Entry of HIV-1 into target cells requires cell-surface CD4 and additional host cell cofactors. A cofactor required for infection with virus adapted for growth in transformed T-cell lines was recently identified and named fusin. However, fusin does not promote entry of macrophage-tropic viruses, which are believed to be the key pathogenic strains in vivo. The principal cofactor for entry mediated by the envelope glycoproteins of primary macrophage-tropic strains of HIV-1 is CC-CKR-5, a receptor for the beta-chemokines RANTES, MIP-1alpha and MIP-1beta.

Notch signaling is a central mechanism for controlling embryogenesis. However, its in vivo function during mesenchymal cell differentiation, and specifically, in bone homeostasis remains largely unknown. Here, we show that osteoblast-specific gain of Notch function causes severe osteosclerosis due to increased proliferation of immature osteoblasts. Under these pathological conditions, Notch stimulates early osteoblastic proliferation by up-regulating Cyclin D, Cyclin E, and Osterix. Notch also regulates terminal osteoblastic differentiation by directly binding Runx2 and repressing its transactivation function. In contrast, loss of all physiologic Notch signaling in osteoblasts, generated by deletion of Presenilin 1 and 2 in bone, is associated with late onset, age-related osteoporosis resulting from increased osteoblast-dependent osteoclastic activity due to decreased production of Osteoprotegerin. Together, these findings highlight the potential dimorphic effects of Notch signaling in bone homeostasis and may provide direction for novel therapeutic applications.Evolutionarily conserved Notch signaling plays a critical role in cell fate determination, and various developmental processes by translating cell-cell interactions into specific transcriptional programs 1, 2 . Temporal and spatial modulation of this pathway can significantly affect proliferation, differentiation and apoptotic events 3 . Moreover, the timing of Notch signaling can lead to diverse effects within the same cell lineage 4, 5 . In mammals, activation of up to four Notch receptors by membrane-bound ligands initiates a process leading to presenilin-mediated cleavage and release of the Notch intracellular domain (NICD) from the membrane that then traffics to the nucleus. NICD subsequently regulates the expression of genes in cooperation with the transcription factor RBP-Jκ and Mastermind-like proteins.The observation that mutations in the Notch ligand Delta homologue-3 (Dll-3) and γ-secretase Presenilin1 both cause axial skeletal phenotypes originally linked Notch signaling with skeletal development 6, 7 . Recently, several in vitro studies with conflicting results implicated the Notch pathway in the regulation of osteoblast differentiation, but the in vivo role of Notch signaling in bone homeostasis still remains unknown 8-12 .Corresponding Author: Brendan Lee, M.D., Ph.D., One Baylor Plaza, Rm 635E, Houston, Tx 77030,, Email E-mail: blee@bcm.tmc.edu. In this study, we investigate the tissue, cellular, and molecular consequences of both gain and loss of function of Notch signaling in committed osteoblasts. NIH Public Access RESULTS Gain of function of Notch signaling results in severe osteosclerosisTo determine the pathological consequences of in vivo gain of Notch function during bone formation and homeostasis, we generated transgenic mice expressing the Notch1 intracellular domain (N1ICD) under the control of the type I collagen (Col1a1) promoter (Suppl. Fig. 1a,b). Here, gain of Notch function would occur in committed osteoblastic ce...

The 5' ends of trypanosome mRNAs consist of an identical sequence of 35 nucleotides. This "mini-exon" sequence is derived from the 5' end of a 137 nucleotide RNA (medRNA). The remainder of each mRNA is derived from a protein-coding exon that is not linked to the mini-exon. We propose that medRNA is spliced in trans to de-novo-initiated transcripts of protein-coding genes. This trans splicing model predicts that the downstream portion of medRNA will be part of a branched structure and then be released as a free product (minRNA). We demonstrate that significant levels of minRNA exist in trypanosome RNA. Furthermore, minRNA can be released from high molecular weight RNA by a HeLa cell S100 "debranching" extract. We conclude that trans splicing is the physiological process by which mature mRNA molecules are synthesized in trypanosomes.

Because of their ability to transduce nondividing cells, human immunodeficiency virus type 1 (HIV)-based vectors have great potential for the therapeutic delivery of genes to cells. We describe here a systematic study of the packaging limit of HIV-based vectors. Restriction endonuclease-generated bacterial chromosomal DNA fragments of different lengths were cloned at three different positions within a lentiviral vector. Vesicular stomatitis virus G protein (VSV G) pseudotyped lentiviral particles were prepared and the different clones were titered on mammalian cells. We observed that the restriction endonuclease site positions at the 5' and 3' ends of the genome were superior with regard to insertional capacity of foreign DNA. In all cases, viral titers decreased semi-logarithmically with increasing vector length. There appears to be no absolute packaging limit because measurable titers were obtained even when the proviral length was in excess of 18 kb. The reduction in titer appears to occur at the level of viral encapsidation, although we cannot exclude limitations in nuclear export of proviral RNA. These results suggest that HIV-based vectors may have a secondary advantage over oncoretroviral vectors because of their greater packaging limit, although the very low titers of the larger vectors will be of limited utility.