Pseudomonas aeruginosa delivers the toxin ExoU to eukaryotic cells via a type III secretion system. Intoxication with ExoU is associated with lung injury, bacterial dissemination and sepsis in animal model and human infections. To search for ExoU targets in a genetically tractable system, we used controlled expression of the toxin in Saccharomyces cerevisiae. ExoU was cytotoxic for yeast and caused a vacuolar fragmentation phenotype. Inhibitors of human calcium-independent (iPLA 2 ) and cytosolic phospholipase A 2 (cPLA 2 ) lipase activity reduce the cytotoxicity of ExoU. The catalytic domains of patatin, iPLA 2 and cPLA 2 align or are similar to ExoU sequences. Sitespeci®c mutagenesis of predicted catalytic residues (ExoUS142A or ExoUD344A) eliminated toxicity. ExoU expression in yeast resulted in an accumulation of free palmitic acid, changes in the phospholipid pro®les and reduction of radiolabeled neutral lipids. ExoUS142A and ExoUD344A expressed in yeast failed to release palmitic acid. Recombinant ExoU demonstrated lipase activity in vitro, but only in the presence of a yeast extract. From these data we conclude that ExoU is a lipase that requires activation or modi®cation by eukaryotic factors.

No abstract

The prominence of vasoactive intestinal peptide (VIP) in rodent thymic neurons suggested that this potent mediator of T cell functions may alter developmental responses of thymocytes to T cell receptor (TCR) -dependent stimulation. CD4+8+ DPK cells derived from a thymic lymphoma of a TCR transgenic mouse respond to pigeon cytochrome C (PCC) antigen in association with distinct I-E MHC II haplotypes on antigen-presenting cells (APCs) by differentiating into CD4+8- T cells. The specific recognition of VIP by two types of homologous G-protein-coupled receptors (VIPR1 and VIPR2) on DPK cells was attributable predominantly to VIPR1 before and to VIPR2 after exposure to APCs and PCC, as assessed by quantification of the respective mRNAs. PCC-evoked differentiation of DPK cells was enhanced significantly by 1 to 100 nM VIP after 3 to 4 days. The effects of VIP analogs with VIPR type selectivity implied that VIP enhancement of differentiation of DPK cells was mediated principally by VIPR2. Differential reduction in the expression of each type of VIPR by transfection of DPK cells with plasmids encoding the respective antisense mRNAs confirmed the central role of VIPR2 in VIP-enhanced conversion to CD4+8- T cells. The suppression of DPK cell differentiation by inhibitors of adenylyl cyclase and protein kinase A suggested a transductional role for VIP-elicited increases in [cAMP]i. That the changes in frequency of CD4+8+ and CD4+8- DPK cells reflected principally differentiation was supported by the lack of consistent differences between the two subsets in the effects of VIP and VIPR2 agonist on cell number, viability, apoptosis, and proliferation. VIP may be one endogenous mediator that explains the unique thymic microenvironment for topographically specific development of T cells.

The major immunoregulatory effects of vasoactive intestinal peptide (VIP) are mediated by structurally distinct type I (VIPR1) and II (VIPR2) G protein-associated receptors on many different types of immune cells. VIP is released in functionally relevant concentrations during many immunologic and inflammatory responses. Mast cells (VIPR1), macrophages (VIPR1 and VIPR2), B cells, and T cells (VIPR1, VIPR2, or VIPR1 and VIPR2) recognize and respond to VIP in patterns that are controlled by the relative levels of expression of VIPR1 and VIPR2. VIPR2 transduces human T-cell chemotaxis, expression of matrix metalloproteinases (MMPs) 2 and 9 and consequently basement membrane and connective tissue transmigration, while signaling suppression of proliferation and cytokine production. In contrast, VIPR1 fails to transduce T-cell chemotaxis but mediates suppression of chemotaxis and MMP expression elicited by some cytokines and chemokines. The relative representation of each type of VIPR, which is presumed to be under cytokine control, thus may determine T-cell responses to VIP and other immune mediators in tissue compartments innervated by VIPergic nerves.

Engrailed (En) protein expression in neurons of the mesothoracic and metathoracic ganglia of the adult grasshopper, Schistocerca americana, was examined by immunohistochemistry. Each neuromere had a dorsally located cluster of En-positive neurons within the dorsal unpaired median (DUM) group, comprising one cluster in the mesothoracic ganglion (T2) and four clusters in the metathoracic ganglion, one for each component neuromere (T3, A1, A2, A3). Ventrally, En-positive neurons occurred in the posterior one-third of each neuromere. In T2 and T3, three ventral groups of neurons were labeled bilaterally. In the abdominal neuromeres, many fewer ventral neurons were En-positive. These also were bilaterally symmetrical, but did not occur in patterns that allowed assignment of homology with the T2 and T3 groups. Altogether, En-positive neurons comprised roughly 10% of the ganglionic populations. In the bilateral groups, as in the DUM groups, En expression was restricted to interneurons, consistent with the suggestion that En expression contributes to some aspect of interneuronal phenotype. En-positive neurons in the DUM groups also expressed gamma-aminobutyric acid (GABA) immunoreactivity. Further study showed that all neurons in one En-positive bilateral group and some neurons in another bilateral group were GABA immunoreactive, but that neurons in a third bilateral group were En-positive only. Additionally, several discrete clusters of neurons were GABA-immunoreactive but En-negative. A provisional morphological scheme is presented, which relates the several neuronal clusters to their likely neuroblasts of origin, as a basis for further research into the composition of neuronal lineages. J. Comp. Neurol. 440:85-96, 2001.

We report that the homeodomain protein Engrailed (En) is differentially expressed by neuronal type. Expression was examined within identified midline neurons in T3, A1, and A2 neuromeres of the adult grasshopper by using immunohistochemistry. All save a few neurons in the adult dorsal unpaired median (DUM) group arise embryonically from a single precursor, the median neuroblast. DUM neurons are efferent neurons, local interneurons, or intersegmental interneurons, recognizable as such by their distinct morphologies and neurotransmitter phenotypes. We show that interneurons are En-positive, whereas efferents are En-negative. In the T3 DUM group, the 70 or so interneurons contained cytoplasmic immunoreactivity for gamma-aminobutyric acid (GABA) and glutamate decarboxylase. In double-labeling experiments, all GABA-immunoreactive neurons were also En-positive, and all En-positive neurons contained GABA immunoreactivity. In complementary experiments, the 20 or so efferents in the T3 DUM group, which are octopaminergic, were selectively labeled with a histological marker and then processed to reveal En immunoreactivity. No efferents in the group were En-positive. The abdominal DUM groups contain fewer neurons, but the same dichotomy of labeling was found. The En pattern is established during embryogenesis, with the type-specific pattern apparent by stage 90% of development, the earliest stage examined here. The differential expression of En in the embryo and its continued expression in the adult nervous system suggest a role in the development and maintenance of neuronal phenotype. Morphological differences between efferents and interneurons are discussed in light of a hypothesis that En mediates differential expression of cell adhesion or cell-affinity molecules.

We report that the homeodomain protein Engrailed (En) is differentially expressed by neuronal type. Expression was examined within identified midline neurons in T3, A1, and A2 neuromeres of the adult grasshopper by using immunohistochemistry. All save a few neurons in the adult dorsal unpaired median (DUM) group arise embryonically from a single precursor, the median neuroblast. DUM neurons are efferent neurons, local interneurons, or intersegmental interneurons, recognizable as such by their distinct morphologies and neurotransmitter phenotypes. We show that interneurons are En-positive, whereas efferents are En-negative. In the T3 DUM group, the 70 or so interneurons contained cytoplasmic immunoreactivity for gamma-aminobutyric acid (GABA) and glutamate decarboxylase. In double-labeling experiments, all GABA-immunoreactive neurons were also En-positive, and all En-positive neurons contained GABA immunoreactivity. In complementary experiments, the 20 or so efferents in the T3 DUM group, which are octopaminergic, were selectively labeled with a histological marker and then processed to reveal En immunoreactivity. No efferents in the group were En-positive. The abdominal DUM groups contain fewer neurons, but the same dichotomy of labeling was found. The En pattern is established during embryogenesis, with the type-specific pattern apparent by stage 90% of development, the earliest stage examined here. The differential expression of En in the embryo and its continued expression in the adult nervous system suggest a role in the development and maintenance of neuronal phenotype. Morphological differences between efferents and interneurons are discussed in light of a hypothesis that En mediates differential expression of cell adhesion or cell-affinity molecules.

The prominence of vasoactive intestinal peptide (VIP) in rodent thymic neurons suggested that this potent mediator of T cell functions may alter developmental responses of thymocytes to T cell receptor (TCR) -dependent stimulation. CD4 / 8 / DPK cells derived from a thymic lymphoma of a TCR transgenic mouse respond to pigeon cytochrome C (PCC) antigen in association with distinct I-E MHC II haplotypes on antigen-presenting cells (APCs) by differentiating into CD4 / 8 0 T cells. The specific recognition of VIP by two types of homologous G-protein-coupled receptors (VIPR1 and VIPR2) on DPK cells was attributable predominantly to VIPR1 before and to VIPR2 after exposure to APCs and PCC, as assessed by quantification of the respective mRNAs. PCC-evoked differentiation of DPK cells was enhanced significantly by 1 to 100 nM VIP after 3 to 4 days. The effects of VIP analogs with VIPR type selectivity implied that VIP enhancement of differentiation of DPK cells was mediated principally by VIPR2. Differential reduction in the expression of each type of VIPR by transfection of DPK cells with plasmids encoding the respective antisense mRNAs confirmed the central role of VIPR2 in VIP-enhanced conversion to CD4 / 8 0 T cells. The suppression of DPK cell differentiation by inhibitors of adenylyl cyclase and protein kinase A suggested a transductional role for VIP-elicited increases in [cAMP] i . That the changes in frequency of CD4 / 8 / and CD4 / 8 0 DPK cells reflected principally differentiation was supported by the lack of consistent differences between the two subsets in the effects of VIP and VIPR2 agonist on cell number, viability, apoptosis, and proliferation. VIP may be one endogenous mediator that explains the unique thymic microenvironment for topographically specific development of T cells.