Dissociation of differentiation and proliferation in the primary induction of cytolytic T lymphocytes by alloantigens

The requirement for DNA synthesis in the induction of cytolytic T lymphocytes (CTL) by alloantigens has been investigated. C57BL/6 splenic T cells purified by passage on nylon wool columns were stimulated in vitro in mixed leukocyte culture (MLC) and assayed for cytotoxicity against 51Cr-labeled target cells. With this system, CTL activity was detectable after 24 hr of MLC and reached high levels after 48 hr. Addition of cytosine arabinoside (ARA-C) or hydroxyurea to such cultures at concentrations that were sufficient to inhibit DNA synthesis by greater than 98% did not reduce CTL activity measured after 24 hr; however, the increase in activity that occurred between 24 and 48 hr in control cultures was strongly reduced (or abolished) by these drugs. Velocity sedimentation analysis of MLC cells activated for 48 hr in the presence of ARA-C further revealed that CTL precursor lymphocytes had enlarged into medium- to large-sized CTL under these conditions. These studies provide direct evidence that the primary induction of CTL by alloantigens can be dissociated into a differentiation step, which occurs within 24 hr in the absence of DNA synthesis and is accompanied by blast transformation, and a subsequent proliferation.     

Effect of benzyl alcohol on lipid bilayers. A comparisons of bilayer systems.

The effect of the small anesthetic molecule, benzyl alcohol, on the structure of various bilayer system has been studied by optical, electrical, and x-ray diffraction techniques. We find that the modifications in bilayer thickness caused by benzyl alcohol differ dramatically for planar (or black lipid) bilayers containing solvent, planar bilayers containing little or no solvent, and vesicular bilayers. Benzyl alcohol increases the thickness of planar bilayers containing n-alkane solvents, yet decreases the thickness of "solvent-free" planar bilayers. The effect of benzyl alcohol on vesicular bilayers below the phase transition temperature also depends on whether solvent is present in the bilayers. Without solvent, gel-state bilayers are reduced in thickness by benzyl alcohol, whereas in the presence of solvent, the thickness is unchanged. Above the phase transition temperature, benzyl alcohol has no measurable effect on vesicular bilayer thickness, whether solvent is present or not. These results indicate that different model membrane systems respond quite differently to a particular anesthetic.     

Restimulation of cytolytic T-lymphocytes in long-term mixed leukocyte cultures. 1. Physical and proliferative characteristics of cytolytic T-lymphocytes restimulated at low cell density.

Some physical and proliferative characteristics of cytolytic thymus-derived lymphocytes (CTL) have been investigated in long-term mixed leukocyte cultures (MLC). Velocity sedimentation analysis of MLC cells restimulated by homologous alloantigens at low responding cell density indicated that a shift from large cycling CTL to much smaller (probably non-cycling) CTL occurred between the third and sixth day in secondary cultures. This change in physical characteristics as a function of growth phase was accompanied by a parallel change in the responsiveness of secondary MLC cells to a further alloantigenic stimulus; restimulated Day 3 secondary cells gave rise to a transient CTL response (peaking after 2–3 days) whereas the response of restimulated Day 6 secondary cells increased for 4 days and reached much higher peak levels. Repeated stimulation of MLC cells under the latter conditions led to dramatic increases in both CTL activity and viable cell number. In particular, four sequential restimulations at 7-day intervals resulted in a calculated absolute increase of approximately 500,000-fold in both parameters. Cells derived from such extensive proliferation retained their original lytic specificity and were uniquely T cells as determined by surface markers. These results raise interesting questions regarding the extent and regulation of CTL proliferation in MLC.     

Aryl hydrocarbon hydroxylase activity in the fungus Cunninghamella bainieri: evidence for the presence of cytochrome P-450.

The aryl hydrocarbon hydroxylase (AHH) enzyme from the fungus Cunninghamella bainieri has been characterized. It is NADPH dependent and exhibits a pH optimum near 7.8. It is inhibited by CO, SKF 525-A, and metyrapone, but cyanide shows no inhibitory effect. These data, together with the pattern of inhibition and stimulation shown by metal ions, suggest that the fungal AHH activity is due to a cytochrome P-450. About 25% of the hydroxylase activity remains in the supernatant while the remainder precipitates after centrifugation at 100,00g for 2.5 h. The 100,000g supernatant was further fractionated by (NH₄)₂SO₄ precipitation. A NADPH-dependent cytochrome c reductase is concentrated mainly in the 100,000g supernatant, and a cytochrome c oxidase is present mainly in the 100,000g pellet. The cytochrome c reductase is essential for AHH activity as shown by the inhibition of AHH activity with cytochrome c and dichloroindophenol. Solubilization of a portion of the 100,000g pellet in aqueous digitonin followed by dithionite reduction and addition of CO resulted in the observation of a maximum absorbance at 450 nm characteristic of cytochrome P-450.     

Light-induced glutamate transport in Halobacterium halobium envelope vesicles. I. Kinetics of the light-dependent and the sodium-gradient-dependent uptake.

During illumination Halobacterium halobium cell envelope vesicles accumulate [3H]glutamate by an apparently unidirectional transport system. The driving force for the active transport originates from the light-dependent translocation of protons by bacteriorhodopsin and is due to a transmembrane electrical potential rather than a pH difference. Transport of glutamate against high concentration gradients is also achieved in the dark, with high outside/inside Na+ gradients. Transport in both cases proceeds with similar kinetics and shows a requirement for Na+ on the outside and for K+ on the inside of the vesicles. The unidirectional nature of glutamate transport seems to be due to the low permeability of the membranes to the anionic glutamate, and to the differential cation requirement of the carrier on the two sides of the membrane for substrate translocation. Thus, glutamate gradients can be collapsed in the dark either by lowering the intravesicle pH (with nigericin, or carbonyl cyanide p-trifluoromethoxyphenylhydrazone plus valinomycin), or by reversing the cation balance across the membranes, i.e., providing NaCl on the inside and KCl on the outside of the vesicles. In contrast to the case of light-dependent glutamate transport, the initial rates of Na+-gradient-dependent transport are not depressed when an opposing diffusion potential is introduced by adding the membrane-permeant cation, triphenylmethylphosphonium bromide. Therefore, it appears that, although the electrical potential must be the primary source of energy for the light-dependent transport, the translocation step itself is electrically neutral.     

Phospholipid exchange between bilayer membrane vesicles.

The turbidity of lipid vesicles, freshly prepared by sonicating purified dimyristoyllecithin (DML) in dilute KCl solutions, was measured as a function of time at various temperatures. A sharp maximum in the rate of increase of turbidity is found just above the crystal:liquid-crystal phase transition temperature (Tm). The initial rate of turbidity increase is first order with respect to DML concentration. Electron and light microscopy reveal large vesicles which are not present before incubation or after incubation at temperatures far from the Tm. When temperature, rather than time, is the independent variable, a sharp drop in turbidity is seen at the Tm. The magnitude of this drop and the temperature at which it occurs were used to measure the rate of lipid transfer between vesicles composed of different lipids. A mixture of DML vesicles and dipalmitoyllecithin (DPL) vesicles exhibits sharp drops in turbidity at 24 and 41 degrees, the corresponding Tm's. With time, the magnitude of the transition at 24 degrees decreases while that which was originally at 41 degrees moves to lower temperatures and increases in magnitude. At equilibrium there is a single transition at 32.5 degrees characteristic of vesicles composed of equimolar DPL and DML. The rate at which equilibrium is approached increases at around 24 degrees and again around 41 degrees. These observations indicate that vesicles are in equilibrium with monomolecular lipid, the concentration of the latter being higher the shorter the lipid acyl group or the smaller the vesicle. DML molecules are therefore lost from small vesicles to large vesicles (DML system) or lost from DML vesicles to DML-DPL vesicles (mixed system). When DML vesicles containing a few percent brain gangliosides were studied, different behavior was observed; the initial rate of increase of turbidity becomes second order in lipid concentration, and the rate constant increases with increasing concentrations of KCl. The kinetic order, coupled with the fact that electrolyte reduces intervesicle electrostatic repulsion, argues that in this situation the mechanism of vesicle growth requires vesicle collision.     

Lipid vesicle-cell interactions. III. Introduction of a new antigenic determinant into erythrocyte membranes

The introduction of a new antigenic determinant, 2,4-dinitrophenyl-aminocaproyl-phosphatidylethanolamine (DNP-Cap-PE), into the surface membranes of intact human erythrocytes is described. Fresh cells were incubated in the presence of liposomes composed of 10% DNP-Cap-PE, 5% stearylamine, 20% lysolecithin, and 65% lecithin. Such liposome-treated erythrocytes are shown to be susceptible to immune lysis by anti-DNP serum in the presence of complement. Uptake of DNP-Cap-PE by erythrocyte membranes is also demonstrated by immunofluorescence using indirect staining with rabbit anti-DNP serum followed by fluroescein-conjugated goat anti-rabbit IgG and by electron microscopy using ferritin-conjugated antibody. Antigen uptake did not occur at low temperatures or from vesicles lacking lysolecithin and stearylamine. Fluorescence microscopy shows that the antigen-antibody complexes are free to diffuse over the cell surface, eventually coalescing into a single area on the cell membrane. Electron microscopy suggests that a substantial proportion of the lipid antigen is incorporated by fusion of vesicles with the cell membrane. There are indications that vesicle treatment causes a small proportion of cells to invaginate.     

Characterization of effector lymphocytes associated with immunity to murine sarcoma virus (MSV) induced tumors. I. Physical properties of cytolytic T lymphocytes generated in vitro and of their immediate progenitors.

Cytolytic T lymphocytes (CTL) were generated in secondary mixed leukocyte-tumor cell cultures (MLTC) with syngeneic RB1-5 tumor cells as stimulating cells and with responding spleen cells from regressor mice that had rejected a murine sarcoma virus (MSV)-induced tumor. CTL precursor cells were found to be exclusively of thymic origin and non-T cells were apparently not required for CTL generation. When the size variations of CTL from syngeneic MLTC were analyzed by velocity sedimentation it appeared that a transition from small precursor cells to larger effector cells occurred during the first 5 days in culture; this change in cell size was then followed by a shift toward small-sized cells. Furthermore, the CTL generated in syngeneic MLTC in the MSV tumor immune system were compared with those CTL obtained in allogeneic mixed leukocyte cultures (MLC) and were shown to exhibit fundamental similarities.     

Studies of insulin release and rat pancreatic islet metabolism with diastereomers of D-glucose

Studies of insulin release with diastereomers and other analogues of D-glucose demonstrated that only sugars which undergo oxidation to CO₂ stimulate insulin release by the pancreatic islet. None of the non-metabolizable diastereomers of glucose stimulated insulin release in the presence of a substimulatory concentration of glucose for fuel. Although 5.5 mM glucose formed 77% as much CO₂ as 16.7 mM mannose and twice that of 16.7 mM fructose, 5.5 mM glucose did not stimulate insulin release whereas 16.7 mM mannose and fructose did stimulate insulin release. These results indicate that the important stimulus for glucose-induced insulin release involves metabolism of glucose, but that the stimulus does not involve solely a fuel function of glucose.