Selectively labeling the heterologous protein in Escherichia coli for NMR studies: A strategy to speed up NMR spectroscopy

Nuclear magnetic resonance is an important tool for high-resolution structural studies of proteins. It demands high protein concentration and high purity; however, the expression of proteins at high levels often leads to protein aggregation and the protein purification step can correspond to a high percentage of the overall time in the structural determination process, In the present article we show that the step of sample optimization can be simplified by selective labeling the heterologous protein expressed in Escherichia coli by the use of rifampicin, Yeast thioredoxin and a coix transcription factor Opaque 2 leucine zipper (LZ) were used to show the effectiveness of the protocol. The H-1/N-15 heteronuclear correlation two-dimensional NMR spectrum (HMQC) of the selective N-15-labeled thioredoxin without any purification is remarkably similar to the spectrum of the purified protein, The method has high yields and a good H-1/N-15 HMQC spectrum can be obtained with 50 mi of Mo growth medium. Opaque 2 LZ, a difficult protein due to the lower expression level and high hydrophobicity, was also probed. The N-15-edited spectrum of Opaque 2 LZ showed only the resonances of the protein of heterologous expression (Opaque 2 LZ) while the H-1 spectrum shows several other resonances from other proteins of the cell lysate, The demand for a fast methodology for structural determination is increasing with the advent of genome/proteome projects. Selective labeling the heterologous protein can speed up NMR structural studies as well as NMR-based drug screening. This methodology is especially effective for difficult proteins such as hydrophobic transcription factors, membrane proteins, and others, (C) 2001 Academic Press.148114214

Nandrolone and resistance training induce heart remodeling: Role of fetal genes and implications for cardiac pathophysiology

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Aims: This study was conducted to assess the isolated and combined effects of nandrolone and resistance training on cardiac morphology, function, and mRNA expression of pathological cardiac hypertrophy markers. Main methods: Wistar rats were randomly divided into four groups and submitted to 6 weeks of treatment with nandrolone and/or resistance training. Cardiac parameters were determined by echocardiography. Heart was analyzed for collagen infiltration. Real-time RT-PCR was used to assess the pathological cardiac hypertrophy markers. Key findings: Both resistance training and nandrolone induced cardiac hypertrophy. Nandrolone increased the cardiac collagen content, and reduced the cardiac index in non-trained and trained groups, when compared with the respective vehicle-treated groups. Nandrolone reduced the ratio of maximum early to late transmitral flow velocity in non-trained and trained groups, when compared with the respective vehicle-treated groups. Nandrolone reduced the alpha-myosin heavy chain gene expression in both non-trained and trained groups, when compared with the respective vehicle-treated groups. Training reduced the beta-myosin heavy chain gene expression in the groups treated with vehicle and nandrolone. Only the association between training and nandrolone increased the expression of the skeletal alpha-actin gene and atrial natriuretic peptide in the left ventricle. Significance: This study indicated that nandrolone, whether associated with resistance training or not, induces cardiac hypertrophy, which is associated with enhanced collagen content, re-expression of fetal genes the in left ventricle, and impaired diastolic and systolic function. (C) 2011 Elsevier Inc. All rights reserved.8917-18631637Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)FAEPEX/UNICAMPConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)FAPESP [05/60284-6