Arginine transport in human erythroid cells: discrimination of CAT1 and 4F2hc/y+LAT2 roles

Since arginine metabolites, such as nitric oxide and polyamines, influence the expression of genes involved in erythroid differentiation, the transport of the cationic amino acid may play an important role in erythroid cells. However, available data only concern the presence in these cells of CAT1 transporter (system y⁺), while no information exists on the role of the heterodimeric transporters of system y⁺L (4F2hc/y⁺LAT1 and 4F2hc/y⁺LAT2) which operates transmembrane arginine fluxes cis-inhibited by neutral amino acids in the presence of sodium. Using erythroleukemia K562 cells and normal erythroid precursors, we demonstrate here that arginine transport in human erythroid cells is due to the additive contributions of a leucine-sensitive and leucine-insensitive component. In both cell types, leucine inhibition of arginine influx is much less evident in the absence of sodium, a hallmark of system y⁺L. In K562 cells, N-ethylmaleimide, a known inhibitor of CAT transporters (system y⁺), suppresses only a fraction of arginine influx corresponding to leucine-insensitive uptake. Moreover, in Xenopus oocytes coexpressing 4F2hc and y⁺LAT2, leucine exerts a marked inhibition of arginine transport, partially dependent on sodium, while no inhibition is seen in oocytes expressing CAT1. Lastly, silencing of SLC7A6, the gene for y⁺LAT2, lowers arginine transport and doubles the intracellular content of the cationic amino acid in K562 cells. We conclude that arginine transport in human erythroid cells is due to both system y⁺ (CAT1 transporter) and system y⁺L (4F2hc/y⁺LAT2 isoform), which mainly contribute, respectively, to the influx and to the efflux of the cationic amino acid.     

Recycling of aromatic amino acids via TAT1 allows efflux of neutral amino acids via LAT2-4F2hc exchanger

The rate of amino acid efflux from individual cells needs to be adapted to cellular demands and plays a central role for the control of extracellular amino acid homeostasis. A particular example of such an outward amino acid transport is the basolateral efflux from transporting epithelial cells located in the small intestine and kidney proximal tubule. Because LAT2-4F2hc (Slc7a8–Slc3a2), the best known basolateral neutral amino acid transporter of these epithelial cells, functions as an obligatory exchanger, we tested whether TAT1 (Slc16a10), the aromatic amino-acid facilitated diffusion transporter, might allow amino acid efflux via this exchanger by recycling its influx substrates. In this study, we show by immunofluorescence that TAT1 and LAT2 indeed colocalize in the early kidney proximal tubule. Using the Xenopus laevis oocytes expression system, we show that l-glutamine is released from oocytes into an amino-acid-free medium only when both transporters are coexpressed. High-performance liquid chromatography analysis reveals that several other neutral amino acids are released as well. The transport function of both TAT1 and LAT2-4F2hc is necessary for this efflux, as coexpression of functionally inactive but surface-expressed mutants is ineffective. Based on negative results of coimmunoprecipitation and crosslinking experiments, the physical interaction of these transporters does not appear to be required. Furthermore, replacement of TAT1 or LAT2-4F2hc by the facilitated diffusion transporter LAT4 or the obligatory exchanger LAT1, respectively, supports similar functional cooperation. Taken together, the results suggest that the aromatic amino acid diffusion pathway TAT1 can control neutral amino acid efflux via neighboring exchanger LAT2-4F2hc, by recycling its aromatic influx substrates.     

LPIN1 genetic variation is associated with rosiglitazone response in type 2 diabetic patients

Lipin1 protein, a product of the LPIN1 gene, is required for normal adipose tissue development and metabolism. Lipin1 deficiency results in immature adipocyte development in cases of mouse fatty liver dystrophy and human lipodystrophy. Recently, pioglitazone has been reported to increase human adipocyte lipin1 expression. We evaluated the effects of LPIN1 polymorphisms on rosiglitazone response in patients with type 2 diabetes (T2DM). A total of 262 patients were treated with 12 weeks of rosiglitazone (4 mg/day) in addition to their previous drug regimen medications. Six single nucleotide polymorphisms (SNPs) at the LPIN1 locus were genotyped: rs11693809, rs10192566, rs2278513, rs2577262, rs2716610, and rs1050800. Because rs11693809, rs10192566, and rs2278513 are in nearly complete linkage disequilibrium (D'>0.958, r(2) >0.882), we analyzed rs10192566, rs2577262, rs2716610, and rs1050800. Rs10192566 was significantly associated with rosiglitazone treatment response. Patients with the G allele in rs10192566 had a larger decrease in fasting plasma glucose, 2-h postprandial glucose, and HbA1c than those without. This genetic effect remained significant after adjustment for age, sex, and initial body weight. No other SNPs were associated with response. These data suggest that LPIN1 genetic variations can affect rosiglitazone treatment response in T2DM.     

Sapropterin therapy increases stability of blood phenylalanine levels in patients with BH4-responsive phenylketonuria (PKU)

It has recently been demonstrated that variability in blood phenylalanine levels is inversely correlated with IQ and is a better predictor of IQ in early and continuously treated patients with phenylketonuria (PKU) than mean blood phenylalanine levels. This suggests that stability of blood phenylalanine should be a therapeutic goal in patients with PKU. The purpose of this study was to determine if treatment with sapropterin in patients with BH4-responsive PKU would increase the stability of blood phenylalanine levels. The records of all patients treated with sapropterin in the PKU Clinic at Children's Memorial Hospital in Chicago were examined retrospectively. Patients were included in the study if they were responsive to sapropterin during a 2- to 4-week challenge (reduction of blood phenylalanine level of at least 25% after 2 weeks of therapy or, in the case of patients with well-controlled blood phenylalanine at the time of testing, increased dietary phenylalanine tolerance by 4 weeks of treatment). A total of 37 subjects were eligible for inclusion (16 male; 21 female); the mean age was 12.6 years (range, 1.5–32.0). The total number of observations (phenylalanine levels) for all subjects was 1391 with a mean of 39 per subject (range, 13–96). Linear mixed modeling was utilized to estimate variances of the blood phenylalanine before (pre) and after (post) starting sapropterin. Likelihood ratio test was performed using SAS 9.1. Means and standard deviations for phenylalanine as estimated by the model were 6.67 mg/dl (4.20) and post 5.16 (3.78). The mean level post-sapropterin was significantly lower (p = .0002). The within-subject variances (mean and SD) of phenylalanine were: pre 6.897 (2.62) and post 4.799 (2.19). These two variances are significantly different with a p = .0017. We conclude that sapropterin therapy results in increased stability of blood phenylalanine levels. This effect is likely to improve cognitive outcome in BH4-responsive patients with PKU.     

Significance of System L Amino Acid Transporter 1 (LAT-1) and 4F2 Heavy Chain (4F2hc) Expression in Human Developing Intestines

To clarify the significance of expression of system L amino acid transporter 1 (LAT1) and 4F2 heavy chain (4F2hc) in the developing intestine, immunohistochemical investigation and molecular analysis were performed in the human embryonic and/or fetal intestines, ranging from 28–30 days to 34–35 weeks gestation. The molecular analysis for the expression of LAT1 and 4F2hc mRNAs was done in the pure epithelial cell samples prepared after laser assisted microdissection. The immunoreactivities against LAT1 and 4F2hc were detected along the basolateral cell membrane of the primitive gut epithelium at 28–30 days gestation. According to advance in gestational age of up to 24–25 weeks gestation, the immunoreactivity of LAT1 was predominantly observed in the supranuclear cytplasmic localization with a granular or dot-like staining pattern. Up to 8–9 weeks gestation, the immunoreactivity of 4F2hc showed almost the same as that of LAT1. However, after the age of 12–13 weeks gestation, the immunoreactivity of 4F2hc was predominantly localized along the cell membrane of apical surface of the epithelial cells. No apical and linear membranous localization of LAT1 was observed until nearly 20 weeks gestation. In the late gestational stage, both the immunoreactivities against LAT1 and 4F2hc were localized along the apical surface of the epithelial cells. In conclusion, the expression of LAT1 and 4F2hc in early developing intestine suggests they have a more important role in cell proliferation rather than functional differentiation. The predominant cytoplasmic localization of LAT1 during mid-fetal life seems to be largely inactive as amino acid transporter. On the other hand, the apical and linear membranous co-localization of LAT1 and 4F2hc in the late fetal life suggests that these molecules may play a role as a functional amino acid transporter in the fetal intestinal epithelium.     

TLR4 genetic variation is associated with inflammatory responses in Gram-positive sepsis

Objectives

To identify important pathogen recognition receptor (PRR) pathways regulating innate immune responses and outcome in Staphylococcus aureus sepsis.

Tyr-MIF-1 binding in brain is not altered by ligands selective for the GABAA/benzodiazepine receptor

Binding of benzodiazepines to the benzodiazepine gamma-aminobutyric acid (GABA) receptor-chloride channel complex has been shown to be altered by Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2). This raised the possibility of allosteric binding interactions between Tyr-MIF-1 sites and the GABAA receptor complex. We tested this possibility in rat brain by examining the binding of Tyr-MIF-1 to brain membranes in the presence of clonazepam, GABA, a combination of clonazepam and GABA, RO15788, or picrotoxinin. None of the tested substances affected Tyr-MIF-1 binding. We also tested mouse cortex for changes in Tyr-MIF-1 binding in the presence of ligands that bind to the GABA/benzodiazepine/chloride channel complex. Clonazepam, flunitrazepam, RO15788, and picrotoxinin at concentrations ranging from 10(-13) to 10(-5) M, each in the absence or presence of GABA at concentrations ranging from 10(-9) to 10(-5) M, each in the absence or presence of GABA at concentrations ranging from 10(-9) to 10(-6) M, did not significantly alter the binding of Tyr-MIF-1. The results indicate that simple bidirectional allosteric interactions between Tyr-MIF-1 binding sites and benzodiazepine, GABA or chloride channel binding sites are not likely to be the mechanism by which Tyr-MIF-1 affects binding at this complex.     

Hereditary haemorrhagic telangiectasia with extensive liver involvement is not caused by either HHT1 or HHT2.

Hereditary haemorrhagic telangiectasia (HHT) is a genetically heterogeneous dominant disorder. Two disease loci have been mapped to chromosomes 9q3 and 12q. In a large pedigree, with an unusually high number of patients with liver vascular malformations, both previously mapped loci have been excluded. The loci for two other inherited vascular malformation diseases, cerebral cavernous malformations and multiple cutaneous and mucosal venous malformations, have also been excluded. Thus we conclude that at least a third, as yet unmapped, HHT locus does exist, possibly associated with high frequency of liver involvement.     

PLCL1 rs7595412 variation is not associated with hip bone size variation in postmenopausal Danish women

Background  

Bone size (BS) variation is under strong genetic control and plays an important role in determining bone strength and fracture risk. Recently, a genome-wide association study identified polymorphisms associated with hip BS variation in the PLCL1 (phospholipase c-like 1) locus. Carriers of the major A allele of the most significant polymorphism, rs7595412, have around 17% larger hip BS than non-carriers. We therefore hypothesized that this polymorphism may also influence postmenopausal complications.     

E2F1 is not essential for apoptosis induced by potassium deprivation in cerebellar granule neurons

Cerebellar granule neurons (CGNs) undergo apoptosis when deprived of depolarizing concentration of potassium. A key regulator of cell cycle, E2F1, was believed to play a role in CGN apoptosis induced by potassium deprivation. However, here we demonstrated that although E2F1 was upregulated in wild type CGNs following potassium deprivation, CGNs that derived from E2F1 knockout mice underwent apoptosis at a similar rate as the wild type. Analysis of the apoptotic neurons revealed no difference in the activation of caspase-3 in E2F1 null and wild type CGNs. Furthermore, knockdown of E2F1 expression by RNA interference failed to attenuate the apoptosis of CGNs induced by potassium deprivation. Taken together, our results suggested that E2F1 is not essential for apoptosis induced by potassium deprivation in CGNs.     

Molecular genetics of PKU in Eastern Europe: A nonsense mutation associated with haplotype 4 of the phenylalanine hydroxylase gene

Phenylketonuria (PKU) is a genetic disorder secondary to a deficiency of hepatic phenyalanine hydroxylase (PAH). Several mutations in thePAH gene have recently been reported, and linkage disequilibrium was observed between RFLP haplotypes and specific mutations. A new molecular lesion has been identified in exon 7 of thePAH gene in a Hungarian PKU patient by direct sequencing of PCR-amplified DNA. The C-to-T transition causes the substitution of Arg²⁴³ to a termination codon, and the mutant allele is associated with haplotype 4 of thePAH gene. The mutation is present in two of nine mutant haplotype 4 alleles among Eastern Europeans and is not present among Western Europeans and Asians. The rarity of this mutant allele and its restricted geographic distribution suggest that the mutational event occurred recently on a normal haplotype 4 background in Eastern Europe.     

IFIH1-GCA-KCNH7 locus is not associated with genetic susceptibility to multiple sclerosis in French patients

A recent investigation reported, for the first time, an association between variants in the IFIH1-GCA-KCNH7 locus and multiple sclerosis (MS). We sought to replicate this genetic association in MS with a new independent MS cohort composed of French Caucasian MS trio families. The two most significant IFIH1 single nucleotide polymorphisms, rs1990760 and rs2068330, reported as involved in MS susceptibility, were genotyped in 591 French Caucasian MS trio families, and analyzed using the transmission/disequilibrium test. No association with MS was found (rs1990760, P=0.45 and rs2068330, P=0.27). Similarly, no significant association was detected after stratification for HLA-DRB1^*1501 carriers. Reasons that may explain this discrepancy between the original report and our study are discussed.     

Exhaled carbon monoxide concentration is not elevated in patients with inflammatory bowel disease

The present study was initiated to examine whether the concentration of CO in the breath is elevated in patients with inflammatory bowel disease (IBD). Twenty-three clinically stable patients with IBD in the outpatient clinic (11 with Crohn’s disease, 12 with ulcerative colitis), who are non-smokers and non-passive smokers, were selected and the concentration of CO in their breath was measured using a breath gas analyser (TRI lyser mBA-3000). The concentration of CO in the breath of 23 patients with IBD was 2.5±0.9 (1.1–4.3) ppm. This concentration comes within the range of standard values in our previous reports (2.5±2.2 ppm). Any significant difference was not observed between 2.4±0.9 (1.5–4.3) ppm for the 11 Crohn’s disease patients and the 2.6±1.0 (1.1–3.9) ppm for the 12 ulcerative colitis patients. The results suggest that clinically stable patients with IBD do not show high values for concentration of CO in the breath.     

Inter-individual variation in the mutagenic activation of 2-acetylaminofluorene by human liver in relation to animal metabolic models

A relatively large, reproducible inter-individual variationwas found in the ability of 17 human liver S9 samples to mediatethe mutagenicity of 2-acetylaminofluorene (2AAF) in Salmonellatyphimurium strain TA98. In an animal model, variation in metabolicactivation of 2AAF did not appear to relate to the phenotypeof debrisoquine 4-hydroxylase since hepatic S9 from poor andextensive metabolizer phenotypes (female DA and female Wistarrat, respectively) mediated the mutagenicity of this aromaticamide equally well. Approximately onethird of human liver samplesexhibited an ability to detoxify 2AAF in a modified bacterialmutagenicity assay in a manner similar to that shown by guineapig (but not rat or rabbit) S9. However, only in 2/14 humanpreparations was the detoxification inhibited by 8-hydroxyquinolinewhich has previously been recognized as an inhibitor of a detoxifying‘transoxygenation’ in guinea pig liver. The resultssupport a growing body of evidence for inter-individual variationin human carcinogen metabolism which may be important in determiningsusceptibility to chemical carcinogenesis.     

Natural genetic variation caused by small insertions and deletions in the human genome

Human genetic variation is expected to play a central role in personalized medicine. Yet only a fraction of the natural genetic variation that is harbored by humans has been discovered to date. Here we report almost 2 million small insertions and deletions (INDELs) that range from 1 bp to 10,000 bp in length in the genomes of 79 diverse humans. These variants include 819,363 small INDELs that map to human genes. Small INDELs frequently were found in the coding exons of these genes, and several lines of evidence indicate that such variation is a major determinant of human biological diversity. Microarray-based genotyping experiments revealed several interesting observations regarding the population genetics of small INDEL variation. For example, we found that many of our INDELs had high levels of linkage disequilibrium (LD) with both HapMap SNPs and with high-scoring SNPs from genome-wide association studies. Overall, our study indicates that small INDEL variation is likely to be a key factor underlying inherited traits and diseases in humans.     

Glycerophosphoethanolamine concentration is elevated in brain of patients with dominantly inherited olivopontocerebellar atrophy

We measured the concentration of glycerophosphoethanolamine (GPEA), a membrane breakdown product, in autopsied brain of 10 patients with dominantly inherited olivopontocerebellar atrophy (OPCA), a cerebellar ataxia disorder. As compared with the controls, mean GPEA levels were significantly elevated by 37–69% in 11 of the 15 brain areas examined, including extracerebellar brain regions in which no neuronal cell loss could be detected by semiquantitative estimation. Our data suggest the possibility of altered membrane phospholipid metabolism in OPCA which could be a contributing factor in the neuronal cell death.     

Copy-number variation in BMPR2 is not associated with the pathogenesis of pulmonary arterial hypertension

Background  

Copy-number variations (CNVs) are structural variations in the genome involving 1 kb to 3 mb of DNA. CNV has been reported within intron 1 of the BMPR2 gene. We propose that CNV could affect phenotype in familial and/or sporadic pulmonary arterial hypertension (PAH) by altering gene expression.     

Downregulation of the hedgehog receptor PTCH1 in colorectal serrated adenocarcinomas is not caused by PTCH1 mutations

Colorectal serrated adenocarcinoma forms about 15–20% of colorectal carcinomas. We have previously shown that downregulation of PTCH1 is distinctive for this type of colorectal cancer. In several other tumor types, somatic inactivating PTCH1 mutations have been shown to lead to aberrant Hedgehog signaling, but in colorectal cancer the role of PTCH1 mutations has not been thoroughly studied. Here, we have analyzed the mutation status of PTCH1 in a series of 33 colorectal serrated adenocarcinomas by sequencing all 23 coding exons. We detected 11 previously known SNPs and eight new alterations. The latter included five synonymous changes and two previously unknown missense variations, somatic M319V, and germline V1231A. V1231A was also present in population controls and likely represents polymorphism. The somatic M319V variant does not appear to be an attractive candidate for a disease-associated mutation because in silico analyses did not support the pathogenic nature of the change. A somatic, intronic 1-bp deletion was detected in a short poly(T) stretch in two microsatellite unstable tumors. None of the three changes had predicted effect on splicing when analyzed in silico. Our results did not reveal any clearly deleterious inactivating PTCH1 mutations in our collection of colorectal serrated adenocarcinomas. This suggests that other mechanisms are involved in the observed downregulation of the PTCH1 gene. These might include, e.g., constantly active MAPK signaling by KRAS or BRAF mutations or silencing of PTCH1 by hypermethylation, and further studies are needed to reveal these mechanisms.