A collaborative exercise on mRNA profiling for the identification of blood was organized by the European DNA Profiling Group (EDNAP). Seven blood samples and one blood dilution series were analyzed by the participating laboratories for the reportedly blood-specific markers HBB, SPTB and PBGD, using different kits, chemistries and instrumentation. The results demonstrate that HBB is expressed abundantly in blood, SPTB moderately and PBGD significantly less. All but one of the 16 participating laboratories were able to successfully isolate and detect RNA from the dried bloodstains even though a majority of the laboratories had no prior experience with RNA. Despite some expected variation in sensitivity between laboratories, the method proved to be reproducible and sensitive using different analysis strategies. The results of this collaborative exercise support the potential use of mRNA profiling as an alternative to conventional serological tests.
Blood Stains , DNA Fingerprinting/methods , RNA, Messenger/blood , White People/genetics , Biomarkers/blood , Cooperative Behavior , DNA Fingerprinting/instrumentation , Electrophoresis, Capillary , Humans , Hydroxymethylbilane Synthase/analysis , Limit of Detection , Nucleic Acid Amplification Techniques , RNA/blood , RNA/isolation & purification , RNA, Messenger/chemistry , Reagent Kits, Diagnostic/statistics & numerical data , Reproducibility of Results , Reverse Transcriptase Polymerase Chain Reaction/methods , Sensitivity and Specificity , Spectrin/analysis , beta-Globins/analysis
Several types of fatty acid-binding proteins are found in mammalian cells. Cultured endothelial cells from bovine aorta were shown to contain exclusively the cardiac-type fatty acid-binding protein (cFABP) with a mean concentration of 90 ng cFABP/mg extract protein. Only small variations were observed from passage to passage. In pulse-chase labeling experiments with L-[35S]methionine, a half-life of 4.0 d was measured for cFABP which is about two times longer than the average half-life of the extracted proteins. These data imply that in aortic endothelial cells cFABP is not subject to short-term regulation. However, addition of clofibric acid to the culture medium led to a shortening of the half-life of cFABP, which was compensated for by an increase in its biosynthesis. The turnover of the bulk of extract proteins remained unchanged when the cells were challenged with clofibric acid.
Aorta/metabolism , Carrier Proteins/metabolism , Clofibric Acid/pharmacology , Endothelium, Vascular/metabolism , Neoplasm Proteins , Animals , Aorta/cytology , Aorta/drug effects , Cattle , Cells, Cultured , Dose-Response Relationship, Drug , Endothelium, Vascular/drug effects , Fatty Acid-Binding Proteins , Half-Life
The mature part of the chloroplast triose phosphate-phosphate translocator was cloned into the yeast expression vector pEVP11. This construct was used to transform cells from both Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe. The chloroplast translocator protein was functionally expressed in the transformed yeast cells and represented about 1-2% of the Sch. pombe cell membrane protein. It was localized to mitochondrial membranes and/or membranes of the rough endoplasmic reticulum. In order to purify the recombinant translocator protein, a sequence encoding a C-terminal tag of six histidine residues was introduced into the corresponding cDNA. The expressed histidine-tagged translocator protein was purified from the transformed yeast cells under nondenaturing conditions to apparent homogeneity by a single-step affinity chromatography using a Ni2+. nitrilotriacetic acid resin. Both the expressed triose phosphate translocator and the recombinant histidine-tagged protein possess substrate specificities identical to those of the authentic chloroplast protein, providing definitive evidence for its identity as the triose phosphate translocator and further disproving its assignment as the receptor for chloroplast protein import. The yeast expression system in combination with the Ni2+. nitrilotriacetic acid chromatography thus provides a valuable tool for the production of purified membrane proteins in a functional state.
