Investigation into the role of phosphatidylserine in modifying the susceptibility of human lymphocytes to secretory phospholipase A2 using cells deficient in the expression of scramblase

Normal human lymphocytes resisted the hydrolytic action of secretory phospholipase A₂ but became susceptible to the enzyme following treatment with a calcium ionophore, ionomycin. To test the hypothesis that this susceptibility requires exposure of the anionic lipid phosphatidylserine on the external face of the cell membrane, experiments were repeated with a human Burkitt's lymphoma cell line (Raji cells). In contrast to normal lymphocytes or S49 mouse lymphoma cells, most of the Raji cells (83%) did not translocate phosphatidylserine to the cell surface upon treatment with ionomycin. Those few that did display exposed phosphatidylserine were hydrolyzed immediately upon addition of phospholipase A₂. Interestingly, the remaining cells were also completely susceptible to the enzyme but were hydrolyzed at a slower rate and after a latency of about 100 s. In contradistinction to the defect in phosphatidylserine translocation, Raji cells did display other physical membrane changes upon ionomycin treatment that may be relevant to hydrolysis by phospholipase A₂. These changes were detected by merocyanine 540 and trimethylammonium diphenylhexatriene fluorescence and were common among normal lymphocytes, S49 cells, and Raji cells. The levels of these latter effects corresponded well with the relative rates of hydrolysis among the three cell lines. These results suggested that while phosphatidylserine enhances the rate of cell membrane hydrolysis by secretory phospholipase A₂, it is not an absolute requirement. Other physical properties such as membrane order contribute to the level of membrane susceptibility to the enzyme independent of phosphatidylserine.     

Kasugamycin resistant mutants of Bacillus stearothermophilus lacking the enzyme for the methylation of two adjacent adenosines in 16S ribosomal RNA

Several mutants of B. stearothermophilus have been isolated that are resistant to the antibiotic kasugamycin. One of these is shown to lack dimethylation of two adjacent adenosines in the 16S ribosomal RNA. All mutants that were analyzed biochemically lack the enzyme that is able to methylate this site. Ribosomal sensitivity and resistance to kasugamycin in B. stearothermophilus is therefore, like in E. coli, closely connected with dimethylation of the adenosines.     

Featured Article: Isolation,characterization, and cultivation of human hepatocytes and non-parenchymal liver cells

Primary human hepatocytes (PHH) are considered to be the gold standard for in vitro testing of xenobiotic metabolism and hepatotoxicity. However, PHH cultivation in 2D mono-cultures leads to dedifferentiation and a loss of function. It is well known that hepatic non-parenchymal cells (NPC), such as Kupffer cells (KC), liver endothelial cells (LEC), and hepatic stellate cells (HSC), play a central role in the maintenance of PHH functions. The aims of the present study were to establish a protocol for the simultaneous isolation of human PHH and NPC from the same tissue specimen and to test their suitability for in vitro co-culture. Human PHH and NPC were isolated from tissue obtained by partial liver resection by a two-step EDTA/collagenase perfusion technique. The obtained cell fractions were purified by Percoll density gradient centrifugation. KC, LEC, and HSC contained in the NPC fraction were separated using specific adherence properties and magnetic activated cell sorting (MACS®). Identified NPC revealed a yield of 1.9 × 10⁶ KC, 2.7 × 10⁵ LEC and 4.7 × 10⁵ HSC per gram liver tissue, showing viabilities >90%. Characterization of these NPC showed that all populations went through an activation process, which influenced the cell fate. The activation of KC strongly depended on the tissue quality and donor anamnesis. KC became activated in culture in association with a loss of viability within 4–5 days. LEC lost specific features during culture, while HSC went through a transformation process into myofibroblasts. The testing of different culture conditions for HSC demonstrated that they can attenuate, but not prevent dedifferentiation in vitro. In conclusion, the method described allows the isolation and separation of PHH and NPC in high quality and quantity from the same donor.