Palmitic acid conjugates of poly-L-lysine (PLL-PA) were prepared, and their ability to deliver plasmid DNA into human skin fibroblasts was evaluated in vitro. The conjugates were capable of condensing a 4.7 kb plasmid DNA into 50-200 nm particles (mean +/- SD = 112 +/- 34 nm), which were slightly smaller than the particles formed by PLL (mean +/- SD = 126 +/- 51 nm). Both PLL and PLL-PA were readily taken up by the cells, but PLL-PA delivered the plasmid DNA into a higher proportion of cells. DNA delivery was found to be reduced by endocytosis inhibitor Brefeldin A, suggesting an active mechanism of particle uptake. Using enhanced green fluorescent protein (EGFP) as a reporter gene, PLL-PA was found to give the highest number of EGFP-positive cells among several carriers tested, including polyethyleneimine, Lipofectamine-2000, and an adenovirus. Although some carriers gave a higher percentage of EGFP-positive cells than PLL-PA, they were also associated with higher toxicities. We conclude that PLL-PA is a promising gene carrier for non-viral modification of human fibroblasts.

As assessed by incorporation into liposomes and by adsorption to octyl-Sepharose, the integrity of the membrane anchor for the purified tetrameric forms of alkaline phosphatase from human liver and placenta was intact. Any treatment that resulted in a dimeric enzyme precluded incorporation and adsorption. An intact anchor also allowed incorporation into red cell ghosts. The addition of hydrophobic proteins inhibited incorporation into liposomes to varying degrees. Alkaline phosphatase was 100% releasable from liposomes and red cell ghosts by a phospholipase C specific for phosphatidylinositol. There was no appreciable difference in the rates of release of placental and liver alkaline phosphatases, although both were approximately 250 x slower in liposomes and 100 x slower in red cell ghosts than the enzyme's release from a suspension of cultured osteosarcoma cells. Both enzymes were released by phosphatidylinositol phospholipase C as dimers and would not reincorporate or adsorb to octyl-Sepharose. However, the enzyme incorporated, resolubilized by Triton X-100, and cleansed of the detergent by butanol treatment was tetrameric by gradient gel electrophoresis, was hydrophobic, and could reincorporate into fresh liposomes. A monoclonal antibody to liver alkaline phosphatase inhibited the enzyme's incorporation into liposomes, and abolished its release from liposomes and its conversion to dimers by phosphatidylinositol phospholipase C.

No abstract

Apoptotic cell death is widely believed to be the fate of negatively selected cells in the thymus. In this work we have used multiparameter flow cytometry to analyse reductions in DNA content that occur among differentiating human CD3-4-8- multinegative (MN) thymocytes as they acquire CD3/TCR during in vitro culture. DNA content was measured as the intensity of the DNA-binding dye DAPI. The position of the diploid peak was identified by comparison to chicken red blood cells and to unfractionated uncultured thymocytes which have a sharply defined diploid peak. Apoptosis, was defined as a reduction in DNA content. Apoptotic cell death occurred continuously throughout the 7 day culture period and at the latest stages of culture DNA fragmentation was apparent on gels. Although both CD3- and CD3+ progeny became apoptotic, it was more frequent among the CD3+ progeny of the MN thymocytes. Apoptotic progeny included 60-70% CD3+ cells, while progeny remaining diploid were 8-36% CD3+. Fifty five per cent of CD3+ TCR delta 1+ progeny had less than 75% of the diploid DNA content, while for CD3+ TCR delta 1- progeny only 28% were in this category. CD3+ TCR delta 1+ progeny also comprised 66% of the cycling cells at days 6-7 of culture, suggesting a pattern of rapid cell division followed by apoptotic cell death for this subset. A lack of positive selection in culture may trigger apoptosis among the TCR delta 1 progeny. In contrast, TCR alpha beta progeny arising in culture appear to be less susceptible to apoptosis, perhaps due to their lack of CD4 and CD8. The expression of CD45RA and CD45R0 isoforms were assessed on the progeny of MN thymocytes based on their DNA content. Although 30% of apoptotic progeny expressed CD45RA, it was present at relatively low density compared to that on diploid or cycling cells, 55% of which were CD45RAhi. The majority of apoptotic cells expressed neither CD45RA or CD45R0, but were CD45+, indicating the presence of an isoform not detected by our monoclonal antibodies (MoAbs). This is consistent with speculations that apoptotic cell death among thymocytes is preceded by a transition in CD45 isoform expression. These conditions may model early selective events resulting from high avidity TCR engagement that is independent of CD4 and/or CD8.