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
- Fructose-Bisphosphate Aldolase metabolism MeSH
- Cell Line MeSH
- Hep G2 Cells MeSH
- Caco-2 Cells MeSH
- Enzyme Assays methods MeSH
- Phenotype MeSH
- Phosphofructokinases metabolism MeSH
- Phosphoglucomutase metabolism MeSH
- Phosphotransferases (Alcohol Group Acceptor) metabolism MeSH
- Glucosephosphate Dehydrogenase metabolism MeSH
- Glucose metabolism MeSH
- Glycolysis * MeSH
- HEK293 Cells MeSH
- Hexokinase metabolism MeSH
- Humans MeSH
- Carbohydrate Metabolism * MeSH
- Mice MeSH
- Cell Line, Tumor MeSH
- Pilot Projects MeSH
- Feasibility Studies MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article 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
- Alanine Transaminase metabolism MeSH
- Fructose-Bisphosphate Aldolase metabolism MeSH
- Antirheumatic Agents therapeutic use MeSH
- Aspartate Aminotransferases metabolism MeSH
- Dermatomyositis drug therapy metabolism physiopathology MeSH
- Patient Reported Outcome Measures MeSH
- Outcome Assessment, Health Care MeSH
- Creatine Kinase metabolism MeSH
- L-Lactate Dehydrogenase metabolism MeSH
- Humans MeSH
- Logistic Models MeSH
- Polymyositis drug therapy metabolism physiopathology MeSH
- Surveys and Questionnaires MeSH
- Rheumatology MeSH
- Societies, Medical MeSH
- Muscle Strength MeSH
- Treatment Outcome MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Consensus Development Conference MeSH
- Geographicals
- Europe MeSH
- United States 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
- Alanine Transaminase metabolism MeSH
- Fructose-Bisphosphate Aldolase metabolism MeSH
- Antirheumatic Agents therapeutic use MeSH
- Aspartate Aminotransferases metabolism MeSH
- Cyclosporine therapeutic use MeSH
- Dermatomyositis drug therapy metabolism physiopathology MeSH
- Child MeSH
- Glucocorticoids therapeutic use MeSH
- Patient Reported Outcome Measures MeSH
- Outcome Assessment, Health Care MeSH
- Creatine Kinase metabolism MeSH
- L-Lactate Dehydrogenase metabolism MeSH
- Humans MeSH
- Logistic Models MeSH
- Methotrexate therapeutic use MeSH
- Adolescent MeSH
- Prednisone therapeutic use MeSH
- Surveys and Questionnaires MeSH
- Reproducibility of Results MeSH
- Rheumatology MeSH
- Rituximab therapeutic use MeSH
- Societies, Medical MeSH
- Muscle Strength MeSH
- Treatment Outcome MeSH
- Check Tag
- Child MeSH
- Humans MeSH
- Adolescent MeSH
- Publication type
- Journal Article MeSH
- Consensus Development Conference MeSH
- Geographicals
- Europe MeSH
- United States MeSH
- MeSH
- Fructose-Bisphosphate Aldolase genetics MeSH
- Child MeSH
- Fructose standards adverse effects MeSH
- Humans MeSH
- Fruit standards adverse effects MeSH
- Fructose Metabolism, Inborn Errors * diet therapy blood urine physiopathology MeSH
- Check Tag
- Child MeSH
- Humans MeSH
- Male MeSH
- Publication type
- Case Reports 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
- Electrophoresis, Gel, Two-Dimensional methods MeSH
- Fructose-Bisphosphate Aldolase genetics metabolism MeSH
- Biomarkers metabolism MeSH
- Species Specificity MeSH
- Phosphoglycerate Kinase genetics metabolism MeSH
- Phosphopyruvate Hydratase genetics metabolism MeSH
- Glycerolphosphate Dehydrogenase genetics metabolism MeSH
- Isoelectric Focusing methods MeSH
- Isoenzymes genetics metabolism MeSH
- Peptide Mapping methods MeSH
- Peptides genetics metabolism MeSH
- Proteomics methods MeSH
- Fishes classification genetics metabolism MeSH
- Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization methods MeSH
- Spermatozoa metabolism MeSH
- Animals MeSH
- Check Tag
- Male MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Comparative Study MeSH
- Geographicals
- Mississippi MeSH
- Siberia 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
- Fructose-Bisphosphate Aldolase analysis MeSH
- Phosphogluconate Dehydrogenase analysis MeSH
- Clinical Enzyme Tests MeSH
- Humans MeSH
- Uterine Cervical Neoplasms diagnosis enzymology prevention & control MeSH
- Check Tag
- Humans MeSH
- Female MeSH
- Publication type
- English Abstract MeSH
- Journal Article MeSH
- MeSH
- Alanine Transaminase blood MeSH
- Fructose-Bisphosphate Aldolase blood MeSH
- Alkaline Phosphatase blood MeSH
- Aspartate Aminotransferases blood MeSH
- Cholinesterases blood MeSH
- Phosphoric Monoester Hydrolases blood MeSH
- Creatine Kinase blood MeSH
- Acid Phosphatase blood MeSH
- L-Lactate Dehydrogenase blood MeSH
- Humans MeSH
- Bone Neoplasms enzymology MeSH
- Osteomyelitis enzymology MeSH
- Check Tag
- Humans MeSH
- MeSH
- Adenoviridae MeSH
- Fructose-Bisphosphate Aldolase analysis MeSH
- Cytopathogenic Effect, Viral MeSH
- Embryo, Mammalian metabolism MeSH
- Sarcoma, Experimental metabolism MeSH
- Glycolysis MeSH
- Rats MeSH
- Culture Techniques MeSH
- L-Lactate Dehydrogenase analysis MeSH
- Humans MeSH
- Carbohydrate Metabolism MeSH
- Mitosis MeSH
- Cell Transformation, Neoplastic MeSH
- Oncogenic Viruses MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Humans MeSH
- Animals MeSH
