High-affinity Uptake Research Articles

Distinct non-coding RNAs confer root-dependent sense transgene-induced post-transcriptional gene silencing and nitrogen-dependent post-transcriptional regulation to AtAMT1;1 transcripts in Arabidopsis roots.

High-affinity ammonium uptake in roots mediate by AMT1-type ammonium transporters, which are tightly controlled at multiple regulatory levels for adapting various nitrogen availability. For Arabidopsis AtAMT1;1 gene, in addition to the transcriptional and post-translational controls, an organ-dependent and N-dependent post-transcriptional regulation was suggested as an additional regulatory step for fine tuning ammonium uptake, but the underlying mechanisms remain to be elucidated. Here, we showed that degradation of AtAMT1;1 transcript in roots of Pro35s:AtAMT1;1-transformed atamt1;1-1 Arabidopsis plants resulted from RDR6-dependent sense transgene-induced post-transcriptional gene silencing (S-PTGS). The siRNAs for S-PTGS may derive from the aberrant RNA, of which the production was co-determined by sequence feature and excessive expression of AtAMT1;1. Switching to the expression of AtAMT1;1 driven by ProAtUBQ10 or of AtAMT1;1 mutated at two siRNA-targeted hotspots reduced AtAMT1;1-specific siRNAs and overcame S-PTGS in roots. In roots of these lines, however, the steady-state transcript levels of AtAMT1;1 still significantly decreased under conditions of N-sufficiency compared with N-deficiency, confirming a N-dependent post-transcriptional regulatory manner. A crucial role of the 207-bp 3'-end sequence of AtAMT1;1 was further demonstrated by N-dependent accumulation of chimeric-AtAMT1;1 transcript in T-DNA insertion lines and of GFP-tagged chimeric-AtAMT1;1 transcript in transgenic lines. A novel non-coding RNA (ncRNA), which was highly abundant in N-sufficient roots, may target the above-identified 3'-end region for the degrading AtAMT1;1 transcript. This degradation could be prevented by a mutation on the AtAMT1;1 transcript at a potential cleavage site (+1458). These results suggested two distinct mechanisms of regulating AtAMT1;1 mRNA turnover by ncRNA for strictly control of ammonium uptake in roots.

Investigating Nutrient Limitation Role on Improvement of Growth and Poly(3-Hydroxybutyrate) Accumulation by Burkholderia sacchari LMG 19450 From Xylose as the Sole Carbon Source.

Burkholderia sacchari LMG19450, a non-model organism and a promising microbial platform, was studied to determine nutrient limitation impact on poly(3-hydroxybutyrate) [P(3HB)] production and bacterial growth from xylose, a major hemicellulosic residue. Nitrogen and phosphorus limitations have been studied in a number of cases to enhance PHA accumulation, but not combining xylose and B. sacchari. Within this strategy, it was sought to understand how to control PHA production and even modulate monomer composition. Nitrogen-limited and phosphorus-limited fed-batch experiments in bioreactors were performed to evaluate each one's influence on cell growth and poly(3-hydroxybutyrate) production. The mineral medium composition was defined based on yields calculated from typical results so that nitrogen was available during phosphorus limitation and residual phosphorus was available when limiting nitrogen. Sets of experiments were performed so as to promote cell growth in the first stage (supplied with initial xylose 15 g/L), followed by an accumulation phase, where N or P was the limiting nutrient when xylose was fed in pulses to avoid concentrations lower than 5 g/L. N-limited fed-batch specific cell growth (around 0.19 1/h) and substrate consumption (around 0.24 1/h) rates were higher when compared to phosphorus-limited ones. Xylose to PHA yield was similar in both conditions [0.37 gP(3HB)/gxyl]. We also described pst gene cluster in B. sacchari, responsible for high-affinity phosphate uptake. Obtained phosphorus to biomass yields might evidence polyphosphate accumulation. Results were compared with studies with B. sacchari and other PHA-producing microorganisms. Since it is the first report of the mentioned kinetic parameters for LMG 19450 growing on xylose solely, our results open exciting perspectives to develop an efficient bioprocess strategy with increased P(3HB) production from xylose or xylose-rich substrates.

Pharmacokinetic evaluation of [18F]PR04.MZ for PET/CT imaging and quantification of dopamine transporters in the human brain.

Dopamine transporters (DAT) modulate pre-synaptic dopamine and physiological functions such as movement and reward. DAT also mirrors disease state in neurological disorders, rendering it an essential diagnostic target. [18F]PR04.MZ is a new PET imaging agent for DAT with an improved affinity and selectivity profile, for which we here describe the complete pharmacokinetic evaluation in healthy controls. Thirty-two healthy subjects underwent T1-weighted MRI and dynamic PET scans for 180 min with arterial blood sampling (n = 5) or 90 min without blood sampling (n = 25) after injection of 197.6 ± 12.2 MBq [18F]PR04.MZ. Blood and plasma metabolite analysis were performed. MRI-based normalization of brain images, delineation of VOIs, and kinetic modeling was conducted to determine distribution volumes (Vt) and binding potentials (BPnd). The impact of scan duration was evaluated and repeated PET scans were performed to assess test-retest variability (n = 5). A static imaging protocol has been validated for clinical applications. [18F]PR04.MZ showed rapid metabolization in circulation, very high uptake in striatum and midbrain, and very low non-specific binding. The two-tissue compartment model 2TCM provided best fits for measured time-activity-curves and calculated Vts in putamen, caudate, substantia nigra pars compacta (SNpc), and cerebellar cortex were 11.83, 9.73, 2.12, and 0.57, respectively. All non-invasive models correlated well with BPnd values derived from 2TCM but underestimated DAT availability by about 28-33%. Of those, simplified reference tissue model (SRTM) provided the best fits, lowest Akaike Information Criteria values, and BPnd values of 14.82, 11.95, and 2.63 in putamen, caudate, and SNpc, respectively. BPnd estimates for striatal regions and SNpc were stable between 90 and 130 min post-injection. Test-retest results were excellent, showing low variability in all and excellent reliability in most relevant regions. Static imaging from 60 to 90-min post-injection is a viable alternative for quantification. [18F]PR04.MZ is a PET tracer with very high affinity, selectivity, and specific uptake in striatum and midbrain. 2TCM and SRTM provide good fits, high and stable Vts or BPnds, and good test-retest reliability for precise quantification of DAT in human subjects.

STEM-22. TARGETING PYRIMIDINE SYNTHESIS ACCENTUATES MOLECULAR THERAPY RESPONSE IN GLIOBLASTOMA STEM CELLS

Abstract Glioblastoma stem cells (GSCs) reprogram glucose metabolism by hijacking high-affinity glucose uptake to survive in a nutritionally dynamic microenvironment. Here, we trace metabolic aberrations in GSCs to link core genetic mutations in glioblastoma to dependency on de novo pyrimidine synthesis. Targeting the pyrimidine synthetic rate-limiting step enzyme carbamoyl-phosphate synthetase 2, aspartate transcarbamyolase, dihydroorotase (CAD) or the critical downstream enzyme, dihydroorotate dehydrogenase (DHODH) inhibited GSC survival, self-renewal, and in vivo tumor initiation through the depletion of the pyrimidine nucleotide supply in rodent models. Mutations in EGFR or PTEN generated distinct CAD phosphorylation patterns to activate carbon influx through pyrimidine synthesis. Simultaneous abrogation of tumor-specific driver mutations and DHODH activity with clinically approved inhibitors demonstrated sustained inhibition of metabolic activity of pyrimidine synthesis and GSC tumorigenic capacity. Higher expression of pyrimidine synthesis genes portend poor prognosis of glioblastoma patients. Collectively, our results demonstrate a therapeutic approach of precision medicine through targeting the nexus between driver mutations and metabolic reprogramming in cancer stem cells.

Unique structural features in an Nramp metal transporter impart substrate-specific proton cotransport and a kinetic bias to favor import.

Natural resistance-associated macrophage protein (Nramp) transporters enable uptake of essential transition metal micronutrients in numerous biological contexts. These proteins are believed to function as secondary transporters that harness the electrochemical energy of proton gradients by "coupling" proton and metal transport. Here we use the Deinococcus radiodurans (Dra) Nramp homologue, for which we have determined crystal structures in multiple conformations, to investigate mechanistic details of metal and proton transport. We untangle the proton-metal coupling behavior of DraNramp into two distinct phenomena: ΔpH stimulation of metal transport rates and metal stimulation of proton transport. Surprisingly, metal type influences substrate stoichiometry, leading to manganese-proton cotransport but cadmium uniport, while proton uniport also occurs. Additionally, a physiological negative membrane potential is required for high-affinity metal uptake. To begin to understand how Nramp's structure imparts these properties, we target a conserved salt-bridge network that forms a proton-transport pathway from the metal-binding site to the cytosol. Mutations to this network diminish voltage and ΔpH dependence of metal transport rates, alter substrate selectivity, perturb or eliminate metal-stimulated proton transport, and erode the directional bias favoring outward-to-inward metal transport under physiological-like conditions. Thus, this unique salt-bridge network may help Nramp-family transporters maximize metal uptake and reduce deleterious back-transport of acquired metals. We provide a new mechanistic model for Nramp proton-metal cotransport and propose that functional advantages may arise from deviations from the traditional model of symport.

Targeting Metabolic Reprogramming to Radiosensitize Glioblastoma Stem Cells

Glioblastoma is the most prevalent and lethal primary intrinsic tumor in the central nervous system. Metabolic reprogramming, a hallmark of cancer, promotes tumor cell proliferation and survival. Glioblastoma stem cells (GSCs) reprogram glucose metabolism by hijacking high-affinity glucose uptake to survive in a nutritionally dynamic microenvironment. Understanding the metabolic regulation of GSCs may inform novel therapeutic approaches to improved clinical outcome for glioblastoma patients. In current study, we aim to interrogate the aberrant metabolic pattern in GSCs and develop novel targeted approach to sensitize GSC to radiation. Patient-derived GSCs were validated using functional assays. Metabolomic analysis of GSCs and matched differentiated glioblastoma cells (DGCs) was performed using carbon tracing followed by non-targeted mass spectrometry to identify aberrant metabolic pathways upregulated in GSCs. Whole-genome promoter/enhancer analysis with chromatin immunoprecipitation against histone 3 lysine 27 acetylation followed by deep sequencing (H3K27ac ChIPseq) was used to validate these identified metabolic pathways in GSCs. Quantitative RT-PCR and Western blotting were performed assess the relative expression between GSCs and DGCs. Radiation sensitivity was measured using Cs-137 irradiator. Validation of the stem-like function of target metabolic gene was performed using RNA interference followed by in vitro neurosphere formation assay and in vivo tumorigenesis with xenograft model. We previously showed that increased glucose influx is a key survival mechanism in GSCs. Metabolomic profiling identified the upregulation of metabolites of pyrimidine synthesis pathway in GSCs to channel carbon flow from glucose. The activation of pyrimidine synthetic genes in GSCs was validated with unbiased enrichment analysis of whole-genome promoter/enhancer pattern. Targeting the pyrimidine synthetic rate-limiting step enzyme CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamyolase, dihydroorotase) or the critical downstream enzyme, DHODH (dihydroorotate dehydrogenase) inhibited the survival, self-renewal, and in vivo tumor initiation of GSCs through the depletion of the pyrimidine nucleotide supply. Core glioblastoma driver mutations in EGFR or PTEN generated distinct CAD phosphorylation patterns to activate carbon influx through pyrimidine synthesis. Simultaneous abrogation of tumor-specific driver mutations and DHODH activity with clinically approved inhibitors demonstrated sustained inhibition of GSC tumorigenic capacity and sensitization to radiation. Higher expression of pyrimidine synthesis genes portended a poor prognosis of glioblastoma patients. Our results demonstrate a novel therapeutic approach of sensitizing GSC to radiation through targeting the nexus between driver mutations and metabolic reprogramming in stem-like glioma cells.

The Role of SreA-Mediated Iron Regulation in Maintaining Epichloë festucae-Lolium perenne Symbioses.

In ascomycetes and basidiomycetes, iron-responsive GATA-type transcriptional repressors are involved in regulating iron homeostasis, notably to prevent iron toxicity through control of iron uptake. To date, it has been unknown whether this iron regulator contributes toward mutualistic endosymbiosis of microbes with plants, a system where the endophyte must function within the constraints of an in-host existence, including a dependency on the host for nutrient acquisition. Functional characterization of one such protein, SreA from Epichloë festucae, a fungal endosymbiont of cool-season grasses, indicates that regulation of iron homeostasis processes is important for symbiotic maintenance. The deletion of the sreA gene (ΔsreA) led to iron-dependent aberrant hyphal growth and the gradual loss of endophyte hyphae from perennial ryegrass. SreA negatively regulates the siderophore biosynthesis and high-affinity iron uptake systems of E. festucae, similar to other fungi, resulting in iron accumulation in mutants. Our evidence suggests that SreA is involved in the processes that moderate Epichloë iron acquisition from the plant apoplast, because overharvesting of iron in ΔsreA mutants was detected as premature chlorosis of the host using a hydroponic plant growth assay. E. festucae appears to have a tightly regulated iron management system, involving SreA that balances endophyte growth with its survival and prevents overcompetition with the host for iron in the intercellular niche, thus promoting mutualistic associations. Mutations that interfere with Epichloë iron management negatively affect iron-dependent fungal growth and destabilize mutualistic Epichloë -ryegrass associations.

Impaired hippocampal and thalamic acetylcholine release in P301L tau-transgenic mice

We evaluated acetylcholine release by microdialysis in 10 month old control and JNPL3 mice which carry a mutant tau gene (P301 L). Three brain regions were compared: hippocampus and thalamus which receive cholinergic input from the basal forebrain, and the red nucleus which receives cholinergic projections from brain stem nuclei. Cognitive and motor functions of the mice were largely normal. In microdialysis experiments, we found significant reductions in basal ACh levels in hippocampus and thalamus, but not in the red nucleus. ACh release was impaired most strongly (by 50%) when a physiological stimulus was applied, i.e. exploration of a novel environment, whereas most mice responded adequately with an increase of ACh release upon infusion of scopolamine. A strong reduction of scopolamine–mediated ACh release was seen after amyloid Aß42 peptide was administered into the hippocampus of tau-transgenic mice. Choline acetyltransferase activities were unchanged in tau-transgenic mice but acetylcholinesterase activities were increased in thalamus. Lactate and choline levels were increased in tau-transgenic mice but high-affinity choline uptake was slightly reduced. Our data suggest that even mild to moderate tau pathology in JNPL3 mice is able to depress cholinergic transmission in brain regions that receive input from the basal forebrain via long projection neurons. This impairment may be reinforced by amyloid peptide formation.

A reevaluation of the contribution of NRT1.1 to nitrate uptake in Arabidopsis under low‐nitrate supply

NRT1.1 has been previously characterized as a dual-affinity nitrate transporter in Arabidopsis, though several lines of evidence have raised questions regarding its high-affinity function in nitrate uptake. Here, we show that the induction of NRT2.1- and NRT2.2-mediated nitrate uptake interferes with measurements of the contribution of NRT1.1 to high-affinity uptake using nrt1.1 mutants. Therefore, a nrt1.1/2.1/2.2 triple mutant was generated to reevaluate the role of NRT1.1 in high-affinity nitrate uptake. This triple mutant has a lower rate of nitrate uptake than the nrt2.1/2.2 double mutant under low external nitrate supply, resulting in a lower growth rate than that of the double mutant. Therefore, we conclude that NRT1.1-mediated high-affinity nitrate uptake is necessary for plant growth under low-nitrate conditions.

SAR11 bacteria have a high affinity and multifunctional glycine betaine transporter.

Marine bacterioplankton face stiff competition for limited nutrient resources. SAR11, a ubiquitous clade of very small and highly abundant Alphaproteobacteria, are known to devote much of their energy to synthesizing ATP-binding cassette periplasmic proteins that bind substrates. We hypothesized that their small size and relatively large periplasmic space might enable them to outcompete other bacterioplankton for nutrients. Using uptake experiments with 14 C-glycine betaine, we discovered that two strains of SAR11, Candidatus Pelagibacter sp. HTCC7211 and Cand. P. ubique HTCC1062, have extraordinarily high affinity for glycine betaine (GBT), with half-saturation (K s ) values around 1 nM and specific affinity values between 8 and 14 L mg cell-1 h-1 . Competitive inhibition studies indicated that the GBT transporters in these strains are multifunctional, transporting multiple substrates in addition to GBT. Both strains could use most of the transported compounds for metabolism and ATP production. Our findings indicate that Pelagibacter cells are primarily responsible for the high affinity and multifunctional GBT uptake systems observed in seawater. Maximization of whole-cell affinities may enable these organisms to compete effectively for nutrients during periods when the gross transport capacity of the heterotrophic plankton community exceeds the supply, depressing ambient concentrations.

Trk1-mediated potassium uptake contributes to cell-surface properties and virulence of Candida glabrata

The absence of high-affinity potassium uptake in Candida glabrata, the consequence of the deletion of the TRK1 gene encoding the sole potassium-specific transporter, has a pleiotropic effect. Here, we show that in addition to changes in basic physiological parameters (e.g., membrane potential and intracellular pH) and decreased tolerance to various cell stresses, the loss of high affinity potassium uptake also alters cell-surface properties, such as an increased hydrophobicity and adherence capacity. The loss of an efficient potassium uptake system results in diminished virulence as assessed by two insect host models, Drosophila melanogaster and Galleria mellonella, and experiments with macrophages. Macrophages kill trk1Δ cells more effectively than wild type cells. Consistently, macrophages accrue less damage when co-cultured with trk1Δ mutant cells compared to wild-type cells. We further show that low levels of potassium in the environment increase the adherence of C. glabrata cells to polystyrene and the propensity of C. glabrata cells to form biofilms.

Osmo-ionic regulation and carbonic anhydrase, Na+/K+-ATPase and V-H+-ATPase activities in gills of the ancient freshwater crustacean Aegla schmitti (Anomura) exposed to high salinities

Aeglidae anomuran crabs originated in the sea, but invaded and diversified in southern South American freshwater (FW) streams. We here aimed at examining their tolerance of increased salinity, after a long time of evolution in FW (~33 million years). Aegla schmitti were exposed to FW and dilute seawater of salinities 15, 20, and 25‰ for 1, 5 and 10 days. Mortality in 35‰ was also assessed. Hemolymph osmolality, Na+, K+, Cl−, and Mg2+ ions, and hydration levels of the abdominal muscle were assayed. The activities of the Carbonic Anhydrase (CA), Na+/K+-ATPase (NKA) and V-H+-ATPase (VHA) were also assayed in the gills. A. schmitti preserves osmoregulatory mechanisms of its marine ancestors. It is able to survive in high salinities (25‰) for at least 10 days. Mortality in 35‰ was of 56% after 1 day, and of 100% after 7 days. In 25‰, NaCl is apparently hyporegulated at all times, while hemolymph osmolality rises after 5 days. CA and NKA activities remained unchanged in all experimental conditions, while VHA activity decreased after 10 days in 25‰. Hemolymph NaCl data was compatible with either hyporegulation and/or putative influx of NaCl into cells for regulatory volume increase (RVI). Further studies should deepen the understanding of the roles of low permeabilities and saturation of high affinity uptake systems in truly FW decapods, in their responses to high salinities. Moreover, the fate of extracellular NaCl as secretion in true hypo-regulation and/or influx into cells for RVI should also be investigated.

Generation of Metal-Depleted Conditions for In Vitro Growth of Neisseria gonorrhoeae.

Neisseria gonorrhoeae employs high-affinity metal acquisition systems to obtain necessary nutrients, such as iron (Fe) and zinc (Zn) from the environment. Because growth and replication depend upon successful metal acquisition, these high-affinity uptake systems are important virulence factors. Expression of metal acquisition systems is tightly controlled and preferentially expressed under low-metal conditions. Therefore, in order to optimally produce these transport proteins and study them in vitro, growth media must be deployed that mimic low-metal conditions. This chapter describes the chelators, media, and culturing conditions that can generate low-metal in vitro growth conditions.

A New Mechanism for High-Affinity Uptake of C4-Dicarboxylates in Bacteria Revealed by the Structure of Rhodopseudomonas palustris MatC (RPA3494), a Periplasmic Binding Protein of the Tripartite Tricarboxylate Transporter (TTT) Family

C4-dicarboxylates play a central role in cellular physiology as key metabolic intermediates. Under aerobic conditions, they participate in the citric acid cycle, while in anaerobic bacteria, they are important in energy-conserving fermentation and respiration processes. Ten different families of secondary transporters have been described to participate in C4-dicarboxylate movement across biological membranes, but only one of these utilizes an extracytoplasmic solute binding protein to achieve high-affinity uptake. Here, we identify the MatBAC system from the photosynthetic bacterium Rhodopseudomonas palustris as the first member of the tripartite tricarboxylate transport family to be involved in C4-dicarboxylate transport. Tryptophan fluorescence spectroscopy showed that MatC, the periplasmic binding protein from this system, binds to l- and d-malate with Kd values of 27 and 21 nM, respectively, the highest reported affinity to date for these C4-dicarboxylates, and to succinate (Kd = 110 nM) and fumarate (Kd = 400 nM). The 2.1-Å crystal structure of MatC with bound malate shows a high level of substrate coordination, with participation of two water molecules that bridge hydrogen bonds between the ligand proximal carboxylic group and the main chain of two conserved loops in the protein structure. The substrate coordination in MatC correlates with the binding data and explains the protein's selectivity for different substrates and respective binding affinities. Our results reveal a new function in C4-dicarboxylate transport by members of the poorly characterized tripartite tricarboxylate transport family, which are widely distributed in bacterial genomes but for which details of structure–function relationships and transport mechanisms have been lacking.

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.

Shoot nitrate underlies a perception of nitrogen satiety to trigger local and systemic signaling cascades in Arabidopsis thaliana

ABSTRACT Nitrate is an important ion for plant growth and development. It serves not only as a building block for amino acid synthesis but also as a signaling molecule. Changes in the exogenous nitrate concentrations affect the expression of nitrate-responsive genes within minutes. Following these rapid transcriptional events, nitrate and its downstream organic nitrogen (N) compounds accumulate in the plant body, inducing secondary responses to the internal N level. Nevertheless, the respective roles of nitrate and organic N in triggering plant responses to internal N remain to be clarified. Several studies have implied that internal nitrate levels regulate root N uptake independent of the levels of N assimilation products. However, little is known about the specific effects of internal nitrate levels on plant growth and gene expression. To manipulate the internal nitrate levels independently of internal organic N, we grew wild-type Arabidopsis and a nitrate reductase (NR)-null mutant under a series of modified N conditions. Using their shoots and roots, we performed analyses of plant growth and RNA sequencing. The results showed that elevated shoot nitrate accumulation in the NR-null mutant was accompanied by increased expression of nitrate assimilatory genes in the shoots, decreased gene expression of high-affinity nitrate and ammonium uptake transporters in the roots, and decreased lateral root growth. Furthermore, the genes normally induced by N deficiency were significantly downregulated both in the shoots and roots of the NR-null mutant, compared with the wild-type. Our transcriptional profiling suggests that the transcription factors NLP7 and NIGT mediate a wide range of these transcriptional responses. Taken together, we conclude that shoot nitrate acts as a N satiety signal to trigger local and systemic signaling cascades in A. thaliana. The present study illustrates an adaptive strategy of plants to survive in N-limited environments, depending on the residual nitrate storage.

Zinc-dependent substrate-level phosphorylation powers Salmonella growth under nitrosative stress of the innate host response.

The metabolic processes that enable the replication of intracellular Salmonella under nitrosative stress conditions engendered in the innate response of macrophages are poorly understood. A screen of Salmonella transposon mutants identified the ABC-type high-affinity zinc uptake system ZnuABC as a critical determinant of the adaptation of Salmonella to the nitrosative stress generated by the enzymatic activity of inducible nitric oxide (NO) synthase of mononuclear phagocytic cells. NO limits the virulence of a znuB mutant in an acute murine model of salmonellosis. The ZnuABC transporter is crucial for the glycolytic function of fructose bisphosphate aldolase, thereby fueling growth of Salmonella during nitrosative stress produced in the innate response of macrophages. Our investigations demonstrate that glycolysis mediates resistance of Salmonella to the antimicrobial activity of NO produced in an acute model of infection. The ATP synthesized by substrate-level phosphorylation at the payoff phase of glycolysis and acetate fermentation powers the replication of Salmonella experiencing high levels of nitrosative stress. In contrast, despite its high potential for ATP synthesis, oxidative phosphorylation is a major target of inhibition by NO and contributes little to the antinitrosative defenses of intracellular Salmonella. Our investigations have uncovered a previously unsuspected conjunction between zinc homeostasis, glucose metabolism and cellular energetics in the adaptation of intracellular Salmonella to the reactive nitrogen species synthesized in the innate host response.

Increased Cholinergic Response in α-Synuclein Transgenic Mice (h-α-synL62).

Pathological accumulation of misfolded α-synuclein (α-syn) in the brain plays a key role in the pathogenesis of Parkinson's disease, leading to neuronal dysfunction and motor disorders. The underlying mechanisms linking α-syn aggregations with neurotransmitter disturbance in Parkinson's brains are not well characterized. In the present study, we investigated transgenic mice expressing an aggregation-prone form of full-length human α-syn (h-α-synL62) linked to a signal sequence. These mice display dopamine depletion and progressive motor deficits. We detected accumulation of α-syn in cholinergic interneurons where they are colocalized with choline acetyltransferase. Using microdialysis, we measured acetylcholine levels in the striatum at baseline and during stimulation in the open field and with scopolamine. While no difference between wild-type and transgenic mice was detected in 3 month old mice, striatal acetylcholine levels at 9 months of age were significantly higher in transgenic mice. Concomitantly, high-affinity choline uptake was also increased while choline acetyltransferase and acetylcholine esterase activities were unchanged. The results suggest a disinhibition of acetylcholine release in α-syn transgenic mice.

Role of pvdE Pyoverdine Synthesis in Pseudomonas aeruginosa Keratitis.

Pseudomonas aeruginosa produces pyoverdine, encoded by the pvdE gene, for high-affinity iron uptake from transferrin and lactoferrin. This study investigated the contribution of pyoverdine to P. aeruginosa keratitis pathogenesis using in vitro and in vivo models. The P. aeruginosa strains examined were parental strain PAO1 and isogenic mutant strain pvdE (ΔpvdE) defective in pyoverdine. Bacterial growth in vitro was determined by PAO1 and ΔpvdE optical densities in Luria-Bertani (LB) broth. PAO1 or ΔpvdE (10 colony-forming units/mL) was inoculated onto cultured human corneal epithelial cells (HCECs) for 1 hour. The monolayers were examined for bacterial adhesion and invasion. In addition, the corneas of C57BL/6 mice were infected with PAO1 or ΔpvdE. Corneal virulence was evaluated by determining clinical scores and bacterial counts during infection. The growth of PAO1 and ΔpvdE in LB broth was similar. Although adhesion of ΔpvdE onto HCECs was significantly increased compared with PAO1, the invasive capacity of ΔpvdE was significantly decreased. Clinical scores and bacterial numbers were significantly lower in ΔpvdE-infected eyes compared with PAO1-infected eyes at 6, 24, and 48 hours (P < 0.001). ΔpvdE was not detected in mouse corneas and did not induce corneal opacity at 6, 24, or 48 hours. ΔpvdE lost invasive ability toward HCECs. Moreover, ΔpvdE did not cause keratitis in vivo. Thus, pvdE pyoverdine synthesis has critical roles in proliferation and invasion on ocular surfaces and could be a target for prevention of P. aeruginosa keratitis.

Soluble Aβ42 Acts as Allosteric Activator of the Core Cholinergic Enzyme Choline Acetyltransferase.

Two major questions in the field of Alzheimer-type dementia remain elusive. One is the native function of amyloid-β (Aβ) peptides and the other is an early deficit in the central cholinergic network. Nevertheless, recent evidence suggests that Aβ peptides are involved in the regulation of acetylcholine (ACh) homeostasis either by allosteric activation of ACh-degrading cholinesterases or by inhibiting the high-affinity choline uptake transporter. In the current study, we report that Aβ peptides, in particular Aβ42, allosterically enhances the catalytic rate of the core-cholinergic enzyme choline acetyltransferase (ChAT), responsible for biosynthesis of ACh. Detailed in vitro enzyme kinetic analysis indicated that both soluble Aβ40 and Aβ42 enhanced the catalytic efficiency of ChAT by ∼21% and 26% at physiological concentration ranges found in human cerebrospinal fluid (CSF). Further analyses indicated that activation of ChAT by Aβ was highly specific. Intriguingly, Aβ42 exhibited an EC50 of activation potency at 10-fold lower concentrations compared to Aβ40. The activation was persistent even in the presence of a physiological Aβ 40/42 mixture ratio, expected in human CSF. In conclusion, we report for the first time that Aβ42 peptide acts as allosteric enhancers of ACh-biosynthesizing enzyme ChAT. Together with two previous observations, this points to a complex molecular cross-talk between Aβ and the enzymatic machinery involved in maintaining cellular, synaptic and extra-synaptic ACh homeostasis, warranting further investigation.