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1.  Species selective diazirine positioning in tag-free photoactive quorum sensing probes 
The synthesis and comparison of activities of ‘tag-free’ probes with diazirines in varying positions is described. Remarkable differences in effects on P. aeruginosa and on human bronchial epithelial cells were observed, supporting efforts to isolate and identify receptors for N-acyl homoserine lactones.
PMCID: PMC3723129  PMID: 23702727
2.  The bacterial quorum-sensing signal molecule N-3-oxo-dodecanoyl-L-homoserine lactone reciprocally modulates pro- and anti-inflammatory cytokines in activated macrophages 
The bacterial N-3-oxo-dodecanoyl-L-homoserine lactone (C12) has critical roles in both inter-bacterial communication and inter-kingdom signaling. The ability of C12 to down-regulate production of the key pro-inflammatory cytokine tumor necrosis factor α (TNFα) in stimulated macrophages was suggested to contribute to the establishment of chronic infections by opportunistic Gram-negative bacteria, such as Pseudomonas aeruginosa. We show that in contrast to TNFα suppression, C12 amplifies production of the major anti-inflammatory cytokine interleukin-10 (IL-10) in lipopolysaccharide (LPS)-stimulated murine RAW264.7 macrophages as well as peritoneal macrophages. Furthermore, C12 increases IL-10 mRNA level and IL-10 promoter reporter activity in LPS-stimulated RAW264.7 macrophages, indicating that C12 modulates IL-10 expression at the transcriptional level. Finally, C12 substantially potentiated LPS-stimulated NFκB DNA-binding level, and prolonged p38 MAP kinase phosphorylation in the RAW264.7 macrophages, suggesting that increased transcriptional activity of NFκB and/or p38-activated transcription factors serves to up-regulate IL-10 production in macrophages exposed to both LPS and C12. These findings reveal another part of the complex array of host transitions through which opportunistic bacteria down-regulate immune responses in order to flourish and establish a chronic infection.
PMCID: PMC3691282  PMID: 23720811
3.  Immunomodulation and the quorum sensing molecule 3-oxo-C12-homoserine lactone: The importance of chemical scaffolding for probe development† 
As a guide for chemical probe design, focused analogue synthetic studies were undertaken upon the lactone ring of 3-oxo-C12-homoserine lactone. We have concluded that hydrolytic instability of the heterocyclic ring is pivotal for its ability to modulate immune signaling and probe preparation was aligned with these findings.
PMCID: PMC3566768  PMID: 23328974
4.  The use of small molecule probes to study spatially separated stimulus-induced signaling pathways 
Simultaneous activation of signaling pathways requires dynamic assembly of higher-order protein complexes at the cytoplasmic domains of membrane-associated receptors in a stimulus-specific manner. Here, using the paradigm of cellular activation through cytokine and innate immune receptors, we demonstrate the proof-of-principle application of small molecule probes for the dissection of receptor-proximal signaling processes, such as activation of the transcription factor NF- B and the protein kinase p38.
PMCID: PMC3288742  PMID: 22300658
NF-kB pathway; Toll-like-receptor; Selective inhibitor; Innate immunity; Tumor Necrosis Factor
5.  Synthesis of “clickable” acylhomoserine lactone quorum sensing probes: unanticipated effects on mammalian cell activation 
Alkynyl- and azido-tagged 3-oxo-C12-acylhomoserine lactone probes have been synthesized to examine their potential utility as probes for discovering the mammalian protein target of the Pseudomonas aeruginosa autoinducer, 3-oxo-C12-acylhomoserine lactone. Although such substitutions are commonly believed to be quite conservative, from these studies, we have uncovered a drastic difference in activity between the alkynyl- and azido-modified compounds, and provide an example where such structural modification has proved to be much less than conservative.
PMCID: PMC3081916  PMID: 21190852
Acylhomoserine lactones; Click chemistry; Pseudomonas aeruginosa; Quorum sensing
6.  Defining the Mode of Action of Tetramic Acid Antibacterials Derived from Pseudomonas aeruginosa Quorum Sensing Signals 
Journal of the American Chemical Society  2009;131(40):14473-14479.
In Nature, bacteria rarely exist as single, isolated entities, but rather as communities comprised of many other species including higher host organisms. To survive in these competitive environments, microorganisms have developed elaborate tactics such as the formation of biofilms and the production of antimicrobial toxins. Recently, it was discovered that the Gram-negative bacterium Pseudomonas aeruginosa, an opportunistic human pathogen, produces an antibiotic, 3-(1-hydroxydecylidene)-5-(2-hydroxyethyl)pyrrolidine-2,4-dione (C12-TA), derived from one of its quorum sensing molecules. Here, we present a comprehensive study of the expanded spectrum of C12-TA antibacterial activity against microbial competitors encountered by P. aeruginosa in Nature as well as significant human pathogens. The mechanism of action of C12-TA was also elucidated and C12-TA was found to dissipate both the membrane potential and pH gradient of Gram-positive bacteria, correlating well with cell death. Notably, in stark contrast to its parent molecule 3-oxo-dodecanoyl homoserine lactone (3-oxo-C12-HSL), neither activation of cellular stress pathways nor cytotoxicity was observed in human cells treated with C12-TA. Our results suggest that the QS machinery of P. aeruginosa has evolved for a dual-function, both to signal others of the same species, and also to defend against both host immunity and competing bacteria. Because of the broad-spectrum antibacterial activity, established mode of action, lack of rapid resistance development, and tolerance by human cells, the C12-TA scaffold may also serve as a new lead compound for the development of antimicrobial therapeutics.
PMCID: PMC2760024  PMID: 19807189
7.  Regulation of the MEF2 Family of Transcription Factors by p38† 
Molecular and Cellular Biology  1999;19(1):21-30.
Members of the MEF2 family of transcription factors bind as homo- and heterodimers to the MEF2 site found in the promoter regions of numerous muscle-specific, growth- or stress-induced genes. We showed previously that the transactivation activity of MEF2C is stimulated by p38 mitogen-activated protein (MAP) kinase. In this study, we examined the potential role of the p38 MAP kinase pathway in regulating the other MEF2 family members. We found that MEF2A, but not MEF2B or MEF2D, is a substrate for p38. Among the four p38 group members, p38 is the most potent kinase for MEF2A. Threonines 312 and 319 within the transcription activation domain of MEF2A are the regulatory sites phosphorylated by p38. Phosphorylation of MEF2A in a MEF2A-MEF2D heterodimer enhances MEF2-dependent gene expression. These results demonstrate that the MAP kinase signaling pathway can discriminate between different MEF2 isoforms and can regulate MEF2-dependent genes through posttranslational activation of preexisting MEF2 protein.
PMCID: PMC83862  PMID: 9858528

Results 1-7 (7)