The bacterium Escherichia coli O157:H7 is a worldwide threat to public health and has been implicated in many outbreaks of haemorrhagic colitis, some of which included fatalities caused by haemolytic uraemic syndrome. Close to 75,000 cases of O157:H7 infection are now estimated to occur annually in the United States. The severity of disease, the lack of effective treatment and the potential for large-scale outbreaks from contaminated food supplies have propelled intensive research on the pathogenesis and detection of E. coli O157:H7 (ref. 4). Here we have sequenced the genome of E. coli O157:H7 to identify candidate genes responsible for pathogenesis, to develop better methods of strain detection and to advance our understanding of the evolution of E. coli, through comparison with the genome of the non-pathogenic laboratory strain E. coli K-12 (ref. 5). We find that lateral gene transfer is far more extensive than previously anticipated. In fact, 1,387 new genes encoded in strain-specific clusters of diverse sizes were found in O157:H7. These include candidate virulence factors, alternative metabolic capacities, several prophages and other new functions--all of which could be targets for surveillance.

Genetic screens in Drosophila have identified p50(cdc37) to be an essential component of the sevenless receptor/mitogen-activated kinase protein (MAPK) signaling pathway, but neither the function nor the target of p50(cdc37) in this pathway has been defined. In this study, we examined the role of p50(cdc37) and its Hsp90 chaperone partner in Raf/Mek/MAPK signaling biochemically. We found that coexpression of wild-type p50(cdc37) with Raf-1 resulted in robust and dose-dependent activation of Raf-1 in Sf9 cells. In addition, p50(cdc37) greatly potentiated v-Src-mediated Raf-1 activation. Moreover, we found that p50(cdc37) is the primary determinant of Hsp90 recruitment to Raf-1. Overexpression of a p50(cdc37) mutant which is unable to recruit Hsp90 into the Raf-1 complex inhibited Raf-1 and MAPK activation by growth factors. Similarly, pretreatment with geldanamycin (GA), an Hsp90-specific inhibitor, prevented both the association of Raf-1 with the p50(cdc37)-Hsp90 heterodimer and Raf-1 kinase activation by serum. Activation of Raf-1 via baculovirus coexpression with oncogenic Src or Ras in Sf9 cells was also strongly inhibited by dominant negative p50(cdc37) or by GA. Thus, formation of a ternary Raf-1-p50(cdc37)-Hsp90 complex is crucial for Raf-1 activity and MAPK pathway signaling. These results provide the first biochemical evidence for the requirement of the p50(cdc37)-Hsp90 complex in protein kinase regulation and for Raf-1 function in particular.

Transient receptor potential (TRP) cation-selective channels are an emerging class of proteins that are involved in a variety of important biological functions including pain transduction, thermosensation, mechanoregulation, and vasorelaxation. Utilizing a bioinformatics approach, we have identified the full-length human TRPM3 (hTRPM3) as a member of the TRP family. Following the identification of the founding member of this family, dTRP, which is from a Drosophila mutant with abnormal visual signal transduction (2), mammalian homologues have been cloned and all of them contain a six-transmembrane domain followed by a TRP motif (XWKFXR). Based on homology, they are divided into three subfamilies: TRPC (canonical), TRPV (vanilloid), and TRPM (melastatin) (3). Members of the TRPM subfamily have unusually long cytoplasmic tails at both ends of the channel domain, and some of the family members have an enzyme domain in the C-terminal region. Despite their similarities of structure, TRPMs have different ion-conductive properties, activation mechanisms, and putative biological functions. TRPM1 is down-regulated in metastatic melanomas (4). TRPM2 is a Ca 2ϩ -permeable channel that contains an ADP-ribose pyrophosphatase domain and can be activated by ADP-ribose, NAD (5, 6), and changes in redox status (7). The TRPM2 gene is mapped to the chromosome region linked to bipolar affective disorder, nonsyndromic hereditary deafness, Knobloch syndrome, and holosencephaly (8). Two splice variants of TRPM4 have been described. TRPM4a is predominantly a Ca 2ϩ -permeable channel (9); whereas TRPM4b conducts monovalent cations upon activation by changes in intracellular Ca 2ϩ (10). TRPM5 is associated with Beckwith-Wiedemann syndrome and a predisposition to neoplasias (11). TRPM7, another bifunctional protein, has kinase activity in addition to its ion channel activity. TRPM7 is regulated by Mg 2ϩ -ATP and/or inositol 1,4,5-disphosphate and is required for cell viability (12-14). TRPM8 is up-regulated in prostate cancer and other malignancies (15). Recently, it has been shown to be a receptor that senses cold stimuli (16,17).Using a bioinformatics approach, we have identified a member of the human TRPM subfamily that we have called hTRPM3, consistent with the unified TRP nomenclature (3). hTRPM3 contains long N and C termini, although it does not contain any additional enzymatic features. hTRPM3 mRNA is expressed primarily in kidney with lower levels in brain, testis, and spinal cord. When expressed in HEK 293 cells, hTRPM3 is co-localized with the plasma membrane and is capable of mediating Ca 2ϩ entry. This hTRPM3-mediated Ca 2ϩ conductance * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.The nucleotide sequence (s)

To understand the mechanism of activation of the IB kinase (IKK) complex in the tumor necrosis factor (TNF) receptor 1 pathway, we examined the possibility that oligomerization of the IKK complex triggered by ligandinduced trimerization of the TNF receptor 1 complex is responsible for activation of the IKKs. Gel filtration analysis of the IKK complex revealed that TNF␣ stimulation induces a large increase in the size of this complex, suggesting oligomerization. Substitution of the Cterminal region of IKK␥, which interacts with RIP, with a truncated DR4 lacking its cytoplasmic death domain, produced a molecule that could induce IKK and NF-B activation in cells in response to TRAIL. Enforced oligomerization of the N terminus of IKK␥ or truncated IKK␣ or IKK␤ lacking their serine-cluster domains can also induce IKK and NF-B activation. These data suggest that IKK␥ functions as a signaling adaptor between the upstream regulators such as RIP and the IKKs and that oligomerization of the IKK complex by upstream regulators is a critical step in activation of this complex.In unstimulated cells, the transcription factor NF-B is sequestered in the cytoplasm through interaction with inhibitory proteins known as IBs (1). Upon stimulation by proinflammatory cytokines like TNF␣, IBs undergo phosphorylation at specific serine residues by kinases known as IB kinases (IKKs).1 Phosphorylation marks IBs for ubiquitination and degradation via the proteosome pathway (2-5). Degradation of IBs allows the liberated NF-B to translocate to the nucleus and activate the transcription of target genes (6).The IB kinase activity is present as a large (700 -900 kDa) complex that includes two kinases designated IKK␣ and IKK␤ (7-11) and a noncatalytic subunit termed IKK␥ (also called NEMO, IKKAP1, or FIP-3) (12-15), whose function in physiologic signaling remains unclear. Genetic and biochemical studies have shown that IKK␥ is essential for IKK activation by at least six different stimuli, including those generated by TNF␣ and interleukin-1 (12,16,17). Additional proteins found to interact with the IKK complex include MEK kinase (MEKK1), NF-B-inducing kinase (NIK), receptor-interacting protein (RIP), and IKK complex-associated protein (15, 18 -23). IKK␣ and IKK␤ share significant sequence homology and contain three identical structural domains, namely a protein kinase domain at their N termini, a leucine zipper (LZ) and a helixloop-helix (HLH) motif at their C termini. IKK␥ does not contain a catalytic domain and is composed of three large ␣-helical regions, including a leucine zipper.Purified recombinant IKK␣ and IKK␤ are both able to phosphorylate IB␣ and IB␤ and can form homo-and heterodimers through their LZ motifs. Mutations interfering with this dimerization abolish kinase activity (11,24). No IKK activity can be elicited in vivo in IKK␥-deficient cells after treatment with TNF␣ or interleukin-1 (12,16,17). Moreover, IKK complexes assembled in vivo in cells expressing a truncated IKK␥ lacking its C-terminal LZ were not responsive to cytokin...