Using NMR spectroscopy, we studied purified, human T lymphocytes in a serum-free medium. Purified cells were entrapped inside agarose beads and induced to proliferate by the mitogens phorbol-12-myristate-13-acetate and ionomycin. T lymphocytes in standard culture and inside agarose beads exhibit comparable viability, and similar extent and kinetics of DNA synthesis and interleukin-2 secretion. 31P-NMR revealed decreased phosphomonoester and increased phosphodiester content in cells stimulated for two days or longer. 13C-glucose utilization and 13C-lactate production rates showed that 85% of the utilized glucose was converted to lactate. 1H-NMR spectra of the perfusing media indicated that lactate was also produced from substrates other than glucose or glycogen. Glucose accounted for 25% of the lactate produced by quiescent cells, and for 67% of lactate production by stimulated cells. Glycolysis was enhanced 6-fold within the first 2 hours following stimulation, and 15-fold by 48 or 96 h. Aerobic lactate production was increased 3-fold by 48 h, with only a minor enhancement during the first 12 h of stimulation. Our results indicate a shift from mostly aerobic to mostly anaerobic lactate production in T lymphocytes within the first 90 min of the G0 to G1 transition during cell cycle progression.

A technique for the entrapment of the unicellular algae Dunaliella salina in agarose beads and their perfusion during NMR measurements is presented. The trapped cells maintained their ability to proliferate under normal growth conditions, and remained viable and stable under steady-state conditions for long periods during NMR measurements. Following osmotic shock in the dark, prominent changes were observed in the intracellular level of ATP and polyphosphates, but little to no changes in the intracellular pH or orthoposphate content. When cells were subjected to hyperosmotic shock, the ATP level decreased. The content of NMR-visible polyphosphates decreased as well, presumably due to the production of longer, NMR-invisible structures. Following hypoosmotic shock, the ATP content increased and longer polyphosphates were broken down to shorter, more mobile polymers.Dunaliella (Volvocales, Chlorophyceae) is a unicellular, motile green alga, which lacks a rigid cell wall. Dunaliella has the capacity to adapt to a wide range of salt concentrations (0.1 -5.5 M NaCl), adjusting to the extracellular osmotic pressure by accumulating glycerol as an osmolyte and compatible solute. At constant salinity, the turnover rate of the glycerol pool is relatively slow [l]. When subjected to a hypoosmotic or hyperosmotic shock, the cells react within seconds like osmometers, swelling or shrinking, respectively, due to very rapid water fluxes. This is followed by a metabolic phase, which lasts about two hours, during which the cell carries out massive glycerol synthesis or elimination [2] in order to regain its original volume. It was established that the main immediate carbon source for glycerol production in the light (and the only one in the dark), and the end product of the glycerol elimination process is starch [3, 41. A cycle of glycerol metabolism has been proposed [5, 61, but the initial signal triggering glycerol metabolism and the control points of glycerol -starch interconversion are not yet resolved.NMR techniques were recently employed in studies aimed at understanding the mechanism of osmoregulation and its control. Such techniques monitor intracellular components in vivo, non-invasively and in real time. Living Dunaliella cells were studied using 31P, I3C and 23Na NMR [7-131. In vivo NMR techniques require very high cell densities for obtaining a good signal-to-noise ratio within a reasonable time. Means must therefore be developed to maintain such high cell densities under conditions which closely simulate normal growth conditions. This can be achieved by trapping of the cells and continuous perfusion with fresh medium of a controlled composition. Perfusion also permits the study of the response of the cells to changes in the extracellular environment in real time, without removing the cells from the cavity of the NMR .magnet.

Polylysine induced rapid aggregation of large unilamellar vesicles composed of phosphatidylcholine-cardiolipin (1:1 molar ratio) but not their fusion. Application of the terbium-dipicolinic acid fusion assay showed that addition of polylysine at nanomolar concentrations enabled a significant lowering of the Ca2+ threshold concentration for vesicle fusion from 9 to 1 mM. Analysis of the kinetics of fusion with a mass-action kinetic model showed that polylysine enhanced significantly the rate of aggregation but affected only slightly the rate of fusion per se. Maximal enhancement of overall fusion rates occurred at a charge ratio (polylysine/cardiolipin) of about 0.5. At larger polylysine concentrations, e.g., at charge ratios greater than 3, polylysine inhibited vesicle fusion.

The Intracellular Na' concentration in the halotolerant alga Dunaliella salina was measured in intact cells by 2'Na-NMR spectroscopy, utilizing the dysprosium tripolyphosphate complex as a sodium shift reagent, and was found to be 88 ± 28 millimolar. Intracellular sodium ion content and intracellular volume were the same, within the experimental error, in cells adapted to grow in media containing between 0.1 and 4.0 molar NaCl. These values assume extracellular and intracellular NMR visibilities of the 'Na nuclei of 100 and 40%, respectively. The relaxation rate of intracellular sodium was enhanced with increasing salinity of the growth medium, in parallel to the intracellular osmosity due to the presence of glycerol, indicating that Na' ions and glycerol are codistribbuted within the cell volume.Members ofthe genus Dunaliella (division Chlorophycophyta, order Volvocales, family Polyblepharidaceae) have the capacity to tolerate and adapt to a very wide range of salt concentrations (0.1-5.5 M NaCl). This is achieved through the ability of the algae to survive the initial osmotic stress and then adjust their intracellular solute content to the new level required for the maintenance of a similar cell volume. Glycerol has been established to be the major osmoregulator and compatible solute in