Background RNA interference (RNAi) is a post-transcriptional RNA degradation process, which has become a very useful tool in gene function studies and gene therapy applications. Long-term cellular expression of small interfering RNA (siRNA) molecules required for many gene therapy applications can be achieved by lentiviral vectors (LVs). The two most commonly used promoters to drive the short hairpin RNA (shRNA) expression are the human U6 small nuclear promoter (U6) and the human H1 promoter (H1).

Anhidrotic ectodermal dysplasia (EDA) is an X-linked disorder characterized by abnormal development of ectoderm and its appendices. The EDA gene encodes different isoforms of ectodysplasin, a transmembrane protein. The two longest isoforms, ectodysplasin-A1 and -A2, which differ by an insertion of two amino acids, are trimeric type II membrane proteins with an extracellular portion containing a short collagenous domain and a TNF ligand motif in the C-terminal region. We show that ectodysplasin is released from cells to the culture medium. Deletion constructs were used to localize the cleavage site and show that the putative recognition sequence of a furin-like enzyme is needed for the cleavage. Some EDA patients have missense mutations affecting this recognition sequence, suggesting that cleavage has biological significance in vivo. EDAR, a recently cloned member of the TNFR family and the product of the downless gene, is able to co-precipitate ectodysplasin, confirming that they form a ligand-receptor pair. In situ hybridization and immunostaining studies show that ectodysplasin and EDAR are expressed in adjacent or partially overlapping layers in the developing human skin. We conclude that as a soluble ligand, ectodysplasin is able to interact with EDAR and mediate signals needed for the development of ectodermal appendages.

Alterations of T-cell receptor signaling by human immunodeficiency virus type 1 (HIV-1) Nef involve its association with a highly active subpopulation of p21-activated kinase 2 (PAK2) within a dynamic signalosome assembled in detergent-insoluble membrane microdomains. Nef-PAK2 complexes contain the GTPases Rac and Cdc42 as well as a factor providing guanine nucleotide exchange factor (GEF) activity for Rac/Cdc42. However, the identity of this GEF has remained controversial. Previous studies suggested the association of Nef with at least three independent GEFs, Vav, DOCK2/ELMO1, and ␤Pix. Here we used a broad panel of approaches to address which of these GEFs is involved in the functional interaction of Nef with PAK2 activity.

Shortage of oxygen is one of the prime stress conditions in tissues. In this study, we looked for microRNAs expressed during hypoxia and showed that miR-210 expression was upregulated in response to hypoxia in vitro and in vivo. An active form of the HIF-1a induced the expression of miR-210, showing the involvement of the HIF-1 signaling pathway in miR-210 gene transcription. Furthermore, miR-210 was shown to bind to the predicted target sites of ephrin-A3 or neuronal pentraxin 1, causing repression in luciferase reporter activity. Contrary to the microRNA-mediated repression hypothesis, ephrin-A3 was expressed at very high levels in post-ischemic mouse hippocampus in vivo. Thus, the regulatory effects of miR-210 on its targets in vivo need to be further characterized.

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