Identification of macrophage migration inhibitory factor as a potent endothelial cell growth-promoting agent released by ectopic human endometrial cells.

The growth of endometrial cells in ectopic locations (endometriosis) is dependent on the establishment of an adequate blood supply. Neovascularization (angiogenesis) is therefore a vital step toward the progression of this disease. We first revealed the presence of a potent mitogenic activity for human endothelial cells in the culture medium of immortalized human endometriotic cells. The activity, measured by the level of [(3)H]-thymidine incorporation into the DNA of human coronary artery endothelial cells, was then purified by anion exchange high-performance liquid chromatography. Electrophoretic analysis of one of the bioactive fractions that markedly enhanced endothelial cell proliferation showed three distinct bands with apparent molecular masses of 15.8, 12.6, and 6.5 kDa. N-terminal microsequencing of an internal peptide from the 12. 6-kDa protein showed 100% homology with human macrophage migration inhibitory factor (MIF). The protein was positively identified as MIF by Western blot analysis using a specific anti-MIF antibody. Anti-MIF antibody inhibited the bioactivity found in the evaluated fraction and the conditioned medium of primary endometriotic cell cultures, and commercial recombinant human MIF displayed a high mitogenic activity for endothelial cells. Our findings reveal that MIF is released by endometriotic cells and acts as a potent mitogenic factor for human endothelial cells in vitro. This may have a considerable interest, in view of the crucial role of angiogenesis in ectopic endometrial cell growth and activity and in numerous tissues undergoing dynamic physiological changes, such as human endometrium.

High throughput and economical mutation detection and RFLP analysis using a minimethod for DNA preparation from whole blood and acrylamide gel electrophoresis.

We report a simple, rapid, and high throughput method which allows the simultaneous processing of multiple whole blood samples for routine DNA purification and analysis. The method is based on a microscale DNA preparation and digestion using minimal amounts of reagents and handling. The amount of material necessary for a Southern blot analysis (5-7 micrograms) is obtained from 200 microliters of whole blood. All steps involved in DNA preparation and restriction digestion are processed in a single 1.5-ml Eppendorf tube. DNA preparation is performed using a salting out procedure with a proteinase K digestion step but no phenol/chloroform extraction. Restricted fragments are separated by electrophoresis through polyacrylamide slab gels followed by electrotransfer to nylon membranes. By varying the electrophoresis parameters (V/cm or duration), fragments of interest up to 12 kb length can be separated with high resolution. At least 80 samples can be processed at once per DNA preparation, and multiples of this number depend on the available equipment. This economical and rapid method allows routine DNA analysis for mutation or RFLP detection to be performed on a large scale which is a mandatory feature in any DNA-based population screening program. In addition, the DNA purified by the minimethod can be used as an economical source for PCR analysis.