RESUMO
After completing the thio-substitution with Lawesson's reagent, ethanol was found to be effective in the decomposition of the inherent stoichiometric six-membered-ring byproduct from the Lawesson's reagent to a highly polarized diethyl thiophosphonate. The treatment significantly simplified the following chromatography purification of the desired thioamide in a small scale preparation. As scaling up the preparation of two pincer-type thioamides, we have successfully developed a convenient process with ethylene glycol to replace ethanol during the workup, including a traditional phase separation, extraction, and recrystallization. The newly developed chromatography-free procedure did not generate P-containing aqueous waste, and only organic effluents were discharged. It is believed that the optimized procedure offers the great opportunity of applying the Lawesson's reagent for various thio-substitution reactions on a large scale.
RESUMO
In the last 10 years, more and more attention has been focused on SNIP1 (Smad nuclear interacting protein 1), which functions as a transcriptional coactivator. We report here that through quantitative real-time PCR analysis in 18 different human tissues, SNIP1 was found to be expressed ubiquitously. When overexpressed in HeLa cells, SNIP1-EGFP fused protein exhibited a nuclear localization with a characteristic subnuclear distribution in speckles or formed larger discrete nuclear bodies in some cells. Reporter gene assay showed that overexpression of SNIP1 in HEK 293 cells or H1299 cells strongly activated the HSE signaling pathway. Moreover, SNIP1 could selectively regulate the transcription of HSP70A1A and HSP27. Taken together, our findings suggest that SNIP1 might also be a positive regulator of HSE signaling pathway.
Assuntos
Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Elementos de Resposta , Transdução de Sinais , Linhagem Celular Tumoral , Genes Reporter , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/análise , Proteínas de Ligação a RNA , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Recombinantes de Fusão/metabolismo , Transcrição GênicaRESUMO
Members of the transforming growth factor-ß (TGF-ß) superfamily have significant roles in the regulation of a wide variety of physiological processes. In our present work, phylogenetic tree analysis showed that human GDF3 (Growth and differentiation factor 3) and human GDF1 formed a subgroup of closely related molecules. Through quantitative real-time PCR analysis in different human tissues, GDF1 and GDF3 expression level had a big difference in brain. GDF3 could activate downstream signaling through associating with ALK7 (Activin receptor-like kinase 7) in a Cripto-dependent fashion. A CHO cell line stably transfected with the encoding sequence of GDF3, named CHO-GDF3, was established. Western blotting analysis demonstrated that GDF3 protein could be secreted into the medium from CHO cells and immunofluorescence experiment showed that GDF3 was mainly distributed in cytoplasm of the stable cell line, the primary hippocampal neurons, and brain tissues. Furthermore, the conditioned medium from CHO-GDF3 could reduce PC12 cell growth and induce cell differentiation. All these findings bring new insights into the functional study of GDF3.
Assuntos
Encéfalo/metabolismo , Diferenciação Celular/efeitos dos fármacos , Fator 3 de Diferenciação de Crescimento/farmacologia , Receptores de Ativinas Tipo I/metabolismo , Animais , Encéfalo/citologia , Química Encefálica , Células CHO , Cricetinae , Meios de Cultivo Condicionados/farmacologia , Fator 1 de Diferenciação de Crescimento , Fator 3 de Diferenciação de Crescimento/metabolismo , Humanos , Células PC12 , Ratos , Transdução de Sinais , Distribuição TecidualRESUMO
Recent studies have shown that, in mammals, the highly expressed genes have shorter gene length and their protein products have relatively lower evolutionary rates. However, the global relationship between genes' expression level and their features such as tissue-specificity, function and protein subcellular localization has not been investigated extensively, especially in mammalian. In order to solve it, we analysed 8,570 genes across 46 human tissues. Our results suggest that widely expressed genes have higher mean expression levels than tissue-specific ones and genes encoding zinc-finger proteins have low expression levels similar to that of DNA-binding proteins. In the analysis of protein subcellular localization, it is shown that nuclear and Golgi apparatus proteins have lower mean expression levels than those of mitochondria, endoplasmic reticulum and membrane proteins, while genes encoding cytoplasm and extracellular components display the highest expression levels. When comparing the gene expression levels and the number of expressed genes in different tissues, we found that some tissues have less active genes while single gene encodes relatively more transcripts. Taken together, gene expression levels are clearly correlated with their tissue-specificity, function and protein subcellular localization, and are highly conserved during evolution.
Assuntos
Evolução Molecular , Perfilação da Expressão Gênica/métodos , Expressão Gênica , Especificidade de Órgãos/genética , Proteínas/metabolismo , Membrana Celular/metabolismo , Citoplasma/metabolismo , Matriz Extracelular/metabolismo , Genes/genética , Humanos , Organelas/metabolismo , Proteínas/genéticaRESUMO
A nickel-catalyzed reductive decyanation of aromatic nitriles has been developed, in which the readily available and abundant ethanol was applied as the hydride donor. Various functional groups on the aromatic rings, such as alkoxyl, amino, imino and amide, were compatible in this catalytic protocol. Heteroaryl, benzylic and alkenyl nitriles were also tolerated. Mechanistic investigation indicated that ethanol provided hydride efficiently via ß-hydride elimination in this reductive decyanation.
RESUMO
We previously identified the human HMBOX1 (homeobox containing 1) gene, which represents a distinct group of HNF transcription factors, and is a potential transcription repressor with highly expression in pancreas. In our present work, we found that there exists a splicing variant of HMBOX1, designated HMBOX1b. Compared to HMBOX1, HMBOX1b encodes a 304 amino acids protein that shares the N-terminal region but has no homeodomain and the C-terminal region because of an alternative exon 7 which results in reading frame shifting. Unlike the highly pancreatic expression of HMBOX1, HMBOX1b was ubiquitous expressed in all human tissues detected by RT-PCR. Immunofluorescence staining showed that HMBOX1b accumulated in both cytoplasm and nucleus, and transcriptional reporter assays indicated that HMBOX1b only retained faint transcriptional repressive activity. Taken together, our findings suggest a distinct role of HMBOX1b, and the control of mRNA splicing might be involved in homeobox genes regulation.
Assuntos
Processamento Alternativo/genética , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/genética , Proteínas Repressoras/metabolismo , Transcrição Gênica , Sequência de Aminoácidos , Linhagem Celular , Clonagem Molecular , Biologia Computacional , Células Eucarióticas/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Humanos , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Transporte Proteico , Frações Subcelulares/metabolismoRESUMO
The cyclin-dependent kinases (CDKs) are a family of serine/threonine kinases, playing an essential role in regulating cell-cycle progression. In our present work, human CDK5 and a novel CDK5 splicing variant, named as CDK5-SV, were cloned from the cDNA library of human testis. CDK5-SV lacking the exon 7 of CDK5 encodes a protein of 260 amino acids. Through RT-PCR analysis in different human tissues, CDK5-SV was found to be expressed in testis, skeletal muscle, colon, bone marrow and ovary, while CDK5 was ubiquitously expressed. Immunofluorescence experiment in HeLa cells showed that the subcellular localizations of CDK5-SV and CDK5 were totally different. CDK5 mainly located in the cytoplasm, while CDK5-SV accumulated in nucleus. Reporter gene assay showed that when co-transfected with beta-catenin, CDK5 and CDK5-SV could both strongly inhibit the Wnt/beta-catenin signaling pathway. Consistently, CDK5-SV could also interact with beta-catenin as CDK5 does. Taken together, our findings suggest that CDK5-SV might also be a negative regulator of Wnt/beta-catenin signaling pathway.
Assuntos
Processamento Alternativo/genética , Quinase 5 Dependente de Ciclina/genética , Quinase 5 Dependente de Ciclina/metabolismo , Transdução de Sinais , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Clonagem Molecular , Biologia Computacional , Quinase 5 Dependente de Ciclina/química , Células Eucarióticas/enzimologia , Perfilação da Expressão Gênica , Humanos , Dados de Sequência Molecular , Ligação Proteica , Transporte Proteico , Frações Subcelulares/enzimologiaRESUMO
PURPOSE: The aim of this study is to investigate whether GDF3 is related to the progression of human breast cancer and the effects of GDF3 on breast cancer cells. METHODS: The expression of GDF3 in 24 breast cancer specimens paired with corresponding neighboring nontumorous tissue was studied by Western blot. Breast cancer cells were treated with different concentrations of recombinant human GDF3 protein. Using lentivirus containing sh-RNA, we knocked down the expression of GDF3. Soft agar assay was performed to explore the effects of GDF3 on colony formation. Different anti-tumor drugs dealt with MCF-7 cells stably expressing GDF3. RESULTS: We found that GDF3 expression level was significantly down-regulated in breast cancer tissues compared to the surrounding nontumorous tissues. GDF3 proteins could inhibit the proliferation of MCF-7 and T47D cells. We also found that the knockdown of GDF3 resulted in the promotion of colony formation and enhanced the ability of anchorage-independent cell growth in soft agar. Furthermore, overexpression of GDF3 could promote the apoptosis induced by Taxol. CONCLUSIONS: Our data indicated that GDF3 expression is significantly decreased in human breast cancer tissues, and reconstitution of GDF3 in breast cancer may be a potential therapeutic approach to inhibit aggressive growth of breast cancer.