Factors influencing the lipid composition and fluidity of red cell membranes in vitro: production of red cells possessing more than two cholesterols per phospholipid
Abstract
Electron microscopic studies on dried isolated red cell ghosts have been reported to show lesions associated with cell membranes in paroxysmal nocturnal hemoglobinuria (PNH). In this study, carbon-platinum replicas of membranes of freeze-cleaved, partially hydrated PNH red cells and isolated PNH cell ghosts failed to confirm the existence of these abnormalities. This suggests that the previously described lesions are the products of drying artifacts, although they may reflect hidden structural differences between PNH and normal red cell membranes.
Earlier observations of ours have suggested that, under in vitro conditions resembling those under which platelets function haemostatically in vivo, their activation is promoted by the red cells. Seme of the evidence suggested that this is through limited haemolysis with release of ADP. However, newly determined time relationships make this uncertain. Could red cells provide ADP without haemolysis?Crtheir flow properties affect the process more? To analyse the problem, we are determining dependence of red cell deformability on membrane constitution; and release of haemoglobin and adeninenucleotides under different conditions. Ten percent human red cell suspensions in physiological salines flow under constant pressures through 2, 3, 4 and 5 pm micropore filters, the flow rate measured continuously with an electronic balance. Initial flow rates are increased by fluidising agents, eg. ethanol, and decreased by agents with opposite effect. Our results are consistent with the new hypothesis of S.J. Singer on the mode of action of amphipathic agents, such as chlorpromazine, on red cell membranes.
Stationary cultures of Claviceps purpurea (Fr.) Tul. grown 50 days on a liquid medium containing mannitol, ammonium succinate, and salts produced small amounts of ergot alkaloids.Ergometrine, ergometrinine, ergotamine, ergotaminine, ergocornine, ergocorninine, ergocryptine, agroclavine, and penniclavine were identified by paper chromatography of culture extracts. The selection of strains, the composition of the medium, and the length of the incubation period appeared to be important factors influencing the production of alkaloids.
The proportion of spectrin tetramers and dimers in 4 degrees C low ionic strength extracts of red cell membranes of 9 subjects with 4 different variants of hereditary elliptocytosis (HE) and 2 subjects with hereditary spherocytosis (HS) was determined by nondenaturing gel electrophoresis. Such extracts reflect the native oligomeric state of spectrin in the red cell membrane. In two hemolytic HE variants (an unclassified adult with increased thermal sensitivity of red cells and an infant also showing increased thermal sensitivity of red cells), the proportion of dimers was increased, whereas the remaining subjects had values within the control range. Conversion of spectrin tetramers to dimers under isotonic conditions at 37 degrees C, or spectrin dimers to tetramers at 30 degrees C, resulted in a high proportion of dimers in the above two HE variants, as well as in a third variant with probable mild HE and sporadic hemolysis. The mother of the infant with elliptocytosis and increased thermal sensitivity of red cells, although hematologically normal, had an increased proportion of dimers in 4 degrees C low ionic strength extracts of her red cell membranes. These findings reflect an underlying primary or secondary abnormality of spectrin in these subjects that affects the association state of spectrin in the red cell membrane. Their exact relationship to the pathogenesis of the elliptical shape of the red cell, or to the presence of hemolysis, is at present unclear.
Factors Influencing the in vitro Production of Staphylococcal Coagulase