Novel 6-alkyl-bridged 4-arylalkylpiperazin-1-yl derivatives of azepino[4,3-b]indol-1(2H)-one as potent BChE-selective inhibitors showing protective effects against neurodegenerative insults.

Due to the putative role of butyrylcholinesterase (BChE) in regulation of acetylcholine levels and functions in the late stages of the Alzheimer's disease (AD), the potential of selective inhibitors (BChEIs) has been envisaged as an alternative to administration of acetylcholinesterase inhibitors (AChEIs). Starting from our recent findings, herein the synthesis and in vitro evaluation of cholinesterase (ChE) inhibition of a novel series of some twenty 3,4,5,6-tetrahydroazepino[4,3-b]indol-1(2H)-one derivatives, bearing at the indole nitrogen diverse alkyl-bridged 4-arylalkylpiperazin-1-yl chains, are reported. The length of the spacers, as well as the type of arylalkyl group affected the enzyme inhibition potency and BChE/AChE selectivity. Two compounds, namely 14c (IC50 = 163 nM) and 14d (IC50 = 65 nM), bearing at the nitrogen atom in position 6 a n-pentyl- or n-heptyl-bridged 4-phenethylpiperazin-1-yl chains, respectively, proved to be highly potent mixed-type inhibitors of both equine and human BChE isoforms, showing more than two order magnitude of selectivity over AChE. The study of binding kinetics through surface plasmon resonance (SPR) highlighted differences in their BChE residence times (8 and 47 s for 14c and 14d, respectively). Moreover, 14c and 14d proved to hit other mechanisms known to trigger neurodegeneration underlying AD and other CNS disorders. Unlike 14c, compound 14d proved also capable of inhibiting by more than 60% the in vitro self-induced aggregation of neurotoxic amyloid-ß (Aß) peptide at 100 µM concentration. On the other hand, 14c was slightly better than 14d in counteracting, at 1 and 10 µM concentration, glutamate excitotoxicity, due to over-excitation of NMDA receptors, and hydrogen peroxide-induced oxidative stress assessed in neuroblastoma cell line SH-SY5Y. This paper is dedicated to Prof. Marcello Ferappi, former dean of the Faculty of Pharmacy of the University of Bari, in the occasion of his 90th birthday.

Novel 6-hydroxybenzothiazol-2-carboxamides as potent and selective monoamine oxidase B inhibitors endowed with neuroprotective activity.

In neurodegenerative diseases, using a single molecule that can exert multiple effects to modify the disease may have superior activity over the classical "one molecule-one target" approach. Herein, we describe the discovery of 6-hydroxybenzothiazol-2-carboxamides as highly potent and selective MAO-B inhibitors. Variation of the amide substituent led to several potent compounds having diverse side chains with cyclohexylamide 40 displaying the highest potency towards MAO-B (IC50 = 11 nM). To discover new compounds with extended efficacy against neurotoxic mechanisms in neurodegenerative diseases, MAO-B inhibitors were screened against PHF6, R3 tau, cellular tau and α-synuclein (α-syn) aggregation. We identified the phenethylamide 30 as a multipotent inhibitor of MAO-B (IC50 = 41 nM) and α-syn and tau aggregation. It showed no cytotoxic effects on SH-SY5Y neuroblastoma cells, while also providing neuroprotection against toxicities induced by α-syn and tau. The evaluation of key physicochemical and in vitro-ADME properties revealed a great potential as drug-like small molecules with multitarget neuroprotective activity.

Neuroprotective Effect of Resveratrol against Manganese-Induced Oxidative Stress and Matrix Metalloproteinase-9 in an "In Vivo" Model of Neurotoxicity.

Chronic exposure to manganese (Mn) leads to its accumulation in the central nervous system (CNS) and neurotoxicity with not well-known mechanisms. We investigated the involvement of matrix metalloproteinase (MMP)-2 and -9 in Mn neurotoxicity in an in vivo model of rats treated through an intraperitoneal injection, for 4 weeks, with 50 mg/kg of MnCl2 in the presence or in the absence of 30 mg/kg of resveratrol (RSV). A loss of weight was observed in Mn-treated rats compared with untreated and RSV-treated rats. A progressive recovery of body weight was detected in rats co-treated with Mn and RSV. The analysis of brain homogenates indicated that RSV counteracted the Mn-induced increase in MMP-9 levels and reactive oxygen species production as well as the Mn-induced decrease in superoxide dismutase activity and glutathione content. In conclusion, Mn exposure, resulting in MMP-9 induction with mechanisms related to oxidative stress, represents a risk factor for the development of CNS diseases.

hERG stereoselective modulation by mexiletine-derived ureas: Molecular docking study, synthesis, and biological evaluation.

Long QT syndrome (LQTS) is a disorder of cardiac electrophysiology resulting in life-threatening arrhythmias; nowadays, only a few drugs are available for the management of LQTS. Focusing our attention on LQT2, one of the most common subtypes of LQTS caused by mutations in the human ether-à-go-go-related gene (hERG), in the present work, the stereoselectivity of the recently discovered mexiletine-derived urea 8 was investigated on the hERG potassium channel. According to preliminary in silico predictions, in vitro studies revealed a stereoselective behavior, with the meso form showing the greatest hERG opening activity. In addition, functional studies on guinea pig isolated left atria, aorta, and ileum demonstrated that 8 does not present any cardiac or intestinal liability in our ex vivo studies. Due to its overall profile, (R,S)-8 paves the way for the design and development of a new series of compounds potentially useful in the treatment of both congenital and drug-induced forms of LQTS.

Bioisosteric replacement based on 1,2,4-oxadiazoles in the discovery of 1H-indazole-bearing neuroprotective MAO B inhibitors.

Following a hybridization strategy, a series of 5-substituted-1H-indazoles were designed and evaluated in vitro as inhibitors of human monoamine oxidase (hMAO) A and B. Among structural modifications, the bioisostere-based introduction of 1,2,4-oxadiazole ring returned the most potent and selective human MAO B inhibitor (compound 20, IC50 = 52 nM, SI > 192). The most promising inhibitors were studied in cell-based neuroprotection models of SH-SY5Y and astrocytes line against H2O2. Moreover, preliminary drug-like features (aqueous solubility at pH 7.4; hydrolytic stability at acidic and neutral pH) were assessed for selected 1,2,4-oxadiazoles and compared to amide analogues through RP-HPLC methods. Molecular docking simulations highlighted the crucial role of molecular flexibility in providing a better shape complementarity for compound 20 within MAO B enzymatic cleft than rigid analogue 18. Enzymatic kinetics analysis along with thermal stability curves (Tm shift = +2.9 °C) provided clues of a tight-binding mechanism for hMAO B inhibition by 20.

Thioxanthenone-based derivatives as multitarget therapeutic leads for Alzheimer's disease.

A set of twenty-five thioxanthene-9-one and xanthene-9-one derivatives, that were previously shown to inhibit cholinesterases (ChEs) and amyloid ß (Aß40) aggregation, were evaluated for the inhibition of tau protein aggregation. All compounds exhibited a good activity, and eight of them (5-8, 10, 14, 15 and 20) shared comparable low micromolar inhibitory potency versus Aß40 aggregation and human acetylcholinesterase (AChE), while inhibiting human butyrylcholinesterase (BChE) even at submicromolar concentration. Compound 20 showed outstanding biological data, inhibiting tau protein and Aß40 aggregation with IC50 = 1.8 and 1.3 µM, respectively. Moreover, at 0.1-10 µM it also exhibited neuroprotective activity against tau toxicity induced by okadoic acid in human neuroblastoma SH-SY5Y cells, that was comparable to that of estradiol and PD38. In preliminary toxicity studies, these interesting results for compound 20 are somewhat conflicting with a narrow safety window. However, compound 10, although endowed with a little lower potency for tau and Aß aggregation inhibition additionally demonstrated good inhibition of ChEs and rather low cytotoxicity. Compound 4 is also worth of note for its high potency as hBChE inhibitor (IC50 = 7 nM) and for the three order of magnitude selectivity versus hAChE. Molecular modelling studies were performed to explain the different behavior of compounds 4 and 20 towards hBChE. The observed balance of the inhibitory potencies versus the relevant targets indicates the thioxanthene-9-one derivatives as potential MTDLs for AD therapy, provided that the safety window will be improved by further structural variations, currently under investigation.

New Insights Regarding Hemin Inhibition of the Purified Rat Brain 2-Oxoglutarate Carrier and Relationships with Mitochondrial Dysfunction.

A kinetic analysis of the transport assays on the purified rat brain 2-oxoglutarate/malate carrier (OGC) was performed starting from our recent results reporting about a competitive inhibitory behavior of hemin, a physiological porphyrin derivative, on the OGC reconstituted in an active form into proteoliposomes. The newly provided transport data and the elaboration of the kinetic equations show evidence that hemin exerts a mechanism of partially competitive inhibition, coupled with the formation of a ternary complex hemin-carrier substrate, when hemin targets the OGC from the matrix face. A possible interpretation of the provided kinetic analysis, which is supported by computational studies, could indicate the existence of a binding region responsible for the inhibition of the OGC and supposedly involved in the regulation of OGC activity. The proposed regulatory binding site is located on OGC mitochondrial matrix loops, where hemin could establish specific interactions with residues involved in the substrate recognition and/or conformational changes responsible for the translocation of mitochondrial carrier substrates. The regulatory binding site would be placed about 6 Å below the substrate binding site of the OGC, facing the mitochondrial matrix, and would allow the simultaneous binding of hemin and 2-oxoglutarate or malate to different regions of the carrier. Overall, the presented experimental and computational analyses help to shed light on the possible existence of the hemin-carrier substrate ternary complex, confirming the ability of the OGC to bind porphyrin derivatives, and in particular hemin, with possible consequences for the mitochondrial redox state mediated by the malate/aspartate shuttle led by the mitochondrial carriers OGC and AGC.

Microwave-Assisted Extraction of Bioactive Compounds from Lentil Wastes: Antioxidant Activity Evaluation and Metabolomic Characterization.

The recovery of industrial by-products is part of the zero-waste circular economy. Lentil seed coats are generally considered to be a waste by-product. However, this low-value by-product is rich in bioactive compounds and may be considered an eco-friendly source of health-promoting phytochemicals. For the first time, a sustainable microwave-assisted extraction technique was applied, and a solvent screening was carried out to enhance the bioactive compound content and the antioxidant activity of green and red lentil hull extracts. With respect to green lentil hull extracts that were obtained with different solvents, the aqueous extract of the red lentil seed coats showed the highest total phenolic and total flavonoid content (TPC = 28.3 ± 0.1 mg GAE/g dry weight, TFC = 1.89 ± 0.01 mg CE/100 mg dry weight, respectively), as well as the highest antioxidant activity, both in terms of the free radical scavenging activity (ABTS, 39.06 ± 0.73 mg TE/g dry weight; DPPH, IC50 = 0.39 µg/mL) and the protection of the neuroblastoma cell line (SH-SY5Y, IC50 = 10.1 ± 0.6 µg/mL), the latter of which has never been investigated so far. Furthermore, a metabolite discovery analysis was for the first time performed on the aqueous extracts of both cultivars using an HPLC separation which was coupled with an Orbitrap-based high-Resolution Mass Spectrometry technique.

Imaging Intron Evolution.

Intron evolution may be readily imaged through the combined use of the "dot plot" function of the NCBI BLAST, aligning two sequences at a time, and the Vertebrate "Multiz" alignment and conservation tool of the UCSC Genome Browser. With the NCBI BLAST, an ideal alignment of two highly conserved sequences generates a diagonal straight line in the plot from the lower left corner to the upper right corner. Gaps in this line correspond to non-conserved sections. In addition, the dot plot of the alignment of a sequence with the same sequence after the removal of the Transposable Elements (TEs) can be observed along the diagonal gaps that correspond to the sites of TE insertion. The UCSC Genome Browser can graph, along the entire sequence of a single gene, the level of overall conservation in vertebrates. This level can be compared with the conservation level of the gene in one or more selected vertebrate species. As an example, we show the graphic analysis of the intron conservation in two genes: the mitochondrial solute carrier 21 (SLC25A21) and the growth hormone receptor (GHR), whose coding sequences are conserved through vertebrates, while their introns show dramatic changes in nucleotide composition and even length. In the SLC25A21, a few short but significant nucleotide sequences are conserved in zebrafish, Xenopus and humans, and the rate of conservation steadily increases from chicken/human to mouse/human alignments. In the GHR, a less conserved gene, the earlier indication of intron conservation is a small signal in chicken/human alignment. The UCSC tool may simultaneously display the conservation level of a gene in different vertebrates, with reference to the level of overall conservation in Vertebrates. It is shown that, at least in SLC25A21, the sites of higher conservation are not always coincident in chicken and zebrafish nor are the sites of higher vertebrate conservation.

Evidence for Non-Essential Salt Bridges in the M-Gates of Mitochondrial Carrier Proteins.

Mitochondrial carriers, which transport metabolites, nucleotides, and cofactors across the mitochondrial inner membrane, have six transmembrane α-helices enclosing a translocation pore with a central substrate binding site whose access is controlled by a cytoplasmic and a matrix gate (M-gate). The salt bridges formed by the three PX[DE]XX[RK] motifs located on the odd-numbered transmembrane α-helices greatly contribute to closing the M-gate. We have measured the transport rates of cysteine mutants of the charged residue positions in the PX[DE]XX[RK] motifs of the bovine oxoglutarate carrier, the yeast GTP/GDP carrier, and the yeast NAD+ transporter, which all lack one of these charged residues. Most single substitutions, including those of the non-charged and unpaired charged residues, completely inactivated transport. Double mutations of charged pairs showed that all three carriers contain salt bridges non-essential for activity. Two double substitutions of these non-essential charge pairs exhibited higher transport rates than their corresponding single mutants, whereas swapping the charged residues in these positions did not increase activity. The results demonstrate that some of the residues in the charged residue positions of the PX[DE]XX[KR] motifs are important for reasons other than forming salt bridges, probably for playing specific roles related to the substrate interaction-mediated conformational changes leading to the M-gate opening/closing.

Probing Fluorinated Motifs onto Dual AChE-MAO B Inhibitors: Rational Design, Synthesis, Biological Evaluation, and Early-ADME Studies.

Bioisosteric H/F or CH2OH/CF2H replacement was introduced in coumarin derivatives previously characterized as dual AChE-MAO B inhibitors to probe the effects on both inhibitory potency and drug-likeness. Along with in vitro screening, we investigated early-ADME parameters related to solubility and lipophilicity (Sol7.4, CHI7.4, log D7.4), oral bioavailability and central nervous system (CNS) penetration (PAMPA-HDM and PAMPA-blood-brain barrier (BBB) assays, Caco-2 bidirectional transport study), and metabolic liability (half-lives and clearance in microsomes, inhibition of CYP3A4). Both specific and nonspecific tissue toxicities were determined in SH-SY5Y and HepG2 lines, respectively. Compound 15 bearing a -CF2H motif emerged as a water-soluble, orally bioavailable CNS-permeant potent inhibitor of both human AChE (IC50 = 550 nM) and MAO B (IC50 = 8.2 nM, B/A selectivity > 1200). Moreover, 15 behaved as a safe and metabolically stable neuroprotective agent, devoid of cytochrome liability.

Chemical characterisation of PM10 from ship emissions: a study on samples from hydrofoil exhaust stacks.

A chemical characterization of PM10 collected at hydrofoil exhaust stacks was performed conducting two on-board measuring campaigns, with the aim of assessing the ship emission impact on PM10 collected in the coastal area of Naples (Southern Italy) and providing information about the characteristics of this important PM emission source.Samples were analysed determining the contribution of different chemical parameters to PM10's mass, which consisted of polycyclic aromatic hydrocarbons (PAHs) (0.10 ± 0.12%), total carbon (61.9% ± 20.0%, with 40.4% of organic carbon, OC, and 21.5% of elemental carbon, EC) and elemental fraction (0.44% ± 1.00%). Differences in terms of composition and chemical parameter profiles were observed between samples collected during offshore navigation (Off) and samples collected during shunting operations (SO), the latter of higher concern on a local scale. For SO samples, lower contributions of OC and EC were observed (39.7% and 19.6% respectively) compared to Off samples (41.5% and 24.2%), and an increase in terms of elements (from 0.32 to 0.51%) and PAHs (from 0.06 to 0.12%) concentrations was observed. In addition, enrichment factors (EFs) for some elements such as V, Zn, Cd, Cu, Ag and Hg as well as PAHs profile varied significantly between SO and Off. Data presented here were compared with data on chemical composition of PM10 sampled in a tunnel, in a background site and in an urban site in the city of Naples. Results indicated that shipping activities contributed significantly to the emission of V and, in some extent, Zn and Cd; in addition, PAH profiles indicated a greater contribution to urban PM10 from vehicular traffic than shipping emissions. These results can significantly contribute to the correct evaluation of the influence of shipping emission on PM10 generation in urban coastal areas and can be a useful reference for similar studies. The coastal area of Naples is an important example of the coexistence of residential, touristic and natural areas with pollutants emission sources including, among the others, shipping emissions. In this and similar contexts, it is important to distinguish the contribution of each emission source to clearly define environmental control policies.

The Interaction of Hemin, a Porphyrin Derivative, with the Purified Rat Brain 2-Oxoglutarate Carrier.

The mitochondrial 2-oxoglutarate carrier (OGC), isolated and purified from rat brain mitochondria, was reconstituted into proteoliposomes to study the interaction with hemin, a porphyrin derivative, which may result from the breakdown of heme-containing proteins and plays a key role in several metabolic pathways. By kinetic approaches, on the basis of the single binding centre gated pore mechanism, we analyzed the effect of hemin on the transport rate of OGC in uptake and efflux experiments in proteoliposomes reconstituted in the presence of the substrate 2-oxoglutarate. Overall, our experimental data fit the hypothesis that hemin operates a competitive inhibition in the 0.5-10 µM concentration range. As a consequence of the OGC inhibition, the malate/aspartate shuttle might be impaired, causing an alteration of mitochondrial function. Hence, considering that the metabolism of porphyrins implies both cytoplasmic and mitochondrial processes, OGC may participate in the regulation of porphyrin derivatives availability and the related metabolic pathways that depend on them (such as oxidative phosphorylation and apoptosis). For the sake of clarity, a simplified model based on induced-fit molecular docking supported the in vitro transport assays findings that hemin was as good as 2-oxoglutarate to bind the carrier by engaging specific ionic hydrogen bond interactions with a number of key residues known for participating in the similarly located mitochondrial carrier substrate binding site.

First Attempt to Couple Proteomics with the AhR Reporter Gene Bioassay in Soil Pollution Monitoring and Assessment.

A topsoil sample obtained from a highly industrialized area (Taranto, Italy) was tested on the DR-CALUX® cell line and the exposed cells processed with proteomic and bioinformatics analyses. The presence of polyhalogenated compounds in the topsoil extracts was confirmed by GC-MS/MS analysis. Proteomic analysis of the cells exposed to the topsoil extracts identified 43 differential proteins. Enrichment analysis highlighted biological processes, such as the cellular response to a chemical stimulus, stress, and inorganic substances; regulation of translation; regulation of apoptotic process; and the response to organonitrogen compounds in light of particular drugs and compounds, extrapolated by bioinformatics all linked to the identified protein modifications. Our results confirm and reflect the complex epidemiological situation occurring among Taranto inhabitants and underline the need to further investigate the presence and sources of inferred chemicals in soils. The combination of bioassays and proteomics reveals a more complex scenario of chemicals able to affect cellular pathways and leading to toxicities rather than those identified by only bioassays and related chemical analysis. This combined approach turns out to be a promising tool for soil risk assessment and deserves further investigation and developments for soil monitoring and risk assessment.

Particle-Bound PAHs and Elements in a Highly Industrialized City in Southern Italy: PM2.5 Chemical Characterization and Source Apportionment after the Implementation of Governmental Measures for Air Pollution Mitigation and Control.

The present study was aimed at determining airborne concentrations of PAHs, Nitro-/Oxy-PAHs and elements in industrial and urban areas of Taranto, a site of environmental risk in Southern Italy, after the issue of strategic measures for air pollution mitigation and control by the Italian Environment Ministry in 2012. A PM2.5 sampling campaign was carried out from 9 to 28 December 2014 at eight receptor sites, two placed in the urban settlement and five included in the high spatial resolution fence monitoring network of the biggest European steel plant. The integration of collected data with meteorological parameters and source apportionment analysis by Positive Matrix Factorization and bivariate polar plots allowed to discriminate among emission sources and estimate their contributions. Evidence on the effect of distinct processes (homogenization, sintering) occurring inside the steel plant on airborne concentrations of PAHs and selected elements was provided. The impact of emissions from the steel plant "core" on the surrounding area was observed at receptor sites downwind to it. Moreover, the extent of the effectiveness of mitigation measures, partially applied at the moment of study's beginning, was demonstrated by mean and peak pollutant concentrations at all receptor sites up to one order of magnitude lower than those documented prior to 2012.

Uncoupling proteins 1 and 2 (UCP1 and UCP2) from Arabidopsis thaliana are mitochondrial transporters of aspartate, glutamate, and dicarboxylates.

The Arabidopsis thaliana genome contains 58 members of the solute carrier family SLC25, also called the mitochondrial carrier family, many of which have been shown to transport specific metabolites, nucleotides, and cofactors across the mitochondrial membrane. Here, two Arabidopsis members of this family, AtUCP1 and AtUCP2, which were previously thought to be uncoupling proteins and hence named UCP1/PUMP1 and UCP2/PUMP2, respectively, are assigned with a novel function. They were expressed in bacteria, purified, and reconstituted in phospholipid vesicles. Their transport properties demonstrate that they transport amino acids (aspartate, glutamate, cysteine sulfinate, and cysteate), dicarboxylates (malate, oxaloacetate, and 2-oxoglutarate), phosphate, sulfate, and thiosulfate. Transport was saturable and inhibited by mercurials and other mitochondrial carrier inhibitors to various degrees. AtUCP1 and AtUCP2 catalyzed a fast counterexchange transport as well as a low uniport of substrates, with transport rates of AtUCP1 being much higher than those of AtUCP2 in both cases. The aspartate/glutamate heteroexchange mediated by AtUCP1 and AtUCP2 is electroneutral, in contrast to that mediated by the mammalian mitochondrial aspartate glutamate carrier. Furthermore, both carriers were found to be targeted to mitochondria. Metabolite profiling of single and double knockouts shows changes in organic acid and amino acid levels. Notably, AtUCP1 and AtUCP2 are the first reported mitochondrial carriers in Arabidopsis to transport aspartate and glutamate. It is proposed that the primary function of AtUCP1 and AtUCP2 is to catalyze an aspartateout/glutamatein exchange across the mitochondrial membrane and thereby contribute to the export of reducing equivalents from the mitochondria in photorespiration.

Mitochondrial ATP-Mg/phosphate carriers transport divalent inorganic cations in complex with ATP.

The ATP-Mg/phosphate carriers (APCs) modulate the intramitochondrial adenine nucleotide pool size. In this study the concentration-dependent effects of Mg2+ and other divalent cations (Me2+) on the transport of [3H]ATP in liposomes reconstituted with purified human and Arabidopsis APCs (hAPCs and AtAPCs, respectively, including some lacking their N-terminal domains) have been investigated. The transport of Me2+ mediated by these proteins was also measured. In the presence of a low external concentration of [3H]ATP (12 µM) and increasing concentrations of Me2+, Mg2+ stimulated the activity (measured as initial transport rate of [3H]ATP) of hAPCs and decreased that of AtAPCs; Fe2+ and Zn2+ stimulated markedly hAPCs and moderately AtAPCs; Ca2+ and Mn2+ markedly AtAPCs and moderately hAPCs; and Cu2+ decreased the activity of both hAPCs and AtAPCs. All the Me2+-dependent effects correlated well with the amount of ATP-Me complex present. The transport of [14C]AMP, which has a much lower ability of complexation than ATP, was not affected by the presence of the Me2+ tested, except Cu2+. Furthermore, the transport of [3H]ATP catalyzed by the ATP/ADP carrier, which is known to transport only free ATP and ADP, was inhibited by all the Me2+ tested in an inverse relationship with the formation of the ATP-Me complex. Finally, direct measurements of Mg2+, Mn2+, Fe2+, Zn2+ and Cu2+ showed that they are cotransported with ATP by both hAPCs and AtAPCs. It is likely that in vivo APCs transport free ATP and ATP-Mg complex to different degrees, and probably trace amounts of other Me2+ in complex with ATP.

Functional characterization and organ distribution of three mitochondrial ATP-Mg/Pi carriers in Arabidopsis thaliana.

The Arabidopsis thaliana genome contains 58 membrane proteins belonging to the mitochondrial carrier family. Three members of this family, here named AtAPC1, AtAPC2, and AtAPC3, exhibit high structural similarities to the human mitochondrial ATP-Mg(2+)/phosphate carriers. Under normal physiological conditions the AtAPC1 gene was expressed at least five times more than the other two AtAPC genes in flower, leaf, stem, root and seedlings. However, in stress conditions the expression levels of AtAPC1 and AtAPC3 change. Direct transport assays with recombinant and reconstituted AtAPC1, AtAPC2 and AtAPC3 showed that they transport phosphate, AMP, ADP, ATP, adenosine 5'-phosphosulfate and, to a lesser extent, other nucleotides. AtAPC2 and AtAPC3 also had the ability to transport sulfate and thiosulfate. All three AtAPCs catalyzed a counter-exchange transport that was saturable and inhibited by pyridoxal-5'-phosphate. The transport activities of AtAPCs were also inhibited by the addition of EDTA or EGTA and stimulated by the addition of Ca(2+). Given that phosphate and sulfate can be recycled via their own specific carriers, these findings indicate that AtAPCs can catalyze net transfer of adenine nucleotides across the inner mitochondrial membrane in exchange for phosphate (or sulfate), and that this transport is regulated both at the transcriptional level and by Ca(2+).

Mitochondrial transporters for ornithine and related amino acids: a review.

Among the members of the mitochondrial carrier family, there are transporters that catalyze the translocation of ornithine and related substrates, such as arginine, homoarginine, lysine, histidine, and citrulline, across the inner mitochondrial membrane. The mitochondrial carriers ORC1, ORC2, and SLC25A29 from Homo sapiens, BAC1 and BAC2 from Arabidopsis thaliana, and Ort1p from Saccharomyces cerevisiae have been biochemically characterized by transport assays in liposomes. All of them transport ornithine and amino acids with side chains terminating at least with one amine. There are, however, marked differences in their substrate specificities including their affinity for ornithine (KM values in the mM to µM range). These differences are most likely reflected by minor differences in the substrate binding sites of these carriers. The physiological role of the above-mentioned mitochondrial carriers is to link several metabolic pathways that take place partly in the cytosol and partly in the mitochondrial matrix and to provide basic amino acids for mitochondrial translation. In the liver, human ORC1 catalyzes the citrulline/ornithine exchange across the mitochondrial inner membrane, which is required for the urea cycle. Human ORC1, ORC2, and SLC25A29 are likely to be involved in the biosynthesis and transport of arginine, which can be used as a precursor for the synthesis of NO, agmatine, polyamines, creatine, glutamine, glutamate, and proline, as well as in the degradation of basic amino acids. BAC1 and BAC2 are implicated in some processes similar to those of their human counterparts and in nitrogen and amino acid metabolism linked to stress conditions and the development of plants. Ort1p is involved in the biosynthesis of arginine and polyamines in yeast.