RESUMO
Most protocols used to study the dynamics of calcium (Ca2+) in the malaria parasite are based on dyes, which are invasive and do not allow discrimination between the signal from the host cell and the parasite. To avoid this pitfall, we have generated a parasite line expressing the genetically encoded calcium sensor GCaMP3. The PfGCaMP3 parasite line is an innovative tool for studying spontaneous intracellular Ca2+ oscillations without external markers. Using this parasite line, we demonstrate the occurrence of spontaneous Ca2+ oscillations in the ring, trophozoite, and schizont stages in Plasmodium falciparum. Using the Fourier transform to fluorescence intensity data extracted from different experiments, we observe cytosolic Ca2+ fluctuations. These spontaneous cytosolic Ca2+ oscillations occur in the three intraerythrocytic stages of the parasite, with most oscillations occurring in the ring and trophozoite stages. A control parasite line expressing only a GFP control did not reveal such fluctuations, demonstrating the specificity of the observations. Our results clearly show dynamic, spontaneous Ca2+ oscillations during the asexual stage in P. falciparum, independent from external stimuli.
RESUMO
Chagas disease vectors can ingest several times their own volume in blood with each meal. This ad libitum feeding causes an intense process of diuresis, inducing the insect to eliminate a large quantity of urine and faeces. To ensure diuresis, the speed of circulation of the haemolymph is increased. The Triatominae circulatory system is quite simple, including the dorsal vessel, which pumps haemolymph in an anterograde direction. The return is caused by peristaltic contractions of the anterior midgut. Triatominae insects can spend several weeks without feeding, meaning that most of the time, the insect is in a resting condition. Although the mechanisms controlling the circulation of the haemolymph during post-prandial diuresis have been largely analysed, the mechanisms controlling it during resting conditions are poorly understood. In this study, we analysed several canonical pathways (i.e. L-type VGCC, GPCR, RyR, IP3R) and a novel system represented by the recently characterized Piezo proteins. Our results show that during the resting condition, haemolymph circulation depends on a cross-talk between myogenic activity, inhibitory and stimulatory cellular messengers, and Piezo proteins. This report also unveils for the first time the existence of a putative Piezo protein in Hemiptera.
Assuntos
Hemolinfa , Rhodnius , Animais , Rhodnius/fisiologia , Proteínas de Insetos/metabolismo , Insetos Vetores/fisiologia , Doença de Chagas/transmissão , Descanso/fisiologiaRESUMO
In the last two decades, several working groups in the international psychoanalytic community have been interested in the development of systematic tools for psychodynamic diagnosis, case formulation and treatment planning. Such psychodynamic diagnostic manuals are efforts to systematically integrate an enormous and rich amount of historically partialized and dispersed information, but which constitute the substantial contribution of psychoanalysis to the field of mental health. The aim of the present article is to provide an updated review on this kind of systematic tools for diagnosis, case formulation and therapeutic planning, designed for the field of psychodynamic approaches. To this end, we describe the aims and structure of: 1) the Psychodynamic Diagnostic Manual 2 (PDM-2), 2) the Operationalized Psychodynamic Diagnosis (OPD-2/OPD-3) and 3) the Operationalized Psychodynamic Diagnosis for Children and adolescents 2 (OPD-CA-2). The contributions of these current tools to clinical practice and empirical research are discussed, as well as the need to disseminate these types of instruments in our regional context.
En las últimas dos décadas, diversos grupos de trabajo de la comunidad psicoanalítica internacional se han interesado por el desarrollo de herramientas sistemáticas para el diagnóstico, la formulación de los casos y la planificación del tratamiento psicodinámico. Este tipo de manuales diagnósticos psicodinámicos son esfuerzos de integración sistemática de una enorme y rica cantidad de información históricamente parcializada y dispersa, pero que constituye el aporte sustancial del psicoanálisis al campo de la salud mental. El objetivo del presente artículo es ofrecer una revisión actualizada sobre esta clase de herramientas sistemáticas de diagnóstico, formulación del caso y planificación terapéutica, diseñadas para el campo de los abordajes psicodinámicos. A estos fines, se describe la estructura y los objetivos de: 1) el Manual Diagnóstico Psicodinámico 2 (PDM-2), 2) el Diagnóstico Psicodinámico Operacionalizado (OPD-2/OPD-3) y 3) el Diagnóstico Psicodinámico Operacionalizado Infanto-Juvenil 2 (OPD-IJ-2).Se discuten las contribuciones de estas herramientas actuales para la práctica clínica y la investigación empírica, así como la necesidad de difundir este tipo de instrumentos en nuestro contexto regional.
Assuntos
Transtornos Mentais , Psicoterapia Psicodinâmica , Humanos , Psicoterapia Psicodinâmica/métodos , Transtornos Mentais/terapia , Transtornos Mentais/diagnósticoRESUMO
BACKGROUND: Exposure of humans and animals to heavy metals is increasing day-by-day; thus, lead even today remains of significant public health concern. According to CDC, blood lead reference value (BLRV) ranges from 3.5 µg/dl to 5 µg/dl in adults. Recently, almost 2.6% decline in male fertility per year has been reported but the cause is not well established. Lead (Pb2+) affects the size of testis, semen quality, and secretory functions of prostate. But the molecular mechanism(s) of lead toxicity in sperm cells is not clear. Thus, present study was undertaken to evaluate the adverse effects of lead acetate at environmentally relevant exposure levels (0.5, 5, 10 and 20 ppm) on functional and molecular dynamics of spermatozoa of bucks following in vitro exposure for 15 min and 3 h. RESULTS: Lead significantly decreased motility, viable count, and motion kinematic patterns of spermatozoa like curvilinear velocity, straight-line velocity, average path velocity, beat cross frequency and maximum amplitude of head lateral displacement even at 5 ppm concentration. Pb2+ modulated intracellular cAMP and Ca2+ levels in sperm cells through L-type calcium channels and induced spontaneous or premature acrosome reaction (AR) by increasing tyrosine phosphorylation of sperm proteins and downregulated mitochondrial transmembrane potential. Lead significantly increased DNA damage and apoptosis as well. Electron microscopy studies revealed Pb2+ -induced deleterious effects on plasma membrane of head and acrosome including collapsed cristae in mitochondria. CONCLUSIONS: Pb2+ not only mimics Ca2+ but also affects cellular targets involved in generation of cAMP, mitochondrial transmembrane potential, and ionic exchange. Lead seems to interact with Ca2+ channels because of charge similarity and probably enters the sperm cell through these channels and results in hyperpolarization. Our findings also indicate lead-induced TP and intracellular Ca2+ release in spermatozoa which in turn may be responsible for premature acrosome exocytosis which is essential feature of capacitation for fertilization. Thus, lead seems to reduce the fertilizing capacity of spermatozoa even at 0.5 ppm concentrations.
Assuntos
Reação Acrossômica , Acrossomo , Cálcio , Chumbo , Motilidade dos Espermatozoides , Espermatozoides , Masculino , Espermatozoides/efeitos dos fármacos , Cálcio/metabolismo , Motilidade dos Espermatozoides/efeitos dos fármacos , Animais , Acrossomo/efeitos dos fármacos , Chumbo/toxicidade , Reação Acrossômica/efeitos dos fármacos , AMP Cíclico/metabolismo , Bovinos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Análise do Sêmen , Dano ao DNA/efeitos dos fármacos , Compostos Organometálicos/toxicidade , Compostos Organometálicos/farmacologiaRESUMO
Lithium therapy received approval during the 1970s, and it has been used for its antidepressant, antimanic, and anti-suicidal effects for acute and long-term prophylaxis and treatment of bipolar disorder (BPD). These properties have been well established; however, the molecular and cellular mechanisms remain controversial. In the past few years, many studies demonstrated that at the cellular level, lithium acts as a regulator of neurogenesis, aging, and Ca2+ homeostasis. At the molecular level, lithium modulates aging by inhibiting glycogen synthase kinase-3ß (GSK-3ß), and the phosphatidylinositol (PI) cycle; latter, lithium specifically inhibits inositol production, acting as a non-competitive inhibitor of inositol monophosphatase (IMPase). Mitochondria and peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) have been related to lithium activity, and its regulation is mediated by GSK-3ß degradation and inhibition. Lithium also impacts Ca2+ homeostasis in the mitochondria modulating the function of the lithium-permeable mitochondrial Na+-Ca2+exchanger (NCLX), affecting Ca2+ efflux from the mitochondrial matrix to the endoplasmic reticulum (ER). A close relationship between the protease Omi, GSK-3ß, and PGC-1α has also been established. The purpose of this review is to summarize some of the intracellular mechanisms related to lithium activity and how, through them, neuronal aging could be controlled.
Assuntos
Senescência Celular , Compostos de Lítio , Neurônios , Neurônios/efeitos dos fármacos , Compostos de Lítio/farmacologia , Fármacos Neuroprotetores/farmacologia , Enzimas/metabolismo , Inositol/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Cálcio/metabolismo , Humanos , Animais , Senescência Celular/efeitos dos fármacosRESUMO
Pannexin1 hemichannels (Panx1 HCs) are found in the membrane of most mammalian cells and communicate the intracellular and extracellular spaces, enabling the passive transfer of ions and small molecules. They are involved in physiological and pathophysiological conditions. During apoptosis, the C-terminal tail of Panx1 is proteolytically cleaved, but the permeability features of hemichannels and their role in cell death remain elusive. To address these topics, HeLa cells transfected with full-length human Panx1 (fl-hPanx1) or C-terminal truncated hPanx1 (Δ371hPanx1) were exposed to alkaline extracellular saline solution, increasing the activity of Panx1 HCs. The Δ371hPanx1 HC was permeable to DAPI and Etd+, but not to propidium iodide, whereas fl-hPanx1 HC was only permeable to DAPI. Furthermore, the cytoplasmic Ca2+ signal increased only in Δ371hPanx1 cells, which was supported by bioinformatics approaches. The influx of Ca2+ through Δ371hPanx1 HCs was necessary to promote cell death up to about 95% of cells, whereas the exposure to alkaline saline solution without Ca2+ failed to induce cell death, and the Ca2+ ionophore A23187 promoted more than 80% cell death even in fl-hPanx1 transfectants. Moreover, cell death was prevented with carbenoxolone or 10Panx1 in Δ371hPanx1 cells, whereas it was undetectable in HeLa Panx1-/- cells. Pretreatment with Ferrostatin-1 and necrostatin-1 did not prevent cell death, suggesting that ferroptosis or necroptosis was not involved. In comparison, zVAD-FMK, a pancaspase inhibitor, reduced death by ~60%, suggesting the involvement of apoptosis. Therefore, alkaline pH increases the activity of Δ371hPanx1HCs, leading to a critical intracellular free-Ca2+ overload that promotes cell death.
Assuntos
Cálcio , Conexinas , Proteínas do Tecido Nervoso , Humanos , Conexinas/metabolismo , Conexinas/genética , Células HeLa , Cálcio/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/genética , Apoptose , Morte Celular , Sinalização do CálcioRESUMO
Our research aimed to elucidate the mechanism by which aurintricarboxylic acid (ATA) inhibits plasma membrane Ca2+-ATPase (PMCA), a crucial enzyme responsible for calcium transport. Given the pivotal role of PMCA in cellular calcium homeostasis, understanding how it is inhibited by ATA holds significant implications for potentially regulating physiopathological cellular processes in which this pump is involved. Our experimental findings revealed that ATA employs multiple modes of action to inhibit PMCA activity, which are influenced by ATP but also by the presence of calcium and magnesium ions. Specifically, magnesium appears to enhance this inhibitory effect. Our experimental and in-silico results suggest that, unlike those reported in other proteins, ATA complexed with magnesium (ATA·Mg) is the molecule that inhibits PMCA. In summary, our study presents a novel perspective and establishes a solid foundation for future research efforts aimed at the development of new pharmacological molecules both for PMCA and other proteins.
Assuntos
Ácido Aurintricarboxílico , Cálcio , Magnésio , ATPases Transportadoras de Cálcio da Membrana Plasmática , Magnésio/metabolismo , Magnésio/farmacologia , Ácido Aurintricarboxílico/farmacologia , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , ATPases Transportadoras de Cálcio da Membrana Plasmática/antagonistas & inibidores , Cálcio/metabolismo , Trifosfato de Adenosina/metabolismo , Membrana Celular/metabolismo , Membrana Celular/efeitos dos fármacos , Animais , HumanosRESUMO
The Cupressaceae family includes species considered to be medicinal. Their essential oil is used for headaches, colds, cough, and bronchitis. Cedar trees like Chamaecyparis lawsoniana (C. lawsoniana) are commonly found in urban areas. We investigated whether C. lawsoniana exerts some of its effects by modifying airway smooth muscle (ASM) contractility. The leaves of C. lawsoniana (363 g) were pulverized mechanically, and extracts were obtained by successive maceration 1:10 (w:w) with methanol/CHCl3. Guinea pig tracheal rings were contracted with KCl, tetraethylammonium (TEA), histamine (HIS), or carbachol (Cch) in organ baths. In the Cch experiments, tissues were pre-incubated with D-600, an antagonist of L-type voltage-dependent Ca2+ channels (L-VDCC) before the addition of C. lawsoniana. Interestingly, at different concentrations, C. lawsoniana diminished the tracheal contractions induced by KCl, TEA, HIS, and Cch. In ASM cells, C. lawsoniana significantly diminished L-type Ca2+ currents. ASM cells stimulated with Cch produced a transient Ca2+ peak followed by a sustained plateau maintained by L-VDCC and store-operated Ca2+ channels (SOCC). C. lawsoniana almost abolished this last response. These results show that C. lawsoniana, and its active metabolite quercetin, relax the ASM by inhibiting the L-VDCC and SOCC; further studies must be performed to obtain the complete set of metabolites of the extract and study at length their pharmacological properties.
Assuntos
Cálcio , Chamaecyparis , Contração Muscular , Músculo Liso , Extratos Vegetais , Quercetina , Traqueia , Animais , Cobaias , Músculo Liso/efeitos dos fármacos , Músculo Liso/metabolismo , Contração Muscular/efeitos dos fármacos , Quercetina/farmacologia , Quercetina/química , Traqueia/efeitos dos fármacos , Traqueia/metabolismo , Extratos Vegetais/farmacologia , Extratos Vegetais/química , Chamaecyparis/química , Cálcio/metabolismo , Masculino , Bloqueadores dos Canais de Cálcio/farmacologia , Histamina/metabolismo , Canais de Cálcio Tipo L/metabolismo , Folhas de Planta/químicaRESUMO
OBJECTIVE: This study aimed to investigate the effect of Esketamine (ESK) on the Hypoxia/Reoxygenation (H/R) injury of cardiomyocytes by regulating TRPV1 and inhibiting the concentration of intracellular Ca2+. METHODS: The H/R injury model of H9c2 cardiomyocytes was established after 4h hypoxia and 6h reoxygenation. H9c2 cells were treated with different concentrations of ESK or TRPV1 agonist capsaicin (10 µM) or TRPV1 inhibitor capsazepine (1 µM). Cell viability was detected by CCK-8 method, and apoptosis by flow cytometry. Intracellular Ca2+ concentration was evaluated by Fluo-4 AM. LDH, MDA, SOD, and GSH-Px were detected with corresponding commercial kits. TRPV1 and p-TRPV1 proteins were detected by Western blot. RESULTS: After H/R, H9c2 cell viability decreased, apoptosis increased, intracellular Ca2+ concentration increased, LDH and MDA levels increased, SOD and GSH-Px levels decreased, and p-TRPV1 expression increased. ESK treatment rescued these changes induced by H/R. After up-regulating TRPV1, the protective effect of ESK on H/R injury of H9c2 cells was weakened, while down-regulating TRPV1 could further protect against H/R injury. CONCLUSION: ESK alleviates H/R injury of cardiomyocytes by regulating TRPV1 expression and inhibiting intracellular Ca2+ concentration.
Assuntos
Apoptose , Cálcio , Capsaicina/análogos & derivados , Sobrevivência Celular , Ketamina , Miócitos Cardíacos , Canais de Cátion TRPV , Canais de Cátion TRPV/metabolismo , Canais de Cátion TRPV/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Cálcio/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Animais , Ketamina/farmacologia , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/tratamento farmacológico , Ratos , Capsaicina/farmacologia , Hipóxia Celular/efeitos dos fármacos , Linhagem Celular , Citometria de Fluxo , Estresse Oxidativo/efeitos dos fármacos , Western BlottingRESUMO
L-type calcium channels are essential for the excitation-contraction coupling in cardiac muscle. The CaV1.2 channel is the most predominant isoform in the ventricle which consists of a multi-subunit membrane complex that includes the CaV1.2 pore-forming subunit and auxiliary subunits like CaVα2δ and CaVß2b. The CaV1.2 channel's C-terminus undergoes proteolytic cleavage, and the distal C-terminal domain (DCtermD) associates with the channel core through two domains known as proximal and distal C-terminal regulatory domain (PCRD and DCRD, respectively). The interaction between the DCtermD and the remaining C-terminus reduces the channel activity and modifies voltage- and calcium-dependent inactivation mechanisms, leading to an autoinhibitory effect. In this study, we investigate how the interaction between DCRD and PCRD affects the inactivation processes and CaV1.2 activity. We expressed a 14-amino acid peptide miming the DCRD-PCRD interaction sequence in both heterologous systems and cardiomyocytes. Our results show that overexpression of this small peptide can displace the DCtermD and replicate the effects of the entire DCtermD on voltage-dependent inactivation and channel inhibition. However, the effect on calcium-dependent inactivation requires the full DCtermD and is prevented by overexpression of calmodulin. In conclusion, our results suggest that the interaction between DCRD and PCRD is sufficient to bring about the current inhibition and alter the voltage-dependent inactivation, possibly in an allosteric manner. Additionally, our data suggest that the DCtermD competitively modifies the calcium-dependent mechanism. The identified peptide sequence provides a valuable tool for further dissecting the molecular mechanisms that regulate L-type calcium channels' basal activity in cardiomyocytes.