OBJECTIVE: Enzymatic fingerprinting of key enzymes of glucose metabolism is a valuable analysis tool in cell physiological phenotyping of plant samples. Yet, a similar approach for mammalian cell line samples is missing. In this study, we applied semi-high throughput enzyme activity assays that were originally designed for plant samples and tested their feasibility in extracts of six frequently used mammalian cell lines (Caco2, HaCaT, C2C12, HEK293, HepG2 and INS-1E). RESULTS: Enzyme activities for aldolase, hexokinase, glucose-6-phosphate dehydrogenase, phosphoglucoisomerase, phosphoglucomutase, phosphofructokinase could be detected in samples of one or more mammalian cell lines. We characterized effects of sample dilution, assay temperature and repeated freeze-thaw cycles causing potential biases. After careful selection of experimental parameters, the presented semi-high throughput methods could be established as useful tool for physiological phenotyping of cultured mammalian cells.
- MeSH
- aldolasa metabolismus MeSH
- buněčné linie MeSH
- buňky Hep G2 MeSH
- Caco-2 buňky MeSH
- enzymatické testy metody MeSH
- fenotyp MeSH
- fosfofruktokinasy metabolismus MeSH
- fosfoglukomutasa metabolismus MeSH
- fosfotransferasy s alkoholovou skupinou jako akceptorem metabolismus MeSH
- glukosa-6-fosfátdehydrogenasa metabolismus MeSH
- glukosa metabolismus MeSH
- glykolýza * MeSH
- HEK293 buňky MeSH
- hexokinasa metabolismus MeSH
- lidé MeSH
- metabolismus sacharidů * MeSH
- myši MeSH
- nádorové buněčné linie MeSH
- pilotní projekty MeSH
- studie proveditelnosti MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
OBJECTIVE: To develop response criteria for adult dermatomyositis (DM) and polymyositis (PM). METHODS: Expert surveys, logistic regression, and conjoint analysis were used to develop 287 definitions using core set measures. Myositis experts rated greater improvement among multiple pairwise scenarios in conjoint analysis surveys, where different levels of improvement in 2 core set measures were presented. The PAPRIKA (Potentially All Pairwise Rankings of All Possible Alternatives) method determined the relative weights of core set measures and conjoint analysis definitions. The performance characteristics of the definitions were evaluated on patient profiles using expert consensus (gold standard) and were validated using data from a clinical trial. The nominal group technique was used to reach consensus. RESULTS: Consensus was reached for a conjoint analysis-based continuous model using absolute percent change in core set measures (physician, patient, and extramuscular global activity, muscle strength, Health Assessment Questionnaire, and muscle enzyme levels). A total improvement score (range 0-100), determined by summing scores for each core set measure, was based on improvement in and relative weight of each core set measure. Thresholds for minimal, moderate, and major improvement were ≥20, ≥40, and ≥60 points in the total improvement score. The same criteria were chosen for juvenile DM, with different improvement thresholds. Sensitivity and specificity in DM/PM patient cohorts were 85% and 92%, 90% and 96%, and 92% and 98% for minimal, moderate, and major improvement, respectively. Definitions were validated in the clinical trial analysis for differentiating the physician rating of improvement (P < 0.001). CONCLUSION: The response criteria for adult DM/PM consisted of the conjoint analysis model based on absolute percent change in 6 core set measures, with thresholds for minimal, moderate, and major improvement.
- MeSH
- alanintransaminasa metabolismus MeSH
- aldolasa metabolismus MeSH
- antirevmatika terapeutické užití MeSH
- aspartátaminotransferasy metabolismus MeSH
- dermatomyozitida farmakoterapie metabolismus patofyziologie MeSH
- hodnocení výsledků péče pacientem MeSH
- hodnocení výsledků zdravotní péče MeSH
- kreatinkinasa metabolismus MeSH
- L-laktátdehydrogenasa metabolismus MeSH
- lidé MeSH
- logistické modely MeSH
- polymyozitida farmakoterapie metabolismus patofyziologie MeSH
- průzkumy a dotazníky MeSH
- revmatologie MeSH
- společnosti lékařské MeSH
- svalová síla MeSH
- výsledek terapie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- konsensus - konference MeSH
- Geografické názvy
- Evropa MeSH
- Spojené státy americké MeSH
OBJECTIVE: To develop response criteria for juvenile dermatomyositis (DM). METHODS: We analyzed the performance of 312 definitions that used core set measures from either the International Myositis Assessment and Clinical Studies Group (IMACS) or the Paediatric Rheumatology International Trials Organisation (PRINTO) and were derived from natural history data and a conjoint analysis survey. They were further validated using data from the PRINTO trial of prednisone alone compared to prednisone with methotrexate or cyclosporine and the Rituximab in Myositis (RIM) trial. At a consensus conference, experts considered 14 top candidate criteria based on their performance characteristics and clinical face validity, using nominal group technique. RESULTS: Consensus was reached for a conjoint analysis-based continuous model with a total improvement score of 0-100, using absolute percent change in core set measures of minimal (≥30), moderate (≥45), and major (≥70) improvement. The same criteria were chosen for adult DM/polymyositis, with differing thresholds for improvement. The sensitivity and specificity were 89% and 91-98% for minimal improvement, 92-94% and 94-99% for moderate improvement, and 91-98% and 85-86% for major improvement, respectively, in juvenile DM patient cohorts using the IMACS and PRINTO core set measures. These criteria were validated in the PRINTO trial for differentiating between treatment arms for minimal and moderate improvement (P = 0.009-0.057) and in the RIM trial for significantly differentiating the physician's rating for improvement (P < 0.006). CONCLUSION: The response criteria for juvenile DM consisted of a conjoint analysis-based model using a continuous improvement score based on absolute percent change in core set measures, with thresholds for minimal, moderate, and major improvement.
- MeSH
- alanintransaminasa metabolismus MeSH
- aldolasa metabolismus MeSH
- antirevmatika terapeutické užití MeSH
- aspartátaminotransferasy metabolismus MeSH
- cyklosporin terapeutické užití MeSH
- dermatomyozitida farmakoterapie metabolismus patofyziologie MeSH
- dítě MeSH
- glukokortikoidy terapeutické užití MeSH
- hodnocení výsledků péče pacientem MeSH
- hodnocení výsledků zdravotní péče MeSH
- kreatinkinasa metabolismus MeSH
- L-laktátdehydrogenasa metabolismus MeSH
- lidé MeSH
- logistické modely MeSH
- methotrexát terapeutické užití MeSH
- mladiství MeSH
- prednison terapeutické užití MeSH
- průzkumy a dotazníky MeSH
- reprodukovatelnost výsledků MeSH
- revmatologie MeSH
- rituximab terapeutické užití MeSH
- společnosti lékařské MeSH
- svalová síla MeSH
- výsledek terapie MeSH
- Check Tag
- dítě MeSH
- lidé MeSH
- mladiství MeSH
- Publikační typ
- časopisecké články MeSH
- konsensus - konference MeSH
- Geografické názvy
- Evropa MeSH
- Spojené státy americké MeSH
- MeSH
- aldolasa genetika MeSH
- dítě MeSH
- fruktosa normy škodlivé účinky MeSH
- lidé MeSH
- ovoce normy škodlivé účinky MeSH
- vrozené poruchy metabolismu fruktózy * dietoterapie krev moč patofyziologie MeSH
- Check Tag
- dítě MeSH
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- kazuistiky MeSH
Sturgeon and paddlefish (Acipenseriformes), the source of roe consumed as caviar, are a unique and commercially valuable group of ancient fishes. In this study, comparative proteomics was used to analyze protein profiles of spermatozoa from five sturgeon species and one paddlefish: Siberian sturgeon (Acipenser baerii), sterlet (A. ruthenus), Russian sturgeon (A. gueldenstaedtii), starry sturgeon (A. stellatus), beluga (Huso huso), and Mississippi paddlefish (Polyodon spathula). Protein profiles of spermatozoa were determined by isoelectric focusing and two-dimensional electrophoresis (2-DE) high-resolution gels. The peptides, previously selected by 2-DE analysis as potentially species-specific, were obtained by "in-gel" tryptic digestion, followed by matrix-associated laser desorption/ionization time-of-flight/mass spectrometry (MALDI-TOF/MS). Among the 23 protein spots selected, 14 were identified as isoforms of enolase B present in all species, but with different isoelectric points or molecular mass. Exceptions were A. ruthenus and H. huso, species with a close phylogenetic relationship. Glycerol-3-phosphate dehydrogenase was detected exclusively in P. spathula. Phosphoglycerate kinase was detected only in A. ruthenus and H. huso, and 3 additional proteins (fructose bisphosphate aldolase A-2, glycogen phosphorylase type IV and glyceraldehyde-3-phosphate dehydrogenase) were found exclusively in A. gueldenstaedtii and H. huso. This study points to the application of proteomics for differential characterization and comparative studies of acipenseriform species at the molecular level.
- MeSH
- 2D gelová elektroforéza metody MeSH
- aldolasa genetika metabolismus MeSH
- biologické markery metabolismus MeSH
- druhová specificita MeSH
- fosfoglycerátkinasa genetika metabolismus MeSH
- fosfopyruváthydratasa genetika metabolismus MeSH
- glycerolfosfátdehydrogenasa genetika metabolismus MeSH
- isoelektrická fokusace metody MeSH
- izoenzymy genetika metabolismus MeSH
- peptidové mapování metody MeSH
- peptidy genetika metabolismus MeSH
- proteomika metody MeSH
- ryby klasifikace genetika metabolismus MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice metody MeSH
- spermie metabolismus MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
- Geografické názvy
- Mississippi MeSH
- Sibiř MeSH
We have shown previously that iron deprivation significantly stimulates the uptake of non-transferrin ferric iron from ferric citrate by erythroleukemia K562 cells and that this stimulation depends on protein synthesis. However, we have not detected increased expression of any known iron transport protein (Kovar J. et al. (2006) Blood Cells Mol Dis 37:95-99). Therefore, in order to identify membrane proteins of K562 cells with increased expression under iron deprivation, we employed the isolation of membrane proteins by two-phase partitioning system, protein separation by high-resolution 2D electrophoresis, computer differential analysis, and tandem mass spectrometry. Employing these techniques we identified two proteins with statistically significant upregulation, i.e., aldolase A (ALDA) and voltage-dependent anion channel 2 (VDAC2). The upregulation of aldolase A and VDAC2 in K562 cells under iron deprivation was also confirmed by western blot analysis. This is the first time when the control of aldolase A and VDAC2 levels by iron status of the cell is demonstrated.
- MeSH
- alanintransaminasa krev MeSH
- aldolasa krev MeSH
- alkalická fosfatasa krev MeSH
- aspartátaminotransferasy krev MeSH
- cholinesterasy krev MeSH
- fosfatasy krev MeSH
- kreatinkinasa krev MeSH
- kyselá fosfatasa krev MeSH
- L-laktátdehydrogenasa krev MeSH
- lidé MeSH
- nádory kostí enzymologie MeSH
- osteomyelitida enzymologie MeSH
- Check Tag
- lidé MeSH
Acta medica Scandinavica, ISSN 0001-3101 suppl. 542
243 s. : il., tab. ; 24 cm
