Maternal Calorie Restriction Induces a Transcriptional Cytoprotective Response in Embryonic Liver Partially Dependent on Nrf2.

BACKGROUND: Calorie restriction is known to enhance Nrf2 signaling and longevity in adult mice, partially by reducing reactive oxygen species, but calorie restriction during pregnancy leads to intrauterine growth retardation. The latter is associated with fetal reprogramming leading to increased incidence of obesity, metabolic syndrome and diabetes in adult life. Transcription factor Nrf2 is a central regulator of the antioxidant response and its crosstalk with metabolic pathways is emerging. We hypothesized that the Nrf2 pathway is induced in embryos during calorie restriction in pregnant mothers. METHODS: From gestational day 10 up to day 16, 50% of the necessary mouse diet was provided to Nrf2 heterozygous pregnant females with fathers being of the same genotype. Embryos were harvested at the end of gestational day 16 and fetal liver was used for qRT-PCR and assessment of oxidative stress (OS). RESULTS: Intrauterine calorie restriction led to upregulation of mRNA expression of antioxidant genes (Nqo1, Gsta1, Gsta4) and of genes related to integrated stress response (Chac1, Ddit3) in WT embryos. The expression of a key gluconeogenic (G6pase) and two lipogenic genes (Acacb, Fasn) was repressed in calorie-restricted embryos. In Nrf2 knockout embryos, the induction of Nqo1 and Gsta1 genes was abrogated while that of Gsta4 was preserved, indicating an at least partially Nrf2-dependent induction of antioxidant genes after in utero calorie restriction. Measures of OS showed no difference (superoxide radical and malondialdehyde) or a small decrease (thiobarbituric reactive substances) in calorie-restricted WT embryos. CONCLUSIONS: Calorie restriction during pregnancy elicits the transcriptional induction of cytoprotective/antioxidant genes in the fetal liver, which is at least partially Nrf2-dependent, with a physiological significance that warrants further investigation.

A quantitative western blot technique using TMB: Comparison with the conventional technique.

Numerous molecular biological experiments performed throughout the world require the detection or quantification of a protein of interest. Western blotting is one of the most popular techniques used for this purpose and offers quantitative information with the aid of specialized software. However, its dependence on the picture that is captured, and the background and the absence of a common protocol prevent the technique from being completely quantitative. To overcome these obstacles, we present a simple and reliable assay that is similar to the regular technique, with the exception of the last stage of band visualization and quantification. We propose that small pieces of the blot that include the protein of interest can be cut and dipped in a small volume of 3,3',5,5'-tetramethylbenzidine solution, giving a colorimetric signal with linear dependence on the quantity of the protein. The reaction is stopped with H2 SO4 , and the signal is measured in a plate reader. This modification shows high linearity without additional costs and can be applied for both purified proteins and proteins found in a lysate. The results obtained with our proposed technique were compared with those obtained by the conventional method and proved to be more reliable.

Tobacco-specific nitrosamines: A literature review.

Cigarette smoke is a complex mixture of chemicals, including several tobacco-specific nitrosamines (TSNA). Most TSNA are formed in tobacco during the post-harvest period, while a number are produced when a cigarette is burned. Considerable evidence supports the role of TSNA important causative factors for cancers of the lung, pancreas, esophagus, and oral cavity in people who use tobacco products. Of the known TSNA, nicotine-derived nitrosamine ketone (NNK) and N-nitrosonornicotine (NNN) are the most carcinogenic. Other TSNA include N'-nitrosoanatabine (NAT) and N-nitrosoanabasine (NAB). New tobacco products (e.g., e-cigarettes) designed to attract consumers who are concerned about the health effects of tobacco have been appearing on the market. Several studies have reported that certain TSNA have been detected in the replacement liquids and vapour of e-cigarettes, but the levels are generally considerably lower than in tobacco cigarettes. Additionally, the FDA recently announced its intention to regulate TSNA in e-cigarettes, cigar tobacco and pipe tobacco. With the rise of new technologies for reducing the use of tobacco products-such as e-cigarettes- to evaluate exposure levels to these harmful chemicals over time, researchers will be monitoring levels of TSNA in the body as a result of the use of these devices.

TA-GC cloning: A new simple and versatile technique for the directional cloning of PCR products for recombinant protein expression.

During the last few decades, the recombinant protein expression finds more and more applications. The cloning of protein-coding genes into expression vectors is required to be directional for proper expression, and versatile in order to facilitate gene insertion in multiple different vectors for expression tests. In this study, the TA-GC cloning method is proposed, as a new, simple and efficient method for the directional cloning of protein-coding genes in expression vectors. The presented method features several advantages over existing methods, which tend to be relatively more labour intensive, inflexible or expensive. The proposed method relies on the complementarity between single A- and G-overhangs of the protein-coding gene, obtained after a short incubation with T4 DNA polymerase, and T and C overhangs of the novel vector pET-BccI, created after digestion with the restriction endonuclease BccI. The novel protein-expression vector pET-BccI also facilitates the screening of transformed colonies for recombinant transformants. Evaluation experiments of the proposed TA-GC cloning method showed that 81% of the transformed colonies contained recombinant pET-BccI plasmids, and 98% of the recombinant colonies expressed the desired protein. This demonstrates that TA-GC cloning could be a valuable method for cloning protein-coding genes in expression vectors.

Utilizing the virus-induced blocking of apoptosis in an easy baculovirus titration method.

Baculovirus-mediated protein expression is a robust experimental technique for producing recombinant higher-eukaryotic proteins because it combines high yields with considerable post-translational modification capabilities. In this expression system, the determination of the titer of recombinant baculovirus stocks is important to achieve the correct multiplicity of infection for effective amplification of the virus and high expression of the target protein. To overcome the drawbacks of existing titration methods (e.g., plaque assay, real-time PCR), we present a simple and reliable assay that uses the ability of baculoviruses to block apoptosis in their host cells to accurately titrate virus samples. Briefly, after incubation with serial dilutions of baculovirus samples, Sf9 cells were UV irradiated and, after apoptosis induction, they were viewed via microscopy; the presence of cluster(s) of infected cells as islets indicated blocked apoptosis. Subsequently, baculovirus titers were calculated through the determination of the 50% endpoint dilution. The method is simple, inexpensive, and does not require unique laboratory equipment, consumables or expertise; moreover, it is versatile enough to be adapted for the titration of every virus species that can block apoptosis in any culturable host cells which undergo apoptosis under specific conditions.

Purification and functional characterization of a truncated human α4ß2 nicotinic acetylcholine receptor.

Nicotinic acetylcholine receptors (nAChR) are abundant in the brain and are essential in cognitive function, learning and memory. Previous efforts on α4ß2 nAChR had been focused on functional and pharmacological characterization, where high expression yield is not essential. For structural studies though, large amounts of pure protein is important but heterologous overexpression of membrane proteins can be a burdensome task, especially if high amounts are required. In the current study, a truncated mutant of the human α4ß2 nAChR was designed in order to improve expression and solubility and to obtain material suitable for high resolution structural studies. We showed that the wild type α4ß2 nAChR presented low expression and solubilization yield both of which were improved with the truncated construct. The truncated nAChR showed similar binding profile to the wild type, was purified by a two-step chromatography and isolated in high purity and adequate quantity.

Preferential production of IgG1, IL-4 and IL-10 in MuSK-immunized mice.

Myasthenia gravis (MG) is an autoimmune disease characterized by muscle weakness associated with acetylcholine receptor (AChR), muscle-specific receptor kinase (MuSK) or low-density lipoprotein receptor-related protein 4 (LRP4)-antibodies. MuSK-antibodies are predominantly of the non-complement fixing IgG4 isotype. The MuSK associated experimental autoimmune myasthenia gravis (EAMG) model was established in mice to investigate immunoglobulin (Ig) and cytokine responses related with MuSK immunity. C57BL/6 (B6) mice immunized with 30µg of recombinant human MuSK in incomplete or complete Freund's adjuvant (CFA) showed significant EAMG susceptibility (>80% incidence). Although mice immunized with 10µg of MuSK had lower EAMG incidence (14.3%), serum MuSK-antibody levels were comparable to mice immunized with 30µg MuSK. While MuSK immunization stimulated production of all antibody isotypes, non-complement fixing IgG1 was the dominant anti-MuSK Ig isotype in both sera and neuromuscular junctions. Moreover, MuSK immunized IgG1 knockout mice showed very low serum MuSK-antibody levels. Sera and MuSK-stimulated lymph node cell supernatants of MuSK immunized mice showed significantly higher levels of IL-4 and IL-10 (but not IFN-γ and IL-12), than those of CFA immunized mice. Our results suggest that through activation of Th2-type cells, anti-MuSK immunity promotes production of IL-4, which in turn activates anti-MuSK IgG1, the mouse analog of human IgG4. These findings might provide clues for the pathogenesis of other IgG4-related diseases as well as development of disease specific treatment methods (e.g. specific IgG4 inhibitors) for MuSK-related MG.

Expression of a highly antigenic and native-like folded extracellular domain of the human α1 subunit of muscle nicotinic acetylcholine receptor, suitable for use in antigen specific therapies for Myasthenia Gravis.

We describe the expression of the extracellular domain of the human α1 nicotinic acetylcholine receptor (nAChR) in lepidopteran insect cells (i-α1-ECD) and its suitability for use in antigen-specific therapies for Myasthenia Gravis (MG). Compared to the previously expressed protein in P. pastoris (y-α1-ECD), i-α1-ECD had a 2-fold increased expression yield, bound anti-nAChR monoclonal antibodies and autoantibodies from MG patients two to several-fold more efficiently and resulted in a secondary structure closer to that of the crystal structure of mouse α1-ECD. Our results indicate that i-α1-ECD is an improved protein for use in antigen-specific MG therapeutic strategies.