The alpha-chemokine stromal cell-derived factor (SDF)-1alpha binds to the seven transmembrane G-protein-coupled CXCR-4 receptor and acts to modulate cell migration and proliferation. The signaling pathways that mediate the effects of SDF-1alpha are not well characterized. We studied events following SDF-1alpha binding to CXCR-4 in a model murine pre-B cell line transfected with human CXCR-4. There was enhanced tyrosine phosphorylation and association of components of focal adhesion complexes such as the related adhesion focal tyrosine kinase, paxillin, and Crk. We also observed activation of phosphatidylinositol 3-kinase. Wortmannin, a selective inhibitor of phosphatidylinositol 3-kinase, partially inhibited the SDF-1alpha-induced migration and tyrosine phosphorylation of paxillin. SDF-1alpha treatment selectively activated p44/42 mitogen-activated protein kinase (Erk 1 and Erk 2) and its upstream kinase mitogen-activated protein kinase kinase but not p38 mitogen-activated protein kinase, c-Jun amino-terminal kinase or mitogen activated protein kinase kinase. We also observed that SDF-1alpha treatment increased NF-kappaB activity in nuclear extracts from the CXCR-4 transfectants. Taken together, these studies revealed that SDF-1alpha activates distinct signaling pathways that may mediate cell growth, migration, and transcriptional activation.

Inhalation of fungal spores (conidia) occurs commonly and, in specific circumstances, can result in invasive disease. We investigated the murine inflammatory response to conidia of Aspergillus fumigatus, the most common invasive mold in immunocompromised hosts. In contrast to dormant spores, germinating conidia induce neutrophil recruitment to the airways and TNF-alpha/MIP-2 secretion by alveolar macrophages. Fungal beta-glucans act as a trigger for the induction of these inflammatory responses through their time-dependent exposure on the surface of germinating conidia. Dectin-1, an innate immune receptor that recognizes fungal beta-glucans, is recruited in vivo to alveolar macrophage phagosomes that have internalized conidia with exposed beta-glucans. Antibody-mediated blockade of Dectin-1 partially inhibits TNF-alpha/MIP-2 induction by metabolically active conidia. TLR-2- and MyD88-mediated signals provide an additive contribution to macrophage activation by germinating conidia. Selective responsiveness to germinating conidia provides the innate immune system with a mechanism to restrict inflammatory responses to metabolically active, potentially invasive fungal spores.

Hepatocellular carcinoma (HCC) is one of the most deadly cancers worldwide and has no effective treatment, yet the molecular basis of hepatocarcinogenesis remains largely unknown. Here we report findings from a whole-genome sequencing (WGS) study of 88 matched HCC tumor/normal pairs, 81 of which are Hepatitis B virus (HBV) positive, seeking to identify genetically altered genes and pathways implicated in HBV-associated HCC. We find beta-catenin to be the most frequently mutated oncogene (15.9%) and TP53 the most frequently mutated tumor suppressor (35.2%). The Wnt/beta-catenin and JAK/STAT pathways, altered in 62.5% and 45.5% of cases, respectively, are likely to act as two major oncogenic drivers in HCC. This study also identifies several prevalent and potentially actionable mutations, including activating mutations of Janus kinase 1 (JAK1), in 9.1% of patients and provides a path toward therapeutic intervention of the disease.

Sulforaphane (SFN), a compound found at high levels in broccoli and broccoli sprouts, is a potent inducer of phase 2 detoxification enzymes and inhibits tumorigenesis in animal models. SFN also has a marked effect on cell cycle checkpoint controls and cell survival and/or apoptosis in various cancer cells, through mechanisms that are poorly understood. We tested the hypothesis that SFN acts as an inhibitor of histone deacetylase (HDAC). In human embryonic kidney 293 cells, SFN dose-dependently increased the activity of a beta-catenin-responsive reporter (TOPflash), without altering beta-catenin or HDAC protein levels. Cytoplasmic and nuclear extracts from these cells had diminished HDAC activity, and both global and localized histone acetylation was increased, compared with untreated controls. Studies with SFN and with media from SFN-treated cells indicated that the parent compound was not responsible for the inhibition of HDAC, and this was confirmed using an inhibitor of glutathione S-transferase, which blocked the first step in the metabolism of SFN, via the mercapturic acid pathway. Whereas SFN and its glutathione conjugate (SFN-GSH) had little or no effect, the two major metabolites SFN-cysteine and SFN-N-acetylcysteine were effective HDAC inhibitors in vitro. Finally, several of these findings were recapitulated in HCT116 human colorectal cancer cells: SFN dose-dependently increased TOPflash reporter activity and inhibited HDAC activity, there was an increase in acetylated histones and in p21(Cip1/Waf1), and chromatin immunoprecipitation assays revealed an increase in acetylated histones bound to the P21 promoter. Collectively, these findings suggest that SFN may be effective as a tumor-suppressing agent and as a chemotherapeutic agent, alone or in combination with other HDAC inhibitors currently undergoing clinical trials.

BACKGROUND

RT-qPCR is a preferred method for rapid and reliable quantification of gene expression studies. Appropriate application of RT-qPCR in such studies requires the use of reference gene(s) as an internal control to normalize mRNA levels between different samples for an exact comparison of gene expression level. However, recent studies have shown that no single reference gene is universal for all experiments. Thus, the identification of high quality reference gene(s) is of paramount importance for the interpretation of data generated by RT-qPCR. Only a few studies on reference genes have been done in plants and none in peach (Prunus persica L. Batsch). Therefore, the present study was conducted to identify suitable reference gene(s) for normalization of gene expression in peach.

RESULTS

In this work, eleven reference genes were investigated in different peach samples using RT-qPCR with SYBR green. These genes are: actin 2/7 (ACT), cyclophilin (CYP2), RNA polymerase II (RP II), phospholipase A2 (PLA2), ribosomal protein L13 (RPL13), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 18S ribosomal RNA (18S rRNA), tubblin beta (TUB), tubblin alpha (TUA), translation elongation factor 2 (TEF2) and ubiquitin 10 (UBQ10). All eleven reference genes displayed a wide range of Cq values in all samples, indicating that they expressed variably. The stability of these genes except for RPL13 was determined by three different descriptive statistics, geNorm, NormFinder and BestKeeper, which produced highly comparable results.

CONCLUSIONS

Our study demonstrates that expression stability varied greatly between genes studied in peach. Based on the results from geNorm, NormFinder and BestKeeper analyses, for all the sample pools analyzed, TEF2, UBQ10 and RP II were found to be the most suitable reference genes with a very high statistical reliability, and TEF2 and RP II for the other sample series, while 18S rRNA, RPL13 and PLA2 were unsuitable as internal controls. GAPDH and ACT also performed poorly and were less stable in our analysis. To achieve accurate comparison of levels of gene expression, two or more reference genes must be used for data normalization. The combinations of TEF2/UBQ10/RP II and TEF2/RP II were suggested for use in all samples and subsets, respectively.

The receptor for advanced glycation endproducts (RAGE) mediates responses to cell danger and stress. When bound by its many ligands (which include advanced glycation endproducts, certain members of the S100/calgranulin family, extracellular high-mobility group box 1, the integrin Mac-1, amyloid beta-peptide and fibrils), RAGE activates programs responsible for acute and chronic inflammation. RAGE is therefore also involved in cancer progression, diabetes, atherosclerosis, and Alzheimer's disease. RAGE has several isoforms deriving from alternative splicing, including a soluble form called endogenous secretory RAGE (esRAGE). We show here that most soluble RAGE, either produced by cell lines or present in human blood, is not recognized by an anti-esRAGE antibody. Cells transfected with the cDNA for full-length RAGE, and thus not expressing esRAGE, produce a form of soluble RAGE, cleaved RAGE (cRAGE) that derives from proteolytic cleavage of the membrane-bound molecules and acts as a decoy receptor. By screening chemical inhibitors and genetically modified mouse embryonic fibroblasts (MEFs), we identify the sheddase ADAM10 as a membrane protease responsible for RAGE cleavage. Binding of its ligand HMGB1 promotes RAGE shedding. Our data do not disprove the interpretation that high levels of soluble forms of RAGE protect against chronic inflammation, but rather suggest that they correlate with high levels of ongoing inflammation.

Protein kinase B (PKB) has emerged as the focal point for many signal transduction pathways, regulating multiple cellular processes such as glucose metabolism, transcription, apoptosis, cell proliferation, angiogenesis, and cell motility. In addition to acting as a kinase toward many substrates involved in these processes, PKB forms complexes with other proteins that are not substrates, but rather act as modulators of PKB activity and function. In this review, we discuss the implications of these data in understanding the multitude of functions predicted for PKB in cells.

The cell nucleus is a highly compartmentalized organelle harbouring a variety of dynamic membraneless nuclear bodies. How these subnuclear domains are established and maintained is not well understood. Here, we investigate the molecular mechanism of how one nuclear body, the paraspeckle, is assembled and organized. Paraspeckles are discrete ribonucleoprotein bodies found in mammalian cells and implicated in nuclear retention of hyperedited mRNAs. We developed a live-cell imaging system that allows for the inducible transcription of Men ɛ/β (also known as Neat1; ref. 12) noncoding RNAs (ncRNAs) and the direct visualization of the recruitment of paraspeckle proteins. Using this system, we demonstrate that Men ɛ/β ncRNAs are essential to initiate the de novo assembly of paraspeckles. These newly formed structures effectively harbour nuclear-retained mRNAs confirming that they are bona fide functional paraspeckles. By three independent approaches, we show that it is the act of Men ɛ/β transcription, but not ncRNAs alone, that regulates paraspeckle maintenance. Finally, fluorescence recovery after photobleaching (FRAP) analyses supported a critical structural role for Men ɛ/β ncRNAs in paraspeckle organization. This study establishes a model in which Men ɛ/β ncRNAs serve as a platform to recruit proteins to assemble paraspeckles.

SOX genes encode a family of high-mobility group transcription factors that play critical roles in organogenesis. The functional specificity of different SOX proteins and the tissue specificity of a particular SOX factor are largely determined by the differential partnership of SOX transcription factors with other transcription regulators, many of which have not yet been discovered. Virtually all members of the SOX family have been found to be deregulated in a wide variety of tumors. However, little is known about the cellular and molecular behaviors involved in the oncogenic potential of SOX proteins. Using cell culture experiments, tissue analysis, molecular profiling, and animal studies, we report here that SOX2 promotes cell proliferation and tumorigenesis by facilitating the G(1)/S transition and through its transcription regulation of the CCND1 gene in breast cancer cells. In addition, we identified beta-catenin as the transcription partner for SOX2 and demonstrated that SOX2 and beta-catenin act in synergy in the transcription regulation of CCND1 in breast cancer cells. Our experiments not only determined a role for SOX2 in mammary tumorigenesis but also revealed another activity of the multifunctional protein, beta-catenin.

The metabolic nuclear receptors act as metabolic and toxicological sensors, enabling the organism to quickly adapt to environmental changes by inducing the appropriate metabolic genes and pathways. Ligands for these metabolic receptors are compounds from dietary origin, intermediates in metabolic pathways, drugs, or other environmental factors that, unlike classical nuclear receptor ligands, are present in high concentrations. Metabolic receptors are master regulators integrating the homeostatic control of (a) energy and glucose metabolism through peroxisome proliferator-activated receptor gamma (PPARgamma); (b) fatty acid, triglyceride, and lipoprotein metabolism via PPARalpha, beta/delta, and gamma; (c) reverse cholesterol transport and cholesterol absorption through the liver X receptors (LXRs) and liver receptor homolog-1 (LRH-1); (d) bile acid metabolism through the farnesol X receptor (FXR), LXRs, LRH-1; and (e) the defense against xeno- and endobiotics by the pregnane X receptor/steroid and xenobiotic receptor (PXR/SXR). The transcriptional control of these metabolic circuits requires coordination between these metabolic receptors and other transcription factors and coregulators. Altered signaling by this subset of receptors, either through chronic ligand excess or genetic factors, may cause an imbalance in these homeostatic circuits and contribute to the pathogenesis of common metabolic diseases such as obesity, insulin resistance and type 2 diabetes, hyperlipidemia and atherosclerosis, and gallbladder disease. Further studies should exploit the fact that many of these nuclear receptors are designed to respond to small molecules and turn them into therapeutic targets for the treatment of these disorders.

Stimulated human monocytes/macrophages are a source of mediators such as tumor necrosis factor alpha (TNF-alpha), interleukin 1 (IL-1), and prostaglandin E2 (PGE2), which can modulate inflammatory and immune reactions. Therefore, the ability to control the production of such mediators by monocytes/macrophages may have therapeutic benefits, and it has been proposed that glucocorticoids may act in this way. Purified human monocytes, when stimulated in vitro with lipopolysaccharide (LPS) or with LPS and gamma interferon (IFN-gamma), produce TNF-alpha, IL-1, and PGE2. Cotreatment of stimulated cells with the purified human lymphokine, interleukin 4 (IL-4 greater than or equal to 0.1-0.5 unit/ml; 12-60 pM) dramatically blocked the increased levels of these three mediators; for TNF-alpha and IL-1, the inhibition was manifest at the level of mRNA. Thus, IL-4 can suppress some parameters of monocyte activation and, as for B cells, have opposite effects to IFN-gamma. The effects of IL-4 on human monocytes are similar to those obtained with the glucocorticoid dexamethasone (0.1 microM).

The transforming growth factor-beta (TGF-beta) superfamily contains a variety of growth factors which all share common sequence elements and structural motifs. These proteins are known to exert a wide spectrum of biological responses on a large variety of cell types in both vertebrates and invertebrates. Many of them have important functions during embryonic development in pattern formation and tissue specification, and in adult tissues, they are involved in processes such as wound healing, bone repair, and bone remodeling. The family is divided into two general branches: the BMP/GDF and the TGF-beta/Activin/Nodal branches, whose members have diverse, often complementary effects. It is obvious that an orchestered regulation of different actions of these proteins is necessary for proper functioning. The TGF-beta family members act by binding extracellularly to a complex of serine/threonine kinase receptors, which consequently activate Smad molecules by phosphorylation. These Smads translocate to the nucleus, where they modulate transcription of specific genes. Three levels by which this signaling pathway is regulated could be distinguished. First, a control mechanism exists in the intracellular space, where inhibitory Smads and Smurfs prevent further signaling and activation of target genes. Second, at the membrane site, the pseudoreceptor BAMBI/Nma is able to inhibit further signaling within the cells. Finally, a range of extracellular mediators are identified which modulate the functioning of members of the TGF-beta superfamily. Here, we review the insights in the extracellular regulation of members of the BMP subfamily of secreted growth factors with a major emphasis on vertebrate BMP modulation.

Recently, a positive and a negative elongation factor, implicated in 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) inhibition of transcription elongation, has been identified. P-TEFb is a positive transcription elongation factor and the DRB-sensitive kinase that phosphorylates the C-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II). PITALRE, a member of the Cdc2 family of protein kinases, is the catalytic subunit of P-TEFb. DSIF is a human homolog of the yeast Spt4-Spt5 complex and renders elongation of transcription sensitive to DRB. DRB sensitivity-inducing factor (DSIF) binds to RNA Pol II and may directly regulate elongation. Here we show a functional interaction between P-TEFb and DSIF. The reduction of P-TEFb activity induced by either DRB, antibody against PITALRE, or immunodepletion resulted in a negative effect of DSIF on transcription. DSIF acts at an early phase of elongation, and the prior action of P-TEFb makes transcription resistant to DSIF. The state of phosphorylation of CTD determines the DSIF-RNA Pol II interaction, and may provide a direct link between P-TEFb and DSIF. Taken together, this study reveals a molecular basis for DRB action and suggests that P-TEFb stimulates elongation by alleviating the negative action of DSIF.

Sphingosine 1-phosphate (S1P), a multifunctional lipid mediator, regulates lymphocyte trafficking, vascular permeability, and angiogenesis by activation of the S1P1 receptor. This receptor is activated by FTY720-P, a phosphorylated derivative of the immunosuppressant and vasoactive compound FTY720. However, in contrast to the natural ligand S1P, FTY720-P appears to act as a functional antagonist, even though the mechanisms involved are poorly understood. In this study, we investigated the fate of endogenously expressed S1P1 receptor in agonist-activated human umbilical vein endothelial cells and human embryonic kidney 293 cells expressing green fluorescent protein-tagged S1P1. We show that FTY720-P is more potent than S1P at inducing receptor degradation. Pretreatment with an antagonist of S1P1, VPC 44116, prevented receptor internalization and degradation. FTY720-P did not induce degradation of internalization-deficient S1P1 receptor mutants. Further, small interfering RNA-mediated down-regulation of G protein-coupled receptor kinase-2 and beta-arrestins abolished FTY720-P-induced S1P1 receptor degradation. These data suggest that agonist-induced phosphorylation of S1P1 and subsequent endocytosis are required for FTY720-P-induced degradation of the receptor. S1P1 degradation is blocked by MG132, a proteasomal inhibitor. Indeed, FTY720-P strongly induced polyubiquitinylation of S1P1 receptor, whereas S1P at concentrations that induced complete internalization was not as efficient, suggesting that receptor internalization is required but not sufficient for ubiquitinylation and degradation. We propose that the ability of FTY720-P to target the S1P1 receptor to the ubiquitinylation and proteasomal degradation pathway may at least in part underlie its immunosuppressive and anti-angiogenic properties.

Eukaryotic protein-encoding genes possess poly(A) signals that define the end of the messenger RNA and mediate downstream transcriptional termination by RNA polymerase II (Pol II). Termination could occur through an 'anti-termination' mechanism whereby elongation factors dissociate when the poly(A) signal is encountered, producing termination-competent Pol II. An alternative 'torpedo' model postulated that poly(A) site cleavage provides an unprotected RNA 5' end that is degraded by 5' ->> 3' exonuclease activities (torpedoes) and so induces dissociation of Pol II from the DNA template. This model has been questioned because unprocessed transcripts read all the way to the site of transcriptional termination before upstream polyadenylation. However, nascent transcripts located 1 kilobase downstream of the human beta-globin gene poly(A) signal are associated with a co-transcriptional cleavage (CoTC) activity that acts with the poly(A) signal to elicit efficient transcriptional termination. The CoTC sequence is an autocatalytic RNA structure that undergoes rapid self-cleavage. Here we show that CoTC acts as a precursor to termination by presenting a free RNA 5' end that is recognized by the human 5' ->> 3' exonuclease Xrn2. Degradation of the downstream cleavage product by Xrn2 results in transcriptional termination, as envisaged in the torpedo model.

Selective inhibition of T cell costimulation using the Baction between CD40 and its T cell-based ligand CD154 (CD40L) have been shown in rodents to act synergistically with CTLA4-Ig. It has thus been hypothesized that these agents might be capable of inducing long-term acceptance of allografted tissues in primates. To test this hypothesis in a relevant preclinical model, CTLA4-Ig and the CD40L-specific monoclonal antibody 5C8 were tested in rhesus monkeys. Both agents effectively inhibited rhesus mixed lymphocyte reactions, but the combination was 100 times more effective than either drug alone. Renal allografts were transplanted into nephectomized rhesus monkeys shown to be disparate at major histocompatibility complex class I and class II loci. Control animals rejected in 5-8 days. Brief induction doses of CTLA4-Ig or 5C8 alone significantly prolonged rejection-free survival (20-98 days). Two of four animals treated with both agents experienced extended (>150 days) rejection-free allograft survival. Two animals treated with 5C8 alone and one animal treated with both 5C8 and CTLA4-Ig experienced late, biopsy-proven rejection, but a repeat course of their induction regimen successfully restored normal graft function. Neither drug affected peripheral T cell or B cell counts. There were no clinically evident side effects or rejections during treatment. We conclude that CTLA4-Ig and 5C8 can both prevent and reverse acute allograft rejection, significantly prolonging the survival of major histocompatibility complex-mismatched renal allografts in primates without the need for chronic immunosuppression.

The human beta-interferon gene is regulated by an inducible enhancer element. Analysis of the effect of deletions within this element on beta-interferon transcription indicates that this enhancer is under negative control. Deletion of sequences from the 3' end of the enhancer leads to a dramatic increase in the basal level of beta-interferon mRNA and a decrease in the induction ratio. The remaining 5' region of the enhancer can act as a strong constitutive transcription element, and it shares considerable homology with sequences known to be required for the activity of constitutive viral enhancers. We conclude that the beta-interferon enhancer consists of a constitutive transcription element and a negative regulatory sequence that prevents enhancer activity prior to induction. Thus, derepression of a constitutive transcription element appears to play a key role in the control of human beta-interferon gene expression.

Several lines of evidence are compatible with the hypothesis that Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA-2) or leader protein (EBNA-LP) affects expression of the EBV latent infection membrane protein LMP1. We now demonstrate the following. (i) Acute transfection and expression of EBNA-2 under control of simian virus 40 or Moloney murine leukemia virus promoters resulted in increased LMP1 expression in P3HR-1-infected Burkitt's lymphoma cells and the P3HR-1 or Daudi cell line. (ii) Transfection and expression of EBNA-LP alone had no effect on LMP1 expression and did not act synergistically with EBNA-2 to affect LMP1 expression. (iii) LMP1 expression in Daudi and P3HR-1-infected cells was controlled at the mRNA level, and EBNA-2 expression in Daudi cells increased LMP1 mRNA. (iv) No other EBV genes were required for EBNA-2 transactivation of LMP1 since cotransfection of recombinant EBNA-2 expression vectors and genomic LMP1 DNA fragments enhanced LMP1 expression in the EBV-negative B-lymphoma cell lines BJAB, Louckes, and BL30. (v) An EBNA-2-responsive element was found within the -512 to +40 LMP1 DNA since this DNA linked to a chloramphenicol acetyltransferase reporter gene was transactivated by cotransfection with an EBNA-2 expression vector. (vi) The EBV type 2 EBNA-2 transactivated LMP1 as well as the EBV type 1 EBNA-2. (vii) Two deletions within the EBNA-2 gene which rendered EBV transformation incompetent did not transactivate LMP1, whereas a transformation-competent EBNA-2 deletion mutant did transactivate LMP1. LMP1 is a potent effector of B-lymphocyte activation and can act synergistically with EBNA-2 to induce cellular CD23 gene expression. Thus, EBNA-2 transactivation of LMP1 amplifies the biological impact of EBNA-2 and underscores its central role in EBV-induced growth transformation.

In the 1970's great strides were made in understanding the mechanism of action of amphotericin B and nystatin: the formation of transmembrane pores was clearly demonstrated in planar lipid monolayers, in multilamellar phospholipid vesicles and in Acholeplasma laidlawii cells and the importance of the presence and of the nature of the membrane sterol was analyzed. For polyene antibiotics with shorter chains, a mechanism of membrane disruption was proposed. However, recently obtained data on unilamellar vesicles have complicated the situation. It has been shown that: membranes in the gel state (which is not common in cells), even if they do not contain sterols may be made permeable by polyene antibiotics, several mechanisms may operate, simultaneously or sequentially, depending on the antibiotic/lipid ratio, the time elapsed after mixing and the mode of addition of the antibiotic, there is a rapid exchange of the antibiotic molecules between the vesicles. Although pore formation is apparently involved in the toxicity of amphotericin B and nystatin, it is not the sole factor which contributes to cell death, since K+ leakage induced by these antibiotics is separate from their lethal action. The peroxidation of membrane lipids, which has been demonstrated for erythrocytes and Candida albicans cells in the presence of amphotericin B, may play a determining role in toxicity concurrently with colloid osmotic effect. On the other hand, it has been shown that the action of polyene antibiotics on cells is not always detrimental: at sub-lethal concentrations these drugs stimulate either the activity of some membrane enzymes or cellular metabolism. In particular, some cells of the immune system are stimulated. Furthermore, polyene antibiotics may act synergistically with other drugs, such as antitumor or antifungal compounds. This may occur either by an increased incorporation of the drug, under the influence of a polyene antibiotic-induced change of membrane potential, for example, or by a direct interaction of both drugs. That fungal membranes contain ergosterol while mammalian cell membranes contain cholesterol, has generally been considered the basis for the selective toxicity of amphotericin B and nystatin for fungi. Actually, in vitro studies have not always borne out this assumption, thereby casting doubt on the use of polyene antibiotics as antifungal agents in mammalian cell culture media.(ABSTRACT TRUNCATED AT 400 WORDS)

Nuclear factor kappa B (NF-kappaB) transcription factors regulate several important physiological processes, including inflammation and immune responses, cell growth, apoptosis, and the expression of certain viral genes. Therefore, the NF-kappaB signaling pathway has also provided a focus for pharmacological intervention, primarily in situations of chronic inflammation or in cancer, where the pathway is often constitutively active and plays a key role in the disease. Now that many of the molecular details of the NF-kappaB pathway are known, it is clear that modulators of this pathway can act at several levels. As described herein, over 750 inhibitors of the NF-kappaB pathway have been identified, including a variety of natural and synthetic molecules. These compounds include antioxidants, peptides, small RNA/DNA, microbial and viral proteins, small molecules, and engineered dominant-negative or constitutively active polypeptides. Several of these molecules act as general inhibitors of NF-kappaB induction, whereas others inhibit specific pathways of induction. In addition, some compounds appear to target multiple steps in the NF-kappaB pathway. Compounds designed as specific NF-kappaB inhibitors are not yet in clinical use, but they are likely to be developed as treatments for certain cancers and neurodegenerative and inflammatory diseases. Moreover, the therapeutic and preventative effects of many natural products may, at least in part, be due to their ability to inhibit NF-kappaB.

Eukaryotic genome complexity necessitates boundary and insulator elements to partition genomic content into distinct domains. We show that inverted repeat (IR) boundary elements flanking the fission yeast mating-type heterochromatin domain contain B-box sequences, which prevent heterochromatin from spreading into neighboring euchromatic regions by recruiting transcription factor TFIIIC complex without RNA polymerase III (Pol III). Genome-wide analysis reveals TFIIIC with Pol III at all tRNA genes, many of which cluster at pericentromeric heterochromatin domain boundaries. However, a single tRNA(phe) gene with modest TFIIIC enrichment is insufficient to serve as boundary and requires RNAi-associated element to restrain heterochromatin spreading. Remarkably, we found TFIIIC localization without Pol III at many sites located between divergent promoters. These sites appear to act as chromosome-organizing clamps by tethering distant loci to the nuclear periphery, at which TFIIIC is concentrated into several distinct bodies. Our analyses uncover a general genome organization mechanism involving conserved TFIIIC complex.

The expression of alpha (immediate-early) genes of cytomegalovirus is regulated via a complex enhancer that consists of several different repeat elements. We describe here the autoinduction of expression from the alpha promoter-enhancer by the most abundant alpha gene product, a 491-amino-acid nuclear phosphoprotein referred to as ie1. We defined the 18-base-pair repeat element within the alpha enhancer as the signal through which ie1 acts to regulate gene expression. This element contains an NF kappa B site that may play an important role in ie1 autoregulation. Our analysis, which relied on deletions through the enhancer as well as reconstitution of responsiveness to a promoter with synthetic 18-base-pair repeats, strongly implicated ie1 in the transcriptional transactivation of the alpha promoter through its enhancer.

CD4 + CD25 + regulatory T cells (Tregs) are potent suppressors, playing important roles in autoimmunity and transplantation tolerance. Understanding the signals necessary for the generation and expansion of Tregs is important for clinical cellular therapy, but only limited progress has been made. Recent reports suggest a role for TGF-beta in the generation of Tregs from CD4 + CD25 - precursors, but the mechanism remains unknown. Here, we demonstrate that TGF-betaact like conventional Tregs. The generation of Foxp3 + Tregs requires stimulation of the T-cell receptor, the IL-2R and the TGF-beta receptor. More importantly, strong costimulation through CD28 prevents Foxp3 expression and suppressive function in an IL-4-dependent manner. Furthermore, TGF-beta-driven Tregs inhibit innate inflammatory responses to syngeneic transplanted pancreatic islets and enhance islet transplant survival. Thus, TGF-beta is a key regulator of the signaling pathways that initiate and maintain Foxp3 expression and suppressive function in CD4 + CD25 - precursors. TGF-beta and signaling through TGF-beta receptor, CD28 costimulation and IL-4 may be key components for the manipulation of Treg. The de novo generation of Foxp3 + cells from CD4 + cells has the potential to be used for treatment of autoimmune diseases and induction of transplant tolerance.

Transforming growth factor beta activated kinase-1 (TAK1), a member of the mitogen-activated protein kinase kinase kinase family, has emerged as a key regulator of signal transduction cascades leading to the activation of the transcription factors nuclear factor-kappa B (NF-kappaB) and activator protein-1 (AP-1). Stimulation of cells with cytokines and microbial pathogens results in the activation of TAK1, which subsequently activates the I-kappa B kinase complex (IKK) and mitogen-activated protein (MAP) kinases, culminating in the activation of NF-kappaB and AP-1, respectively. Recent studies have shown that polyubiquitination of signalling proteins through lysine (Lys)-63-linked polyubiquitin chains plays an important role in the activation of TAK1 and IKK. Unlike Lys-48-linked polyubiquitination, which normally targets proteins for degradation by the proteasome, Lys-63-linked polyubiquitin chains act as scaffolds to assemble protein kinase complexes and mediate their activation through proteasome-independent mechanisms. The concept of ubiquitin-mediated activation of protein kinases is supported by the discoveries of ubiquitination and deubiquitination enzymes as well as ubiquitin-binding proteins that function upstream of TAK1 and IKK. Recent biochemical and genetic studies provide further insights into the mechanism and function of ubiquitin signalling and these advances will be the focus of this review.