ABSTRACT
The zebrafish pronephric tubule consists of proximal and distal segments and a collecting duct. The proximal segment is subdivided into the neck, proximal convoluted tubule (PCT) and proximal straight tubule (PST) segments. The distal segment consists of the distal-early (DE) and distal-late (DL) segments. How the proximal and distal segments develop along the anteroposterior axis is poorly understood. Here we show that knockdown of taz in zebrafish caused shortening and a significant reduction in the number of principal cells of the PST-DE segment, and proximalization of the pronephric tubule in 24 hpf embryos. RA treatment expanded the pronephric proximal domain in normal embryos as in taz morphants, an effect that was further enhanced upon exposure of taz morphants to RA. The early pronephric defects in 24 hpf taz morphants led to the failure of anterior pronephric tubule migration and convolution, and to PCT dilation and cyst formation in older embryos. In situ hybridization showed weak and transient expression of taz at the bud stage in the intermediate mesoderm, the source of pronephric progenitors. The present findings show that Taz is required in the anteroposterior patterning of the pronephric progenitor domain in the intermediate mesoderm, acting in part by regulating RA signaling in the pronephric progenitor field in the intermediate mesoderm.
Subject(s)
Intracellular Signaling Peptides and Proteins/physiology , Kidney Tubules/embryology , Pronephros/embryology , Zebrafish Proteins/physiology , Zebrafish/embryology , Zebrafish/physiology , Animals , Animals, Genetically Modified , Body Patterning/drug effects , Body Patterning/genetics , Body Patterning/physiology , Cell Count , Gene Expression Regulation, Developmental , Gene Knockdown Techniques , Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Intracellular Signaling Peptides and Proteins/genetics , Kidney Tubules/cytology , Kidney Tubules/metabolism , Mesoderm/cytology , Mesoderm/embryology , Mesoderm/metabolism , Pronephros/cytology , Pronephros/metabolism , Transcriptional Coactivator with PDZ-Binding Motif Proteins , Tretinoin/metabolism , Tretinoin/pharmacology , Zebrafish/genetics , Zebrafish Proteins/antagonists & inhibitors , Zebrafish Proteins/geneticsABSTRACT
Allograft rejection remains a major limitation to successful solid organ transplantation. Here, we investigated the biosynthesis and bioactions of the pro-resolving mediators lipoxin A(4) and resolvin E1 in host responses to organ transplantation. In samples obtained during screening bronchoscopy after human lung transplantation, bronchoalveolar lavage fluid levels of lipoxin A(4) were increased in association with the severity of allograft rejection that was graded independently by clinical pathology. Lipoxin A(4) significantly inhibited calcineurin activation in human neutrophils, and lipoxin A(4) stable analogs prevented acute rejection of vascularized cardiac and renal allografts. Transgenic animals expressing human lipoxin A(4) receptors revealed important sites of action in host tissues for lipoxin A(4)'s protective effects. Resolvin E1 displays counter-regulatory actions for leukocytes, in part, via increased lipoxin A(4) biosynthesis, yet RvE1 administered (1µg, iv) to donor (days -1 and 0) and recipient mice (days -1, 0 and +4) was even more potent than a lipoxin stable analog (1µg, iv) in prolonging renal allograft survival (median survival time=74.0 days with RvE1 and 37.5 days with a LXA(4) analog). Together, these results highlight the potential for pro-resolving mediators in prolonging survival of solid organ transplants.
Subject(s)
Eicosapentaenoic Acid/analogs & derivatives , Graft Rejection/physiopathology , Lipoxins/biosynthesis , Animals , Bronchoalveolar Lavage Fluid/chemistry , Cyclosporine/pharmacology , Eicosapentaenoic Acid/biosynthesis , Graft Rejection/metabolism , Graft Survival/drug effects , Heart Transplantation , Humans , Kidney Transplantation , Lung Transplantation , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Transgenic , Neutrophil Activation/drug effects , Receptors, Formyl Peptide/biosynthesis , Receptors, Formyl Peptide/geneticsABSTRACT
Anthrax is caused by the gram-positive, spore-forming bacterium, Bacillus anthracis. Anthrax receptors play a crucial role in the pathogenesis of the anthrax disease. Anthrax toxin receptor ATR/TEM8 VWA domain is responsible for the binding of protective antigen (PA) of B. anthracis, and thus an attractive target for structure-based drug therapies. However, the production of soluble and functional ATR/TEM8 VWA domain currently requires the use of mammalian expression systems. In this work, we expressed the ATR/TEM8 VWA domain as a fusion protein in Escherichia coli. Recombinant ATR/TEM8 VWA domain has been purified to homogeneity, and its identity has been verified by both N-terminal protein microsequencing and mass spectrometry. The purified ATR/TEM8 VWA domain exhibits very high affinity to PA based on BIAcore assay. Moreover, like the domain expressed in mammalian system, the bacterially expressed ATR/TEM8 VWA domain can block cytotoxicity induced by anthrax toxins, suggesting that the bacterially expressed ATR/TEM8 VWA domain is properly folded and fully functional.
