Cloning of the mouse gene encoding plasma glutathione peroxidase: organization, sequence and chromosomal localization.

Using a reverse transcription coupled to PCR amplification strategy, with degenerated primers localized in highly conserved domains of known glutathione peroxidase (GPX) proteins, we have generated, from mouse epididymal RNA, a cDNA fragment which was subsequently used to isolate a genomic clone encoding mouse plasma GPX (GPX3). GPX3 is a major enzyme in reducing lipid hydroperoxides and hydrogen peroxide in plasma. We confirm here that the mouse epididymis is a new site of expression of GPX3 and report, together with the sequence, the structural analysis and the chromosomal localization of the mouse GPX3 single-copy gene to chromosome 11.

Spontaneously immortalized epithelial cells from mouse caput epididymidis.

We report here on the characterization of tissue-culture cell lines derived from primary cultures of the mouse caput epididymidis epithelium. The cell lines were spontaneously immortalized without the use of transforming oncogenes. In defined conditions, our epididymal cells adopted various morphological features that resembles that of the in vivo epididymis epithelium such as a polarized organization and the presence of junctional structures at their apical/lateral membranes as revealed by electron microscopy analyses. Flow cytometry analysis revealed that we were dealing with homogenous cell populations that had reached a near-tetraploid state. RT-PCR assays were used in order to show that several genes that can be considered as markers of in vivo caput epididymidis epithelium activity were expressed in our cell lines confirming that these cells were indeed in a differentiated state close to their endogenous state.

In vivo 23Na nuclear magnetic resonance study of maintenance of a sodium gradient in the ruminal bacterium Fibrobacter succinogenes S85.

Sodium gradients (DeltapNa) were measured in resting cells of Fibrobacter succinogenes by in vivo 23Na nuclear magnetic resonance using Tm(DOTP)5- [thulium(III) 1,4,7,10-tetraazacyclododecane-N',N",N"'-tetramethylenephosphonate] as the shift reagent. This bacterium was able to maintain a DeltapNa of -55 to -40 mV for extracellular sodium concentrations ranging from 30 to 200 mM. Depletion of Na+ ions during the washing steps led to irreversible damage (modification of glucose metabolism and inability to maintain a sodium gradient).

Structural organization, chromosomal localization, expression and phylogenetic evaluation of mouse glutathione peroxidase encoding genes.

We have reported earlier the cloning and the chromosomal localization of 2 GPX-encoding sequences expressed differentially within the mouse epididymis, gpx5 and gpx3. Here, we have mapped on the mouse chromosomes the third known murine GPX-encoding gene, the cytosolic GPX or gpx1. We have compared the degree of identity of the 3 GPX proteins, the respective organization of the 3 corresponding single copy genes and, using degenerated oligonucleotides designed in highly conserved domains of the proteins, we have analyzed the expression of GPX-encoding genes in the mouse epididymis as well as in control tissues known to express GPX proteins (the liver for GPX1 and the kidney for GPX3). The 3 genes characterized to date were found expressed in each of the tissues tested but in a highly tissue-restricted manner. Nucleotidic sequences comparisons were carried out on GPX-encoding sequences from various species and were used to draw a dendrogram. Phylogenetic evaluation of the sequence information, as well as the chromosomal localizations, suggest that the GPX genes have evolved by duplication events followed by random insertions from a single ancestral gene.

Human herpesvirus 6 (HHV-6) is a helper virus for adeno-associated virus type 2 (AAV-2) and the AAV-2 rep gene homologue in HHV-6 can mediate AAV-2 DNA replication and regulate gene expression.

We have previously described the apparent acquisition by human herpesvirus 6 (HHV-6) of the multifunctional rep gene of the helper-dependent human parvovirus adeno-associated virus type 2 (AAV-2). We report here that HHV-6 is a full helper virus for AAV-2 replication, suggesting a mechanism for transfer of the rep gene between the two viruses by recombination of replicative intermediates. The HHV-6 rep gene cloned under control of the human cytomegalovirus immediate early promoter complemented replication of a rep-deficient AAV-2 genome. In cotransfection experiments with heterologous promoters linked to the CAT reporter gene, HHV-6 rep activated the human immunodeficiency virus (HIV) long terminal repeat (LTR) in fibroblast cell lines but not in T-cells. In contrast, AAV-2 rep inhibited HIV LTR activity in both fibroblast and T-cell lines. The effect of HHV-6 and AAV-2 rep genes on the HIV LTR was independent of the NF-kappa B, Sp1, and TATA box elements. These results suggest that HHV-6 Rep is a multifunctional regulatory protein with properties related to, but distinct from, those of AAV-2 Rep.

The PEA3 protein of the Ets oncogene family is a putative transcriptional modulator of the mouse epididymis-specific glutathione peroxidase gene gpx5.

This report presents data that suggest that the tissue-restricted polyoma enhancer activator protein (PEA3) of the Ets oncogene family of DNA-binding proteins is a putative modulator of the epididymis-specific glutathione peroxidase 5 gene gpx5. Northern and polymerase chain reactions on reverse-transcribed epididymal RNAs were used to show that the PEA3 factor is spatially and temporally expressed within the mouse epididymis in a manner consistent with gpx5 characteristics of expression. Then, using contransfection experiments carried out in heterologous tissue-culture cells with various deletions of the gpx5 promoter driving a CAT reporter gene, we have shown that the transcriptional activity of the gpx5 promoter is modulated by the presence of the PEA3 protein. Subsequently, we have shown using gel-shift assays that DNA sequences located within the 5' flanking region of the gpx5 gene have the ability to bind specifically to the PEA3 protein. Finally, using Northern assays we present data that suggest that PEA3 mRNA accumulation in the mouse caput epididymidis is controlled by androgens and testicular factors. Altogether, these results strongly suggest that the PEA3 factor might participate in the transcriptional control of the murine epididymis caput-specific gpx5 gene.

GPx3: the plasma-type glutathione peroxidase is expressed under androgenic control in the mouse epididymis and vas deferens.

We report here-using northern experiments, western blotting, and immunohistochemistry-on the findings that the plasma type glutathione peroxidase, GPx3, a major enzyme in reducing lipid hydroperoxides and hydrogen peroxide in plasma, is also expressed at significant levels in tissues of the male genital tract including epididymis and vas deferens. Within the epididymis and the kidney, the accumulation of the GPx3 mRNA and protein were investigated during postnatal development and found to be temporally regulated in a tissue-specific manner. Furthermore, we show here that androgen withdrawal by castration down regulates the expression of the GPx3 gene both in the epididymis and vas deferens while GPx3 expression in the kidney was found to be androgen-independent. Finally, immunohistochemistry data reveals that within the epididymis GPx3 distribution is quite peculiar suggesting the existence in this organ of complex traductional and/or transcriptional regulatory processes.

Characterization, regulation of the expression and putative roles of two glutathione peroxidase proteins found in the mouse epididymis.

Two glutathione peroxidase genes (gpx5 and gpx3) were found to be expressed in the mouse epididymis. Gpx5 was shown to be epididymis specific and restricted to the caput epididymidis, while gpx3 was found to be expressed in a wide array of tissues including the caput, corpus and cauda epididymides. Both single copy genes are regulated by androgens as well as being developmentally regulated during postnatal ontogenesis of the epididymis. In this report data collected to date concerning the mechanisms by which these genes are regulated in the mouse epididymis are summarized. The putative roles of these antioxidant enzymes in the sperm maturation process are discussed.