After intravascular delivery of genetically marked adult mouse bone marrow into lethally irradiated normal adult hosts, donor-derived cells expressing neuronal proteins (neuronal phenotypes) developed in the central nervous system. Flow cytometry revealed a population of donor-derived cells in the brain with characteristics distinct from bone marrow. Confocal microscopy of individual cells showed that hundreds of marrow-derived cells in brain sections expressed gene products typical of neurons (NeuN, 200-kilodalton neurofilament, and class III beta-tubulin) and were able to activate the transcription factor cAMP response element-binding protein (CREB). The generation of neuronal phenotypes in the adult brain 1 to 6 months after an adult bone marrow transplant demonstrates a remarkable plasticity of adult tissues with potential clinical applications.

This study addresses the binding of ions and the permeation of substrates during function of the GABA transporter GAT1. GAT1 was expressed in Xenopus oocytes and studied electrophysiologically as well as with [3 H]GABA flux; GAT1 was also expressed in mammalian cells and studied with [3 H]GABA and [ 3 H]tiagabine binding. Voltage jumps, Na ϩ and Cl Ϫ concentration jumps, and exposure to high-affinity blockers (NO-05-711 and SKF-100330A) all produce capacitive charge movements. Occlusive interactions among these three types of perturbations show that they all measure the same population of charges. The concentration dependences of the charge movements reveal (1) that two Na ϩ ions interact with the transporter even in the absence of GABA, and (2) that Cl Ϫ facilitates the binding of Na ϩ . Comparison between the charge movements and the transport-associated current shows that this initial Na ϩ -transporter interaction limits the overall transport rate when [GABA] is saturating. However, two classes of manipulation-treatment with high-affinity uptake blockers and the W68L mutation-"lock" Na ϩ onto the transporter by slowing or preventing the subsequent events that release the substrates to the intracellular medium. The Na ϩ substitutes Li ϩ and Cs ϩ do not support charge movements, but they can permeate the transporter in an uncoupled manner. Our results (1) support the hypothesis that efficient removal of synaptic transmitter by the GABA transporter GAT1 depends on the previous binding of Na ϩ and Cl Ϫ , and (2) indicate the important role of the conserved putative transmembrane domain 1 in interactions with the permeant substrates.

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IntroductionWilms ' tumour (WT; nephroblastoma) is the most frequent tumour of the genitourinary tract in children and rated fourth in overall incidence among childhood cancers [1] Abstract During development, renal stem cells reside in the nephrogenic blastema. Wilms' tumour (WT), a common childhood malignancy, is suggested to arise from the nephrogenic blastema that undergoes partial differentiation and as such is an attractive model to study renal stem cells leading to cancer initiation and maintenance. Previously we have made use of blastema-enriched WT stem-like xenografts propagated in vivo to define a 'WT-stem' signature set, which includes cell surface markers convenient for cell isolation (frizzled homolog 2 [Drosophila] -FZD2, FZD7, G-protein coupled receptor 39, activin receptor type 2B, neural cell adhesion molecule -NCAM).We show by fluorescenceactivated cell sorting analysis of sphere-forming heterogeneous primary WT cultures that most of these markers and other stem cell surface antigens (haematopoietic, CD133, CD34, mesenchymal, CD105, CD90, CD44; cancer, CD133, MDR1; hESC, CD24 and putative renal, cadherin 11)

Recent studies indicate a dual epigenetic role of the Polycomb group (PcG) proteins in self-renewal of stem cells and oncogenesis. Their elevation in our previous human kidney microarray screen led us examine whether they participate in processes involving normal and malignant renal progenitors. We therefore analyzed the expression of the PcG genes (EZH2, BMI-1, EED, SUZ12) in relation to that of the nephric-progenitor genes (WT1, PAX2, SALL1, SIX2, CITED1) using real-time polymerase chain reaction and methylation assays during renal development, regeneration, and tumorigenesis. Although all of the nephric-progenitor genes were shown to be developmentally regulated, analysis of polycomb gene expression during murine nephrogenesis and in an in vitro induction model of the nephrogenic mesenchyme indicated dynamic regulation only for EZH2 in the normal renal progenitor population. In contrast, induction of adult kidney regeneration by ischemia/reperfusion injury resulted primarily in rapid elevation of BMI-1, whereas EZH2 was silenced. Analysis of renal tumorigenesis in stem cell-like tumor xenografts established by serial passage of Wilms' tumor (WT) in immunodeficient mice showed cooperative upregulation of all PcG genes. This was accompanied by upregulation of WT1, PAX2, and SALL1 but downregulation of SIX2. Accordingly, methylation-specific quantitative polymerase chain reaction demonstrated promoter hypomethylation of WT1, PAX2, and SIX2 in primary WT and fetal kidneys, whereas progressive WT xenografts showed hypermethylation of SIX2, possibly leading to loss of renal differentiation. PcG genes vary in expression during renal development, regeneration, and tumorigenesis. We suggest a link between polycomb activation and epigenetic alterations of the renal progenitor population in initiation and progression of renal cancer. STEM

Abstract:The function of PrP C , the cellular prion protein (PrP), is still unknown. Like other glycophosphatidylinositol-anchored proteins, PrP resides on Triton-insoluble, cholesterol-rich membranous microdomains, termed rafts. We have recently shown that the activity and subcellular localization of the neuronal isoform of nitric oxide synthase (nNOS) are impaired in adult PrP 0/0 mice as well as in scrapie-infected mice. In this study, we sought to determine whether PrP and nNOS are part of the same functional complex and, if so, to identify additional components of such a complex. To this aim, we looked for proteins that coimmunoprecipitated with PrP in the presence of detergents either that completely dissociate rafts, to identify stronger interactions, or that preserve the raft structure, to identify weaker interactions. Using this detergent-dependent immunoprecipitation protocol we found that PrP interacts strongly with dystroglycan, a transmembrane protein that is the core of the dystrophinglycoprotein complex (DGC). Additional results suggest that PrP also interacts with additional members of the DGC, including nNOS. PrP coprecipitated only with established presynaptic proteins, consistent with recent findings suggesting that PrP is a presynaptic protein. Key Words: Prion protein-Neuronal nitric oxide synthaseDystrophin-glycoprotein complex. J. Neurochem. 75, 1889Neurochem. 75, -1897Neurochem. 75, (2000.PrP C , the cellular prion protein (PrP), is the normal isoform of PrP Sc , the protein that is believed to be the major if not the only component of the prion. Prions cause transmissible neurodegenerative diseases such as scrapie and bovine spongiform encephalopathy (Prusiner, 1991). Both PrP isoforms share the same amino acid sequence, but, in contrast to PrP C , which is sensitive to protease digestion, PrP Sc digestion with proteinase K results in a protease-resistant peptide termed PrP27-30 (Baldwin et al., 1995).Although the cellular function of the normal PrP remains unresolved, convincing numbers of studies have shown that PrP C binds copper specifically and may even play a role in the cellular response to oxidative stress. In particular, it was shown that cerebellar cells from PrP 0/0 mice are more sensitive to copper toxicity and oxidative stress than cerebellar cells from wild-type mice (Brown et al., 1998). In addition, sperm cells originating from PrP 0/0 mice were more sensitive to copper toxicity than sperm cells from wild-type mice (Shaked et al., 1999). It was also recently suggested that PrP possesses a copperdependent superoxide dismutase activity (Brown et al., 1999).PrP binding proteins were looked for by several methods (Oesch et al., 1990;Oesch, 1994;Kurschner and Morgan, 1995;Rieger et al., 1997;Yehiely et al., 1997). However, none of the proteins identified was shown to interact with any of the PrP isoforms under physiological conditions or to influence in any way the pathogenesis of prion diseases.It is still unknown whether the function of PrP C is fulfilled during prion in...

Adenosine to Inosine (A-to-I) RNA editing is a site-specific modification of RNA transcripts, catalyzed by members of the ADAR (Adenosine Deaminase Acting on RNA) protein family. RNA editing occurs in human RNA in thousands of different sites. Some of the sites are located in protein-coding regions but the majority is found in non-coding regions, such as 3′UTRs, 5′UTRs and introns - mainly in Alu elements. While editing is found in all tissues, the highest levels of editing are found in the brain. It was shown that editing levels within protein-coding regions are increased during embryogenesis and after birth and that RNA editing is crucial for organism viability as well as for normal development. In this study we characterized the A-to-I RNA editing phenomenon during neuronal and spontaneous differentiation of human embryonic stem cells (hESCs). We identified high editing levels of Alu repetitive elements in hESCs and demonstrated a global decrease in editing levels of non-coding Alu sites when hESCs are differentiating, particularly into the neural lineage. Using RNA interference, we showed that the elevated editing levels of Alu elements in undifferentiated hESCs are highly dependent on ADAR1. DNA microarray analysis showed that ADAR1 knockdown has a global effect on gene expression in hESCs and leads to a significant increase in RNA expression levels of genes involved in differentiation and development processes, including neurogenesis. Taken together, we speculate that A-to-I editing of Alu sequences plays a role in the regulation of hESC early differentiation decisions.

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