Effectiveness, against tuberculosis, of pseudo-ternary complexes: Peptide-DNA-cationic liposome

We report the effects of a synthetic peptide designed to act as a nuclear localization signal on the treatment of tuberculosis. The peptide contains 21 amino acid residues with the following specific domains: nuclear localization signal from SV 40T, cationic shuttle sequence, and cysteamide group at the C-terminus. The peptide was complexed with the plasmid DNAhsp65 and incorporated into cationic liposomes, forming a pseudo-ternary complex. The same cationic liposomes, composed of egg chicken L-alpha-phosphatidylcholine, 1,2-dioleoyl-3-trimethylammonium-propane, and 1,2-dioleoyl-3-trimethylammonium-propane (2:1:1 M), were previously evaluated as a gene carrier for tuberculosis immunization protocols with DNAhsp65. The pseudo-ternary complex presented a controlled size (250 nm), spherical-like shape, and various lamellae in liposomes as evaluated by transmission electron microscopy. An assay of fluorescence probe accessibility confirmed insertion of the peptide/DNA into the liposome structure. Peptide addition conferred no cytotoxicity in vitro, and similar therapeutic effects against tuberculosis were seen with four times less DNA compared with naked DNA treatment. Taken together, the results indicate that the pseudo-ternary complex is a promising gene vaccine for tuberculosis treatment. This work contributes to the development of multifunctional nanostructures in the search for strategies for in vivo DNA delivery. (C) 2011 Elsevier Inc. All rights reserved.

Germline-Specific MATH-BTB Substrate Adaptor MAB1 Regulates Spindle Length and Nuclei Identity in Maize

Germline and early embryo development constitute ideal model systems to study the establishment of polarity, cell identity, and asymmetric cell divisions (ACDs) in plants. We describe here the function of the MATH-BTB domain protein MAB1 that is exclusively expressed in the germ lineages and the zygote of maize (Zea mays). mab1 (RNA interference [RNAi]) mutant plants display chromosome segregation defects and short spindles during meiosis that cause insufficient separation and migration of nuclei. After the meiosis-to-mitosis transition, two attached nuclei of similar identity are formed in mab1 (RNAi) mutants leading to an arrest of further germline development. Transient expression studies of MAB1 in tobacco (Nicotiana tabacum) Bright Yellow-2 cells revealed a cell cycle-dependent nuclear localization pattern but no direct colocalization with the spindle apparatus. MAB1 is able to form homodimers and interacts with the E3 ubiquitin ligase component Cullin 3a (CUL3a) in the cytoplasm, likely as a substrate-specific adapter protein. The microtubule-severing subunit p60 of katanin was identified as a candidate substrate for MAB1, suggesting that MAB1 resembles the animal key ACD regulator Maternal Effect Lethal 26 (MEL-26). In summary, our findings provide further evidence for the importance of posttranslational regulation for asymmetric divisions and germline progression in plants and identified an unstable key protein that seems to be involved in regulating the stability of a spindle apparatus regulator(s).

Development of a recombinant fusion protein based on the dynein light chain LC8 for non-viral gene delivery

The low efficiency of gene transfer is a recurrent problem in DNA vaccine development and gene therapy studies using non-viral vectors such as plasmid DNA (pDNA). This is mainly due to the fact that during their traffic to the target cell's nuclei, plasmid vectors must overcome a series of physical, enzymatic and diffusional barriers. The main objective of this work is the development of recombinant proteins specifically designed for pDNA delivery, which take advantage of molecular motors like dynein, for the transport of cargos from the periphery to the centrosome of mammalian cells. A DNA binding sequence was fused to the N-terminus of the recombinant human dynein light chain LC8. Expression studies indicated that the fusion protein was correctly expressed in soluble form using E. coli BL21(DE3) strain. As expected, gel permeation assays found the purified protein mainly present as dimers, the functional oligomeric state of LC8. Gel retardation assays and atomic force microscopy proved the ability of the fusion protein to interact and condense pDNA. Zeta potential measurements indicated that LC8 with DNA binding domain (LD4) has an enhanced capacity to interact and condense pDNA, generating positively charged complexes. Transfection of cultured HeLa cells confirmed the ability of the LD4 to facilitate pDNA uptake and indicate the involvement of the retrograde transport in the intracellular trafficking of pDNA: LD4 complexes. Finally, cytotoxicity studies demonstrated a very low toxicity of the fusion protein vector, indicating the potential for in vivo applications. The study presented here is part of an effort to develop new modular shuttle proteins able to take advantage of strategies used by viruses to infect mammalian cells, aiming to provide new tools for gene therapy and DNA vaccination studies. (C) 2012 Elsevier B.V. All rights reserved.

The intronic long noncoding RNA ANRASSF1 recruits PRC2 to the RASSF1A promoter, reducing the expression of RASSF1A and increasing cell proliferation

The down-regulation of the tumor-suppressor gene RASSF1A has been shown to increase cell proliferation in several tumors. RASSF1A expression is regulated through epigenetic events involving the polycomb repressive complex 2 (PRC2); however, the molecular mechanisms modulating the recruitment of this epigenetic modifier to the RASSF1 locus remain largely unknown. Here, we identify and characterize ANRASSF1, an endogenous unspliced long noncoding RNA (lncRNA) that is transcribed from the opposite strand on the RASSF1 gene locus in several cell lines and tissues and binds PRC2. ANRASSF1 is transcribed through RNA polymerase II and is 5'-capped and polyadenylated; it exhibits nuclear localization and has a shorter half-life compared with other lncRNAs that bind PRC2. ANRASSF1 endogenous expression is higher in breast and prostate tumor cell lines compared with non-tumor, and an opposite pattern is observed for RASSF1A. ANRASSF1 ectopic overexpression reduces RASSF1A abundance and increases the proliferation of HeLa cells, whereas ANRASSF1 silencing causes the opposite effects. These changes in ANRASSF1 levels do not affect the RASSF1C isoform abundance. ANRASSF1 overexpression causes a marked increase in both PRC2 occupancy and histone H3K27me3 repressive marks, specifically at the RASSF1A promoter region. No effect of ANRASSF1 overexpression was detected on PRC2 occupancy and histone H3K27me3 at the promoter regions of RASSF1C and the four other neighboring genes, including two well-characterized tumor suppressor genes. Additionally, we demonstrated that ANRASSF1 forms an RNA/DNA hybrid and recruits PRC2 to the RASSF1A promoter. Together, these results demonstrate a novel mechanism of epigenetic repression of the RASSF1A tumor suppressor gene involving antisense unspliced lncRNA, in which ANRASSF1 selectively represses the expression of the RASSF1 isoform overlapping the antisense transcript in a location-specific manner. In a broader perspective, our findings suggest that other non-characterized unspliced intronic lncRNAs transcribed in the human genome might contribute to a location-specific epigenetic modulation of genes.

Glucocorticoid and Estrogen Receptors Are Reduced in Mitochondria of Lung Epithelial Cells in Asthma

Mitochondrial glucocorticoid (mtGR) and estrogen (mtER) receptors participate in the coordination of the cell's energy requirement and in the mitochondrial oxidative phosphorylation enzyme (OXPHOS) biosynthesis, affecting reactive oxygen species (ROS) generation and induction of apoptosis. Although activation of mtGR and mtER is known to trigger anti-inflammatory signals, little information exists on the presence of these receptors in lung tissue and their role in respiratory physiology and disease. Using a mouse model of allergic airway inflammation disease and applying confocal microscopy, subcellular fractionation, and Western blot analysis we showed mitochondrial localization of GR alpha and ER beta in lung tissue. Allergic airway inflammation caused reduction in mtGR alpha, mtER beta, and OXPHOS enzyme biosynthesis in lung cells mitochondria and particularly in bronchial epithelial cells mitochondria, which was accompanied by decrease in lung mitochondrial mass and induction of apoptosis. Confirmation and validation of the reduction of the mitochondrial receptors in lung epithelial cells in human asthma was achieved by analyzing autopsies from fatal asthma cases. The presence of the mitochondrial GR alpha and ER beta in lung tissue cells and especially their reduction in bronchial epithelial cells during allergic airway inflammation suggests a crucial role of these receptors in the regulation of mitochondrial function in asthma, implicating their involvement in the pathophysiology of the disease.

E-cadherin in canine mast cell tumors: Decreased expression and altered subcellular localization in Grade 3 tumors

Mast cell tumors (MCTs) are the most frequent round cell tumors in dogs and comprise approximately 21% of all canine cutaneous tumors. MCTs are highly invasive and metastatic corresponding to the histological grade. E-cadherin is an adhesion molecule expressed in epithelial cells and although it is an epithelial cellular marker, studies have shown expression of E-cadherin in canine round cell tumors. To better characterize the expression pattern of E-cadherin in several different histological grades of MCTs in dogs, the expression and localization of the adhesion molecule was investigated using immunohistochemistry. For this purpose, 18 cutaneous MCTs were classified into three histological grades, 1, 2 or 3. Clinical history and follow-up data were available for all of the dogs. Cytoplasmic and nuclear expressions of E-cadherin in all three types of tumors were verified by immunostaining using two different antibodies. There was decreased E-cadherin expression in the more aggressive MCTs (Grade 3), suggesting an association between E-cadherin and tumor aggressiveness. Additionally, the loss of E-cadherin expression in either the cytoplasm or nucleus in more aggressive and undifferentiated tumor types confirmed the importance of cellular adhesion in tumor behavior. (C) 2012 Published by Elsevier Ltd.

Mutational analyses of the signals involved in the subcellular location of DSCR1

Abstract Background Down syndrome is the most frequent genetic disorder in humans. Rare cases involving partial trisomy of chromosome 21 allowed a small chromosomal region common to all carriers, called Down Syndrome Critical Region (DSCR), to be determined. The DSCR1 gene was identified in this region and is expressed preferentially in the brain, heart and skeletal muscle. Recent studies have shown that DSCR1 belongs to a family of proteins that binds and inhibits calcineurin, a serine-threonine phosphatase. The work reported on herein consisted of a study of the subcellular location of DSCR1 and DSCR1-mutated forms by fusion with a green fluorescent protein, using various cell lines, including human. Results The protein's location was preferentially nuclear, independently of the isoform, cell line and insertion in the GFP's N- or C-terminal. A segment in the C-terminal, which is important in the location of the protein, was identified by deletion. On the other hand, site-directed mutational analyses have indicated the involvement of some serine and threonine residues in this event. Conclusion In this paper, we discuss the identification of amino acids which can be important for subcellular location of DSCR1. The involvement of residues that are prone to phosphorylation suggests that the location and function of DSCR1 may be regulated by kinases and/or phosphatases.