Resonance energy transfer studies of the mechanisms of microclustering of lentil lectin membrane receptors on HeLa cells

The association and dissociation mechanisms of lectin membrane receptor microclustering on HeLa cells have been studied by measuring resonance energy transfer between fluoresceinated and rhodaminated lentil lectin. Compounds known to affect membrane receptor mobility, such as Ca²⁺ ions, methylamine, cytochalasin D and nocodazole, did not modify the association kinetics nor the maximal energy transfer values at 4 and 37 °C. Dissociation of the membrane receptor microclusters was followed by measuring the temporal decrease in energy transfer values at 4 °C after preincubation for different time intervals at 37 °C. The rate of dissociation of the lectin receptors decreased in the presence of Ca²⁺ ions (10⁻³ M) and after cross-linking with anti-lectin antibodies. An increase was observed in the presence of cytochalasin D (10⁻⁶ M) and, to a lesser extent, of methylamine (10⁻² M). When cytochalasin D and methylamine were combined at subliminal concentrations, a partial synergistic effect was observed. Nocodazole (10⁻⁶ M) had no effect. The results suggest that the association of lectin membrane receptors in microclusters is mediated only by physicochemical parameters. Ca²⁺ ions, cytochalasin D (microfilaments) and methylamine (transglutaminase)-sensitive components appear, however, to play an important role in the stabilization of the receptor microclusters.     

Reversible uncoupling of energy transfer between phycobilins and chlorophyll in Anacystis nidulans. Light stimulation of cold-induced phycobilisome detachment

Phycobilin fluorescence of Anacystis nidulans grown at 28°C increases substantially upon cooling below 10°C. A maximal increase is found around ?5°C and amounts to 300%, with almost complete reversibility upon re-warming. Illumination with actinic light leads to considerable stimulation of the cold-induced phycobilin fluorescence increase. Analysis of the light stimulation phenomenon reveals: (1) Actinic illumination shifts the fluorescence-temperature characteristic by about 3°C upwards on the T-axis. At temperatures below 5°C the light stimulating effect becomes smaller again and fluorescence-temperature characteristics measured at high and low light intensity converge around ?5°C. (2) In the 13-8°C region a large (up to 100%) light-induced phycobilin fluorescence increase is observed, while only negligible changes occur in the dark. (3) 3-(3,4-Dichlorophenyl)-1,1-dimethyl urea (DCMU) as well as uncouplers inhibit the light stimulation, which hence depends on coupled electron transport.In agreement with previous work (Schreiber, U. (1979) FEBS Lett. 107, 4–9) it is concluded that illumination enhances cold-induced phycobilisome detachment by increasing the net negative charge at the outer surface of the thylakoid membrane. The possible role of a fluid → ordered transition of membrane lipids (Murata, N. and Fork, D.C. (1975) Plant Physiol. 56, 791–796) is discussed.     

Activated-sludge process dynamics with continuous total organic carbon and oxygen uptake measurements

The dynamic responses of a bench-scale activated-sludge process to step changes and square-wave inputs in the feed flow and concentration were measured. Instrumentation permitted the continuous measurement of the oxygen uptake rate and dissolved organic carbon responses. Notable were the sensitivity of the oxygen uptake rate to process changes and the reliability of the dynamic oxygen electrode method. The responses were found to be greatly influenced by the organic loading, FS₀/XV, which was incorporated into a load-dependent kinetics model. Simulations showd good agreement with experiment in the case of the square-wave disturbances. Because of the changing and complex nature of the activated sludge it was necessary to reestimate the parameter set for each run.     

Discriminant analysis on cells from developing squamous cancer of the respiratory tract

Cytologic preparations made from the tracheobronchial tree taken by the Schreiber catheter have been scanned by three color microphotometry. The digitized cell images were processed by the analytical cytodiagnostic programs of the TICAS system. Cells were sorted into two control groups and five groups of increasing atypia ranging from normal epithelium to invasive squamous cell carcinoma. Standard statistical tests, including Wilk's Lambda, Rao's V, and the Kruskal-Wallis tests are performed on these subsets of cell image features. This study demonstrates that discriminant analyses permit differentiation between normal cells and those from marked atypia or carcinoma and that the classification achieves a high degree of agreement with visual assignment.     

Heat-induced changes of chlorophyll fluorescence in isolated chloroplasts and related heat-damage at the pigment level

The heat-induced changes of chlorophyll fluorescence excitation and emission properties were studied in isolated chloroplasts of Larrea divaricata Cav. An analysis of the temperature dependency of fluorescence, under Fo and Fmax conditions, of temperature-jump fluorescence induction kinetics, and of 77 degrees K emission spectra of preheated chloroplasts revealed two major components in the heat-induced fluorescence changes: (1) a fluorescence rise, reflecting the block of Photosystem II reaction centers; and (2) a fluorescence decrease, caused by the functional separation of light-harvesting pigment protein complex from the rest of the pigment system. Preferential excitation of chlorophyll a around 420 nm, produced a predominant fluorescence rise. Preferential excitation of chlorophyll b, at 480 nm, gives a predominant fluorescence decrease. It is proposed that the overlapping of the fluorescence decrease on the somewhat faster fluorescence rise, results in the biphasic fluorescence rise kinetics observed in isolated chloroplasts. Both the rise component and the decay component are affected by the thermal stability of the chloroplasts, acquired during growth of the plants in different thermal environments. Mg2+ enhances the stability against heat-damage expressed in the decrease component, but has no effect on the rise component. Heat pretreatment leads to a decrease of the variable fluorescence in the light-induced 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) rise curve, but no change in half-rise time is observed. It is concluded that the block of Photosystem II reaction centers precedes the loss of the light-harvesting pigment protein complex. However, the approximately antiparallel heat-induced Fmax decrease and Fo increase suggest a common cause for the two events. A heat-induced perturbation of the thylakoid membrane is discussed.     

The ultrastructure of normal and glycerol treated muscle in the ghost crab,Ocypode cursor

The ultrastructure of normal and glycerol treated fibers of the closer muscle of the ghost crab, Ocypode cursor, was studiedmthe muscle is composed of presumably phasic (short sarcomeres) and tonic (long sarcomeres) fibers, the latter greatly predominating. Horseradish peroxidase (HRP) was used as an extracellular tracer to delineate the tubular system (TS), and to determine to what extent this system becomes detached from the extracellular space as a result of glycerol treatment. Sarcolemmal clefts invade deeply into the muscle at Z-lines and I-bands; tubules invaginate into the muscle from the clefts and from the surface sarcolemma at the Z-lines, A-I overlaps and A-bands. A tubules are in frequent diadic or tetradic contact with the sarcoplasmic reticulum (SR), whereas Z tubules appear to be randomly associated with SR, terminal cisterns (TC) and Z-line fibrils. When HRP was administered to normal muscle, black reaction product was found adjacent to the outer surface of the sarcolemma, within the clefts and within profiles of the TS throughout the tissue. In glycerol treated muscle peripheral vacuolation frequently occurred; black reaction product penetrated only as far as the vacuoles and into dilated Z-line tubules, but was virtually absent from the rest of the TS. This lack of continuity between the extracellular space and the A tubules indicated disruption or constriction of the A tubules as a result of glycerol treatment, although Z tubule contact with the extracellular space appeared unimpaired. These findings provide ultrastructural correlates of the electrophysiological changes produced by glycerol treatment of the closer muscle of the ghost crab (Papir, 1973), namely, interference with excitation-contraction (e-c) coupling. The random association of the Z tubules with SR and TC, and their resistance to disruption by glycerol treatment, tend to endorse the claims that the Z tubules in crustacean muscle are not directly involved in e-c coupling (Brandt et al., 1965; Peachey, 1967; Selverston, 1967).