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Human proteins MTO1 and GTPBP3 are thought to jointly catalyze the modification of the wobble uridine in mitochondrial tRNAs. Defects in each protein cause infantile hypertrophic cardiomyopathy with lactic acidosis. However, the underlying mechanisms are mostly unknown. Using fibroblasts from an MTO1 patient and MTO1 silenced cells, we found that the MTO1 deficiency is associated with a metabolic reprogramming mediated by inactivation of AMPK, down regulation of the uncoupling protein 2 (UCP2) and transcription factor PPARγ, and activation of the hypoxia inducible factor 1 (HIF-1). As a result, glycolysis and oxidative phosphorylation are uncoupled, while fatty acid metabolism is altered, leading to accumulation of lipid droplets in MTO1 fibroblasts. Unexpectedly, this response is different from that triggered by the GTPBP3 defect, as GTPBP3-depleted cells exhibit AMPK activation, increased levels of UCP2 and PPARγ, and inactivation of HIF-1. In addition, fatty acid oxidation and respiration are stimulated in these cells. Therefore, the HIF-PPARγ-UCP2-AMPK axis is operating differently in MTO1- and GTPBP3-defective cells, which strongly suggests that one of these proteins has an additional role, besides mitochondrial-tRNA modification. This work provides new and useful information on the molecular basis of the MTO1 and GTPBP3 defects and on putative targets for therapeutic intervention.

The MnmE-MnmG complex of Escherichia coli uses either ammonium or glycine as a substrate to incorporate the 5-aminomethyl or 5-carboxymethylaminomethyl group into the wobble uridine of certain tRNAs. Both modifications can be converted into a 5-methylaminomethyl group by the independent oxidoreductase and methyltransferase activities of MnmC, which respectively reside in the MnmC(o) and MnmC(m) domains of this bifunctional enzyme. MnmE and MnmG, but not MnmC, are evolutionarily conserved. Bacillus subtilis lacks genes encoding MnmC(o) and/or MnmC(m) homologs. The glycine pathway has been considered predominant in this typical gram-positive species because only the 5-carboxymethylaminomethyl group has been detected in tRNA Lys UUU and bulk tRNA to date. Here, we show that the 5-methylaminomethyl modification is prevalent in B. subtilis tRNA Gln . Our data indicate that B. subtilis has evolved MnmC(o)- and MnmC(m)-like activities that reside in non MnmC homologous protein(s), which suggests that both activities provide some sort of biological advantage.UUG and tRNA Glu UUC . Our data indicate that B. subtilis has evolved MnmC(o)- and MnmC(m)-like activities that reside in non MnmC homologous protein(s), which suggests that both activities provide some sort of biological advantage.

In this review, we show how chromaffin cells have contributed to evaluate neuroprotective compounds with diverse mechanisms of action. Chromaffin cells are considered paraneurons, as they share many common features with neurons: (i) they synthesize, store, and release neurotransmitters upon stimulation and (ii) they express voltage-dependent calcium, sodium, and potassium channels, in addition to a wide variety of receptors. All these characteristics, together with the fact that primary cultures from bovine adrenal glands or chromaffin cells from the tumor pheochromocytoma cell line PC12 are easy to culture, make them an ideal model to study neurotoxic mechanisms and neuroprotective drugs. In the first part of this review, we will analyze the different cytotoxicity models related to calcium dyshomeostasis and neurodegenerative disorders like Alzheimer's or Parkinson's. Along the second part of the review, we describe how different classes of drugs have been evaluated in chromaffin cells to determine their neuroprotective profile in different neurodegenerative-related models.

Introduction: Global developmental delay (GDD) and intellectual disability (ID) are frequent reasons for consultation in paediatric neurology departments. Nowadays, array comparative genomic hybridisation (array-CGH) is one of the most widely used techniques for diagnosing these disorders. Our purpose was to determine the phenotypic features associated with pathological results in this genetic test., Methods: We conducted a blind study of the epidemiological, clinical, anthropometric, and morphological features of 80 patients with unexplained ID to determine which features were associated with pathological results in array-CGH., Results: Pathological results were found in 27.5% of the patients. Factors associated with pathological results in array-CGH were a family history of GDD/ID (OR = 12.1), congenital malformations (OR = 5.33), having more than 3 facial dysmorphic features (OR = 20.9), and hypotonia (OR = 3.25)., Conclusions: Our findings are consistent with those reported by other published series. We therefore conclude that the probability of having pathological results in array-CGH increases with the presence of any of the features mentioned above in patients with ID/GDD., (Copyright © 2016 Sociedad Española de Neurología. Publicado por Elsevier España, S.L.U. All rights reserved.)

Mitochondrial diseases due to mutations in the mitochondrial (mt) DNA are heterogeneous in clinical manifestations but usually include OXPHOS dysfunction. Mechanisms by which OXPHOS dysfunction contributes to the disease phenotype invoke, apart from cell energy deficit, maladaptive responses to mitochondria-to-nucleus retrograde signaling. Here we used five different cybrid models of mtDNA diseases to demonstrate that the expression of the nuclear-encoded mt-tRNA modification enzymes TRMU, GTPBP3 and MTO1 varies in response to specific pathological mtDNA mutations, thus altering the modification status of mt-tRNAs. Importantly, we demonstrated that the expression of TRMU, GTPBP3 and MTO1 is regulated by different miRNAs, which are induced by retrograde signals like ROS and Ca 2+ via different pathways. Our data suggest that the up- or down-regulation of the mt-tRNA modification enzymes is part of a cellular response to cope with a stoichiometric imbalance between mtDNA- and nuclear-encoded OXPHOS subunits. However, this miRNA-mediated response fails to provide full protection from the OXPHOS dysfunction; rather, it appears to aggravate the phenotype since transfection of the mutant cybrids with miRNA antagonists improves the energetic state of the cells, which opens up options for new therapeutic approaches.

Several oxidative phosphorylation (OXPHOS) diseases are caused by defects in the post-transcriptional modification of mitochondrial tRNAs (mt-tRNAs). Mutations in MTO1 or GTPBP3 impair the modification of the wobble uridine at position 5 of the pyrimidine ring and cause heart failure. Mutations in TRMU affect modification at position 2 and cause liver disease. Presently, the molecular basis of the diseases and why mutations in the different genes lead to such different clinical symptoms is poorly understood. Here we use Caenorhabditis elegans as a model organism to investigate how defects in the TRMU, GTPBP3 and MTO1 orthologues (designated as mttu-1, mtcu-1, and mtcu-2, respectively) exert their effects. We found that whereas the inactivation of each C. elegans gene is associated with a mild OXPHOS dysfunction, mutations in mtcu-1 or mtcu-2 cause changes in the expression of metabolic and mitochondrial stress response genes that are quite different from those caused by mttu-1 mutations. Our data suggest that retrograde signaling promotes defect-specific metabolic reprogramming, which is able to rescue the OXPHOS dysfunction in the single mutants by stimulating the oxidative tricarboxylic acid cycle flux through complex II. This adaptive response, however, appears to be associated with a biological cost since the single mutant worms exhibit thermosensitivity and decreased fertility and, in the case of mttu-1, longer reproductive cycle. Notably, mttu-1 worms also exhibit increased lifespan. We further show that mtcu-1; mttu-1 and mtcu-2; mttu-1 double mutants display severe growth defects and sterility. The animal models presented here support the idea that the pathological states in humans may initially develop not as a direct consequence of a bioenergetic defect, but from the cell's maladaptive response to the hypomodification status of mt-tRNAs. Our work highlights the important association of the defect-specific metabolic rewiring with the pathological phenotype, which must be taken into consideration in exploring specific therapeutic interventions.

Previously, we have described N-Bz-L-Glu[NH-2-(1-benzylpiperidin-4-yl)ethyl]-O-nHex (IQM9.21, L-1) as an interesting multifunctional neuroprotective compound for the potential treatment of neurodegenerative diseases. Here, we describe new derivatives and different synthetic routes, such as chemoenzymatic and solid-phase synthesis, aiming to improve the previously described route in solution. The lipase-catalysed amidation of L- and D-Glu diesters with N-benzyl-4-(2-aminoethyl)piperidine has been studied, using Candida antarctica and Mucor miehei lipases. In all cases, the α-amidated compound was obtained as the main product, pointing out that regioselectivity was independent of the reacting Glu enantiomer and the nature of the lipase. An efficient solid-phase route has been used to produce new donepezil-based L- and D-Glu derivatives, resulting in good yield. At micromolar concentrations, the new compounds inhibited human cholinesterases and protected neurons against toxic insults related to Alzheimer's disease and cerebral ischemia. The CNS-permeable compounds N-Cbz-L-Glu(OEt)-[NH-2-(1-benzylpiperidin-4-yl)ethyl] (L-3) and L-1 blocked the voltage-dependent calcium channels and L-3 protected rat hippocampal slices against oxygen-glucose deprivation, becoming promising anti-Alzheimer and anti-stroke lead compounds., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

GTPBP3 is an evolutionary conserved protein presumably involved in mitochondrial tRNA (mt-tRNA) modification. In humans, GTPBP3 mutations cause hypertrophic cardiomyopathy with lactic acidosis, and have been associated with a defect in mitochondrial translation, yet the pathomechanism remains unclear. Here we use a GTPBP3 stable-silencing model (shGTPBP3 cells) for a further characterization of the phenotype conferred by the GTPBP3 defect. We experimentally show for the first time that GTPBP3 depletion is associated with an mt-tRNA hypomodification status, as mt-tRNAs from shGTPBP3 cells were more sensitive to digestion by angiogenin than tRNAs from control cells. Despite the effect of stable silencing of GTPBP3 on global mitochondrial translation being rather mild, the steady-state levels and activity of Complex I, and cellular ATP levels were 50% of those found in the controls. Notably, the ATPase activity of Complex V increased by about 40% in GTPBP3 depleted cells suggesting that mitochondria consume ATP to maintain the membrane potential. Moreover, shGTPBP3 cells exhibited enhanced antioxidant capacity and a nearly 2-fold increase in the uncoupling protein UCP2 levels. Our data indicate that stable silencing of GTPBP3 triggers an AMPK-dependent retrograde signaling pathway that down-regulates the expression of the NDUFAF3 and NDUFAF4 Complex I assembly factors and the mitochondrial pyruvate carrier (MPC), while up-regulating the expression of UCP2. We also found that genes involved in glycolysis and oxidation of fatty acids are up-regulated. These data are compatible with a model in which high UCP2 levels, together with a reduction in pyruvate transport due to the down-regulation of MPC, promote a shift from pyruvate to fatty acid oxidation, and to an uncoupling of glycolysis and oxidative phosphorylation. These metabolic alterations, and the low ATP levels, may negatively affect heart function.

The neuroprotective profile of the dibenzothiadiazepine ITH12410/SC058 (2-chloro-5,6-dihydro-5,6-diacetyldibenzo[b,f][1,4,5]thiadiazepine) against several neurotoxicity models related to neurodegenerative diseases is herein described. ITH12410/SC058 protected SH-SY5Y cells against the loss of cell viability elicited by amyloid beta peptide and okadaic acid, a selective inhibitor of phosphoprotein phosphatase 2A that induces neurofibrillary tangle formation. Furthermore, ITH12410/SC058 is neuroprotective against several in vitro models of oxidative stress, that is, H2O2 exposure or incubation with rotenone plus oligomycin A in SH-SY5Y cells, and oxygen and glucose deprivation followed by reoxygenation in rat hippocampal slices. By contrast, ITH12410/SC058 was unable to significantly protect SH-SY5Y neuroblastoma cells against the toxicity elicited by Ca(2+) overload. Our results confirm the hypothesis that the dibenzothiadiazepine ITH12410/SC058 features its neuroprotective actions in a multitarget fashion, and is a promising drug for the treatment of neurodegenerative diseases.

In this work we describe a new family of dibenzo[1,4,5]thiadiazepines (2-12) that showed an interesting in vitro biological profile, namely neuroprotective and antioxidant properties, as well as blockade of cytosolic calcium entry. They showed no cytotoxic effects and the majority were predicted as CNS-permeable compounds. In human neuroblastoma cells they displayed good neuroprotective properties against mitochondrial oxidative stress which, in many cases, almost reached the full protection (>90%) when compounds were incubated with cells 24 h before the addition of toxic stressors. In co-incubation conditions these figures were smaller, although some compounds maintained an interesting level of neuroprotection, higher than 50%. Four selected compounds (2, 5, 8, and 11) were found to be effective antioxidant agents by sequestering mitochondrial radical oxygen species (ROS). Moreover, compound 2 showed a remarkable calcium-channel modulating activity. The interest of these compounds is increased by the fact that dibenzo[1,4,5]thiadiazepine is a barely known structure that is not difficult to synthesize and presents very few described derivatives, opening a new and broad line of research in Medicinal Chemistry., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)

Here, we describe a new family of melatonin-N,N-dibenzyl(N-methyl)amine hybrids that show a balanced multifunctional profile covering neurogenic, antioxidant, cholinergic, and neuroprotective properties at low-micromolar concentrations. They promote maturation of neural stem cells into a neuronal phenotype and thus they could contribute to CNS repair. They also protect neural cells against mitochondrial oxidative stress, show antioxidant properties, and inhibit human acetylcholinesterase (AChE). Moreover, they displace propidium from the peripheral anionic site of AChE, preventing the β-amyloid aggregation promoted by AChE. In addition, they show low cell toxicity and can penetrate into the CNS. This multifunctional profile highlights these melatonin-N,N-dibenzyl(N-methyl)amine hybrids as useful prototypes in the research of innovative drugs for Alzheimer's disease.

Introduction: Aminoglycosides like streptomycin are well-known for binding at specific regions of ribosome RNA and then acting as translation inhibitors. Nowadays, several pathogens have been detected to acquire an undefined strategy involving mutation at non structural ribosome genes like those acting as RNA methylases. rsmG is one of those genes which encodes an AdoMet-dependent methyltransferase responsible for the synthesis of m 7 G527 in the 530 loop of bacterial 16S rRNA. This loop is universally conserved, plays a key role in ribosomal accuracy, and is a target for streptomycin binding. Loss of the m 7 G527 modification confers low-level streptomycin resistance and may affect ribosomal functioning., Objectives: After taking into account genetic information indicating that some clinical isolates of human pathogens show streptomycin resistance associated with mutations at rsmG , we decided to explore new hot spots for mutation capable of impairing the RsmG in vivo function and of promoting low-level streptomycin resistance., Materials and Methods: To gain insights into the molecular and genetic mechanism of acquiring this aminoglycoside resistance phenotype and the emergence of high-level streptomycin resistance in rsmG mutants, we mutated Escherichia coli rsmG and also performed a genotyping study on rpsL from several isolates showing the ability to grow at higher streptomycin concentrations than parental strains., Results: We found that the mutations at rpsL were preferentially present in these mutants, and we observed a clear synergy between rsmG and rpsL genes to induce streptomycin resistance., Conclusion: We contribute to understand a common mechanism that is probably transferable to other ribosome RNA methylase genes responsible for modifications at central sites for ribosome function.

Posttranscriptional modification of the uridine located at the wobble position (U34) of tRNAs is crucial for optimization of translation. Defects in the U34 modification of mitochondrial-tRNAs are associated with a group of rare diseases collectively characterized by the impairment of the oxidative phosphorylation system. Retrograde signaling pathways from mitochondria to nucleus are involved in the pathophysiology of these diseases. These pathways may be triggered by not only the disturbance of the mitochondrial (mt) translation caused by hypomodification of tRNAs, but also as a result of nonconventional roles of mt-tRNAs and mt-tRNA-modifying enzymes. The evolutionary conservation of these enzymes supports their importance for cell and organismal functions. Interestingly, bacterial and eukaryotic cells respond to stress by altering the expression or activity of these tRNA-modifying enzymes, which leads to changes in the modification status of tRNAs. This review summarizes recent findings about these enzymes and sets them within the previous data context.

ITH12246 (ethyl 5-amino-2-methyl-6,7,8,9-tetrahydrobenzo[b][1,8]naphthyridine-3-carboxylate) is a 1,8-naphthyridine described to feature an interesting neuroprotective profile in in vitro models of Alzheimer's disease. These effects were proposed to be due in part to a regulatory action on protein phosphatase 2A inhibition, as it prevented binding of its inhibitor okadaic acid. We decided to investigate the pharmacological properties of ITH12246, evaluating its ability to counteract the memory impairment evoked by scopolamine, a muscarinic antagonist described to promote memory loss, as well as to reduce the infarct volume in mice suffering phototrombosis. Prior to conducting these experiments, we confirmed its in vitro neuroprotective activity against both oxidative stress and Ca(2+) overload-derived excitotoxicity, using SH-SY5Y neuroblastoma cells and rat hippocampal slices. Using a predictive model of blood-brain barrier crossing, it seems that the passage of ITH12246 is not hindered. Its potential hepatotoxicity was observed only at very high concentrations, from 0.1 mM. ITH12246, at the concentration of 10 mg/kg i.p., was able to improve the memory index of mice treated with scopolamine, from 0.22 to 0.35, in a similar fashion to the well-known Alzheimer's disease drug galantamine 2.5 mg/kg. On the other hand, ITH12246, at the concentration of 2.5 mg/kg, reduced the phototrombosis-triggered infarct volume by 67%. In the same experimental conditions, 15 mg/kg melatonin, used as control standard, reduced the infarct volume by 30%. All of these findings allow us to consider ITH12246 as a new potential drug for the treatment of neurodegenerative diseases, which would act as a multifactorial neuroprotectant.

MnmE is a homodimeric multi-domain GTPase involved in tRNA modification. This protein differs from Ras-like GTPases in its low affinity for guanine nucleotides and mechanism of activation, which occurs by a cis, nucleotide- and potassium-dependent dimerization of its G-domains. Moreover, MnmE requires GTP hydrolysis to be functionally active. However, how GTP hydrolysis drives tRNA modification and how the MnmE GTPase cycle is regulated remains unresolved. Here, the kinetics of the MnmE GTPase cycle was studied under single-turnover conditions using stopped- and quench-flow techniques. We found that the G-domain dissociation is the rate-limiting step of the overall reaction. Mutational analysis and fast kinetics assays revealed that GTP hydrolysis, G-domain dissociation and Pi release can be uncoupled and that G-domain dissociation is directly responsible for the 'ON' state of MnmE. Thus, MnmE provides a new paradigm of how the ON/OFF cycling of GTPases may regulate a cellular process. We also demonstrate that the MnmE GTPase cycle is negatively controlled by the reaction products GDP and Pi. This feedback mechanism may prevent inefficacious GTP hydrolysis in vivo. We propose a biological model whereby a conformational change triggered by tRNA binding is required to remove product inhibition and initiate a new GTPase/tRNA-modification cycle.

ITH33/IQM9.21 is a novel compound belonging to a family of glutamic acid derivatives, synthesized under the hypothesis implying that multitarget ligands may provide more efficient neuroprotection than single-targeted compounds. In rat hippocampal slices, oxygen plus glucose deprivation followed by re-oxygenation (OGD/Reox) elicited 42% cell death. At 1 μM, ITH33/IQM9.21 mitigated this damage by 26% and by 55% at 3 μM. OGD/Reox also elicited mitochondrial depolarization, overproduction of reactive oxygen species (ROS), enhanced expression of nitric oxide synthase (iNOS) and reduction of GSH levels. These changes were almost fully prevented when 3 μM ITH33/IQM9.21 was present during slice treatment with OGD/Reox. In isolated hippocampal neurons, ITH33/IQM9.21 reduced [Ca(2+)](c) transients induced by a high K(+) depolarizing solution or glutamate. In a photothrombotic model of stroke in mice, intraperitoneal injection of ITH33/IQM9.21 at 1.25 mg/kg, 2.5 mg/kg or 5 mg/kg given before and during 2 days after stroke induction, reduced infarct volume by over 45%. Furthermore, when the compound was administered 1 h post-stroke, a similar effect was observed. In conclusion, these in vitro and in vivo results suggest that ITH33/IQM9.21 exhibits neuroprotective effects to protect the vulnerable neurons at the ischemic penumbra by an effective and multifaceted mechanism, mediated by reduction of Ca(2+) overload, providing mitochondrial protection and antioxidant actions., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Modifying RNA enzymes are highly specific for substrate-rRNA or tRNA-and the target position. In Escherichia coli, there are very few multisite acting enzymes, and only one rRNA/tRNA dual-specificity enzyme, pseudouridine synthase RluA, has been identified to date. Among the tRNA-modifying enzymes, the methyltransferase responsible for the m(2)A synthesis at purine 37 in a tRNA set still remains unknown. m(2)A is also present at position 2503 in the peptidyl transferase center of 23S RNA, where it is introduced by RlmN, a radical S-adenosyl-L-methionine (SAM) enzyme. Here, we show that E. coli RlmN is a dual-specificity enzyme that catalyzes methylation of both rRNA and tRNA. The ΔrlmN mutant lacks m(2)A in both RNA types, whereas the expression of recombinant RlmN from a plasmid introduced into this mutant restores tRNA modification. Moreover, RlmN performs m(2)A(37) synthesis in vitro using a tRNA chimera as a substrate. This chimera has also proved useful to characterize some tRNA identity determinants for RlmN and other tRNA modification enzymes. Our data suggest that RlmN works in a late step during tRNA maturation by recognizing a precise 3D structure of tRNA. RlmN inactivation increases the misreading of a UAG stop codon. Since loss of m(2)A(37) from tRNA is expected to produce a hyperaccurate phenotype, we believe that the error-prone phenotype exhibited by the ΔrlmN mutant is due to loss of m(2)A from 23S rRNA and, accordingly, that the m(2)A2503 modification plays a crucial role in the proofreading step occurring at the peptidyl transferase center.

Benzothiazepine CGP37157 is widely used as tool to explore the role of mitochondria in cell Ca(2+) handling, by its blocking effect of the mitochondria Na(+)/Ca(2+) exchanger. Recently, CGP37157 has shown to exhibit neuroprotective properties. In the trend to improve its neuroprotection profile, we have synthesized ITH12505, an isosteric analogue having a methyl instead of chlorine at C2' of the phenyl ring. ITH12505 has exerted neuroprotective properties similar to CGP37157 in chromaffin cells and hippocampal slices stressed with veratridine. Also, both compounds afforded neuroprotection in hippocampal slices stressed with glutamate. However, while ITH12505 elicited protection in SH-SY5Y cells stressed with oligomycin A/rotenone, CGP37157 was ineffective. In hippocampal slices subjected to oxygen/glucose deprivation plus reoxygenation, ITH12505 offered protection at 3-30 μM, while CGP37157 only protected at 30 μM. Both compounds caused blockade of Ca(2+) channels in high K(+)-depolarized SH-SY5Y cells. An in vitro experiment for assaying central nervous system penetration (PAMPA-BBB; parallel artificial membrane permeability assay for blood-brain barrier) revealed that both compounds could cross the blood-brain barrier, thus reaching their biological targets in the central nervous system. In conclusion, by causing a mild isosteric replacement in the benzothiazepine CGP37157, we have obtained ITH12505, with improved neuroprotective properties. These findings may inspire the design and synthesis of new benzothiazepines targeting mitochondrial Na(+)/Ca(2+) exchanger and L-type voltage-dependent Ca(2+) channels, having antioxidant properties.

Among all RNAs, tRNA exhibits the largest number and the widest variety of post-transcriptional modifications. Modifications within the anticodon stem loop, mainly at the wobble position and purine-37, collectively contribute to stabilize the codon-anticodon pairing, maintain the translational reading frame, facilitate the engagement of the ribosomal decoding site and enable translocation of tRNA from the A-site to the P-site of the ribosome. Modifications at the wobble uridine (U34) of tRNAs reading two degenerate codons ending in purine are complex and result from the activity of two multi-enzyme pathways, the IscS-MnmA and MnmEG pathways, which independently work on positions 2 and 5 of the U34 pyrimidine ring, respectively, and from a third pathway, controlled by TrmL (YibK), that modifies the 2'-hydroxyl group of the ribose. MnmEG is the only common pathway to all the mentioned tRNAs, and involves the GTP- and FAD-dependent activity of the MnmEG complex and, in some cases, the activity of the bifunctional enzyme MnmC. The Escherichia coli MnmEG complex catalyzes the incorporation of an aminomethyl group into the C5 atom of U34 using methylene-tetrahydrofolate and glycine or ammonium as donors. The reaction requires GTP hydrolysis, probably to assemble the active site of the enzyme or to carry out substrate recognition. Inactivation of the evolutionarily conserved MnmEG pathway produces a pleiotropic phenotype in bacteria and mitochondrial dysfunction in human cell lines. While the IscS-MnmA pathway and the MnmA-mediated thiouridylation reaction are relatively well understood, we have limited information on the reactions mediated by the MnmEG, MnmC and TrmL enzymes and on the precise role of proteins MnmE and MnmG in the MnmEG complex activity. This review summarizes the present state of knowledge on these pathways and what we still need to know, with special emphasis on the MnmEG pathway., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)

RsmG is an AdoMet-dependent methyltransferase responsible for the synthesis of m(7)G527 in the 530 loop of bacterial 16S rRNA. This loop is universally conserved, plays a key role in ribosomal accuracy, and is a target for streptomycin binding. Loss of the m(7)G527 modification confers low-level streptomycin resistance and may affect ribosomal functioning. Here, we explore the mechanisms controlling RsmG expression and activity, which may somehow respond to the demand set by the amount of rRNA. We confirm that rsmG is the second member in a bicistronic operon and demonstrate that rsmG also has its own promoter, which appears, in actively growing cells, as a control device to offset both the relatively low stability of RsmG and inhibition of the operon promoter. RsmG levels decrease under conditions that down-regulate rRNA synthesis. However, coordination between rRNA and RsmG expression does not seem to occur at the level of transcription initiation. Instead, it might depend on the activity of an inverted repeated region, located between the rsmG promoter and ribosome binding site, which we show to work as a weak transcriptional terminator. To gain insights into the enzymatic mechanism of RsmG, highly conserved residues were mutated and the abilities of the resulting proteins to confer streptomycin resistance, to modify rRNA, and to bind AdoMet were explored. Our data demonstrate for the first time the critical importance of some residues located in the active site of Escherichia coli RsmG for the m(7)G modification process and suggest a role for them in rRNA binding and catalysis.

The synthesis and pharmacological analyses of a number of pyrazolo[3,4-b]quinoline and benzo[b]pyrazolo[4,3-g][1,8]naphthyridine derivatives are reported. We have synthesized the diversely substituted tacrine analogues 1-6, by Friedländer-type reaction of readily available o-amino-1-methyl-pyrazole-dicarbonitriles with cyclohexanone. The biological evaluation showed that pyrazolotacrines 1-6 are inhibitors of Electrophorus electricus acetylcholinesterase (EeAChE), in the micromolar range, and quite selective in respect to serum horse butyrylcholinesterase (eqBuChE) inhibition; the most interesting inhibitor is N-(5-amino-1-methyl-6,7,8,9-tetrahydro-1H-benzo[b]pyrazolo[4,3-g][1,8]naphthyridin-3-yl)acetamide (5) [IC(50) (EeAChE) = 0.069 ± 0.006 μM; IC(50) (eqBuChE) = 6.3 ± 0.6 μM]. Kinetic studies showed that compound 5 is a mixed-type inhibitor of EeAChE (K(i) = 155 nM). Inhibitor 5 showed a 45% neuroprotection value against rotenone/oligomycin A-induced neuronal death., (Copyright © 2011 Elsevier Masson SAS. All rights reserved.)

We have previously reported the multifunctional profile of N-(3-chloro-10H-phenothiazin-10-yl)-3-(dimethylamino)propanamide (1) as an effective neuroprotectant and selective butyrylcholinesterase inhibitor. In this paper, we have developed a series of N-acylaminophenothiazines obtained from our compound library or newly synthesised. At micro- and sub-micromolar concentrations, these compounds selectively inhibited butyrylcholinesterase (BuChE), protected neurons against damage caused by both exogenous and mitochondrial free radicals, showed low toxicity, and could penetrate into the CNS. In addition, N-(3-chloro-10H-phenothiazin-10-yl)-2-(pyrrolidin-1-yl)acetamide (11) modulated the cytosolic calcium concentration and protected human neuroblastoma cells against several toxics, such as calcium overload induced by an L-type Ca2+-channel agonist, tau-hyperphosphorylation induced by okadaic acid and Aβ peptide., (Copyright © 2011 Elsevier Masson SAS. All rights reserved.)

The synthesis, structure, theoretical and experimental in vitro antioxidant properties using the DPPH, ORAC, and benzoic acid, as well as preliminary in vitro pharmacological activities of (Z)-α-aryl and heteroaryl N-alkyl-nitrones 6-15, 18, 19, 21, and 23, is reported. In the in vitro antioxidant activity, for the DPPH radical test, only nitrones bearing free phenol groups gave the best RSA (%) values, nitrones 13 and 14 showing the highest values in this assay. In the ORAC analysis, the most potent radical scavenger was nitrone indole 21, followed by the N-benzyl benzene-type nitrones 10 and 15. Interestingly enough, the archetypal nitrone 7 (PBN) gave a low RSA value (1.4%) in the DPPH test, or was inactive in the ORAC assay. Concerning the ability to scavenge the hydroxyl radical, all the nitrones studied proved active in this experiment, showing high values in the 94-97% range, the most potent being nitrone 14. The theoretical calculations for the prediction of the antioxidant power, and the potential of ionization confirm that nitrones 9 and 10 are among the best compounds in electron transfer processes, a result that is also in good agreement with the experimental values in the DPPH assay. The calculated energy values for the reaction of ROS (hydroxyl, peroxyl) with the nitrones predict that the most favourable adduct-spin will take place between nitrones 9, 10, and 21, a fact that would be in agreement with their experimentally observed scavenger ability. The in vitro pharmacological analysis showed that the neuroprotective profile of the target molecules was in general low, with values ranging from 0% to 18.7%, in human neuroblastoma cells stressed with a mixture of rotenone/oligomycin-A, being nitrones 18, and 6-8 the most potent, as they show values in the range 24-18.4%., (Crown Copyright © 2010. Published by Elsevier Ltd. All rights reserved.)

The synthesis, biological assessment, and molecular modelling of new tacrine analogues 11-22 is described. Compounds 11-22 have been obtained by Friedländer-type reaction of 2-aminopyridine-3-carbonitriles 1-10 with cyclohexanone or 1-benzyl-4-piperidone. The biological evaluation showed that some of these molecules were good AChE inhibitors, in the nanomolar range, and quite selective regarding the inhibition of BuChE, the most potent being 5-amino-2-(dimethylamino)-6,7,8,9-tetrahydrobenzo[1,8-b]-naphthyridine-3-carbonitrile (11) [IC(50) (EeAChE: 14nM); IC(50) (eqBuChE: 5.2μM]. Kinetic studies on the easily available and potent anticholinesterasic compound 5-amino-2-(methoxy)-6,7,8,9-tetrahydrobenzo[1,8-b]-naphthyridine-3-carbonitrile (16) [IC(50) (EeAChE: 64nM); IC(50) (eqBuChE: 9.6μM] showed that this compound is a mixed-type inhibitor (K(i)=69.2nM) of EeAChE. Molecular modelling on inhibitor 16 confirms that this compound, as expected and similarly to tacrine, binds at the catalytic active site of EeAChE. The neuroprotective profile of molecules 11-22 has been investigated in SH-SY5Y neuroblastoma cells stressed with a mixture of oligomycin-A/rotenone. Compound 16 was also able to rescue by 50% cell death induced by okadaic acid in SH-SY5Y cells. From these results we conclude that the neuroprotective profile of these molecules is moderate, the most potent being compounds 12 and 17 which reduced cell death by 29%. Compound 16 does not affect ACh- nor K(+)-induced calcium signals in bovine chromaffin cells. Consequently, tacrine analogues 11-22 can be considered attractive therapeutic molecules on two key pharmacological targets playing key roles in the progression of Alzheimer, that is, cholinergic dysfunction and oxidative stress, as well as in neuronal cerebrovascular diseases., (Copyright © 2010. Published by Elsevier Ltd.)

From an in-house library of compounds, five phenothiazines and one dibenzothiadiazepine were selected to be tested in neuroprotective and cholinergic assays. Three of them, derived from the N-alkylphenothiazine, the N-acylaminophenothiazine, and the 1,4,5-dibenzo[b,f]thiadiazepine system, protected human neuroblastoma cells against oxidative stress generated by both exogenous and mitochondrial free radicals. They could also penetrate the CNS, according to an in vitro blood-brain barrier model, and an N-acylaminophenothiazine derivative behaved as a selective inhibitor of butyrylcholinesterase. Free radical capture and/or promotion of antioxidant protein biosynthesis are mechanisms that can be implicated in their neuroprotective actions. Due to their excellent pharmacological properties and the fact that they were not biologically explored in the past, one N-acylaminophenothiazine and one 1,4,5-dibenzo[b,f]thiadiazepine have been selected to develop two new series that are currently in progress., (Copyright © 2010 Elsevier Masson SAS. All rights reserved.)

Transfer RNAs are the most densely modified nucleic acid molecules in living cells. In Escherichia coli, more than 30 nucleoside modifications have been characterized, ranging from methylations and pseudouridylations to more complex additions that require multiple enzymatic steps. Most of the modifying enzymes have been identified, although a few notable exceptions include the 2'-O-methyltransferase(s) that methylate the ribose at the nucleotide 34 wobble position in the two leucyl isoacceptors tRNA(Leu)(CmAA) and tRNA(Leu)(cmnm5UmAA). Here, we have used a comparative genomics approach to uncover candidate E. coli genes for the missing enzyme(s). Transfer RNAs from null mutants for candidate genes were analyzed by mass spectrometry and revealed that inactivation of yibK leads to loss of 2'-O-methylation at position 34 in both tRNA(Leu)(CmAA) and tRNA(Leu)(cmnm5UmAA). Loss of YibK methylation reduces the efficiency of codon-wobble base interaction, as demonstrated in an amber suppressor supP system. Inactivation of yibK had no detectable effect on steady-state growth rate, although a distinct disadvantage was noted in multiple-round, mixed-population growth experiments, suggesting that the ability to recover from the stationary phase was impaired. Methylation is restored in vivo by complementing with a recombinant copy of yibK. Despite being one of the smallest characterized α/β knot proteins, YibK independently catalyzes the methyl transfer from S-adenosyl-L-methionine to the 2'-OH of the wobble nucleotide; YibK recognition of this target requires a pyridine at position 34 and N⁶-(isopentenyl)-2-methylthioadenosine at position 37. YibK is one of the last remaining E. coli tRNA modification enzymes to be identified and is now renamed TrmL.

The synthesis, molecular modeling, and pharmacological analysis of new multipotent simple, and readily available 2-aminopyridine-3,5-dicarbonitriles (3-20), and 2-chloropyridine-3,5-dicarbonitriles (21-28), prepared from 2-amino-6-chloropyridine-3,5-dicarbonitrile (1) and 2-amino-6-chloro-4-phenylpyridine-3,5-dicarbonitrile (2) is described. The biological evaluation showed that some of these molecules were modest inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE), in the micromolar range. The 2-amino (3, 4), and 2-chloro derivatives 21-23, 25, 26 were AChE selective inhibitors, whereas 2-amino derivatives 5, 14 proved to be selective for BuChE. Only inhibitor 24 was equipotent for both cholinesterases. Kinetic studies on compound 23 showed that this compound is a mixed-type inhibitor of AChE showing a K(i) of 6.33 microM. No clear SAR can be obtained form these data, but apparently, compounds bearing small groups such as the N,N'-dimethylamino or the pyrrolidino, regardless of the presence of a 2-amino, or 6-chloro substituent in the pyridine ring, preferentially inhibit AChE. Molecular modeling on inhibitors 4, 5, 22, and 23 has been carried out to give a better insight into the binding mode on the catalytic active site (CAS), and peripheral anionic site (PAS) of AChE. The most important differences in the observed binding relay on the modifications of the group at C2, as the amino group forms two hydrogen bonds that direct the binding mode, while in the case of compounds with a chlorine atom, this is not possible. The neuroprotective profile of these molecules has been investigated. In the LDH test, only compounds 26, 3, 22, and 24 showed neuroprotection with values in the range 37.8-31.6% in SH-SY5Y neuroblastoma cells stressed with a mixture of oligomycin-A/rotenone, but in the MTT test only compound 17 (32.9%) showed a similar profile. Consequently, these compounds can be considered as attractive multipotent therapeutic molecules on two key pharmacological receptors playing key roles in the progress of Alzheimer, that is, cholinergic dysfunction and oxidative stress, and neuronal vascular diseases., (Copyright 2010 Elsevier Ltd. All rights reserved.)

1,8-Naphthyridine derivatives related to 17 (ITH4012), a neuroprotective compound reported by our research group, have been synthesized. In general, they have shown better inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) than most tacrine derivatives previously synthesized in our laboratory. The compounds presented an interesting neuroprotective profile in SH-SY5Y neuroblastoma cells stressed with rotenone/oligomycin A. Moreover, compound 14 (ethyl 5-amino-2-methyl-6,7,8,9-tetrahydrobenzo[b][1,8]naphthyridine-3-carboxylate) also caused protection in cells stressed with okadaic acid (OA) or amyloid beta 1-42 peptide (Abeta(1-42)). Interestingly, compound 14 prevented the OA-induced PP2A inhibition, one of the enzymes implicated in tau dephosphorylation. This compound also exhibited neuroprotection against neurotoxicity elicited by oxygen and glucose deprivation in hippocampal slices. Because these stressors caused neuronal damage related to physiopathological hallmarks found in the brain of Alzheimer's disease (AD) patients, we conclude that compound 14 deserves further in vivo studies in AD models to test its therapeutic potential in this disease.

Tacrine and PBT2 (an 8-hydroxyquinoline derivative) are well-known drugs that inhibit cholinesterases and decrease beta-amyloid (Abeta) levels by complexation of redox-active metals, respectively. In this work, novel tacrine-8-hydroxyquinoline hybrids have been designed, synthesized, and evaluated as potential multifunctional drugs for the treatment of Alzheimer's disease. At nano- and subnanomolar concentrations they inhibit human acetyl- and butyrylcholinesterase (AChE and BuChE), being more potent than tacrine. They also displace propidium iodide from the peripheral anionic site of AChE and thus could be able to inhibit Abeta aggregation promoted by AChE. They show better antioxidant properties than Trolox, the aromatic portion of vitamin E responsible for radical capture, and display neuroprotective properties against mitochondrial free radicals. In addition, they selectively complex Cu(II), show low cell toxicity, and could be able to penetrate the CNS, according to an in vitro blood-brain barrier model.

The cysteine desulfurase IscS is a highly conserved master enzyme initiating sulfur transfer via persulfide to a range of acceptor proteins involved in Fe-S cluster assembly, tRNA modifications, and sulfur-containing cofactor biosynthesis. Several IscS-interacting partners including IscU, a scaffold for Fe-S cluster assembly; TusA, the first member of a sulfur relay leading to sulfur incorporation into the wobble uridine of several tRNAs; ThiI, involved in tRNA modification and thiamine biosynthesis; and rhodanese RhdA are sulfur acceptors. Other proteins, such as CyaY/frataxin and IscX, also bind to IscS, but their functional roles are not directly related to sulfur transfer. We have determined the crystal structures of IscS-IscU and IscS-TusA complexes providing the first insight into their different modes of binding and the mechanism of sulfur transfer. Exhaustive mutational analysis of the IscS surface allowed us to map the binding sites of various partner proteins and to determine the functional and biochemical role of selected IscS and TusA residues. IscS interacts with its partners through an extensive surface area centered on the active site Cys328. The structures indicate that the acceptor proteins approach Cys328 from different directions and suggest that the conformational plasticity of a long loop containing this cysteine is essential for the ability of IscS to transfer sulfur to multiple acceptor proteins. The sulfur acceptors can only bind to IscS one at a time, while frataxin and IscX can form a ternary complex with IscU and IscS. Our data support the role of frataxin as an iron donor for IscU to form the Fe-S clusters., Competing Interests: The authors have declared that no competing interests exist.

The MnmE-MnmG complex is involved in tRNA modification. We have determined the crystal structure of Escherichia coli MnmG at 2.4-A resolution, mutated highly conserved residues with putative roles in flavin adenine dinucleotide (FAD) or tRNA binding and MnmE interaction, and analyzed the effects of these mutations in vivo and in vitro. Limited trypsinolysis of MnmG suggests significant conformational changes upon FAD binding.

Novel multifunctional compounds have been designed, synthesized, and evaluated as potential drugs for the treatment of Alzheimer's disease (AD). With an L-glutamic moiety as a suitable biocompatible linker, three pharmacophoric groups were joined: (1) an N-benzylpiperidine fragment selected to inhibit acetylcholinesterase by interacting with the catalytic active site (CAS), (2) an N-protecting group of the amino acid, capable of interacting with the acetylcholinesterase (AChE)-peripheral anionic site (PAS) and protecting neurons against oxidative stress, and (3) a lipophilic alkyl ester that would facilitate penetration into the central nervous system by crossing the blood-brain barrier. At submicromolar concentration, they inhibit AChE and butyrylcholinesterase (BuChE) of human origin, displace the binding of propidium iodide from the PAS of AChE, and could thus inhibit Abeta aggregation promoted by AChE. They also display neuroprotective properties against mitochondrial free radicals, show low toxicity, and could be able to penetrate into the CNS.

Tacripyrines (1-14) have been designed by combining an AChE inhibitor (tacrine) with a calcium antagonist such as nimodipine and are targeted to develop a multitarget therapeutic strategy to confront AD. Tacripyrines are selective and potent AChE inhibitors in the nanomolar range. The mixed type inhibition of hAChE activity of compound 11 (IC(50) 105 +/- 15 nM) is associated to a 30.7 +/- 8.6% inhibition of the proaggregating action of AChE on the Abeta and a moderate inhibition of Abeta self-aggregation (34.9 +/- 5.4%). Molecular modeling indicates that binding of compound 11 to the AChE PAS mainly involves the (R)-11 enantiomer, which also agrees with the noncompetitive inhibition mechanism exhibited by p-methoxytacripyrine 11. Tacripyrines are neuroprotective agents, show moderate Ca(2+) channel blocking effect, and cross the blood-brain barrier, emerging as lead candidates for treating AD.

Tacrine-melatonin hybrids were designed and synthesized as new multifunctional drug candidates for Alzheimer's disease. These compounds may simultaneously palliate intellectual deficits and protect the brain against both beta-amyloid (A beta) peptide and oxidative stress. They show improved cholinergic and antioxidant properties, and are more potent and selective inhibitors of human acetylcholinesterase (hAChE) than tacrine. They also capture free radicals better than melatonin. Molecular modeling studies show that these hybrids target both the catalytic active site (CAS) and the peripheral anionic site (PAS) of AChE. At sub-micromolar concentrations they efficiently displace the binding of propidium iodide from the PAS and could thus inhibit A beta peptide aggregation promoted by AChE. Moreover, they also inhibit A beta self-aggregation and display neuroprotective properties in a human neuroblastoma line against cell death induced by various toxic insults, such as A beta(25-35), H(2)O(2), and rotenone. Finally, they exhibit low toxicity and may be able to penetrate the central nervous system according to an in vitro parallel artificial membrane permeability assay for the blood-brain barrier (PAMPA-BBB).

Marine sponges are becoming a rich source of potential new medicines. NP04634 is a synthetic derivative of 11,19 dideoxyfistularin, a natural product of the Mediterranean sponge Aplysina cavernicola. We report the cytoprotective effects of this new compound in isolated bovine chromaffin cells exposed to cytotoxic stimuli that have been related to neuronal cell death, i.e. Ca(2+) overload and mitochondrial dysfunction. Cell death was achieved by: (i) causing Ca(2+) overload through voltage-dependent calcium channels by exposing the cells to 30 mM K(+), 5 mM Ca(2+) plus 0.3 microM FPL64176 (an L-type Ca(2+)-channel activator); (ii) incubating the cells with veratridine, causing cytosolic Ca(2+) concentration ([Ca(2+)](c)) oscillations and mitochondrial disruption; and (iii) blocking mitochondrial complexes I and V using a combination of 30 microM rotenone and 10 microM oligomycin. At 10 microM, NP04634 caused significant protection against 30K(+)/5Ca(2+)/FPL-induced toxicity. NP04634 caused a concentration-dependent reduction in [Ca(2+)](c) induced by 70 mM K(+) in cells loaded with Fluo-4; maximum blockade was 67% at 30 microM. Veratridine caused continuous [Ca(2+)](c) oscillations that translated into 43.4+/-2% cell death. In this model, NP04634 caused 42% and 67% protection at 3 and 10 microM, respectively. NP04634 reduced [Ca(2+)](c) oscillations and mitochondrial depolarization caused by veratridine. NP04634 at 10 microM also protected against mitochondrial disruption caused by rotenone plus oligomycin. In conclusion, NP04634 is a novel compound of marine origin with cytoprotective properties that might have potential therapeutic implications under pathological circumstances involving Ca(2+) overload and mitochondrial disruption, such as in certain neurodegenerative diseases and/or stroke.

Forefoot varus is a static deformity not easy to assess clinically. If left uncorrected, it is thought to affect both the posture of the patient and the kinematics of their lower limbs, and even the spine. Three-dimensional gait assessment could help to confirm forefoot varus diagnosis and provide objective evidence of the functional adaptive mechanisms postulated in the literature. The recently available Oxford Foot Model was used, simultaneously with a conventional lower limb model, to compare the kinematics of 10 forefoot varus children (aged 8-13) and 11 healthy controls (aged 7-13) during gait. Data acquisition was performed using a six-camera motion capture system, with a total of 27 reflective markers. A patient-by-patient comparison with the controls suggested several compensation patterns, although statistically significant differences were found only for the mean values of hip adduction/abduction during load response and midstance and hip flexion/extension during pre-swing. A multivariate statistical technique was used to determine which of the measured variables better separated both groups. The best discriminant model presented here includes hip adduction/abduction during load response, hindfoot/tibia inversion/eversion during pre-swing, hindfoot/tibia dorsiflexion/plantar flexion during load response and arch height during midstance, providing a rate of correct classification of 81%. The results could not fully confirm the kinematic relationships suggested in the literature. The small degree of forefoot varus deformity present in the patient group could have prevented other variables from becoming discriminant. A larger patient sample would help determine the possible different compensatory patterns to different degrees of forefoot varus.

Human GTPBP3 is an evolutionarily conserved, multidomain protein involved in mitochondrial tRNA modification. Characterization of its biochemical properties and the phenotype conferred by GTPBP3 inactivation is crucial to understanding the role of this protein in tRNA maturation and its effects on mitochondrial respiration. We show that the two most abundant GTPBP3 isoforms exhibit moderate affinity for guanine nucleotides like their bacterial homologue, MnmE, although they hydrolyze GTP at a 100-fold lower rate. This suggests that regulation of the GTPase activity, essential for the tRNA modification function of MnmE, is different in GTPBP3. In fact, potassium-induced dimerization of the G domain leads to stimulation of the GTPase activity in MnmE but not in GTPBP3. The GTPBP3 N-terminal domain mediates a potassium-independent dimerization, which appears as an evolutionarily conserved property of the protein family, probably related to the construction of the binding site for the one-carbon-unit donor in the modification reaction. Partial inactivation of GTPBP3 by small interfering RNA reduces oxygen consumption, ATP production, and mitochondrial protein synthesis, while the degradation of these proteins slightly increases. It also results in mitochondria with defective membrane potential and increased superoxide levels. These phenotypic traits suggest that GTPBP3 defects contribute to the pathogenesis of some oxidative phosphorylation diseases.

In this communication, we describe the synthesis and biological evaluation of tacripyrimedones 1-5, a series of new tacrine-1,4-dihydropyridine hybrids bearing the general structure of 11-amino-12-aryl-3,3-dimethyl-3,4,5,7,8,9,10,12-octahydrodibenzo[b,g][1,8]naphthyridine-1(2H)-one. These multifunctional compounds are moderately potent and selective AChEIs, with no activity toward BuChE. Kinetic analysis and molecular modeling studies point out that the new compounds preferentially bind the peripheral anionic site of AChE. In addition, compounds 1-5 show an excellent neuroprotective profile, and a moderate blocking effect of L-type voltage-dependent calcium channels due to the mitigation of [Ca(2+)] elevation elicited by K(+) depolarization. Therefore, they represent a new family of molecules with potential therapeutic application for the treatment of Alzheimer's disease.

The synthesis and pharmacology of 6-amino-1,4-dihydropyridines, such as ethyl 6-amino-4-aryl-5-cyano-1,4-dihydro-2-methyl-3-pyridinecarboxylic acids (3-16) and 2-amino-4-aryl-7,7-dimethyl-5-oxo-1,4,5,6,7,8-hexahydro-3-quinolinenitriles (17-21) are described. Compounds 18 and 21, at the concentration of 0.3 microM, proved to be the best blockers of the [Ca(2+)] overload induced by depolarization with high [K(+)] of SH-SY5Y neuroblastoma cells, with values of 63.8% and 50.4%, respectively. Most of the compounds induced a remarkable neuroprotective effect against toxicity caused by high [K(+)]-elicited [Ca(2+)] overload, and against H(2)O(2)-generated free radicals, in SH-SY5Y cells.

Alzheimer's disease (AD) is associated with a gradual loss of attention and memory that has been related to impairment of brain cholinergic neurotransmission, particularly a deficit of cholinergic neurons. The first therapeutic target that has demonstrated therapeutic efficacy on cognition, behaviour and functional daily activities has been the inhibition of acetylcholinesterase. The acetylcholinesterase inhibitors used to treat AD patients at present are donepezil, rivastigmine and galantamine. This review summarises the current state of the art concerning the pharmacology of galantamine, focusing on the most important details of its possibilities as an acetylcholinesterase inhibitor, an allosteric potentiator of neuronal nicotinic receptors for acetylcholine, a modulator of neurotransmitter release, and an agent causing neuroprotection through an antiapoptotic action. In so doing, galantamine will be discussed in the context of the treatment of dementia, both of AD type and of mixed vascular-Alzheimer type.

We investigated the mechanism of the neuroprotective properties of chondroitin sulfate (CS), an endogenous perineuronal net glycosaminoglycan, in human neuroblastoma SH-SY5Y cells subjected to oxidative stress. Preincubation with CS for 24 h afforded concentration-dependent protection against H2O2-induced toxicity (50 microM for 24 h) measured as lactic dehydrogenase released to the incubation media; cell death was prevented at the concentrations of 600 and 1000 microM. Cell death caused by a combination of 10 microM rotenone plus 1 microM oligomycin-A (Rot/oligo) was also reduced by CS at concentrations ranging from 0.3 to 100 microM; in this toxicity model, maximum protection was achieved at 3 microM (48%). No significant protection was observed in a cell death model of Ca2+ overload (70 mM K+, for 24 h). H2O2 and Rot/oligo generated reactive oxygen species (ROS) measured as an increase in the fluorescence of dichlorofluorescein diacetate-loaded cells. CS drastically reduced ROS generation induced by both H2O2 (extracellular ROS) and Rot/oligo (intracellular ROS). CS also increased the expression of phosphorylated Akt and heme oxygenase-1 by 2-fold. The protective effects of CS were prevented by chelerythrine, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), cycloheximide, and Sn(IV)-protoporphyrin IX. Taken together, these results show that CS can protect SH-SY5Y cells under oxidative stress conditions by activating protein kinase C, which phosphorylates Akt that, via the phosphatidylinositol 3-kinase/Akt pathway, induces the synthesis of the antioxidant protein heme oxygenase-1.

The hypothesis that K(+) channels and cell depolarization are involved in neuronal death and neuroprotection was tested in bovine chromaffin cells subjected to two treatment periods: the first period (preconditioning period) lasted 6 to 48 h and consisted of treatment with high K(+) solutions or with tetraethylammonium (TEA), a K(+) channel blocker; the second period consisted of incubation with veratridine for 24 h, to cause cell damage. Preconditioning with high K(+) (20-80 mM) or TEA (10-30 mM) for 24 h caused 20-60% cytoprotection against veratridine-induced cell death in bovine chromaffin cells. The absence of Ca(2+) ions during the first 9 h of an 18-h preconditioning period abolished the cytoprotection. Preconditioning with K(+) or TEA increased by 2.5-fold the expression of brain-derived neurotrophic factor and by nearly 2-fold the expression of the antiapoptotic protein Bcl-2. However, preconditioning did not modify the veratridine-evoked Ca(2+) signal. High K(+) shifted the Em by about 10 mV and TEA evoked a transient burst of action potentials superimposed on a sustained depolarization. We conclude that preconditioning may protect chromaffin cells from death by blocking K(+) channels that depolarize the cell and cause a cytosolic Ca(2+) signal, leading to enhanced expression of BDNF and Bcl-2.

In this work we describe the synthesis and biological evaluation of the tacrine-1,4-dihydropyridine (DHP) hybrids (3-11). These multipotent molecules are the result of the juxtaposition of an acetylcholinesterase inhibitor (AChEI) such as tacrine (1) and a 1,4-DHP such as nimodipine (2). Compounds 3-11 are very selective and potent AChEIs and show an excellent neuroprotective profile and a moderate Ca2+ channel blockade effect. Consequently, these molecules are new potential drugs for the treatment of Alzheimer's disease.

The synthesis and the biological evaluation (neuroprotection, voltage dependent calcium channel blockade, AChE/BuChE inhibitory activity and propidium binding) of new multipotent tetracyclic tacrine analogues (5-13) are described. Compounds 7, 8 and 11 showed a significant neuroprotective effect on neuroblastoma cells subjected to Ca(2+) overload or free radical induced toxicity. These compounds are modest AChE inhibitors [the best inhibitor (11) is 50-fold less potent than tacrine], but proved to be very selective, as for most of them no BuChE inhibition was observed. In addition, the propidium displacement experiments showed that these compounds bind AChE to the peripheral anionic site (PAS) of AChE and, consequently, are potential agents that can prevent the aggregation of beta-amyloid. Overall, compound 8 is a modest and selective AChE inhibitor, but an efficient neuroprotective agent against 70mM K(+) and 60microM H(2)O(2). Based on these results, some of these molecules can be considered as lead candidates for the further development of anti-Alzheimer drugs.

The synthesis and biological evaluation of ethyl 5-amino-4-(3-pyridyl)-2-methyl-6,7,8,9-tetrahydro-4H-pyrano[2,3-b]quinoline-3-carboxylates (9-11) is described. We have found that these compounds inhibit AChE with a mild potency, mitigates the [Ca(2+)](c) triggered by high K(+), and cause neuroprotection against Ca(2+) overloading and free radical-induced neuronal death.

A gas chromatography/negative chemical ionization-mass spectrometry (GC/NCI-MS) method has been developed for the simultaneous determination of the fungicides captan and folpet in khaki (persimmon; flesh and peel) and cauliflower. Samples were extracted with acetone in the presence of 0.1 M zinc acetate solution in order to avoid degradation of fungicides and were purified using solid-phase extraction with divinylbenzene polymeric cartridges. Purified extracts were evaporated and dissolved in hexane prior to injection into the GC/NCI-MS system. Isotope-labeled captan and folpet were used as surrogate/internal standards, and quantification was performed using matrix-matched calibration. The method showed linear response in the concentration range tested (50-2500 ng/mL). The method was fully validated with untreated blank samples of khaki (flesh and peel) and cauliflower spiked at 0.05 and 0.5 mg/kg. Satisfactory recoveries between 82 and 106% and relative standard deviations lower than 11% in all cases (n = 5) were obtained. The limit of detection for both compounds were estimated to be 0.01 mg/kg. The developed method has been applied to treated and untreated samples collected from residue trials.

Field trial studies have been performed with captan and fenitrothion on cauliflower to propose maximum residue limits and to study the dissipation of the pesticides. Residue levels have been determined at different times following good laboratory practice using gas chromatography with mass spectrometric detection. The behaviour of residue levels of these compounds after household processing has been analysed using gas chromatography with electron capture detection. Seven days after treatment, residue levels of captan could be detected, but not of fenitrothion. The half-lives of dissipation for captan and fenitrothion were calculated as 0.9 and 1.8 days respectively. Washing did not significantly affect the residual amounts of captan and fenitrothion observed in raw vegetables; however, after cooking, captan had degraded completely, whereas residue levels of fenitrothion were not modified significantly.