Egr-1, a new downstream molecule of Epstein-Barr virus latent membrane protein 1

To investigate the effect of Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) on human cancer cells, we sought to identify and analyze potential target genes that were differentially expressed in the presence and absence of LMP1. Our cDNA microarray analysis revealed that expression of early growth response gene-1 (Egr-1) was increased by LMP1 expression in MCF7 and Jurkat cells. An NFkappaB inhibitor (SN50) antagonized LMP1-induced enhancement of Egr-1 expression, indicating that LMP1 induced Egr-1 via NFkappaB. Furthermore, three lines of evidence indicated that Egr-1 was required for LMP1-induced cancer cell survival. First, Egr-1 expression enhanced the survival of doxorubicin-treated MCF7 cells. Second, inhibition of Egr-1 expression by siRNA (siEgr-1) effectively suppressed LMP-1-induced survival of MCF7 cells. Third, Egr-1 knockdown decreased LMP1-induced expression of Bfl-1. Similar relationships among EBV infection, Egr-1 and drug resistance were also observed in tissues of peripheral T-cell lymphoma-unspecified (PTCL-u) patients.     

Structure-function relationships in a bacterial DING protein

A recombinant DING protein from Pseudomonas fluorescens has been previously shown to have a phosphate-binding site, and to be mitogenic for human cells. Here we report the three-dimensional structure of the protein, confirming a close similarity to the "Venus flytrap" structure seen in other human and bacterial phosphate-binding proteins. Site-directed mutagenesis confirms the role of a key residue involved in phosphate binding, and that the mitogenic activity is not dependent on this property. Deletion of one of the two hinged domains that constitute the Venus flytrap also eliminates phosphate binding whilst enhancing mitogenic activity.     

Cell cycle arrest of stamen initials in maize sex determination

The maize sex determination pathway results in the arrest of stamen in ear spikelets and the abortion of pistils in both the tassel spikelets and in the secondary florets of ear spikelets. Arrested stamen cells showed no signs of DNA fragmentation, an absence of CYCLIN B expression, and an accumulation of the negative cell cycle regulator WEE1 RNA.     

The balance between the novel protein target of wingless and the Drosophila Rho-associated kinase pathway regulates planar cell polarity in the Drosophila wing

Planar cell polarity (PCP) signaling is mediated by the serpentine receptor Frizzled (Fz) and transduced by Dishevelled (Dsh). Wingless (Wg) signaling utilizes Drosophila Frizzled 2 (DFz2) as a receptor and also requires Dsh for transducing signals to regulate cell proliferation and differentiation in many developmental contexts. Distinct pathways are activated downstream of Dsh in Wg- and Fz-signaling pathways. Recently, a number of genes, which have essential roles as downstream components of PCP signaling, have been identified in Drosophila. They include the small GTPase RhoA/Rho1, its downstream effector Drosophila rho-associated kinase (Drok), and a number of genes such as inturned (in) and fuzzy (fy), whose biochemical functions are unclear. RhoA and Drok provide a link from Fz/Dsh signaling to the modulation of actin cytoskeleton. Here we report the identification of the novel gene target of wingless (tow) by enhancer trap screening. tow expression is negatively regulated by Wg signaling in wing imaginal discs, and the balance between tow and the Drok pathway regulates wing-hair morphogenesis. A loss-of-function mutation in tow does not result in a distinct phenotype. Genetic interaction and gain-of-function studies provide evidence that Tow acts downstream of Fz/Dsh and plays a role in restricting the number of hairs that wing cells form.     

Quantitative trait loci for the circadian clock in Neurospora crassa

Neurospora crassa has been a model organism for the study of circadian clocks for the past four decades. Among natural accessions of Neurospora crassa, there is significant variation in clock phenotypes. In an attempt to investigate natural allelic variants contributing to quantitative variation, we used a quantitative trait loci mapping approach to analyze three independent mapping populations whose progenitors were collected from geographically isolated locations. Two circadian clock phenotypes, free-running period and entrained phase, were evaluated in the 188 F(1) progeny of each mapping population. To identify the clock QTL, we applied two QTL mapping analyses: composite interval mapping (CIM) and Bayesian multiple QTL analysis (BMQ). When controlling false positive rates < or =0.05, BMQ appears to be the more sensitive of the two approaches. BMQ confirmed most of the QTL from CIM (18 QTL) and identified 23 additional QTL. While 13 QTL colocalize with previously identified clock genes, we identified 30 QTL that were not linked with any previously characterized clock genes. These are candidate regions where clock genes may be located and are expected to lead to new insights in clock regulation.     

Interleukin-1 (IL-1)-induced TAK1-dependent Versus MEKK3-dependent NFkappaB activation pathways bifurcate at IL-1 receptor-associated kinase modification

Interleukin-1 (IL-1) receptor-associated kinase (IRAK) is phosphorylated after it is recruited to the receptor, subsequently ubiquitinated, and eventually degraded upon IL-1 stimulation. Although a point mutation changing lysine 134 to arginine (K134R) in IRAK abolished IL-1-induced IRAK ubiquitination and degradation, mutations of serines and threonines adjacent to lysine 134 to alanines ((S/T)A (131-144)) reduced IL-1-induced IRAK phosphorylation and abolished IRAK ubiquitination. Through the study of these IRAK modification mutants, we uncovered two parallel IL-1-mediated signaling pathways for NFkappaB activation, TAK1-dependent and MEKK3-dependent, respectively. These two pathways bifurcate at the level of IRAK modification. The TAK1-dependent pathway leads to IKKalpha/beta phosphorylation and IKKbeta activation, resulting in classical NFkappaB activation through IkappaBalpha phosphorylation and degradation. The TAK1-independent MEKK3-dependent pathway involves IKKgamma phosphorylation and IKKalpha activation, resulting in NFkappaB activation through IkappaBalpha phosphorylation and subsequent dissociation from NFkappaB but without IkappaBalpha degradation. These results provide significant insight to our further understanding of NFkappaB activation pathways.     

Further evidence for two functional forms of prothrombinase each specific for either of the two prothrombin activation cleavages

Previous work showed that prothrombin derivatives cleavable only at Arg-320 (rMZ) or Arg-271 (rP2) are partial, rather than competitive, inhibitors of prothrombin activation by prothrombinase. A "ping-pong"-like model, which posits two equilibrating forms of prothrombinase, explained the inhibition pattern. The present studies were undertaken to further investigate this putative mechanism. Two models were developed, one allowing for one form of the enzyme and the other allowing for two forms. Both models also allowed channeling and ratcheting. The models were fit to full time courses of prothrombin, meizothrombin, prethrombin-2, and the B-chain. In the absence of ratcheting and channeling, neither model fits the data. In their presence, however, both models fit very well, and thus they could not be distinguished. Therefore, inhibition of rMZ activation by rP2 was studied. Inhibition was partial and the two-form model fit the data with randomly distributed residuals, whereas the one-form model did not. Initial rates of fluorescein-labeled prothrombin cleavage in the presence of various prothrombin derivatives reported by Brufatto and Nesheim (Brufatto, N., and Nesheim, M. E. (2003) J. Biol. Chem. 278, 6755-6764) were also analyzed using the two models. The two-form model fit the partial inhibition data well, whereas the one-form model did not. In addition, prothrombin at varying concentrations was activated, and subsequently, the initial rates were plotted with respect to the initial prothrombin concentration. When compared with the expected initial rates as determined by the simulation of the models, the two-form model fit the observed rates better than the one-form model. The results obtained here further support the existence of two functional forms of prothrombinase.     

Food intake regulates oleoylethanolamide formation and degradation in the proximal small intestine

Oleoylethanolamide (OEA) is a lipid mediator that inhibits food intake by activating the nuclear receptor peroxisome proliferator-activated receptor-alpha. In the rodent small intestine OEA levels decrease during food deprivation and increase upon refeeding, suggesting that endogenous OEA may participate in the regulation of satiety. Here we show that feeding stimulates OEA mobilization in the mucosal layer of rat duodenum and jejunum but not in the serosal layer from the same intestinal segments in other sections of the gastrointestinal tract (stomach, ileum, colon) or in a broad series of internal organs and tissues (e.g. liver, brain, heart, plasma). Feeding also increases the levels of other unsaturated fatty acid ethanolamides (FAEs) (e.g. linoleoylethanolamide) without affecting those of saturated FAEs (e.g. palmitoylethanolamide). Feeding-induced OEA mobilization is accompanied by enhanced accumulation of OEA-generating N-acylphosphatidylethanolamines (NAPEs) increased activity and expression of the OEA-synthesizing enzyme NAPE-phospholipase D, and decreased activity and expression of the OEAdegrading enzyme fatty acid amide hydrolase. Immunostaining studies revealed that NAPE-phospholipase D and fatty acid amide hydrolase are expressed in intestinal enterocytes and lamina propria cells. Collectively, these results indicate that nutrient availability controls OEA mobilization in the mucosa of the proximal intestine through a concerted regulation of OEA biosynthesis and degradation.