Production of a reporter transgenic pig for monitoring Cre recombinase activity

The pig is thought to be the most suitable non-human source of organs for xenotransplantation and is widely used as a model of human disease. Using pigs as disease models requires the design of conditional Cre recombinase-loxP gene modifications, which, in turn, requires a Cre-expressing pig with defined patterns of expression controlled by the use of a tissue-specific promoter. In order to monitor Cre recombinant expression in vivo, it is important to create a reporter strain. We have generated reporter a pig that is based on a single vector that drives the ubiquitous expression of the enhanced green fluorescent protein (EGFP). The EGFP gene is expressed only after Cre-mediated excision of loxP-flanked stop sequences. These reporter transgenic pigs will be of great value for monitoring Cre recombinase activity in vivo.     

The Ski protein can inhibit ligand induced RARα and HDAC3 degradation in the retinoic acid signaling pathway

Recent data has implicated the Ski protein as being a physiologically relevant negative regulator of signaling by retinoic acid (RA). The mechanism by which Ski represses RA signaling is unknown. Co-immunoprecipitation and immunofluorescence assay showed that Ski and RARα are in the same complex in both the absence and presence of RA, which makes Ski different from other corepressors. We determined that Ski can stabilize RARα and HDAC3. These results suggest that Ski represses RA signaling by stabilizing corepressor complex.     

New pulsed EPR methods and their application to characterize mitochondrial complex I

Electron Paramagnetic Resonance (EPR) spectroscopy is the method of choice to study paramagnetic cofactors that often play an important role as active centers in electron transfer processes in biological systems. However, in many cases more than one paramagnetic species is contributing to the observed EPR spectrum, making the analysis of individual contributions difficult and in some cases impossible. With time-domain techniques it is possible to exploit differences in the relaxation behavior of different paramagnetic species to distinguish between them and separate their individual spectral contribution. Here we give an overview of the use of pulsed EPR spectroscopy to study the iron-sulfur clusters of NADH:ubiquinone oxidoreductase (complex I). While FeS cluster N1 can be studied individually at a temperature of 30 K, this is not possible for FeS cluster N2 due to its severe spectral overlap with cluster N1. In this case Relaxation Filtered Hyperfine (REFINE) spectroscopy can be used to separate the overlapping spectra based on differences in their relaxation behavior.     

Study of modifiers factors associated to mitochondrial mutations in individuals with hearing impairment

Hearing impairment is the most prevalent sensorial deficit in the general population. Congenital deafness occurs in about 1 in 1000 live births, of which approximately 50% has hereditary cause in development countries. Non-syndromic deafness can be caused by mutations in both nuclear and mitochondrial genes. Mutations in mtDNA have been associated with aminoglycoside-induced and non-syndromic deafness in many families worldwide. However, the nuclear background influences the phenotypic expression of these pathogenic mutations. Indeed, it has been proposed that nuclear modifier genes modulate the phenotypic manifestation of the mitochondrial A1555G mutation in the MTRNR1 gene. The both putative nuclear modifiers genes TRMU and MTO1 encoding a highly conserved mitochondrial related to tRNA modification. It has been hypothesizes that human TRMU and also MTO1 nuclear genes may modulate the phenotypic manifestation of deafness-associated mitochondrial mutations. The aim of this work was to elucidate the contribution of mitochondrial mutations, nuclear modifier genes mutations and aminoglycoside exposure in the deafness phenotype. Our findings suggest that the genetic background of individuals may play an important role in the pathogenesis of deafness-associated with mitochondrial mutation and aminoglycoside-induced.     

Centrosome attachment to the C. elegans male pronucleus is dependent on the surface area of the nuclear envelope

A close association must be maintained between the male pronucleus and the centrosomes during pronuclear migration. In C. elegans, simultaneous depletion of inner nuclear membrane LEM proteins EMR-1 and LEM-2, depletion of the nuclear lamina proteins LMN-1 or BAF-1, or the depletion of nuclear import components leads to embryonic lethality with small pronuclei. Here, a novel centrosome detachment phenotype in C. elegans zygotes is described. Zygotes with defects in the nuclear envelope had small pronuclei with a single centrosome detached from the male pronucleus. ZYG-12, SUN-1, and LIS-1, which function at the nuclear envelope with dynein to attach centrosomes, were observed at normal concentrations on the nuclear envelope of pronuclei with detached centrosomes. Analysis of time-lapse images showed that as mutant pronuclei grew in surface area, they captured detached centrosomes. Larger tetraploid or smaller histone::mCherry pronuclei suppressed or enhanced the centrosome detachment phenotype respectively. In embryos fertilized with anucleated sperm, only one centrosome was captured by small female pronuclei, suggesting the mechanism of capture is dependent on the surface area of the outer nuclear membrane available to interact with aster microtubules. We propose that the limiting factor for centrosome attachment to the surface of abnormally small pronuclei is dynein.     

Characterization of the role of full-length CRMP3 and its calpain-cleaved product in inhibiting microtubule polymerization and neurite outgrowth

Collapsin response mediator proteins (CRMPs) are key modulators of cytoskeletons during neurite outgrowth in response to chemorepulsive guidance molecules. However, their roles in adult injured neurons are not well understood. We previously demonstrated that CRMP3 underwent calcium-dependent N-terminal protein cleavage during excitotoxicity-induced neurite retraction and neuronal death. Here, we report findings that the full-length CRMP3 inhibits tubulin polymerization and neurite outgrowth in cultured mature cerebellar granule neurons, while the N-terminal truncated CRMP3 underwent nuclear translocation and caused a significant nuclear condensation. The N-terminal truncated CRMP3 underwent nuclear translocation through nuclear pores. Nuclear protein pull-down assay and mass spectrometry analysis showed that the N-terminal truncated CRMP3 was associated with nuclear vimentin. In fact, nuclear-localized CRMP3 co-localized with vimentin during glutamate-induced excitotoxicity. However, the association between the truncated CRMP3 and vimentin was not critical for nuclear condensation and neurite outgrowth since over-expression of truncated CRMP3 in vimentin null neurons did not alleviate nuclear condensation and neurite outgrowth inhibition. Together, these studies showed CRMP3's role in attenuating neurite outgrowth possibility through inhibiting microtubule polymerization, and also revealed its novel association with vimentin during nuclear condensation prior to neuronal death.     

Molecular Dissection of Bombyx mori Nucleopolyhedrovirus orf8 Gene

Viruses including baculoviruses are obligatory parasites, as their genomes do not encode all the proteins required for replication. Therefore, viruses have evolved to exploit the behavior and the physiology of their hosts and often eoevolved with their hosts over millions of years. Recent comparative analyses of complete genome sequences of baculoviruses revealed the patterns of gene acquisitions and losses that have occurred during baculovirus evolution. In addition, knowledge of virus genes has also provided understanding of the mechanism of baculovirus infection including replication, species-specific virulence and host range. The Bm8 gene of Bombyx mori nucleopolyhedrovirus (NPV) and its homologues are found only in group I NPV genomes. The Autographa californica NPV Acl6 gene is a homologue of Bm8 and, encodes a viral structural protein. It has been shown that Bm8/Ac 16 interacts with baculoviral and cellular proteins. Bm8/Ac 16 interacts with baculoviral IE1 that is facilitated by coiled coil domains, and the interaction with IE1 is important for Bin8 function. Ac16 also forms a complex with viral FP25 and cellular actin and associates with membranes via palmitoylation. These data suggested that this gene family encodes a multifunctional protein that accomplishes specific needs of group INPVs.