Mitochondrial membrane permeabilization during the apoptotic process.

Apoptosis may be viewed as a triphasic process. During the pre-mitochondrial initiation phase, very different pro-apoptotic signal transduction or damage pathways can be activated. These pathways then converge on the mitochondrion, where they cause the permeabilization of the inner and/or outer membranes with consequent release of soluble intermembrane proteins into the cytosol. The process of mitochondrial membrane permeabilization would constitute the decision/effector phase of the apoptotic process. During the post-mitochondrial degradation phase downstream caspases and nucleases are activated and the cell acquires an apoptotic morphology. Recently, a number of different second messengers (calcium, ceramide derivatives, nitric oxide, reactive oxygen species) and pro-apoptotic proteins (Bax, Bak, Bid, and caspases) have been found to directly compromise the barrier function of mitochondrial membranes, when added to isolated mitochondria. The effects of several among these agents are mediated at least in part via the permeability transition pore complex (PTPC), a composite channel in which members of the Bcl-2 family interact with sessile transmembrane proteins such as the adenine nucleotide translocator. These findings suggest that the PTPC may constitute a pharmacological target for chemotherapy and cytoprotection.

Concerning the separation of mammalian cells in immobilized metal ion affinity partitioning systems: a matter of selectivity.

The effect of introducing an immobilized metal ion ligand in the lower phase of the PEG/Dextran system was studied on the erythrocytes and lymphocytes partition. The ligand in the lower phase was added as an insoluble form [Sepharose-IDA-M(II)] with or without a ligand in the upper phase. We first checked that the addition of the insoluble ligand in the system did not affect the phase volume and settling, and also that Sepharose-IDA-M(II) partitioned strictly in the lower phase. Then we studied the partition of cells with various concentrations of ligand in the lower and upper phases. We clearly demonstrate here that the partition in immobilized metal ion affinity partitioning (IMAP) systems is correlated with the affinity between the cell surface and the ligand. Cells are attracted to the ligand-containing phase. This fact is important not only for the greater understanding of IMAP, but could also for the separation of some types of cells.

Study of human cord blood lymphocytes by immobilized metal ion affinity partitioning.

The potential of immobilized metal ion affinity partitioning (IMAP) using dextran-PEG+PEG-IDA-M(II) systems to separate mononuclear cells from cord blood has been evaluated. The distribution of B cells, T cells, monocytes and hematopoietic stem cells between PEG and dextran phases was determined by flow cytometry with fluorochrome-labelled specific antibodies. Comparing these values with the post-Ficoll repartition resulted in the determination of enrichment factors, for each subpopulation, in the different phases. We were able to distinguish the partition pattern of B cells, T cells, monocytes and stem cells in different IMAP systems. Their partition was affected by the nature and the concentration of the metal used, but no specificity in distribution for the subpopulations was found.