We show that the transcription factor RBP-J kappa/Su(H) interacts directly with a novel intracellular domain of the cell-surface receptor Notch. RBP-J kappa/Su(H) does not appear to interact with Notch via the CDC10/ankyrin repeats implicated in previous studies.

In response to ionizing radiation (IR), the tumor suppressor p53 is stabilized and promotes either cell cycle arrest or apoptosis. Chk2 activated by IR contributes to this stabilization, possibly by direct phosphorylation. Like p53, Chk2 is mutated in patients with Li-Fraumeni syndrome. Since the ataxia telangiectasia mutated (ATM) gene is required for IR-induced activation of Chk2, it has been assumed that ATM and Chk2 act in a linear pathway leading to p53 activation. To clarify the role of Chk2 in tumorigenesis, we generated genetargeted Chk2-deficient mice. Unlike ATM ؊/؊ and p53 ؊/؊ mice, Chk2 ؊/؊ mice do not spontaneously develop tumors, although Chk2 does suppress 7,12-dimethylbenzanthracene-induced skin tumors. Tissues from Chk2 ؊/؊ mice, including those from the thymus, central nervous system, fibroblasts, epidermis, and hair follicles, show significant defects in IR-induced apoptosis or impaired G 1 /S arrest. Quantitative comparison of the G 1 /S checkpoint, apoptosis, and expression of p53 proteins in Chk2 ؊/؊ versus ATM ؊/؊ thymocytes suggested that Chk2 can regulate p53-dependent apoptosis in an ATM-independent manner. IR-induced apoptosis was restored in Chk2 ؊/؊ thymocytes by reintroduction of the wild-type Chk2 gene but not by a Chk2 gene in which the sites phosphorylated by ATM and ataxia telangiectasia and rad3 ؉ related (ATR) were mutated to alanine. ATR may thus selectively contribute to p53-mediated apoptosis. These data indicate that distinct pathways regulate the activation of p53 leading to cell cycle arrest or apoptosis.

Epstein‐Barr virus nuclear antigen 2 (EBNA‐2) plays a crucial role in B cell immortalization by Epstein‐Barr virus (EBV), most probably by its ability to transactivate several cellular and viral genes. Recently, we showed that EBNA‐2 interacts with the TP1 promoter of EBV through a cellular protein. In this report we provide evidence that this protein is recombination signal binding protein (RBP)‐J kappa, highly conserved in evolution, and originally isolated by its ability to bind to the J kappa‐type V(D)J recombination signal sequence. To identify the cellular protein interacting with the TP1 promoter, we performed electrophoretic mobility shift assays using binding sequences of known transcription factors, that carry partial homology to the crucial sequences of the EBNA‐2 responsive element (EBNA‐2RE), as competitor. Competition assays revealed the RBP‐J kappa recognition site as a very efficient competitor of cellular TP1 promoter binding protein. In parallel, we purified the protein to homogeneity from Raji cells by two ion‐exchange columns and affinity purification using the EBNA‐2RE coupled to magnetic beads. Affinity purified fractions separated on SDS‐PAGE revealed a single predominant band after silver staining which was recognized by anti‐RBP‐J kappa monoclonal antibody. These purified fractions exhibited binding specificity for EBNA‐2RE and EBNA‐2. In vitro‐translated murine RBP‐2 cDNA reacted with EBNA‐2RE and EBNA‐2 in the same fashion as the affinity purified protein. The interaction between RBP‐J kappa and EBNA‐2 is a prerequisite for EBNA‐2‐mediated transactivation of the TP1 promoter.

By random transposon TnS insertions, we previously identified six virulence-associated SaIl fragments, B, D, F, G, H, and P, in the 230-kilobase plasmid pMYSH6000 of Shigellaflexneri 2a. In this study, we analyzed the sites of 134 independent TnS insertions on four contiguous Sall fragments, B, P, H, and D, of pMYSH6000 and identified five virulence-associated regions; four were associated with inducing a positive Sereny test (Ser), invasion into epithelial cells (Inv), binding to Congo red (Pcr), and inhibition of bacterial growth (Igr), and one was associated with the Ser and Inv but not with the Pcr or Igr phenotypes. Hybridization studies revealed that these virulence-associated DNA regions were highly conserved among 15 other virulence plasmids of four species of Shigella and enteroinvasive Escherichuz coli. These data indicate that at least seven separate genetic determinants on the virulence plasmid are required for fuDl expression of the virulence phenotype of shigeliae.Shigellae are enteroinvasive bacteria that cause bacillary dysentery in humans and monkeys. These organisms invade colonic epithelial cells, multiply intracellularly, and spread to adjacent cells (9). The genetic determinants required for these abilities are located on at least three separate sites of the chromosome (3-5, 18) and on a 100-to 140-megadalton (MDa) plasmid. Commonly, plasmids of shigellae and enteroinvasive Escherichia coli (EIEC) contain genetic regions that are required in the early steps of the invasion process (18, 19); loss of the plasmid or deletion of an essential region consistently leads to loss of virulence (13,21).Expression of virulence in shigellae is dependent on temperature (12). Shigellae grown at 37°C are fully virulent, whereas bacteria grown at 30°C neither invade epithelial cells (12) nor provoke keratoconjunctivitis in guinea pigs (24). Making use of this property, Hale et al. (6) identified at least seven plasmid-coded, virulence-associated peptides produced by Shigella flexneri 2a and 5 and EIEC strain 0143. By Western blot (immunoblot) analysis of extracts of whole cells, four peptides of 78, 62, 43, and 38 kDa were recognized by convalescent-phase monkey antisera. These workers proposed that these proteins function as components of the invasion phenotype and are expressed on the bacterial surface. Oaks et al. (15) identified an additional plasmid-encoded surface peptide of 140 kDa which was also specifically recognized by convalescent-phase human or monkey sera. To identify the genetic regions associated with invasion, Maurelli et al. (14) shotgun-cloned Sau3A digests of the plasmid DNA into a cosmid vector, which was subsequently introduced into plasmid-free S. flexneri 5. A clone containing a 37-kilobase (kb) minimum sequence necessary for invasion was isolated. This recombinant clone also produced the four virulence-associated peptides described by Hale et al. (6). A DNA fragment coding for three antigenic proteins of 57, 43, and 39 kDa was cloned into a A expression vector by Buysse et al. (1). T...

A plasmid of about 140 megadaltons has been associated with the invasiveness of Shigella flexneri. Upon subculturing in liquid media of fully virulent isolates of Shigella flexneri 2a YSH6000, which contains only a 230-kilobase-pair (kbp) plasmid in addition to 3.3-and 4.2-kbp cryptic plasmids characteristic to all S. flexneri strains, loss of invasiveness, loss of Congo red binding activity (Pcr), and complete loss of, or a deletion, or even a single-site IS insertion in the plasmid occurred simultaneously. This was ascribed to the fact that, once a noninvasive Pcrcell has emerged, it overgrows the wild type as a consequence of its selective advantage in artificial media. A deletion map of the 230-kbp plasmid was made by analyzing Sall digests of 39 deletion derivatives plus 1 formed by insertion of an IS1-like element in independently isolated, noninvasive Pcrmutants. Of 39 deletion derivatives, 16 belonged to a single type, and 6 belonged to another, suggesting deletion hot spots. The deletion map was confirmed and extended by analyzing 359 Sail-generated partial digests of the wild-type plasmid cloned into pBR322. Three copies of IS1-like elements were found on three different Sall fragments by Southern hybridization. Segments required for the Pcr+ phenotype seemed to occur at several different locations in the plasmid. Each of 28 representative Pcrmutants were negative by the Sereny test. Hence, many, or possibly all, Pcr determinants were required for full virulence.

On the virulence plasmid of Shigella flexneri the virG region required for cell-to-cell spread of the bacterium encodes a 130 kiloDalton (kD) antigen and Region-2 essential for the bacterial invasion of epithelial cells encodes 57, 43 and 39 kD antigens. The expression of these four antigens is positively regulated by the 30 kD protein encoded by virF, whose nucleotide sequence had been determined and which was previously found to be essential for virulence. An approximately 3.8 kilobase (kb) RNA transcript is found to be transcribed by the virG region and is positively regulated by the virF protein resulting in increased production of the 130 kD antigen. The virF sequence is conserved among all shigellae and enteroinvasive Escherichia coli.

Electrides are ionic compounds in which electrons act as anions. These compounds are expected to have interesting properties arising from their exotic structure. The fatal drawbacks of the thermal and chemical instability of organic electrides were resolved by the synthesis of a room temperature (RT) stable electride using single crystalline 12CaO.7Al2O3 (C12A7) with a nanoporous structure and the chemical treatments for a long duration. However, an innovative fabrication method is obviously required for practical applications such as cold electron-emitter and thermionic devices. Herein we report a simple synthesis for polycrystalline C12A7 electrides with a moderate electronic conductivity via a strongly reducing C12A7 "melt", i.e., direct solidification of the melt or crystallization of the transparent glass. Generation of carrier electrons and precipitation of the C12A7 phase from the strongly reducing melt and glass are likely associated with the incorporation of carbon-related anions for stabilizing the C12A7 phase and keeping the mobile electrons in C12A7. These findings will be broadly utilized for applications by mass production in a desired shape and dimension, facilitating the research of electrides.

No abstract