Proline Uptake Research Articles

Cis-4-[18F]fluoro-L-proline Molecular Imaging Experimental Liver Fibrosis.

Introduction: Early-stage liver fibrosis is potentially reversible, but difficult to diagnose. Clinical management would be enhanced by the development of a non-invasive imaging technique able to identify hepatic injury early, before end-stage fibrosis ensues. The analog of the amino acid proline, cis-4-[18F]fluoro-L-proline ([18F]fluoro-proline), which targets collagenogenesis in hepatic stellate cells (HSC), was used to detect fibrosis.Methods: Acute steatohepatitis was induced in experimental animals by liquid ethanol diet for 8 weeks, intra-gastric binge feedings every 10th day along with lipopolysaccharide (LPS) injection. The control animals received control diet for 8 weeks and an equivalent volume of saline on the same schedule as the acute steatohepatitis model. First, in vitro cellular experiments were carried out to assess [3H]proline uptake by HSC, hepatocytes and Kupffer cells derived from rats with acute steatohepatitis (n = 14) and controls (n = 14). Next, ex vivo liver experiments were done to investigate unlabeled proline-mediated collagen synthesis and its associated proline transporter expression in acute steatohepatitis (n = 5) and controls (n = 5). Last, in vivo dynamic and static [18F]fluoro-proline micro-PET/CT imaging was performed in animal models of acute steatohepatitis (n = 7) and control (n = 7) mice.Results: [3H]proline uptake was 5-fold higher in the HSCs of steatohepatitis rats than controls after incubation of up to 60 min. There was an excellent correlation between [3H]proline uptake and liver collagen expression (r-value > 0.90, p < 0.05). Subsequent liver tissue studies demonstrated 2–3-fold higher proline transporter expression in acute steatohepatitis animals than in controls, and proline-related collagen synthesis was blocked by this transporter inhibitor. In vivo micro-PET/CT studies with [18F]fluoro-proline showed 2–3-fold higher uptake in the livers of acute steatohepatitis mice than in controls. There was an excellent correlation between [18F]fluoro-proline uptake and liver collagen expression in the livers of acute steatohepatitis mice (r-value = 0.97, p < 0.001).Conclusion: [18F]fluoro-proline localizes in the liver and correlates with collagenogenesis in acute steatohepatitis with a signal intensity that is sufficiently high to allow imaging with micro-PET/CT. Thus, [18F]fluoro-proline could serve as a PET imaging biomarker for detecting early-stage liver fibrosis.

Functional Consequences of Low Activity of Transport System A for Neutral Amino Acids in Human Bone Marrow Mesenchymal Stem Cells.

In cultured human fibroblasts, SNAT transporters (System A) account for the accumulation of non-essential neutral amino acids, are adaptively up-regulated upon amino acid deprivation and play a major role in cell volume recovery upon hypertonic stress. No information is instead available on the expression and activity of SNAT transporters in human bone marrow mesenchymal stromal cells (MSC), although they are increasingly investigated for their staminal and immunomodulatory properties and used for several therapeutic applications. The uptake of glutamine and proline, two substrates of SNAT1 and SNAT2 transporters, was measured in primary human MSC and an MSC line. The amino acid analogue MeAIB, a specific substrate of these carriers, has been used to selectively inhibit SNAT-dependent transport of glutamine and, through its sodium-dependent transport, as an indicator of SNAT1/2 activity. SNAT1/2 expression and localization were assessed with RT-PCR and confocal microscopy, respectively. Cell volume was assessed from urea distribution space. In all these experiments, primary human fibroblasts were used as the positive control for SNAT expression and activity. Compared with fibroblasts, MSC have a lower SNAT1 expression and hardly detectable membrane localization of both SNAT1 and SNAT2. Moreover, they exhibit no sodium-dependent MeAIB uptake or MeAIB-inhibitable glutamine transport, and exhibit a lower ability to accumulate glutamine and proline than fibroblasts. MSC exhibited an only marginal increase in MeAIB transport upon amino acid starvation and did not recover cell volume after hypertonic stress. In conclusion, the activity of SNAT transporters is low in human MSC. MSC adaptation to amino acid shortage is expected to rely on intracellular synthesis, given the absence of an effective up-regulation of the SNAT transporters.

Crystal violet structural analogues identified by in silico drug repositioning present anti-Trypanosoma cruzi activity through inhibition of proline transporter TcAAAP069.

BackgroundCrystal violet (CV) was used for several years in blood banks to eliminate the parasite Trypanosoma cruzi in endemic areas in order to prevent transfusion-transmitted Chagas disease. One mechanism of action described for CV involves inhibition of proline uptake. In T. cruzi, proline is essential for host cell infection and intracellular differentiation among other processes, and can be obtained through the proline permease TcAAAP069.Methodology/Principal findingsCV inhibited proline transporter TcAAAP069 and parasites overexpressing this permease were 47-fold more sensitive to this compound than control parasites. Using CV as reference molecule, loratadine, cyproheptadine, olanzapine and clofazimine were identified as structurally related compounds to CV (structural analogues) by in silico drug repurposing through a similarity-based virtual screening protocol. All these already-approved drugs for clinical use inhibited TcAAAP069 activity with different efficacies and also presented trypanocidal action in epimastigotes, trypomastigotes and amastigotes of the Y, CL Brener and Dm28c T. cruzi strains. Finally, a synergistic effect between benznidazole and the CV chemical analogues was evidenced by combination and dose-reduction indexes values in epimastigotes and trypomastigotes of the Y strain.Conclusions/SignificanceLoratadine, cyproheptadine and clofazimine inhibit TcAAAP069 proline transporter and also present trypanocidal effect against all T. cruzi life stages in strains from three different DTUs. These CV structural analogues could be a starting point to design therapeutic alternatives to treat Chagas disease by finding new indications for old drugs. This approach, called drug repurposing is a recommended strategy by the World Health Organization to treat neglected diseases, like Chagas disease, and combination therapy may improve the possibility of success of repositioned drugs.

Cultivation at high osmotic pressure confers ubiquinone 8–independent protection of respiration on Escherichia coli

Cultivation at high osmotic pressure confers ubiquinone 8–independent protection of respiration on Escherichia coli

Effect of the deubiquitination enzyme gene UBP6 on the stress-responsive transcription factor Msn2-mediated control of the amino acid permease Gnp1 in yeast

In the yeast Saccharomyces cerevisiae, the transcriptional factor Msn2 plays an essential role in response to a variety of environmental stresses by activating the transcription of many genes that contain the stress-responsive elements in the promoters. We previously reported that overexpression of the MSN2 gene confers tolerance to various stresses in industrial yeast strains. Recently, the overexpression of MSN2 was shown to increase the amount of the amino acid permease Gnp1 on the plasma membrane, leading to the increased uptake of proline into the cell, suggesting a novel link between the Msn2-mediated stress response and amino acid homeostasis in yeast. Here, we found that overexpression of MSN2 increased ubiquitinated protein levels with reduced free ubiquitin. Among deubiquitinating enzymes (DUBs), it was revealed that the loss of Ubp6 depleted the free ubiquitin level and decreased tolerance to the toxic amino acid analogues. The overexpression of UBP6 in MSN2-overexpressing cells clearly complemented the impaired tolerance towards the toxic amino acid analogues. Both the protein level and the plasma-membrane localization of Gnp1 were increased in ubp6-deleted cells, as shown in MSN2-overexpressing cells. These results suggest that an excess level of Msn2 impairs endocytic degradation of Gnp1 through dysfunction of Ubp6 and other DUBs.

Involvement of the stress-responsive transcription factor gene MSN2 in the control of amino acid uptake in Saccharomyces cerevisiae.

The transcriptional factor Msn2 plays a pivotal role in response to environmental stresses by activating the transcription of stress-responsive genes in Saccharomyces cerevisiae. Our previous studies demonstrate that intracellular proline acts as a key protectant against various stresses. It is unknown, however, whether Msn2 is involved in proline homeostasis in S. cerevisiae cells. We here found that MSN2-overexpressing (MSN2-OE) cells showed higher sensitivity to a toxic analogue of proline, l-azetidine-2-carboxylic acid (AZC), as well as to the other amino acid toxic analogues, than wild-type cells. Overexpression of MSN2 increased the intracellular content of AZC, suggesting that Msn2 positively regulates the uptake of proline. Among the known proline permease genes, GNP1 was shown to play a predominant role in the AZC toxicity. Based on quantitative real-time PCR and western blot analyses, the overexpression of MSN2 did not induce any increases in the transcript levels of GNP1 or the other proline permease genes, while the amount of the Gnp1 protein was markedly increased in MSN2-OE cells. Microscopic observation suggested that the endocytic degradation of Gnp1 was impaired in MSN2-OE cells. Thus, this study sheds light on a novel link between the Msn2-mediated global stress response and the amino acid homeostasis in S. cerevisiae.

Exogenous proline has favorable effects on growth and browning suppression in rice but not in tobacco

Proline is one of the amino acids that compose proteins and has various roles under non-stress and stress conditions. In this study, we investigated the effect of proline on the growth and browning of two plants, tobacco and rice, by exogenous application and endogenous increase of proline. Exogenous proline had a different effect on the growth and browning between tobacco and rice: proline affected negatively the growth of tobacco seedlings and favorably that of rice seedlings. In addition, proline prevented browning only in rice cultured cells, consistent with the increase of proline contents, but not in tobacco BY-2 cells. These results might be due to the difference of exogenous proline uptake activity in these cells. From the Lineweaver-Burk plots, proline inhibited polyphenol oxidase activity in vitro, which is a major factor of enzymatic browning in plants, by affecting the enzyme-substrate complex. Proline could suppress the browning of the plant callus by inhibition of PPO activity.

Proteomics Reveals Multiple Phenotypes Associated with N-linked Glycosylation in Campylobacter jejuni

Campylobacter jejuni is a major gastrointestinal pathogen generally acquired via consumption of poorly prepared poultry. N-linked protein glycosylation encoded by the pgl gene cluster targets >80 membrane proteins and is required for both nonsymptomatic chicken colonization and full human virulence. Despite this, the biological functions of N-glycosylation remain unknown. We examined the effects of pgl gene deletion on the C. jejuni proteome using label-based liquid chromatography/tandem mass spectrometry (LC-MS/MS) and validation using data independent acquisition (DIA-SWATH-MS). We quantified 1359 proteins corresponding to ∼84% of the C. jejuni NCTC 11168 genome, and 1080 of these were validated by DIA-SWATH-MS. Deletion of the pglB oligosaccharyltransferase (ΔpglB) resulted in a significant change in abundance of 185 proteins, 137 of which were restored to their wild-type levels by reintroduction of pglB (Δaaz.batpglB::ΔpglB). Deletion of pglB was associated with significantly reduced abundances of pgl targets and increased stress-related proteins, including ClpB, GroEL, GroES, GrpE and DnaK. pglB mutants demonstrated reduced survival following temperature (4 °C and 46 °C) and osmotic (150 mm NaCl) shock and altered biofilm phenotypes compared with wild-type C. jejuni Targeted metabolomics established that pgl negative C. jejuni switched from aspartate (Asp) to proline (Pro) uptake and accumulated intracellular succinate related to proteome changes including elevated PutP/PutA (proline transport and utilization), and reduced DctA/DcuB (aspartate import and succinate export, respectively). ΔpglB chemotaxis to some substrates (Asp, glutamate, succinate and α-ketoglutarate) was reduced and associated with altered abundance of transducer-like (Tlp) proteins. Glycosylation negative C. jejuni were depleted of all respiration-associated proteins that allow the use of alternative electron acceptors under low oxygen. We demonstrate for the first time that N-glycosylation is required for a specific enzyme activity (Nap nitrate reductase) that is associated with reduced abundance of the NapAB glycoproteins. These data indicate a multifactorial role for N-glycosylation in C. jejuni physiology.

Dual Role of the C-Terminal Domain in Osmosensing by Bacterial Osmolyte Transporter ProP

ProP is a member of the major facilitator superfamily, a proton-osmolyte symporter, and an osmosensing transporter. ProP proteins share extended cytoplasmic carboxyl terminal domains (CTDs) implicated in osmosensing. The CTDs of the best characterized, group A ProP orthologs, terminate in sequences that form intermolecular, antiparallel α-helical coiled coils (e.g., ProPEc, from Escherichia coli). Group B orthologs lack that feature (e.g., ProPXc, from Xanthomonas campestris). ProPXc was expressed and characterized in E. coli to further elucidate the role of the coiled coil in osmosensing. The activity of ProPXc was a sigmoid function of the osmolality in cells and proteoliposomes. ProPEc and ProPXc attained similar activities at the same expression level in E. coli. ProPEc transports proline and glycine betaine with comparable high affinities at low osmolality. In contrast, proline weakly inhibited high-affinity glycine-betaine uptake via ProPXc. The KM for proline uptake via ProPEc increases dramatically with the osmolality. The KM for glycine-betaine uptake via ProPXc did not. Thus, ProPXc is an osmosensing transporter, and the C-terminal coiled coil is not essential for osmosensing. The role of CTD-membrane interaction in osmosensing was examined further. As for ProPEc, the ProPXc CTD co-sedimented with liposomes comprising E. coli phospholipid. Molecular dynamics simulations illustrated association of the monomeric ProPEc CTD with the membrane surface. Comparison with the available NMR structure for the homodimeric coiled coil formed by the ProPEc-CTD suggested that membrane association and homodimeric coiled-coil formation by that peptide are mutually exclusive. The membrane fluidity in liposomes comprising E. coli phospholipid decreased with increasing osmolality in the range relevant for ProP activation. These data support the proposal that ProP activates as cellular dehydration increases cytoplasmic cation concentration, releasing the CTD from the membrane surface. For group A orthologs, this also favors α-helical coiled-coil formation that stabilizes the transporter in an active form.

Arabidopsis AMINO ACID PERMEASE1 Contributes to Salt Stress-Induced Proline Uptake from Exogenous Sources.

Stress-induced proline accumulation in plants is thought to result primarily from enhanced proline biosynthesis and decreased proline degradation. To identify regulatory components involved in proline transport, we screened for Arabidopsis thaliana T-DNA mutants with enhanced tolerance to toxic levels of exogenous proline (45 mM). We isolated the proline resistant 1-1 (pre1-1) mutant and map-based cloning identified PRE1 as AMINO ACID PERMEASE1 (AAP1, At1g58360), which encodes a plasma membrane-localized amino acid permease. AAP1 expression is induced by salt stress and abscisic acid, but not by proline. In pre1-1 mutants, a 19-nucleotide deletion in the AAP1 coding region produced a premature stop codon. When grown on proline-containing medium, pre1-1 mutants accumulated significantly less proline than did the wild type. Under salt stress, proline uptake decreased significantly in pre1-1 mutants. By contrast, proline uptake increased significantly in the wild type. These results suggest that AAP1 functions in the increase of proline uptake during salt stress. In addition, proline uptake promotes salt tolerance in Arabidopsis seedlings. We conclude that plants can increase proline accumulation by AtAAP1-mediated proline uptake from exogenous source, which help to improve the salt tolerance of seedlings.

Role of Proline in Pathogen and Host Interactions.

Proline metabolism has complex roles in a variety of biological processes, including cell signaling, stress protection, and energy production. Proline also contributes to the pathogenesis of various disease-causing organisms. Understanding the mechanisms of how pathogens utilize proline is important for developing new strategies against infectious diseases. Recent Advances: The ability of pathogens to acquire amino acids is critical during infection. Besides protein biosynthesis, some amino acids, such as proline, serve as a carbon, nitrogen, or energy source in bacterial and protozoa pathogens. The role of proline during infection depends on the physiology of the host/pathogen interactions. Some pathogens rely on proline as a critical respiratory substrate, whereas others exploit proline for stress protection. Disruption of proline metabolism and uptake has been shown to significantly attenuate virulence of certain pathogens, whereas in other pathogens the importance of proline during infection is not known. Inhibiting proline metabolism and transport may be a useful therapeutic strategy against some pathogens. Developing specific inhibitors to avoid off-target effects in the host, however, will be challenging. Also, potential treatments that target proline metabolism should consider the impact on intracellular levels of Δ1-pyrroline-5-carboxylate, a metabolite intermediate that can have opposing effects on pathogenesis. Further characterization of how proline metabolism is regulated during infection would provide new insights into the role of proline in pathogenesis. Biochemical and structural characterization of proline metabolic enzymes from different pathogens could lead to new tools for exploring proline metabolism during infection and possibly new therapeutic compounds.

Process optimization for enhancing production of cis-4-hydroxy-l-proline by engineered Escherichia coli

BackgroundUnderstanding the bioprocess limitations is critical for the efficient design of biocatalysts to facilitate process feasibility and improve process economics. In this study, a proline hydroxylation process with recombinant Escherichia coli expressing l-proline cis-4-hydroxylase (SmP4H) was investigated. The factors that influencing the metabolism of microbial hosts and process economics were focused on for the optimization of cis-4-hydroxy-l-proline (CHOP) production.ResultsIn recombinant E. coli, SmP4H synthesis limitation was observed. After the optimization of expression system, CHOP production was improved in accordance with the enhanced SmP4H synthesis. Furthermore, the effects of the regulation of proline uptake and metabolism on whole-cell catalytic activity were investigated. The improved CHOP production by repressing putA gene responsible for l-proline degradation or overexpressing l-proline transporter putP on CHOP production suggested the important role of substrate uptake and metabolism on the whole-cell biocatalyst efficiency. Through genetically modifying these factors, the biocatalyst activity was significantly improved, and CHOP production was increased by twofold. Meanwhile, to further improve process economics, a two-strain coupling whole-cell system was established to supply co-substrate (α-ketoglutarate, α-KG) with a cheaper chemical l-glutamate as a starting material, and 13.5 g/L of CHOP was successfully produced.ConclusionsIn this study, SmP4H expression, and l-proline uptake and degradation, were uncovered as the hurdles for microbial production of CHOP. Accordingly, the whole-cell biocatalysts were metabolically engineered for enhancing CHOP production. Meanwhile, a two-strain biotransformation system for CHOP biosynthesis was developed aiming at supplying α-KG more economically. Our work provided valuable insights into the design of recombinant microorganism to improve the biotransformation efficiency that catalyzed by Fe(II)/α-KG-dependent dioxygenase.

Alleviation of Drought Stress in White Clover after Inoculation with Arbuscular Mycorrhizal Fungi

White clover is extremely susceptive to drought stress (DS), while it is not clear whether arbuscular mycorrhizal fungi (AMF) enhance drought tolerance of the plant. This study was carried out to evaluate effects of two AMF species, Funneliformis mosseae and Paraglomus occultum, on flavonoid, soluble protein, proline, and nutrient uptake in roots of white clover under well-watered (WW) and DS conditions. Root colonization by F. mosseae and P. occultum was heavily decreased by 7-week DS treatment. Mycorrhizal plants showed considerably greater biomass production in shoot, root, and total (shoot+root) than non-mycorrhizal plants, irrespective of soil water status. AMF inoculation led to significantly higher root soluble protein and proline accumulation under WW and DS and root flavonoid level under DS, regardless of AMF species. Root N, P, K and Cu concentrations were dramatically increased by mycorrhization under WW and DS, and root Ca, Mg, Fe, and Mn levels were significantly higher in AMF plants than in non-AMF plants under WW. It concluded that AMF strongly enhanced plant growth and drought tolerance of white clover by greater nutrient absorption and protective substances (soluble protein, proline, and flavonoid) accumulation.

Importance of the High-Expression of Proline Transporter PutP to the Adaptation of Escherichia coli to High Salinity.

The effect of the amount of the proline transporter PutP expression on the mechanism of adaptation of E. coli cells to high salinity was analyzed. The PutP gene derived from the E. coli expression plasmid was introduced into the E. coli cell, and a high PutP expression strain was developed. At 1.2 M NaCl culture condition, the growth of normal E. coli cells was inhibited, whereas high ProP expression cells showed growth under 2.5 M NaCl conditions. The uptake of proline by E. coli as a compatible solute and substrate for metabolization was in good accordance with those seen in cell growth. These data suggested that the amount of the proline transporter PutP expression played an important role in the adaptation of E. coli cells to high saline conditions.

Genomes of Candidatus Wolbachia bourtzisii wDacA and Candidatus Wolbachia pipientis wDacB from the Cochineal Insect Dactylopius coccus (Hemiptera: Dactylopiidae).

Dactylopius species, known as cochineal insects, are the source of the carminic acid dye used worldwide. The presence of two Wolbachia strains in Dactylopius coccus from Mexico was revealed by PCR amplification of wsp and sequencing of 16S rRNA genes. A metagenome analysis recovered the genome sequences of Candidatus Wolbachia bourtzisii wDacA (supergroup A) and Candidatus Wolbachia pipientis wDacB (supergroup B). Genome read coverage, as well as 16S rRNA clone sequencing, revealed that wDacB was more abundant than wDacA. The strains shared similar predicted metabolic capabilities that are common to Wolbachia, including riboflavin, ubiquinone, and heme biosynthesis, but lacked other vitamin and cofactor biosynthesis as well as glycolysis, the oxidative pentose phosphate pathway, and sugar uptake systems. A complete tricarboxylic acid cycle and gluconeogenesis were predicted as well as limited amino acid biosynthesis. Uptake and catabolism of proline were evidenced in Dactylopius Wolbachia strains. Both strains possessed WO-like phage regions and type I and type IV secretion systems. Several efflux systems found suggested the existence of metal toxicity within their host. Besides already described putative virulence factors like ankyrin domain proteins, VlrC homologs, and patatin-like proteins, putative novel virulence factors related to those found in intracellular pathogens like Legionella and Mycobacterium are highlighted for the first time in Wolbachia. Candidate genes identified in other Wolbachia that are likely involved in cytoplasmic incompatibility were found in wDacB but not in wDacA.

Trans-intestinal absorption rates differ between free amino acids during larval development in Atlantic herring (Clupea harengus)

Trans-intestinal absorption (TIA) rates for the free amino acids (FAA) alanine, proline, and glutamic acid were measured in two developmental phases of Atlantic herring larvae: Early (14–16day-post-hatch) and Late (37–39day-post-hatch). TIA rates were determined by measuring net uptake of a 14C-radiolabeled FAA tracer solution into the body after administration of a single bolus to the digestive tract by tube-feeding. Tracer solutions (concentration range: 0.02 to 100mM) were administered at <50% of gut volume (GV) and the response of TIA rate to increasing administered dose (pmol FAA ind−1) determined. GV was determined as the maximum volume of a tube-fed liquid bolus that could be retained in the digestive tract of a fasted larva without causing any evacuation from the mouth or anus, and was dependent on larval standard length. Our data revealed that alanine, proline and glutamic acid have different TIA rates (pmolmin−1), and results were consistent at both larval stages. At the same administered dose (pmol ind−1), the TIA rate for glutamic acid was only 8–25% of that of alanine and 15–30% of proline in both larval stages. In Early and Late larvae, the dose-dependent increase in proline and glutamic acid uptake was significantly more rapid than for alanine. Hence, alanine was the most rapidly absorbed FAA at low dose, followed by proline and then glutamic acid, but proline became more rapidly absorbed than alanine at the highest doses, in both stages. In conclusion, the study demonstrated that FAA representing neutral, imino and acidic classes, have different rates of absorption. Across a broad range of concentrations, Atlantic herring larvae have a much lower capacity to deliver glutamic acid from the intestinal lumen to body tissues relative to alanine and proline. Statement of relevanceThis paper provides important and quantitative information to understand uptake of different classes of dietary free amino acids in larval stages of fish. We use herring as model since it gives the best combination of size, transparency, robustness, gut morphology for in vivo tube feeding. The data is relevant for formulation of starter diets.

Adaptive responses to cefotaxime treatment in ESBL-producing Escherichia coli and the possible use of significantly regulated pathways as novel secondary targets.

The aim of the study was to determine how ESBL-producing Escherichia coli change the expression of metabolic and biosynthesis genes when adapting to inhibitory concentrations of cefotaxime. Secondly, it was investigated whether significantly regulated pathways constitute putative secondary targets that can be used to combat the resistant bacteria. Strains of E. coli MG1655 encoding blaCTX-M-1 from an IncI1 plasmid and from the chromosome were challenged with cefotaxime corresponding to inhibitory concentrations, and transcriptional patterns were compared with growth without or with very low concentrations of cefotaxime by RNA sequencing. Significantly regulated pathways were inhibited with suitable inhibitors, or genes encoding the enzymes of the regulated pathways were knocked out. The ability of the bacteria to grow in the presence of cefotaxime was determined. Chequerboard assays were utilized to confirm synergies between treatments. Genes belonging to 16 different functional gene classes were significantly regulated. Protein and peptidoglycan syntheses were up-regulated and low concentrations of chloramphenicol or d-cycloserine, targeting these systems, strongly reduced the MIC of cefotaxime (>32-fold). Inhibition and/or mutations in other genes that were significantly regulated, belonging to energy synthesis, purine synthesis, proline uptake or potassium uptake, also rendered the resistant bacteria more susceptible to cefotaxime. The results show that ESBL-producing E. coli adapt to treatment with cefotaxime by changing their gene expression patterns and furthermore that targeting regulated adaptive pathways may be a suitable way to identify targets for drugs that will specifically inhibit the resistant bacteria.

Trypanosoma cruzi Proline Transport Presents a Cell Density-dependent Regulation.

Trypanosoma cruzi, the etiological agent of Chagas disease, uses proline as its main carbon source, essential for parasite growth and stage differentiation in epimastigotes and amastigotes. Since proline is mainly obtained from extracellular medium by transport proteins, in this work we studied the regulation of the T. cruzi proline transporter TcAAAP069. Proline uptake and intracellular concentration presented oscillations during epimastigote growth phases, increasing during the early exponential phase (322pmol/min) and decreasing to undetectable levels during the late exponential phase. Transporter expression rate correlated with proline uptake, and its subcellular localization alternated from both, the plasma membrane and close to the flagellar pocket, when the transport is higher, to only the flagellar pocket region, when the transport decreased until proline uptake and TcAAAP069 protein became undetectable at the end of the growth curve. Interestingly, when parasites were treated with conditioned medium or were concentrated to artificially increase the culture density, the proline transport was completely abolished resembling the effects observed in late exponential phase. These data highlight for the first time the existence of a density-associated regulation of relevant physiological processes such as proline metabolism.

Proline Absorption and SGK1 Expression are Inhibited in Intestinal Tis7 Transgenic Mice

Background/Aims: Expression of the transcriptional co-regulator tis7 is markedly increased in the adaptive small intestine in a mouse model of short bowel syndrome. Transgenic mice with enterocytic overexpression of tis7 (tis7^tg) have accelerated triglyceride absorption, with increased adiposity yet reduced skeletal muscle mass. To further explore this phenotype, we examined whether tis7 also regulates amino acid and carbohydrate absorption. Methods: Small intestinal glucose and amino acid uptake were quantified in wild type (WT) and tis7^tg mice. Amino acid transporter expression was assessed by qRT-PCR and immunoblot. Apical cell surface transporter expression was quantified by cell surface biotinylation. Results: Active glucose uptake rates were unchanged. Uptake of proline but not leucine was significantly reduced in tis7^tg vs. WT jejunum. Expression of serum and glucocorticoid-induced kinase 1 (SGK1), a solute carrier activator, was inhibited in tis7^tg jejunum. Apical membrane expression of the proline transporter SLC6A20 was reduced in tis7^tg jejunum. Conclusions: Tis7 overexpression in enterocytes inhibits proline uptake, associated with decreased expression of activated SGK1 and reduced cell surface expression of SLC6A20. Consistent with the observed tis7^tg phenotype, tis7 overexpression increases triglyceride absorption but has adverse effects on the uptake of selected amino acids. Tis7 has pleiotropic effects on nutrient absorption.

High Levels of Dietary Supplement Vitamins A, C and E are Absorbed in the Small Intestine and Protect Nutrient Transport Against Chronic Gamma Irradiation.

We examined nutrient transport in the intestines of mice exposed to chronic low-LET 137Cs gamma rays. The mice were whole-body irradiated for 3 days at dose rates of 0, 0.13 and 0.20 Gy/h, for total dose delivery of 0, 9.6 or 14.4 Gy, respectively. The mice were fed either a control diet or a diet supplemented with high levels of vitamins A, C and E. Our results showed that nutrient transport was perturbed by the chronic irradiation conditions. However, no apparent alteration of the macroscopic intestinal structures of the small intestine were observed up to day 10 after initiating irradiation. Jejunal fructose uptake measured in vitro was strongly affected by the chronic irradiation, whereas uptake of proline, carnosine and the bile acid taurocholate in the ileum was less affected. D-glucose transport did not appear to be inhibited significantly by either 9.6 or 14.4 Gy exposure. In the 14.4 Gy irradiated groups, the diet supplemented with high levels of vitamins A, C and E increased intestinal transport of fructose compared to the control diet (day 10; t test, P = 0.032), which correlated with elevated levels of vitamins A, C and E in the plasma and jejunal enterocytes. Our earlier studies with mice exposed acutely to 137Cs gamma rays demonstrated significant protection for transport of fructose, glucose, proline and carnosine. Taken together, these results suggest that high levels of vitamins A, C and E dietary supplements help preserve intestinal nutrient transport when intestines are irradiated chronically or acutely with low-LET gamma rays.