Mycobacterium avium subspecies paratuberculosis (MAP), the causative agent of Johne disease in cattle and other ruminants, is proposed to be at least one of the causes of Crohn disease in humans. MAP and Mycobacterium avium subspecies avium, a closely related opportunistic environmental bacterium, share 95% of their genes and exhibit homologies of more than 99% between these genes. The identification of molecules specific for MAP is essential for understanding its pathogenicity and for development of useful diagnostic tools. The application of gas chromatography, mass spectrometry, and nuclear magnetic resonance led to the structural identification Mycobacterium avium subspecies paratuberculosis (MAP)2 and Mycobacterium avium subspecies avium (MAA) are closely related subspecies. They share 95% of their genes and exhibit homologies of more than 99% between these genes (1). MAP can be distinguished from MAA by the presence of IS900; however, there is a very similar repetitive element (IS901) in MAA. MAP also differs from MAA by its growth characteristics: 1) MAP grows more slowly, with a generation time of 22-26 h compared with 10 -12 h for MAA, and 2) MAP requires the siderophore Mycobactin J for growth (MAA can synthesize this siderophore) (2, 3). MAP also lacks the antigenic glycopeptidolipids that serve as the basis for serotyping the M. avium complex, although rough colony variants of MAA are also devoid of these molecules. However, none of these differences helps to explain why MAP occupies a specific biological niche distinct from MAA. MAP is the causative agent of Johne disease in cattle and other ruminants and is proposed to be at least one of the causes of Crohn disease in humans (2). Both diseases are presented as a chronic inflammation of the bowel. MAA is an environmental bacterium found in soil and water that causes opportunistic infections in humans, including lymphadenitis in children, "hot tub lung" in the general population, and systemic infection in immunocompromised individuals (4). MAP-specific traits have recently been defined by sequencing the genomes of strains of each subspecies (1) and by identifying subspecies-specific genes (5, 6), but more investigations are necessary especially in the fields of proteomics, lipidomics, glycomics, and gene regulation. Lipidomics is a promising area of study because members of the family Mycobacteriaceae contain large numbers of complex lipids in their cell wall, and we recently identified several cell envelope and culture filtrate lipids present in MAP strain K-10 but absent from MAA strain 2151.3 Here we report the chemical structure and seroreactivity of Para-LP-01, a major cell wall-associated MAPspecific lipopeptide. EXPERIMENTAL PROCEDURESChemical Reagents-All chemical reagents were of the highest grade from Sigma unless otherwise specified.Bacterial Growth-MAP strain K-10 is a bovine isolate from Nebraska that was provided by V. Kapur (University of Minnesota). MAA strain 2151 is a human sputum isolate (7). They were grown on Middlebrook 7H11 ag...

POSH (Plenty of SH3 domains) binds to activated Rac and promotes apoptosis by acting as a scaffold to assemble a signal transduction pathway leading from Rac to JNK activation. Overexpression of POSH induces apoptosis in a variety of cell types, but apoptosis can be prevented by co-expressing the pro-survival protein kinase Akt. We report here that POSH is a direct substrate for phosphorylation by Akt in vivo and in vitro, and we identify a major site of Akt phosphorylation as serine 304 of POSH, which lies within the Rac-binding domain. We further show that phosphorylation of POSH results in a decreased ability to bind activated Rac, as does phosphomimetic S304D and S304E mutation of POSH. S304D mutant POSH also shows a strongly reduced ability to induce apoptosis. These findings identify a novel mechanism by which Akt promotes cell survival. POSH3 (Plenty of SH3 domains) is a recently discovered proapoptotic protein that appears to be widely expressed in multiple cell types, although at low levels. POSH was first identified as a binding partner of activated Rac and has been shown to act as a scaffolding protein in a kinase cascade signaling pathway that leads to apoptotic cell death (1, 2). In this pathway, Rac activates one of the mixed lineage kinases (MLKs, a group of MAPKKKs), which in turn phosphorylate and activate MKK4 and/or MKK7 (which are MAPKKs) which then phosphorylate and activate c-Jun N-terminal kinases (JNKs, one group of MAPKs) (2). Activated JNKs induce release of cytochrome c from mitochondria and trigger subsequent apoptosis. POSH directly binds Rac, MLK, and another scaffold protein, JIP (JNK-interacting protein), which in turn binds MKK4/7 and JNK, to facilitate this pathway. This multiprotein signaling assembly has been termed PJAC, for POSH-JIP apoptotic complex (3). The role of POSH as a scaffold for this signaling complex appears to be critical; in an apoptotic model involving withdrawal of nerve growth factor from cultures of neuronally differentiated PC12 cells or rat primary sympathetic neurons, apoptosis was dramatically reduced by pretreatment with POSH short interfering RNA or antisense oligonucleotides (4).The decision of a cell to undergo apoptosis is not undertaken lightly; apoptotic pathways are subject to regulation at many levels, and cells must integrate a variety of pro-apoptotic and anti-apoptotic signals. Regulation of the PJAC apoptotic pathway appears to follow this pattern, with multiple regulatory interactions. One form of regulation of PJAC may lie in the expression level of POSH protein within cells. POSH is maintained within healthy cells at very low levels, at least in part by POSH auto-ubiquitination and proteasomal degradation (5). Increasing the level of POSH protein by microinjection or ectopic expression induces apoptosis in a variety of cell types (1, 2, 4, 6 -9).Another potential regulator of the POSH-JIP apoptotic complex appears to be the pro-survival kinase Akt, also known as protein kinase B. Three closely related Akt genes exist (AKT1-3) that have b...

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