No abstract

Axl receptor tyrosine kinase exists as a transmembrane protein and as a soluble molecule. We show that constitutive and phorbol 12-myristate 13-acetate-induced generation of soluble Axl (sAxl) involves the activity of disintegrin-like metalloproteinase 10 (ADAM10). Spontaneous and inducible Axl cleavage was inhibited by the broad-spectrum metalloproteinase inhibitor GM6001 and by hydroxamate GW280264X, which is capable of blocking ADAM10 and ADAM17. Furthermore, murine fibroblasts deficient in ADAM10 expression exhibited a significant reduction in constitutive and inducible Axl shedding, whereas reconstitution of ADAM10 restored sAxl production, suggesting that ADAM10-mediated proteolysis constitutes a major mechanism for sAxl generation in mice. Partially overlapping 14-amino-acid stretch deletions in the membrane-proximal region of Axl dramatically affected sAxl generation, indicating that these regions are involved in regulating the access of the protease to the cleavage site. Importantly, relatively high circulating levels of sAxl are present in mouse sera in a heterocomplex with Axl ligand Gas6. Conversely, two other family members, Tyro3 and Mer, were not detected in mouse sera and conditioned medium. sAxl is constitutively released by murine primary cells such as dendritic and transformed cell lines. Upon immobilization, sAxl promoted cell migration and induced the phosphorylation of Axl and phosphatidylinositol 3-kinase. Thus, ADAM10-mediated generation of sAxl might play an important role in diverse biological processes.Receptor tyrosine kinases (RTKs) play fundamental roles in diverse cell functions, including proliferation, differentiation, survival, migration, and metabolism (16). Axl RTK (also known as Ark, Ufo, and Tyro7) is the prototype of a family of transmembrane receptors, which also includes Tyro3 (also known as Sky, Brt, Etk, Tif, Dtk, and Rse) and Mer (c-Eyk, Nyk, and Tyro12) (34,44,64). They share a distinct molecular structure characterized by two immunoglobulin-like motifs and two fibronectin type III repeats in their extracellular domain and a cytoplasmic domain that contains a conserved catalytic kinase region (34, 44). Axl, Tyro3, and Mer are variably expressed in neural, lymphoid, vascular, and reproductive tissues and in different primary cells and tumor cell lines (11,41,42). Mutant mice that lack these three receptors have a defective phagocytic clearance of apoptotic cells and impaired spermatogenesis (41) and develop a severe lymphoproliferative disorder accompanied by broad-spectrum autoimmunity (42).A common heterophilic ligand for these RTK family members is Gas6, a vitamin K-dependent protein that is widely secreted by most tissues, including the lungs, intestine, and vascular endothelium (43). Gas6 is the product of growth arrest-specific gene 6, which was initially cloned from serumstarved fibroblasts and shares about 44% sequence identity and similar domain organization with protein S, a negative regulator of blood coagulation (48). Recent studies indicate that the Gas6/A...

Natural isolates of Bacillus subtilis are often difficult to transform due to their low genetic competence levels. Here we describe two methods that stimulate natural transformation. The first method uses plasmid pGSP12, which expresses the competence transcription factor ComK and stimulates competence development about 100-fold. The second method stimulates Campbell-type recombination of DNA ligation mixtures in B. subtilis by the addition of polyethylene glycol. We employed these novel methods to study the regulation of the synthetases for the lipopeptide antibiotics mycosubtilin (myc) and surfactin (srfA) in B. subtilis strain ATCC 6633. By means of lacZ reporter fusions, it was shown that the expression of srfA is >100 times lower in strain ATCC 6633 than in the laboratory strain B. subtilis 168. Expression of the myc operon was highest in rich medium, whereas srfA expression reached maximal levels in minimal medium. Further genetic analyses showed that the srfA operon is mainly regulated by the response regulator ComA, while the myc operon is primarily regulated by the transition-state regulator AbrB. Although there is in vitro evidence for a synergistic activity of mycosubtilin and surfactin, the expression of both lipopeptide antibiotics is clearly not coordinated.

No abstract

Interleukin 15 (IL-15) is a pleiotropic cytokine that is hardly detectable in biological fluids. Here, we show that IL-15 forms functional heterocomplexes with soluble high affinity IL-15 receptor ␣ (IL-15R␣) chain in mouse serum and cell-conditioned medium, which prevents IL-15 detection by ELISA. We also demonstrate that two soluble IL-15R␣ (sIL-15R␣) sushi domain isoforms are generated through a novel alternative splicing mechanism within the IL-15R␣ gene. These isoforms potentiate IL-15 action by promoting the IL-15-mediated proliferation of the CTLL cell line and interferon ␥ production by murine NK cells, which suggests a role in IL-15 transpresentation. Conversely, a full-length sIL-15R␣ ectodomain released by tumor necrosis factor-␣-converting enzyme (TACE)-dependent proteolysis inhibits IL-15 activity. Thus, a dual mechanism of sIL-15R␣ generation exists in mice, giving rise to polypeptides with distinct properties, which regulate IL-15 function.

No abstract

IL‐15, a T‐cell growth factor, has been shown to be increased in inflammatory bowel disease (IBD). It has been suggested that neutralization of IL‐15 could protect from T cell‐dependent autoimmune inflammation. On the other hand, an anti‐apoptotic effect of IL‐15 has been demonstrated in kidney epithelial cells during nephritis. We therefore tested the role of IL‐15 in two different experimental models of colitis in vivo, and in models of intestinal epithelial cell (IEC) apoptosis in vitro. IL‐15 blockade in chronic dextran sulphate sodium‐induced colitis resulted in aggravation of the disease with a significantly 2.1‐fold increased epithelial damage score compared to controls. TUNEL staining clearly revealed increased apoptosis. IL‐6, TNF and IFN‐γ secretion by mesenteric lymph node cells were increased. In the T cell‐dependent SCID transfer model of colitis IL‐15 neutralization reduced the inflammatory infiltration and proinflammatory cytokine production. Despite that, the intestinal epithelial damage was not reduced. In vitro, IL‐15 pre‐incubation prevented up to 75% of CH11 antibody‐induced apoptosis in SW‐480 cells and reduced caspase‐3 activity. According to this, endogenously produced IL‐15 in chronic colitis does not only act as a proinflammatory cytokine but has at the same time the potential to reduce mucosal damage by preventing IEC apoptosis.

The following should appear as the last sentence of Materials and Methods. "The two primer pair sequences used for determination of the expression levels of IL-15R␣ and IL-15 in human monocytes and macrophages are the following: IL-15R␣RT forward primer, 5Ј-ATG TCC GTG GAA CAC GCA GAC-3Ј; IL-15R␣RT reverse primer, 5Ј-CCT CTT CTC AGT CGT CTT TAG-3Ј; IL-15RT forward primer, 5Ј-TAG CCA GCC CAT ACA AGA TC-3Ј; IL-15RT reverse primer, 5Ј-TCA AGA AGT GTT GAT GAA CAT-3Ј." We regret any inconvenience that this omission may have caused.