Quantifying Biomass Changes of Single CD8+ T Cells during Antigen Specific Cytotoxicity

Existing approaches that quantify cytotoxic T cell responses rely on bulk or surrogate measurements which impede the direct identification of single activated T cells of interest. Single cell microscopy or flow cytometry methodologies typically rely on fluorescent labeling, which limits applicability to primary cells such as human derived T lymphocytes. Here, we introduce a quantitative method to track single T lymphocyte mediated cytotoxic events within a mixed population of cells using live cell interferometry (LCI), a label-free microscopy technique that maintains cell viability. LCI quantifies the mass distribution within individual cells by measuring the phase shift caused by the interaction of light with intracellular biomass. Using LCI, we imaged cytotoxic T cells killing cognate target cells. In addition to a characteristic target cell mass decrease of 20–60% over 1–4 h following attack by a T cell, there was a significant 4-fold increase in T cell mass accumulation rate at the start of the cytotoxic event and a 2–3 fold increase in T cell mass relative to the mass of unresponsive T cells. Direct, label-free measurement of CD8+ T and target cell mass changes provides a kinetic, quantitative assessment of T cell activation and a relatively rapid approach to identify specific, activated patient-derived T cells for applications in cancer immunotherapy.     

The oxidation-reduction potential of P-700 in chloroplast lamellae and subchloroplast particles

The oxidation-reduction potential of P-700 has been determined in chloroplast lamellae and in subchloroplast particles by measuring the magnitude of flash-induced absorption changes at 820 or at 703 nm (due to the oxidation of P-700) in the presence of known concentrations of potassium ferro- and ferricyanide. A midpoint potential of about +490 mV was determined in chloroplast lamellae and in particles prepared with digitonin (D-144) or Triton (TSF-1). A lower potential was determined with Photosystem I particles obtained after harsher treatments with Triton or a mixture of detergents. The potential is even lower in chlorophyll-protein complex I particles prepared with sodium dodecyl sulfate (about +430 mV). Very similar values were determined from oxidized minus reduced spectra measured with a double-beam spectrophotometer. Titrations were also made with D-144 and TSF-I particles, with 66% glyeerol in the buffer, at 21 °C and at 77 °K. P-700 was found to be half-oxidized at ferricyanide/ferrocyanide ratios of about 60 at 21 °C and of about 1 at 77 °K. This shows that the redox equilibrium is largely perturbed by the cooling process.     

Gibberellic Acid effects on greening in pea seedlings

The effect of gibberellic acid (GA) on light-induced greening of etiolated pea plants (Pisum sativum [L.] cultivars Alaska and Progress) was characterized. Progress, a GA-deficient dwarf of Alaska, was found to accumulate chlorophyll and light harvesting chlorophyll protein associated with photosystem II (LHC-II) more rapidly than Alaska, Alaska treated with GA, or Progress treated with GA. A slightly lower chlorophyll content was noted after 24 hours of light induced greening for Alaska treated with GA relative to untreated Alaska. GA-treated Progress, Alaska, and GA-treated Alaska all gave essentially identical patterns for LHC-II accumulation. Similar patterns of LHC-II mRNA induction were found in all four treatments indicating that differences in mRNA induction did not cause differences in LHC-II accumulation. Chlorophyll and LHC-II accumulation in each treatment followed the same patterns of accumulation and a significant correlation (at the 0.01 level of significance) was found between chlorophyll and LHC-II content. Since Progress treated with GA accumulated LHC-II and chlorophyll in a manner similar to that of Alaska, it is clear that GA alters the process of greening either directly or indirectly.     

Characterization of four herbicide-resistant mutants of Rhodopseudomonas viridis by genetic analysis, electron paramagnetic resonance, and optical spectroscopy

Herbicides of the triazine class block electron transfer in the photosynthetic reaction centers of purple bacteria and PSII of higher plants. They are thought to act by competing with one of the electron acceptors, the secondary quinone, QB, for its binding site. Several mutants of the purple bacterium Rhodopseudomonas viridis resistant to terbutryn [2-(methylthio)-4-(ethylamino)-6-(tert-butylamino)-s-triazine] have been isolated by their ability to grow photosynthetically in the presence of the herbicide. Sequence analysis of the genes coding for the L and M subunits of the reaction center showed that four different mutants were obtained, two of them being double mutated: T1 (SerL223----Ala and ArgL217----His), T3 (PheL216----Ser and ValM263----Phe), T4 (TyrL222----Phe), and T6 (PheL216----Ser). The residues L223 and L216 are involved in binding of QB, whereas L217 and L222 are not. M263 is part of the binding pocket of the primary quinone, QA. The affinity of the reaction centers for terbutryn and the electron transfer inhibitor o-phenanthroline, determined via the biphasic charge recombination after one flash, is decreased for all mutants. The affinity for ubiquinone 9 is also decreased, except in T1. Characterization by EPR spectroscopy showed that the QB.-Fe2+ signal of T4, having a g = 1.93 peak, is different from the signals obtained with the wild type and the other mutants but very similar to those of Rhodospirillum rubrum and PSII. The results obtained by the combination of these different techniques are discussed with respect to the three-dimensional structure of the wild type and the mode of binding of ubiquinone, terbutryn, and o-phenanthroline as determined by X-ray structure analysis.     

Visual optics in toads (Bufo americanus)

Aspects of visual optics were investigated in the American toad (Bufo americanus). The development of the refractive state of the eye during metamorphosis was followed with IR photoretinoscopy. Frozen sections documented the changes in optical parameters before and after metamorphosis. There is a difference in light sensitivity between juvenile and adult toads. Binocular accommodation in adult toads was observed. 1. IR photoretinoscopic measurements showed that the refractive state of the eye changed very rapidly during metamorphosis, about 10 D/h while the animal entered the terrestrial habitat. 2. Frozen sections showed that the almost spherical lens in a tadpole eye had flattened in a just metamorphosed toad's eye while at the same time the distance of the lens to the retina had decreased. However, the morphological measurements were not sufficiently sensitive to record the relatively small changes in ocular dimensions that were responsible for the rapid changes in refractive state during metamorphosis. 3. Schematic eyes, with homogeneous and non homogeneous lenses, were constructed for tadpoles, juvenile toads, and adult toads. 4. Nonparaxial raytracing studies in schematic eyes suggested that the lenses of animals of the three developmental stages tadpole, juvenile toad, and adult are not homogeneous but have a refractive index gradient. The raytracing studies indicated that the refractive index gradient is different for the different developmental stages, being highest in the tadpole lens. 5. The observations of toads during feeding behavior at different light levels showed an increased light sensitivity in the adult nocturnal toads in contrast to the juvenile animals, which are diurnal. The increased light sensitivity could partly be explained with an increase in aperture and an increase in red rod outer segments. To fully explain the higher light sensitivity in adult toads, changes in neuronal parameters had to be assumed. 6. Retinoscopic measurements of the resting refractive state in the adult toad showed a hyperopic defocus of about +8 D. By subtracting the measurement artefact for retinoscopy, the true resting focus was found to be nearly emmetropic. 7. The amount of natural accommodation in adult toads during normal feeding behavior was investigated with IR photoretinoscopy. Binocular accommodation of about 8 D was observed.     

Thromboxane receptor antagonism combined with thromboxane synthase inhibition. 6. 4-substituted 3-pyridinylalkanoic acids.

(3-Pyridinyl)alkanoic acids substituted at the 4-position with an (arylsulfonamido)alkyl group were synthesized and found to behave as platelet thromboxane receptor antagonists (TxRAs) and thromboxane synthase inhibitors (TxSIs). The compounds behaved as agonists at the vascular receptor for thromboxane A₂.     

Functional role of vitamin K in photosystem I of the cyanobacterium Synechocystis 6803

The function of vitamin K1 in the primary electron-transfer processes of photosystem I (PS I) was investigated in the cyanobacterium Synechocystis 6803. A preparation of purified PS I was found to contain two vitamin K1's per reaction center. One vitamin K1 was removed by extraction with hexane, and further extraction using hexane including 0.3% methanol resulted in a preparation devoid of vitamin K1. The hexane-extracted PS I was functional in the photoreduction of NADP+, but the PS I after extraction using hexane-methanol was totally inactive. Activity was restored by using exogenous vitamin K1 plus the hexane extract. Vitamin K3 would not substitute. The room temperature recombination kinetics of the PS I extracted with hexane were not significantly modified. However, following the removal of both vitamin K1's, the 20-ms recombination between P-700+ and P-430- was replaced by a dominant relaxation (t 1/2 = 30 ns) due to recombination of the primary biradical P-700+ A0- and a slower component originating from the P-700 triplet. This kinetic behavior was consistent with an interruption of forward electron transfer to the acceptor A1. Addition of either vitamin K1 or vitamin K3 to such preparations resulted in restoration of the slow kinetic phase (greater than 2 ms), indicating significant competition by the two exogenous quinones for electron transfer from A0-. In the case of vitamin K3, this change in the kinetics induced by vitamin K1, suggesting successful reconstitution of the acceptor site A1. These data support the hypothesis that acceptor A1 is vitamin K1 and is a component of the electron-transfer pathway for NADP+ reduction.     

Electron Transfer in the Photosynthetic Membrane: Influence of PH and Surface Potential on the P-680 Reduction Kinetics

The primary electron donor P-680 of the Photosystem-II reaction center was photoxidized by a short flash given after dark adaptation of photosynthetic membranes in which oxygen evolution was inhibited. The P-680⁺ reduction rate was measured under different conditions of pH and salt concentration by following the recovery of the absorption change at 820 nm. As previously reported for Tris-washed chloroplasts (Conjeaud, H., and P. Mathis, 1980, Biochim. Biophys. Acta, 590:353-359) a fast phase of P-680⁺ reduction slows down as the bulk pH decreases. When salt concentration increases, this fast phase becomes faster for pH above 4.5-5 and slower below. A quantitative interpretation is proposed in which the P-680⁺ reduction kinetics by the secondary electron donor Z are controlled by the local pH. This pH, at the membrane level, can be calculated using the Gouy-Chapman theory. A good fit of the results requires to assume that the surface charge density of the inside of the membrane, near the Photosystem-II reaction center, is positive at low pH values and becomes negative as the pH increases, with a local isoelectric point ~4.8. These results lead us to propose a functional scheme in which a pH-dependent proton release is coupled to the electron transfer between secondary and primary donors of Photosystem-II. The H⁺/e ratio varies from 1 at low pH to 0 at high pH, with a real pK ~6.5 for the protonatable species.