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1.
Int J Mol Sci ; 24(5)2023 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-36901815

RESUMO

The chemical diversity of sphingolipids in plants allows the assignment of specific roles to special molecular species. These roles include NaCl receptors for glycosylinositolphosphoceramides or second messengers for long-chain bases (LCBs), free or in their acylated forms. Such signaling function has been associated with plant immunity, with an apparent connection to mitogen-activated protein kinase 6 (MPK6) and reactive oxygen species (ROS). This work used in planta assays with mutants and fumonisin B1 (FB1) to generate varying levels of endogenous sphingolipids. This was complemented with in planta pathogenicity tests using virulent and avirulent Pseudomonas syringae strains. Our results indicate that the surge of specific free LCBs and ceramides induced by FB1 or an avirulent strain trigger a biphasic ROS production. The first transient phase is partially produced by NADPH oxidase, and the second is sustained and is related to programmed cell death. MPK6 acts downstream of LCB buildup and upstream of late ROS and is required to selectively inhibit the growth of the avirulent but not the virulent strain. Altogether, these results provide evidence that a LCB- MPK6- ROS signaling pathway contributes differentially to the two forms of immunity described in plants, upregulating the defense scheme of a non-compatible interaction.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Espécies Reativas de Oxigênio/metabolismo , Morte Celular , Transdução de Sinais , Esfingolipídeos/metabolismo , Regulação da Expressão Gênica de Plantas
2.
Biochem Biophys Res Commun ; 524(4): 1030-1036, 2020 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-32063359

RESUMO

The hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are encoded by a family of four genes (HCN1-4). All isoforms are expressed in the heart, HCN4 being the most abundant in the sinoatrial node (SAN). HCN channels are responsible for the "funny" current (If) associated with the generation and autonomic control of the diastolic depolarization phase of cardiac action potential. In this work we performed a proteomic analysis of HCN4 transfected in HEK293 cells. Most of the identified proteins in the HCN4 network belonged to mitochondria. The subcellular localization of HCN channels was predicted in plasma membrane, mitochondria and nucleus. Experimentally, HCN2 (full-length, truncated), HCN3 (full-length, truncated) and HCN4 (truncated) were detected in rat heart mitochondria by immunoblotting. If sensitive to ZD7288, was recorded by patch-clamp in mitoplasts from cardiomyocytes. Mitochondrial membrane potential (ΔΨm) assessment in H9c2 cells revealed that ZD7288 induced almost 50% higher hyperpolarization respect to control at 30 min. Furthermore, ZD7288 reduced oxygen consumption attributed to ATP synthesis in H9c2 cells. In conclusion, we identify for the first time functional HCN channels in mammalian cardiac mitochondria and demonstrate their impact on ΔΨm and respiration.


Assuntos
Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Potencial da Membrana Mitocondrial , Mitocôndrias Cardíacas/metabolismo , Consumo de Oxigênio , Animais , Linhagem Celular , Células HEK293 , Humanos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/análise , Camundongos Endogâmicos BALB C , Miócitos Cardíacos/metabolismo , Ratos Wistar
3.
Eur J Neurosci ; 49(5): 604-622, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-29797362

RESUMO

The purpose of this review is to bridge the gap between earlier literature on striatal cholinergic interneurons and mechanisms of microcircuit interaction demonstrated with the use of newly available tools. It is well known that the main source of the high level of acetylcholine in the striatum, compared to other brain regions, is the cholinergic interneurons. These interneurons provide an extensive local innervation that suggests they may be a key modulator of striatal microcircuits. Supporting this idea requires the consideration of functional properties of these interneurons, their influence on medium spiny neurons, other interneurons, and interactions with other synaptic regulators. Here, we underline the effects of intrastriatal and extrastriatal afferents onto cholinergic interneurons and discuss the activation of pre- and postsynaptic muscarinic and nicotinic receptors that participate in the modulation of intrastriatal neuronal interactions. We further address recent findings about corelease of other transmitters in cholinergic interneurons and actions of these interneurons in striosome and matrix compartments. In addition, we summarize recent evidence on acetylcholine-mediated striatal synaptic plasticity and propose roles for cholinergic interneurons in normal striatal physiology. A short examination of their role in neurological disorders such as Parkinson's, Huntington's, and Tourette's pathologies and dystonia is also included.


Assuntos
Doenças dos Gânglios da Base/fisiopatologia , Neurônios Colinérgicos/fisiologia , Corpo Estriado/fisiologia , Interneurônios/fisiologia , Rede Nervosa/fisiologia , Plasticidade Neuronal/fisiologia , Animais , Humanos
4.
Int J Mol Sci ; 20(20)2019 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-31601020

RESUMO

Hyperpolarization-activated cationic HCN channels comprise four members (HCN1-4) that control dendritic integration, synaptic transmission and action potential firing. In the kidney, HCN1, HCN2 and HCN3 are differentially expressed and contribute to the transport of sodium, potassium (K+) and ammonium into the nephrons. HCN3 is regulated by K+ diets in the kidney. In this work we performed a proteomic analysis of HCN3 expressed in human embryonic kidney cells (HEK293 cells). More than 50% of the interacting proteins belonged to mitochondria. Therefore, we explored the presence of HCN channels in kidney mitochondria. By immunoblotting and immunogold electron microscopy HCN3 protein expression was found in rat kidney mitochondria; it was also confirmed in human kidney. Patch-clamp recordings of renal mitochondria and mitochondria from HEK293 cells overexpressing HCN1, HCN2 and HCN3 channels, stained with MitoTracker Green FM, indicated that only HCN3 could produce inwardly K+ currents that were inhibited by ZD7288, a specific blocker of HCN channels. Furthermore, ZD7288 caused inhibition of the oxygen consumption coupled to ATP synthesis and hyperpolarization of the inner mitochondrial membrane. In conclusion, we show for the first time that pacemaker HCN channels contribute to K+ transport in mitochondria facilitating the activity of the respiratory chain and ATP synthesis by controlling the inner mitochondrial membrane potential.


Assuntos
Rim/metabolismo , Mitocôndrias/metabolismo , Canais de Potássio/metabolismo , Potenciais de Ação , Respiração Celular , Cromatografia Líquida , Ativação do Canal Iônico , Mitocôndrias/genética , Nucleotídeos Cíclicos/metabolismo , Proteoma , Proteômica/métodos , Espectrometria de Massas em Tandem
5.
BMC Neurosci ; 19(1): 42, 2018 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-30012109

RESUMO

BACKGROUND: Striatal fast-spiking interneurons (FSI) are a subset of GABAergic cells that express calcium-binding protein parvalbumin (PV). They provide feed-forward inhibition to striatal projection neurons (SPNs), receive cortical, thalamic and dopaminergic inputs and are coupled together by electrical and chemical synapses, being important components of the striatal circuitry. It is known that dopamine (DA) depolarizes FSI via D1-class DA receptors, but no studies about the ionic mechanism of this action have been reported. Here we ask about the ion channels that are the effectors of DA actions. This work studies their Ca2+ currents. RESULTS: Whole-cell recordings in acutely dissociated and identified FSI from PV-Cre transgenic mice were used to show that FSI express an array of voltage gated Ca2+ channel classes: CaV1, CaV2.1, CaV2.2, CaV2.3 and CaV3. However, CaV1 Ca2+ channel carries most of the whole-cell Ca2+ current in FSI. Activation of D1-like class of DA receptors by the D1-receptor selective agonist SKF-81297 (SKF) enhances whole-cell Ca2+ currents through CaV1 channels modulation. A previous block of CaV1 channels with nicardipine occludes the action of the DA-agonist, suggesting that no other Ca2+ channel is modulated by D1-receptor activation. Bath application of SKF in brain slices increases the firing rate and activity of FSI as measured with both whole-cell and Ca2+ imaging recordings. These actions are reduced by nicardipine. CONCLUSIONS: The present work discloses one final effector of DA modulation in FSI. We conclude that the facilitatory action of DA in FSI is in part due to CaV1 Ca2+ channels positive modulation.


Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo L/efeitos dos fármacos , Cálcio/metabolismo , Agonistas de Dopamina/farmacologia , Animais , Corpo Estriado/efeitos dos fármacos , Dopamina/metabolismo , Interneurônios/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Camundongos Transgênicos , Parvalbuminas/metabolismo
6.
Appl Opt ; 57(30): 8841-8844, 2018 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-30461866

RESUMO

Huanglongbing (HLB) is the most destructive disease of citrus worldwide. The disease is caused by the proto-bacteria Candidatus Liberibacter asiaticus and transmitted by the Asian citrus psyllid Diaphorina citri. HLB symptoms are slow to appear while the tree continues to be a source of inoculum. Monitoring tree health and rapid detection of HLB is critical for sustainable citrus production. Currently, scientists are working on developing new techniques for pre-symptomatic detection of HLB, as there is no available method for real-time assessment of tree health. In this study, we demonstrate the rapid and efficient discrimination between healthy and HLB-affected citrus by laser-induced breakdown spectroscopy combined with chemometric analysis. Healthy and HLB-affected trees were differentiated with a high degree of precision. The novelty of this method lies in the fingerprinting of healthy and diseased plants based on their organic and inorganic constituents, and the use of a multi-pulse laser coupled with a microscope to take spectra of the plant phloem.


Assuntos
Citrus/microbiologia , Floema/química , Doenças das Plantas/microbiologia , Rhizobiaceae/isolamento & purificação , Análise Espectral/métodos , Técnicas Bacteriológicas
7.
EMBO J ; 31(18): 3704-17, 2012 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-22903061

RESUMO

Understanding the cellular origin of cancer can help to improve disease prevention and therapeutics. Human plasma cell neoplasias are thought to develop from either differentiated B cells or plasma cells. However, when the expression of Maf oncogenes (associated to human plasma cell neoplasias) is targeted to mouse B cells, the resulting animals fail to reproduce the human disease. Here, to explore early cellular changes that might take place in the development of plasma cell neoplasias, we engineered transgenic mice to express MafB in haematopoietic stem/progenitor cells (HS/PCs). Unexpectedly, we show that plasma cell neoplasias arise in the MafB-transgenic mice. Beyond their clinical resemblance to human disease, these neoplasias highly express genes that are known to be upregulated in human multiple myeloma. Moreover, gene expression profiling revealed that MafB-expressing HS/PCs were more similar to B cells and tumour plasma cells than to any other subset, including wild-type HS/PCs. Consistent with this, genome-scale DNA methylation profiling revealed that MafB imposes an epigenetic program in HS/PCs, and that this program is preserved in mature B cells of MafB-transgenic mice, demonstrating a novel molecular mechanism involved in tumour initiation. Our findings suggest that, mechanistically, the haematopoietic progenitor population can be the target for transformation in MafB-associated plasma cell neoplasias.


Assuntos
Regulação Neoplásica da Expressão Gênica , Fator de Transcrição MafB/metabolismo , Mieloma Múltiplo/metabolismo , Animais , Antígenos CD34/biossíntese , Antígenos Ly/metabolismo , Linfócitos B/metabolismo , Metilação de DNA , DNA Complementar/metabolismo , Epigênese Genética , Perfilação da Expressão Gênica , Biblioteca Gênica , Células-Tronco Hematopoéticas/citologia , Humanos , Hibridização in Situ Fluorescente , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Transgênicos , Mieloma Múltiplo/genética , Translocação Genética
8.
Chem Biodivers ; 13(12): 1730-1737, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27448833

RESUMO

Tabernaemontana alba and Tabernaemontana arborea are Apocynaceae species used in Mexican traditional medicine for which little phytochemical information exists. In this study, preliminary gas chromatography/mass spectrometry analyses of different organs obtained from wild plants of both species identified a total of 10 monoterpenoid indole alkaloids (MIAs) and one simple indole alkaloid, nine of which were reported for the first time in these species. Furthermore, callus cultures were established from T. alba leaf explants and regeneration of whole plants was accomplished via somatic embryogenesis. The anti-addictive MIAs ibogaine and voacangine were then quantified by gas chromatography with flame ionization detection in wild plants of both species, as well as greenhouse-grown plants, in vitro-grown plantlets and embryogenic callus of T. alba. Ibogaine and voacangine were present in most samples taken from the whole plants of both species, with stem and root barks showing the highest concentrations. No alkaloids were detected in callus samples. It was concluded that T. alba and T. arborea are potentially viable sources of ibogaine and voacangine, and that these MIAs can be produced through somatic embryogenesis and whole plant regeneration of T. alba. Approaches to increase MIA yields in whole plants and to achieve alkaloid production directly in cell cultures are discussed.


Assuntos
Ibogaína/análogos & derivados , Ibogaína/análise , Tabernaemontana/química , Ibogaína/biossíntese , México , Especificidade da Espécie
9.
J Neurophysiol ; 113(3): 796-807, 2015 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-25392165

RESUMO

The external globus pallidus (GPe) is central for basal ganglia processing. It expresses muscarinic cholinergic receptors and receives cholinergic afferents from the pedunculopontine nuclei (PPN) and other regions. The role of these receptors and afferents is unknown. Muscarinic M1-type receptors are expressed by synapses from striatal projection neurons (SPNs). Because axons from SPNs project to the GPe, one hypothesis is that striatopallidal GABAergic terminals may be modulated by M1 receptors. Alternatively, some M1 receptors may be postsynaptic in some pallidal neurons. Evidence of muscarinic modulation in any of these elements would suggest that cholinergic afferents from the PPN, or other sources, could modulate the function of the GPe. In this study, we show this evidence using striatopallidal slice preparations: after field stimulation in the striatum, the cholinergic muscarinic receptor agonist muscarine significantly reduced the amplitude of inhibitory postsynaptic currents (IPSCs) from synapses that exhibited short-term synaptic facilitation. This inhibition was associated with significant increases in paired-pulse facilitation, and quantal content was proportional to IPSC amplitude. These actions were blocked by atropine, pirenzepine, and mamba toxin-7, suggesting that receptors involved were M1. In addition, we found that some pallidal neurons have functional postsynaptic M1 receptors. Moreover, some evoked IPSCs exhibited short-term depression and a different kind of modulation: they were indirectly modulated by muscarine via the activation of presynaptic cannabinoid CB1 receptors. Thus pallidal synapses presenting distinct forms of short-term plasticity were modulated differently.


Assuntos
Globo Pálido/fisiologia , Potenciais Pós-Sinápticos Inibidores , Receptor Muscarínico M1/metabolismo , Sinapses/metabolismo , Animais , Atropina/farmacologia , Neurônios Colinérgicos/efeitos dos fármacos , Neurônios Colinérgicos/metabolismo , Neurônios Colinérgicos/fisiologia , Globo Pálido/citologia , Peptídeos e Proteínas de Sinalização Intercelular , Muscarina/farmacologia , Agonistas Muscarínicos/farmacologia , Antagonistas Muscarínicos/farmacologia , Peptídeos/farmacologia , Pirenzepina/farmacologia , Ratos , Ratos Wistar , Receptor CB1 de Canabinoide/metabolismo , Receptor Muscarínico M1/agonistas , Receptor Muscarínico M1/antagonistas & inibidores , Sinapses/efeitos dos fármacos , Sinapses/fisiologia
10.
Proc Natl Acad Sci U S A ; 109(26): 10534-9, 2012 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-22689981

RESUMO

Chromosomal translocations involving the MALT1 gene are hallmarks of mucosa-associated lymphoid tissue (MALT) lymphoma. To date, targeting these translocations to mouse B cells has failed to reproduce human disease. Here, we induced MALT1 expression in mouse Sca1(+)Lin(-) hematopoietic stem/progenitor cells, which showed NF-κB activation and early lymphoid priming, being selectively skewed toward B-cell differentiation. These cells accumulated in extranodal tissues and gave rise to clonal tumors recapitulating the principal clinical, biological, and molecular genetic features of MALT lymphoma. Deletion of p53 gene accelerated tumor onset and induced transformation of MALT lymphoma to activated B-cell diffuse large-cell lymphoma (ABC-DLBCL). Treatment of MALT1-induced lymphomas with a specific inhibitor of MALT1 proteolytic activity decreased cell viability, indicating that endogenous Malt1 signaling was required for tumor cell survival. Our study shows that human-like lymphomas can be modeled in mice by targeting MALT1 expression to hematopoietic stem/progenitor cells, demonstrating the oncogenic role of MALT1 in lymphomagenesis. Furthermore, this work establishes a molecular link between MALT lymphoma and ABC-DLBCL, and provides mouse models to test MALT1 inhibitors. Finally, our results suggest that hematopoietic stem/progenitor cells may be involved in the pathogenesis of human mature B-cell lymphomas.


Assuntos
Caspases/genética , Células-Tronco Hematopoéticas/metabolismo , Linfoma/patologia , Proteínas de Neoplasias/genética , Oncogenes , Animais , Humanos , Camundongos , Camundongos Transgênicos , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa , NF-kappa B/metabolismo , Transcrição Gênica
11.
Pharmacol Res Perspect ; 12(3): e1224, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38864106

RESUMO

Coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2n first appeared in Wuhan, China in 2019. Soon after, it was declared a pandemic by the World Health Organization. The health crisis imposed by a new virus and its rapid spread worldwide prompted the fast development of vaccines. For the first time in human history, two vaccines based on recombinant genetic material technology were approved for human use. These mRNA vaccines were applied in massive immunization programs around the world, followed by other vaccines based on more traditional approaches. Even though all vaccines were tested in clinical trials prior to their general administration, serious adverse events, usually of very low incidence, were mostly identified after application of millions of doses. Establishing a direct correlation (the cause-effect paradigm) between vaccination and the appearance of adverse effects has proven challenging. This review focuses on the main adverse effects observed after vaccination, including anaphylaxis, myocarditis, vaccine-induced thrombotic thrombocytopenia, Guillain-Barré syndrome, and transverse myelitis reported in the context of COVID-19 vaccination. We highlight the symptoms, laboratory tests required for an adequate diagnosis, and briefly outline the recommended treatments for these adverse effects. The aim of this work is to increase awareness among healthcare personnel about the serious adverse events that may arise post-vaccination. Regardless of the ongoing discussion about the safety of COVID-19 vaccination, these adverse effects must be identified promptly and treated effectively to reduce the risk of complications.


Assuntos
Vacinas contra COVID-19 , COVID-19 , Humanos , Vacinas contra COVID-19/efeitos adversos , COVID-19/prevenção & controle , COVID-19/epidemiologia , Incidência , Vacinação/efeitos adversos , Anafilaxia/induzido quimicamente , Anafilaxia/etiologia , SARS-CoV-2/imunologia , Síndrome de Guillain-Barré/etiologia , Miocardite/etiologia , Miocardite/induzido quimicamente
12.
EMBO J ; 28(1): 8-20, 2009 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-19037256

RESUMO

In human cancers, all cancerous cells carry the oncogenic genetic lesions. However, to elucidate whether cancer is a stem cell-driven tissue, we have developed a strategy to limit oncogene expression to the stem cell compartment in a transgenic mouse setting. Here, we focus on the effects of the BCR-ABLp210 oncogene, associated with chronic myeloid leukaemia (CML) in humans. We show that CML phenotype and biology can be established in mice by restricting BCR-ABLp210 expression to stem cell antigen 1 (Sca1)(+) cells. The course of the disease in Sca1-BCR-ABLp210 mice was not modified on STI571 treatment. However, BCR-ABLp210-induced CML is reversible through the unique elimination of the cancer stem cells (CSCs). Overall, our data show that oncogene expression in Sca1(+) cells is all that is required to fully reprogramme it, giving rise to a full-blown, oncogene-specified tumour with all its mature cellular diversity, and that elimination of the CSCs is enough to eradicate the whole tumour.


Assuntos
Expressão Gênica , Genes abl/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Células-Tronco , Animais , Ataxina-1 , Ataxinas , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas do Tecido Nervoso/análise , Proteínas Nucleares/análise , Análise de Sobrevida
13.
Am J Pathol ; 181(5): 1879-88, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22982190

RESUMO

The pathogenesis of diffuse large B-cell lymphoma (DLBCL) remains partially unknown. The analysis of the B-cell receptor of the malignant cells could contribute to a better understanding of the DLBCL biology. We studied the molecular features of the immunoglobulin heavy chain (IGH) rearrangements in 165 patients diagnosed with DLBCL not otherwise specified. Clonal IGH rearrangements were amplified according to the BIOMED-2 protocol and PCR products were sequenced directly. We also analyzed the criteria for stereotyped patterns in all complete IGHV-IGHD-IGHJ (V-D-J) sequences. Complete V-D-J rearrangements were identified in 130 of 165 patients. Most cases (89%) were highly mutated, but 12 sequences were truly unmutated or minimally mutated. Three genes, IGHV4-34, IGHV3-23, and IGHV4-39, accounted for one third of the whole cohort, including an overrepresentation of IGHV4-34 (15.5% overall). Interestingly, all IGHV4-34 rearrangements and all unmutated sequences belonged to the nongerminal center B-cell-like (non-GCB) subtype. Overall, we found three cases following the current criteria for stereotyped heavy chain VH CDR3 sequences, two of them belonging to subsets previously described in CLL. IGHV gene repertoire is remarkably biased, implying an antigen-driven origin in DLBCL. The particular features in the sequence of the immunoglobulins suggest the existence of particular subgroups within the non-GCB subtype.


Assuntos
Antígenos de Neoplasias/imunologia , Rearranjo Gênico de Cadeia Pesada de Linfócito B/genética , Centro Germinativo/imunologia , Cadeias Pesadas de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Sequência de Aminoácidos , Aminoácidos/genética , Células Clonais , Regiões Determinantes de Complementaridade/química , Regiões Determinantes de Complementaridade/genética , Feminino , Humanos , Imuno-Histoquímica , Linfoma Difuso de Grandes Células B/classificação , Linfoma Difuso de Grandes Células B/diagnóstico , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Mutação/genética , Hipermutação Somática de Imunoglobulina/genética , Hipermutação Somática de Imunoglobulina/imunologia , Recombinação V(D)J/genética , Recombinação V(D)J/imunologia , Adulto Jovem
14.
Materials (Basel) ; 16(6)2023 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-36984283

RESUMO

Lack of standardization of the retention phase has led to many studies of stability of movements and characteristic of retainers, disregarding the enamel repercussions of fixed retention on this phase. This study aimed to analyze different methods of enamel polishing after detachment of orthodontic retainers. Forty-five healthy premolars were divided into three groups according to the polishing bur after debonding, and four specimens without intervention were used as control. A 0.038 × 0.015 inches gold chain was bonded between the premolars and then removed. The adhesive remnant was removed with three types of burs according to the study groups (Group 1: white stone at high speed; Group 2: high-speed handpiece with a 30-blade tungsten carbide bur; Group 3: low-speed handpiece and a 30-blade tungsten bur). After debonding and polishing, all samples were analyzed with a confocal microscopy on surface roughness parameters: Sa: Arithmetic mean of the height of the surface. Rq: Square mean of the height of the surface and Sz: Maximum surface height. Mechanical tests were carried out to determine the bonding stress of the retention adhered to the teeth using an electromechanical testing machine. The adhesion stress was 8.23 MPa (±0.87). The quality of the refinement of the enamel after debonding is essential in order to preserve its integrity. The use of the 30-blade tungsten carbide bur provides a smooth enamel surface after polishing.

15.
Cell Calcium ; 116: 102800, 2023 12.
Artigo em Inglês | MEDLINE | ID: mdl-37776645

RESUMO

We have studied Danio rerio (Zebrafish) TRPA1 channel using a method that combines single channel electrophysiological and optical recordings to evaluate lateral mobility and channel gating simultaneously in single channels. TRPA1 channel activation by two distinct chemical ligands: allyl isothiocyanate (AITC) and TRPswitch B, results in substantial reduction of channel lateral mobility at the plasma membrane. Incubation with the cholesterol sequestering agent methyl-ß-cyclodextrin (MßCD), prevents the reduction on lateral mobility induced by the two chemical agonists. This results strongly suggest that the open conformation of TRPA1 modulates channel lateral mobility probably by facilitating the insertion of the channel into cholesterol-enriched domains at the plasma membrane.


Assuntos
Canais de Potencial de Receptor Transitório , Animais , Canal de Cátion TRPA1 , Canais de Potencial de Receptor Transitório/metabolismo , Peixe-Zebra/metabolismo , Fenômenos Eletrofisiológicos , Colesterol
16.
Viruses ; 15(2)2023 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-36851625

RESUMO

Baculoviridae is a large family of arthropod-infective viruses. Recombinant baculoviruses have many applications, the best known is as a system for large scale protein production in combination with insect cell cultures. More recently recombinant baculoviruses have been utilized for the display of proteins of interest with applications in medicine. In the present review we analyze the different strategies for the display of proteins and peptides on the surface of recombinant baculoviruses and provide some examples of the different proteins displayed. We analyze briefly the commercially available systems for recombinant baculovirus production and display and discuss the future of this emerging and powerful technology.


Assuntos
Artrópodes , Baculoviridae , Animais , Baculoviridae/genética , Peptídeos/genética , Técnicas de Cultura de Células
17.
Blood ; 115(1): 107-21, 2010 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-19887673

RESUMO

Cytokine genes are targets of multiple epigenetic mechanisms in T lymphocytes. 5-azacytidine (5-azaC) is a nucleoside-based DNA methyltransferase inhibitor that induces demethylation and gene reactivation. In the current study, we analyzed the effect of 5-azaC in T-cell function and observed that 5-azaC inhibits T-cell proliferation and activation, blocking cell cycle in the G(0) to G(1) phase and decreasing the production of proinflammatory cytokines such as tumor necrosis factor-alpha and interferon-gamma. This effect was not attributable to a proapoptotic effect of the drug but to the down-regulation of genes involved in T-cell cycle progression and activation such as CCNG2, MTCP1, CD58, and ADK and up-regulation of genes that induce cell-growth arrest, such as DCUN1D2, U2AF2, GADD45B, or p53. A longer exposure to the drug leads to demethylation of FOXP3 promoter, overexpression of FOXP3, and expansion of regulatory T cells. Finally, the administration of 5-azaC after transplantation prevented the development of graft-versus-host disease, leading to a significant increase in survival in a fully mismatched bone marrow transplantation mouse model. In conclusion, the current study shows the effect of 5-azaC in T lymphocytes and illustrates its role in the allogeneic transplantation setting as an immunomodulatory drug, describing new pathways that must be explored to prevent graft-versus-host disease.


Assuntos
Azacitidina/farmacologia , Transplante de Medula Óssea , Fatores Imunológicos/farmacologia , Animais , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Metilação de DNA/efeitos dos fármacos , Feminino , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Doença Enxerto-Hospedeiro/imunologia , Humanos , Imunidade/efeitos dos fármacos , Imunidade/genética , Ativação Linfocitária/efeitos dos fármacos , Contagem de Linfócitos , Masculino , Camundongos , Regiões Promotoras Genéticas/genética , Baço/citologia , Baço/efeitos dos fármacos , Análise de Sobrevida , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/efeitos dos fármacos , Transplante Homólogo
18.
J Neuroendocrinol ; 34(9): e13164, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35666232

RESUMO

Hypothalamic arginine vasopressin (AVP)-containing magnocellular neurosecretory neurons (AVPMNN) emit collaterals to synaptically innervate limbic regions influencing learning, motivational behaviour, and fear responses. Here, we characterize the dynamics of expression changes of two key determinants for synaptic strength, the postsynaptic density (PSD) proteins AMPAR subunit GluA1 and PSD scaffolding protein 95 (PSD95), in response to in vivo manipulations of AVPMNN neuronal activation state, or exposure to exogenous AVP ex vivo. Both long-term water deprivation in vivo, which powerfully upregulates AVPMNN metabolic activity, and exogenous AVP application ex vivo, in brain slices, significantly increased GluA1 and PSD95 expression as measured by western blotting, in brain regions reportedly receiving direct ascending innervations from AVPMNN (i.e., ventral hippocampus, amygdala and lateral habenula). By contrast, the visual cortex, a region not observed to receive AVPMNN projections, showed no such changes. Ex vivo application of V1a and V1b antagonists to ventral hippocampal slices ablated the AVP stimulated increase in postsynaptic protein expression measured by western blotting. Using a modified expansion microscopy technique, we were able to quantitatively assess the significant augmentation of PSD95 and GLUA1 densities in subcellular compartments in locus coeruleus tyrosine hydroxylase immunopositive fibres, adjacent to AVP axon terminals. Our data strongly suggest that the AVPMNN ascending system plays a role in the regulation of the excitability of targeted neuronal circuits through upregulation of key postsynaptic density proteins corresponding to excitatory synapses.


Assuntos
Sinapses , Tirosina 3-Mono-Oxigenase , Arginina Vasopressina/metabolismo , Hipocampo/metabolismo , Hipotálamo/metabolismo , Sinapses/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismo
19.
ASN Neuro ; 14: 17590914221102075, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36050845

RESUMO

SUMMARY STATEMENT: A2A receptor required previous D2 receptor activation to modulate Ca2+ currents. Istradefylline decreases pramipexole modulation on Ca2+ currents. Istradefylline reduces A2A + neurons activity in striatial microcircuit, but pramipexole failed to further reduce neuronal activity.


Assuntos
Dopamina , Transtornos Parkinsonianos , Adenosina , Animais , Transtornos Parkinsonianos/tratamento farmacológico , Pramipexol , Receptores de Dopamina D2/fisiologia , Roedores
20.
Biol Blood Marrow Transplant ; 17(2): 270-3, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20800691

RESUMO

Graft-versus-host disease (GVHD) is the major cause of morbidity and mortality among patients undergoing allogeneic hematopoietic stem cell transplantation. Although animal models have been clearly established for the study of skin, liver, and gut, currently there is no equivalent experiemental model for analyzing ocular involvement, which is rather common, especially among patients diagnosed with chronic GVHD. In the current study we have developed a murine model of ocular GVHD and, for the first time, we describe the histopathologic features involving cornea and limbus, which could play a role in the physiopathology of the disease at the ocular level. Our results represent a major finding that allows us to define a model for evaluating new therapeutic strategies for treating ocular GVHD prior to their use in clinical setting.


Assuntos
Doenças da Córnea/etiologia , Modelos Animais de Doenças , Doença Enxerto-Hospedeiro/fisiopatologia , Limbo da Córnea , Animais , Apoptose , Transplante de Medula Óssea/efeitos adversos , Caspase 3/metabolismo , Túnica Conjuntiva/metabolismo , Túnica Conjuntiva/patologia , Córnea/patologia , Doenças da Córnea/patologia , Feminino , Doença Enxerto-Hospedeiro/metabolismo , Doença Enxerto-Hospedeiro/patologia , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Limbo da Córnea/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Irradiação Corporal Total/efeitos adversos
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