Amino Acid Transport System Research Articles

Renaissance of (18)F-FDG positron emission tomography in the imaging of pheochromocytoma/paraganglioma.

Renaissance of (18)F-FDG positron emission tomography in the imaging of pheochromocytoma/paraganglioma.

Uptake of Betaine into Mouse Cumulus-Oocyte Complexes via the SLC7A6 Isoform of y+L Transporter1

Betaine (N,N,N-trimethylglycine) has previously been shown to function in cell volume homeostasis in early mouse embryos and also to be a key donor to the methyl pool in the blastocyst. A betaine transporter (SLC6A20A or SIT1) has been shown to be activated after fertilization, but there is no saturable betaine uptake in mouse oocytes or eggs. Unexpectedly, the same high level of betaine is present in mature metaphase II (MII) eggs as is found in one-cell embryos despite the lack of transport in oocytes or eggs. Significant saturable betaine transport is, however, present in intact cumulus-oocyte complexes (COCs). This transport system has an affinity for betaine of ∼227 μM. The inhibition profile indicates that betaine transport by COCs could be completely blocked by methionine, proline, leucine, lysine, and arginine, and transport is dependent on Na(+) but not Cl(-). This is consistent with transport by a y+L-type amino acid transport system. Both transcripts and protein of one y+L isoform, SLC7A6 (y+LAT2), are present in COCs, with little or no expression in isolated germinal vesicle (GV)-stage oocytes, MII eggs, or one-cell embryos. Betaine accumulated by COCs is transferred into the enclosed GV oocyte, which requires functional gap junctions. Thus, at least a portion of the endogenous betaine in MII eggs could be derived from transport into cumulus cells and subsequent transfer into the enclosed oocyte before gap junction closure during meiotic maturation. The oocyte-derived betaine then could be regulated and supplemented by the SIT1 transporter that arises in the embryo after fertilization.

System A amino acid transporter SNAT2 shows subtype-specific affinity for betaine and hyperosmotic inducibility in placental trophoblasts

Betaine uptake is induced by hypertonic stress in a placental trophoblast cell line, and involvement of amino acid transport system A was proposed. Here, we aimed to identify the subtype(s) of system A that mediates hypertonicity-induced betaine uptake. Measurement of [14C]betaine uptake by HEK293 cells transiently transfected with human or rat sodium-coupled neutral amino acid transporters (SNATs), SNAT1, SNAT2 and SNAT4 revealed that only human and rat SNAT2 have betaine uptake activity. The Michaelis constants (Km) of betaine uptake by human and rat SNAT2 were estimated to be 5.3mM and 4.6mM, respectively. Betaine exclusively inhibited the uptake activity of SNAT2 among the rat system A subtypes. We found that rat SNAT1, SNAT2 and SNAT4 were expressed at the mRNA level under isotonic conditions, while expression of SNAT2 and SNAT4 was induced by hypertonicity in TR-TBT 18d-1 cells. Western blot analyses revealed that SNAT2 expression on plasma membrane of TR-TBT 18d-1 cells was more potently induced by hypertonicity than that in total cell lysate. Immunocytochemistry confirmed the induction of SNAT2 expression in TR-TBT 18d-1 cells exposed to hypertonic conditions and indicated that SNAT2 was localized on the plasma membrane in these cells. Our results indicate that SNAT2 transports betaine, and that tonicity-sensitive SNAT2 expression may be involved in regulation of betaine concentration in placental trophoblasts.

Automated synthesis of 18F-fluoropropoxytryptophan for amino acid transporter system imaging.

Objective. This study was to develop a cGMP grade of [18F]fluoropropoxytryptophan (18F-FTP) to assess tryptophan transporters using an automated synthesizer. Methods. Tosylpropoxytryptophan (Ts-TP) was reacted with K18F/kryptofix complex. After column purification, solvent evaporation, and hydrolysis, the identity and purity of the product were validated by radio-TLC (1M-ammonium acetate : methanol = 4 : 1) and HPLC (C-18 column, methanol : water = 7 : 3) analyses. In vitro cellular uptake of 18F-FTP and 18F-FDG was performed in human prostate cancer cells. PET imaging studies were performed with 18F-FTP and 18F-FDG in prostate and small cell lung tumor-bearing mice (3.7 MBq/mouse, iv). Results. Radio-TLC and HPLC analyses of 18F-FTP showed that the Rf and Rt values were 0.9 and 9 min, respectively. Radiochemical purity was >99%. The radiochemical yield was 37.7% (EOS 90 min, decay corrected). Cellular uptake of 18F-FTP and 18F-FDG showed enhanced uptake as a function of incubation time. PET imaging studies showed that 18F-FTP had less tumor uptake than 18F-FDG in prostate cancer model. However, 18F-FTP had more uptake than 18F-FDG in small cell lung cancer model. Conclusion. 18F-FTP could be synthesized with high radiochemical yield. Assessment of upregulated transporters activity by 18F-FTP may provide potential applications in differential diagnosis and prediction of early treatment response.

Intracellular boron accumulation in CHO-K1 cells using amino acid transport control

BPA used in BNCT has a similar structure to some essential amino acids and is transported into tumor cells by amino acid transport systems. Previous study groups have tried various techniques of loading BPA to increase intracellular boron concentration. CHO-K1 cells demonstrate system L (LAT1) activity and are suitable for specifying the transport system of a neutral amino acid. In this study, we examined the intracellular accumulation of boron in CHO-K1 cells by amino acid transport control, which involves co-loading with L-type amino acid esters. Intracellular boron accumulation in CHO-K1 cells showed the greatest increased upon co-loading 1.0mM BPA, with 1.0mM l-Tyr-O-Et and incubating for 60min. This increase is caused by activation of a system L amino acid exchanger between BPA and l-Tyr. The amino acid esters are metabolized to amino acids by intracellular hydrolytic enzymes that increase the concentrations of intracellular amino acids and stimulate exchange transportation. We expect that this amino acid transport control will be useful for enhancing intracellular boron accumulation.

ChemInform Abstract: Fluorine‐18 Labeled Amino Acids for Oncologic Imaging with Positron Emission Tomography

AbstractReview: 155 refs.

RBE4 cells are highly resistant to paraquat‐induced cytotoxicity: studies on uptake and efflux mechanisms

Paraquat (PQ) is a widely used, highly toxic and non-selective contact herbicide, which has been associated with central neurotoxic effects, namely the development of Parkinson's disease, but whose effects to the blood-brain barrier (BBB) itself have rarely been studied. This work studied the mechanisms of PQ uptake and efflux in a rat's BBB cell model, the RBE4 cells. PQ is believed to enter cells using the basic or neutral amino acid or polyamine transport systems or through the choline-uptake system. In contrast, PQ efflux from cells is reported to be mediated by P-glycoprotein. Therefore, we evaluated PQ-induced cytotoxicity and the effect of some substrates/blockers of these transporters (such as arginine, L-valine, putrescine, hemicholinium-3 and GF120918) on such cytotoxicity. RBE4 cells were shown to be extremely resistant to PQ after 24 h of exposure; even at concentrations as high as 50 mM approximately 45% of the cells remained viable. Prolonging exposure until 48 h elicited significant cytotoxicity only for PQ concentrations above 5 mM. Although hemicholinium-3, a choline-uptake system inhibitor, significantly protected cells against PQ-induced toxicity, none of the effects were observed for arginine, L-valine or putrescine. Meanwhile, inhibiting the efflux pump P-glycoprotein using GF120918 significantly enhanced PQ-induced cytotoxicity. In conclusion, PQ used the choline-uptake system, instead of the transporters for the basic or neutral amino acids or for the polyamines, to enter RBE4 cells. P-glycoprotein extrudes PQ back to the extracellular medium. However, this efflux mechanism only partially explains the observed RBE4 resistance to PQ.

A new treatment for human malignant melanoma targeting L-type amino acid transporter 1 (LAT1): A pilot study in a canine model

L-type amino acid transporter 1 (LAT1), an isoform of amino acid transport system L, transports branched or aromatic amino acids essential for fundamental cellular activities such as cellular growth, proliferation and maintenance. This amino acid transporter recently has received attention because of its preferential and up-regulated expression in a variety of human tumors in contrast to its limited distribution and low-level expression in normal tissues. In this study, we explored the feasibility of using LAT1 inhibitor as a new therapeutic agent for human malignant melanomas (MM) using canine spontaneous MM as a model for human MM. A comparative study of LAT expression was performed in 48 normal tissues, 25MM tissues and five cell lines established from MM. The study observed LAT1 mRNA levels from MM tissues and cell lines that were significantly (P<0.01) higher than in normal tissues. Additionally, MM with distant metastasis showed a higher expression than those without distant metastasis. Functional analysis of LAT1 was performed on one of the five cell lines, CMeC-1. [3H]l-Leucine uptake and cellular growth activities in CMeC-1 were inhibited in a dose-dependent manner by selective LAT1 inhibitors (2-amino-2-norbornane-carboxylic acid, BCH and melphalan, LPM). Inhibitory growth activities of various conventional anti-cancer drugs, including carboplatin, cyclophosphamide, dacarbazine, doxorubicin, mitoxantrone, nimustine, vinblastine and vincristine, were significantly (P<0.05) enhanced by combination use with BCH or LPM. These findings suggest that LAT1 could be a new therapeutic target for MM.

SNAT2-specific transport function of betaine among amino acid transport system A subtypes

s / Placenta 34 (2013) A1–A99 A46 P1.102. SNAT2-SPECIFIC TRANSPORT FUNCTION OF BETAINE AMONG AMINO ACID TRANSPORT SYSTEM A SUBTYPES Hiromi Eguchi, Tomohiro Nishimura, Mariko Usuda, Sayaka Suda, Masatoshi Tomi, Emi Nakashima Keio University, Minato-ku, Tokyo, Japan Objectives: Betaineworks as an osmolyte and amethyl donor and is thereby thought toprotect fetal placenta againstosmotic stress andhypomethylation. We have observed the osmosensitive uptake of betaine by rat placental trophoblast cells ismediatedbysystemAsodium-coupledneutral aminoacid transporter (SNAT) 2 (1). SystemA consists of at least three subtypes, namely SNAT1, SNAT2, and SNAT4. The purposeof the present study is to evaluate the affinity of betaine for system A subtypes in human and rat. Methods: The uptake of [14C]betaine, [3H]glycine, and [14C]a-methylaminoisobutyric acid (MeAIB) was measured in human and rat SNAT-overexpressingHEK293cells and comparedwith those inmock-transfected cells. Results: The initial uptake rate of [14C]betaine in human and rat SNAT2tranfected cells was significantly greater than that in mock-transfected cells, while SNAT1and SNAT4-mediated uptakes of [14C]betaine were not observed. On the other hand, the uptakes of [3H]glycine and/or [14C]MeAIB, typical system A substrates, were observed in SNAT1-, SNAT2-, and SNAT4transfected cells. The affinity of [14C]betaine to SNAT2 was low with Km values of approximately 5 mM in both human and rat. Conclusion: The present study clearly showed that SNAT2 only accepts betaine as a substrate, although differences in the substrate profiles among system A transporters have been believed to be subtle. This finding will help us to understand the subtype specific roles of three system A transporter subtypes expressed in placental syncytiotrophoblasts. Reference: (1)Nishimura et al., Placenta 31, 1003-1009 (2010). http://dx.doi.org/10.1016/j.placenta.2013.06.139 P1.103. CYTOPROTECTIVE PROPERTIES OF HYPOTAURINE BY SCAVENGING HYDROXYL RADICALS IN PLACENTAL TROPHOBLAST CELLS Hikari Araki , Duereh Mariam , Tomohiro Nishimura , Yuki Sugita , Kei Higuchi , Masatoshi Tomi , Emi Nakashima 1 Keio University, Minato-ku,Tokyo, Japan; 2 Teikyo University, Itabashi-

L-type amino acid transporter 1 (LAT1): A new therapeutic target for canine mammary gland tumour

L-type amino acid transporter 1 (LAT1), an isoform of amino acid transport system L, transports branched or aromatic amino acids essential for fundamental cellular activities, such as cellular growth, proliferation and maintenance. LAT1 has recently received attention because of its preferential and upregulated expression in a variety of human tumours which is in contrast to its limited distribution and low-level expression in normal tissues. In this study, the feasibility of using an LAT1 inhibitor as a new therapeutic agent was explored for mammary gland tumours (MGT). [3H]l-leucine uptake by CHM, a cell line established from MGT, and effects on cell growth were analysed in the presence or absence of two LAT1 inhibitors, namely, BCH (2-amino-2-norbornane-carboxylic acids) or melphalan (LPM).[3H]l-leucine uptake and cellular growth activities in CHM were inhibited in a dose-dependent manner by both LAT1 inhibitors. The inhibitory growth activities of various conventional anti-cancer drugs used for MGT treatment, including carboplatin, cyclophosphamide, doxorubicin, mitoxantrone, vinblastine and vincristine, were significantly enhanced by combining use with BCH or LPM. The findings suggest that LAT1 could be a new therapeutic target for canine MGT.

EFFECTS OF DIFFERENT NITROGEN SOURCES ON GROWTH AND LIPID ACCUMULATION OF A HETEROTROPHIC MICROALGAE-CHORELLA VULGARIS

Chlorella strains have great potential as a resource for production of biofuels due to their relatively fast growth rate and easy cultivation.Heterotrophic cultivation of Chlorella has been developed due to higher biomass concentration and accumulation of much higher lipid content than classical photoautotrophic culture.Instead of using glucose,using low cost materials,such as starch or cellulose-hydrolyzed solution,is a good strategy to reduce the cost of culture medium and the cost of microalgae-based biofuels from heterotrophic fermentation of Chlorella.Potato starch is relatively cheap material in comparison with cornstarch or cassava starch,hence it could be an ideal carbon source for cultivating Chlorella.In a number of oil-producing microalgal species,Chlorella has the capacity of accumulating much higher proportion of fatty acids by nitrogen starvation.The objective of this study was to investigate the effects of various nitrogen resources on cell growth and lipid accumulation in heterotrophic Chlorella vulgaris using potato starch hydrolysate(PSH) as the sole carbon source.In order to illustrate the influence of various nitrogen sources on biomass and lipid content in C.vulgaris,the effects of these nitrogen source including inorganic and organic nitrogen source such as alanine or tyrosine on cell growth and lipid accumulation of C.vulgaris were examined.As the sole carbon source PSH was added to SE medium for algal cultivation prior to addition of all nitrogen sources.NaNO3 and alanine were added to yield starting concentrations of 1.5 mmol /L,3.0 mmol/L and 6.0 mmol/L,respectively.Tyrosine was added to yield starting concentrations of 0.75 mmol/L,1.5 mmol /L and 3.0 mmol/L,respectively.Continuous cultivation of C.vulgaris was carried out under dark condition for 10 days.The cell number,special growth rate,cellular chlorophyll content,neutral lipid content and total lipid content of C.vulgaris under present cultural condition were determined throughout the entire experimental period.The results showed that:(1) Higher chlorophyll content and biomass production were observed with higher nitrate-nitrogen concentration,although low concentrations of nitrate-nitrogen can achieve high lipid accumulation in C.vulgaris.The highest biomass productivity was obtained at 1.5 mmol/L sodium nitrate,the biomass of 2.65 g/L,lipid concentration of 51.21% and the total lipid production of 1.36 g/L were achieved.(2) When alanine was used as the sole nitrogen source,it was also observed that the cell growth and total chlorophyll content increased but the lipid content reduced.(3) A low level of biomass production was observed while adding tyrosine as a nitrogen source;however,cell enlargement and high chlorophyll content were achieved under this condition,resulting in high lipid content of 38.78%—47.02%.Based on above results,it was concluded that the unicellular alga C.vulgaris was able to respond to nitrogen nutrient changes by inducing specific amino acid transport systems,and it seemed to have developed more specific mechanisms of amino-acid uptake and lipid production along with subtle regulatory phenomena such as induction by nitrogen starvation.Furthermore,tyrosine,as the part of the third uptake system induced by glucose transport system,can also enhance continually cellular lipid accumulation in C.vulgaris.

Activation of SNAT1/SLC38A1 in human breast cancer: correlation with p-Akt overexpression

BackgroundSNAT1 is a subtype of the amino acid transport system A that has been implicated to play a potential role in cancer development and progression, yet its role in breast cancer remains unclear. In present study, we detected SNAT1 expression in breast cancers and explored its underlying mechanism in promoting breast carcinogenesis.MethodsRT-PCR and Western blotting were performed to analyze the transcription and protein levels of SNAT1 in breast cancer cell lines and fresh tissues. Tissue microarray blocks containing breast cancer specimens obtained from 210 patients were constructed. Expression of SNAT1 in these specimens was analyzed using immunohistochemical studies. SNAT1 was down-regulated by SNAT1-shRNA in breast cancer cells and the functional significance was measured.ResultsSNAT1 was up-regulated in breast cancer cell lines and breast cancer tissues. Overexpression of SNAT1 was observed in 127 cases (60.5%). Expression of SNAT1 was significantly associated with tumor size, nodal metastasis, advanced disease stage, Ki-67, and ER status. Suppression of endogenous SNAT1 leads to cell growth inhibition, cell cycle arrest, and apoptosis of 4T1 cells and lowered the phosphorylation level of Akt. SNAT1 expression correlated significantly with p-Akt expression in human breast cancer samples.ConclusionsThe cross-talk between Akt signaling and SNAT1 might play a critical role in the development and progression of breast cancer, providing an important molecular basis for novel diagnostic markers and new attractive targets in the treatment of breast cancer patients.

Radiosynthesis and biological evaluation of 5-(3-[18F]Fluoropropyloxy)-L-tryptophan for tumor PET imaging

[(18)F]FDG PET has difficulty distinguishing tumor from inflammation in the clinic because of the same high uptake in nonmalignant and inflammatory tissue. In contrast, amino acid tracers do not accumulate in inflamed tissues and thus provide an excellent opportunity for their use in clinical cancer imaging. In this study, we developed a new amino acid tracer 5-(3-[(18)F]Fluoropropyloxy)-L-tryptophan ([(18)F]-L-FPTP) by two-step reactions and performed its biologic evaluation. [(18)F]-L-FPTP was prepared by [(18)F]fluoropropylation of 5-hydroxy-L-tryptophan disodium salt and purification on C18 cartridges. The biodistribution of [(18)F]-L-FPTP was determined in normal mice and the incorporation of [(18)F]-L-FPTP into tissue proteins was investigated. In vitro competitive inhibition experiments were performed with Hepa1-6 hepatoma cell lines. [(18)F]-L-FPTP PET imaging was performed on tumor-bearing and inflammation mice and compared with [(18)F]-L-FEHTP PET. The overall uncorrected radiochemical yield of [(18)F]-L-FPTP was 21.1 ± 4.4% with a synthesis time of 60 min, the radiochemical purity was more than 99%. Biodistribution studies demonstrate high uptake of [(18)F]-L-FPTP in liver, kidney, pancreas, and blood at the early phase, and fast clearance in most tissues over the whole observed time. The uptake studies in Hepa1-6 cells suggest that [(18)F]-L-FPTP is transported by the amino acid transport system B(0,+), LAT2 and ASC. [(18)F]-L-FPTP displays good stability and is not incorporated into proteins in vitro. PET imaging shows that [(18)F]-L-FPTP can be a better potential PET tracer for differentiating tumor from inflammation than [(18)F]FDG and 5-(3-[(18)F]fluoroethyloxy)-L-tryptophan ([(18)F]-L-FEHTP), with high [(18)F]-L-FPTP uptake ratio (2.53) of tumor to inflammation at 60 min postinjection. Using [(18)F]fluoropropyl derivatives as intermediates, the new tracer [(18)F]-L-FPTP was achieved with good yield and radiochemical purity, and the biological evaluation results of [(18)F]-L-FPTP showed that it was a hopeful tracer for PET tumor imaging.

Genetic engineering of Escherichia coli to enhance production of l-tryptophan

Reducing the accumulation of acetate in Escherichia coli cultures can decrease carbon efflux as by-products and reduce acetate toxicity, and therefore enable high cell density cultivation. The concentration of intracellular amino acids can be decreased by genetic modifications of the corresponding amino acid transport systems. This can increase the levels of amino acids in the fermentation broth by decreasing the feedback inhibition on the corresponding biosynthetic pathways. Here, the effects of genetic manipulation of phosphate acetyltransferase (pta), high affinity tryptophan transporter (mtr) and aromatic amino acid exporter (yddG) on L-tryptophan production were investigated. The pta mutants accumulated less acetate and showed higher capacity for producing L-tryptophan as compared with the parental strain. The strains lacking mtr, or overexpressed yddG, or with the both mtr knockout and yddG overexpression, accumulated lower concentrations of intracellular tryptophan but higher production of extracellular L-tryptophan. In the L-tryptohan fed-batch fermentation of an E. coli derived from TRTH0709/pMEL03 having deletion of pta-mtr and overexpression of yddG in a 30-L fermentor, the maximum concentration of L-tryptophan (48.68g/L) was obtained, which represented a 15.96% increase as compared with the parental strain. Acetate accumulated to a concentration of 0.95g/L. The intracellular concentration of L-tryptophan was low, and the glucose conversion rate reached a high level of 21.87%, which was increased by 15.53% as compared with the parent strain.

Different Influence of Intracellular Glutamine Depletion on Glutamate Uptake Mediated by Amino Acid Transport Systems; EAATs and X-c, in Placental (BeWo) Cells

Different Influence of Intracellular Glutamine Depletion on Glutamate Uptake Mediated by Amino Acid Transport Systems; EAATs and X-c, in Placental (BeWo) Cells

Fluorine-18 Labeled Amino Acids for Oncologic Imaging with Positron Emission Tomography

(18)F-labeled amino acids are an important class of imaging agents for positron emission tomography (PET) that target the increased rates of amino acid transport by many tumor cells. This class of tracers is structurally diverse, and the biological and imaging properties of a given (18)F-labeled amino acid depends largely upon its mechanism of transport. The system L amino acid transport system has been a major focus of tracer development in this field, but more recently (18)F-labeled amino acids have been developed for other transporters including system A, glutamine, glutamate and cationic amino acid transport systems. Radiolabeled amino acids are best established for brain tumor imaging, but there are emerging applications in other types of cancer such as neuroendocrine tumors and prostate cancer. This review provides an overview of (18)F-labeled amino acids for oncologic imaging in terms of design considerations, radiosynthetic methods, and key clinical applications.

Renal amino acid transport systems and essential hypertension

Several clinical and animal studies suggest that "blood pressure goes with the kidney," that is, a normotensive recipient of a kidney genetically programmed for hypertension will develop hypertension. Intrarenal dopamine plays an important role in the pathogenesis of hypertension by regulating epithelial sodium transport. The candidate transport systems for L-DOPA, the source for dopamine, include the sodium-dependent systems B(0), B(0,+), and y(+)L, and the sodium-independent systems L (LAT1 and LAT2) and b(0,+). Renal LAT2 is overexpressed in the prehypertensive spontaneously hypertensive rat (SHR), which might contribute to enhanced L-DOPA uptake in the proximal tubule and increased dopamine production, as an attempt to overcome the defect in D1 receptor function. On the other hand, it has been recently reported that impaired arginine transport contributes to low renal nitric oxide bioavailability observed in the SHR renal medulla. Here we review the importance of renal amino acid transporters in the kidney and highlight pathophysiological changes in the expression and regulation of these transporters in essential hypertension. The study of the regulation of renal amino acid transporters may help to define the underlying mechanisms predisposing individuals to an increased risk for development of hypertension.

Amino acid absorption by lobster (Homarus americanus) intestine using dual‐label radioisotopic tracer techniques

Amino acids are transported from the lumen of an intestine to the blood across epithelial mucosal and serosal membranes by specific amino acid carrier proteins found within the membrane as well as a transport process involving bis complex formation between the amino acids and metal ions resulting in a dipeptide‐like substrate. In this study the absorption of 3H‐L‐leucine and 35S‐L‐methionine across lobster intestine were simultaneously followed in the presence and absence of Zn metal ions using dual‐label radioisotopic tracer techniques allowing the analysis of independent amino acid fluxes in single experiments. The radiolabelled amino acids were perfused through in vitro lobster intestines and rates of isotope appearance were periodically monitored to estimate trans‐intestinal fluxes of each amino acid. In the presence of luminal Zn, both 3H‐L‐leucine and 35S‐L‐methionine fluxes across the tissue were significantly stimulated. There was no significant evidence that the amino acids competed with each other for carrier transport in the absence of Zn, suggesting separate amino acid transport systems for these substrates. On‐going experiments will determine if bis‐complexes of these amino acids, involving Zn, enhance amino acid flux rates to the serosa, and if there are competitive interactions between the complexes. Agriculture and Food Research Initiative Competitive USDA Grant no. 2010–65206‐20617.

Dual effect of nickel on L-arginine/nitric oxide system in RAW 264.7 macrophages

The immunogenic mechanisms of the potent contact allergen nickel are not completely clear. Nitric oxide (NO) serves as a fundamental signalling and effector molecule in the immune system, but little is known about its possible role in immune reactions elicited by nickel. We investigated the effects of nickel on the L-arginine/inducible NO synthase (iNOS) system in a murine macrophage cell line, RAW 264.7. Both LPS-stimulated and non-stimulated RAW 264.7 cells were incubated in the presence of 0–100μM nickel sulphate for 24h. Subsequently, NO production, iNOS protein expression, L-arginine uptake and gene expression of iNOS and cationic amino acid transporter systems (CAT) were measured. We found that 100μM NiSO4 increased LPS-induced nitrite production as well as the formation of [3H]-L-citrulline from [3H]-L-arginine in the RAW 264.7 cells. Correspondingly, the expression of iNOS gene and protein was also remarkably enhanced. Nevertheless, nickel had an inhibitory effect on L-arginine transport which disappeared gradually upon LPS-stimulation in parallel with an increase in NO output. LPS was found to significantly amplify CAT-3 as well as CAT-2 mRNA expression, mirroring the increase in L-arginine transport. In the range of 1–10μM, NiSO4 did not have any additional effect on CAT mRNA expression, but at 100μM it was able to enhance CAT-1 and CAT-3 mRNA expression upon LPS stimulation.Our data indicate that nickel interferes with macrophages' L-arginine/NOS system on multiple levels. Considering the potent biological effects of NO, these influences may contribute to nickel toxicity.

Proper Fatty Acid Composition Rather than an Ionizable Lipid Amine Is Required for Full Transport Function of Lactose Permease from Escherichia coli

Energy-dependent uphill transport but not energy-independent downhill transport by lactose permease (LacY) is impaired when expressed in Escherichia coli cells or reconstituted in liposomes lacking phosphatidylethanolamine (PE) and containing only anionic phospholipids. The absence of PE results in inversion of the N-terminal half and misfolding of periplasmic domain P7, which are required for uphill transport of substrates. Replacement of PE in vitro by lipids with no net charge (phosphatidylcholine (PC), monoglucosyl diacylglycerol (GlcDAG), or diglucosyl diacylglycerol (GlcGlcDAG)) supported wild type transmembrane topology of the N-terminal half of LacY. The restoration of uphill transport in vitro was dependent on LacY native topology and proper folding of P7. Support of uphill transport by net neutral lipids in vitro (PE > PC ≫ GlcDAG ≠ GlcGlcDAG provided that PE or PC contained one saturated fatty acid) paralleled the results observed previously in vivo (PE = PC > GlcDAG ≠ GlcGlcDAG). Therefore, a free amino group is not required for uphill transport as previously concluded based on the lack of in vitro uphill transport when fully unsaturated PC replaced E. coli-derived PE. A close correlation was observed in vivo and in vitro between the ability of LacY to carry out uphill transport, the native conformation of P7, and the lipid headgroup and fatty acid composition. Therefore, the headgroup and the fatty acid composition of lipids are important for defining LacY topological organization and catalytically important structural features, further illustrating the direct role of lipids, independent of other cellular factors, in defining membrane protein structure/function.