Molecular characterization and in silico analysis of RNA polymerase alpha subunit gene (rpoA) in Date Palm (Phoenix dactylifera L.) cv. Deglet Nour

The rpoA gene coding for the ??-subunit of DNA-dependent RNA polymerase located in the chloroplastic genome of date palm has been characterized from the elite cultivar Deglet Nour by cloning and sequencing of the PCR amplification product. The full length of rpoA-Pd (Phoenix dactylifera) gene was 1014 bp. The comparison in Genbank showed that the rpoA gene has a 100% homology with the Khalas cultivar of date palm and a strong homology with Oil Palm (99%). The deduced protein full length is 337 amino acid corresponding to 38,692 Da polypeptide. It contained an Alpha N-terminal domain (alpha-NTD) between 1 to 233 (aa) and Alpha C-terminal domain (alpha-CTD) between 266 to 337 (aa). Initially, we have compared the sequences of the full-length DNA rpoA gene from Date Palm and Oil palm, 15 mutations have been detected, 4 do not affect amino acid sequences. A multiple alignment of protein sequences of Date Palm and other plants shows 6 mutations specific for palms family and one is specific to monocots species. A multiple alignment of 35 nucleotide sequences from different plant species shows 3 SNPs specific to Date Palm, 6 SNPs specific to Palms family and 6 other to monocot species. The phylogenetic analysis performed in this work shows a strong similarity between Pd-rpoA and rpoA genes from other plant species, but it shows a great divergence with the rpoA of E. coli. To explain whether the separation of the two clades was due to selection pressure. We calculate the ratio Ka/Ks for different species. A synteny analyses of rpoA genes was effected, a high genomic synteny is observed for the ropA in all the species included in this study.     

RhoA and Rho kinase mediate cyclosporine A and sirolimus-induced barrier tightening in renal proximal tubular cells

The regulation and maintenance of the paracellular transport in renal tubular epithelia is vital for kidney functions. Combination of the immunosuppressant drugs cyclosporine A (CsA) and sirolimus (SRL) exerts powerful immunosuppression, but also causes nephrotoxicity. We have previously shown that CsA and SRL elevate transepithelial resistance (TER) in kidney tubular cells partly through MEK/ERK1/2. In this work we examined the hypothesis that the RhoA pathway may also be mediating effects of CsA and SRL. We show that CsA and the CsA/SRL combination activated RhoA, induced cofilin phosphorylation and promoted stress fiber generation. The Rho kinase (ROK) inhibitor, Y27632, prevented CsA and CsA/SRL-induced cofilin phosphorylation and actin remodelling, reduced the TER increase and prevented the rise in claudin-7 levels caused by the drugs. Expression of the exchange factor GEF-H1/lfc was elevated in cells treated with CsA and CsA/SRL. GEF-H1 silencing inhibited RhoA activation by ≈50%, and potently reduced cofilin phosphorylation and stress fiber formation induced by CsA and CsA/SRL. However, GEF-H1 downregulation did not prevent the TER change. Thus the Rho/Rho kinase pathway was involved in mediating CsA and CsA/SRL-induced cytoskeleton rearrangement and TER changes via claudin-7 expression. Our data however point to differential regulation of Rho activation involved in central cytoskeleton remodelling, that is GEF-H1-dependent and junctional permeability that does not require GEF-H1.     

Impact of single-nucleotide polymorphisms at the TP53-binding and responsive promoter region of BCL2 gene in modulating the phenotypic variability of LGMD2C patients

Apoptosis of skeletal muscle fibers is a well-known event occurring in patients suffering from muscular dystrophies. In this study, we hypothesized that functional polymorphisms in genes involved in the mitochondrial apoptotic pathway might modulate the apoptotic capacity underlying the muscle loss and contributing to intrafamilial and interfamilial variable phenotypes in LGMD2C (Limb Girdle Muscular Dystrophy type 2C) patients sharing the same c.521delT mutation in SGCG gene. Detection of apoptosis was confirmed on muscle biopsies taken from LGMD2C patients using the TUNEL method. We genotyped then ten potentially functional SNPs in TP53, BCL-2 and BAX genes involved in the mitochondrial apoptotic pathway. Potential genotype-dependent Bcl-2 and p53 protein expressed in skeletal muscle was investigated using western blot and ELISA assays. The result showed that muscle cells carrying the TP53-R72R and TP53-16?bp del/del genotypes displayed an increased p53 level which could be more effective in inducing apoptosis by activation of the pro-apoptotic gene expression. In addition, the BCL2-938 AA genotype was associated with increased Bcl-2 protein expression in muscle from LGMD2C patients compared to -938CC genotype, while there was no evidence of significant difference in the BAX haplotype. Our findings suggest that increased Bcl-2 protein expression may counteract pro-apoptotic pathways and thus reduce the muscle loss. To the best of our knowledge, this is a pioneer study evaluating the role of apoptotic BCL-2 and TP53 genes in contributing to the phenotypic manifestation of c.521delT mutation in LGMD2C patients. Larger studies are needed to validate these findings.     

Mutational analysis of the mitochondrial tRNALeu(UUR) gene in Tunisian patients with mitochondrial diseases

The mitochondrial tRNA(Leu(UUR)) gene (MTTL) is a hot spot for pathogenic mutations that are associated with mitochondrial diseases with various clinical features. Among these mutations, the A3243G mutation was associated with various types of mitochondrial multisystem disorders, such as MIDD, MELAS, MERRF, PEO, hypertrophic cardiomyopathy, and a subtype of Leigh syndrome. We screened 128 Tunisian patients for the A3243G mutation in the mitochondrial tRNA(Leu(UUR)) gene. This screening was carried out using PCR-RFLP with the restriction endonuclease ApaI. None of the 128 patients or the 100 controls tested were found to carry the mitochondrial A3243G mutation in the tRNA(Leu(UUR)) gene in homoplasmic or heteroplasmic form. After direct sequencing of the entire mitochondrial tRNA(Leu(UUR)) gene and a part of the mitochondrial NADH dehydrogenase 1, we found neither mutations nor polymorphisms in the MTTL1 gene in the tested patients and controls, and we confirmed the absence of the A3243G mutation in this gene. We also found a T3396C transition in the ND1 gene in one family with NSHL which was absent in the other patients and in 100 controls. Neither polymorphisms nor other mutations were found in the mitochondrial tRNA(Leu(UUR)) gene in the tested patients.     

Novel sequence variations in LAMA2 andSGCG genes modulating cis-acting regulatory elements and RNA secondary structure

In this study, we detected new sequence variations in LAMA2 and SGCG genes in 5 ethnic populations, and analysed their effect on enhancer composition and mRNA structure. PCR amplification and DNA sequencing were performed and followed by bioinformatics analyses using ESEfinder as well as MFOLD software. We found 3 novel sequence variations in the LAMA2 (c.3174+22_23insAT and c.6085 +12delA) and SGCG (c. (*) 102A/C) genes. These variations were present in 210 tested healthy controls from Tunisian, Moroccan, Algerian, Lebanese and French populations suggesting that they represent novel polymorphisms within LAMA2 and SGCG genes sequences. ESEfinder showed that the c. (*) 102A/C substitution created a new exon splicing enhancer in the 3'UTR of SGCG genes, whereas the c.6085 +12delA deletion was situated in the base pairing region between LAMA2 mRNA and the U1snRNA spliceosomal components. The RNA structure analyses showed that both variations modulated RNA secondary structure. Our results are suggestive of correlations between mRNA folding and the recruitment of spliceosomal components mediating splicing, including SR proteins. The contribution of common sequence variations to mRNA structural and functional diversity will contribute to a better study of gene expression.     

Exosome biogenesis,bioactivities and functions as new delivery systems of natural compounds

A rapidly growing body of experimental evidence has begun to shed light on the wide ranging molecular mechanisms which modulate intra- and inter-cellular communications. A substantial quantity of the available knowledge has only been uncovered in recent years, and we are learning that donor cells release nanovesicles, known as exosomes, which regulate the cellular behavior of recipient cells following uptake. Based on the impressive capacity of exosomes in delivering their “payload”, different therapeutic agents, are currently being tested using this delivery method for more effective therapy. This review summarizes the most recent developments in exosome bioactivities and discusses the biochemical nature of exosomes and their biogenesis. It also summarizes the use of exosomes as delivery vehicles for drugs and natural compounds to the targeted site.     

Akt1 contains a functional leucine-rich nuclear export sequence

Nuclear Akt1 expression and Akt activation are common in cancer invasion. However, the mechanisms for this association and its causal role in invasion are uncertain. In an effort to identify potential mechanisms for regulating Akt subcellular localization, we analyzed the Akt gene sequences and identified a highly conserved leucine-rich potential nuclear export sequence (NES). Initial experiments demonstrated that leptomycin B induced nuclear Akt1 localization. Transient expression experiments demonstrated that, in comparison to wild-type Akt1, NES-mutated (AKT/NES) Akt1 has reduced interactions with CRM-1 and persistent nuclear localization. Subsequent stable transfection experiments in Akt1-/- fibroblasts confirmed that expression of AKT/NES resulted in persistent nuclear localization and activation1. Finally, stable expression of AKT/NES in Akt1-/- fibroblasts was sufficient to enhance cell migration in vitro. Thus, Akt1 contains a functional NES and mutation of the NES results in nuclear-predominant Akt1 activation that is sufficient to induce migration.     

Characterization of a B220+ lymphoid cell subpopulation with immune modulatory functions in nasal-associated lymphoid tissues

Complex mechanisms operate on mucosal tissues to regulate immune responsiveness and tolerance. When the lymphocyte subpopulations from murine nasal-associated lymphoid tissues (NALT) were characterized, we observed an accumulation of B220(low)CD3(low)CD4(-)CD8(-)CD19(-)c-Kit(+) cells. TCR transgenic mice and athymic mice were used for monitoring T cell lineage and the presence of extrathymic T cell precursors. The majority of cells from NALT exhibited a T cell precursor phenotype (CD4(-)CD8(-)CD19(-)c-Kit(+)). Fas-independent apoptosis was their main mechanism of cell death. We also demonstrated that B220(low)CD4(-)CD8(-)CD19(-) cells from NALT exhibited the potential to down-regulate the activation of mature T cells. However, the innate immunity receptor TLR2 was also highly expressed by this cell subpopulation. Moreover, nasal stimulation with a TLR2/6 agonist resulted in a partial activation of the double-negative cells. These results suggest that the immune responses in NALT may be in part modulated by a cell subpopulation that maintains a tolerogenic milieu by its proapoptotic status and suppressive activity, which can be reverted through stimulation of a TLR signaling cascade.