Long interspersed element (LINE) 1 retrotransposons are major genomic parasites that represent Ϸ17% of the human genome. The LINE-1 ORF2 protein is also responsible for the mobility of Alu elements, which constitute a further Ϸ11% of genomic DNA. Representative members of each element class remain mobile, and deleterious retrotransposition events can induce spontaneous genetic diseases. Here, we demonstrate that APOBEC3A and APOBEC3B, two members of the APOBEC3 family of human innate antiretroviral resistance factors, can enter the nucleus, where LINE-1 and Alu reverse transcription occurs, and specifically inhibit both LINE-1 and Alu retrotransposition. These data suggest that the APOBEC3 protein family may have evolved, at least in part, to defend the integrity of the human genome against endogenous retrotransposons.APOBEC3 protein ͉ mutagenesis ͉ retrotransposon ͉ genome stability ͉ intrinsic immunity
We report a fully defined synthetic polymer coating, poly[2-(methacryloyloxy)ethyl dimethyl-(3-sulfopropyl)ammonium hydroxide] (PMEDSAH), which sustains long-term human embryonic stem (hES) cell growth in several different culture media, including commercially available defined media. The development of a standardized, controllable and sustainable culture matrix for hES cells is an essential step in elucidating mechanisms that control hES cell behavior and in optimizing conditions for biomedical applications of hES cells.
Differentiation of the pluripotent neuroepithelium into neurons and glia is accomplished by the interaction of growth factors and cell-type restricted transcription factors. One approach to obtaining a particular neuronal phenotype is by recapitulating the expression of these factors in embryonic stem (ES) cells. Toward the eventual goal of auditory nerve replacement, the aim of the current investigation was to generate auditory nerve-like glutamatergic neurons from ES cells. Transient expression of Neurog1 promoted widespread neuronal differentiation in vitro; when supplemented with brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF), 75% of ES cell-derived neurons attained a glutamatergic phenotype after 5 d in vitro. Mouse ES cells were also placed into deafened guinea pig cochleae and Neurog1 expression was induced for 48 h followed by 26 d of BDNF/GDNF infusion. In vivo differentiation resulted in 50 -75% of ES cells bearing markers of early neurons, and a majority of these cells had a glutamatergic phenotype. This is the first study to report a high percentage of ES cell differentiation into a glutamatergic phenotype and sets the stage for cell replacement of auditory nerve.
SummaryWe demonstrate that dissociated human pluripotent stem cells (PSCs) are intrinsically programmed to form lumens. PSCs form two-cell cysts with a shared apical domain within 20 hr of plating; these cysts collapse to form monolayers after 5 days. Expression of pluripotency markers is maintained throughout this time. In two-cell cysts, an apical domain, marked by EZRIN and atypical PKCζ, is surrounded by apically targeted organelles (early endosomes and Golgi). Molecularly, actin polymerization, regulated by ARP2/3 and mammalian diaphanous-related formin 1 (MDIA), promotes lumen formation, whereas actin contraction, mediated by MYOSIN-II, inhibits this process. Finally, we show that lumenal shape can be manipulated in bioengineered micro-wells. Since lumen formation is an indispensable step in early mammalian development, this system can provide a powerful model for investigation of this process in a controlled environment. Overall, our data establish that lumenogenesis is a fundamental cell biological property of human PSCs.
Hepsin was previously identified as a putative cell-surface serine protease. When hepatoma cells were treated with anti-hepsin antibodies, their growth was substantially arrested, suggesting the requirement of hepsin molecules present at the cell surface for normal cell growth. This was further supported by a gross inhibition of cell growth with hepsinspecific antisense oligonucleotides. Upon treatment of cells with antisense oligonucleotides, rapid reduction in cellular hepsin was observed. This reduction in cellular hepsin levels was accompanied by drastic morphological changes. Various tissues in the developing mouse embryo showed greatly elevated hepsin levels in regions of active proliferation. These results indicate that hepsin plays an essential role in cell growth and maintenance of cell morphology.Cell-surface serine proteases have been known to play important roles in various cell functions (1). Our current understanding of this class of plasma membrane proteins, however, is significantly limited. Hepsin is a putative membrane-associated serine protease of 51 kDa (2). It is synthesized as a single polypeptide chain of 417 amino acid residues with a 27-residue-long internal hydrophobic sequence (2, 3). Hepsin is located primarily in the plasma membrane with its trypsin-type protease module (the C-terminal half) at the external surface of cells. This molecular orientation makes hepsin particularly interesting since very little is known about the biological roles of such serine proteases in spite of their predicted importance in cell growth. Hepsin is present at significant levels in many different types of mammalian cells such as human hepatoma cells (HepG2 and PLC/PRF/5 cells), mammary cancer cells (MCF784 and T470), peripheral nerve cells (PC12), and baby hamster kidney cells, but at undetectable levels in some types of cells such as human umbilical cord as well as rat capillary endothelial cells. Hepsin is produced in most tissues but at a particularly high level in the liver (2).In the present report, we describe the importance of hepsin for mammalian cell growth. Experimental evidence indicates that the expression of hepsin is necessary for normal cell growth and morphology. MATERIALS AND METHODSEffects of Anti-Hepsin Antibodies and Antisense Oligonucleotides on the Growth of PLC/PRF/5 Cells. To test the importance of hepsin for cell growth, a set of antisense oligonucleotides and their thioate derivatives were prepared. Synthetic phosphodiester oligonucleotides including sense strand (SS-pd-oligo237, 5'-GGCAGTGACATGGCGCA-GAAG-3') and antisense strand (AS-pd-oligo237, 5'-CTTCTGCGCCATGTCACTGCC-3') of hepsin, which areThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. purified on reverse-phase high-performance liquid chromatography, corresponded to the.5' end region of hepsin cDNA (nt 237-257) with the first in-frame ATG at the center (3...
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