This paper describes the synthesis of 14C-labeled glycosphingolipids using the reverse hydrolysis reaction (condensation) of sphingolipid ceramide N-deacylase. It was found that 50-70% of 14C-fatty acids were incorporated into various lyso-glycosphingolipids when a mixture of lyso-glycosphingolipids and fatty acids was incubated at 37 degrees C with 1 mU of the enzyme for 20 h in 1 ml of 25 mM phosphate buffer, pH 6.0-7.0, containing 0-0.1% Triton X-100. The optimum concentration of lyso-glycosphingolipids was 100-400 microM depending on the species of lyso-form when [14C]stearic acid was used at the concentration of 100 microM. Free 14C-fatty acids and lyso-glycosphingolipids were separated from the synthesized 14C-glycosphingolipids by using a Sep-Pak Plus Silica and a Sep-Pak CM or a QMA cartridge, respectively. After treatment of 14C-glycosphingolipids with endoglycoceramidase or sphingolipid ceramide N-deacylase, digestion products were clearly separated from the parent glycosphingolipids on TLC and determined using an image analyzer with a sensitivity 100 times higher than that using non-radiolabeled substrates. Using this method, we found endoglycoceramidase activity in a seaflower, Condylactis sp., for the first time.
Sphingolipid ceramide N-deacylase is an enzyme capable of hydrolyzing the N-acyl linkages of ceramides of various sphingolipids. Recently, it was found that the enzyme catalyzes the reverse hydrolysis reaction in which free fatty acids are condensed to lyso-sphingolipids to produce sphingolipids. This paper describes a simple method for the synthesis of fluorescence-labeled sphingolipids utilizing the condensation reaction of the enzyme. N-TFAc-aminododecanoic acids were efficiently condensed by the enzyme to the lyso-forms of GM1 and sphingomyelin in glycine buffer (pH 10). The reaction products, N-TFAc-amino-GM1 and sphingomyelin, were obtained with overall yields of 60%. The purified products were identified to be omega-amino-GM1 and omega-amino-sphingomyelin, respectively, by TLC and FAB-MS or ESI-LC/MS analysis after removal of the N-TFAc by mild alkaline treatment. NBD-labeled GM1 and sphingomyelin were prepared from omega-amino-GM1 and omega-amino-sphingomyelin by coupling with 4-fluoro-NBD. These fluorescence-labeled substrates, C12-NBD-GM1 and C12-NBD-sphingomyelin, were hydrolyzed by endoglycoceramidase and sphingomyelinase, respectively, to produce NBD-dodecanoylsphingosines, but were resistant to hydrolysis by sphingolipid ceramide N-deacylase. C12-NBD-sphingomyelin was found to be a better substrate than the commercially available C6-NBD-sphingomyelin for the assay of sphingomyelinase from various sources. We also describe a new method to detect GM1-binding proteins using fluorescence-labeled GM1.
Gene silencing using small interfering RNA (siRNA) is not established in avian species. The present study was performed to evaluate RNA interference (RNAi) in the chicken embryo by using a dual fluorescence reporter assay, a plasmid encoding green fluorescent protein (GFP) and a plasmid encoding red fluorescent protein (RFP). The siRNA targeting the GFP mRNA sequence (GFP-siRNA) with both plasmids was introduced into cultured cells and whole embryos by lipofection and microelectroporation, respectively. GFP- and RFP-expressed cells and embryos were observed under fluorescent microscopy and analyzed by flow cytometer, and their mRNAs were analyzed by reverse transcription-PCR (RT-PCR). The strong fluorescence was observed by introducing both plasmids into cells. The intensity of the green fluorescence generated by GFP was greatly suppressed by introducing GFP-siRNA. RT-PCR analysis showed that introducing GFP-siRNA also decreased GFP mRNA levels. In contrast to GFP, the intensity of the red fluorescence generated by RFP and the RFP mRNA levels remained unchanged. In whole embryos, also, introducing GFP-siRNA specifically suppressed GFP expression, and the suppression was maintained for at least 72 h. Consequently, it was concluded that the gene silencing using siRNA is applicable to analyzing the function of genes of interest during avian embryogenesis.
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