The gypsy element of Drosophila differs from most LTR retrotransposons in containing a third open reading frame that resembles retroviral env genes. The protein encoded by ORF3 is glycosylated and processed, like all retroviral envelope proteins. The protein is expressed at high levels in fly strains in which gypsy elements are active. In these strains the protein is found primarily in viral particles. When larvae of fly strains in which gypsy is normally inactive are exposed to sucrose gradient fractions containing these particles, a high level of gypsy insertion activity is observed in their progeny. Thus, gypsy has the expected properties of an insect retrovirus. [Key Words: Drosophila; mutagenesis; transposable element; insect retrovirus] Received May 6, 1994; revised version accepted July 11, 1994.The gypsy element of Drosophila melanogaster has been classified traditionally as a long terminal repeat (LTR) retrotransposon; however, it is one of a small group of LTR retrotransposons from insects that are unusual in that they contain three open reading frames (ORFs). In these retroelements, the first two ORFs correspond to retroviral gag and poi, whereas ORF3 is of unknown function but corresponds in size and genomic location to retroviral env (Fig. 1). All elements with three ORFs described so far are from insects, either Drosophila or the lepidopteran Trichoplusia ni. Recent results have shown that in two elements encoding an ORF3, gypsy and tom, a subgenomic mRNA similar in structure to retroviral env mRNAs can be found (P61isson et al. 1994;Tanda et al. 1994). In the case of gypsy, this transcript is observed only in certain strains in which gypsy transpositional activity is high (P41isson et al. 1994). The primary sequences of the encoded ORF3 proteins of these elements are quite variable and show no obvious similarity to retroviral Env proteins. However, retroviral Env proteins are themselves very variable in primary sequence. Like retroviral Env proteins, the proteins encoded by "retrotransposon" ORF3s contain a putative transmembrane domain near their carboxyl terminus, multiple putative N-glycosylation sites, and putative protease cleavage sites (resembling the cleavage sites in a variety of retroviral Env proteins) at conserved positions (Fig. 1 }. These features of gypsy and the other ORF3-containing insect retrotransposons have prompted the suggestion that these elements may represent endogenous insect retro- viruses (Boeke 1988;Boeke and Corces 1989;Coffin 1993).To address the question of whether gypsy represents a virus or a transposon more directly, we have turned to the use of Drosophila strains that show genetic instabilities associated with high-frequency insertion by gypsy elements. These strains are characterized by a large number of full-length gypsy elements in the euchromatin, appearance of gypsy insertion mutations at high frequency, and the presence of both large amounts of gypsy full-length RNA and spliced ORF3 mRNA (P61isson et al. 1994; N. Prud'homme, M. Gans, M. Masson, C. Ter...

Gypsy displays striking similarities to vertebrate retroviruses, including the presence of a yet uncharacterized additional open reading frame (ORF3) and the recent evidence for infectivity. It is mobilized with high frequency in the germline of the progeny of females homozygous for the flamenco permissive mutation. We report the characterization of a gypsy subgenomic ORF3 RNA encoding typical retroviral envelope proteins. In females, env expression is strongly repressed by one copy of the non‐permissive allele of flamenco. A less dramatic reduction in the accumulation of other transcripts and retrotranscripts is also observed. These effects correlate well with the inhibition of gypsy transposition in the progeny of these females, and are therefore likely to be responsible for this phenomenon. The effects of flamenco on gypsy expression are apparently restricted to the somatic follicle cells that surround the maternal germline. Moreover, permissive follicle cells display a typically polarized distribution of gypsy RNAs and envelope proteins, both being mainly accumulated at the apical pole, close to the oocyte. We propose a model suggesting that gypsy germinal transposition might occur only in individuals that have maternally inherited enveloped gypsy particles due to infection of the maternal germline by the soma.

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Hypercholesterolemia-related endothelial cell dysfunction and decreased endothelium-derived nitric oxide formation may account for impaired angiogenesis and subsequent erectile dysfunction. Angiopoietin-1 (Ang1) is a critical angiogenic factor for vascular maturation and enhances vascular endothelial growth factor (VEGF)-induced angiogenesis in a complementary manner. We hypothesized that combined adenovirus-delivered human Ang1 (ad-Ang1) and VEGF165 (ad-VEGF165) gene transfer might promote angiogenesis cooperatively in a rat model of hypercholesterolemic erectile dysfunction and result in a recovery of erectile function. Ad-Ang1 and ad-VEGF165 were injected either alone or in combination into the corpus cavernosum of the penis. Combined gene transfer of both ad-Ang1 and ad-VEGF165 significantly increased cavernous angiogenesis, eNOS phosphorylation, and cGMP expression compared with that in the groups treated with either therapy alone. Erectile function, as evaluated by electrical stimulation of the cavernous nerve 2 and 8 weeks after treatment, was completely restored in the combined treatment group, whereas intracavernous injection of either ad-Ang1 or ad-VEGF165 alone elicited partial improvement. The results indicate that combined application of angiogenic factors may enhance cavernous angiogenesis cooperatively by reinforcing the endothelium both structurally and functionally, which results in an additive effect on erectile function in hypercholesterolemic rats.

We have previously demonstrated that hypoxia stimulates adipose-derived stem cells (ASCs) through the generation of reactive oxygen species (ROS). However, the precise mechanism involved in the ROS generation by ASCs is not well understood. We sought to investigate in this work: (1) which subtype of NADPH oxidase (Nox) is primarily expressed in ASCs; (2) where Nox4 is localized in ASCs; and (3) whether silencing of Nox4 attenuates hypoxia-enhanced function of ASC. We used 2¢,7¢-dichlorofluorescin diacetate (DCF-DA) as an indicator of ROS generation and found that the fluorescence intensity of DCF-DA was significantly increased after hypoxia exposure (2% oxygen). In addition, hypoxia enhanced the proliferation and migration of ASCs and upregulated the mRNA expression of Oct4 and Rex1. Quantitative analysis of mRNA expression of Nox family in ASCs demonstrated that Nox4 is primarily expressed in ASCs, while immunofluorescence assay showed that Nox4 is mainly localized in the perinuclear region and overlaps with Mitotracker, a mitochondria marker. Silencing of Nox4 by siRNA treatment downregulated the RNA and protein expression of Nox4, which significantly reduced the ROS generation under hypoxia. In addition, Nox4 silencing significantly reduced the proliferation and migration of ASCs and downregulated the mRNA expression of Oct4 and Rex1. Phosphorylation of platelet-derived growth factor receptor-b, AKT, and ERK1/2 also diminished following Nox4 silencing. In a nutshell, these results suggest that Nox4 is primarily expressed in ASCs and plays a pivotal role in the hypoxia-enhanced stimulation of ASCs.

Introduction It has been suggested that transforming growth factor-β1 (TGF-β1) plays an important role in the pathogenesis of diabetes-induced erectile dysfunction. Aim To investigate the expression and activity of Smad transcriptional factors, the key molecules for the initiation of TGF-β-mediated fibrosis, in the penis of streptozotocin (STZ)-induced diabetic rats. Methods Fifty-two 8-week-old Sprague–Dawley rats were used and divided into control and diabetic groups. Diabetes was induced by an intravenous injection of STZ. Main Outcome Measures Eight weeks later, erectile function was measured by electrical stimulation of the cavernous nerve (N = 12 per group). The penis was harvested and stained with Masson trichrome or antibody to TGF-β1, phospho-Smad2 (P-Smad2), smooth muscle α-actin, and factor VIII (N = 12 per group). Penis specimens from a separate group of animals were used for TGF-β1 enzyme-linked immunosorbent assay (ELISA), P-Smad2/Smad2, phospho-Smad3 (P-Smad3)/Smad3, fibronectin, collagen I, and collagen IV western blot, or hydroxyproline determination. Results Erectile function was significantly reduced in diabetic rats compared with that in controls. The expression of TGF-β1, P-Smad2, and P-Smad3 protein evaluated by ELISA or western blot was higher in diabetic rats than in controls. Compared with that in control rats, P-Smad2 expression was higher mainly in smooth muscle cells and fibroblasts of diabetic rats, whereas no significant differences were noted in endothelial cells or in the dorsal nerve bundle. Cavernous smooth muscle and endothelial cell contents were lower in diabetic rats than in controls. Cavernous fibronectin, collagen IV, and hydroxyproline content was significantly higher in diabetic rats than in controls. Conclusion Upregulation of TGF-β1 and activation of the Smad signaling pathway in the penis of diabetic rats might play important roles in diabetes-induced structural changes and deterioration of erectile function.