Dendritic cells (DCs) are antigen-presenting cells that play a major role in initiating primary immune responses. We have utilized two independent approaches, DNA microarrays and proteomics, to analyze the expression profile of human CD14؉ blood monocytes and their derived DCs. Analysis of gene expression changes at the RNA level using oligonucleotide microarrays complementary to 6300 human genes showed that ϳ40% of the genes were expressed in DCs. A total of 255 genes (4%) were found to be regulated during DC differentiation or maturation. Most of these genes were not previously associated with DCs and included genes encoding secreted proteins as well as genes involved in cell adhesion, signaling, and lipid metabolism. Protein analysis of the same cell populations was done using two-dimensional gel electrophoresis. A total of 900 distinct protein spots were included, and 4% of them exhibited quantitative changes during DC differentiation and maturation. Differentially expressed proteins were identified by mass spectrometry and found to represent proteins with Ca 2؉ binding, fatty acid binding, or chaperone activities as well as proteins involved in cell motility. In addition, proteomic analysis provided an assessment of post-translational modifications. The chaperone protein, calreticulin, was found to undergo cleavage, yielding a novel form. The combined oligonucleotide microarray and proteomic approaches have uncovered novel genes associated with DC differentiation and maturation and has allowed analysis of post-translational modifications of specific proteins as part of these processes.

Rapamycin has been shown to affect translation. We have utilized two complementary approaches to identify genes that are predominantly affected by rapamycin in Jurkat T cells. One was to compare levels of polysomebound and total RNA using oligonucleotide microarrays complementary to 6,300 human genes. Another was to determine protein synthesis levels using two-dimensional PAGE. Analysis of expression changes at the polysome-bound RNA levels showed that translation of most of the expressed genes was partially reduced following rapamycin treatment. However, translation of 136 genes (6% of the expressed genes) was totally inhibited. This group included genes encoding RNA-binding proteins and several proteasome subunit members. Translation of a set of 159 genes (7%) was largely unaffected by rapamycin treatment. These genes included transcription factors, kinases, phosphatases, and members of the RAS superfamily. Analysis of [35 S]methionine-labeled proteins from the same cell populations using two-dimensional PAGE showed that the integrated intensity of 111 of 830 protein spots changed in rapamycin-treated cells by at least 3-fold (70 increased, 41 decreased). We identified 22 affected protein spots representing protein products of 16 genes. The combined microarray and proteomic approach has uncovered novel genes affected by rapamycin that may be involved in its immunosuppressive effect and other genes that are not affected at the level of translation in a context of general inhibition of cap-dependent translation.Rapamycin is a macrolide antibiotic originally isolated from Streptomyces hygroscopicus (1). It is a potent immunosuppressant with therapeutic applications in the prevention of organ allograft rejection and in the treatment of autoimmune disease (2-6). The importance of rapamycin as an immunosuppressant drug has focused attention on its mechanism of action. Rapamycin has a similar biochemical structure to cyclosporin A and FK506. However, unlike cyclosporin A and FK506, rapamycin is not a calcineurin inhibitor (7). The primary mode of immunosuppressive action of rapamycin is an antiproliferative action reflecting the ability of the drug to disrupt signaling by T cell growth-promoting lymphokines such as IL-2 1 and IL-4 (8). The growth-inhibitory effects of rapamycin are not limited to T cells, since this drug inhibits the proliferation of many mammalian cell types as well as that of yeast cells (9).Rapamycin blocks progression of the cell cycle at the G 1 phase by binding to FKBP12 (FK506-binding protein) (10, 11). The rapamycin-FKBP12 complex inhibits mTOR (mammalian target of rapamycin), also referred to as FRAP (FKBP-rapamycin-associated protein) (9). Targets of mTOR include 4E-BP1 and the 40 S ribosomal protein S6 kinase, p70 s6k (12-16). Rapamycin-induced inhibition of p70 s6k activity and subsequent dephosphorylation of the ribosomal S6 protein lead to a selective translational repression of mRNA containing a polypyrimidine-rich tract (TOP) motif at their 5Ј terminus (17). 4E-BP1 is a small heat-and...

The specific and sensitive definitions of disease bracket the true values; the relatively small range indicates that the current study provides good estimations of incidence and prevalence of Parkinson's disease for recent years in France.

4E-BP1 plays a major role in translation by inhibiting cap-dependent translation initiation. Several reports have investigated the regulation of 4E-BP1 phosphorylation, which varies along with cell differentiation and upon various stimulations, but very little is known about the regulation of its expression. In a first part, we show that the expression of 4E-BP1 protein and transcript decreases in hematopoietic cell lines cultivated in the presence of phorbol 12-myristate 13-acetate (PMA). This decrease depends on the activation of the ERK/ mitogen-activated protein kinases. 4E-BP1 expression also decreases when the p38/mitogen-activated protein kinase pathway is activated by granulocyte/macrophage colony-stimulating factor but to a lesser extent than with PMA. In a second part, we examine how 4e-bp1 promoter activity is regulated. PMA and granulocyte/ macrophage colony-stimulating factor induce Egr-1 expression through ERK and p38 activation, respectively. Using a dominant negative mutant of Egr, ZnEgr, we show that this transcription factor is responsible for the inhibition of 4e-bp1 promoter activity. In a third part we show that histidine decarboxylase, whose activity and expression are inversely correlated with 4E-BP1 expression, is a potential target for the translational machinery. These data (i) are the first evidence of a new role of ERK and p38 on the translational machinery and (ii) demonstrate that 4E-BP1 is a new target for Egr-1.