SummaryWe have analysed the capacity of the 11 phase-variable, opacity-associated (Opa) proteins encoded by Neisseria gonorrhoeae MS11 to mediate traversal across polarized monolayers of the human colonic carcinoma T84 cell line. Gonococci expressing either the heparan sulphate proteoglycan (HSPG) binding Opa protein (Opa 50 ) or no Opa protein (Opa ¹ ) did not interact with the apical pole of T84 monolayers, whereas the 10 variant Opa proteins previously shown to bind CD66 receptors were found to mediate efficient gonococcal adherence and transepithelial traversal. Consistent with this, T84 cells were shown by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunoblotting to co-express CD66a (BGP), CD66c (NCA) and CD66e (CEA). The recruitment of CD66 receptors by Opa-expressing gonococci indicates their involvement in mediating adherence to the surface of T84 cells, and these bacterial interactions could be inhibited completely using polyclonal antibodies cross-reacting with all of the CD66 proteins co-expressed on T84 cells. Consistent results were obtained when Opa proteins were expressed in Escherichia coli, suggesting that the Opa-CD66 interaction is sufficient to mediate bacterial traversal. Transcytosis of Opa-expressing N. gonorrhoeae or E. coli did not disrupt the barrier function of infected monolayers, as indicated by a sustained transepithelial electrical resistance (TEER) throughout the course of infection, and confocal laser scanning and electron microscopy both suggest a transcellular rather than a paracellular route of traversal across the monolayers. Parallels between the results seen here and previous work done with organ cultures confirm that T84 monolayers provide a valid model for studying neisserial interactions with the mucosal surface, and suggest that CD66 receptors contribute to this process in vivo.

The sesquiterpene lactone helenalin is a potent antiinflammatory drug whose molecular mechanism of action remains unclear despite numerous investigations. We have previously shown that helenalin and other sesquiterpene lactones selectively inhibit activation of the transcription factor NF-B, a central mediator of the human immune response. These drugs must target a central step in NF-B pathway, since they inhibit NF-B induction by four different stimuli. It has previously been reported that sesquiterpene lactones exert their effect by inhibiting degradation of IB, the inhibitory subunit of NF-B. These data contradicted our report that IB is not detectable in helenalin-treated, ocadaic acid-stimulated cells. Here we use confocal laser scanning microscopy to demonstrate the presence of IBreleased, nuclear NF-B in helenalin-treated, tumor necrosis factor-␣ stimulated cells. These data show that neither IB degradation nor NF-B nuclear translocation are inhibited by helenalin. Rather, we provide evidence that helenalin selectively alkylates the p65 subunit of NF-B. This sesquiterpene lactone is the first anti-inflammatory agent shown to exert its effect by directly modifying NF-B.

The clinical course of Cystic Fibrosis is characterized by recurrent pulmonary infections which ultimately lead to death by respiratory failure. The most common CF causing mutation, deltaF508-CFTR, produces an incorrectly folded protein, which accumulates within the endoplasmic reticulum. However, the molecular mechanism by which the deltaF508-CFTR protein facilitates pulmonary infection and inflammation remains unclear. Here we show that the expression of deltaF508-CFTR causes a constitutive activation of the pro-inflammatory transcription factor NF-kappaB by eliciting an ER stress reaction, the ER-overload response. This endogenous NF-kappaB activation stimulates the transcription of pro-inflammatory cytokines thereby commencing an inflammatory cascade within the CF lung.

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Cytohesins are a family of highly homologous guanine nucleotide exchange factors (GEFs) that act on ADP-ribosylation factors (ARFs). The small ARF-GEFs are involved in integrin signaling, actin cytoskeleton remodeling, and vesicle transport. Here, we selected and applied a specific inhibitor for ARF nucleotide-binding site opener (ARNO)͞cytohesin-2, an RNA aptamer that clearly discriminates between cytohesin-1 and cytohesin-2. This reagent bound to an N-terminal segment of cytohesin-2 and did not inhibit ARF-GEF function in vitro. When transfected into HeLa cells, it persisted for at least 6 h without requiring stabilization. Its effect in vivo was to down-regulate gene expression mediated through the serumresponse element and knockdown mitogen-activated protein kinase activation, indicating that cytohesin-2 acts by means of mitogen-activated protein kinase signaling. We conclude that the N-terminal coiled-coil and parts of the Sec7 domain of cytohesin-2 are required for serum-mediated transcriptional activation in nonimmune cells, whereas cytohesin-1 is not. Our results indicate that intramer technology can be used not only for assigning novel biological functions to proteins or protein domains but also to prove nonredundancy of highly homologous proteins.