Sphingosine phosphate lyase expression is essential for normal development in Caenorhabditis elegans

Sphingolipids are ubiquitous membrane constituents whose metabolites function as signaling molecules in eukaryotic cells. Sphingosine 1-phosphate, a key sphingolipid second messenger, regulates proliferation, motility, invasiveness, and programmed cell death. These effects of sphingosine 1-phosphate and similar phosphorylated sphingoid bases have been observed in organisms as diverse as yeast and humans. Intracellular levels of sphingosine 1-phosphate are tightly regulated by the actions of sphingosine kinase, which is responsible for its synthesis and sphingosine-1-phosphate phosphatase and sphingosine phosphate lyase, the two enzymes responsible for its catabolism. In this study, we describe the cloning of the Caenorhabditis elegans sphingosine phosphate lyase gene along with its functional expression in Saccharomyces cerevisiae. Promoter analysis indicates tissue-specific and developmental regulation of sphingosine phosphate lyase gene expression. Inhibition of C. elegans sphingosine phosphate lyase expression by RNA interference causes accumulation of phosphorylated and unphosphorylated long-chain bases and leads to poor feeding, delayed growth, reproductive abnormalities, and intestinal damage similar to the effects seen with exposure to Bacillus thuringiensis toxin. Our results show that sphingosine phosphate lyase is an essential gene in C. elegans and suggest that the sphingolipid degradative pathway plays a conserved role in regulating animal development.     

Rapid upregulation of endothelial P-selectin expression via reactive oxygen species generation

Endothelial cell ICAM-1 upregulation in response to TNF-alpha is mediated in part by reactive oxygen species (ROS) generated by the endothelial membrane-associated NADPH oxidase and occurs maximally after 4 h as the synthesis of new protein is required. However, thrombin-stimulated P-selectin upregulation is bimodal, the first peak occurring within minutes. We hypothesize that this early peak, which results from the release of preformed P-selectin from within Weibel-Palade bodies, is mediated in part by ROS generated from the endothelial membrane-associated xanthine oxidase. We found that this rapid expression of P-selectin on the surface of endothelial cells was accompanied by qualitatively parallel increases in ROS generation. Both P-selectin expression and ROS generation were inhibited, dose dependently, by the exogenous administration of disparate cell-permeable antioxidants and also by the inhibition of either of the known membrane-associated ROS-generating enzymes NADPH oxidase or xanthine oxidase. This rapid, posttranslational cell signaling response, mediated by ROS generated not only by the classical NADPH oxidase but also by xanthine oxidase, may well represent an important physiological trigger of the microvascular inflammatory response.     

Regulation of cyclic adenosine 3',5'- monophosphate signaling and pulsatile neurosecretion by Gi-coupled plasma membrane estrogen receptors in immortalized gonadotrophin-releasing hormone neurons

Immortalized GnRH neurons (GT1-7) express receptors for estrogen [estrogen receptor-alpha and-13(ERa and ERI3)] and progesterone (progesterone receptor A) and exhibit positive immunostaining for both intracellular and plasma membrane ERs. Exposure of GT1-7 cells to picomolar estradiol concentrations for 5-60 min caused rapid, sustained,and dose-dependent inhibition of cAMP production. In contrast, treatment with nanomolar estradiol concentrations for 60 min increased cAMP production. The inhibitory and stimulatory actions of estradiol on cAMP formation were abolished by the ER antagonist, ICI 182,780. The estradiol-induced inhibition of cAMP production was prevented by treatment with pertussis toxin, consistent with coupling of the plasma membrane ER to an inhibitory G protein. Coimmunoprecipitation studies demonstrated an estradiol-regulated stimulatory interaction between ERa and G,3 that was prevented by the ER antagonist, ICI 182,780. Exposure of perifused GT1-7 cells and hypothalamic neurons to picomolar estradiol levels increased the GnRH peak interval, shortened peak duration, and increased peak amplitude. These findings indicate that occupancy of the plasma membrane-associated ERs expressed in GT1-7 neurons by physio-logical estradiol levels causes activation of a G, protein and modulates cAMP signaling and neuropeptide secretion.     

Multigene DNA prime-boost vaccines for SHIV89.6P

We assessed four prime-boost vaccine regimens with a Gene Gun component for SHIV89.6P in Macaca nemestrina. A dosing experiment using beta-galactosidase plasmid showed that 30 or 45 shots per dose elicited higher titer antibody than smaller doses. For SHIV89.6P, we administered a six-plasmid vaccine capable of producing non-infectious virions in vivo in combination with either vaccinia recombinants or inactivated virus. DNA prime/vaccinia boost, or the reverse, elicited strong immune responses. The SHIV89.6P challenge virus was grown in M. nemestrina peripheral blood mononuclear cells and titered in vivo intrarectally. As has been observed for SHIV89.6P in M. mulatta, the infected M. nemestrina experienced rapid and severe loss of circulating CD4+ T cells. Vaccinated macaques were challenged three weeks after the last boost. DNA prime/vaccina boost or vaccina prime/DNA boost protected 11/12 animals from acute CD4+ T cell depletion and disease, while other regimens were not effective.     

Antiendotoxin activity of protegrin analog IB-367 alone or in combination with piperacillin in different animal models of septic shock

The therapeutic efficacy of protegrin peptide IB-367 was investigated in three rat models of septic shock: (i) rats injected intraperitoneally with 1mg Escherichia coli 0111:B4 lipopolysaccharide, (ii) rats given an intraperitoneal injection of 2 X 10(10) CFU of E. coli ATCC 25922, and (iii) rats in which intra-abdominal sepsis was induced via cecal ligation and puncture. All animals were randomized to receive parenterally isotonic sodium chloride solution, 1mg/kg of IB-367, 60mg/kg piperacillin and 1mg/kg of IB-367 plus 60mg/kg piperacillin. The peptide demonstrated lower level of antimicrobial activity than piperacillin, nevertheless it exhibited the dual properties of antimicrobial and antiendotoxin agent. Finally IB-367 and piperacillin association showed to be the most effective therapeutic approach.     

Sply regulation of sphingolipid signaling molecules is essential for Drosophila development

Sphingosine-1-phosphate is a sphingolipid metabolite that regulates cell proliferation, migration and apoptosis through specific signaling pathways. Sphingosine-1-phosphate lyase catalyzes the conversion of sphingosine-1-phosphate to ethanolamine phosphate and a fatty aldehyde. We report the cloning of the Drosophila sphingosine-1-phosphate lyase gene (Sply) and demonstrate its importance for adult muscle development and integrity, reproduction and larval viability. Sply expression is temporally regulated, with onset of expression during mid-embryogenesis. Sply null mutants accumulate both phosphorylated and unphosphorylated sphingoid bases and exhibit semi-lethality, increased apoptosis in developing embryos, diminished egg-laying, and gross pattern abnormalities in dorsal longitudinal flight muscles. These defects are corrected by restoring Sply expression or by introduction of a suppressor mutation that diminishes sphingolipid synthesis and accumulation of sphingolipid intermediates. This is the first demonstration of novel and complex developmental pathologies directly linked to a disruption of sphingolipid catabolism in metazoans.