Lysine Transport Research Articles

Engineering a Lysine-ON Riboswitch for Metabolic Control of Lysine Production in Corynebacterium glutamicum.

Riboswitches are natural RNA elements that regulate gene expression by binding a ligand. Here, we demonstrate the possibility of altering a natural lysine-OFF riboswitch from Eschericia coli (ECRS) to a synthetic lysine-ON riboswitch and using it for metabolic control. To this end, a lysine-ON riboswitch library was constructed using tetA-based dual genetic selection. After screening the library, the functionality of the selected lysine-ON riboswitches was examined using a report gene, lacZ. Selected lysine-ON riboswitches were introduced into the lysE gene (encoding a lysine transport protein) of Corynebacterium glutamicum and used to achieve dynamic control of lysine transport in a recombinant lysine-producing strain, C. glutamicum LPECRS, which bears a deregulated aspartokinase and a lysine-OFF riboswitch for dynamic control of the enzyme citrate synthase. Batch fermentation results of the strains showed that the C. glutamicum LPECRS strain with an additional lysine-ON riboswitch for the control of lysE achieved a 21% increase in the yield of lysine compared to that of the C. glutamicum LPECRS strain and even a 89% increase in yield compared to that of the strain with deregulated aspartokinase. This work provides a useful approach to generate lysine-ON riboswitches for C. glutamicum metabolic engineering and demonstrates for the first time a synergetic effect of lysine-ON and -OFF riboswitches for improving lysine production in this industrially important microorganism. The approach can be used to dynamically control other genes and can be applied to other microorganisms.

Cystinuria: current concepts and future directions.

Cystinuria, an autosomic recessive genetic disorder is an uncommon cause of nephrolithiasis characterized by an impairment of transport of cystine, ornithine, lysine, and arginine (COLA). Of these, only cystine is insoluble enough to cause stone formation. Although a classification exists that categorizes the disease depending on chromosomal mutation, this does not currently alter management which consists of increased fluid intake, urine alkalinization, reduced sodium intake and, if warranted, cystine-binding thiol drugs. Cystine stones are relatively resistant to fragmentation. Intrinsic characteristics on imaging may help in planning surgical treatment. Finally, advances in crystal growth inhibition are encouraging as they may provide a new tool to treat this condition which although uncommon, is treatable and has been associated with lower quality of life and renal function compared to other stone formers.

Quinone-amino acid conjugates targeting Leishmania amino acid transporters.

The aim of the present study was to investigate the feasibility of targeting Leishmania transporters via appropriately designed chemical probes. Leishmania donovani, the parasite that causes visceral leishmaniasis, is auxotrophic for arginine and lysine and has specific transporters (LdAAP3 and LdAAP7) to import these nutrients. Probes 1–15 were originated by conjugating cytotoxic quinone fragments (II and III) with amino acids (i.e. arginine and lysine) by means of an amide linkage. The toxicity of the synthesized conjugates against Leishmania extracellular (promastigotes) and intracellular (amastigotes) forms was investigated, as well their inhibition of the relevant amino acid transporters. We observed that some conjugates indeed displayed toxicity against the parasites; in particular, 7 was identified as the most potent derivative (at concentrations of 1 µg/mL and 2.5 µg/mL residual cell viability was reduced to 15% and 48% in promastigotes and amastigotes, respectively). Notably, 6, while retaining the cytotoxic activity of quinone II, displayed no toxicity against mammalian THP1 cells. Transport assays indicated that the novel conjugates inhibited transport activity of lysine, arginine and proline transporters. Furthermore, our analyses suggested that the toxic conjugates might be translocated by the transporters into the cells. The non-toxic probes that inhibited transport competed with the natural substrates for binding to the transporters without being translocated. Thus, it is likely that 6, by exploiting amino acid transporters, can selectively deliver its toxic effects to Leishmania cells. This work provides the first evidence that amino acid transporters of the human pathogen Leishmania might be modulated by small molecules, and warrants their further investigation from drug discovery and chemical biology perspectives.

Novel therapy for pyridoxine dependent epilepsy due to ALDH7A1 genetic defect: l-arginine supplementation alternative to lysine-restricted diet

Background and hypothesisPyridoxine dependent epilepsy (PDE) due to mutations in the ALDH7A1 gene (PDE-ALDH7A1) is caused by α-aminoadipic-semialdehyde-dehydrogenase enzyme deficiency in the lysine pathway resulting in the accumulation of α-aminoadipic acid semialdehyde (α-AASA). Classical presentation is neonatal intractable seizures with a dramatic response to pyridoxine. Pyridoxine therapy does not prevent developmental delays in the majority of the patients. We hypothesized that l-arginine supplementation will decrease accumulation of α-AASA by competitive inhibition of lysine transport into the central nervous system and improve neurodevelopmental and neurocognitive functions in PDE-ALDH7A1. MethodsA 12-year-old male with PDE-ALDH7A1 was treated with l-arginine supplementation as an innovative therapy. Treatment outcome was monitored by cerebral-spinal-fluid (CSF) α-AASA measurements at baseline, 6th and 12th months of therapy. Neuropsychological assessments were performed at baseline and 12th months of therapy. Resultsl-arginine therapy was well tolerated without side effects. CSF α-AASA was decreased 57% at 12th months of therapy. Neuropsychological assessments revealed improvements in general abilities index from 108 to 116 and improvements in verbal and motor functioning at 12th months of therapy. ConclusionThe short-term treatment outcome of this novel l-arginine supplementation therapy for PDE-ALDH7A1 was successful for biochemical and neurocognitive improvements.

New Insights into the Interplay Between the Lysine Transporter LysP and the pH Sensor CadC in Escherichia Coli

The coordination of signal transduction and substrate transport represents a sophisticated way to integrate information on metabolite fluxes into transcriptional regulation. This widely distributed process involves protein-protein interactions between two integral membrane proteins. Here we report new insights into the molecular mechanism of the regulatory interplay between the lysine-specific permease LysP and the membrane-integrated pH sensor CadC, which together induce lysine-dependent adaptation of E. coli under acidic stress. In vivo analyses revealed that, in the absence of either stimulus, the two proteins form a stable association, which is modulated by lysine and low pH. In addition to its transmembrane helix, the periplasmic domain of CadC also participated in the interaction. Site-directed mutagenesis pinpointed Arg265 and Arg268 in CadC as well as Asp275 and Asp278 in LysP as potential periplasmic interaction sites. Moreover, a systematic analysis of 100 LysP variants with single-site replacements indicated that the lysine signal is transduced from co-sensor to sensor via lysine-dependent conformational changes (upon substrate binding and/or transport) of LysP. Our results suggest a scenario in which CadC is inhibited by LysP via intramembrane and periplasmic contacts under non-inducing conditions. Upon induction, lysine-dependent conformational changes in LysP transduce the lysine signal via a direct conformational coupling to CadC without resolving the interaction completely. Moreover, concomitant pH-dependent protonation of periplasmic amino acids in both proteins dissolves their electrostatic connections resulting in further destabilization of the CadC/LysP interaction.

Alterations of arginine in Lysinuric Protein Intolerance (LPI) macrophages

Lysinuric Protein Intolerance (LPI) is an inherited transport defect of cationic amino acids (arginine, lysine and ornithine) transport at the basolateral membrane of intestinal and renal tubular cells caused by mutations in SLC7A7 encoding for System y+L‐related y+LAT1 protein. The severe clinical course of this disorder suggests that LPI should be considered a severe multisystem disease with features proper of ‘metabolic’ disorders. In particular, immune dysfunction could be attributed to an altered metabolism of nitric oxide (NO) secondary to abnormal arginine intracellular metabolism. To address this hypothesis we measured arginine transport/metabolism in monocyte‐derived macrophages (MDM), obtained from peripheral blood monocytes isolated from 5 LPI patients. In these cells the efflux of 3H‐arginine through System y+L was markedly lower than in normal MDM, with a decrease that ranged from 60 to 75%. Moreover, SLC7A7 silencing in a human monocytic cell line (THP‐1 cells) caused a significant increase of intracellular arginine content. These results indicate that arginine entrapping takes place in LPI monocytes‐macrophages, supporting the hypothesis that NO overproduction observed in vivo may be due to an abnormal intracellular metabolism of arginine.Supported by CLIMB (Children living with inherited metabolic diseases, Crewe, UK)

Molecular and clinical evaluation of Turkish patients with lysinuric protein intolerance

Lysinuric protein intolerance is an autosomal recessive metabolic disorder caused by defective transport of the cationic amino acids lysine, arginine and ornithine in the epithelial cells of the basolateral membrane in the small intestine and renal tubules. Mutations in the solute carrier family 7, member 7, SLC7A7, gene cause this multisystemic disease with a variety of clinical symptoms such as hepatosplenomegaly, osteoporosis, hypotonia, developmental delay, pulmonary insufficiency or end-stage renal disease. In the present study, genomic structure of SLC7A7 in six Turkish patients with lysinuric protein intolerance was examined in order to detect disease causing mutations by denaturing high pressure liquid chromatography and direct sequencing. Four novel mutations were identified in SLC7A7: c.223insGTC, p.Val74_Ile75insVal; c.283insTGG, p.Glu94_Thr95insTrp; c.344_347delTTGC, p.Leu115LeufsX53; and c.1099insT, p.Ile367TyrfsX16. Clinical and biochemical findings were evaluated together with these molecular analyses.

Cloning, Expression, and Functional Characterization of Secondary Amino Acid Transporters of Lactococcus lactis

Fourteen genes encoding putative secondary amino acid transporters were identified in the genomes of Lactococcus lactis subsp. cremoris strains MG1363 and SK11 and L. lactis subsp. lactis strains IL1403 and KF147, 12 of which were common to all four strains. Amino acid uptake in L. lactis cells overexpressing the genes revealed transporters specific for histidine, lysine, arginine, agmatine, putrescine, aromatic amino acids, acidic amino acids, serine, and branched-chain amino acids. Substrate specificities were demonstrated by inhibition profiles determined in the presence of excesses of the other amino acids. Four knockout mutants, lacking the lysine transporter LysP, the histidine transporter HisP (formerly LysQ), the acidic amino acid transporter AcaP (YlcA), or the aromatic amino acid transporter FywP (YsjA), were constructed. The LysP, HisP, and FywP deletion mutants showed drastically decreased rates of uptake of the corresponding substrates at low concentrations. The same was observed for the AcaP mutant with aspartate but not with glutamate. In rich M17 medium, the deletion of none of the transporters affected growth. In contrast, the deletion of the HisP, AcaP, and FywP transporters did affect growth in a defined medium with free amino acids as the sole amino acid source. HisP was essential at low histidine concentrations, and AcaP was essential in the absence of glutamine. FywP appeared to play a role in retaining intracellularly synthesized aromatic amino acids when these were not added to the medium. Finally, HisP, AcaP, and FywP did not play a role in the excretion of accumulated histidine, glutamate, or phenylalanine, respectively, indicating the involvement of other transporters.

Single-Molecule Studies of the Lysine Riboswitch Reveal Effector-Dependent Conformational Dynamics of the Aptamer Domain

The lysine riboswitch is a cis-acting RNA genetic regulatory element found in the leader sequence of bacterial mRNAs coding for proteins related to biosynthesis or transport of lysine. Structural analysis of the lysine-binding aptamer domain of this RNA has revealed that it completely encapsulates the ligand and therefore must undergo a structural opening/closing upon interaction with lysine. In this work, single-molecule fluorescence resonance energy transfer (FRET) methods are used to monitor these ligand-induced structural transitions that are central to lysine riboswitch function. Specifically, a model FRET system has been developed for characterizing the lysine dissociation constant as well as the opening/closing rate constants for the Bacillus subtilis lysC aptamer domain. These techniques permit measurement of the dissociation constant (K(D)) for lysine binding of 1.7(5) mM and opening/closing rate constants of 1.4(3) s(-1) and 0.203(7) s(-1), respectively. These rates predict an apparent dissociation constant for lysine binding (K(D,apparent)) of 0.25(9) mM at near physiological ionic strength, which differs markedly from previous reports.

Cationic Amino Acid Uptake Constitutes a Metabolic Regulation Mechanism and Occurs in the Flagellar Pocket of Trypanosoma cruzi

Trypanosomatids' amino acid permeases are key proteins in parasite metabolism since they participate in the adaptation of parasites to different environments. Here, we report that TcAAP3, a member of a Trypanosoma cruzi multigene family of permeases, is a bona fide arginine transporter. Most higher eukaryotic cells incorporate cationic amino acids through a single transporter. In contrast, T. cruzi can recognize and transport cationic amino acids by mono-specific permeases since a 100-fold molar excess of lysine could not affect the arginine transport in parasites that over-express the arginine permease (TcAAP3 epimastigotes). In order to test if the permease activity regulates downstream processes of the arginine metabolism, the expression of the single T. cruzi enzyme that uses arginine as substrate, arginine kinase, was evaluated in TcAAP3 epimastigotes. In this parasite model, intracellular arginine concentration increases 4-folds and ATP level remains constant until cultures reach the stationary phase of growth, with decreases of about 6-folds in respect to the controls. Interestingly, Western Blot analysis demonstrated that arginine kinase is significantly down-regulated during the stationary phase of growth in TcAAP3 epimastigotes. This decrease could represent a compensatory mechanism for the increase in ATP consumption as a consequence of the displacement of the reaction equilibrium of arginine kinase, when the intracellular arginine concentration augments and the glucose from the medium is exhausted. Using immunofluorescence techniques we also determined that TcAAP3 and the specific lysine transporter TcAAP7 co-localize in a specialized region of the plasma membrane named flagellar pocket, staining a single locus close to the flagellar pocket collar. Taken together these data suggest that arginine transport is closely related to arginine metabolism and cell energy balance. The clinical relevance of studying trypanosomatids' permeases relies on the possibility of using these molecules as a route of entry of therapeutic drugs.

Safety, efficacy and physiological actions of a lysine-free, arginine-rich formula to treat glutaryl-CoA dehydrogenase deficiency: Focus on cerebral amino acid influx

Striatal degeneration from glutaryl-CoA dehydrogenase deficiency (glutaric aciduria type 1, GA1) is associated with cerebral formation and entrapment of glutaryl-CoA and its derivatives that depend on cerebral lysine influx. In 2006 we designed a lysine-free study formula enriched with arginine to selectively block lysine transport across cerebral endothelia and thereby limit glutaryl-CoA production by brain. Between 2006 and present, we treated twelve consecutive children with study formula (LYSx group) while holding all other treatment practices constant. Clinical and biochemical outcomes were compared to 25 GA1 patients (PROx group) treated between 1995 and 2005 with natural protein restriction (dietary lysine/arginine ratio of 1.7±0.3mg:mg). We used published kinetic parameters of the y+and LAT1 blood–brain barrier transporters to model the influx of amino acids into the brain. Arginine fortification to achieve a mean dietary lysine/arginine ratio of 0.7±0.2mg:mg was neuroprotective. All 12 LYSx patients are physically and neurologically healthy after 28 aggregate patient-years of follow up (current ages 28±21months) and there were no adverse events related to formula use. This represents a 36% reduction of neurological risk (95% confidence interval 14–52%, p=0.018) that we can directly attribute to altered amino acid intake. During the first year of life, 20% lower lysine intake and two-fold higher arginine intake by LYSx patients were associated with 50% lower plasma lysine, 3-fold lower plasma lysine/arginine concentration ratio, 42% lower mean calculated cerebral lysine influx, 54% higher calculated cerebral arginine influx, 15–26% higher calculated cerebral influx of several anaplerotic precursors (isoleucine, threonine, methionine, and leucine), 50% less 3-hydroxyglutarate excretion, and a 3-fold lower hospitalization rate (0.8 versus 2.3 hospitalizations per patient per year). The relationship between arginine fortification and plasma lysine indicates that transport competition exists at both cerebrovascular and gastrointestinal barriers, suggesting their co-administration is key to efficacy. Monitoring the ratio between lysine and arginine in diet and plasma may prove a useful strategy for treating children with GA1.

Transport of Free and Peptide‐Bound Glycated Amino Acids: Synthesis, Transepithelial Flux at Caco‐2 Cell Monolayers, and Interaction with Apical Membrane Transport Proteins

In glycation reactions, the side chains of protein-bound nucleophilic amino acids such as lysine and arginine are post-translationally modified to a variety of derivatives also known as Maillard reaction products (MRPs). Considerable amounts of MRPs are taken up in food. Here we have studied the interactions of free and dipeptide-bound MRPs with intestinal transport systems. Free and dipeptide-bound derivatives of N(6)-(1-fructosyl)lysine (FL), N(6)-(carboxymethyl)lysine (CML), N(6)-(1-carboxyethyl)lysine (CEL), formyline, argpyrimidine, and methylglyoxal-derived hydroimidazolone 1 (MG-H1) were synthesized. The inhibition of L-[(3)H]lysine and [(14) C]glycylsarcosine uptakes was measured in Caco-2 cells which express the H(+)/peptide transporter PEPT1 and lysine transport system(s). Glycated amino acids always displayed lower affinities than their unmodified analogues towards the L-[(3)H]lysine transporter(s). In contrast, all glycated dipeptides except Ala-FL were medium- to high-affinity inhibitors of [(14)C]Gly-Sar uptake. The transepithelial flux of the derivatives across Caco-2 cell monolayers was determined. Free amino acids and intact peptides derived from CML and CEL were translocated to very small extents. Application of peptide-bound MRPs, however, led to elevation (up to 80-fold) of the net flux and intracellular accumulation of glycated amino acids, which were hydrolyzed from the dipeptides inside the cells. We conclude 1) that free MRPs are not substrates for the intestinal lysine transporter(s), and 2) that dietary MRPs are absorbed into intestinal cells in the form of dipeptides, most likely by the peptide transporter PEPT1. After hydrolysis, hydrophobic glycated amino acids such as pyrraline, formyline, maltosine, and argpyrimidine undergo basolateral efflux, most likely by simple diffusion down their concentration gradients.

Lysine transporters in human trypanosomatid pathogens

In previous studies we characterized arginine transporter genes from Trypanosoma cruzi and Leishmania donovani, the etiological agents of chagas disease and kala azar, respectively, both fatal diseases in humans. Unlike arginine transporters in higher eukaryotes that transport also lysine, these parasite transporters translocate only arginine. This phenomenon prompted us to identify and characterize parasite lysine transporters. Here we demonstrate that LdAAP7 and TcAAP7 encode lysine-specific permeases in L. donovani and T. cruzi, respectively. These two lysine permeases are both members of the large amino acid/auxin permease family and share certain biochemical properties, such as specificity and Km. However, we evidence that LdAAP7 and TcAAP7 differ in their regulation and localization, such differences are likely a reflection of the dissimilar L. donovani and T. cruzi life cycles. Failed attempts to delete both alleles of LdAAP7 support the premise that this is an essential gene that encodes the only lysine permeases expressed in L. donovani promastigotes and T. cruzi epimastigotes, respectively.

NO control: nitric oxide directly regulates substrate delivery to NOS. Focus on “Nitric oxide can acutely modulate its biosynthesis through a negative feedback mechanism on l-arginine transport in cardiac myocytes”

although the amino acid arginine is commonly associated with nitric oxide (NO) production via NO synthase (NOS), it also participates in the synthesis of urea, creatine, creatinine, agmatine, polyamines, as well as overall protein synthesis. Furthermore, it also influences hormone release (insulin,

Functional characterization of the chicken cationic amino acid transporter-2 isoforms

Utilization of lysine and arginine by mammalian skeletal muscle is due in large part to the system y(+) cationic amino acid transporters (CAT), particularly CAT-2A. The chicken CAT-2 (cCAT-2) gene is alternatively spliced to produce three isoforms (cCAT-2A/B/C). Chicken (Gallus gallus) CAT-2 isoforms were transiently and stably expressed in mammalian cell lines to determine cCAT-2 isoform cellular localization and transport properties. The cCAT-2A protein localized to the plasma membrane and the cCAT-2B protein localized to the cytoplasm juxtaposed to the plasma membrane. The cCAT-2C protein localized non-specifically throughout the cytoplasm. The cCAT-2A protein exhibited saturable transport. The K(t) of cCAT-2A for lysine using transient transfection was 2.6mM and the V(max) was 11.9pmol/mg protein/min. The K(t) of cCAT-2A for lysine using stable transfection was 7.9mM and the V(max) was 12.8pmol/mg protein/min. Transient and stable transfections of cCAT-2B or cCAT-2C resulted in no lysine or arginine transport. These data indicate that cCAT-2A is a low affinity, high velocity transporter for lysine and arginine and is the cCAT-2 isoform responsible for lysine and arginine transport in avian skeletal muscle.

Avt5p is required for vacuolar uptake of amino acids in the fission yeast Schizosaccharomycespombe

We identified SPBC1685.07c of Schizosaccharomyces pombe as a novel vacuolar protein, Avt5p, with similarity to vacuolar amino acid transporters Avt5p from Saccharomyces cerevisiae. Avt5p localizes to the vacuolar membrane and upon disruption of avt5, uptake of histidine, glutamate, tyrosine, arginine, lysine or serine was impaired. During nitrogen starvation, the transient increase of vacuolar lysine transport observed for wild-type cells still occurred in the mutant cells, however, uptake of glutamate did not significantly increase in response to nitrogen starvation. Our results show that under diverse growth conditions Avt5p is involved in vacuolar transport of a selective set of amino acids.

Combined hyperlipidemia in patients with lysinuric protein intolerance

Lysinuric protein intolerance (LPI) is an autosomal recessive disorder characterized by defective transport of cationic amino acids lysine, arginine, and ornithine. Low plasma concentrations of arginine and ornithine lead to impaired urea cycle function and, subsequently, decreased protein tolerance. Patients often develop natural aversion to protein-rich foods, which may predispose them to nutritional problems. The objective of this retrospective study was to investigate lipid values and efficacy of lipid-lowering therapy in patients with LPI. Serum total and high-density-lipoprotein (HDL)-cholesterol and triglyceride concentrations were analyzed in 39 Finnish LPI patients (14 males) aged 3-64 years. Dietary intakes were analyzed from food records. Mean [standard deviation (SD)] serum and HDL-cholesterol and triglyceride concentrations were 7.16 (2.13) mmol/l, 1.21 (0.58) mmol/l, and 4.0 (2.4) mmol/l, respectively. Patients with renal dysfunction had marginally higher total cholesterol and significantly higher triglyceride concentration than patients without renal impairment. Twenty-two patients were started on 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors (atorvastatin or simvastatin). After 6 months, serum cholesterol and triglyceride concentrations had decreased by 32% (p < 0.001), whereas HDL-cholesterol had increased by 13% (p = 0.016). Serum cholesterol and triglyceride values are markedly elevated in LPI patients. Although the mechanism of combined hyperlipidemia remains unknown and is not explained by fat consumption, hyperlipidemia is clearly progressive with age, suggesting that starting statin therapy early is probably beneficial. Statins are well-tolerated and efficacious in LPI.

Inotropic effects of l-lysine in the mammalian heart

We studied the effects of L-lysine in cardiac preparations of mice and men. Of note, L-lysine increased force of contraction in a concentration- and time-dependent manner in isolated electrically paced left atrium of mouse and in human right atrium. It further increased heart rate and left ventricular pressure in the isolated perfused mouse heart. In isolated adult mouse cardiomyocytes, the contractility as assessed by edge detection was increased as well as the Ca(2+) transients after electrically pacing by field stimulation. However, using the patch clamp technique, no effect of L-lysine on action potential duration from a constant holding potential or on current through L-type calcium channels could be observed. However, L-lysine led to a depolarization of unclamped cells. Furthermore, effects of L-lysine were stereospecific, as they were not elicited by D-lysine. The inotropic effects of L-lysine were not abrogated by additionally applied L-ornithine or L-arginine (known inhibitors of lysine transport). However, L-lysine (5 mM) shifted the concentration-response curve for a positive inotropic effect of 5-hydroxytryptamine (5-HT; serotonin) in atrium of transgenic mice (with cardiac specific overexpression of 5-HT(4) receptors) to higher concentrations. In summary, we describe a novel positive inotropic effect of an essential amino acid, L-lysine, in the mammalian heart. One might speculate that L-lysine treatment under certain conditions could sustain cardiac performance. Moreover, L-lysine is able to block, at least in part, cardiac 5-HT(4) receptors.

Transport of Free and Peptide-Bound Pyrraline at Intestinal and Renal Epithelial Cells

Pyrraline is a quantitatively dominating glycation compound of the advanced Maillard reaction in foods and can be found in urine after consumption of pyrraline-containing food items. The purpose of this study was to investigate the transport of pyrraline and its dipeptide derivatives alanylpyrraline (Ala-Pyrr) and pyrralylalanine (Pyrr-Ala) at intestinal and renal cell lines. Pyrraline inhibited the l-[(3)H]lysine uptake with IC(50) values of 0.3 mM (Caco-2 cells) and 3.5 mM (OK cells), respectively, but not the uptake of [(14)C]Gly-Sar (Caco-2 and SKPT cells). In contrast, Ala-Pyrr strongly inhibited the uptake of [(14)C]Gly-Sar in Caco-2 and SKPT cells with IC(50) values of 0.19 and 0.017 mM, respectively. Pyrr-Ala inhibited the carrier-mediated uptake of [(14)C]Gly-Sar in Caco-2 and SKPT cells by 50% at concentrations of 0.03 and 0.008 mM, respectively. The transepithelial flux of peptide-bound pyrraline across Caco-2 cell monolayers was up to 15-fold higher compared to the flux of free pyrraline. We conclude that free pyrraline is not a substrate for the intestinal lysine transporter and that the absorption of dietary pyrraline occurs most likely in the form of dipeptides rather than as the free amino acid.

Five Novel Mutations in Cystinuria Genes SLC3A1 and SLC7A9

Five Novel Mutations in Cystinuria Genes SLC3A1 and SLC7A9Cystinuria is an autosomal recessive disorder that is characterized by impaired transport of cystine, lysine, ornithine and arginine in the proximal renal tubule and epithelial cells of the gastrointestinal tract. The transport of these amino acids is mediated by the rBAT/b0,+AT transporter, the subunits of which are encoded by the genes SLC3A1, located on chromosome 2p16.3-21, and SLC7A9, located on chromosome 19q12-13.1. Based on the urinary cystine excretion patterns of obligate heterozygotes, cystinuria is classified into type I (normal amino acid urinary pattern in heterozygotes) and non type I (a variable degree of urinary hyper excretion of cystine and dibasic amino acids in heterozygotes). On the basis of genetic aspects, cystinuria is classified into type A, is caused by mutations in both alleles of SLC3A1; type B, caused by mutations in both alleles of SLC7A9 and type AB, is caused by one mutation in SLC3A1 and one mutation in SLC7A9. Here we present two novel mutations in the SLC3A1 gene (C242R and L573X), which were found in patients from Serbia, and three in the SLC7A9 gene (G73R, V375I, 1048-1051 delACTC), found in patients from Serbia, Macedonia and Turkey, respectively.