Leucine uptake was not chloride-dependent, as replacement of NaCl by sodium gluconate (-Cl) did not significantly alter transport activity (P40.1). Transport of amino acids by SLC6A19 was driven by membrane potential, as the addition of 50 mM KCl (+ K) to the transport buffer reduced leucine uptake by 58%. (b) Uptake (pmol per 15 min) of 14 C-labeled amino acids in oocytes transfected with SLC6A19 cRNA or controls injected with water. The specificity for neutral amino acids is shown in this experiment, which was done three times. Phenylalanine seemed to be the best substrate, followed by leucine, glutamine and alanine. Glutamate and arginine were not actively transported.

Background: Hemochromatosis in white subjects is mostly due to homozygosity for the common C282Y substitution in HFE. Although clinical symptoms are preventable by early detection of the genetic predisposition and prophylactic treatment, population screening is not currently advocated because of the discrepancy between the common mutation prevalence and apparently lower frequency of clinical disease. This study compared screening for hemochromatosis in subjects with or without a family history. Methods:We assessed disease expression by clinical evaluation and liver biopsy in 672 essentially asymptomatic C282Y homozygous subjects identified by either family screening or health checks. We also observed a subgroup of untreated homozygotes with normal serum ferritin levels for up to 24 years.Results: Prevalence of hepatic iron overload and fibrosis were comparable between the 2 groups. Disease-related conditions were more common in male subjects identified by health checks, but they were older. Hepatic iron overload (grades 2-4) was present in 56% and 34.5% of male and female subjects, respectively; hepatic fibrosis (stages 2-4) in 18.4% and 5.4%; and cirrhosis in 5.6% and 1.9%. Hepatic fibrosis and cirrhosis correlated significantly with the hepatic iron concentration, and except in cases of cirrhosis, there was a 7.5-fold reduction in the mean fibrosis score after phlebotomy. All subjects with cirrhosis were asymptomatic.Conclusions: Screening for hemochromatosis in apparently healthy subjects homozygous for the C282Y mutation with or without a family history reveals comparable levels of hepatic iron overload and disease. Significant hepatic fibrosis is frequently found in asymptomatic subjects with hemochromatosis and, except when cirrhosis is present, is reversed by iron removal.

There are two functional Omega class glutathione transferase (GST) genes in humans. GSTO1 is polymorphic with several coding region alleles, including an A140D substitution, a potential deletion of E155 and an E208K substitution. GSTO2 is also polymorphic with an N142D substitution in the coding region. We investigated the effect of these variations on the enzyme's thioltransferase, dehydroascorbate reductase, monomethylarsonate reductase and dimethylarsonate reductase activities. Variant proteins were expressed in Escherichia coli and purified by Ni-agarose affinity chromatography. GSTO2-2 was insoluble and had to be dissolved and refolded from 8 M urea. The A140D and E208K substitutions in GSTO1-1 did not alter specific activity. The deletion of E155 caused a two- to three-fold increase in the specific activity with each substrate. This deletion also caused a significant decrease in the enzyme's heat stability. The E155 deletion has been linked to abnormal arsenic excretion patterns; however, the available data do not clearly identify the cause of this abnormality. We found that GSTO2-2 has activity with the same substrates as GSTO1-1, and the dehydroascorbate reductase activity of GSTO2-2 is approximately 70-100-fold higher than that of GSTO1-1. The polymorphic N142D substitution had no effect on the specific activity of the enzyme with any substrate. The most notable feature of GSTO2-2 was its very high dehydroascorbate reductase activity, which suggests that GSTO2-2 may significantly protect against oxidative stress by recycling ascorbate. A defect in ascorbate metabolism may provide a common mechanism by which the Omega class GSTs influence the age-at-onset of Alzheimer's and Parkinson's diseases.