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1.
Psychiatr Danub ; 31(3): 347-354, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31596828

RESUMO

BACKGROUND: The role of sirtuins as a pathogenetic element of some mental disorders is becoming increasingly more common. They participate in many cellular processes, such as ageing, transcription, apoptosis, inflammatory processes, post-translational modification of proteins, gene transcription silencing, activation of DNA repair mechanisms, and regulation of many metabolic processes. The aim of this paper is to verify the statistical hypothesis assuming the difference in expression at the level of mRNA in genes for sirtuins 1-7 between patients with recurrent depressive disorders (rDD) and patients from the control group, and the hypothesis assuming the relation between the expression at the level of mRNA for these genes and clinical variables in the course of recurrent depressive disorders. SUBJECTS AND METHODS: A total of 198 individuals took part in the study (rDD gropup, N=99; control group, N=99). RESULTS: SIR-1 and SIR-6 expression at the mRNA level was significantly higher among the people with rDD as compared to the subjects from the control group. A reversed relationship was observed for SIR-2, SIR-3, SIR-4 and SIR-5. Statistically significant correlations were observed only in the case of SIR-1 and the number of depression episodes (negative relationship), as well as SIR-5 and the severity of depression measured by the Hamilton Depression Rating Scale (positive relationship). CONCLUSIONS: Expression at the mRNA level for selected sirtuins is a factor that significantly differentiates people with depressive episodes from healthy ones. SIR-1 and SIR-6 expression at the mRNA level was significantly higher among the people with depression as compared to the subjects from the control group. A reversed relationship (also statistically significant) was observed for SIR-2, SIR-3, SIR-4 and SIR-5.


Assuntos
Transtorno Depressivo Maior/genética , Regulação da Expressão Gênica , Sirtuínas/genética , Doença Crônica , Humanos , RNA Mensageiro/análise , RNA Mensageiro/genética
2.
Nat Methods ; 16(9): 862-865, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31471614

RESUMO

Fluorogenic RNA aptamers bind and activate the fluorescence of otherwise nonfluorescent dyes. However, fluorogenic aptamers are limited by the small number of fluorogenic dyes suitable for use in live cells. In this communication, fluorogenic proteins whose fluorescence is activated by RNA aptamers are described. Fluorogenic proteins are highly unstable until they bind RNA aptamers inserted into messenger RNAs, resulting in fluorescent RNA-protein complexes that enable live imaging of mRNA in living cells.


Assuntos
Aptâmeros de Nucleotídeos/metabolismo , Fluorescência , Corantes Fluorescentes/química , Microscopia de Fluorescência/métodos , Imagem Molecular/métodos , RNA Mensageiro/análise , Aptâmeros de Nucleotídeos/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Células HEK293 , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo
3.
Exp Parasitol ; 206: 107767, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31520603

RESUMO

Schistosoma mansoni eggs can influence immune responses directed at them, and the mechanisms by which this is achieved are being unravelled. Going towards, developing effective tools for the study of how S. mansoni influences naïve T cells, we have developed S. mansoni eggs expressing chicken ovalbumin (OVA), using a lentiviral transduction system. Indeed, such a parasite could be used in conjunction with cells from OT-II transgenic mice as a source of naïve, antigen-specific T cells. The expression of the transgenic protein was confirmed by real-time RT-PCR of OVA-specific mRNA and western blotting using polyclonal antibodies specific for OVA. T cells from OT-II transgenic mice expressing a T cell receptor specific for the OVA323-339 peptide recognised the OVA-transduced S. mansoni eggs. Using flow cytometry on CFSE-labelled OT-II splenocytes, we demonstrated that OVA-transduced eggs elicit higher OT-II proliferative responses than untransduced eggs. The OT-II T cells also produced TNF-α and IFN-γ following exposure to OVA-transduced eggs. In addition, moderate amounts of IL-6 and IL-17A were also detected. In contrast, no IL-10, IL-4 and IL-2 were detected in cultures, whether the cells were stimulated with transduced or untransduced eggs. Thus, the cytokine signatures showed the transfected eggs induced predominantly a Th1 response, with a small amount of IL-6 and IL-17.


Assuntos
Ovalbumina/análise , Receptores de Antígenos de Linfócitos T/imunologia , Schistosoma mansoni/metabolismo , Linfócitos T/imunologia , Animais , Western Blotting , Galinhas , Citocinas/análise , Citocinas/metabolismo , Eletroforese em Gel de Ágar , Feminino , Citometria de Fluxo , Interleucina-17/análise , Interleucina-17/metabolismo , Interleucina-2/análise , Interleucina-2/metabolismo , Interleucina-6/análise , Interleucina-6/metabolismo , Fígado/parasitologia , Linfonodos/citologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Ovalbumina/genética , Ovalbumina/imunologia , Ovalbumina/metabolismo , Óvulo/metabolismo , RNA Mensageiro/análise , RNA Mensageiro/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Antígenos de Linfócitos T/genética , Transcrição Reversa , Schistosoma mansoni/genética , Schistosoma mansoni/crescimento & desenvolvimento , Baço/citologia , Linfócitos T/citologia
4.
Exp Parasitol ; 204: 107727, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31344389

RESUMO

BACKGROUND: Trypanosoma rangeli is a protozoan parasite that is non-virulent to the mammalian host and is morphologically and genomically related to Trypanosoma cruzi, whose proliferation within the mammalian host is controversially discussed. OBJECTIVES: We aimed to investigate the T. rangeli cell cycle in vitro and in vivo by characterizing the timespan of the parasite life cycle and by proposing a molecular marker to assess cytokinesis. METHODOLOGY: The morphological events and their timing during the cell cycle of T. rangeli epimastigotes were assessed using DNA staining, flagellum labelling and bromodeoxyuridine incorporation. Messenger RNA levels of four genes previously associated with the cell cycle of trypanosomatids (AUK1, PLK, MOB1 and TRACK) were evaluated in the different T. rangeli forms. FINDINGS: T. rangeli epimastigotes completed the cell cycle in vitro in 20.8 h. PLK emerged as a potential molecular marker for cell division, as its mRNA levels were significantly increased in exponentially growing epimastigotes compared with growth-arrested parasites or in vitro-differentiated trypomastigotes. PLK expression in T. rangeli can be detected near the flagellum protrusion site, reinforcing its role in the cell cycle. Interestingly, T. rangeli bloodstream trypomastigotes exhibited very low mRNA levels of PLK and were almost entirely composed of parasites in G1 phase. MAIN CONCLUSIONS: Our work is the first to describe the T. rangeli cell cycle in vitro and proposes that PLK mRNA levels could be a useful tool to investigate the T. rangeli ability to proliferate within the mammalian host bloodstream.


Assuntos
Proteínas de Ciclo Celular/genética , Ciclo Celular/genética , Citocinese/fisiologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , RNA Mensageiro/análise , Trypanosoma rangeli/citologia , Animais , Bromodesoxiuridina/metabolismo , Ciclo Celular/efeitos dos fármacos , Citocinese/genética , DNA de Protozoário/química , DNA de Protozoário/isolamento & purificação , Citometria de Fluxo , Imunofluorescência , Hidroxiureia/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Inibidores da Síntese de Ácido Nucleico/farmacologia , RNA de Protozoário/genética , RNA de Protozoário/isolamento & purificação , Fatores de Tempo , Trypanosoma rangeli/efeitos dos fármacos , Trypanosoma rangeli/enzimologia , Trypanosoma rangeli/genética , Tripanossomíase/parasitologia
5.
Biol Res ; 52(1): 35, 2019 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-31296259

RESUMO

BACKGROUND: Non-small cell lung cancer (NSCLC) is one of the leading causes of death in the world. NSCLC diagnosed at an early stage can be highly curable with a positive prognosis, but biomarker limitations make it difficult to diagnose lung cancer at an early stage. To identify biomarkers for lung cancer development, we previously focused on the oncogenic roles of transcription factor TFAP2C in lung cancers and revealed the molecular mechanism of several oncogenes in lung tumorigenesis based on TFAP2C-related microarray analysis. RESULTS: In this study, we analyzed microarray data to identify tumor suppressor genes and nine genes downregulated by TFAP2C were screened. Among the nine genes, we focused on growth arrest and DNA-damage-inducible beta (GADD45B) and phorbol-12-myristate-13-acetate-induced protein 1 (PMAIP1) as representative TFAP2C-regulated tumor suppressor genes. It was observed that overexpressed TFAP2C resulted in inhibition of GADD45B and PMAIP1 expressions at both the mRNA and protein levels in NSCLC cells. In addition, downregulation of GADD45B and PMAIP1 by TFAP2C promoted cell proliferation and cell motility, which are closely associated with NSCLC tumorigenesis. CONCLUSION: This study indicates that GADD45B and PMAIP1 could be promising tumor suppressors for NSCLC and might be useful as prognostic markers for use in NSCLC therapy.


Assuntos
Antígenos de Diferenciação/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Proliferação de Células/genética , Regulação para Baixo/genética , Neoplasias Pulmonares/genética , Fator de Transcrição AP-2/genética , Biomarcadores Tumorais/análise , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor/fisiologia , Humanos , RNA Mensageiro/análise , RNA Interferente Pequeno/análise
6.
Nature ; 571(7765): 424-428, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31292544

RESUMO

N6-methyladenosine (m6A) is the most prevalent modified nucleotide in mRNA1,2, with around 25% of mRNAs containing at least one m6A. Methylation of mRNA to form m6A is required for diverse cellular and physiological processes3. Although the presence of m6A in an mRNA can affect its fate in different ways, it is unclear how m6A directs this process and why the effects of m6A can vary in different cellular contexts. Here we show that the cytosolic m6A-binding proteins-YTHDF1, YTHDF2 and YTHDF3-undergo liquid-liquid phase separation in vitro and in cells. This phase separation is markedly enhanced by mRNAs that contain multiple, but not single, m6A residues. Polymethylated mRNAs act as a multivalent scaffold for the binding of YTHDF proteins, juxtaposing their low-complexity domains and thereby leading to phase separation. The resulting mRNA-YTHDF complexes then partition into different endogenous phase-separated compartments, such as P-bodies, stress granules or neuronal RNA granules. m6A-mRNA is subject to compartment-specific regulation, including a reduction in the stability and translation of mRNA. These studies reveal that the number and distribution of m6A sites in cellular mRNAs can regulate and influence the composition of the phase-separated transcriptome, and suggest that the cellular properties of m6A-modified mRNAs are governed by liquid-liquid phase separation principles.


Assuntos
Adenosina/análogos & derivados , Compartimento Celular , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Adenosina/metabolismo , Animais , Transporte Biológico , Linhagem Celular , Grânulos Citoplasmáticos/química , Grânulos Citoplasmáticos/metabolismo , Humanos , Metilação , Metiltransferases/deficiência , Camundongos , Transição de Fase , RNA Mensageiro/análise , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Estresse Fisiológico
7.
J Dairy Sci ; 102(9): 8305-8318, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31301838

RESUMO

Although choline requirements are unknown, enhanced postruminal supply may decrease liver triacylglycerol (TAG) storage and increase flux through the methionine cycle, helping cows during a negative energy balance (NEB). The objective was to investigate effects of postruminal choline supply during NEB on hepatic activity of betaine-homocysteine methyltransferase (BHMT), methionine synthase (MTR), methionine adenosyltransferase, transcription of enzymes, and metabolite concentrations in the methionine cycle. Ten primiparous rumen-cannulated Holstein cows (158 ± 24 d postpartum) were used in a replicated 5 × 5 Latin square design with 4-d treatment periods and 10 d of recovery (14 d/period). Treatments were unrestricted intake with abomasal infusion of water (A0), restricted intake (R; 60% of net energy for lactation requirements to induce NEB) with abomasal infusion of water (R0) or R plus abomasal infusion of 6.25, 12.5, or 25 g/d of choline ion. Liver tissue was collected on d 5 after the infusions ended, blood on d 1 to 5, and milk on d 1 to 4. Statistical contrasts were A0 versus R0 (CONT1) and tests of linear (L), quadratic (Q), and cubic (C) effects of choline dose. Plasma choline increased with R (CONT1) and choline (L). Although R decreased milk yield (CONT1), choline increased milk yield and liver phosphatidylcholine (PC), but decreased TAG (L). No differences were observed in plasma PC or very-low-density lipoprotein concentrations with R or choline. Activity and mRNA abundance of BHMT were greater with R (CONT1) and increased with choline (L). Although activity of MTR was lower with R (CONT1), it tended to increase with choline (L). No effect of R was detected for activity of methionine adenosyltransferase, but it changed cubically across dose of choline. Those responses were associated with linear increases in the concentrations of liver tissue (+13%) and plasma methionine concentrations. The mRNA abundance of CPT1A, SLC22A5, APOA5, and APOB, genes associated with fatty acid oxidation and lipoprotein metabolism, was upregulated by choline (Q). Overall, enhanced supply of choline during NEB increases hepatic activity of BHMT and MTR to regenerate methionine and PC, partly to help clear TAG. The relevance of these effects during the periparturient period merits further research.


Assuntos
5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/metabolismo , Betaína-Homocisteína S-Metiltransferase/metabolismo , Bovinos/metabolismo , Colina/administração & dosagem , Metabolismo Energético/efeitos dos fármacos , Fígado/metabolismo , Metionina/metabolismo , Abomaso/efeitos dos fármacos , Animais , Betaína-Homocisteína S-Metiltransferase/genética , Colina/sangue , Ácidos Graxos/metabolismo , Feminino , Lactação/efeitos dos fármacos , Metabolismo dos Lipídeos/efeitos dos fármacos , Lipoproteínas/metabolismo , Fígado/efeitos dos fármacos , Fígado/enzimologia , Metionina/sangue , Oxirredução , Parto/metabolismo , Gravidez , RNA Mensageiro/análise
8.
J Dairy Sci ; 102(9): 8513-8526, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31255268

RESUMO

Intensive milk feeding and butyrate supplementation in calves stimulate body growth and affect gastrointestinal development. The aim of the present study was to investigate the synergistic effects of ad libitum milk replacer (MR) feeding and butyrate supplementation of MR on rumen and small intestinal growth and on gene expression in the small intestine related to growth and energy metabolism at weaning. Male Holstein calves (n = 32) received colostrum from birth to d 3 of age and MR either ad libitum (Adl) or restrictively (Res; 6 L of MR/d; 12.5% solids) with (AdlB+, ResB+) or without (AdlB-, ResB-) 0.24% butyrate from d 4 until wk 8 of age. From wk 9 to 10, all calves were weaned and were fed 2 L/d until the end of the trial. Concentrate, hay, and water were freely available. At d 80, calves were slaughtered, volatile fatty acids were measured in rumen fluid, and rumen and small intestine samples were taken for histomorphometric measurements. The expression of mRNA associated with the local insulin-like growth factor (IGF) system and glucose metabolism as well as lactase and maltase activities were measured in the intestinal mucosa. The small intestine was 3 m longer in Adl than in Res. In the atrium ruminis, papilla width was greater in Res than in Adl. Villus circumference, cut surface, and height in the duodenum, proximal jejunum, and ileum were greater in Adl than in Res and in the proximal, mid, and distal jejunum and ileum were greater in calves treated with butyrate. Crypt depth in the duodenum and proximal jejunum was greater in Adl than in Res and in the ileum was smaller in calves treated with butyrate. The villus height:crypt depth ratio was greatest in AdlB+ calves. In the proximal and mid jejunum, IGF1 mRNA abundance was lower in calves treated with butyrate. In the proximal jejunum, INSR mRNA abundance was greater in Res than in Adl. The abundance of PCK2 mRNA was greater in Res than in Adl in the duodenum and was greatest in ResB- in the mid jejunum. Lactase activity tended to be greater in Res than in Adl and after butyrate treatment in the proximal jejunum. The results indicated an elevated growth of the small intestinal mucosa at weaning due to intensive milk feeding and butyrate supplementation, and the local IGF system was involved in intestinal growth regulation. Rumen development was not affected by butyrate supplementation of MR and was slightly delayed due to ad libitum MR feeding.


Assuntos
Butiratos/administração & dosagem , Bovinos/crescimento & desenvolvimento , Dieta/veterinária , Trato Gastrointestinal/crescimento & desenvolvimento , Substitutos do Leite/administração & dosagem , Rúmen/crescimento & desenvolvimento , Animais , Colostro , Suplementos Nutricionais , Ácidos Graxos Voláteis/análise , Feminino , Trato Gastrointestinal/química , Fator de Crescimento Insulin-Like I , Lactase/metabolismo , Masculino , Leite/metabolismo , Gravidez , RNA Mensageiro/análise , Proteínas Recombinantes , Rúmen/química , Somatomedinas/genética , Desmame
9.
Gen Physiol Biophys ; 38(4): 365-368, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31219430

RESUMO

The role of vascular endothelial growth factor (VEGF) in chronic stress and neurodevelopmental disorders is of growing research interest. Here we show that post-weaning isolation rearing of rats decreased gene expression of VEGF in the hippocampus. Gene expression of VEGF upstream regulator fibroblast growth factor-2 (FGF-2) or its downstream mediator endothelial nitric oxide synthase (eNOS) was unchanged. Other signaling pathways appear to be involved in isolation-induced reduction in VEGF gene expression. Sex differences in VEGF and eNOS gene expression with significantly higher mRNA levels in females than males were revealed.


Assuntos
Regulação para Baixo , Hipocampo/metabolismo , Isolamento Social , Fator A de Crescimento do Endotélio Vascular/genética , Desmame , Animais , Feminino , Fator 2 de Crescimento de Fibroblastos/genética , Masculino , Óxido Nítrico Sintase Tipo III/genética , RNA Mensageiro/análise , RNA Mensageiro/genética , Ratos
10.
Sci Data ; 6(1): 94, 2019 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-31209217

RESUMO

Transcript levels powerfully influence cell behavior and phenotype and are carefully regulated at several steps. Recently developed single cell approaches such as RNA single molecule fluorescence in-situ hybridization (smFISH) have produced advances in our understanding of how these steps work within the cell. In comparison to single-cell sequencing, smFISH provides more accurate quantification of RNA levels. Additionally, transcript subcellular localization is directly visualized, enabling the analysis of transcription (initiation and elongation), RNA export and degradation. As part of our efforts to investigate how this type of analysis can generate improved models of gene expression, we used smFISH to quantify the kinetic expression of STL1 and CTT1 mRNAs in single Saccharomyces cerevisiae cells upon 0.2 and 0.4 M NaCl osmotic stress. In this Data Descriptor, we outline our procedure along with our data in the form of raw images and processed mRNA counts. We discuss how these data can be used to develop single cell modelling approaches, to study fundamental processes in transcription regulation and develop single cell image processing approaches.


Assuntos
Hibridização in Situ Fluorescente , RNA Fúngico , RNA Mensageiro , Saccharomyces cerevisiae/genética , Análise de Célula Única , Proteínas de Membrana Transportadoras/análise , Proteínas de Membrana Transportadoras/genética , RNA Fúngico/análise , RNA Fúngico/genética , RNA Mensageiro/análise , RNA Mensageiro/genética , Proteínas de Saccharomyces cerevisiae/análise , Proteínas de Saccharomyces cerevisiae/genética , Análise de Célula Única/métodos , Análise de Célula Única/normas
11.
Histochem Cell Biol ; 152(2): 155-166, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31111198

RESUMO

Trace amine-associated receptors are G protein-coupled receptors of which TAAR1 is the most well-studied. Recently, Vattai et al. (J Cancer Res Clin Oncol 143:1637-1647 https://doi.org/10.1007/s00432-017-2420-8 , 2017) reported that expression of TAAR1 may be a marker of breast cancer (BC) survival, with a positive correlation also suggested between TAAR1 expression and HER2 positivity. Neither a role for TAAR1 in breast tissue, nor in cancer, had previously been suspected. We, therefore, sought to provide independent validation and to further examine these putative relationships. First, a bioinformatic analysis on 58 total samples including normal breast tissue, BC-related cell lines, and tumour samples representing different BC sub-types found no clear correlation between TAAR1 mRNA levels and any BC subtype, including HER2 + . We next confirmed the bioinformatics data correlated to protein expression using a well validated anti-human TAAR1 antibody. TAAR1 mRNA levels correlated with the relative intensity of immunofluorescence staining in six BC cell lines (MCF-7, T47D, MDA-MB-231, SKBR3, MDA-MB-468, BT-474), but not in the MCF-10A immortalized mammary gland line, which had high mRNA but low protein levels. As expected, TAAR1 protein was intracellular in all cell lines. Surprisingly MCF-7, SKBR3, and MDA-MB-468 showed pronounced nuclear localization. The relative protein expression in MCF-7, MDA-MB-231, and MCF-10A lines was further confirmed by semi-quantitative flow cytometry. Finally, we demonstrate that the commercially available anti-TAAR1 antibody has poor selectivity, which likely explains the lack of correlation with the previous study. Therefore, while we clearly demonstrate variable expression and sub-cellular localization of TAAR1 across BC cell lines, we find no evidence for association with BC subtype.


Assuntos
Neoplasias da Mama/genética , Receptores Acoplados a Proteínas-G/análise , Receptores Acoplados a Proteínas-G/genética , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/metabolismo , Linhagem Celular , Biologia Computacional , Feminino , Citometria de Fluxo , Imunofluorescência , Humanos , RNA Mensageiro/análise , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo
12.
Cell Physiol Biochem ; 52(6): 1484-1502, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31099508

RESUMO

BACKGROUND/AIMS: The transient receptor potential cation channel subfamily C member 6 (TRPC6) is a Ca2+-permeable nonselective cation channel and has received recent attention because of its capability to promote chronic kidney disease (CKD). The aims of this study were (i) to examine whether deletion of TRPC6 impacts on renal fibrosis and inflammatory cell infiltration in an early CKD model of unilateral ureter obstruction (UUO) in mice; and (ii) whether TRPC6-deficiency as well as UUO affect the regulation of TRPC expression in murine kidneys. METHODS: Wild-type (WT), Trpc6-knockout (Trpc6-/-) and New Zealand obese (NZO) mice underwent sham operation or unilateral ureteral obstruction (UUO). The kidneys were harvested 7 days after surgery. We examined renal fibrosis and inflammatory cell infiltration by histological and immunohistochemical staining. The mRNA expression of TRPC members and markers of fibrosis and inflammation in kidney were assessed by using real-time quantitative reverse transcription PCR. RESULTS: Histological and immunohistochemical analyses revealed less inflammatory cell infiltration (F4/80 and CD3) in UUO kidneys of Trpc6-/- mice compared to UUO kidneys of WT mice as well as less fibrosis. Genomic deletion of TRPC6 also affected the expression of pro-fibrotic genes in UUO Trpc6-/- kidneys compared to UUO WT kidneys while the expression of pro-inflammatory genes did not differ. UUO caused marked up-regulation of Trpc6 and down-regulation of Trpc1 mRNA in kidneys of WT and NZO mice. Trpc3 mRNA expression was significantly elevated in kidneys of Trpc6-/- mice underwent UUO while the levels did not change in kidneys of neither WT nor in NZO mice underwent UUO. CONCLUSION: TRPC6 contributes to renal fibrosis and immune cell infiltration in the UUO mouse model. Therefore, inhibition of TRPC6 emerges as a promising novel therapeutic strategy for treatment of chronic kidney failure in chronic obstructive nephropathy. However, confounding genomic and non-genomic effects of other TRPC channels should be taken into consideration to fully comprehend the renoprotective potential of targeting TRPC6 therapeutically under chronic kidney damaging conditions.


Assuntos
Regulação da Expressão Gênica , Rim/patologia , Canais de Cátion TRPC/genética , Obstrução Ureteral/genética , Animais , Modelos Animais de Doenças , Fibrose , Deleção de Genes , Inflamação/complicações , Inflamação/genética , Inflamação/patologia , Rim/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Obesos , RNA Mensageiro/análise , RNA Mensageiro/genética , Insuficiência Renal Crônica/complicações , Insuficiência Renal Crônica/genética , Insuficiência Renal Crônica/patologia , Regulação para Cima , Obstrução Ureteral/complicações , Obstrução Ureteral/patologia
13.
Eur J Endocrinol ; 181(1): 69-78, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-31096184

RESUMO

Objective: The pathophysiology of aldosterone-producing adenomas (APAs) has been intensively investigated using genetic and epigenetic approaches. However, the role of miRNAs in APA is not fully understood. The present study profiled miRNAs in APAs as an exploratory approach to elucidate their pathophysiological roles in APAs. Design: Tissues of APAs and other adrenocortical adenomas were obtained from patients who underwent adrenalectomy. Methods: Candidate miRNAs differentially detected from samples were examined by whole miRNA sequencing. The expression of candidate miRNAs in APA tissues were further validated by real-time quantitative polymerase chain reaction (qPCR). Further, differential miRNA expression between APAs with and without KCNJ5 somatic mutations was examined. Prediction of miRNA target genes was performed by bioinformatics analysis. For specific miRNAs, correlation analysis between the levels of their target genes and CYP11B2 was analyzed in APA tissues. Results: Our study determined differential expression of six miRNAs in APA or APA with KCNJ5 mutations. We further demonstrated that miR299 levels were negatively correlated with mRNA levels of CACNB2, which encodes the beta-subunit of the L-type calcium channel. Additionally, we found significant correlations among miR299, CACNB2, and CYP11B2 levels in APA tissues. Conclusions: Our study suggests the possible pathophysiological involvement of specific miRNAs in calcium signaling and aldosterone hypersecretion in APAs. Further studies, including in vitro analyses, are required to clarify these findings.


Assuntos
Adenoma Adrenocortical/genética , Aldosterona/biossíntese , Citocromo P-450 CYP11B2/genética , Perfilação da Expressão Gênica , MicroRNAs/fisiologia , Adrenalectomia , Adenoma Adrenocortical/metabolismo , Idoso , Canais de Cálcio Tipo L/genética , Feminino , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Expressão Gênica/fisiologia , Humanos , Masculino , MicroRNAs/análise , Pessoa de Meia-Idade , Mutação , RNA Mensageiro/análise , Reação em Cadeia da Polimerase em Tempo Real
14.
J Dairy Sci ; 102(7): 6263-6275, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31103297

RESUMO

Domestic yaks (Bos grunniens) and domestic Taurus cattle (Bos taurus) are closely related. An interesting phenomenon in interspecific crossings is male sterility in the F1 hybrid (yattle) and F2 backcross, with no late meiotic cells or spermatids in the seminiferous tubules. The mammalian Y chromosome is crucial for spermatogenesis and male fertility. This study investigated the copy number variations and mRNA of Y-transitional region genes TSPY2 (testis specific protein, Y-linked 2 and testis-specific Y-encoded protein 3-like) and PRAMEY (preferentially expressed antigen in melanoma, Y-linked), and Y-ampliconic region genes TSPY (testis-specific Y-encoded protein 1-like), ZNF280BY (zinc finger protein 280B, Y-linked) and HSFY (heat-shock transcription factor, Y-linked) in mature testes from Taurus cattle, yaks, and yattle. Phylogenetic trees divided 33 copies of TSPY into major 2 types (TSPY-T1 and TSPY-T2), 19 copies of TSPY2 into 2 types (TSPY2-T1 and T2), and 8 copies of PRAMEY into 4 types (PRAMEY-T1 to T4). Searching by the Basic Local Alignment Search Tool of the TSPY2 coding sequences in GenBank revealed that TSPY2 was conserved in Bovidae. The TSPY2-T2 sequences were absent, whereas PRAMEY-T2 and PRAMEY-T4 were amplified on the yak Y chromosome. The average copy numbers of TSPY-T2 and ZNF280BY were significantly different between cattle and yaks. The TSPY-T2, TSPY2, PRAMEY, ZNF280BY, and HSFY genes were uniquely or predominantly expressed in testes. Reverse-transcription quantitative PCR showed that the TSPY-T2, PRAMEY-T2, HSFY, ZNF280BY, protamine 1 (PRM1), and protamine 2 (PRM2) genes were almost not expressed in yattle. The PRM1 and PRM2 genes are used as positive markers for spermatozoa. Thus, our results showed that the genomic structure of the Y-transitional and Y-ampliconic region differed between Taurus cattle and yaks. Dysregulated expression of Y-ampliconic region genes TSPY-T2, HSPY, ZNF280BY, and Y-transitional region gene PRAMEY-T2 may be associated with hybrid male sterility in yattle.


Assuntos
Antígenos de Neoplasias/genética , Bovinos/genética , Proteínas de Ciclo Celular/genética , Ligação Genética/genética , Hibridização Genética/genética , Cromossomo Y/genética , Animais , Cruzamentos Genéticos , Variações do Número de Cópias de DNA , Expressão Gênica , Regulação da Expressão Gênica , Variação Genética/genética , Infertilidade Masculina/genética , Masculino , Filogenia , RNA Mensageiro/análise , Espermatogênese/genética , Testículo/metabolismo
15.
Acta Cir Bras ; 34(4): e201900403, 2019 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-31038583

RESUMO

PURPOSE: To investigate the long non-coding RNAs (lncRNAs) profile on renal ischemia reperfusion in a mouse model. METHODS: Microarray analysis was used to study the expression of misregulated lncRNA in a mouse model of renal ischemia reperfusion(I/R) with long ischemia time. Quantitative real-time PCR (qPCR) was used to verify the expression of selected lncRNAs and mRNAs.The potential functions of the lncRNA was analyzed by bioinformatics tools and databases. RESULTS: Kidney function was impaired in I/R group compared to the normal group. Analysis showed that a total of 2267 lncRNAs and 2341 messenger RNAs (mRNAs) were significantly expressed in I/R group (≥2.0-fold, p < 0.05).The qPCR result showed that lncRNAs and mRNAs expression were consistent with the microarray analysis. The co-expression network profile analysis based on five validated lncRNAs and 203 interacted mRNAs showed it existed a total of 208 nodes and 333 connections. The GO and KEEG pathway analysis results showed that multiple lncRNAs are involved the mechanism of I/R. CONCLUSION: Multiple lncRNAs are involved in the mechanism of I/R.These analysis results will help us to further understand the mechanism of I/R and promote the new methods targeted at lncRNA to improve I/R injury.


Assuntos
Rim/irrigação sanguínea , RNA Longo não Codificante/análise , RNA Mensageiro/análise , Traumatismo por Reperfusão/genética , Animais , Regulação para Baixo , Expressão Gênica , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase em Tempo Real , Valores de Referência , Análise Serial de Tecidos/métodos , Regulação para Cima
16.
Nature ; 570(7761): 332-337, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31042697

RESUMO

Alzheimer's disease is a pervasive neurodegenerative disorder, the molecular complexity of which remains poorly understood. Here, we analysed 80,660 single-nucleus transcriptomes from the prefrontal cortex of 48 individuals with varying degrees of Alzheimer's disease pathology. Across six major brain cell types, we identified transcriptionally distinct subpopulations, including those associated with pathology and characterized by regulators of myelination, inflammation, and neuron survival. The strongest disease-associated changes appeared early in pathological progression and were highly cell-type specific, whereas genes upregulated at late stages were common across cell types and primarily involved in the global stress response. Notably, we found that female cells were overrepresented in disease-associated subpopulations, and that transcriptional responses were substantially different between sexes in several cell types, including oligodendrocytes. Overall, myelination-related processes were recurrently perturbed in multiple cell types, suggesting that myelination has a key role in Alzheimer's disease pathophysiology. Our single-cell transcriptomic resource provides a blueprint for interrogating the molecular and cellular basis of Alzheimer's disease.


Assuntos
Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Análise de Célula Única , Transcriptoma , Envelhecimento/genética , Envelhecimento/patologia , Progressão da Doença , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Especificidade de Órgãos , Córtex Pré-Frontal/metabolismo , Córtex Pré-Frontal/patologia , RNA Mensageiro/análise , RNA Mensageiro/genética , Análise de Sequência de RNA , Caracteres Sexuais
17.
RNA ; 25(8): 1038-1046, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31064786

RESUMO

Visualization of gene expression at single RNA molecular level represents a great challenge to both imaging technologies and molecular engineering. Here we show a single molecule chromogenic in situ hybridization (smCISH) assay that enables counting and localizing individual RNA molecules in fixed cells and tissue under bright-field microscopy. Our method is based on in situ padlock probe assays directly using RNA as a ligation template and rolling circle amplification combined with enzyme catalyzed chromogenic reaction for amplification product visualization. We show potential applications of our method by detecting gene expression variations in single cells, subcellular localization information of expressed genes, and gene expression heterogeneity in formalin-fixed, paraffin-embedded tissue sections. This facile and straightforward method can in principle be applied to any type of RNA molecules in different samples.


Assuntos
Compostos Cromogênicos/química , RNA Mensageiro/análise , Imagem Individual de Molécula/métodos , Animais , Expressão Gênica , Humanos , Hibridização in Situ Fluorescente , RNA Mensageiro/química , Inclusão do Tecido , Fixação de Tecidos
18.
Sci Total Environ ; 677: 590-598, 2019 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-31071664

RESUMO

Metals and heavy metals are natural contaminants with an increasing presence in aquatic ecosystems as a result of human activities. Although they are mixed in the water, research is usually focused on analyzing them in isolation, so there is a lack of knowledge about their combined effects. The aim of this work was to assess the damage produced by mixtures of cadmium and copper, two frequent metals used in industry, in the harlequin midge Chironomus riparius (Diptera). The effects of acute doses of cadmium and copper were evaluated in fourth instar larvae by analyzing the mRNA levels of six genes related to apoptosis (DRONC, IAP1), immune system (PO1, Defensin), stress (Gp93), and copper homeostasis (Ctr1). DRONC, Ctr1, and IAP1 transcripts are described here for first time in this species. Individual fourth instar larvae were submitted to 10 µM, 1 µM and 0.1 µM of CdCl2 or CuCl2, and mixture. The employed individuals came from different egg masses. Real-time PCR analysis showed a complex pattern of alterations in transcriptional activity for two genes, DRONC and Gp93, while the rest of them did not show any statistically significant differences. The effector caspase DRONC showed upregulation with the highest concentration tested of the mixture. In case of gp93, chaperone involved in regulation of immune response, differences in expression levels were found with 1 and 10 µM Cu and 0.1 and 10 µM of mixtures, compared to control samples. These results suggest that mixtures affect the transcriptional activity differently and produce changes in apoptosis and stress processes, although it is also possible that Gp93 alteration could be related to the immune system since it is homologous to human protein Gp96, which has been related with Toll-like receptors. In conclusion, cadmium and copper mixtures can affect the population by affecting the ability of larvae to respond to the infection and the apoptosis, an important process in the metamorphosis of insects.


Assuntos
Apoptose/efeitos dos fármacos , Cloreto de Cádmio/efeitos adversos , Chironomidae/efeitos dos fármacos , Cobre/efeitos adversos , Proteínas de Insetos/genética , Transcrição Genética/efeitos dos fármacos , Poluentes Químicos da Água/efeitos adversos , Animais , Apoptose/genética , Chironomidae/genética , Chironomidae/crescimento & desenvolvimento , Chironomidae/fisiologia , Relação Dose-Resposta a Droga , Proteínas de Insetos/metabolismo , Larva/efeitos dos fármacos , Larva/fisiologia , RNA Mensageiro/análise , Reação em Cadeia da Polimerase em Tempo Real , Regulação para Cima
19.
Med Sci Monit ; 25: 2386-2396, 2019 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-30938333

RESUMO

BACKGROUND Cisplatin-resistant gastric cancer (GC) occurs in patients with GC treated with cisplatin-based chemotherapy, which results in disease progression and early recurrence during the treatment. MATERIAL AND METHODS To understand the initiation and developmental mechanism underlying cisplatin-resistant GC, we developed cisplatin-resistant SGC7901 cells (SGC7901/DDP) from the parental cells (SGC7901/S) by continuous exposure to increasing concentrations of cisplatin and subjected these 2 cell lines to RNA sequencing analysis. The data were verified by quantitative polymerase chain reaction and their functional role was evaluated by cell counting kit 8 assay and cell apoptosis and cell cycle flow cytometric analysis. Bioinformatics analysis was performed to classify the differentially-expressed genes (DEGs) involved in the development of cisplatin resistance. RESULTS In comparison with SGC7901/S cells, SGC7901/DDP cells showed a total of 3165 DEGs (2014 upregulated and 1151 downregulated, fold change ≥2, and adjusted P value <0.001). qRT-PCR confirmed the reliability of the RNA sequencing results. Depletion of the top 5 upregulated mRNAs reversed the resistant index, increased apoptotic SGC7901/DDP cells, and arrested the cells at G2/M phase. Gene ontology analysis revealed that the DEGs mainly regulate metabolic process, immune system, locomotion, cell adhesion, cell growth, cell death, cytoskeleton organization, cell binding, signal transducing activity, and antioxidant activity. Kyoto Encyclopedia of Genes and Genomes analysis showed that the DEGs were mainly involved in the PI3K-Akt signaling pathway, Rap1 signaling pathway, proteoglycans in cancer, regulation of actin cytoskeleton, and pathways in cancer. CONCLUSIONS The present study is the first to interrogate mRNAs profiles in human GC cells with cisplatin resistance using RNA sequencing, which may assist in discovering potential therapeutic targets for cisplatin-resistant GC patients.


Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Perfilação da Expressão Gênica/métodos , Neoplasias Gástricas/genética , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cisplatino/metabolismo , Cisplatino/farmacologia , Resistência a Múltiplos Medicamentos/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , RNA Mensageiro/análise , Reprodutibilidade dos Testes , Transcriptoma/genética
20.
Eur J Med Chem ; 173: 99-106, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-30991278

RESUMO

Diagnosis and treatment of breast cancer can be greatly enhanced and personalized based on the quantitative detection of mRNA markers. Here, we targeted the development of a fluorescent oligonucleotide probe to detect specifically the HER-2 mRNA breast cancer marker. We have selected the chromophore of the Green Fluorescent Protein (GFP), 4-hydroxybenzylidene imidazolinone (HBI), as a fluorophore covalently bound to an oligonucleotide probe and potentially capable of intercalating within a probe-mRNA duplex. We first synthesized the two-ring scaffold of the HBI chromophore 5 and coupled it to 2'-deoxyuridine at C5-position via a 7-atom-spacer, to give 4. Indeed, in the highly viscous glycerol used to mimic the reduced conformational flexibility of the intercalated HBI, chromophore 4 displayed a quantum yield of 0.29 and brightness of 20600 M-1cm-1, while no fluorescent signal was observed in methanol. Next, we synthesized a 20-mer oligonucleotide probe incorporating 4 at position 6 (5'-CCCGTUTCAACAGGAGTTTC-3'), ONHBI, targeting nucleotides 1233-1253 of HER-2 mRNA. A 16-fold enhancement of ONHBI emission intensity upon hybridization with the complementary RNA vs that of the oligonucleotide probe alone indicated the presence of target oligonucleotide and proved the intercalation of the chromophore (quantum yield 0.52; brightness 23500 M-1cm-1). Even more, an 11-fold enhancement of ONHBI emission (quantum yield 0.50; brightness 23200 M-1cm-1) was observed when the probe was mixed with total RNA extract from a human cell line that has high levels of HER2 mRNA expression. Thus, we propose ONHBI as a promising probe potentially useful for the sensitive and specific detection of HER2 mRNA breast cancer marker.


Assuntos
Biomarcadores Tumorais/análise , Neoplasias da Mama/diagnóstico por imagem , Proteínas de Fluorescência Verde/química , Sondas de Oligonucleotídeos/química , RNA Mensageiro/análise , Receptor ErbB-2/análise , Linhagem Celular Tumoral , Feminino , Humanos , Estrutura Molecular , Sondas de Oligonucleotídeos/síntese química , Espectrometria de Fluorescência
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