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1.
Pediatr Diabetes ; 21(5): 791-799, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32181961

RESUMO

OBJECTIVE: To compare the duration (hours until HCO 3 - ≥ 15 mmol/L) of diabetic ketoacidosis (DKA) episodes that are the first manifestation of new type 1 diabetes (NT1D) and those that are a complication in patients with previously diagnosed type 1 diabetes (PT1D). METHODS: A multicenter retrospective cohort study was designed. The duration of DKA was measured from the start of the treatment. The primary outcome was the comparison of the time needed in each group to reach HCO 3 - ≥ 15 mmol/L. The secondary outcomes were the comparison of the time to reach pH ≥ 7.3 and length of hospital stay in each group. Data were analyzed with a bivariate analysis of the variables vs primary outcome. Then, a regression model was analyzed. Results There were 305 episodes included (NT1D: 115, PT1D: 190). DKA in the NT1D group lasted longer (NT1D 20 (16-19) vs PT1D 12 (8-16), hours, P < .01) with a significant difference in each level of DKA severity. This group also took longer to reach pH ≥ 7.3 (NT1D 16 (12-22) vs PT1D 9 (6-12), hours, P < .01) and had a longer hospital stay (NT1D 9 (6-12) vs PT1D 7 (4-10), hours, P < .01). CONCLUSION: The duration of DKA is longer in patients with NT1D regardless of characteristics like DKA severity, duration of symptoms, and type of treatments received.

2.
Sci Rep ; 10(1): 4804, 2020 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-32179786

RESUMO

Glycine receptors (GlyRs) are anion-permeable pentameric ligand-gated ion channels (pLGICs). The GlyR activation is critical for the control of key neurophysiological functions, such as motor coordination, respiratory control, muscle tone and pain processing. The relevance of the GlyR function is further highlighted by the presence of abnormal glycinergic inhibition in many pathophysiological states, such as hyperekplexia, epilepsy, autism and chronic pain. In this context, previous studies have shown that the functional inhibition of  GlyRs containing the α3 subunit is a pivotal mechanism of pain hypersensitivity. This pathway involves the activation of EP2 receptors and the subsequent PKA-dependent phosphorylation of α3GlyRs within the intracellular domain (ICD), which decrease the GlyR-associated currents and enhance neuronal excitability. Despite the importance of this mechanism of glycinergic dis-inhibition associated with dysfunctional α3GlyRs, our current understanding of the molecular events involved is limited. Here, we report that the activation of PKA signaling pathway decreases the unitary conductance of α3GlyRs. We show in addition that the substitution of the PKA-targeted serine with a negatively charged residue within the ICD of α3GlyRs and of chimeric receptors combining bacterial GLIC and α3GlyR was sufficient to generate receptors with reduced conductance. Thus, our findings reveal a potential biophysical mechanism of glycinergic dis-inhibition and suggest that post-translational modifications of the ICD, such as phosphorylation, may shape the conductance of other pLGICs.

3.
J Chem Inf Model ; 60(2): 995-1004, 2020 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-31876421

RESUMO

Neuroligin-1 (NL1) is a postsynaptic cell adhesion protein that plays a crucial role in synapsis and signaling between neurons. Due to its clustered distribution in synaptic clefts, NL1 appears as a novel potential site for synaptic targeting purposes. In this work, in silico protein topography analysis was employed to identify two prospective binding sites on the NL1 dimer surface in the 2:2 synaptic adhesion complex with ß-neurexin (PDB code 3B3Q ). Receptor-based rational design, cell-penetrating capability prediction, molecular docking, molecular dynamics simulations, and binding free energy calculations were used to identify five heptapeptides candidates with favorable predicted profiles as non cell-penetrating NL1-binding agents. Preliminary in vitro colocalization assays with NL1-transfected HEK 293 cells confirmed that peptides remain in the extracellular space without inducing detectable changes in cell morphology. The highest NL1-colocatization capability was attained by the peptide ADEAIVA, which appears as a promising candidate for the future development of specific NL1-targeting systems as part of synapse-directed therapies against central nervous system diseases.

4.
Front Pharmacol ; 10: 331, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31024303

RESUMO

Glycine receptors (GlyRs) are chloride-permeable pentameric ligand-gated ion channels. The inhibitory activity of GlyRs is essential for many physiological processes, such as motor control and respiration. In addition, several pathological states, such as hyperekplexia, epilepsy, and chronic pain, are associated with abnormal glycinergic inhibition. Recent studies have pointed out that positive allosteric modulators targeting the GlyR α3 subunit (α3GlyR) displayed beneficial effects in chronic pain models. Interestingly, previous electrophysiological studies have shown that tropeines, which are a family of synthetic antagonists of the serotonin type 3 receptors (5-HT3Rs), potentiate the activity of GlyRs conformed by α1 subunits. However, despite its importance as a pharmacological target in chronic pain, it is currently unknown whether the α3GlyR function is modulated by tropeines. Using electrophysiological techniques and molecular docking simulations, here we show that tropeines are inhibitors of the α3GlyR function. Tropisetron, a prototypical tropeine, exerted concentration-dependent inhibitory effects on α3GlyRs at the low micromolar range. In addition, three other tropeines showed similar effects. Single-channel recordings show that tropisetron inhibition is associated with a decrease in the open probability of the ion channel. Molecular docking assays suggest that tropeines preferentially bind to an agonist-free, closed state of the ion channel. The tropeine binding occurs in a discrete pocket around the vicinity of the orthosteric site within the extracellular domain of α3GlyR. Thus, our results describe the pharmacological modulation of tropeines on α3GlyRs. These findings may contribute to the development of GlyR-selective tropeine derivatives for basic and/or clinical applications.

5.
J Alzheimers Dis ; 67(1): 343-356, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30584148

RESUMO

Alzheimer's disease (AD) is a neurodegenerative pathology, which is characterized by progressive and irreversible cognitive impairment. Most of the neuronal perturbations described in AD can be associated with soluble amyloid- ß oligomers (SO-Aß). There is a large amount of evidence demonstrating the neuroprotective effect of Nicotine neurotransmission in AD, mainly through nicotinic acetylcholine receptor (nAChR) activation and antiapoptotic PI3K/Akt/Bcl-2 pathway signaling. Using HPLC and GC/MS, we isolated and characterized two alkaloids obtained from C. scoparius, Lupanine (Lup), and 17- oxo-sparteine (17- ox), and examined their neuroprotective properties in a cellular model of SO-Aß toxicity. Our results showed that Lup and 17- ox (both at 0.03µM) prevented SO-Aß-induced toxicity in PC12 cells (Lup: 64±7%; 17- ox: 57±6%). Similar results were seen in hippocampal neurons where these alkaloids prevented SO-Aß neurotoxicity (Lup: 57±2%; 17- ox: 52±3%) and increased the frequency of spontaneous calcium transients (Lup: 60±4%; 17- Ox: 40±3%), suggesting an enhancing effect on neural network activity and synaptic activity potentiation. All of the neuroprotective effects elicited by both alkaloids were completely blocked by α-bungarotoxin. Additionally, we observed that the presence of both Lup and 17- ox increased Akt phosphorylation levels (52±4% and 35±7%, respectively) in cells treated with SO-Aß (3 h). Taken together, our results suggest that the activation of nAChR by Lup and 17- ox induces neuroprotection in different cellular models, and appears to be an interesting target for the development of new pharmacological tools and strategies against AD.


Assuntos
Peptídeos beta-Amiloides/antagonistas & inibidores , Peptídeos beta-Amiloides/toxicidade , Cytisus/química , Fármacos Neuroprotetores/farmacologia , Receptores Nicotínicos/efeitos dos fármacos , Esparteína/análogos & derivados , Esparteína/farmacologia , Animais , Sinalização do Cálcio/efeitos dos fármacos , Células HEK293 , Hipocampo/patologia , Humanos , Camundongos Endogâmicos C57BL , Rede Nervosa/efeitos dos fármacos , Neurônios/patologia , Proteína Oncogênica v-akt/metabolismo , Células PC12 , Ratos , Esparteína/química , Esparteína/isolamento & purificação , Sinapses/efeitos dos fármacos
6.
Mar Drugs ; 16(12)2018 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-30545061

RESUMO

Marine biotoxins in fish and shellfish can cause several symptoms in consumers, such as diarrhea, amnesia, or even death by paralysis. Monitoring programs are in place for testing shellfish on a regular basis. In some countries testing is performed using the so-called mouse bioassay, an assay that faces ethical concerns not only because of animal distress, but also because it lacks specificity and results in high amounts of false positives. In Europe, for lipophilic marine biotoxins (LMBs), a chemical analytical method using LC-MS/MS was developed as an alternative and is now the reference method. However, safety is often questioned when relying solely on such a method, and as a result, the mouse bioassay might still be used. In this study the use of a cell-based assay for screening, i.e., the neuro-2a assay, in combination with the official LC-MS/MS method was investigated as a new alternative strategy for the detection and quantification of LMBs. To this end, samples that had been tested previously with the mouse bioassay were analyzed in the neuro-2a bioassay and the LC-MS/MS method. The neuro-2a bioassay was able to detect all LMBs at the regulatory levels and all samples that tested positive in the mouse bioassay were also suspect in the neuro-2a bioassay. In most cases, these samples contained toxin levels (yessotoxins) that explain the outcome of the bioassay but did not exceed the established maximum permitted levels.


Assuntos
Alternativas aos Testes com Animais/métodos , Bioensaio/métodos , Toxinas Marinhas/análise , Intoxicação por Frutos do Mar/prevenção & controle , Frutos do Mar/análise , Alternativas aos Testes com Animais/instrumentação , Animais , Bioensaio/instrumentação , Bivalves , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão/instrumentação , Cromatografia Líquida de Alta Pressão/métodos , Reações Falso-Positivas , Toxinas Marinhas/toxicidade , Camundongos , Oxocinas/análise , Oxocinas/toxicidade , Frutos do Mar/toxicidade , Intoxicação por Frutos do Mar/etiologia , Espectrometria de Massas em Tandem/instrumentação , Espectrometria de Massas em Tandem/métodos
7.
Nanomedicine ; 14(7): 2227-2234, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30048814

RESUMO

Understanding the molecular features responsible for the plasma kinetics of surface-modified polyamido amine (PAMAM) dendrimers is critical to explore novel biomedical applications for these nanomaterials. In this report, polyethylene glycol (PEG) and folic acid (FA) were employed to obtain partially-substituted PAMAM dendrimers as model biocompatible nanomaterials with different size, charge and surface functionality. Cytotoxicity assays on HEK cells at 1-500 µM concentration confirmed that PEG and FA incorporation increased the cell viability of PAMAM-based nanomaterials. Measurements of plasma kinetics in vivo revealed that PEG-PAMAM has an extended circulation time in mice blood (71.7 min) over native PAMAM (53.3 min) and FA-PAMAM (41.8 min). Molecular dynamics simulations revealed a direct relationship between circulation time and dendrimer size, thus providing valuable evidence to increase understanding about the modulation of functional properties of PAMAM-based systems through surface modification, and to guide future efforts on the rational design of novel biomedical nanomaterials.


Assuntos
Apoptose , Dendrímeros/farmacologia , Portadores de Fármacos/química , Plasma/metabolismo , Animais , Proliferação de Células , Dendrímeros/farmacocinética , Ácido Fólico/química , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Simulação de Dinâmica Molecular
8.
Neuropharmacology ; 128: 366-378, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29079292

RESUMO

The most common cause of dementia is Alzheimer's disease. The etiology of the disease is unknown, although considerable evidence suggests a critical role for the soluble oligomers of amyloid beta peptide (Aß). Because Aß increases the expression of purinergic receptors (P2XRs) in vitro and in vivo, we studied the functional correlation between long-term exposure to Aß and the ability of P2XRs to modulate network synaptic tone. We used electrophysiological recordings and Ca2+ microfluorimetry to assess the effects of chronic exposure (24 h) to Aß oligomers (0.5 µM) together with known inhibitors of P2XRs, such as PPADS and apyrase on synaptic function. Changes in the expression of P2XR were quantified using RT-qPCR. We observed changes in the expression of P2X1R, P2X7R and an increase in P2X2R; and also in protein levels in PC12 cells (143%) and hippocampal neurons (120%) with Aß. In parallel, the reduction on the frequency and amplitude of mEPSCs (72% and 35%, respectively) were prevented by P2XR inhibition using a low PPADS concentration. Additionally, the current amplitude and intracellular Ca2+ signals evoked by extracellular ATP were increased (70% and 75%, respectively), suggesting an over activation of purinergic neurotransmission in cells pre-treated with Aß. Taken together, our findings suggest that Aß disrupts the main components of synaptic transmission at both pre- and post-synaptic sites, and induces changes in the expression of key P2XRs, especially P2X2R; changing the neuromodulator function of the purinergic tone that could involve the P2X2R as a key factor for cytotoxic mechanisms. These results identify novel targets for the treatment of dementia and other diseases characterized by increased purinergic transmission.


Assuntos
Peptídeos beta-Amiloides/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Receptores Purinérgicos P2X/metabolismo , Trifosfato de Adenosina/farmacologia , Peptídeos beta-Amiloides/química , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Córtex Cerebral/citologia , Proteína 4 Homóloga a Disks-Large/metabolismo , Embrião de Mamíferos , Feminino , Proteínas Associadas aos Microtúbulos/metabolismo , Neurotransmissores/farmacologia , Técnicas de Patch-Clamp , Inibidores da Agregação de Plaquetas/farmacologia , Gravidez , Fosfato de Piridoxal/análogos & derivados , Fosfato de Piridoxal/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores Purinérgicos P2X/genética
9.
Nanomaterials (Basel) ; 8(1)2017 Dec 25.
Artigo em Inglês | MEDLINE | ID: mdl-29295581

RESUMO

One of the most studied nanocarriers for drug delivery are polyamidoamine (PAMAM) dendrimers. However, the alterations produced by PAMAM dendrimers in neuronal function have not been thoroughly investigated, and important aspects such as effects on synaptic transmission remain unexplored. We focused on the neuronal activity disruption induced by dendrimers and the possibility to prevent these effects by surface chemical modifications. Therefore, we studied the effects of fourth generation PAMAM with unmodified positively charged surface (G4) in hippocampal neurons, and compared the results with dendrimers functionalized in 25% of their surface groups with folate (PFO25) and polyethylene glycol (PPEG25). G4 dendrimers significantly reduced cell viability at 1 µM, which was attenuated by both chemical modifications, PPEG25 being the less cytotoxic. Patch clamp recordings demonstrated that G4 induced a 7.5-fold increment in capacitive currents as a measure of membrane permeability. Moreover, treatment with this dendrimer increased intracellular Ca2+ by 8-fold with a complete disruption of transients pattern, having as consequence that G4 treatment increased the synaptic vesicle release and frequency of synaptic events by 2.4- and 3-fold, respectively. PFO25 and PPEG25 treatments did not alter membrane permeability, total Ca2+ intake, synaptic vesicle release or synaptic activity frequency. These results demonstrate that cationic G4 dendrimers have neurotoxic effects and induce alterations in normal synaptic activity, which are generated by the augmentation of membrane permeability and a subsequent intracellular Ca2+ increase. Interestingly, these toxic effects and synaptic alterations are prevented by the modification of 25% of PAMAM surface with either folate or polyethylene glycol.

10.
Mol Pharm ; 13(10): 3395-3403, 2016 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-27556289

RESUMO

Polyamidoamine (PAMAM) dendrimers are hyperbranched macromolecules which have been described as one of the most promising drug nanocarrier systems. A key process to understand is their cellular internalization mechanism because of its direct influence on their intracellular distribution, association with organelles, entry kinetics, and cargo release. Despite that internalization mechanisms of dendrimers have been studied in different cell types, in the case of neurons they are not completely described. Considering the relevance of central nervous system (CNS) diseases and neuropharmacology, the aim of this report is to describe the molecular internalization mechanism of different PAMAM-based dendrimer systems in hippocampal neurons. Four dendrimers based on fourth generation PAMAM with different surface properties were studied: unmodified G4, with a positively charged surface; PP50, with a substitution of the 50% of amino surface groups with polyethylene glycol neutral groups; PAc, with a substitution of the 30% of amino surface groups with acrylate anionic groups; and PFO, decorated with folic acid groups in a 25% of total terminal groups. Confocal images show that both G4 and PFO are able to enter the neurons, but not PP50 and PAc. Colocalization study with specific endocytosis markers and specific endocytosis inhibitor assay demonstrate that clathrin-mediated endocytosis would be the main internalization mechanism for G4, whereas clathrin- and caveolae-mediated endocytosis would be implicated in PFO internalization. These results show the existence of different internalization mechanisms for PAMAM dendrimers in neurons and the possibility to control their internalization properties with specific chemical modifications.


Assuntos
Dendrímeros/metabolismo , Hipocampo/citologia , Neurônios/metabolismo , Animais , Células Cultivadas , Endocitose/fisiologia , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal
11.
J Biol Chem ; 291(36): 18791-8, 2016 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-27402845

RESUMO

The acute intoxicating effects of ethanol in the central nervous system result from the modulation of several molecular targets. It is widely accepted that ethanol enhances the activity of the glycine receptor (GlyR), thus enhancing inhibitory neurotransmission, leading to motor effects, sedation, and respiratory depression. We previously reported that small peptides interfered with the binding of Gßγ to the GlyR and consequently inhibited the ethanol-induced potentiation of the receptor. Now, using virtual screening, we identified a subset of small molecules capable of interacting with the binding site of Gßγ. One of these compounds, M554, inhibited the ethanol potentiation of the GlyR in both evoked currents and synaptic transmission in vitro When this compound was tested in vivo in mice treated with ethanol (1-3.5 g/kg), it was found to induce a faster recovery of motor incoordination in rotarod experiments and a shorter sedative effect in loss of righting reflex assays. This study describes a novel molecule that might be relevant for the design of useful therapeutic compounds in the treatment of acute alcohol intoxication.


Assuntos
Intoxicação Alcoólica/tratamento farmacológico , Etanol/efeitos adversos , Subunidades beta da Proteína de Ligação ao GTP/antagonistas & inibidores , Subunidades gama da Proteína de Ligação ao GTP/antagonistas & inibidores , Peptídeos , Receptores da Glicina/antagonistas & inibidores , Transmissão Sináptica/efeitos dos fármacos , Intoxicação Alcoólica/metabolismo , Animais , Etanol/farmacologia , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Células HEK293 , Humanos , Camundongos , Peptídeos/química , Peptídeos/farmacologia , Receptores da Glicina/metabolismo
12.
Mater Sci Eng C Mater Biol Appl ; 65: 164-71, 2016 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-27157739

RESUMO

PAMAM-grafted TiO2 nanotubes (PAMAM-TiO2NT) have been synthesized and evaluated as new drug nanocarriers, using curcumin (CUR), methotrexate (MTX), and silibinin (SIL) as model therapeutic compounds. TiO2NT were surface-modified using a silane coupling agent and subsequently conjugated with PAMAM dendrimer of the third generation. The characterization of PAMAM-TiO2NT nanomaterials was performed by FTIR, TEM, N2 adsorption-desorption isotherms, XRD, and TGA techniques, which accounted for a 2.6wt.% of PAMAM grafting in the prepared materials. The drug loading capacity, drug release properties, and cytotoxicity of PAMAM-TiO2NT showed a significant improvement compared to pristine TiO2NT, thus revealing the promising properties of these new materials for drug delivery purposes.


Assuntos
Dendrímeros/química , Portadores de Fármacos/química , Nanotubos/química , Preparações Farmacêuticas/química , Titânio/química , Sobrevivência Celular/efeitos dos fármacos , Curcumina/química , Curcumina/metabolismo , Curcumina/toxicidade , Liberação Controlada de Fármacos , Células HeLa , Humanos , Metotrexato/química , Metotrexato/metabolismo , Metotrexato/toxicidade , Microscopia Eletrônica de Transmissão , Preparações Farmacêuticas/metabolismo , Silibina , Silimarina/química , Silimarina/metabolismo , Silimarina/toxicidade , Espectroscopia de Infravermelho com Transformada de Fourier
13.
Br J Pharmacol ; 173(14): 2263-77, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27128379

RESUMO

BACKGROUND AND PURPOSE: Gelsemine is one of the principal alkaloids produced by the Gelsemium genus of plants belonging to the Loganiaceae family. The extracts of these plants have been used for many years, for a variety of medicinal purposes. Coincidentally, recent studies have shown that gelsemine exerts anxiolytic and analgesic effects on behavioural models. Several lines of evidence have suggested that these beneficial actions were dependent on glycine receptors, which are inhibitory neurotransmitter-gated ion channels of the CNS. However, it is currently unknown whether gelsemine can directly modulate the function of glycine receptors. EXPERIMENTAL APPROACH: We examined the functional effects of gelsemine on glycine receptors expressed in transfected HEK293 cells and in cultured spinal neurons by electrophysiological techniques. KEY RESULTS: Gelsemine directly modulated recombinant and native glycine receptors and exerted conformation-specific and subunit-selective effects. Gelsemine modulation was voltage-independent and was associated with differential changes in the apparent affinity for glycine and in the open probability of the ion channel. In addition, the alkaloid preferentially targeted glycine receptors in spinal neurons and showed only minor effects on GABAA and AMPA receptors. Furthermore, gelsemine significantly diminished the frequency of glycinergic and glutamatergic synaptic events without altering the amplitude. CONCLUSIONS AND IMPLICATIONS: Our results provide a pharmacological basis to explain, at least in part, the glycine receptor-dependent, beneficial and toxic effects of gelsemine in animals and humans. In addition, the pharmacological profile of gelsemine may open new approaches to the development of subunit-selective modulators of glycine receptors.


Assuntos
Alcaloides/farmacologia , Receptores da Glicina/metabolismo , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Feminino , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Gravidez , Receptores da Glicina/química , Relação Estrutura-Atividade
14.
Harmful Algae ; 55: 238-249, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-28073537

RESUMO

The detection of sparse Alexandrium catenella-resting cysts in sediments of southern Chilean fjords has cast doubts on their importance in the recurrence of massive toxic dinoflagellate blooms in the region. The role of resting cysts and the existence of different regional Chilean populations was studied by culturing and genetic approaches to define: (1) cyst production; (2) dormancy period; (3) excystment success; (4) offspring viability and (5) strain mating compatibility. This study newly revealed a short cyst dormancy (minimum 69 days), the role of key abiotic factors (in decreasing order salinity, irradiance, temperature and nutrients) controlling cyst germination (max. 60%) and germling growth rates (up to 0.36-0.52div.day-1). Amplified fragment length polymorphism (AFLP) characterization showed significant differences in genetic distances (GD) among A. catenella populations that were primarily determined by the geographical origin of isolates and most likely driven by oceanographic dispersal barriers. A complex heterothallic mating system pointed to variable reproductive compatibility (RCs) among Chilean strains that was high among northern (Los Lagos/North Aysén) and southern populations (Magallanes), but limited among the genetically differentiated central (South Aysén) populations. Field cyst surveys after a massive 2009 bloom event revealed the existence of exceptional high cyst densities in particular areas of the fjords (max. 14.627cystscm-3), which contrast with low cyst concentrations (<221.3cystscm-3) detected by previous oceanographic campaigns. In conclusion, the present study suggests that A. catenella resting cysts play a more important role in the success of this species in Chilean fjords than previously thought. Results from in vitro experiments suggest that pelagic-benthic processes can maintain year-round low vegetative cell concentrations in the water column, but also can explain the detection of high cysts aggregations after the 2009-bloom event. Regional drivers that lead to massive outbreaks, however, are still unknown but potential scenarios are discussed.


Assuntos
Dinoflagelados/fisiologia , Eutrofização/fisiologia , Análise do Polimorfismo de Comprimento de Fragmentos Amplificados , Chile , Dinoflagelados/citologia , Estuários
15.
16.
Pharmacol Res ; 101: 18-29, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26158502

RESUMO

It is well accepted that ethanol is able to produce major health and economic problems associated to its abuse. Because of its intoxicating and addictive properties, it is necessary to analyze its effect in the central nervous system. However, we are only now learning about the mechanisms controlling the modification of important membrane proteins such as ligand-activated ion channels by ethanol. Furthermore, only recently are these effects being correlated to behavioral changes. Current studies show that the glycine receptor (GlyR) is a susceptible target for low concentrations of ethanol (5-40mM). GlyRs are relevant for the effects of ethanol because they are found in the spinal cord and brain stem where they primarily express the α1 subunit. More recently, the presence of GlyRs was described in higher regions, such as the hippocampus and nucleus accumbens, with a prevalence of α2/α3 subunits. Here, we review data on the following aspects of ethanol effects on GlyRs: (1) direct interaction of ethanol with amino acids in the extracellular or transmembrane domains, and indirect mechanisms through the activation of signal transduction pathways; (2) analysis of α2 and α3 subunits having different sensitivities to ethanol which allows the identification of structural requirements for ethanol modulation present in the intracellular domain and C-terminal region; (3) Genetically modified knock-in mice for α1 GlyRs that have an impaired interaction with G protein and demonstrate reduced ethanol sensitivity without changes in glycinergic transmission; and (4) GlyRs as potential therapeutic targets.


Assuntos
Comportamento/efeitos dos fármacos , Etanol/farmacologia , Receptores da Glicina/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacos , Anestésicos Gerais/farmacologia , Animais , Comportamento/fisiologia , Etanol/toxicidade , Humanos , Camundongos , Camundongos Mutantes , Modelos Neurológicos , Receptores da Glicina/química , Receptores da Glicina/genética , Receptores da Glicina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transmissão Sináptica/fisiologia
17.
J Pharmacol Exp Ther ; 352(1): 148-55, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25339760

RESUMO

The α1-subunit containing glycine receptors (GlyRs) is potentiated by ethanol, in part, by intracellular Gßγ actions. Previous studies have suggested that molecular requirements in the large intracellular domain are involved; however, the lack of structural data about this region has made it difficult to describe a detailed mechanism. Using circular dichroism and molecular modeling, we generated a full model of the α1-GlyR, which includes the large intracellular domain and provides new information on structural requirements for allosteric modulation by ethanol and Gßγ. The data strongly suggest the existence of an α-helical conformation in the regions near transmembrane (TM)-3 and TM4 of the large intracellular domain. The secondary structure in the N-terminal region of the large intracellular domain near TM3 appeared critical for ethanol action, and this was tested using the homologous domain of the γ2-subunit of the GABAA receptor predicted to have little helical conformation. This region of γ2 was able to bind Gßγ and form a functional channel when combined with α1-GlyR, but it was not sensitive to ethanol. Mutations in the N- and C-terminal regions introduced to replace corresponding amino acids of the α1-GlyR sequence restored the ability to be modulated by ethanol and Gßγ. Recovery of the sensitivity to ethanol was associated with the existence of a helical conformation similar to α1-GlyR, thus being an essential secondary structural requirement for GlyR modulation by ethanol and G protein.


Assuntos
Etanol/farmacologia , Subunidades beta da Proteína de Ligação ao GTP/farmacologia , Subunidades gama da Proteína de Ligação ao GTP/farmacologia , Espaço Intracelular/metabolismo , Receptores da Glicina/química , Receptores da Glicina/metabolismo , Sequência de Aminoácidos , Animais , Relação Dose-Resposta a Droga , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Células HEK293 , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Secundária de Proteína/efeitos dos fármacos , Estrutura Terciária de Proteína/efeitos dos fármacos , Ratos , Receptores de GABA-A/metabolismo
18.
J Alzheimers Dis ; 42(1): 143-55, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24825567

RESUMO

Alzheimer's disease (AD) is a progressive and neurodegenerative disorder and one of the current therapies involves strengthening the cholinergic tone in central synapses. Neuroprotective properties for nicotine have been described in AD, through its actions on nicotinic receptors and the further activation of the PI3K/Akt/Bcl-2 survival pathway. We have tested a quinolizidine alkaloid extract (TM0112) obtained from Teline monspessulanna (L.) K. Koch seeds to evaluate its action on nicotinic acetylcholine receptor (nAChR) in a neuronal AD model. Our data show that PC-12 cells pretreated with amyloid-ß (Aß) peptide for 24 h in presence of TM0112 modified Aß-reduction on cellular viability (Aß = 80 ± 3%; +TM0112 = 113 ± 4%, n = 15). In addition, this effect was blocked with atropine, MLA, and α-BTX (+TM0112+atropine = 87 ± 4%; +TM0112+MLA = 86 ± 4%; +TM0112+α-BTX = 92 ± 3%). Furthermore, similar protective effects were observed in rat cortical neurons (Aß = 63 ± 6%; +TM0112 = 114 ± 8%), but not in HEK293T cells (Aß = 61.4 ± 6.1%; +TM0112 = 62.8 ± 5.2) that do not express α7 nAChR. Moreover, the frequency of synaptic activity in the neuronal network (Aß = 51.6 ± 16.9%; +TM0112 = 210.8 ± 47.9%, n > 10), as well as the intracellular Ca2+ transients were recovered by TM0112 (Aß = 61.4 ± 6.9%; +TM0112 = 112.0 ± 5.7%; n = 3) in rat hippocampal neurons. TM0112 increased P-Akt, up to 250% with respect to control, and elevated Bcl-2/Bax percentage (Aß = 61.0 ± 8.2%; +TM0112 = 105.4 ± 19.5%, n = 4), suggesting a coupling between nAChR activation and an intracellular neuroprotective pathway. Our results suggest that TM0112 could be a new potential source for anti-AD drugs.


Assuntos
Alcaloides/farmacologia , Doença de Alzheimer/tratamento farmacológico , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Quinolizidinas/farmacologia , Receptores Nicotínicos/metabolismo , Doença de Alzheimer/fisiopatologia , Peptídeos beta-Amiloides/toxicidade , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/fisiopatologia , Fabaceae , Células HEK293 , Humanos , Neurônios/fisiologia , Células PC12 , Fragmentos de Peptídeos/toxicidade , Fitoterapia , Extratos Vegetais/farmacologia , Ratos , Ratos Sprague-Dawley , Sementes , Transmissão Sináptica/efeitos dos fármacos
19.
J Biol Chem ; 287(48): 40713-21, 2012 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-23035114

RESUMO

BACKGROUND: Gßγ interaction with GlyR is an important determinant in ethanol potentiation of this channel. RESULTS: A small peptide, RQH(C7), can inhibit ethanol potentiation of GlyR currents. CONCLUSION: Results with RQH(C7) indicate that ethanol mediated potentiation of GlyR is in part by Gßγ activation. SIGNIFICANCE: Molecular interaction between Gßγ and GlyR could be used as a target for pharmacological modification of ethanol effects. Previous studies indicate that ethanol can modulate glycine receptors (GlyR), in part, through Gßγ interaction with basic residues in the intracellular loop. In this study, we show that a seven-amino acid peptide (RQH(C7)), which has the primary structure of a motif in the large intracellular loop of GlyR (GlyR-IL), was able to inhibit the ethanol-elicited potentiation of this channel from 47 ± 2 to 16 ± 4%, without interfering with the effect of Gßγ on GIRK (G protein activated inwardly rectifying potassium channel) activation. RQH(C7) displayed a concentration-dependent effect on ethanol action in evoked and synaptic currents. A fragment of GlyR-IL without the basic amino acids did not interact with Gßγ or inhibit ethanol potentiation of GlyR. In silico analysis using docking and molecular dynamics allowed to identify a region of ~350Å(2) involving aspartic acids 186, 228, and 246 in Gßγ where we propose that RQH(C7) binds and exerts its blocking action on the effect of ethanol in GlyR.


Assuntos
Etanol/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Peptídeos/metabolismo , Receptores da Glicina/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/química , Subunidades beta da Proteína de Ligação ao GTP/genética , Subunidades gama da Proteína de Ligação ao GTP/genética , Células HEK293 , Humanos , Cinética , Peptídeos/química , Ligação Proteica , Receptores da Glicina/química , Receptores da Glicina/genética
20.
Int J Alzheimers Dis ; 2012: 674142, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22482077

RESUMO

Shilajit is a natural substance found mainly in the Himalayas, formed for centuries by the gradual decomposition of certain plants by the action of microorganisms. It is a potent and very safe dietary supplement, restoring the energetic balance and potentially able to prevent several diseases. Recent investigations point to an interesting medical application toward the control of cognitive disorders associated with aging, and cognitive stimulation. Thus, fulvic acid, the main active principle, blocks tau self-aggregation, opening an avenue toward the study of Alzheimer's therapy. In essence, this is a nutraceutical product of demonstrated benefits for human health. Considering the expected impact of shilajit usage in the medical field, especially in the neurological sciences, more investigations at the basic biological level as well as clinical trials are necessary, in order to understand how organic molecules of shilajit and particularly fulvic acid, one of the active principles, and oligoelements act at both the molecular and cellular levels and in the whole organism.

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