Faculty Bibliography

Neutrophils provide an essential defense against bacterial and fungal infection and play a major role in tissue damage during inflammation. Using oligonucleotide microarrays, we have examined the time course of changes in gene expression induced by stimulation with live, opsonized Escherichia coli, soluble lipopolysaccharide, and the chemoattractant formyl-methionyl-leucyl-phenylalanine. The results indicate that activated neutrophils generate a broad and vigorous set of alterations in gene expression. The responses included changes in the levels of transcripts encoding 148 transcription factors and chromatin-remodeling genes and 95 regulators of protein synthesis or stability. Clustering analysis showed distinct temporal patterns with many rapid changes in gene expression within the first hour of exposure. In addition to the temporal clustering of genes, we also observed rather different profiles associated with each stimulus, suggesting that even a nonvirulent organism such as E. coli is able to play a dynamic role in shaping the inflammatory response. Principal component analysis of transcription factor genes demonstrated clear separation of the neutrophil-response clusters from those of resting and stimulated human monocytes. The present study indicates that combinatorial transcriptional regulation including alterations of chromatin structure may play a role in the rapid changes in gene expression that occur in these terminally differentiated cells

Mutations in the human P gene result in oculocutaneous albinism type 2, the most common form of albinism. Mouse melan-p1 melanocytes, cultured from mice null at the homologous pink-eyed dilution (p) locus, exhibit defective melanin production. A variety of compounds including tyrosine, NH4Cl, bafilomycin A1, concanamycin, monensin, and nigericin are capable of restoring melanin synthesis in these cells. In the current study, we investigated the subcellular effects of bafilomycin A1 and monensin treatment of melan-p1 cells. Both agents play two roles in the processing of tyrosinase (Tyr) in melan-p1 cells. First, combined glycosidase digestion and immunoblotting analysis showed that these agents reduce levels of Tyr retained in the endoplasmic reticulum (ER) and facilitate the release of Tyr from the ER to the Golgi. Secondly, treatment with these compounds resulted in the stabilization of Tyr. Surprisingly, induction of melanin synthesis corresponds more closely with diminution of ER-retained Tyr, rather than the absolute amount of Tyr. Our results suggest that bafilomycin A1 and monensin induce melanin synthesis in melan-p1 cells mainly by facilitating Tyr processing from the ER to the Golgi by increasing the pH in either the ER or the ER-Golgi intermediate compartment

Atopic diseases are characterized by Th2 and IgE responses to common environmental and food antigens. In vivo, IgE production depends on interactions between allergen-specific B lymphocytes and Th2 lymphocytes. IgE levels are extremely low in normal individuals, suggesting that IgE production is under strong regulation. One of the reasons behind the lack of atopy in healthy individuals is the activity of regulatory T cells, which prevent naive T helper cell precursors from acquiring a differentiated Th2 phenotype. In addition to naturally occurring regulatory T cells, atopy can be prevented by allergen-specific tolerant/regulatory cells induced through mucosal stimulation, and by mechanisms that directly suppress Iepsilon sterile transcript production on activated B lymphocytes. This article reviews the recent progress on thymic-derived as well as peripherally induced regulatory T cells as they relate to atopy. The latter discussion also includes regulatory T cells that arise through immunotherapy

3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCoAr) provides attractive cues to Drosophila germ cells, guiding them toward the embryonic gonad. However, it remains unclear how HMGCoAr mediates this attraction. In a genomic analysis of the HMGCoAr pathway, we found that the fly genome lacks several enzymes required for cholesterol biosynthesis, ruling out cholesterol and cholesterol-derived proteins as mediators of PGC migration. Genetic analysis of the pathway revealed that two enzymes, farnesyl-diphosphate synthase and geranylgeranyl-diphosphate synthase, required for the production of isoprenoids, act downstream of HMGCoAr in germ cell migration. Consistent with a role in geranylgeranylation, embryos deficient in geranylgeranyl transferase type I show germ cell migration defects. Our data, together with similar findings in zebrafish, implicate an isoprenylated protein in germ cell attraction. The specificity and evolutionary conservation of the HMGCoAr pathway for germ cells suggest that an attractant common to invertebrates and vertebrates guides germ cells in early embryos

Mutation and deletion of the p53 tumor suppressor gene are arguably the most prevalent among the multiple genetic alterations found in human bladder cancer, but these p53 defects are primarily associated with the advanced diseases, and their roles in bladder tumor initiation and in synergizing with oncogenes in tumor progression have yet to be defined. Using the mouse uroplakin II gene promoter, we have targeted into urothelium of transgenic mice a dominant-negative mutant of p53 that lacks the DNA-binding domain but retains the tetramerization domain. Urothelium-expressed p53 mutant binds to and stabilizes the endogenous wild-type p53, induces nuclear abnormality, hyperplasia and occasionally dysplasia, without eliciting frank carcinomas. Concurrent expression of the p53 mutant with an activated Ha-ras, the latter of which alone induces urothelial hyperplasia, fails to accelerate tumor formation. In contrast, the expression of the activated Ha-ras in the absence of p53, as accomplished by crossing the activated Ha-ras transgenic mice with the p53 knockout mice, results in early-onset bladder tumors that are either low-grade superficial papillary or high grade in nature. These results provide the first in vivo experimental evidence that p53 deficiency predisposes the urothelium to hyperproliferation, but is insufficient for bladder tumorigenesis; that the mere reduction of p53 dosage, as produced in transgenic mice expressing the dominant-negative p53 or in heterozygous p53 knockouts, is incapable of synergizing with Ha-ras to induce bladder tumors; and that the complete loss of p53 is a prerequisite for collaborating with activated Ha-ras to promote bladder tumorigenesis

RNApolII-dependent transcription is repressed in primordial germ cells of many animals during early development and is thought to be important for maintenance of germline fate by preventing somatic differentiation. Germ cell transcriptional repression occurs concurrently with inhibition of phosphorylation in the carboxy-terminal domain (CTD) of RNApolII, as well as with chromatin remodeling. The precise mechanisms involved are unknown. Here, we present evidence that a noncoding RNA transcribed by the gene polar granule component (pgc) regulates transcriptional repression in Drosophila germ cells. Germ cells lacking pgc RNA express genes important for differentiation of nearby somatic cells and show premature phosphorylation of RNApolII. We further show that germ cells lacking pgc show increased levels of K4, but not K9 histone H3 methylation, and that the chromatin remodeling Swi/Snf complex is required for a second stage in germ cell transcriptional repression. We propose that a noncoding RNA controls transcription in early germ cells by blocking the transition from preinitiation to transcriptional elongation. We further show that repression of somatic differentiation signals mediated by the Torso receptor-tyrosine kinase is important for germline development

The unique immunological properties of the liver may be due to the function of hepatic dendritic cells (DC). However, liver DC have not been well characterized because of the difficulty in isolating adequate numbers of cells for analysis. Using immunomagnetic bead and flow cytometric cell sorting, we compared freshly isolated murine liver and spleen CD11c+ DC. We found that liver DC are less mature, capture less Ag, and induce less T cell stimulation than spleen DC. Nevertheless, liver DC were able to generate high levels of IL-12 in response to CpG stimulation. We identified four distinct subtypes of liver DC based on the widely used DC subset markers CD8alpha and CD11b. Lymphoid (CD8alpha+CD11b-) and myeloid (CD8alpha-CD11b+) liver DC activated T cells to a similar degree as did their splenic DC counterparts but comprised only 20% of all liver DC. In contrast, the two more prevalent liver DC subsets were only weakly immunostimulatory. Plasmacytoid DC (B220+) accounted for 19% of liver DC, but only 5% of spleen DC. Our findings support the widely held notion that liver DC are generally weak activators of immunity, although they are capable of producing inflammatory cytokines, and certain subtypes potently activate T cells

Transient phosphorylation of the alpha-subunit of translation initiation factor 2 (eIF2alpha) represses translation and activates select gene expression under diverse stressful conditions. Defects in the eIF2alpha phosphorylation-dependent integrated stress response impair resistance to accumulation of malfolded proteins in the endoplasmic reticulum (ER stress), to oxidative stress and to nutrient deprivations. To study the hypothesized protective role of eIF2alpha phosphorylation in isolation of parallel stress signaling pathways, we fused the kinase domain of pancreatic endoplasmic reticulum kinase (PERK), an ER stress-inducible eIF2alpha kinase that is normally activated by dimerization, to a protein module that binds a small dimerizer molecule. The activity of this artificial eIF2alpha kinase, Fv2E-PERK, is subordinate to the dimerizer and is uncoupled from upstream stress signaling. Fv2E-PERK activation enhanced the expression of numerous stress-induced genes and protected cells from the lethal effects of oxidants, peroxynitrite donors and ER stress. Our findings indicate that eIF2alpha phosphorylation can initiate signaling in a cytoprotective gene expression pathway independently of other parallel stress-induced signals and that activation of this pathway can single-handedly promote a stress-resistant preconditioned state

Regulation of gene expression by nutrients in mammals is an important mechanism allowing them to adapt their physiological functions according to the supply of nutrient in the diet. It has been shown recently that amino acids are able to regulate by themselves the expression of numerous genes. CHOP, asparagine synthetase, and IGFBP-1 regulation following AA starvation will be described in this review with special interest in the molecular mechanisms involved.