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1.
Biomolecules ; 13(4)2023 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-37189426

RESUMO

Circular RNAs, as covalently circularized RNA loops, have many unique biochemical properties. Many circRNA biological functions and clinical indications are being continually discovered. Increasingly, circRNAs are being used as a new class of biomarkers, which are potentially superior to linear RNAs due to the unusual cell/tissue/disease specificities and the exonuclease-resistant stabilized circular form in the biofluids. Profiling circRNA expression has been a common step in circRNA research to provide much needed insight into circRNA biology and to facilitate rapid advances in the circRNA field. We will review circRNA microarrays as a practical and effective circRNA profiling technology for regularly equipped biological or clinical research labs, share valuable experiences, and highlight the significant findings from the profiling studies.


Assuntos
RNA Circular , Perfilação da Expressão Gênica , Análise em Microsséries , RNA Circular/genética , RNA Circular/metabolismo
2.
Methods Mol Biol ; 2372: 53-74, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34417743

RESUMO

Arraystar LncRNA microarrays are designed for transcriptome-wide gene expression profiling of both lncRNAs and mRNAs on the same array. The array contents feature comprehensive collections of lncRNAs and include all canonical known coding mRNAs . Each lncRNA transcript is detected by a splice junction specific probe or a unique exon sequence , such that the alternatively spliced transcript isoforms or variants are reliably and accurately detected. The highly optimized experimental protocols and efficient workflow ensure sensitive, robust, and accurate microarray data generation. Standard data analyses are provided for microarray raw data processing, data quality control, gene expression clustering heat map visualization, differentially expressed lncRNAs and mRNAs , lncRNA subcategories, regulatory relationships of lncRNAs with the target mRNAs , gene ontology and pathway analyses. The LncRNA microarrays are a powerful tool for studying lncRNAs in biology and disease, with broad applications in gene expression profiling, gene regulatory mechanism research, lncRNA functional discovery, and biomarker development.


Assuntos
Análise em Microsséries , Perfilação da Expressão Gênica , Ontologia Genética , Redes Reguladoras de Genes , RNA Longo não Codificante/genética , RNA Mensageiro/genética
3.
Methods Mol Biol ; 1402: 43-61, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26721483

RESUMO

Arraystar LncRNA microarrays are designed for global gene expression profiling of both LncRNAs and mRNAs on the same array. The array contents feature comprehensive collections of LncRNAs and include entire sets of known coding mRNAs. Each RNA transcript is detected by a splice junction-specific probe or a unique exon sequence, such that the alternatively spliced transcript isoforms or variants are reliably and accurately detected. The highly optimized experimental protocols and efficient workflow ensure sensitive, robust, and accurate microarray data generation. Standard data analyses are provided for microarray raw data processing, data quality control, gene expression clustering and heat map visualization, differentially expressed LncRNAs and mRNAs, LncRNA subcategories, regulatory relationships of LncRNAs with the mRNAs, gene ontology, and pathway analysis. The LncRNA microarrays are powerful tools for the study of LncRNAs in biology and disease, with broad applications in gene expression profiling, gene regulatory mechanism research, LncRNA functional discovery, and biomarker development.


Assuntos
Perfilação da Expressão Gênica/instrumentação , Análise de Sequência com Séries de Oligonucleotídeos/instrumentação , RNA Longo não Codificante/genética , Animais , Perfilação da Expressão Gênica/métodos , Ontologia Genética , Redes Reguladoras de Genes , Humanos , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Reação em Cadeia da Polimerase/instrumentação , Reação em Cadeia da Polimerase/métodos , Ratos , Software , Espectrofotometria/instrumentação , Espectrofotometria/métodos
4.
J Mol Diagn ; 9(1): 105-12, 2007 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17251343

RESUMO

Mutations in the human ABCC6 gene cause pseudoxanthoma elasticum (PXE), a hereditary disorder that impacts the skin, eyes, and cardiovascular system. Currently, the diagnosis of PXE is based on physical findings and histological examination of a biopsy of affected skin. We have combined two simple, polymerase chain reaction (PCR)-based methods to develop a rapid, reliable genetic assay for the majority of known PXE mutations. After PCR amplification and heteroduplex formation, mutations in exon 24 and exon 28 of the ABCC6 gene were detected with Surveyor nuclease, which cleaves double-stranded DNA at any mismatch site. Mutations originating from deletion of a segment of the ABCC6 gene between exon 23 and exon 29 (ex23_ex29del) were detected by long-range PCR. Size analysis of digestion fragments and long-range PCR products was performed by agarose gel electrophoresis. The methods accurately identified mutations or the absence thereof in 16 affected individuals as confirmed by DNA sequencing. Fifteen patients had one or two point mutations, and two of these individuals carried the ex23_ex29del in their second allele. This mutation detection and mapping strategy provides a simple and reliable genetic assay to assist in diagnosis of PXE, differential diagnosis of PXE-like conditions, and study of PXE genetics.


Assuntos
Técnicas de Diagnóstico Molecular/métodos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Mutação Puntual/genética , Reação em Cadeia da Polimerase/métodos , Pseudoxantoma Elástico/genética , Diagnóstico Diferencial , Eletroforese em Gel de Ágar , Éxons/genética , Humanos , Análise de Sequência de DNA
5.
Pharm Res ; 21(11): 2105-11, 2004 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-15587934

RESUMO

PURPOSE: B-type natriuretic peptide (BNP) has been in clinical use for the treatment of decompensated congestive heart failure. However, BNP has a very short half-life in circulation, which limits its application to acute CHF and requires continuous i.v. infusion. To provide superior pharmacological benefits of BNP to other stages of chronic congestive heart failure and to eliminate problems associated with drug delivery via continuous i.v. infusion, we have designed and evaluated AlbuBNP, a long-acting form of BNP by recombinant fusion to human serum albumin for use in chronic congestive heart failure, post-acute follow-up, and postmyocardial infarction. METHODS: Human BNP (1-32) was seamlessly fused to mature human serum albumin at N-terminus to create AlbuBNP. The bioactivities of AlbuBNP were evaluated by natriuretic peptide receptor-A mediated cGMP activation assay, hemodynamic responses, and plasma cGMP elevation. The pharmacokinetic properties were determined after single i.v. or s.c. bolus injection in C57/BL6 mice. RESULTS: AlbuBNP had approxiamtely the same maximal bioactivity as BNP to activate cGMP in the in vitro NPRA/cGMP assay. The EC50s were 28.4+/-1.2 and 0.46+/-1.1 nM for AlbuBNP and BNP, respectively. In spontaneously hypertensive rats, AlbuBNP lowered both systolic and diastolic blood pressure, having sustainable mean arterial pressure reduction for more than 2 days. Six nmol/kg AlbuBNP i.v. bolus in mice increased plasma cGMP level 5.6-fold over the baseline. The elimination half-life in mice was dramatically increased from 3 min for BNP to 12-19 h for AlbuBNP. CONCLUSIONS: AlbuBNP is bioactive and has desired pharmacokinetic properties for long-term use. It has the potential to be further developed as a new therapeutic option for chronic, acute, and post-acute CHF to alleviate symptoms, improve clinical status, and slow the disease progression by sustained drug exposure via infrequent simple subcutaneous injections.


Assuntos
Insuficiência Cardíaca/tratamento farmacológico , Peptídeo Natriurético Encefálico/uso terapêutico , Proteínas Recombinantes de Fusão/farmacocinética , Proteínas Recombinantes de Fusão/uso terapêutico , Albumina Sérica/uso terapêutico , Sequência de Aminoácidos , Animais , Área Sob a Curva , Pressão Sanguínea/efeitos dos fármacos , GMP Cíclico/metabolismo , Expressão Gênica , Humanos , Imunoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Peptídeo Natriurético Encefálico/química , Peptídeo Natriurético Encefálico/farmacocinética , Ratos , Ratos Endogâmicos SHR , Proteínas Recombinantes de Fusão/química , Albumina Sérica/química
6.
Biochem Biophys Res Commun ; 321(4): 1024-31, 2004 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-15358131

RESUMO

Growth differentiation factor 3 (GDF-3) is structurally a bone morphogenetic protein/growth differentiation factor subfamily member of the TGF-beta superfamily. GDF-3 exhibits highest level of expression in white fat tissue in mice and is greatly induced by high fat diet if fat metabolic pathway is blocked. To identify its biological function, GDF-3 was overexpressed in mice by adenovirus mediated gene transfer. Mice transduced with GDF-3 displayed profound weight gain when fed with high fat diet. The phenotypes included greatly expanded adipose tissue mass, increased body adiposity, highly hypertrophic adipocytes, hepatic steatosis, and elevated plasma leptin. GDF-3 stimulated peroxisome proliferator activated receptor expression in adipocytes, a master nuclear receptor that controls adipogenesis. However, GDF-3 was not involved in blood glucose homeostasis or insulin resistance, a condition associated with obesity. In contrast, similar phenotypes were not observed in GDF-3 mice fed with normal chow, indicating that GDF-3 is only active under high lipid load. Thus, GDF-3 is a new non-diabetic adipogenic factor tightly coupled with fat metabolism.


Assuntos
Tecido Adiposo/crescimento & desenvolvimento , Gorduras na Dieta/administração & dosagem , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Células 3T3-L1 , Adipócitos/citologia , Adipócitos/fisiologia , Animais , Sequência de Bases , Glicemia/metabolismo , Tamanho Celular/genética , Tamanho Celular/fisiologia , Células Cultivadas , Citocinas/genética , Citocinas/fisiologia , DNA/genética , Fígado Gorduroso/etiologia , Fígado Gorduroso/genética , Fígado Gorduroso/patologia , Expressão Gênica , Fator 3 de Diferenciação de Crescimento , Humanos , Insulina/sangue , Peptídeos e Proteínas de Sinalização Intercelular/genética , Leptina/sangue , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptores Citoplasmáticos e Nucleares/genética , Fatores de Transcrição/genética , Transdução Genética , Aumento de Peso/genética , Aumento de Peso/fisiologia
7.
Biochem Biophys Res Commun ; 305(4): 981-8, 2003 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-12767927

RESUMO

We have identified and characterized a novel single span transmembrane leucine-rich repeat protein, synleurin, that renders cells highly sensitive to the activation by cytokines and lipopolysaccharide (LPS). The major part of the extracellular domain consists of a leucine-rich repeats (LRR) cassette. The LRR central core has 12 analogous LRR repeating modules arranged in a seamless tandem array. The LRRs are most homologous to that of chondroadherin, insulin-like growth factor binding proteins, platelet glycoprotein V, slits, and toll-like receptors. Synleurin expression was detected at low levels in many tissues, including smooth muscle, brain, uterus, pancreas, cartilage, adipose, spleen, and testis. When synleurin is ecotopically expressed in transfected cells, the cells exhibit amplified responses to bFGF, EGF, PDGF-B, IGF-1, IGF-2, and LPS. Synleurin gene (slrn) maps to human chromosome at 5q12. The name synleurin reflects its synergistic effect on cytokine stimulation and its prominent leucine-rich repeats.


Assuntos
Substâncias de Crescimento/farmacologia , Leucina/análise , Proteínas de Membrana/química , Proteínas de Membrana/fisiologia , Sequência de Aminoácidos , Cromossomos Humanos Par 5 , Clonagem Molecular , Citocinas/fisiologia , Humanos , Proteínas de Membrana/genética , Dados de Sequência Molecular , Filogenia , Estrutura Terciária de Proteína , Sequências Repetitivas de Aminoácidos , Transdução de Sinais , Distribuição Tecidual , beta Carioferinas
8.
Biochem Biophys Res Commun ; 294(4): 835-42, 2002 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-12061783

RESUMO

A hidden Markov model (HMM) has been used to describe, predict, identify, and generate secretory signal peptide sequences. The relative strengths of artificial secretory signals emitted from the human signal peptide HMM (SP-HMM) correlate with their HMM bit scores as determined by their effectiveness to direct alkaline phosphatase secretion. The nature of the signal strength is in effect the closeness to the consensus. The HMM bit score of 8 is experimentally determined to be the threshold for discriminating signal sequences from non-secretory ones. An artificial SP-HMM generated signal sequence of the maximum model bit score (HMM + 38) was selected as an ideal human signal sequence. This signal peptide (secrecon) directs strong protein secretion and expression. We further ranked the signal strengths of the signal peptides of the known human secretory proteins by SP-HMM bit scores. The applications of high-bit scoring HMM signals in recombinant protein production and protein engineering are discussed.


Assuntos
Peptídeos/metabolismo , Sinais Direcionadores de Proteínas , Linhagem Celular , Humanos , Funções Verossimilhança , Cadeias de Markov , Plasmídeos/metabolismo , Estrutura Terciária de Proteína , Proteínas Recombinantes/metabolismo
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