RESUMO
Reactive oxygen species (ROS), antioxidants and their reduction-oxidation (redox) states all contribute to the redox homeostasis, but glutathione is considered to be the master regulator of it. We aimed to understand the relationship between the redox potential and the diverse glutathione transferase (GST) enzyme family by comparing the stress responses of two tomato cultivars (Solanum lycopersicum 'Moneymaker' and 'Ailsa Craig'). Four-week-old plants were treated by two concentrations of mannitol, NaCl and salicylic acid. The lower H2O2 and malondialdehyde contents indicated higher stress tolerance of 'Moneymaker'. The redox status of roots was characterized by measuring the reduced and oxidized form of ascorbate and glutathione spectrophotometrically after 24 h. The redox potential of 'Ailsa Craig' was more oxidized compared to 'Moneymaker' even under control conditions and became more positive due to treatments. High-throughput quantitative real-time PCR revealed that besides overall higher expression levels, SlGSTs were activated more efficiently in 'Moneymaker' due to stresses, resulting in generally higher GST and glutathione peroxidase activities compared to 'Ailsa Craig'. The expression level of SlGSTs correlated differently, however Pearson's correlation analysis showed usually strong positive correlation between SlGST transcription and glutathione redox potential. The possible redox regulation of SlGST expressions was discussed.
Assuntos
Peróxido de Hidrogênio , Solanum lycopersicum , Antioxidantes , Glutationa/metabolismo , Solanum lycopersicum/metabolismo , Oxirredução , Estresse Oxidativo , Ácido SalicílicoRESUMO
Patch clamp recording of neurons is a labor-intensive and time-consuming procedure. Here, we demonstrate a tool that fully automatically performs electrophysiological recordings in label-free tissue slices. The automation covers the detection of cells in label-free images, calibration of the micropipette movement, approach to the cell with the pipette, formation of the whole-cell configuration, and recording. The cell detection is based on deep learning. The model is trained on a new image database of neurons in unlabeled brain tissue slices. The pipette tip detection and approaching phase use image analysis techniques for precise movements. High-quality measurements are performed on hundreds of human and rodent neurons. We also demonstrate that further molecular and anatomical analysis can be performed on the recorded cells. The software has a diary module that automatically logs patch clamp events. Our tool can multiply the number of daily measurements to help brain research.
Assuntos
Aprendizado Profundo , Neurônios/citologia , Adulto , Idoso , Animais , Automação , Encéfalo/citologia , Eletrofisiologia , Feminino , Humanos , Processamento de Imagem Assistida por Computador , Masculino , Pessoa de Meia-Idade , Neurônios/química , Técnicas de Patch-Clamp , Ratos , Ratos Wistar , Software , Gravação em VídeoRESUMO
Single cell genomics and proteomics with the combination of innovative three-dimensional (3D) cell culture techniques can open new avenues toward the understanding of intra-tumor heterogeneity. Here, we characterize lung cancer markers using single cell mass cytometry to compare different in vitro cell culturing methods: two-dimensional (2D), carrier-free, or bead-based 3D culturing with in vivo xenografts. Proliferation, viability, and cell cycle phase distribution has been investigated. Gene expression analysis enabled the selection of markers that were overexpressed: TMEM45A, SLC16A3, CD66, SLC2A1, CA9, CD24, or repressed: EGFR either in vivo or in long-term 3D cultures. Additionally, TRA-1-60, pan-keratins, CD326, Galectin-3, and CD274, markers with known clinical significance have been investigated at single cell resolution. The described twelve markers convincingly highlighted a unique pattern reflecting intra-tumor heterogeneity of 3D samples and in vivo A549 lung cancer cells. In 3D systems CA9, CD24, and EGFR showed higher expression than in vivo. Multidimensional single cell proteome profiling revealed that 3D cultures represent a transition from 2D to in vivo conditions by intermediate marker expression of TRA-1-60, TMEM45A, pan-keratin, CD326, MCT4, Gal-3, CD66, GLUT1, and CD274. Therefore, 3D cultures of NSCLC cells bearing more putative cancer targets should be used in drug screening as the preferred technique rather than the Petri-dish.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/patologia , Técnicas de Cultura de Células , Citometria de Fluxo , Neoplasias Pulmonares/patologia , Modelos Biológicos , Análise de Célula Única , Células A549 , Carcinoma Pulmonar de Células não Pequenas/genética , Humanos , Neoplasias Pulmonares/genética , Células Tumorais CultivadasRESUMO
BACKGROUND: Here we examined myocardial microRNA (miRNA) expression profile in a sensory neuropathy model with cardiac diastolic dysfunction and aimed to identify key mRNA molecular targets of the differentially expressed miRNAs that may contribute to cardiac dysfunction. METHODS: Male Wistar rats were treated with vehicle or capsaicin for 3 days to induce systemic sensory neuropathy. Seven days later, diastolic dysfunction was detected by echocardiography, and miRNAs were isolated from the whole ventricles. RESULTS: Out of 711 known miRNAs measured by miRNA microarray, the expression of 257 miRNAs was detected in the heart. As compared to vehicle-treated hearts, miR-344b, miR-466b, miR-98, let-7a, miR-1, miR-206, and miR-34b were downregulated, while miR-181a was upregulated as validated also by quantitative real time polymerase chain reaction (qRT-PCR). By an in silico network analysis, we identified common mRNA targets (insulin-like growth factor 1 (IGF-1), solute carrier family 2 facilitated glucose transporter member 12 (SLC2a-12), eukaryotic translation initiation factor 4e (EIF-4e), and Unc-51 like autophagy activating kinase 2 (ULK-2)) targeted by at least three altered miRNAs. Predicted upregulation of these mRNA targets were validated by qRT-PCR. CONCLUSION: This is the first demonstration that sensory neuropathy affects cardiac miRNA expression network targeting IGF-1, SLC2a-12, EIF-4e, and ULK-2, which may contribute to cardiac diastolic dysfunction. These results further support the need for unbiased omics approach followed by in silico prediction and validation of molecular targets to reveal novel pathomechanisms.
Assuntos
Insuficiência Cardíaca Diastólica/etiologia , MicroRNAs/genética , Polineuropatias/complicações , Animais , Capsaicina/toxicidade , Modelos Animais de Doenças , Fator de Iniciação 4E em Eucariotos/genética , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Proteínas Facilitadoras de Transporte de Glucose/genética , Insuficiência Cardíaca Diastólica/genética , Fator de Crescimento Insulin-Like I/genética , Masculino , Polineuropatias/induzido quimicamente , Proteínas Serina-Treonina Quinases/genética , Ratos , Ratos WistarRESUMO
AIMS/HYPOTHESIS: Glucagon-like peptide 1 (GLP-1) receptors are expressed by pancreatic beta cells and GLP-1 receptor signalling promotes insulin secretion. GLP-1 receptor agonists have neural effects and are therapeutically promising for mild cognitive impairment and Alzheimer's disease. Our previous results showed that insulin is released by neurogliaform neurons in the cerebral cortex, but the expression of GLP-1 receptors on insulin-producing neocortical neurons has not been tested. In this study, we aimed to determine whether GLP-1 receptors are present in insulin-containing neurons. METHODS: We harvested the cytoplasm of electrophysiologically and anatomically identified neurogliaform interneurons during patch-clamp recordings performed in slices of rat neocortex. Using single-cell digital PCR, we determined copy numbers of Glp1r mRNA and other key genes in neurogliaform cells harvested in conditions corresponding to hypoglycaemia (0.5 mmol/l glucose) and hyperglycaemia (10 mmol/l glucose). In addition, we performed whole-cell patch-clamp recordings on neurogliaform cells to test the effects of GLP-1 receptor agonists for functional validation of single-cell digital PCR results. RESULTS: Single-cell digital PCR revealed GLP-1 receptor expression in neurogliaform cells and showed that copy numbers of mRNA of the Glp1r gene in hyperglycaemia exceeded those in hypoglycaemia by 9.6 times (p < 0.008). Moreover, single-cell digital PCR confirmed co-expression of Glp1r and Ins2 mRNA in neurogliaform cells. Functional expression of GLP-1 receptors was confirmed with whole-cell patch-clamp electrophysiology, showing a reversible effect of GLP-1 on neurogliaform cells. This effect was prevented by pre-treatment with the GLP-1 receptor-specific antagonist exendin-3(9-39) and was absent in hypoglycaemia. In addition, single-cell digital PCR of neurogliaform cells revealed that the expression of transcription factors (Pdx1, Isl1, Mafb) are important in beta cell development. CONCLUSIONS/INTERPRETATION: Our results provide evidence for the functional expression of GLP-1 receptors in neurons known to release insulin in the cerebral cortex. Hyperglycaemia increases the expression of GLP-1 receptors in neurogliaform cells, suggesting that endogenous incretins and therapeutic GLP-1 receptor agonists might have effects on these neurons, similar to those in pancreatic beta cells.
Assuntos
Córtex Cerebral/metabolismo , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Insulina/metabolismo , Interneurônios/metabolismo , Animais , Citoplasma/metabolismo , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Hiperglicemia/metabolismo , Hipoglicemia/metabolismo , Masculino , Neocórtex/metabolismo , Ratos , Ratos Wistar , Transdução de SinaisRESUMO
Escherichia coli is a well-established and popular host for heterologous expression of proteins. The preference in the choice of synonymous codons (codon bias), however, might differ for the host and the original source of the recombinant protein, constituting a potential bottleneck in production. Codon choice affects the efficiency of translation by a complex and poorly understood mechanism. The availability of certain tRNA species is one of the factors that may curtail the capacity of translation. Here we provide a tRNA-overexpressing strategy that allows the resolution of the codon bias, and boosts the translational capacity of the popular host BL21(DE3) when rare codons are encountered. In the BL21(DE3)-derived strain, called SixPack, copies of the genes corresponding to the six least abundant tRNA species have been assembled in a synthetic fragment and inserted into a rRNA operon. This arrangement, while not interfering with the growth properties of the new strain, allows dynamic control of the transcription of the extra tRNA genes, providing significantly elevated levels of the rare tRNAs in the exponential growth phase. Results from expression assays of a panel of recombinant proteins of diverse origin and codon composition showed that the performance of SixPack surpassed that of the parental BL21(DE3) or a related strain equipped with a rare tRNA-expressing plasmid.
Assuntos
Escherichia coli/genética , Biossíntese de Proteínas/genética , Códon , Escherichia coli/metabolismo , Edição de Genes/métodos , RNA Ribossômico/genética , RNA de Transferência/metabolismo , Proteínas Recombinantes/biossínteseRESUMO
We describe convergent evidence from transcriptomics, morphology, and physiology for a specialized GABAergic neuron subtype in human cortex. Using unbiased single-nucleus RNA sequencing, we identify ten GABAergic interneuron subtypes with combinatorial gene signatures in human cortical layer 1 and characterize a group of human interneurons with anatomical features never described in rodents, having large 'rosehip'-like axonal boutons and compact arborization. These rosehip cells show an immunohistochemical profile (GAD1+CCK+, CNR1-SST-CALB2-PVALB-) matching a single transcriptomically defined cell type whose specific molecular marker signature is not seen in mouse cortex. Rosehip cells in layer 1 make homotypic gap junctions, predominantly target apical dendritic shafts of layer 3 pyramidal neurons, and inhibit backpropagating pyramidal action potentials in microdomains of the dendritic tuft. These cells are therefore positioned for potent local control of distal dendritic computation in cortical pyramidal neurons.
Assuntos
Córtex Cerebral/metabolismo , Córtex Cerebral/ultraestrutura , Neurônios GABAérgicos/metabolismo , Neurônios GABAérgicos/ultraestrutura , Transcriptoma , Adulto , Idoso , Axônios/ultraestrutura , Espinhas Dendríticas/metabolismo , Espinhas Dendríticas/ultraestrutura , Junções Comunicantes/metabolismo , Junções Comunicantes/ultraestrutura , Biblioteca Gênica , Humanos , Masculino , Reação em Cadeia da Polimerase , Terminações Pré-Sinápticas/metabolismo , Terminações Pré-Sinápticas/ultraestrutura , Células Piramidais/metabolismo , Células Piramidais/ultraestrutura , RNA/análise , RNA/genética , Análise de Sequência de RNARESUMO
A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.
RESUMO
Little is known about the molecular mechanism including microRNAs (miRNA) in hypercholesterolemia-induced cardiac dysfunction. We aimed to explore novel hypercholesterolemia-induced pathway alterations in the heart by an unbiased approach based on miRNA omics, target prediction and validation. With miRNA microarray we identified forty-seven upregulated and ten downregulated miRNAs in hypercholesterolemic rat hearts compared to the normocholesterolemic group. Eleven mRNAs with at least 4 interacting upregulated miRNAs were selected by a network theoretical approach, out of which 3 mRNAs (beta-2 adrenergic receptor [Adrb2], calcineurin B type 1 [Ppp3r1] and calcium/calmodulin-dependent serine protein kinase [Cask]) were validated with qRT-PCR and Western blot. In hypercholesterolemic hearts, the expression of Adrb2 mRNA was significantly decreased. ADRB2 and PPP3R1 protein were significantly downregulated in hypercholesterolemic hearts. The direct interaction of Adrb2 with upregulated miRNAs was demonstrated by luciferase reporter assay. Gene ontology analysis revealed that the majority of the predicted mRNA changes may contribute to the hypercholesterolemia-induced cardiac dysfunction. In summary, the present unbiased target prediction approach based on global cardiac miRNA expression profiling revealed for the first time in the literature that both the mRNA and protein product of Adrb2 and PPP3R1 protein are decreased in the hypercholesterolemic heart.
Assuntos
Calcineurina/genética , Hipercolesterolemia/genética , MicroRNAs/genética , Miocárdio/metabolismo , Receptores Adrenérgicos beta 2/genética , Animais , Calcineurina/metabolismo , Regulação para Baixo , Guanilato Quinases/genética , Guanilato Quinases/metabolismo , Células HeLa , Humanos , Hipercolesterolemia/metabolismo , MicroRNAs/metabolismo , Processamento Pós-Transcricional do RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Receptores Adrenérgicos beta 2/metabolismoRESUMO
Quantifying heterogeneities within cell populations is important for many fields including cancer research and neurobiology; however, techniques to isolate individual cells are limited. Here, we describe a high-throughput, non-disruptive, and cost-effective isolation method that is capable of capturing individually targeted cells using widely available techniques. Using high-resolution microscopy, laser microcapture microscopy, image analysis, and machine learning, our technology enables scalable molecular genetic analysis of single cells, targetable by morphology or location within the sample.
Assuntos
Separação Celular/métodos , Processamento de Imagem Assistida por Computador/métodos , Microscopia Confocal/métodos , Análise de Célula Única/métodos , Animais , Células Cultivadas , Perfilação da Expressão Gênica , Humanos , Aprendizado de Máquina , Células Piramidais/citologia , Células Piramidais/metabolismo , Reprodutibilidade dos TestesRESUMO
A series of novel curcuminoids were synthesised for the first time via a Mannich-3CR/organocatalysed Claisen-Schmidt condensation sequence. Structure-activity relationship (SAR) studies were performed by applying viability assays and holographic microscopic imaging to these curcumin analogues for anti-proliferative activity against A549 and H1975 lung adenocarcinoma cells. The TNFα-induced NF-κB inhibition and autophagy induction effects correlated strongly with the cytotoxic potential of the analogues. Significant inhibition of tumour growth was observed when the most potent analogue 44 was added in liposomes at one-sixth of the maximally tolerated dose in the A549 xenograft model. The novel spectrum of activity of these Mannich curcuminoids warrants further preclinical investigations.
Assuntos
Antineoplásicos/farmacologia , Curcumina/farmacologia , Bases de Mannich/farmacologia , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Curcumina/análogos & derivados , Curcumina/química , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Masculino , Bases de Mannich/química , Camundongos , Camundongos SCID , Estrutura Molecular , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/patologia , Relação Estrutura-AtividadeRESUMO
Functional and molecular changes associated with pathophysiological conditions are relatively easily detected based on tissue samples collected from patients. Population specific cellular responses to disease might remain undiscovered in samples taken from organs formed by a multitude of cell types. This is particularly apparent in the human cerebral cortex composed of a yet undefined number of neuron types with a potentially different involvement in disease processes. We combined cellular electrophysiology, anatomy and single cell digital PCR in human neurons identified in situ for the first time to assess mRNA expression and corresponding functional changes in response to edema and increased intracranial pressure. In single pyramidal cells, mRNA copy numbers of AQP1, AQP3, HMOX1, KCNN4, SCN3B and SOD2 increased, while CACNA1B, CRH decreased in edema. In addition, single pyramidal cells increased the copy number of AQP1, HTR5A and KCNS1 mRNAs in response to increased intracranial pressure. In contrast to pyramidal cells, AQP1, HMOX1and KCNN4 remained unchanged in single cell digital PCR performed on fast spiking cells in edema. Corroborating single cell digital PCR results, pharmacological and immunohistochemical results also suggested the presence of KCNN4 encoding the α-subunit of KCa3.1 channels in edema on pyramidal cells, but not on interneurons. We measured the frequency of spontaneous EPSPs on pyramidal cells in both pathophysiological conditions and on fast spiking interneurons in edema and found a significant decrease in each case, which was accompanied by an increase in input resistances on both cell types and by a drop in dendritic spine density on pyramidal cells consistent with a loss of excitatory synapses. Our results identify anatomical and/or physiological changes in human pyramidal and fast spiking cells in edema and increased intracranial pressure revealing cell type specific quantitative changes in gene expression. Some of the edema/increased intracranial pressure modulated and single human pyramidal cell verified gene products identified here might be considered as novel pharmacological targets in cell type specific neuroprotection.
Assuntos
Edema Encefálico/metabolismo , Hipertensão Intracraniana/metabolismo , Neocórtex/metabolismo , Neurônios/metabolismo , Adulto , Edema Encefálico/patologia , Edema Encefálico/cirurgia , Feminino , Regulação da Expressão Gênica , Substância Cinzenta/metabolismo , Substância Cinzenta/patologia , Substância Cinzenta/cirurgia , Humanos , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Hipertensão Intracraniana/patologia , Hipertensão Intracraniana/cirurgia , Pressão Intracraniana/fisiologia , Masculino , Potenciais da Membrana/fisiologia , Pessoa de Meia-Idade , Neocórtex/patologia , Neocórtex/cirurgia , Neurônios/patologia , RNA Mensageiro/metabolismo , Técnicas de Cultura de TecidosRESUMO
C-150 a Mannich-type curcumin derivative, exhibited pronounced cytotoxic effects against eight glioma cell lines at micromolar concentrations. Inhibition of cell proliferation by C-150 was mediated by affecting multiple targets as confirmed at transcription and protein level. C-150 effectively reduced the transcription activation of NFkB, inhibited PKC-alpha which are constitutively over-expressed in glioblastoma. The effects of C-150 on the Akt/ Notch signaling were also demonstrated in a Drosophila tumorigenesis model. C-150 reduced the number of tumors in Drosophila with similar efficacy to mitoxantrone. In an in vivo orthotopic glioma model, C-150 significantly increased the median survival of treated nude rats compared to control animals. The multi-target action of C-150, and its preliminary in vivo efficacy would render this curcumin analogue as a potent clinical candidate against glioblastoma.
Assuntos
Acrilamidas/química , Neoplasias Encefálicas/tratamento farmacológico , Curcumina/análogos & derivados , Curcumina/química , Glioblastoma/tratamento farmacológico , NF-kappa B/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Receptores Notch/antagonistas & inibidores , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Animais , Antineoplásicos/química , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Drosophila melanogaster , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Glioblastoma/metabolismo , Humanos , Concentração Inibidora 50 , Melanoma Experimental , Camundongos , Transplante de Neoplasias , Ratos , Ratos Nus , Receptores Notch/metabolismo , Transdução de Sinais , Transcrição GênicaRESUMO
We aimed to characterize early changes in microRNA expression in acute cardioprotection by ischemic pre- and postconditioning in rat hearts. Hearts isolated from male Wistar rats were subjected to 1) time-matched nonischemic perfusion, 2) ischemia-reperfusion (30 min of coronary occlusion and 120 min of reperfusion), 3) preconditioning (3 × 5 min of coronary occlusion) followed by ischemia-reperfusion, or 4) ischemia-reperfusion with postconditioning (6 × 10 s of global ischemia-reperfusion at the onset of reperfusion). Infarct size was significantly reduced by both interventions. Of 350 different microRNAs assessed by microarray analysis, 147-160 microRNAs showed detectable expression levels. Compared with microRNA alterations induced by ischemia-reperfusion versus time-matched nonischemic controls, five microRNAs were significantly affected by both pre- and postconditioning (microRNA-125b*, microRNA-139-3p, microRNA-320, microRNA-532-3p, and microRNA-188), four microRNAs were significantly affected by preconditioning (microRNA-487b, microRNA-139-5p, microRNA-192, and microRNA-212), and nine microRNAs were significantly affected by postconditioning (microRNA-1, microRNA let-7i, microRNA let-7e, microRNA let-7b, microRNA-181a, microRNA-208, microRNA-328, microRNA-335, and microRNA-503). Expression of randomly selected microRNAs was validated by quantitative real-time PCR. By a systematic comparison of the direction of microRNA expression changes in all groups, we identified microRNAs, specific mimics, or antagomiRs that may have pre- and postconditioning-like cardioprotective effects (protectomiRs). Transfection of selected protectomiRs (mimics of microRNA-139-5p, microRNA-125b*, microRNA let-7b, and inhibitor of microRNA-487b) into cardiac myocytes subjected to simulated ischemia-reperfusion showed a significant cytoprotective effect. This is the first demonstration that the ischemia-reperfusion-induced microRNA expression profile is significantly influenced by both pre- and postconditioning, which shows the involvement of microRNAs in cardioprotective signaling. Moreover, by analysis of microRNA expression patterns in cardioprotection by pre- and postconditioning, specific protectomiRs can be revealed as potential therapeutic tools for the treatment of ischemia-reperfusion injury.
Assuntos
Pós-Condicionamento Isquêmico , Precondicionamento Isquêmico Miocárdico , MicroRNAs/metabolismo , Infarto do Miocárdio/prevenção & controle , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miócitos Cardíacos/metabolismo , Animais , Morte Celular , Células Cultivadas , Modelos Animais de Doenças , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Masculino , Infarto do Miocárdio/genética , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Miócitos Cardíacos/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase em Tempo Real , Reprodutibilidade dos Testes , Fatores de Tempo , TransfecçãoRESUMO
Concentrations of insulin in the brain are severalfold higher than blood plasma levels. Insulin in the brain regulates the metabolism, molecular composition, and cognitive performance of microcircuits and reduces food intake; cerebral insulin levels are altered in diabetes, aging, obesity, and Alzheimer's disease. Released by pancreatic ß cells, insulin passes the blood-brain barrier, but sources of locally released insulin still remain unclear. We find that insulin is strongly expressed in GABAergic neurogliaform cells in the cerebral cortex of the rat detected by single-cell digital PCR. Focal application of glucose or glibenclamide to neurogliaform cells mimics the excitation suppressing effect of external insulin on local microcircuits via insulin receptors. Thus, neurogliaform cells might link GABAergic and insulinergic action in cortical microcircuits.
Assuntos
Insulina/metabolismo , Neocórtex/citologia , Neocórtex/metabolismo , Neuroglia/metabolismo , Neurônios/metabolismo , Animais , Potenciais Pós-Sinápticos Excitadores/fisiologia , Secreção de Insulina , Masculino , Técnicas de Patch-Clamp , Reação em Cadeia da Polimerase , Radioimunoensaio , Ratos , Ratos Wistar , Ácido gama-Aminobutírico/metabolismoRESUMO
Whole-cell patch-clamp recording enables detection of electrophysiological signals from single neurons as well as harvesting of perisomatic RNA through the patch pipette for subsequent gene expression analysis. Amplification and profiling of RNA with traditional quantitative real-time PCR (qRT-PCR) do not provide exact quantitation due to experimental variation caused by the limited amount of nucleic acid in a single cell. Here we describe a protocol for quantifying mRNA or miRNA expression in individual neurons after patch-clamp recording using high-density nanocapillary digital PCR (dPCR). Expression of a known cell-type dependent marker gene (gabrd), as well as oxidative-stress related induction of hspb1 and hmox1 expression, was quantified in individual neurogliaform and pyramidal cells, respectively. The miRNA mir-132, which plays a role in neurodevelopment, was found to be equally expressed in three different types of neurons. The accuracy and sensitivity of this method were further validated using synthetic spike-in templates and by detecting genes with very low levels of expression.
Assuntos
Neurônios/fisiologia , Técnicas de Patch-Clamp/métodos , Reação em Cadeia da Polimerase/métodos , RNA Mensageiro/metabolismo , Análise de Célula Única/métodos , Animais , Células Cultivadas , Perfilação da Expressão Gênica/métodos , Masculino , MicroRNAs/análise , MicroRNAs/genética , MicroRNAs/metabolismo , Neurônios/química , Neurônios/citologia , Neurônios/metabolismo , RNA Mensageiro/análise , RNA Mensageiro/genética , Ratos , Ratos Wistar , Córtex Somatossensorial/citologiaRESUMO
Diet-induced hypercholesterolemia leads to oxidative/nitrative stress and subsequent myocardial dysfunction. However, the regulatory role of microRNAs in this phenomenon is unknown. We aimed to investigate, whether hypercholesterolemia-induced myocardial microRNA alterations play a role in the development of oxidative/nitrative stress and in subsequent cardiac dysfunction. Male Wistar rats were fed with 2% cholesterol/0.25% cholate-enriched or standard diet for 12weeks. Serum and tissue cholesterol levels were significantly elevated by cholesterol-enriched diet. Left ventricular end-diastolic pressure was significantly increased in cholesterol-fed rats both in vivo and in isolated perfused hearts, indicating diastolic dysfunction. Myocardial expression of microRNAs was affected by cholesterol-enriched diet as assessed by microarray analysis. MicroRNA-25 showed a significant down-regulation as detected by microarray analysis and QRT-PCR. In silico target prediction revealed NADPH oxidase 4 (NOX4) as a putative target of microRNA-25. NOX4 protein showed significant up-regulation in the hearts of cholesterol-fed rats, while NOX1 and NOX2 remained unaffected. Cholesterol-feeding significantly increased myocardial oxidative/nitrative stress as assessed by dihydroethidium staining, protein oxidation assay, and nitro-tyrosine ELISA, respectively. Direct binding of microRNA-25 mimic to the 3' UTR region of NOX4 was demonstrated using a luciferase reporter assay. Transfection of a microRNA-25 mimic into primary cardiomyocytes decreased superoxide production, while a microRNA-25 inhibitor resulted in an up-regulation of NOX4 protein and an increase in oxidative stress that was attenuated by the NADPH oxidase inhibitor diphenyleneiodonium. Here we demonstrated for the first time that hypercholesterolemia affects myocardial microRNA expression, and by down-regulating microRNA-25 increases NOX4 expression and consequently oxidative/nitrative stress in the heart. We conclude that hypercholesterolemia-induced microRNA alterations play an important role in the regulation of oxidative/nitrative stress and in consequent myocardial dysfunction.
Assuntos
Cardiopatias/etiologia , Cardiopatias/genética , Hipercolesterolemia/complicações , Hipercolesterolemia/genética , MicroRNAs/metabolismo , NADPH Oxidases/metabolismo , Estresse Oxidativo/fisiologia , Animais , Coração , Cardiopatias/metabolismo , Imuno-Histoquímica , Masculino , MicroRNAs/genética , Microscopia Eletrônica de Transmissão , NADPH Oxidase 4 , NADPH Oxidases/genética , Estresse Oxidativo/genética , Ratos , Ratos WistarRESUMO
BACKGROUND: Reperfusion of ischemic myocardium may contribute to substantial cardiac tissue damage, but the addition of iron chelators, zinc or zinc complexes has been shown to prevent heart from reperfusion injury. We investigated the possible beneficial effects of an iron-chelating and zinc-complexing agent, Q50, in rat models of ischemia/reperfusion (I/R)-induced myocardial infarction and on global reversible myocardial I/R injury after heart transplantation. METHODS AND RESULTS: Rats underwent 45-min myocardial ischemia by left anterior descending coronary artery ligation followed by 24h reperfusion. Vehicle or Q50 (10 mg/kg, IV) were given 5 min before reperfusion. In a heart transplantation model, donor rats received vehicle or Q50 (30 mg/kg, IV) 1h before the onset of ischemia. In myocardial infarcted rats, increased left ventricular end-systolic and end-diastolic volumes were significantly decreased by Q50 post treatment as compared with the sham group. Moreover, in I/R rat hearts, the decreased dP/dtmax and load-independent contractility parameters were significantly increased after Q50. However, Q50 treatment did not reduce infarct size or have any effect on increased plasma cardiac troponin-T-levels. In the rat model of heart transplantation, 1h after reperfusion, decreased left ventricular systolic pressure, dP/dt(max), dP/dt(min) and myocardial ATP content were significantly increased and myocardial protein expression of superoxide dismutase-1 was upregulated after Q50 treatment. CONCLUSIONS: In 2 experimental models of I/R, administration of Q50 improved myocardial function. Its mechanisms of action implicate in part the restoration of myocardial high-energy phosphates and upregulation of antioxidant enzymes.
Assuntos
Quelantes de Ferro/farmacologia , Traumatismo por Reperfusão Miocárdica , Miocárdio/metabolismo , Zinco , Animais , Modelos Animais de Doenças , Masculino , Traumatismo por Reperfusão Miocárdica/sangue , Traumatismo por Reperfusão Miocárdica/tratamento farmacológico , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Ratos , Ratos Endogâmicos Lew , Ratos Sprague-Dawley , Fatores de Tempo , Troponina T/sangueRESUMO
Micro RNAs (miRNA) are an abundant class of small RNAs that regulate the stability and translation of cognate mRNAs. MiRNAs are potential diagnostic markers, moreover, they play an essential role in the development of various heart disesases. In case of limited tissue material, such as, e.g. human biopsies, purification of miRNAs with sufficient yield is critical. Reproducible expression analysis of miRNAs is highly dependent on the quality of the RNA, which is often difficult to achieve from fibrous tissue such as the heart. Several companies developed general purification kits for miRNAs, however, none of them are specialized to fibrotic tissues. Here we describe an optimized miRNA purification protocol that results in high miRNA yield as compared to other methods including trizol-based and column-based protocols. By using our improved protocol, miRNA obtained from heart tissue gave more reproducible results in QRT-PCR analysis and obtained more significant calls (172 vs. 118) during DNA microarray analysis when compared to the commercially available kit. In addition to the heart tissue, the present protocol can be applied to other fibrotic tissues, such as lung or skeletal muscle to isolate high-purity miRNA.
Assuntos
MicroRNAs/química , Miocárdio/química , Animais , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Ratos , Reação em Cadeia da Polimerase em Tempo RealRESUMO
BACKGROUND: Polyunsaturated fatty acids (PUFAs) such as γ-linolenic acid (GLA), arachidonic acid (AA) and docosahexaenoic acid (DHA) have cytotoxic action on glioma cells. RESULTS: We evaluated the cytotoxic action of GLA, AA and DHA on glioma cells with specific reference to the expression of miRNAs. Relative expression of miRNAs were assessed by using high throughput nanocapillary real-time PCR. Most of the miRNA target genes that showed altered expression could be classified as apoptotic genes and were up-regulated by PUFA or temozolomide treatment, while similar treatments resulted in repression of the corresponding mRNAs, such as cox2, irs1, irs2, ccnd1, itgb3, bcl2, sirt1, tp53inp1 and k-ras. CONCLUSIONS: Our results highlight involvement of miRNAs in the induction of apoptosis in glioma cells by fatty acids and temozolomide.