AIMTo establish the methods for determining the activity of sphingosine kinase(SPK) and the content of sphingosine 1-phosphate (S1P) in biological samples.

METHODSThe ECV304 cells were transfected with pcDNA3 vector encoding Flag-labeled SPK gene. The expression of SPK was measured by Western blot assay and the activity of SPK was determined by enzymatic reaction, isotope incorporation and thin-layer chromatography methods. The S1P in biological samples was extracted, digested by alkaline phosphatase and then catalyzed by SPK. The S1P contents were determined according to the amounts of products.

RESULTSSPK gene transfection could enhance the expression and activity of SPK in cells markedly, and the cellular S1P was also increased obviously. HGF stimulation could increase the activity of SPK and cellular S1P in ECV304 cells.

CONCLUSIONMethods for determining the activity of SPK and the content of SPK in biological samples were established.

Cell Line ; Cytophotometry ; Humans ; Isotope Labeling ; Lysophospholipids ; metabolism ; Phosphotransferases (Alcohol Group Acceptor) ; metabolism ; Sphingosine ; analogs & derivatives ; metabolism

Animals ; Cytological Techniques ; methods ; Cytophotometry ; instrumentation ; methods ; Humans ; Pharmacology ; methods ; trends ; Systems Biology ; methods ; Toxicology ; methods ; trends

OBJECTIVE: Using computer image-analyze technique (CIAT) to study changes of geometry parameters in human spleen nuclei and seek a new experimental method to deduce the estimation the postmortem interval (PMI). METHODS: 31 cadavers that known accurate PMI, sampled and smeared respectively every hour within the first 36 hours after death, fixed with cold Carony fixation, stained by Feulgen-van's method, and measured 5 geometry parameters using the image-analyze instrument including Area (A), Mean-Dia (MD), Average Diameter (AD), perimeter (P), Index of density (ID). RESULTS: A, MD, AD and P in the human spleen nuclei have no correlation with the PMI. But ID rose regularly with the prolongation of PMI in 36 hours. There was a definite correlation between ID and the PMI, r=0.983, linear regression equation with PMI (hours) as the dependent variable was calculated for ID. CONCLUSION Geometry parameter ID was proved to be preferable indexes for estimation of PMI in 36 hours.
Adolescent ; Adult ; Cell Nucleus/ultrastructure* ; Cytophotometry/methods* ; DNA/metabolism* ; Female ; Forensic Pathology ; Humans ; Image Processing, Computer-Assisted/methods* ; Male ; Middle Aged ; Spleen/metabolism* ; Time Factors

OBJECTIVE: To investigate the relationship between the DNA degradation in cells and postmortem interval. METHODS: Tissue pieces of the heart, liver, spleen and kidney of one corpse (male, 54 years old, died of mechanical injury, PMI of 6h) were obtained in 6, 12, 24, 48 h after death, fixed in Carnoy fluid, and then paraffin sections were prepared, stained with Feulgen and analyzed by Image analysis technology (IAT). Meanwhile the single-cell suspension of tissues of the man was prepared and inspected by FCM after PI stained. RESULTS: The amount of DNA of in heart, liver and kidney of human had a repid degraded in first 6 hours after death, that in the spleen showed a better relationship between DNA degradation and PMI. The results was verified by FCM and IAT. CONCLUSION The degradation of DNA of and human tissues shows a well relationship with PMI, especially in spleen. It is useful in estimation of PMI.
Cell Nucleus/metabolism* ; DNA/metabolism* ; Flow Cytometry ; Forensic Medicine/methods* ; Humans ; Image Processing, Computer-Assisted/methods* ; Kidney/metabolism* ; Liver/metabolism* ; Male ; Middle Aged ; Myocardium/metabolism* ; Postmortem Changes ; Spleen/metabolism* ; Staining and Labeling/methods* ; Time Factors

Determination of postmortem interval (PMI) is one of the most valuable subjects in forensic practice. It, however, is often very difficult to accurately determine the PMI in daily practice. Forensic DNA technology has recently been used to estimate the PMI. It has certain advantage to traditional methods. This article reviews this technology with respect to its invention, development, advantage, disadvantage, and potential future applications with emphasis on correlation of DNA degradation and PMI.
Animals ; Bone Marrow Cells/metabolism* ; Cell Nucleus/metabolism* ; DNA/metabolism* ; Flow Cytometry ; Forensic Medicine/methods* ; Hepatocytes/metabolism* ; Humans ; Image Processing, Computer-Assisted/methods* ; Myocardium/metabolism* ; Postmortem Changes ; Spleen/metabolism* ; Time Factors

Estimation of postmortem interval (PMI) is one of the difficult problems in forensic medicine. With the development of molecular biological techniques, DNA quantification methods were widely applied in estimating PMI. The postmortem degradation of DNA in different tissues and organs was discussed in this article and the recent DNA quantitative techniques being used for estimating PMI were reviewed. These techniques included single cell gel electrophoresis, Feulgen staining image analysis, flow cytometry.
Animals ; Cell Nucleus/metabolism* ; Comet Assay/methods* ; DNA/analysis* ; DNA Degradation, Necrotic ; Flow Cytometry ; Forensic Pathology ; Humans ; Image Processing, Computer-Assisted ; Kidney/pathology* ; Postmortem Changes ; Rosaniline Dyes ; Spleen/pathology* ; Temperature ; Time Factors

DNA, Neoplasm ; Humans ; Image Cytometry ; Pleural Effusion, Malignant ; diagnosis

No abstract available.
Flow Cytometry* ; Microscopy*

No abstract available.
Flow Cytometry* ; Microscopy*

No abstract available.
DNA* ; Flow Cytometry*