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1.
Methods Mol Biol ; 2230: 367-376, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33197026

RESUMO

Whole mount in situ hybridization is a sensitive method used to characterize the spatial and temporal expression of RNA transcripts throughout an entire tissue. This method is an excellent tool for studying gene expression during embryonic development. Here, we describe a procedure for digoxigenin labeled in situ hybridization on whole embryos.


Assuntos
Embrião de Mamíferos/ultraestrutura , Desenvolvimento Embrionário/efeitos dos fármacos , Hibridização In Situ/métodos , Sondas RNA/farmacologia , Animais , Digoxigenina/farmacologia , Embrião de Mamíferos/diagnóstico por imagem , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Camundongos , Gravidez , Sondas RNA/isolamento & purificação
2.
Anal Chim Acta ; 1079: 146-152, 2019 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-31387705

RESUMO

A novel versatile locked nucleic acid modified molecular beacon probe (LNA-MB) was developed for imaging intracellular precursor miRNAs (pre-miRNAs) and disturbing Dicer-mediated cleavage process. The target recognition reaction between the smart probe and pre-miRNA can not only induce the conformational changes of probe and block the Dicer cleavage site, but also inhibit the cleavage process, and then achieve down-regulation of miRNA expression. Simultaneously, the target recognition reaction broke the fluorescence resonance energy transfer (FRET) between fluorophore donor FAM and acceptor TAMRA, which were labelled on the LNA-MB probe, further induced the relevant change of fluorescence signal, and then achieved imaging analysis of pre-miRNA and inhibition events in situ. Both in vitro and in single living cell studies showed that the versatile probes exhibited a remarkable performance in targeting with pre-miRNA-21, and nearly 65% downregulation of mature miRNA-21 was achieved with 100 nM probes. All investigations demonstrate that the proposed strategy represents a promising alternative for regulating and inhibiting endogenous disease-associated RNAs, then further for achieving therapeutic outcomes in personalized treatments.


Assuntos
Corantes Fluorescentes/química , MicroRNAs/antagonistas & inibidores , MicroRNAs/metabolismo , Sondas RNA/farmacologia , Proteínas Reguladoras de Apoptose/metabolismo , RNA Helicases DEAD-box/metabolismo , Regulação para Baixo/efeitos dos fármacos , Fluoresceínas/química , Transferência Ressonante de Energia de Fluorescência/métodos , Células HeLa , Humanos , MicroRNAs/genética , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Hibridização de Ácido Nucleico , Oligonucleotídeos/genética , Sondas RNA/genética , Proteínas de Ligação a RNA/metabolismo , Rodaminas/química , Ribonuclease III/metabolismo
3.
Chem Commun (Camb) ; 54(54): 7471-7474, 2018 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-29915829

RESUMO

Herein, we describe an extended version of a fluorescence probe for detecting miRNAs through the novel application of a PyA-cluster system. By testing various (CG)n sequences in the middle of the oligonucleotide strand of the probe, we obtained an optimal sequence that formed a double-three-way-junction structure, with two PyA units positioned close together, in the presence of the target miRNA. This system readily detected the locations of target miRNAs in living cells and allowed visualization of structural changes through variations in the color of the fluorescence.


Assuntos
Corantes Fluorescentes/farmacologia , MicroRNAs/análise , Pirenos/farmacologia , Sondas RNA/farmacologia , Animais , Linhagem Celular Tumoral , Fluorescência , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/química , Humanos , Camundongos , MicroRNAs/química , MicroRNAs/genética , Imagem Molecular , Conformação de Ácido Nucleico , Hibridização de Ácido Nucleico , Pirenos/síntese química , Pirenos/química , Sondas RNA/síntese química , Sondas RNA/química , Sondas RNA/genética
4.
Wiley Interdiscip Rev RNA ; 9(4): e1477, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29726113

RESUMO

The recognition of RNA functions beyond canonical protein synthesis has challenged the central dogma of molecular biology. Indeed, RNA is now known to directly regulate many important cellular processes, including transcription, splicing, translation, and epigenetic modifications. The misregulation of these processes in disease has led to an appreciation of RNA as a therapeutic target. This potential was first recognized in bacteria and viruses, but discoveries of new RNA classes following the sequencing of the human genome have invigorated exploration of its disease-related functions in mammals. As stable structure formation is evolving as a hallmark of mammalian RNAs, the prospect of utilizing small molecules to specifically probe the function of RNA structural domains and their interactions is gaining increased recognition. To date, researchers have discovered bioactive small molecules that modulate phenotypes by binding to expanded repeats, microRNAs, G-quadruplex structures, and RNA splice sites in neurological disorders, cancers, and other diseases. The lessons learned from achieving these successes both call for additional studies and encourage exploration of the plethora of mammalian RNAs whose precise mechanisms of action remain to be elucidated. Efforts toward understanding fundamental principles of small molecule-RNA recognition combined with advances in methodology development should pave the way toward targeting emerging RNA classes such as long noncoding RNAs. Together, these endeavors can unlock the full potential of small molecule-based probing of RNA-regulated processes and enable us to discover new biology and underexplored avenues for therapeutic intervention in human disease. This article is categorized under: RNA Methods > RNA Analyses In Vitro and In Silico RNA Interactions with Proteins and Other Molecules > Small Molecule-RNA Interactions RNA in Disease and Development > RNA in Disease.


Assuntos
Sondas RNA/química , Sondas RNA/farmacologia , RNA/química , RNA/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Doenças do Sistema Nervoso/genética , Doenças do Sistema Nervoso/metabolismo , Fenótipo , RNA/genética , Sítios de Splice de RNA/efeitos dos fármacos , Sítios de Splice de RNA/genética
5.
Nanomedicine ; 8(4): 393-8, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22349098

RESUMO

A new concept of tumor imaging is introduced using a siRNA-based probe that is capable of amplifying a specific endogenous fluorescence emission in cancerous tissue. In previous studies, we demonstrated a significant downregulation of Ferrochelatase (FECH) mRNA-expression in colorectal carcinomas leading to the accumulation of protoporphyrin IX (PpIX), a fluorescent metabolite of the heme synthesis. In this article, we report on first in vivo experiments in xenografted nude mice using folate-coupled liposomes or dendritic polyglycerolamine nanoparticles carrying ferrochelatase-siRNA to enhance PpIX-derived fluorescence in the tumor tissue. Tiny tumor foci could be monitored by the emission of PpIX fluorescence in vivo. Due to the omnipresence of the heme synthesis pathway, targeted application of ferrochelatase-siRNA may provide a general means for molecular imaging. FROM THE CLINICAL EDITOR: A new concept of tumor imaging is presented in this paper using a siRNA-based probe detecting protoporphyrin IX (PpIX), a fluorescent metabolite of the heme synthesis previously demonstrated to accumulate in cancer tissue.


Assuntos
Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Ferroquelatase/metabolismo , Fluorescência , Nanopartículas , Proteínas de Neoplasias/metabolismo , Sondas RNA/farmacologia , RNA Mensageiro/metabolismo , RNA Neoplásico/metabolismo , Animais , Neoplasias Colorretais/genética , Feminino , Ferroquelatase/genética , Humanos , Camundongos , Camundongos Nus , Proteínas de Neoplasias/genética , Transplante de Neoplasias , Protoporfirinas/genética , Protoporfirinas/metabolismo , RNA Mensageiro/genética , RNA Neoplásico/genética , Transplante Heterólogo
8.
Eur J Pharmacol ; 494(2-3): 83-90, 2004 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-15212961

RESUMO

Miltirone, a tanshinone isolated from the root of Salvia miltiorrhiza, has been characterized as a low-affinity ligand for central benzodiazepine receptors. We have now shown that this compound bound with low affinity (micromolar range) to central benzodiazepine recognition sites but did not interact with peripheral benzodiazepine receptors. It failed to potentiate Cl(-) currents induced by gamma-aminobutyric acid (GABA) both in Xenopus oocytes expressing recombinant human GABA(A) receptors and in cultured rat hippocampal pyramidal cells, but it inhibited the ability of diazepam to potentiate the effect of GABA in these systems. Miltirone (1-10 microM) also partially inhibited the increase in the abundance of the mRNA for the alpha(4) subunit of the GABA(A) receptor induced by ethanol withdrawal in cultured hippocampal neurons. These results suggest that miltirone might ameliorate the symptoms associated with discontinuation of long-term administration of ethanol or of other positive modulators of the GABA(A) receptor.


Assuntos
Depressores do Sistema Nervoso Central/efeitos adversos , Etanol/efeitos adversos , Hipocampo/metabolismo , Neurônios/metabolismo , Fenantrenos/farmacologia , RNA Mensageiro/biossíntese , Receptores de GABA-A/química , Síndrome de Abstinência a Substâncias/metabolismo , Tranquilizantes/farmacologia , Animais , Ligação Competitiva/efeitos dos fármacos , Células Cultivadas , Córtex Cerebral/metabolismo , DNA Complementar/biossíntese , DNA Complementar/genética , Eletrofisiologia , Flunitrazepam/metabolismo , Antagonistas GABAérgicos/farmacologia , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Isoquinolinas/metabolismo , Masculino , Neurônios/efeitos dos fármacos , Oócitos/metabolismo , Técnicas de Patch-Clamp , Sondas RNA/farmacologia , Ratos , Ratos Sprague-Dawley , Xenopus laevis
9.
Neuropharmacology ; 42(2): 191-8, 2002 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-11804615

RESUMO

The effects of long-term treatment with and subsequent withdrawal of the two hypnotic drugs zaleplon and zolpidem on the abundance of gamma-aminobutyric acid type A (GABA(A)) receptor subunit mRNAs in cultured rat cerebellar granule cells were investigated. Incubation of neurons with either drug at a concentration of 10 microM for 5 days did not significantly affect the amounts of mRNAs encoding the alpha(1), alpha(4), beta(1), beta(2), beta(3), gamma(2)L, or gamma(2)S subunits. As expected, similar treatment with the nonselective benzodiazepine diazepam resulted in a decrease in the abundance of alpha(1), beta(2), gamma(2)L, and gamma(2)S subunit mRNAs as well as an increase in that of the beta(1) subunit mRNA. Withdrawal of zaleplon or zolpidem, like that of diazepam, induced a marked increase in the amount of the alpha(4) subunit mRNA. In addition, discontinuation of treatment with either hypnotic drug resulted in a decrease in the amounts of alpha(1), beta(2), gamma(2)L, and gamma(2)S subunit mRNAs as well as an increase in that of the beta(1) subunit mRNA. These effects of zaleplon and zolpidem on GABA(A) receptor gene expression are consistent with the reduced tolerance liability of these drugs, compared with that of diazepam, as well as with their ability to induce both physical dependence and withdrawal syndrome.


Assuntos
Acetamidas/efeitos adversos , Acetamidas/farmacologia , Hipnóticos e Sedativos/farmacologia , Piridinas/efeitos adversos , Piridinas/farmacologia , Pirimidinas/efeitos adversos , Pirimidinas/farmacologia , Receptores de GABA-A/biossíntese , Receptores de GABA-A/genética , Síndrome de Abstinência a Substâncias/metabolismo , Animais , Células Cultivadas , Diazepam/farmacologia , Moduladores GABAérgicos/farmacologia , Ensaios de Proteção de Nucleases , Sondas RNA/síntese química , Sondas RNA/farmacologia , RNA Mensageiro/biossíntese , Ratos , Receptores de GABA-A/efeitos dos fármacos , Zolpidem
10.
J Clin Endocrinol Metab ; 86(9): 4520-8, 2001 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-11549702

RESUMO

The objectives of this study were to determine whether antiprogestin therapy or the infusion of human CG to mimic blastocyst transit in the baboon alters heparin-binding EGF-like growth factor expression during the window of implantation. During the menstrual cycle, heparin-binding EGF-like growth factor protein accumulation in the glandular epithelium was low in the proliferative phase and increased to maximal expression on d 5 and 10 postovulation. Stromal cells accumulated high levels of heparin-binding EGF-like growth factor in the proliferative phase, which decreased by d 5 postovulation. These transitional changes in both cell types were delayed when cycling baboons were treated with the antiprogestin ZK 137.316 during the luteal phase. The treatment with human CG had no effect on expression of heparin-binding EGF-like growth factor when compared with cycling baboons on d 10 postovulation and was comparable with that observed on d 18 and 22 of pregnancy. However, the superimposition of the antiprogestin with the human CG treatment also decreased expression in the epithelial cells. In summary, heparin-binding EGF-like growth factor accumulation in the epithelial glands is under the influence of progesterone and does not seem to be influenced by the paracrine secretion of trophoblast CG.


Assuntos
Gonadotropina Coriônica/farmacologia , Fator de Crescimento Epidérmico/biossíntese , Papio/fisiologia , Progestinas/antagonistas & inibidores , Animais , Blastocisto/efeitos dos fármacos , Implantação do Embrião/efeitos dos fármacos , Feminino , Fator de Crescimento Semelhante a EGF de Ligação à Heparina , Humanos , Imuno-Histoquímica , Hibridização In Situ , Peptídeos e Proteínas de Sinalização Intercelular , Comunicação Parácrina/efeitos dos fármacos , Comunicação Parácrina/fisiologia , Gravidez , Progesterona/farmacologia , Sondas RNA/farmacologia , Esteroides/farmacologia , Células Estromais/efeitos dos fármacos
11.
J Neurosci Methods ; 104(1): 19-25, 2000 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-11163407

RESUMO

The lamprey has been used as a model for the study of vertebrate neuronal circuitry and spinal cord regeneration. One of the advantages of this preparation is the ability to view the entire CNS in wholemount, including several identified neurons and neuron groups. However, because of difficulties in penetration of molecular reagents past the dense meninx primitiva and glia limitans, there has been no reliable method for in situ hybridization in spinal cord wholemounts. We now describe a protocol that accomplishes this while preserving the structural integrity of the cord. In order to enhance penetration of probes and antibodies, the m. primitiva was surgically stripped from the spinal cord after incubation of the fresh tissue in 0.1% collagenase I. Additional modifications that enhanced hybridization signal include (a) increasing the amount of Tween-20 in the hybridization mix to 2%, instead of the typical 0.2%; (b) carrying out the hybridization for 36 h and applying the anti-digoxigenin antibody to the tissue for 48 h. Using this protocol, we showed that netrin mRNA is expressed in dorsal cells, in medium sized neurons of the lateral gray matter and in the glial/ependymal cells of the spinal cords of lampreys. This method will help to study the expression of molecules of interest in identified neurons and neuronal groups without the need for serial section reconstruction.


Assuntos
Lampreias/metabolismo , Fatores de Crescimento Neural/genética , Neurônios/química , RNA Mensageiro/análise , Medula Espinal/química , Animais , Hibridização In Situ , Técnicas In Vitro , Lampreias/anatomia & histologia , Netrina-1 , Neurônios/citologia , Sondas RNA/farmacologia , Medula Espinal/citologia , Proteínas Supressoras de Tumor
12.
Zygote ; 3(1): 37-44, 1995 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-7542141

RESUMO

Interspersed RNA is an abundant class of cytoplasmic poly(A)+ RNA which contains repetitive elements within mostly heterogeneous single copy sequences. In spite of its quantitative importance in oocytes or eggs (two-thirds of the total poly(A)+ RNA), very little is known about its synthesis, its interaction with other molecules, and its functional significance. Here, we analysed a prevalent family of interspersed RNA (XR family) during Xenopus oogenesis. We found that XR interspersed RNA, unlike extracted interspersed RNA, did not form RNA duplexes in vivo. In small oocytes (stage III), XR RNA interacted with proteins forming rapidly sedimenting ribonucleoprotein particles (RNPs) with a median sedimentation constant of 80S. However, towards the end of oogenesis (stage VI), these XR RNPs changed into smaller particles with a median sedimentation constant of 40S. By analysing the proteins associated with XR RNA sequence, we have identified a 42 kilodalton protein in small oocytes, which was replaced by a 45 kilodalton protein at stage V of oogenesis.


Assuntos
Oócitos/fisiologia , RNA/metabolismo , Xenopus laevis/genética , Xenopus laevis/fisiologia , Animais , Células Cultivadas , Cromatografia/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Embrião não Mamífero/fisiologia , Feminino , Hibridização In Situ , Técnicas In Vitro , Injeções , Oócitos/química , Oócitos/efeitos dos fármacos , Oogênese/genética , Radioisótopos de Fósforo , RNA/genética , Sondas RNA/farmacologia , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Sequências Repetitivas de Ácido Nucleico , Ribonucleases/química , Ribonucleases/metabolismo , Ribonucleoproteínas/química , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/fisiologia , Transcrição Gênica
13.
Fundam Clin Pharmacol ; 9(6): 569-75, 1995.
Artigo em Inglês | MEDLINE | ID: mdl-8808178

RESUMO

In the present study we have reinvestigated the subtype of alpha 2-adrenoceptors expressed in human adipocytes (from subcutaneous and internal fat deposits) by means of radioligand binding using subtype-selective antagonists, and RNase mapping using a set of specific probes prepared from human alpha 2-adrenoceptors genes (alpha 2C2, alpha 2C4 and alpha 2C10). Comparison of the pharmacological properties of the human adipocyte alpha 2-adrenoceptors with those of the different human adrenoceptors expressed in COS-7 cells demonstrated that: i) human adipocyte alpha 2-adrenoceptors displays a KD for [3H]RX821002 and [3H]MK912 identical to that found in COS-7 cells transfected with the alpha 2C10 gene; ii) yohimbine and oxymetazoline is 1,000-fold more potent than prazosin to inhibit [3H]antagonist binding. RNase protection assays on cellular RNA prepared from the three fat deposits showed the presence of substantial amounts of alpha 2C10 transcripts: in contrast, mRNAs from alpha 2C2 and alpha 2C4 genes were undetectable. Altogether these results definitively establish that human adipocytes express only one alpha 2-adrenoceptor which is of the alpha 2A-subtype and encoded by the alpha 2C10 gene.


Assuntos
Adipócitos/metabolismo , Receptores Adrenérgicos alfa 2/metabolismo , Antagonistas Adrenérgicos alfa/farmacocinética , Adulto , Linhagem Celular , Feminino , Humanos , Idazoxano/análogos & derivados , Idazoxano/farmacocinética , Técnicas In Vitro , Membranas/metabolismo , Pessoa de Meia-Idade , Quinolizinas/farmacocinética , RNA/isolamento & purificação , RNA/metabolismo , Sondas RNA/síntese química , Sondas RNA/farmacologia , Ensaio Radioligante , Receptores Adrenérgicos alfa 2/genética , Ribonucleases/metabolismo
14.
EMBO J ; 8(12): 3587-93, 1989 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-2479547

RESUMO

We used a 279 bp cDNA probe derived from a Dictyostelium alpha-actinin genomic sequence to assay the degree of homology between alpha-actinin from slime molds, mammalian and chicken cells. Recognition of this probe by vertebrate cells was shown in Southern and Northern blots, and by antisense RNA-induced depression of endogenous alpha-actinin synthesis in living cells. Micro-injection of Dictyostelium or chicken gizzard alpha-actinin resulted in incorporation of these proteins in stress fibers, peripheral microfilament belts and adhesion sites. Alpha-actinin-injected cells showed a marked, transient reduction of synthesis of the corresponding endogenous protein. These data emphasize the high degree of conservation of alpha-actinin during evolution and show for the first time autoregulation of synthesis for a microfilament protein.


Assuntos
Actinina/biossíntese , Dictyostelium/metabolismo , Homeostase , Sondas RNA/farmacologia , RNA/farmacologia , Citoesqueleto de Actina/metabolismo , Actinina/genética , Actinina/fisiologia , Animais , Células Cultivadas , Galinhas , Dictyostelium/genética , Dictyostelium/fisiologia , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/genética , Proteínas Fúngicas/farmacologia , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , RNA Antissenso , Ratos , Homologia de Sequência do Ácido Nucleico , Transcrição Gênica
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