Studies on pluripotent hematopoietic stem cells (HSCs) have been hindered by lack of a positive marker, comparable to the CD34 marker of hematopoietic progenitor cells (HPCs). In human postnatal hematopoietic tissues, 0.1 to 0.5% of CD34(+) cells expressed vascular endothelial growth factor receptor 2 (VEGFR2, also known as KDR). Pluripotent HSCs were restricted to the CD34+KDR+ cell fraction. Conversely, lineage-committed HPCs were in the CD34+KDR- subset. On the basis of limiting dilution analysis, the HSC frequency in the CD34+KDR+ fraction was 20 percent in bone marrow (BM) by mouse xenograft assay and 25 to 42 percent in BM, peripheral blood, and cord blood by 12-week long-term culture (LTC) assay. The latter values rose to 53 to 63 percent in LTC supplemented with VEGF and to greater than 95 percent for the cell subfraction resistant to growth factor starvation. Thus, KDR is a positive functional marker defining stem cells and distinguishing them from progenitors.
The addictive potential of opioids may be related to their differential ability to induce G protein signaling and endocytosis. We compared the ability of 20 ligands (sampled from the main chemical classes of opioids) to promote the association of and ␦ receptors with G protein or -arrestin 2. Receptor-arrestin binding was monitored by bioluminescence resonance energy transfer (BRET) in intact cells, where pertussis toxin experiments indicated that the interaction was minimally affected by receptor signaling. To assess receptor-G protein coupling without competition from arrestins, we employed a cell-free BRET assay using membranes isolated from cells expressing luminescent receptors and fluorescent G 1 . In this system, the agonistinduced enhancement of BRET (indicating shortening of distance between the two proteins) was G␣-mediated (as shown by sensitivity to pertussis toxin and guanine nucleotides) and yielded data consistent with the known pharmacology of the ligands. We found marked differences of efficacy for G protein and arrestin, with a pattern suggesting more restrictive structural requirements for arrestin efficacy. The analysis of such differences identified a subset of structures showing a marked discrepancy between efficacies for G protein and arrestin. Addictive opiates like morphine and oxymorphone exhibited large differences both at ␦ and receptors. Thus, they were effective agonists for G protein coupling but acted as competitive enkephalins antagonists (␦) or partial agonists () for arrestin. This arrestin-selective antagonism resulted in inhibition of short and long term events mediated by arrestin, such as rapid receptor internalization and down-regulation.Physiological agonists are usually equally efficient in promoting the interaction of receptors with G protein and arrestin, but manmade analogues can show divergent molecular efficacies for the two transducers (1, 2). This phenomenon, often addressed with a pictorial terminology (3-5), has attracted great interest and if better understood might lead to new types of drugs.The differential efficacy of opioids for G protein and arrestin interactions is also important in the mechanism of opiate addiction. As reported earlier, the addictive opiate morphine cannot induce and actually blocks desensitization and G protein uncoupling of ␦-opioid receptors (DOPR) 2 in neuroblastoma or in transfected cells (6, 7). Subsequent work shows that morphine is a poor inducer of rapid arrestin-dependent endocytosis for both ␦ and (MOPR) receptors (8 -11), although one exception is in striatum neurons with high levels of G protein receptor kinases (12).Two theories predict a relation between lack of endocytosis and the addiction liability of opioids, but the proposed explanations are radically different. One sees rapid endocytosis as a means to quench receptor signaling. Thus, the abnormally sustained signaling pattern produced by a drug that cannot internalize the receptor would promote post-receptor compensatory mechanisms, which may be responsible for the...
B-MYB expression is associated with cell proliferation and recent studies have suggested that it promotes the S phase of mammalian cells. Based on its homology to the transcription factors c-MYB and A-MYB, B-MYB is thought to be involved in transcriptional regulation; however, its activity is not detectable in several cell lines. It was postulated that B-MYB function may depend on the presence of a cofactor, and recent studies suggested that B-MYB is phosphorylated specifically during S phase in murine fibroblasts. In this report we provide evidence that the product of the human B-myb gene can be activated in vivo by coexpression with cyclin A or cyclin E. Transfection studies showed that B-MYB was a weak transcriptional activator in SAOS-2 cells and was unable to promote their proliferation. In contrast, overexpression of both B-MYB and cyclin A or cyclin E caused a drastic increase in the number of SAOS-2 cells in S phase. Also, overexpression of cyclin A and cyclin E in SAOS-2 cells enhanced the ability of B-MYB, but not c-MYB, to transactivate various promoters, including the cdc2 promoter, the HIV-1-LTR, and the simian virus 40 minimal promoter. A direct role for cyclin-dependent activation of B-MYB was demonstrated using an in vitro transcription assay. These observations suggest that one mechanism by which cyclin A and E may promote the S phase is through modification and activation of B-MYB.B-MYB is a transcriptional regulator whose activity appears to be associated with cellular proliferation and differentiation (1-5). B-MYB is highly homologous to c-MYB within the DNA-binding domain region, and this correlates with a similar affinity displayed by both proteins for the myb-binding sequence (C͞T)AAC(G͞T)G (reviewed in ref. 6). A large body of evidence implicates B-MYB as a player in cell-cycle progression. First, although B-myb expression is ubiquitous, it is strictly regulated in cycling cells: B-myb transcription is downregulated in quiescent cells and expression is detected, upon reentry into the cell cycle, in late G 1 (7,8). Repression of B-myb transcription in G 0 ͞early G 1 is dependent upon an E2F binding site within the promoter and appears to involve negative regulation by the retinoblastoma-related proteins p107 and p130 (9, 10). B-MYB is a downstream target of growth suppressors such as p107 and p53 (5, 11) and its transcription is induced by E2F-1 (12), whose activity is associated with cell-cycle progression and, possibly, transformation. Overall, these data support the hypothesis that B-MYB may play a role in G 1 ͞S transition or during S phase itself. B-MYB can affect the growth rate of certain, but not all, cell lines, and it can activate or repress transcription depending on the promoter, cell type, and species. (13-15). The parameters regulating the specificity of B-MYB transcriptional activity have not been elucidated; however, there is evidence to suggest that the conserved region of B-MYB protein binds to a set of cellular factors that may be involved in cell-typedependent t...
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