Proline Uptake Research Articles

Leptin regulates sugar and amino acids transport in the human intestinal cell line Caco‐2

Studies in rodents have shown that leptin controls sugars and glutamine entry in the enterocytes by regulating membrane transporters. Here, we have examined the effect of leptin on sugar and amino acids absorption in the human model of intestinal cells Caco-2 and investigated the transporters involved. Substrate uptake experiments were performed in Caco-2 cells, grown on plates, in the presence and the absence of leptin, and the expression of the different transporters in brush border membrane vesicles was analysed by Western blot. Leptin inhibited 0.1 mm α-methyl-D-glucoside uptake after 5 or 30 min treatment and decreased SGLT1 protein abundance in the apical membrane. Uptake of 20 μm glutamine and 0.1 mm phenylalanine was also inhibited by leptin, indicating sensitivity to the hormone of the Na(+) -dependent neutral amino acid transporters ASCT2 and B(0) AT1. This inhibition was accompanied by a reduction in the transporters expression at the brush border membrane. Leptin also inhibited 1 mm proline and β-alanine uptake in Na(+) medium at pH 6, conditions for optimal activity of the H(+) -dependent neutral amino acid transporter PAT1. In this case, abundance of PAT1 in the brush border membrane after leptin treatment was not modified. Interestingly, leptin inhibitory effect on β-alanine uptake was reversed by the PKA inhibitor H-89 suggesting involvement of PKA pathway in leptin's regulation of PAT1 activity. These data show in human intestinal cells that leptin can rapidly control the activity of physiologically relevant transporters for rich-energy molecules, that is, D-glucose (SGLT1) and amino acids (ASCT2, B(0) AT1 and PAT1).

Effect of inoculation with plant growth-promoting bacteria (PGPB) on amelioration of saline stress in maize (Zea mays)

Our objective was to evaluate the role of Azotobacter strains to protect maize plants against salt damage. Four candidate Azotobacter strains were evaluated, and the two most tolerant to salinity (C5 and C9) were selected for further studies. They were phylogenetically related to Azotobacter chroococcum based on their 16S rDNA sequences. Strains were inoculated on maize roots growing in sterilized soil under different salinity conditions (0, 2.93 and 5.85g NaCl/kg soil). After 4 weeks plant biomass (length and weight), ion uptake (Na+, K+, Ca2+, Mg2+), chlorophyll content, and accumulation of proline and polyphenols were evaluated. Strains C5 and C9 fixed nitrogen and solubilized phosphate regardless of NaCl concentration in most cases, while auxins were synthesized by C5 only under conditions of salinity. In pot experiments, plant growth was promoted by bacterial inoculation only at 2.93 and 5.85g NaCl/kg soil (P<0.05). Bacteria improved Na+ exclusion and K+ uptake in maize, thereby increasing their K+/Na+ ratio. Content of polyphenol and chlorophyll was enhanced by inoculation with strains C5 and C9. The concentration of proline in leaves was increased by salinity, but was decreased when C5 and C9 were used as inoculants. The present observations showed that strains C5 and C9 partially alleviated the saline stress in maize, likely through the integration of several mechanisms that improve the plant response. Hence, the use of nitrogen-fixing plant growth-promoting bacteria may represent an important biotechnological approach to decrease the impact of salinity in crops.

Isolation and Preliminary Characterization of Amino Acid Substitution Mutations That Increase the Activity of the Osmoregulated ProP Protein ofSalmonella EntericaSerovar Typhimurium

In Enterobacteriaceae, the ProP protein, which takes up proline and glycine betaine, is subject to a post-translational control mechanism that increases its activity at high osmolarity. In order to investigate the osmoregulatory mechanism of the Salmonella enterica ProP, we devised a positive selection for mutations that conferred increased activity on this protein at low osmolarity. The selection involved the isolation of mutations in a proline auxotroph that resulted in increased accumulation of proline via the ProP system in the presence of glycine betaine, which is a competitive inhibitor of proline uptake by this permease. This selection was performed by first-year undergraduates in two semesters of a research-based laboratory course. The students generated sixteen mutations resulting in six different single amino acids substitutions. They determined the effects of the mutations on the growth rates of the cells in media of high and low osmolarity in the presence of low concentrations of proline or glycine betaine. Furthermore, they identified the mutations by DNA sequencing and displayed the mutated amino acids on a putative three-dimensional structure of the protein. This analysis suggested that all six amino acid substitutions are residues in trans-membrane helices that have been proposed to contribute to the formation of the transport pore, and, thus, may affect the substrate binding site of the protein.

PrcR, a PucR-type transcriptional activator, is essential for proline utilization and mediates proline-responsive expression of the proline utilization operon putBCP in Bacillus subtilis

The soil bacterium Bacillus subtilis can utilize exogenous proline as a sole nitrogen or carbon source. The proline-inducible putBCP (formerly ycgMNO) operon encodes proteins responsible for proline uptake and two-step oxidation of proline to glutamate. We now report that a gene (formerly ycgP, now designated prcR) located downstream of the putBCP operon is essential for B. subtilis cells to utilize proline as a sole nitrogen or carbon source. Disruption of the prcR gene also abolished proline induction of putB transcription. prcR expression is not subject to autoregulation and proline induction. The PrcR protein shows no significant amino acid sequence similarity to the known transcriptional activators for proline utilization genes of other bacteria, but it does show partial amino acid sequence similarity to the transcriptional regulator PucR for the purine degradation genes of B. subtilis. PrcR orthologues of unknown function are present in some other Bacillus species. Primer-extension analysis suggests that both putB and prcR are transcribed by a σ(A)-dependent promoter. Deletion and mutation analysis revealed that an inverted repeat (5'-TTGTGG-N5-CCACAA-3') centred at position -76 relative to the transcriptional initiation site of putB is essential for putB expression. Electrophoretic mobility shift assays showed that the purified His-tagged PrcR was capable of binding specifically to this inverted repeat. Altogether, these results suggest that PrcR is a PucR-type transcriptional activator that mediates expression of the B. subtilis putBCP operon in response to proline availability.

Three-dimensional quantitative structure–activity relationship analyses of substrates of the human proton-coupled amino acid transporter 1 (hPAT1)

The proton-coupled amino acid transporter hPAT1 has recently gained much interest due to its ability to transport small drugs thereby allowing their oral administration. A three-dimensional quantitative structure–activity relationship (3D QSAR) study has been performed on its natural and synthetic substrates employing comparative molecular similarity indices analysis (CoMSIA) to investigate the structural requirements for substrates and to derive a predictive model that may be used for the design of new prodrugs. The cross-validated CoMSIA models have been derived from a training set of 40 compounds and the predictive ability of the resulting models has been evaluated against a test set of 10 compounds. Despite the relatively narrow range of binding affinities (Ki values) reliable statistical models with good predictive power have been obtained. The best CoMSIA model in terms of a proper balance of all statistical terms and the overall contribution of individual properties has been obtained by considering steric, hydrophobic, hydrogen bond donor and acceptor descriptors (qcv2=0.683, r2=0.958 and rPRED2=0.666). The 3D QSAR model provides insight in the interactions between substrates and hPAT1 on the molecular level and allows the prediction of affinity constants of new compounds. A pharmacophore model has been generated from the training set by means of the MOE (molecular operating environment) program. This model has been used as a query for virtual screening to retrieve potential new substrates from the small-molecule, ‘lead-like’ databases of MOE. The affinities of the compounds were predicted and 11 compounds were identified as possible high-affinity substrates. Two selected compounds strongly inhibited the hPAT mediated l-[3H]proline uptake into Caco-2 cells constitutively expressing the transport protein.

Evidence for a sodium-dependent proline and glycine-betaine uptake in the cyanobacterium Nostoc muscorum

The cyanobacterium Nostoc muscorum is able to utilized proline and glycine-betaine as a nitrogen source under unstressed growth conditions. This cyanobacterium when grow in modified Chu No. 10 medium (without Na+) unable to utilized proline and glycine-betaine as a nitrogen source. Spontaneously occurring mutant clones defective in Na+ transport (Na+-R) were isolated and analyzed for proline and glycine-betaine utilization. The mutant phenotype showed normal heterocyst frequency and nitrogenase activity even in the medium containing 1 mM proline or 1 mM glycine-betaine, indicates the role of Na+ for proline/glycine-betaine uptake. The Na+-R mutant showed 100% survival at pH 11 and was simultaneously able to uptake and utilize proline/glycine-betaine at higher alkaline pH. This indicates that proline and glycine-betaine uptake systems are more efficient at higher alkaline pH. Since, the hypersaline environments are rich in Na+ contents and have alkaline pH, therefore it is suggested that the origin and evolution of specific compatible solutes may not depend only on the osmoregulatory role they play, but also on the other ecological factors operating simultaneously in the organism's niche.

Expression and substrate specificity of betaine/proline transporters suggest a novel choline transport mechanism in sugar beet

Proline transporters (ProTs) originally described as highly selective transporters for proline, have been shown to also transport glycinebetaine (betaine). Here we examined and compared the transport properties of Bet/ProTs from betaine accumulating (sugar beet, Amaranthus, and Atriplex,) and non-accumulating ( Arabidopsis) plants. Using a yeast mutant deficient for uptake of proline and betaine, it was shown that all these transporters exhibited higher affinity for betaine than proline. The uptake of betaine and proline was pH-dependent and inhibited by the proton uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP). We also investigated choline transport by using a choline transport-deficient yeast mutant. Results revealed that these transporters exhibited a higher affinity for choline uptake rather than betaine. Uptake of choline by sugar beet BvBet/ProT1 was independent of the proton gradient and the inhibition by CCCP was reduced compared with that for uptake of betaine, suggesting different proton binding properties between the transport of choline and betaine. Additionally, in situ hybridization experiments revealed the localization of sugar beet BvBet/ProT1 in phloem and xylem parenchyma cells.

Mutational and transcriptional analyses of theStaphylococcus aureuslow-affinity proline transporter OpuD duringin vitrogrowth and infection of murine tissues

Staphylococcus aureus continues to be a major health problem. This species' requirement for proline and proline transport from the extracellular environment is not well understood. Here, we identify a S. aureus low-affinity proline transport gene (opuD) with homology to the OpuD protein of Bacillus subtilis. Mutation of the opuD gene caused a significant decline in proline uptake under low-affinity conditions as compared with wild type, but the opuD mutant strain showed no significant attenuation in a murine abscess model of infection. The S. aureus opuD gene was transcriptionally activated during growth in moderate osmolarity media with high levels of proline or glycine betaine independent of SigB. In murine abscesses, the opuD gene was activated at a later time point, whereas the opuD expression dropped over the course of an 18-h period within murine urinary tracts. Transcriptional regulation of opuD in S. aureus appears to be coordinated within this species when grown in moderate to high NaCl environments, but the level of extracellular proline had a marked effect on expression of this proline transport gene. The differential regulation of proline transport genes in S. aureus may be an adaptation for life in a variety of environments, including survival within the human body.

L-Proline nutrition and catabolism in Staphylococcus saprophyticus

Staphylococcus saprophyticus strains ATCC 15305, ATCC 35552, and ATCC 49907 were found to require L-proline but not L-arginine for growth in a defined culture medium. All three strains could utilize L-ornithine as a proline source and contained L-ornithine aminotransferase and Δ(1)-pyrroline-5-carboxylate reductase activities; strains ATCC 35552 and ATCC 49907 could use L-arginine as a proline source and had L-arginase activity. The proline requirement also could be met by L-prolinamide, L-proline methyl ester, and the dipeptides L-alanyl-L-proline and L-leucyl-L-proline. The bacteria exhibited L-proline degradative activity as measured by the formation of Δ(1)-pyrroline-5-carboxylate. The specific activity of proline degradation was not affected by addition of L-proline or NaCl but was highest in strain ATCC 49907 after growth in Mueller-Hinton broth. A membrane fraction from this strain had L-proline dehydrogenase activity as detected both by reaction of Δ(1)-pyrroline-5-carboxylate with 2-aminobenzaldehyde (0.79 nmol min(-1) mg(-1)) and by the proline-dependent reduction of p-iodonitrotetrazolium (20.1 nmol min(-1) mg(-1)). A soluble fraction from this strain had Δ(1)-pyrroline-5-carboxylate dehydrogenase activity (88.8 nmol min(-1) mg(-1)) as determined by the NAD(+)-dependent oxidation of DL-Δ(1)-pyrroline-5-carboxylate. Addition of L-proline to several culture media did not increase the growth rate or final yield of bacteria but did stimulate growth during osmotic stress. When grown with L: -ornithine as the proline source, S. saprophyticus was most susceptible to the proline analogues L-azetidine-2-carboylate, 3,4-dehydro-DL-proline, DL-thiazolidine-2-carboxylate, and L-thiazolidine-4-carboxylate. These results indicate that proline uptake and metabolism may be a potential target of antimicrobial therapy for this organism.

SLC36A4 (hPAT4) Is a High Affinity Amino Acid Transporter When Expressed in Xenopus laevis Oocytes

The SLC36 family of transporters consists of four genes, two of which, SLC36A1 and SLC36A2, have been demonstrated to code for human proton-coupled amino acid transporters or hPATs. Here we report the characterization of the fourth member of the family, SLC36A4 or hPAT4, which when expressed in Xenopus laevis oocytes also encodes a plasma membrane amino acid transporter, but one that is not proton-coupled and has a very high substrate affinity for the amino acids proline and tryptophan. hPAT4 in Xenopus oocytes mediated sodium-independent, electroneutral uptake of [(3)H]proline, with the highest rate of uptake when the uptake medium pH was 7.4 and an affinity of 3.13 μM. Tryptophan was also an excellently transported substrate with a similarly high affinity (1.72 μM). Other amino acids that inhibited [(3)H]proline were isoleucine (K(i) 0.23 mM), glutamine (0.43 mM), methionine (0.44 mM), and alanine (1.48 mM), and with lower affinity, glycine, threonine, and cysteine (K(i) >5 mM for all). Of the amino acids directly tested for transport, only proline, tryptophan, and alanine showed significant uptake, whereas glycine and cysteine did not. Of the non-proteogenic amino acids and drugs tested, only sarcosine produced inhibition (K(i) 1.09 mM), whereas γ-aminobutyric acid (GABA), β-alanine, L-Dopa, D-serine, and δ-aminolevulinic acid were without effect on [(3)H]proline uptake. This characterization of hPAT4 as a very high affinity/low capacity non-proton-coupled amino acid transporter raises questions about its physiological role, especially as the transport characteristics of hPAT4 are very similar to the Drosophila orthologue PATH, an amino acid "transceptor" that plays a role in nutrient sensing.

Gaboxadol has affinity for the proton-coupled amino acid transporter 1, SLC36A1 (hPAT1)—A modelling approach to determine IC50 values of the three ionic species of gaboxadol

The human proton-coupled amino acid transporter, SLC36A1 (hPAT1), is situated in the apical membrane of small intestinal epithelium. It is involved in cellular uptake of amino acids and orally administered drug substances such as δ-aminolevulinic acid, vigabatrin and gaboxadol. Gaboxadol (Gbx) is a selective extrasynaptic GABAA receptor agonist with high oral bioavailability in rat, dog and human. It is a zwitterionic compound with pKa values of 4.3 and 8.1. Dependent on the pH of the solution Gbx will be present as three individual ionic species, i.e. cationic (Gbx+), zwitterionic (Gbx+/−) and anionic (Gbx−). The aim of the present study was to elucidate the individual affinities of Gbx+, Gbx+/− and Gbx− for SLC36A1. The ability of Gbx to concentration-dependently inhibit a SLC36A1 mediated l-[3H]proline uptake was investigated in Caco-2 cell monolayers at apical pH 5.0–6.8. The IC50 values were computed using an in silico model relying on a genetic algorithm. The IC50 values of Gbx+, Gbx+/− and Gbx− were estimated to 2.6mM, 16mM and >1000mM. This indicates that the positive charge is essential for Gbx binding to SLC36A1. The negative charge is tolerated in the zwitterionic form, whereas no affinity is observed for the anionic form.

In planta function of compatible solute transporters of the AtProT family

The three proline transporters of Arabidopsis thaliana (AtProTs) transport the compatible solutes proline and glycine betaine and the stress-induced compound γ-aminobutyric acid when expressed in heterologous systems. The aim of the present study was to show transport and physiological relevance of these three AtProTs in planta. Using single, double, and triple knockout mutants and AtProT-overexpressing lines, proline content, growth on proline, transport of radiolabelled betaine, and expression of AtProT genes and enzymes of proline metabolism were analysed. AtProT2 was shown to facilitate uptake of L- and D-proline as well as [14C]glycine betaine in planta, indicating a role in the import of compatible solutes into the root. Toxic concentrations of L- and D-proline resulted in a drastic growth retardation of AtProT-overexpressing plants, demonstrating the need for a precise regulation of proline uptake and/or distribution. Furthermore evidence is provided that AtProT genes are highly expressed in tissues with elevated proline content—that is, pollen and leaf epidermis.

Amino acid derivatives are substrates or non-transported inhibitors of the amino acid transporter PAT2 (slc36a2)

The H+-coupled amino acid transporter PAT2 (SLC36A2) transports the amino acids proline, glycine, alanine and hydroxyproline. A physiological role played by PAT2 in amino acid reabsorption in the renal proximal tubule is demonstrated by mutations in SLC36A2 that lead to an iminoglycinuric phenotype (imino acid and glycine uria) in humans. A number of proline, GABA and tryptophan derivatives were examined to determine if they function either as transported substrates or non-transported inhibitors of PAT2. The compounds were investigated following heterologous expression of rat PAT2 in Xenopus laevis oocytes. PAT2 function was characterised by: radiotracer uptake and competition (cis-inhibition) studies; radiotracer efflux and trans-stimulation; and measurement of substrate-induced positive inward current by two-electrode voltage-clamp. In general, the proline derivatives appeared to be transported substrates and the relative ability to induce current flow was closely related to the inhibitory effects on PAT2-mediated l-[3H]proline uptake. In contrast, certain heterocyclic GABA derivatives (e.g. l-pipecolic acid) were translocated only slowly. Finally, the tryptophan derivatives inhibited PAT2 function but did not undergo transport. l-Proline uptake was inhibited by 5-hydroxy-l-tryptophan (IC50 1.6±0.4mM), α-methyl-d,l-tryptophan (3.5±1.5mM), l-tryptophan, 1-methyl-l-tryptophan and indole-3-propionic acid. Although neither 5-hydroxy-l-tryptophan nor α-methyl-d,l-tryptophan were able to elicit inward current in PAT2-expressing oocytes both reduced the current evoked by l-proline. 5-Hydroxy-l-tryptophan and α-methyl-d,l-tryptophan were unable to trans-stimulate l-proline efflux from PAT2-expressing oocytes, confirming that the two compounds act as non-transported blockers of PAT2. These two tryptophan derivatives should prove valuable experimental tools in future investigations of the physiological roles of PAT2.

Genistein abolishes nucleoside uptake by cardiac fibroblasts

Genistein, a soy isoflavone, is reported to exert significant beneficial action in several human disorders, which has generated immense interest in the mechanisms underlying its effects on diverse cellular processes. It has anti-proliferative action on many cell types, an effect generally attributed to tyrosine kinase inhibition. In this study, genistein was found to cause total inhibition of [3H]-thymidine incorporation into DNA and a modest reduction in [3H]-proline incorporation into protein in primary cultures of cardiac fibroblasts. The decrease in [3H]-thymidine incorporation was not associated with a decrease in cell proliferation but correlated exactly with low intracellular levels of [3H]-thymidine. Genistein dramatically reduced [3H]-thymidine but not [3H]-proline uptake by these cells in which the equilibrative nucleoside transporter may be the major route of nucleoside uptake. The effect was irreversible and was demonstrable in pulmonary fibroblasts as well. The findings suggest that nucleoside uptake mechanisms may be a novel target of genistein action in cardiac fibroblasts and point to serious limitations in using genistein to assess the role of tyrosine kinase in cell proliferation by the standard technique of [3H]-thymidine incorporation.

Effect of moderate salinity stress treatment on the stimulation of proline uptake and growth in Escherichia coli CSH4 and its mutants under high salinity

Activity of proline uptake in Escherichia coli CSH4 was inhibited in the presence of 1 M NaCl, while it was recovered if the cells were incubated at 30 degrees C for 1 h in a moderate salinity stress (MSS) solution which consists of Davis minimal medium with 5 mM proline and 0.5 M NaCl. Then, an attempt was made to examine whether MSS treatment is also effective on the activity restoration of proline uptake and growth under high salinity for E. coli CSH4 mutants with different combinations of proP, putA, putP, and proU which are related to the transport and metabolization of proline. After MSS treatment, proline uptake was vigorously occurred for the mutants with proline transporter gene proP but not for its deficient ones. For the expression of proline uptake activities of these mutant strains after MSS treatment, PO(4)(3-) in MSS solution is more important than K(+). No growth of strain CSH4 and its mutants without MSS treatment was observed, when cultured in high osmotic medium G (0.8 M NaCl) consisting of 1 mM glycine betaine and Davis minimal medium without potassium phosphate supplemented. After MSS treatment, however, mutant strains lacking proP showed sufficient growth in medium G. Cell growth of proP(+) strains was recognized if MSS treatment was performed in the absence of proline. In conclusion, growth of mutant strains under high-salinity medium G depended on their amount of proline accumulated during MSS treatment, in which K(+) and PO(4)(3-) might play a key role to guarantee their sufficient growth.

Intestinal gaboxadol absorption via PAT1 (SLC36A1): modified absorption in vivo following co‐administration of L‐tryptophan

Gaboxadol has been in development for treatment of chronic pain and insomnia. The clinical use of gaboxadol has revealed that adverse effects seem related to peak serum concentrations. The aim of this study was to investigate the mechanism of intestinal absorption of gaboxadol in vitro and in vivo. In vitro transport investigations were performed in Caco-2 cell monolayers. In vivo pharmacokinetic investigations were conducted in beagle dogs. Gaboxadol doses of 2.5 mg.kg(-1) were given either as an intravenous injection (1.0 mL.kg(-1)) or as an oral solution (5.0 mL.kg(-1)). Gaboxadol may be a substrate of the human proton-coupled amino acid transporter, hPAT1 and it inhibited the hPAT1-mediated L-[(3)H]proline uptake in Caco-2 cell monolayers with an inhibition constant K(i) of 6.6 mmol.L(-1). The transepithelial transport of gaboxadol was polarized in the apical to basolateral direction, and was dependent on gaboxadol concentration and pH of the apical buffer solution. In beagle dogs, the absorption of gaboxadol was almost complete (absolute bioavailability, F(a), of 85.3%) and T(max) was 0.46 h. Oral co-administration with 2.5-150 mg.kg(-1) of the PAT1 inhibitor, L-tryptophan, significantly decreased the absorption rate constant, k(a), and C(max), and increased T(max) of gaboxadol, whereas the area under the curve and clearance of gaboxadol were constant. The absorption of gaboxadol across the luminal membrane of the small intestinal enterocytes is probably mediated by PAT1. This knowledge is useful for reducing gaboxadol absorption rates in order to decrease peak plasma concentrations.

SNAT2 Amino Acid Transporter Is Regulated by Amino Acids of the SLC6 γ-Aminobutyric Acid Transporter Subfamily in Neocortical Neurons and May Play No Role in Delivering Glutamine for Glutamatergic Transmission

System A transporters SNAT1 and SNAT2 mediate uptake of neutral alpha-amino acids (e.g. glutamine, alanine, and proline) and are expressed in central neurons. We tested the hypothesis that SNAT2 is required to support neurotransmitter glutamate synthesis by examining spontaneous excitatory activity after inducing or repressing SNAT2 expression for prolonged periods. We stimulated de novo synthesis of SNAT2 mRNA and increased SNAT2 mRNA stability and total SNAT2 protein and functional activity, whereas SNAT1 expression was unaffected. Increased endogenous SNAT2 expression did not affect spontaneous excitatory action-potential frequency over control. Long term glutamine exposure strongly repressed SNAT2 expression but increased excitatory action-potential frequency. Quantal size was not altered following SNAT2 induction or repression. These results suggest that spontaneous glutamatergic transmission in pyramidal neurons does not rely on SNAT2. To our surprise, repression of SNAT2 activity was not limited to System A substrates. Taurine, gamma-aminobutyric acid, and beta-alanine (substrates of the SLC6 gamma-aminobutyric acid transporter family) repressed SNAT2 expression more potently (10x) than did System A substrates; however, the responses to System A substrates were more rapid. Since ATF4 (activating transcription factor 4) and CCAAT/enhancer-binding protein are known to bind to an amino acid response element within the SNAT2 promoter and mediate induction of SNAT2 in peripheral cell lines, we tested whether either factor was similarly induced by amino acid deprivation in neurons. We found that glutamine and taurine repressed the induction of both transcription factors. Our data revealed that SNAT2 expression is constitutively low in neurons under physiological conditions but potently induced, together with the taurine transporter TauT, in response to depletion of neutral amino acids.

Discovery and characterization of potent small molecule inhibitors of the high affinity proline transporter

The mammalian proline transporter (PROT) is a high affinity Na +/Cl −-dependent transporter expressed in specific regions of the brain. It is homologous to other neurotransmitter transporters such as glycine, norepinephrine, serotonin, and dopamine transporters. PROT is enriched in glutamatergic synaptic terminals and may play an important role in the regulation of excitatory neurotransmission. No non-peptide small molecule inhibitors have been described for this transporter. To study its physiological role in the central nervous system and evaluate its potential as a therapeutic target, we established cell lines that stably express recombinant hPROT and characterized its kinetic properties for proline uptake. We then screened for inhibitors and identified a series of compounds that inhibit hPROT-mediated proline uptake. A known compound, benztropine, was found to inhibit hPROT with an IC 50 of 0.75 μM. A series of novel compounds were also found, one of which, LP-403812, showed an IC 50 of approximately 0.1 μM on both recombinant human and mouse PROT without significant inhibition of glycine and dopamine transporters at concentrations up to 10 μM. This compound also inhibited proline transporter activity of mouse brain synaptosomes with the same potency. These inhibitors provide important tools for the understanding of PROT functions in the brain and may lead to the development of therapeutic agents for certain neurological disorders.

Proline transport and stress tolerance of ammonia-insensitive mutants of the PUT4-encoded proline-specific permease in yeast

The imino amino acid, proline, has roles in both cellular nutrition and response to stress. Proline uptake in Saccharomyces cerevisiae is largely mediated by a high affinity, specific permease, Put4p, and a low affinity general amino acid permease, Gap1p. Both are subject to nitrogen catabolite repression (NCR) and nitrogen catabolite inactivation (NCI). In order for proline to be fully exploited, its transport must be derepressed, as occurs upon depletion of preferred nitrogen sources, and molecular oxygen must be present to allow the first step of catabolism via proline oxidase. This study focuses on the isolation of variants of Put4p, which are insensitive to repression by a preferred nitrogen source (ammonia) and their subsequent effect on proline transport and stress tolerance. Specific amino acid residues in the carboxy-terminal region of Put4p were targeted by site-directed mutagenesis. Substitution at Serine(605), a potential phosphorylation target, led to the amelioration of ammonia-induced down-regulation of Put4p. When combined with a promoter mutation (-160), the S(605)A mutation resulted in increased proline uptake and accumulation. This increase in proline accumulation was associated with increased cell viability in conditions of high temperature and osmotic stress raising possible benefits in industrial fermentation applications.

Platelet derived growth factor-AB enhances knee meniscal cell activity in vitro

Meniscal healing especially in the inner avascular region has always been a major challenge. In this study we investigated the potential for platelet derived growth factor-AB (PDGF-AB) to promote meniscal tissue regeneration in the inner (avascular), middle, and outer (vascular) zones of the meniscus. Various concentrations of PDGF-AB were tested on sheep meniscal cell cultures. We used the radioactive thymidine uptake assay to assess cell proliferation, and the radioactive sulphur and proline uptake assays and Blyscan assay to assess matrix formation. In general, PDGF-AB stimulated both cell proliferation and matrix formation by cells from all meniscal zones. PDGF-AB at a concentration of 100 ng/ml increased cell proliferation and matrix formation by eight and four fold respectively, by fibrochondrocytes cultured from all meniscal zones ( p < 0.001). These results indicate that fibrochondrocytes present within the avascular region of the meniscus have the ability to proliferate and form new matrix when exposed to anabolic cytokines such as PDGF-AB.