Amino Acid Transporter Gene Research Articles

Effect of SLC7A5 on the proliferation of tumor cells and its relationship with transforming growth factor-β1 signal pathway

To explore the biological effects of amino acid transporter gene SLC7A5 (solute carrier family 7, member 5) on tumor cells and the regulatory mechanism at transcriptional level. Methods: The expression of SLC7A5 was examined in human normal tissues and corresponding tumor tissues by Gene Expression Omnibus (GEO) database. The recombinant plasmid of SLC7A5 gene was constructed, and the effect of the SLC7A5 gene on tumor cell proliferation was investigated by methylthiazolyldiphenyl-tetrazolium bromide (MTT) and flow cytometry. SLC7A5 gene promoter and transcription factor binding sites were predicted through bioinformatics analysis, and the gene promoter recombinant plasmid was constructed. Then the dual luciferase reporter gene assay and reverse transcription polymerase chain reaction (RT-PCR) were used to explore the regulation of transforming growth factor-β1 (TGF-β1) signal on SLC7A5 gene expression. Results: The GEO database analysis showed that the distribution of SLC7A5 was tissue specific, and its expression level was significantly higher in the tumor tissues than that in the corresponding normal tissues. The results of MTT and flow cytometry showed that SLC7A5 could promote cell proliferation. Results from the promoter analysis, reporter gene assay and RT-PCR confirmed that TGF-β1 could up-regulate the activity of SLC7A5 promoter and promote the expression of the SLC7A5 gene. Conclusion: SLC7A5 gene plays a role in promoting tumor development, which is regulated by the TGF-β1 signaling pathway.

Gene expression of amino acid transporter in pigeon (Columbia livia) intestine during post-hatch development and its correlation with amino acid in pigeon milk

This study was conducted to evaluate gene expression of the amino acid transporter in post-hatch pigeon small intestine and the association of pigeon milk amino acid with the above transporter's gene expression. A total of 48 pigeon breeding families were randomly allocated to 8 groups of 6 replicates of one parental pigeon pair and 2 squabs. Samples of pigeon milk and duodenum, jejunum, and ileum were collected on d 1, 2, 3, 4, 6, 8, 10, and 14 post hatch. The results showed that levels of crude protein (8.93 to 15.56%) were highest in pigeon milk on an air-dry basis. Amino acid content in pigeon milk remained constant in the first 4 d, declined abruptly at d 6, then increased dramatically from d 8 to 14. There was a significant effect of interaction between age and intestinal segments on those amino acid transporters gene expression. mRNA abundance of ATB0’+, SNAT-2, LAT-4, rBAT, b0’+ AT, EAAT-3 and PAT-1 was highest in the ileum; B0 AT1, asc-1, and IMINO were predominate in the jejunum; and CAT-1 and y+ LAT2 were greatest in the duodenum. Age-related changes of amino acid transporter mRNA was inconsistent. mRNA levels of SNAT-2, rBAT, y+ LAT2, b0’+ AT, and EAAT-3 ascended with age, whereas that of asc-1, CAT-1, and IMINO diminished significantly. Levels of B0 AT1 and PAT-1 mRNA abundance were minimized at d 6. However, few correlations were found between pigeon milk amino acid and the amino acid transporter gene expressions in squab small intestine. Our findings provide a comprehensive elaboration on ontogeny of the amino acid transporter in post-hatch pigeon intestine.

Diurnal rhythm in mRNA expression of genes encoding amino acid transporter and circadian gene cry in intestinal mucosa of piglets

Long chain PUFA contents in plasma and liver both exhibited diurnal rhythms in pigs. However, whether mRNA expression of amino acid transporter and circadian gene Cry in intestinal mucosa is also rhythmic is yet to be known. The purpose of this study aims to investigate the diurnal rhythm in mRNA expression of genes encoding amino acid transporter and whether their rhythm was related to the expression of circadian gene Cry in intestinal mucosa of piglets. Thirty-six piglets (Duroc × Landrace × Large Yorkshire) at the age of 35 days were selected and fed for three weeks, and then samples were collected at 3:00 am (Clo3), 7:00 am (Clo7), 11:00 am (Clo11), 3:00 pm (Clo15), 7:00 pm (Clo19), and 11:00 pm (Clo23) at the age of 56 days. At each time point, small intestinal mucosa samples were collected from duodenum, jejunum, and ileum for detection of mRNA expression of the amino acid transporters and circadian gene Cry. The results showed that mRNA expression of most amino acid transporters in intestinal mucosa was higher at night and lower during the daytime. Expression of SLC1A2, SLC6A20, SLC7A1, and SLC6A14 in duodenal mucosa reached the peak at Clo3 and Clo7; the diurnal rhythm of expression of SLC1A2, SLC6A20, and SLC7A1 was similar to Cry1, while the diurnal rhythm of expression of SLC6A14 had a similar trend to Cry2. Expression of SLC16A10, SLC1A2, and SLC7A1 in jejunal mucosa reached the peak at Clo7, while SLC6A14 reached the peak at Clo3; the diurnal rhythm of expression of SLC1A2 showed a similarity with Cry1, while the diurnal rhythm of expression of SLC16A10, SLC7A1, and SLC6A14 was similar to Cry2. Expression of SLC6A14, SLC6A20, and SLC7A1 in ileal mucosa reached the peak at Clo3; the diurnal rhythm of expression of SLC6A20 has a similarity with Cry1, while the diurnal rhythm of expression of SLC7A1 and SLC6A14 was similar to Cry2. The results suggested that the mRNA expression of most genes encoding amino acid transporters exhibited diurnal rhythms in the intestinal mucosa of piglets, and SLC7A1, SLC6A14, and SLC1A2 have a similar rhythm with circadian clock genes Cry1 and 2, and they reached the peak at Clo3 and Clo7.

PIF* promotes brain re-myelination locally while regulating systemic inflammation- clinically relevant multiple sclerosis M.smegmatis model.

Neurologic disease diagnosis and treatment is challenging. Multiple Sclerosis (MS) is a demyelinating autoimmune disease with few clinical forms and uncertain etiology. Current studies suggest that it is likely caused by infection(s) triggering a systemic immune response resulting in antigen/non-antigen-related autoimmune response in central nervous system (CNS). New therapeutic approaches are needed. Secreted by viable embryos, PreImplantation Factor (PIF) possesses a local and systemic immunity regulatory role. Synthetic PIF (PIF) duplicates endogenous peptide's protective effect in pre-clinical autoimmune and transplantation models. PIF protects against brain hypoxia-ischemia by directly targeting microglia and neurons. In chronic experimental autoimmune encephalitis (EAE) model PIF reverses paralysis while promoting neural repair. Herein we report that PIF directly promotes brain re-myelination and reverses paralysis in relapsing remitting EAE MS model. PIF crosses the blood-brain barrier targeting microglia. Systemically, PIF decreases pro-inflammatory IL23/IL17 cytokines, while preserving CNS-specific T-cell repertoire. Global brain gene analysis revealed that PIF regulates critical Na+/K+/Ca++ ions, amino acid and glucose transport genes expression. Further, PIF modulates oxidative stress, DNA methylation, cell cycle regulation, and protein ubiquitination while regulating multiple genes. In cultured astrocytes, PIF promotes BDNF-myelin synthesis promoter and SLC2A1 (glucose transport) while reducing deleterious E2F5, and HSP90ab1 (oxidative stress) genes expression. In cultured microglia, PIF increases anti-inflammatory IL10 while reducing pro-inflammatory IFNγ expression. Collectively, PIF promotes brain re-myelination and neuroprotection in relapsing remitting EAE MS model. Coupled with ongoing, Fast-Track FDA approved clinical trial, NCT#02239562 (immune disorder), current data supports PIF's translation for neurodegenerative disorders therapy.

Urinary Exosomes Contain MicroRNAs Capable of Paracrine Modulation of Tubular Transporters in Kidney

Exosomes derived from all nephron segments are present in human urine, where their functionality is incompletely understood. Most studies have focused on biomarker discovery rather than exosome function. Through sequencing we identified the miRNA repertoire of urinary exosomes from healthy volunteers; 276 mature miRNAs and 345 pre-miRNAs were identified (43%/7% of reads). Among the most abundant were members of the miR-10, miR-30 and let-7 families. Targets for the identified miRNAs were predicted using five different databases; genes encoding membrane transporters and their regulators were enriched, highlighting the possibility that these miRNAs could modulate key renal tubular functions in a paracrine manner. As proof of concept, cultured renal epithelial cells were exposed to urinary exosomes and cellular exosomal uptake was confirmed; thereafter, reduced levels of the potassium channel ROMK and kinases SGK1 and WNK1 were observed in a human collecting duct cell line, while SPAK was unaltered. In proximal tubular cells, mRNA levels of the amino acid transporter gene SLC38A2 were diminished and reflected in a significant decrement of its encoded protein SNAT2. Protein levels of the kinase SGK1 did not change. Thus we demonstrated a novel potential function for miRNA in urinary exosomes.

Genetic mechanisms of bone digestion and nutrient absorption in the bone-eating worm Osedax japonicus inferred from transcriptome and gene expression analyses

BackgroundBone-eating worms of the genus Osedax (Annelida, Siboglinidae) have adapted to whale fall environments by acquiring a novel characteristic called the root, which branches and penetrates into sunken bones. The worms lack a digestive tract and mouth opening, and it has been suggested that Osedax degrade vertebrate bones and uptake nutrients through acidification and secretion of enzymes from the root. Symbiotic bacteria in the root tissue may have a crucial role in the metabolism of Osedax. However, the molecular mechanisms and cells responsible for bone digestion and nutrient uptake are still unclear, and information on the metabolic interaction between Osedax and symbiotic bacteria is limited.ResultsWe compared transcriptomes from three different RNA samples from the following tissues: trunk + palps, root + ovisac, and larva + male. A Pfam domain enrichment analysis revealed that protease- and transporter-related genes were enriched in the root + ovisac specific genes compared with the total transcriptome. Through targeted gene annotation we found gene family expansions resulting in a remarkably large number of matrix metalloproteinase (mmp) genes in the Osedax compared with other invertebrates. Twelve of these Osedax mmp genes were expressed in the root epidermal cells. Genes encoding various types of transporters, including amino acid, oligopeptide, bicarbonate, and sulfate/carboxylate transporters, were also expressed in root epidermal cells. In addition, amino acid and other metabolite transporter genes were expressed in bacteriocytes. These protease and transporter genes were first expressed in root tissues at the juvenile stage, when the root starts to develop.ConclusionsThe expression of various proteinase and transporter genes in the root epidermis supports the theory that the root epidermal cells are responsible for bone digestion and subsequent nutrient uptake. Expression of transporter genes in the host bacteriocytes suggests the presence of metabolic interaction between Osedax and symbiotic bacteria.

Galectin-1 Reduces Neuroinflammation via Modulation of Nitric Oxide-Arginase Signaling in HIV-1 Transfected Microglia: a Gold Nanoparticle-Galectin-1 "Nanoplex" a Possible Neurotherapeutic?

Galectins are a family of β-galactoside-binding lectins that are important modulators of homeostasis in the central nervous system (CNS). Galectin-1 is a pivotal regulator of microglia activation that alters the immune balance from neurodegeneration to neuroprotection and could have therapeutic relevance in HIV associated neurocognitive disorders (HAND). We have previously shown that galectin-1 treatment decreased oxidative stress in microglia and hypothesize that the mechanism underlying this phenomenon is the cross regulatory interactions between Nitric oxide (NO) and Arginase I activity in microglia. We induced microglial activation and examined the effect of galectin-1 on the expression of various M1/M2 microglial phenotypic markers. Since, TNF-α is associated with activation of microglial cells involved in pathogenesis of neurodegenerative diseases, we treated HIV transfected human microglial cell cultures (CHME-5/HIV) with TNF-α followed by treatment with galectin-1, to examine the galectin-1 mediated neuro-modulatory response. Our results show that treatment of CHME-5/HIV microglia with galectin-1 reduced TNF-α induced oxidative stress by ~40%, and also significantly reduced iNOS gene expression and NO production while correspondingly increasing arginase-1, cationic amino acid transporter (CAT-1) gene expression and arginase activity. Galectin-1 treatment results in shifting microglia polarization from M1 toward the beneficial M2 phenotype which may prevent neurodegeneration and promote neuroprotection. Thus, our data suggests that galectin-1 treatment reduces neuroinflammation in the CNS microenvironment via the modulation of the NO-arginase network in microglia and thus could play a neuroprotective role in HAND. Further, the therapeutic potential of galectin-1 could be enhanced by conjugation of galectin-1 onto gold nanoparticles (Au-NP), resulting in a nanogold-galectin-1 (Au-Gal-1) multivalent complex that will have more clinical translational efficacy than free galectin-1 by virtue of increasing the payload influx.

539: Higher maternal essential amino acid levels are associated with higher placental amino acid transporter gene expression

539: Higher maternal essential amino acid levels are associated with higher placental amino acid transporter gene expression

Candidate adaptive genes associated with lineage divergence: identifying SNPs via next-generation targeted resequencing in mule deer (Odocoileus hemionus).

Mule deer (Odocoileus hemionus) are an excellent nonmodel species for empirically testing hypotheses in landscape and population genomics due to their large population sizes (low genetic drift), relatively continuous distribution, diversity of occupied habitats and phenotypic variation. Because few genomic resources are currently available for this species, we used exon data from a cattle (Bos taurus) reference genome to direct targeted resequencing of 5935 genes in mule deer. We sequenced approximately 3.75Mbp at minimum 20X coverage in each of the seven mule deer, identifying 23204 single nucleotide polymorphisms (SNPs) within, or adjacent to, 6886 exons in 3559 genes. We found 91 SNP loci (from 69 genes) with putatively fixed allele frequency differences between the two major lineages of mule deer (mule deer and black-tailed deer), and our estimate of mean genetic divergence (genome-wide FST =0.123) between these lineages was consistent with previous findings using microsatellite loci. We detected an over-representation of gamete generation and amino acid transport genes among the genes with SNPs exhibiting potentially fixed allele frequency differences between lineages. This targeted resequencing approach using exon capture techniques has identified a suite of loci that can be used in future research to investigate the genomic basis of adaptation and differentiation between black-tailed deer and mule deer. This study also highlights techniques (and an exon capture array) that will facilitate population genomic research in other cervids and nonmodel organisms.

Transcriptome Analysis and Postprandial Expression of Amino Acid Transporter Genes in the Fast Muscles and Gut of Chinese Perch (Siniperca chuatsi).

The characterization of the expression and regulation of growth-related genes in the muscles of Chinese perch is of great interest to aquaculturists because of the commercial value of the species. The transcriptome annotation of the skeletal muscles is a crucial step in muscle growth-related gene analysis. In this study, we generated 52 504 230 reads of mRNA sequence data from the fast muscles of the Chinese perch by using Solexa/Illumina RNA-seq. Twenty-one amino acid transporter genes were annotated by searching protein and gene ontology databases, and postprandial changes in their transcript abundance were assayed after administering a single satiating meal to Chinese perch juveniles (body mass, approximately 100 g), following fasting for 1 week. The gut content of the Chinese perch increased significantly after 1 h and remained high for 6 h following the meal and emptied within 48–96 h. Expression of eight amino acid transporter genes was assayed in the fast muscles through quantitative real-time polymerase chain reaction at 0, 1, 3, 6, 12, 24, 48, and 96 h. Among the genes, five transporter transcripts were markedly up-regulated within 1 h of refeeding, indicating that they may be potential candidate genes involved in the rapid-response signaling system regulating fish myotomal muscle growth. These genes display coordinated regulation favoring the resumption of myogenesis responding to feeding.

Malabsorption and intestinal adaptation after one anastomosis gastric bypass compared with Roux-en-Y gastric bypass in rats.

The technically easier one-anastomosis (mini) gastric bypass (MGB) is associated with similar metabolic improvements and weight loss as the Roux-en-Y gastric bypass (RYGB). However, MGB is controversial and suspected to result in greater malabsorption than RYGB. In this study, we compared macronutrient absorption and intestinal adaptation after MGB or RYGB in rats. Body weight and food intake were monitored and glucose tolerance tests were performed in rats subjected to MGB, RYGB, or sham surgery. Carbohydrate, protein, and lipid absorption was determined by fecal analyses. Intestinal remodeling was evaluated by histology and immunohistochemistry. Peptide and amino acid transporter mRNA levels were measured in the remodeled intestinal mucosa and those of anorexigenic and orexigenic peptides in the hypothalamus. The MGB and RYGB surgeries both resulted in a reduction of body weight and an improvement of glucose tolerance relative to sham rats. Hypothalamic orexigenic neuropeptide gene expression was higher in MGB rats than in RYGB or sham rats. Fecal losses of calories and proteins were greater after MGB than RYGB or sham surgery. Intestinal hyperplasia occurred after MGB and RYGB with increased jejunum diameter, higher villi, and deeper crypts than in sham rats. Peptidase and peptide or amino acid transporter genes were overexpressed in jejunal mucosa from MGB rats but not RYGB rats. In rats, MGB led to greater protein malabsorption and energy loss than RYGB. This malabsorption was not compensated by intestinal overgrowth and increased expression of peptide transporters in the jejunum.

Effect of egg weight on composition, embryonic growth, and expression of amino acid transporter genes in yolk sac membranes and small intestines of the domestic pigeon (Columba livia)

The objective of this study was to investigate the effect of egg weight on the composition of the egg, the growth of the embryo, and the expression of amino acid transporter genes in the yolk sac membranes and small intestines of the domestic pigeon (Columba livia). A total of 240 fertilized eggs were collected and divided into two groups based on the weight of the eggs, light (LE) and heavy (HE). The composition of 20 eggs from each group was measured, and the remaining eggs were weighed and placed in an incubator. On embryonic days (E) 9, 11, 13, and 15 and day of hatch (DOH), 15 embryos/hatchlings from each group were measured for embryonic growth, and samples were collected. The HE had heavier yolk and albumen weights than the LE (P < 0.01). Compared with the LE, the HE had heavier yolk-free embryonic body and yolk sac weights from E13 to DOH (P < 0.05). Additionally, the HE had larger yolk sac membrane weights from E13 to E15 (P < 0.05) and had more residual yolk sac content on DOH than those of the LE (P < 0.01). The yolk absorption was greater for the HE than for the LE from E11 to E13 (P < 0.05). Furthermore, the abundance of CAT2 and PepT1 mRNA in the yolk sac membranes was greater in the HE than in the LE on E13 (P < 0.05). Compared with the LE, the gene expression of EAAT2 in the intestine on E13 was greater in the HE, whereas the expression of EAAT3 was lower in the HE (P < 0.05). Taken together, our results suggest that egg weight influenced the composition of the eggs, embryonic development, and expression of amino acid transporter genes in the yolk sac membranes and small intestines of pigeon embryos.

Glycinergic-Fipronil Uptake Is Mediated by an Amino Acid Carrier System and Induces the Expression of Amino Acid Transporter Genes in Ricinus communis Seedlings.

Phloem-mobile insecticides are efficient for piercing and sucking insect control. Introduction of sugar or amino acid groups to the parent compound can improve the phloem mobility of insecticides, so a glycinergic-fipronil conjugate (GlyF), 2-(3-(3-cyano-1-(2,6-dichloro-4-(trifluoromethyl)phenyl)-4-((trifluoromethyl)sulfinyl)-1H-pyrazole-5-yl)ureido) acetic acid, was designed and synthesized. Although the "Kleier model" predicted that this conjugate is not phloem mobile, GlyF can be continually detected during a 5 h collection of Ricinus communis phloem sap. Furthermore, an R. communis seedling cotyledon disk uptake experiment demonstrates that the uptake of GlyF is sensitive to pH, carbonyl cyanide m-chlorophenylhydrazone (CCCP), temperature, and p-chloromercuribenzenesulfonic acid (pCMBS) and is likely mediated by amino acid carrier system. To explore the roles of amino acid transporters (AATs) in GlyF uptake, a total of 62 AAT genes were identified from the R. communis genome in silico. Phylogenetic analysis revealed that AATs in R. communis were organized into the ATF (amino acid transporter) and APC (amino acid, polyaminem and choline transporter) superfamilies, with five subfamilies in ATF and two in APC. Furthermore, the expression profiles of 20 abundantly expressed AATs (cycle threshold (Ct) values <27) were analyzed at 1, 3, and 6 h after GlyF treatment by RT-qPCR. The results demonstrated that expression levels of four AAT genes, RcLHT6, RcANT15, RcProT2, and RcCAT2, were induced by the GlyF treatment in R. communis seedlings. On the basis of the observation that the expression profile of the four candidate genes is similar to the time course observation for GlyF foliar disk uptake, it is suggested that those four genes are possible candidates involved in the uptake of GlyF. These results contribute to a better understanding of the mechanism of GlyF uptake as well as phloem loading from a molecular biology perspective and facilitate functional characterization of candidate AAT genes in future studies.

Pod removal responsive change in phytohormones and its impact on protein degradation and amino acid transport in source leaves of Brassica napus

To characterize the hormonal regulation of nitrogen remobilization from source to pod filling in Brassica napus, the hormonal level, proteolytic process, and amino acid transport were assessed in mature leaves of pod-removed or control at the early pod-filling stage. Pod (sink) removal decreased salicylic acid (SA), and significantly increased jasmonic acid (JA). The SA/JA ratio decreased with pod removal, accompanied by low degradation of foliar proteins and Rubisco content. A significant decrease in protease activity was observed in pod-removed leaves, confirmed by in-gel staining of protease. Pod removal reduced the expression of four amino acid transporter genes (BnAAP1, BnAAP2, BnAAP4, and BnAAP6) in mature leaves and reduced amino acid loading into phloem. These results indicated that a decrease in SA resulting from pod removal down-regulated nitrogen remobilization accompanied by a decrease in proteolytic activity and amino acid transport in mature leaves at the pod-filling stage.

Genome-Wide Identification, Classification, and Expression Analysis of Amino Acid Transporter Gene Family in Glycine Max.

Amino acid transporters (AATs) play important roles in transporting amino acid across cellular membranes and are essential for plant growth and development. To date, the AAT gene family in soybean (Glycine max L.) has not been characterized. In this study, we identified 189 AAT genes from the entire soybean genomic sequence, and classified them into 12 distinct subfamilies based upon their sequence composition and phylogenetic positions. To further investigate the functions of these genes, we analyzed the chromosome distributions, gene structures, duplication patterns, phylogenetic tree, tissue expression patterns of the 189 AAT genes in soybean. We found that a large number of AAT genes in soybean were expanded via gene duplication, 46 and 36 GmAAT genes were WGD/segmental and tandemly duplicated, respectively. Further comprehensive analyses of the expression profiles of GmAAT genes in various stages of vegetative and reproductive development showed that soybean AAT genes exhibited preferential or distinct expression patterns among different tissues. Overall, our study provides a framework for further analysis of the biological functions of AAT genes in either soybean or other crops.

Expression pattern of peptide and amino acid genes in digestive tract of transporter juvenile turbot (Scophthalmus maximus L.)

Turbot (Scophthalmus maximus L.), a carnivorous fish species with high dietary protein requirement, was chosen to examine the expression pattern of peptide and amino acid transporter genes along its digestive tract which was divided into six segments including stomach, pyloric caeca, rectum, and three equal parts of the remainder of the intestine. The results showed that the expression of two peptide and eleven amino acid transporters genes exhibited distinct patterns. Peptide transporter 1 (PepT1) was rich in proximal intestine while peptide transporter 2 (PepT2) was abundant in distal intestine. A number of neutral and cationic amino acid transporters expressed richly in whole intestine including B0-type amino acid transporter 1 (B0AT1), L-type amino acid transporter 2 (LAT2), T-type amino acid transporter 1 (TAT1), proton-coupled amino acid transporter 1 (PAT1), y+L-type amino acid transporter 1 (y+LAT1), and cationic amino acid transporter 2 (CAT2) while ASC amino acid transporter 2 (ASCT2), sodium-coupled neutral amino acid transporter 2 (SNAT2), and y+L-type amino acid transporter 2 (y+LAT2) abundantly expressed in stomach. In addition, system b0,+ transporters (rBAT and b0,+AT) existed richly in distal intestine. These findings comprehensively characterized the distribution of solute carrier family proteins, which revealed the relative importance of peptide and amino acid absorption through luminal membrane. Our findings are helpful to understand the mechanism of the utilization of dietary protein in fish with a short digestive tract.

Maternal Factors Are Associated with the Expression of Placental Genes Involved in Amino Acid Metabolism and Transport.

IntroductionMaternal environment and lifestyle factors may modify placental function to match the mother’s capacity to support the demands of fetal growth. Much remains to be understood about maternal influences on placental metabolic and amino acid transporter gene expression. We investigated the influences of maternal lifestyle and body composition (e.g. fat and muscle content) on a selection of metabolic and amino acid transporter genes and their associations with fetal growth.MethodsRNA was extracted from 102 term Southampton Women’s Survey placental samples. Expression of nine metabolic, seven exchange, eight accumulative and three facilitated transporter genes was analyzed using quantitative real-time PCR.ResultsIncreased placental LAT2 (p = 0.01), y + LAT2 (p = 0.03), aspartate aminotransferase 2 (p = 0.02) and decreased aspartate aminotransferase 1 (p = 0.04) mRNA expression associated with pre-pregnancy maternal smoking. Placental mRNA expression of TAT1 (p = 0.01), ASCT1 (p = 0.03), mitochondrial branched chain aminotransferase (p = 0.02) and glutamine synthetase (p = 0.05) was positively associated with maternal strenuous exercise. Increased glutamine synthetase mRNA expression (r = 0.20, p = 0.05) associated with higher maternal diet quality (prudent dietary pattern) pre-pregnancy. Lower LAT4 (r = -0.25, p = 0.05) and aspartate aminotransferase 2 mRNA expression (r = -0.28, p = 0.01) associated with higher early pregnancy diet quality. Lower placental ASCT1 mRNA expression associated with measures of increased maternal fat mass, including pre-pregnancy BMI (r = -0.26, p = 0.01). Lower placental mRNA expression of alanine aminotransferase 2 associated with greater neonatal adiposity, for example neonatal subscapular skinfold thickness (r = -0.33, p = 0.001).ConclusionA number of maternal influences have been linked with outcomes in childhood, independently of neonatal size; our finding of associations between placental expression of transporter and metabolic genes and maternal smoking, physical activity and diet raises the possibility that their effects are mediated in part through alterations in placental function. The observed changes in placental gene expression in relation to modifiable maternal factors are important as they could form part of interventions aimed at maintaining a healthy lifestyle for the mother and for optimal fetal development.

Yeast Cyc8p and Tup1p proteins function as coactivators for transcription of Stp1/2p-dependent amino acid transporter genes

The yeast Cyc8p-Tup1p complex is known to serve primarily as a transcriptional corepressor in a variety of biological processes. However, less is known about its function as a coactivator. Herein, we found tryptophan transporter genes, TAT1 and TAT2, that, when overexpressed, suppressed the slow growth of Δcyc8. We observed that the addition of tryptophan to Δcyc8 cultures partially restored cell growth, and the deletion of CYC8 and TUP1 reduced transcriptional levels of TAT1 and TAT2. Tup1p bound to the promoter region of TAT1 and TAT2 genes that were dependent on STP1 and STP2 (encoding DNA-binding activator proteins) for expression. Similarly, transcription of the other Stp1/2p-dependent amino acid transporter (AAT) genes also required CYC8 and TUP1 gene functions. These data indicate that Cyc8p-Tup1p plays a role as a transcriptional coactivator for AAT genes via Stp1/2p activators and that lowering intracellular tryptophan by CYC8 deletion causes slow growth.

The brominated flame retardants TBP-AE and TBP-DBPE antagonize the chicken androgen receptor and act as potential endocrine disrupters in chicken LMH cells

Increased exposure of birds to endocrine disrupting compounds has resulted in developmental and reproductive dysfunctions. We have recently identified the flame retardants, allyl-2,4,6-tribromophenyl ether (TBP-AE), 2-3-dibromopropyl-2,4,6-tribromophenyl ether (TBP-DBPE) and the TBP-DBPE metabolite 2-bromoallyl-2,4,6-tribromophenyl ether (TBP-BAE) as antagonists to both the human androgen receptor (AR) and the zebrafish AR. In the present study, we aimed at determining whether these compounds also interact with the chicken AR. In silico modeling studies showed that TBP-AE, TBP-BAE and TBP-DBPE were able to dock into to the chicken AR ligand-binding pocket. In vitro transfection assays revealed that all three brominated compounds acted as chicken AR antagonists, inhibiting testosterone induced AR activation. In addition, qRT-PCR studies confirmed that they act as AR antagonists and demonstrated that they also alter gene expression patterns of apoptotic, anti-apoptotic, drug metabolizing and amino acid transporter genes. These studies, using chicken LMH cells, suggest that TBP-AE, TBP-BAE and TBP-DBPE are potential endocrine disrupters in chicken.

Abstract 1185: Regulation of neutral amino acid transporters gene expression profile in non-small cell lung cancer

Abstract INTRODUCTION: In a recent study, we showed that SLC1A5, a key glutamine (Gln) transporter, regulates tumor growth and survival in NSCLC. Here we sought to characterize the expression of other Gln transporters in lung cancer and to test whether the inhibition of SLC1A5 activity induced a change in expression of other amino acid transporters in NSCLC. METHODS: Neutral amino acid transporters reported in the cancer literature were evaluated in publically available lung cancer gene expression datasets. Among those, SLC38A1, SLC1A4, SLC1A5, SLC7A5, SLC7A11, SLC7A8 were selected for the in vitro validation. The expression of these genes was evaluated in two lung squamous carcinoma cell lines (HCC15, H226) and one adenocarcinoma cell line (A549) by RT-PCR and then quantified by qPCR, using SYBR® Green Supermix protocol. The expression of the same genes was assessed in response to transient SLC1A5 knockdown in HCC15 and A549 cell lines by qPCR. RESULTS: Amino acid transporters SLC38A1, SLC1A5, SLC7A5 and SLC7A11 are expressed in NSCLCs, a finding that was confirmed in the three cell lines studied. SLC1A4 was found to have the lowest expression and SLC7A8 no expression in any of cell lines. In response to SLC1A5 transient knockdown in A549, SLC38A1, SLC1A4 and SLC7A5 were significantly up-regulated (p&amp;lt;0.02); in contrast, in HCC15, SLC38A1, SLC1A4 and SLC7A5 were significantly down-regulated (p&amp;lt;0.02) and SLC7A11 was up-regulated (p&amp;lt;0.13). CONCLUSION: Our results show that SLC38A1, SLC1A5, SLC7A5 and SLC7A11 have the highest level of gene expression in NSCLC. We identified a possible compensatory mechanism to satisfy Gln transport and cellular function upon SLC1A5 silencing involving different transporters in a cell-specific manner. Compensatory mechanisms in response to SLC1A5 knockdown deserve further investigation as it may have implications in strategies aiming at inhibiting its activity. Further expression, localization and functional studies will follow to better understand changes occurring in lung cancer progression. Key words: Non-small cell lung cancer, SLC1A5, expression, amino acid, transporters Citation Format: Pierre P. Massion, Maria Senosain, Mohamed Hassanein, Xiangming Ji, Jun Qian, Megan hoeksema. Regulation of neutral amino acid transporters gene expression profile in non-small cell lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1185. doi:10.1158/1538-7445.AM2015-1185