Iris N. Serratos

Cannabinoid receptor agonists reduce the short-term mitochondrial dysfunction and oxidative stress linked to excitotoxicity in the rat brain.

The endocannabinoid system (ECS) is involved in a considerable number of physiological processes in the Central Nervous System. Recently, a modulatory role of cannabinoid receptors (CBr) and CBr agonists on the reduction of the N-methyl-d-aspartate receptor (NMDAr) activation has been demonstrated. Quinolinic acid (QUIN), an endogenous analog of glutamate and excitotoxic metabolite produced in the kynurenine pathway (KP), selectively activates NMDAr and has been shown to participate in different neurodegenerative disorders. Since the early pattern of toxicity exerted by this metabolite is relevant to explain the extent of damage that it can produce in the brain, in this work we investigated the effects of the synthetic CBr agonist WIN 55,212-2 (WIN) and other agonists (anandamide or AEA, and CP 55,940 or CP) on early markers of QUIN-induced toxicity in rat striatal cultured cells and rat brain synaptosomes. WIN, AEA and CP exerted protective effects on the QUIN-induced loss of cell viability. WIN also preserved the immunofluorescent signals for neurons and CBr labeling that were decreased by QUIN. The QUIN-induced early mitochondrial dysfunction, lipid peroxidation and reactive oxygen species (ROS) formation were also partially or completely prevented by WIN pretreatment, but not when this CBr agonist was added simultaneously with QUIN to brain synaptosomes. These findings support a neuroprotective and modulatory role of cannabinoids in the early toxic events elicited by agents inducing excitotoxic processes.

Molecular Mechanism of Tau Aggregation Induced by Anionic and Cationic Dyes

Abnormal tau filaments are a hallmark of Alzheimers disease. Anionic dyes such as Congo Red, Thiazine Red, and Thioflavin S are able to induce tau fibrillization in vitro. SH-SY5Y cells were incubated with each dye for seven days leading to intracellular aggregates of tau protein, with different morphological characteristics. Interestingly, these tau aggregates were not observed when the Methylene Blue dye was added to the cell culture. In order to investigate the molecular mechanisms underlying this phenomenon, we developed a computational model for the interaction of the tau paired helical filament (PHF) core with every dye by docking analysis. The polar/electrostatic and nonpolar contribution to the free binding energy in the tau PHF core-anionic dye interaction was determined. We found that the tau PHF core can generate a positive net charge within the binding site localized at residuesLys311 and Lys340 (numbering according to the longest isoform hTau40). These residues are important for the binding affinity of the negative charges present in the anionic dyes causing an electrostatic environment that stabilizes the complex. Tau PHF core protofibril-Congo Red interaction has a stronger binding affinity compared to Thiazine Red or Thioflavin S. By contrast, the cationic dye Methylene Blue does not bind to nor stabilize the tau PHF core protofibrils. These results characterize the driving forces responsible for the binding of tau to anionic dyes leading to their self-aggregation and suggest that Methylene Blue may act as a destabilizing agent of tau aggregates.

Acute restraint stress reduces hippocampal oxidative damage and behavior in rats: Effect of S-allyl cysteine.

AIMS This simple study was designed to investigate whether acute restraint stress can generate changes in behavioral tests and hippocampal endpoints of oxidative stress in rats, and if the antioxidant S-allyl cysteine (SAC) can prevent these alterations. MATERIALS AND METHODS We evaluated motor activity, forced swimming and anxiety behavior, as well as the hippocampal levels of lipid peroxidation and the activities of glutathione-related enzymes in animals submitted to mild immobilization. The effect of SAC (100 mg/kg, i.p.), given to rats every day 30 min before starting the immobilization session, was also investigated. Immobilization (restraint) stress was induced for a period of 6 h per day for five consecutive days. KEY FINDINGS Our results indicate that, under the tested conditions, acute restraint stimulates compensatory behavioral tasks (motor activity, anxiety and forced swimming) to counteract the stressing conditions prevailing, and selectively increased the levels of lipid peroxidation and the enzyme activities of glutathione-S-transferase (GST) and glutathione peroxidase (GPx) in the hippocampus also as adaptive responses. SAC exhibited preventive effects in the stressed group as it improved behavior, reduced lipid peroxidation and prevented the increase of GST and GPx activities, suggesting that this antioxidant blunted primary pro-oxidative stimuli induced by restraint stress. SIGNIFICANCE Findings of this work also confirm that the use of antioxidants such as SAC can provide effective protection against the acute oxidative damage associated with anxiety produced by stressing conditions.

Modeling the Interaction between Quinolinate and the Receptor for Advanced Glycation End Products (RAGE): Relevance for Early Neuropathological Processes

The receptor for advanced glycation end products (RAGE) is a pattern-recognition receptor involved in neurodegenerative and inflammatory disorders. RAGE induces cellular signaling upon binding to a variety of ligands. Evidence suggests that RAGE up-regulation is involved in quinolinate (QUIN)-induced toxicity. We investigated the QUIN-induced toxic events associated with early noxious responses, which might be linked to signaling cascades leading to cell death. The extent of early cellular damage caused by this receptor in the rat striatum was characterized by image processing methods. To document the direct interaction between QUIN and RAGE, we determined the binding constant (Kb) of RAGE (VC1 domain) with QUIN through a fluorescence assay. We modeled possible binding sites of QUIN to the VC1 domain for both rat and human RAGE. QUIN was found to bind at multiple sites to the VC1 dimer, each leading to particular mechanistic scenarios for the signaling evoked by QUIN binding, some of which directly alter RAGE oligomerization. This work contributes to the understanding of the phenomenon of RAGE-QUIN recognition, leading to the modulation of RAGE function.

Early expression of the receptor for advanced glycation end products in a toxic model produced by 6-hydroxydopamine in the rat striatum.

The receptor for advanced glycation end products (RAGE) is commonly involved in different neurodegenerative and inflammatory disorders. The cellular signaling associated to RAGE activation may occur upon binding to different ligands. In this study we investigated whether the toxic model produced by 6-hydroxydopamine (6-OHDA) in rats comprises early noxious responses related to RAGE-mediated signaling cascades. In order to explore a possible interaction between 6-OHDA and RAGE, affinity parameters of RAGE with 6-OHDA were estimated by different means. The possible binding sites of 6-OHDA with the VC1 homodimer for both rat and human RAGE were also modeled. Our results show that the striatal infusion of 6-OHDA recruits RAGE upregulation, as evidenced by an early expression of the receptor. 6-OHDA was also found to bind the VC1 homodimer, although its affinity was moderate when compared to other ligands. This work contributes to the understanding of the role of RAGE activation for 6-OHDA-induced neurotoxicity.

Chlorophyll a Covalently Bonded to Organo-Modified Translucent Silica Xerogels: Optimizing Fluorescence and Maximum Loading

Chlorophyll is a pyrrolic pigment with important optical properties, which is the reason it has been studied for many years. Recently, interest has been rising with respect to this molecule because of its outstanding physicochemical properties, particularly applicable to the design and development of luminescent materials, hybrid sensor systems, and photodynamic therapy devices for the treatment of cancer cells and bacteria. More recently, our research group has been finding evidence for the possibility of preserving these important properties of substrates containing chlorophyll covalently incorporated within solid pore matrices, such as SiO2, TiO2 or ZrO2 synthesized through the sol-gel process. In this work, we study the optical properties of silica xerogels organo-modified on their surface with allyl and phenyl groups and containing different concentrations of chlorophyll bonded to the pore walls, in order to optimize the fluorescence that these macrocyclic species displays in solution. The intention of this investigation was to determine the maximum chlorophyll a concentration at which this molecule can be trapped inside the pores of a given xerogel and to ascertain if this pigment remains trapped as a monomer, a dimer, or aggregate. Allyl and phenyl groups were deposited on the surface of xerogels in view of their important effects on the stability of the molecule, as well as over the fluorescence emission of chlorophyll; however, these organic groups allow the trapping of either chlorophyll a monomers or dimers. The determination of the above parameters allows finding the most adequate systems for subsequent in vitro or in vivo studies. The characterization of the obtained xerogels was performed through spectroscopic absorption, emission and excitation spectra. These hybrid systems can be employed as mimics of natural systems; the entrapment of chlorophyll inside pore matrices indicates that it is possible to exploit some of the most physicochemical properties of trapped chlorophyll for diverse technological applications. The data herein collected suggest the possibility of applying the developed methodology to other active, captive molecules in order to synthesize new hybrid materials with optimized properties, suitable to be applied in diverse technological fields.

Effects of pH on the Association between the Inhibitor Cystatin and the Proteinase Chymopapain

Cysteine proteinases are involved in many aspects of physiological regulation. In humans, some cathepsins have shown another function in addition to their role as lysosomal proteases in intracellular protein degradation; they have been implicated in the pathogenesis of several heart and blood vessel diseases and in cancer development. In this work, we present a fluorometric and computational study of the binding of one representative plant cysteine proteinase, chymopapain, to one of the most studied inhibitors of these proteinases: chicken cystatin. The binding equilibrium constant, Kb, was determined in the pH range between 3.5 and 10.0, revealing a maximum in the affinity at pH 9.0. We constructed an atomic model for the chymopapain-cystatin dimer by docking the individual 3D protein structures; subsequently, the model was refined using a 100 ns NPT molecular dynamics simulation in explicit water. Upon scrutiny of this model, we identified 14 ionizing residues at the interface of the complex using a cutoff distance of 5.0 Å. Using the pKa values predicted with PROPKA and a modified proton-linkage model, we performed a regression analysis on our data to obtain the composite pKavalues for three isoacidic residues. We also calculated the electrostatic component of the binding energy (ΔGb,elec) at different pH values using an implicit solvent model and APBS software. The pH profile of this calculated energy compares well with the experimentally obtained binding energy, ΔGb. We propose that the residues that form an interchain ionic pair, Lys139A from chymopapain and Glu19B from cystatin, as well as Tyr61A and Tyr67A from chymopapain are the main residues responsible for the observed pH dependence in the chymopapain- cystatin affinity.

Exploring interfacial water trapping in protein-ligand complexes with multithermal titration calorimetry

In this work, we examine the hypothesis about how trapped water molecules at the interface between triosephosphate isomerase (TIM) and either of two phosphorylated inhibitors, 2-phosphoglycolate (2PG) or phosphoglycolohydroxamate (PGH), can explain the anomalous highly negative binding heat capacities (ΔCp,b) of both complexes, TIM-2PG and TIM-PGH. We performed fluorimetric titrations of the enzyme with PGH inhibitor under osmotic stress conditions, using various concentrations of either osmolyte: sucrose, ethylene glycol or glycine betaine. We also analyze the binding processes under various stressor concentrations using a novel calorimetric methodology that allows ΔCp,b determinations in single experiments: Multithermal Titration Calorimetry. The binding constant of the TIM-PGH complex decreased gradually with the concentration of all osmolytes, but at diverse extents depending on the osmolyte nature. According to the osmotic stress theory, this decrease indicates that the number of water molecules associated with the enzyme increases with inhibitor binding, i.e. some solvent molecules became trapped. Additionally, the binding heat capacities became less negative at higher osmolyte concentrations, their final values depending on the osmolyte. These effects were also observed in the TIM-2PG complex using sucrose as stressor. Our results strongly suggest that some water molecules became immobilized when the TIM-inhibitor complexes were formed. A computational analysis of the hydration state of the binding site of TIM in both its free state and its complexed form with 2PG or PGH, based on molecular dynamics (MD) simulations in explicit solvent, showed that the binding site effectively immobilized additional water molecules after binding these inhibitors.