Chronic Effect of Aspartame on Ionic Homeostasis and Monoamine Neurotransmitters in the Rat Brain.

Aspartame is one of the most widely used artificial sweeteners globally. Data concerning acute neurotoxicity of aspartame is controversial, and knowledge on its chronic effect is limited. In the current study, we investigated the chronic effects of aspartame on ionic homeostasis and regional monoamine neurotransmitter concentrations in the brain. Our results showed that aspartame at high dose caused a disturbance in ionic homeostasis and induced apoptosis in the brain. We also investigated the effects of aspartame on brain regional monoamine synthesis, and the results revealed that there was a significant decrease of dopamine in corpus striatum and cerebral cortex and of serotonin in corpus striatum. Moreover, aspartame treatment significantly alters the tyrosine hydroxylase activity and amino acids levels in the brain. Our data suggest that chronic use of aspartame may affect electrolyte homeostasis and monoamine neurotransmitter synthesis dose dependently, and this might have a possible effect on cognitive functions.

Glyphosate commercial formulation causes cytotoxicity, oxidative effects, and apoptosis on human cells: differences with its active ingredient.

In the present study, the effects on oxidative balance and cellular end points of glyphosate, aminomethylphosphonic acid (AMPA), and a glyphosate formulation (G formulation) were examined in HepG2 cell line, at dilution levels far below agricultural recommendations. Our results show that G formulation had toxic effects while no effects were found with acid glyphosate and AMPA treatments. Glyphosate formulation exposure produced an increase in reactive oxygen species, nitrotyrosine formation, superoxide dismutase activity, and glutathione (GSH) levels, while no effects were observed for catalase and GSH-S-transferase activities. Also, G formulation triggered caspase 3/7 activation and hence induced apoptosis pathway in this cell line. Aminomethylphosphonic acid exposure produced an increase in GSH levels while no differences were observed in other antioxidant parameters. No effects were observed when the cells were exposed to acid glyphosate. These results confirm that G formulations have adjuvants working together with the active ingredient and causing toxic effects that are not seen with acid glyphosate.

Tyrosine-sulfate isosteres of CCR5 N-terminus as tools for studying HIV-1 entry.

The HIV-1 co-receptor CCR5 possesses sulfo-tyrosine (TYS) residues at its N-terminus (Nt) that are required for binding HIV-1 gp120 and mediating viral entry. By using a 14-residue fragment of CCR5 Nt containing two TYS residues, we recently showed that CCR5 Nt binds gp120 through a conserved region specific for TYS moieties and suggested that this site may represent a target for inhibitors and probes of HIV-1 entry. As peptides containing sulfo-tyrosines are difficult to synthesize and handle due to limited stability of the sulfo-ester moiety, we have now incorporated TYS isosteres into CCR5 Nt analogs and assessed their binding to a complex of gp120-CD4 using saturation transfer difference (STD) NMR and surface plasmon resonance (SPR). STD enhancements for CCR5 Nt peptides containing tyrosine sulfonate (TYSN) in complex with gp120-CD4 were very similar to those observed for sulfated CCR5 Nt peptides indicating comparable modes of binding. STD enhancements for phosphotyrosine-containing CCR5 Nt analogs were greatly diminished consistent with earlier findings showing sulfo-tyrosine to be essential for CCR5 Nt binding to gp120. Tyrosine sulfonate-containing CCR5 peptides exhibited reduced water solubility, limiting their use in assay and probe development. To improve solubility, we designed, synthesized, and incorporated in CCR5 Nt peptide analogs an orthogonally functionalized azido tris(ethylenoxy) l-alanine (l-ate-Ala) residue. Through NMR and SPR experiments, we show a 19-residue TYSN-containing peptide to be a functional, hydrolytically stable CCR5 Nt isostere that was in turn used to develop both SPR-based and ELISA assays to screen for inhibitors of CCR5 binding to gp120-CD4.

Phenelzine reduces the oxidative damage induced by peroxynitrite in plasma lipids and proteins.

Peroxynitrite is a reactive nitrogen species produced in the intravascular compartment from superoxide anion and nitric oxide. Peroxynitrite destroys blood plasma proteins and membranes of red blood cells and of platelets. This explains why excessive production of peroxynitrite contributes to diseases and to ageing. Therapeutics that antagonize peroxynitrite may delay ageing and the progression of disease. We developed an in vitro assay that allows the investigation of the oxidative damage caused by peroxynitrite in the intravascular compartment. This assay correlates the damage with the rate of formation of protein carbonyl groups, 3-nitrotyrosine (3-NT) and thiobarbituric acid reactive substances. Using this assay, we evaluated the ability of phenelzine, a scavenger of reactive aldehydes, to antagonize the effects of peroxynitrite. Herein, we showed that phenelzine significantly decreased the lipid peroxidative damage caused by peroxynitirite in blood plasma and platelets. Moreover, it inhibited carbonyl group and 3-NT formation in blood plasma and platelet proteins.

Polyphenol-Rich Fraction of Parquetina nigrescens Mitigates Dichlorvos-Induced Cardiorenal Dysfunction Through Reduction in Cardiac Nitrotyrosine and Renal p38 Expressions in Wistar Rats.

Parquetina nigrescens is commonly used to treat diseases in humans and animals in developing countries, including Nigeria. This study evaluates the effects of its polyphenol-rich fraction (prf) on dichlorvos-induced cardio- and renal toxicity. There were several factors assessed during this study, including cardiac and renal markers, serum myeloperoxidase and xanthine oxidase, and electrocardiograph (ECG) changes. The changes in electrocardiograph (ECG) were recorded. Immunohistochemistry of cardiac and renal p38 and nitrotyrosine was determined. Dichlorvos exposure caused a significant decrease in L-glutathione (reduced glutathione) and other antioxidant enzymes with increases in malondialdehyde, myeloperoxidase, advanced oxidation protein products, and protein carbonyl levels. It also brought about alterations in microanatomy of the heart and kidneys accompanied by increases in serum creatinine and urea levels. Exposure to dichlorvos induced prolonged QRS interval and shortened QT durations in rats. Immunohistochemistry revealed lower expressions of cardiac nitrotyrosine and renal p38 (mitogen-activated protein kinase; MAPK) in rats treated with prf of P. nigrescens. Combining all, prf of P. nigrescens demonstrated antioxidant as well as protective properties in the heart and kidneys of rats exposed to dichlorvos. It ameliorated dichlorvos-induced cardio- and nephrotoxicity giving credence to its use in ethnomedicine.

Tyrosine agonists reverse the molecular defects associated with dominant-negative mutations in human peroxisome proliferator-activated receptor gamma.

Loss-of-function mutations in the ligand-binding domain of human peroxisome proliferator-activated receptor gamma (PPARgamma) are associated with a novel syndrome characterized by partial lipodystrophy and severe insulin resistance. Here we have further characterized the properties of natural dominant-negative PPARgamma mutants (P467L, V290M) and evaluated the efficacy of putative natural ligands and synthetic thiazolidinedione (TZD) or tyrosine-based (TA) receptor agonists in rescuing mutant receptor function. A range of natural ligands failed to activate the PPARgamma mutants and their transcriptional responses to TZDs (e.g. pioglitazone, rosiglitazone) were markedly attenuated, whereas TAs (e.g. farglitazar) corrected defects in ligand binding and coactivator recruitment by the PPARgamma mutants, restoring transcriptional function comparable with wild-type receptor. Transcriptional silencing via recruitment of corepressor contributes to dominant-negative inhibition of wild type by the P467L and V290M mutants and the introduction of an artificial mutation (L318A) disrupting corepressor interaction abrogated their dominant-negative activity. More complete ligand-dependent corepressor release and reversal of dominant-negative inhibition was achieved with TA than TZD agonists. Modeling suggests a structural basis for these observations: both mutations destabilize helix 12 to favor receptor-corepressor interaction; conversely, farglitazar makes more extensive contacts than rosiglitazone within the ligand-binding pocket, to stabilize helix 12, facilitating corepressor release and transcriptional activation. Farglitazar was a more potent inducer of PPARgamma target gene (aP2) expression in peripheral blood mononuclear cells with the P467L mutation. Having shown that rosiglitazone is of variable and limited efficacy in these subjects, we suggest that TAs may represent a more rational therapeutic approach.

The influence of ATP on the binding of aromatic amino acids to the ligand response domain of the tyrosine repressor of Haemophilus influenzae.

The binding of aromatic amino acids to the ligand response domain of the tyrosine repressor (TyrR) protein (TyrR(lrd)) of Haemophilus influenzae was investigated using circular dichroism and fluorescence spectroscopy. The induced secondary structural changes were unique for each aromatic amino acid and were further influenced by the presence or absence of ATP. Tyrosine was found to have the highest affinity for TyrR(lrd) in the absence of ATP, whereas the affinity for ATP itself increased in the presence of tyrosine. Binding of tyrosine is therefore the conformational trigger for the activation of TyrR whereas ATP is regarded as a conformational co-activator.

(-)-Epicatechin 3-O-gallate ameliorates the damages related to peroxynitrite production by mechanisms distinct from those of other free radical inhibitors.

This study was carried out to elucidate whether the protective activity of (-)-epicatechin 3-O-gallate (ECg) against excessive peroxynitrite (ONOO(-)) production, is distinct from the activity of several well-known free radical inhibitors, the ONOO(-) inhibitors ebselen and uric acid, the superoxide anion (O(2)(-)) scavenger copper zinc superoxide dismutase (CuZnSOD) and the selective inducible nitric oxide synthase inhibitor L-N(6)-(1-iminoethyl)lysine hydrochloride (L-NIL). To generate ONOO(-), male Wistar rats (n = 6/group) were subjected to ischaemia-reperfusion process together with lipopolysaccharide (LPS) injection. Although ECg did not scavenge the ONOO(-) precursors nitric oxide (NO) and O(2)(-), it reduced the 3-nitrotyrosine level, a property similar to that of uric acid, but distinct from L-NIL. In addition, the elevation in myeloperoxidase activity was reversed by the administration of ECg, uric acid and SOD, but not by that of L-NIL. Furthermore, ECg was the more potent scavenger of the ONOO(-) decomposition product, the hydroxyl radical (*OH), than any other free radical inhibitor tested. The LPS plus ischaemia-reperfusion process resulted in renal dysfunction, estimated by measuring the parameters of renal function--serum urea nitrogen and creatinine levels. However, administration of ECg ameliorated renal dysfunction more than that of the other free radical inhibitors. Moreover, ECg reduced the excessive uric acid level, while the others did not, suggesting a property of ECg distinct from the others. Furthermore, proteinuria, which was demonstrated by the low- and high-molecular weight (LMW and HMW) protein bands of the sodium dodecyl sulfate-polyacrylamide gel electrophoresis pattern, caused by LPS plus ischaemia-reperfusion, was attenuated by administration of ECg and L-NIL, after which the HMW band intensities decreased and LMW protein bands were absent. This study indicates that, in an in-vivo model of ONOO(-) generation, ECg, L-NIL and uric acid exert stronger protective activity against ONOO(-)-induced oxidative damage than SOD and ebselen, and that the mechanism whereby ECg protects against ONOO(-) is distinct from that of L-NIL or uric acid.

Regulation of chloride permeability by endogenously produced tyramine in the Drosophila Malpighian tubule.

The Malpighian (renal) tubule of Drosophila melanogaster is a useful model for studying epithelial transport. The purpose of this study was to identify factors responsible for modulating transepithelial chloride conductance in isolated tubules. I have found that tyrosine and several of its metabolites cause an increase in chloride conductance. The most potent of these agonists is tyramine, which is active at low nanomolar concentrations; the pharmacology of this response matches that of the previously published cloned insect tyramine receptor. In addition, the tubule appears capable of synthesizing tyramine from applied tyrosine, as shown by direct measurement of tyrosine decarboxylase activity. Immunohistochemical staining of tubules with an antibody against tyramine indicates that the principal cells are the sites of tyramine production, whereas previous characterization of the regulation of chloride conductance suggests that tyramine acts on the stellate cells. This is the first demonstration of a physiological role for an insect tyramine receptor.