Biochemical and spectroscopic analysis of UV effects in the marine flagellate Cryptomonas maculata

The effects of solar and artifical ultraviolet radiation on the marine cryptoflagellate, Cryptomonas maculata, were studied. Even after short exposure to UV the accessory photosynthetic pigment phycoerythrin is bleached; likewise the fluorescence undergoes significant changes both in amplitude and in the maximal peak wavelength. In parallel, the photosynthetic oxygen production decreases rapidly during exposure. Gel electrophoresis and FPLC of membrane proteins show a significant decrease in chromoproteins after 2 h UV, which is confirmed by fluorescence excitation and emission spectra of the FPLC fractions.Abbreviations APS ammonium persulfate - DCMU 3-(3,4dichlorophenyl)1,1-dimethylurea; Emulphogen, polyoxyethylene 10 tridecyl ether - FPLC fast protein liquid chromatography - PMSF phenylmethylsulfonyl fluoride - SDS sodium dodecylsulfate - SDS PAGE sodium dodecylsulfate polyacrylamide gel electrophoresis - TEMED NN NNtetramethylethylene diamine - UV-A wavelength range between 320 nm and 400 nm - UV-B wavelength range between 280 nm and 320 nm Dedicated to the 60th birthday of Professor Dr. W. Wehrmeyer     

A fluorescent sterol probe study of cholesterol/phospholipid membranes

The behavior of dehydroergosterol in -α-dimyristoylphosphatidylcholine (DMPC) unsonicated multilamellar liposomes was characterized by absorption spectroscopy and fluorescence measurements. Dehydroergosterol exhibited a lowered absorption coefficient in multilamellar liposomes whiel the steady-state fluorescence anisotropy of dehydroergosterol in these membranes decreased significantly with increasing dehydroergosterol concentration, suggesting membrane sterol-sterol interactions. The comparative steady-state anisotropy of 0.9 mole percent dehydroergosterol in multilamellar liposomes was lower than in small unilamellar vesicles suggesting different sterol environments for dehydroergosterol. Dehydroergosterol fluorescence lifetime was relatively independent of membrane sterol content and yielded similar values in sonicated and unsonicated model membranes. In multilamellar liposomes containing 5 mole percent cholesterol, the gel-to-liqui crystalline phase transition of DMPC detected by 0.9 mole percent dehydroergosterol was significantly broadened when compared to the phase transition detected by dehydroergosterol in the absence of membrane cholesterol (Smutzer, G. et al. (1986) Biochim. Biophys. Acta 862, 361–371). In multilamellar liposomes containing 10 mole percent cholesterol, the major fluorescence lifetime of dehydroergosterol did not detect the gel-to-liquid crystalline phase transition of DMPC. Time-correlated fluorescence anisotropy decays of dehydroergosterol in DMPC multilamellar liposomes in the absence and presence of 5 mole percent cholesterol exhibited a single rotational correlation time near one nanosecond that was relatively independent of temperature and low concentrations of membrane cholesterol. The limiting anisotropy of 0.9 mole percent dehydroergosterol decreased above the gel-to-liquid crystalline phase transition in membranes without cholesterol and was not significantly affected by the phase transition in membranes containing 5 mole percent cholesterol. These results suggested hindered rotational diffusion of dehydroergosterol in multilamellar liposomes. Lifetime and time-correlated fluorescence measurements of 0.9 mole percent dehydroergosterol in multilamellar liposomes further suggested this fluorophore was detecting physical properties of the bulk membrane phospholipids in membranes devoid of cholesterol and was detecting sterol-rich regions in membranes of low sterol concentration.     

Rhodamine 123 as a probe of transmembrane potential in isolated rat-liver mitochondria: spectral and metabolic properties

The spectral and metabolic properties of Rhodamine 123, a fluorescent cationic dye used to label mitochondria in living cells, were investigated in suspensions of isolated rat-liver mitochondria. A red shift of Rhodamine 123 absorbance and fluorescence occurred following mitochondrial energization. Fluorescence quenching of as much as 75% also occurred. The red shift and quenching varied linearly with the potassium diffusion potential, but did not respond to ΔpH. These energy-linked changes were accompanied by dye uptake into the matrix space. Concentration ratios, in-to-out, approached 4000:1. A large fraction of internalized dye was bound. At concentrations higher than those needed to record these spectral changes, Rhodamine 123 inhibited ADP-stimulated (State 3) respiration of mitochondria (K_i = 12 μM) and ATPase activity of inverted inner membrane vesicles (K_i = 126 μM) and partially purified F₁-ATPase (K_i = 177 μM). The smaller K_i for coupled mitochondria was accounted for by energy-dependent Rhodamine 123 uptake into the matrix. Above about 20 nmol/mg protein (10 μM), Rhodamine 123 caused rapid swelling of energized mitochondria. Effects on electron-transfer reactions and coupling were small or negligible even at the highest Rhodamine 123 concentrations employed. Δψ-dependent Rhodamine 123 uptake together with Rhodamine 123 binding account for the intense fluorescent staining of mitochondria in living cells. Inhibition of mitochondria ATPase likely accounts for the cytotoxicity of Rhodamine 123. At concentrations which do not inhibit mitochondrial function, Rhodamine 123 is a sensitive and specific probe of Δψ in isolated mitochondria.     

Enhancement of chloroplast energization in Chlorella by treatments inactivating the nitrate-reducing system

Inactivation of the nitrate-reducing system in whole cells of Chlorella vulgaris Bejerinck by darkening, nitrogen starvation, ammonium, or cycloheximide brings cells into a state with a high yield of the millisecond-delayed fluorescence of chlorophyll. Activation of this system by illumination, by adding glucose to dark-adapted cells or nitrate to nitrogen-starved cells brings the cells into a low-yield state. The transitions between the lowand high-yield state induced by alternating light and dark periods are suppressed by tungstate and restored by subsequent molybdate addition. The drop in the delayed-fluorescence yield upon activation of the nitrate-reducing system is associated with the decrease of the amplitude of the electrochemical proton gradient across the thylakoid membrane of the chloroplast, as evidenced by the kinetics of the light-induced adsorption changes at 520 nm. The decrease of the proton gradient may be caused by the electron flow diverting from the cyclic path in photosystem I as a result of the activation of the electron transfer from ferredoxin to nitrite.Abbreviation DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea     

Cytoskeletal changes visualized by fluorescence microscopy during amoeba-to-flagellate and flagellate-to-amoeba transformations inPhysarum polycephalum

Summary Changes in the intracellular distribution of microtubules and microfilaments during amoeba-to-flagellate and flagellate-to-amoeba transformations inPhysarum polycephalum were examined by fluorescence microscopy using anti-tubulin antibody and NBD-phallacidin, respectively. Amoebae contained an extensive microtubular cytoskeleton, which was converted to a flagellar cone structure during transformation to flagellates in liquid medium. When flagellates reverted back to amoebae, this conical structure disintegrated prior to flagella resorption. Amoebae showed some microfilament-enriched domains along the periphery, from which numerous filamentous extrusions, probably pseudopods and filopods, emanated. Flagellates contained a ridge, a sheet-like structure, along their dorsal axis, especially in the earlier stages of flagellation. Another microfilament-enriched thick filamentous structure ran along the dorsal axis, starting from the anterior tip of the cell. This structure apparently coincided spatially with one of the bundles of microtubules. During the reversion to amoebae, other localized microfilaments were transiently observed at the posterior end. A model of cytoskeletal changes in the transformations between these two cell types was proposed.     

Fluorescence decay of pyrene in small and large unilamellar L,α-Dipalmitoylphosphatidylcholine vesicles above and below the phase transition temperature

The fluorescence decays of pyrene in small and large unilamellar L,-dipalmitoylphosphatidylcholine vesicles have been investigated as a function of probe concentration and temperature. When the molar ratio of pyrene to phospholipid equals 1:3000, no excimer emission is observed and the fluorescence decays are mono-exponential. When this ratio is equal to or higher than 1:120, excimer formation is observed.Above the phase transition temperature the observed fluorescence decays of monomer and excimer can be adequately described by a bi-exponential function. The monomer decays can be equally well fitted to a decay law which takes into account a time-dependence in the probe diffusion rate constant. The fluorescence decay kinetics are compatible with the excimer formation scheme which is valid in an isotropic medium. The excimer lifetime and the (apparent) rate constant of excimer formation have been determined as a function of probe concentration at different temperatures above the phase transition temperature. The activation energy of excimer formation is found to be 29.4±1.3 kJ/mol. In small unilamellar vesicles the diffusion constant associated with the pyrene excimer formation process varies from 8.0x10^-7 cm²/s at 40°C to 2.2x10^-6 cm²/s at 70°C.Below the phase transition temperature the monomer decays can be described by a decay law which takes into account a time dependence of the rate constant of excimer formation. The lateral diffusion coefficient of pyrene calculated from the decay fitting parameters of the monomer region varies from 4.0x10^-9 cm²/s at 20°C to 7.9x10^-8 cm²/s at 35°C. No significant difference could be observed between the pyrene fluorescence decay kinetics in small and large unilamellar vesicles.Abbreviations SUV small unilamellar vesicles - LUV large unilamellar vesicles - DPPC dipalmitoylphosphatidylcholine - DMPC dimyristoylphosphatidylcholine - FRAP fluorescence recovery after photobleaching Part of this research has been presented at the 5th international symposium on surfactants in solution. Bordeaux, July 9th–13th 1984     

Suppression of cyclic AMP-induced cell excitation in Dictyostelium discoideum by inhibitors of eicosanoid oxidation

Abstract Receptor-mediated stimulation of Dictyostelium cells by the aggregative chemoattractant cyclic AMP leads to a complex excitatory response resulting in chemotaxis and the synthesis and release of cyclic AMP as the relayed chemotactic signal. However, the mechanism of this stimulus-response coupling is not well understood. In this study, we show that a number of compounds, best known as inhibitors of cyclooxygenase activity in mammalian cells, prevent cyclic AMP receptor-mediated cell excitation and cyclic AMP accumulation in aggregation-competent Dictyostelium cells. These observations suggest that some eicosanoid-like compound(s) may be involved in stimulus-response coupling in this organism, as is the case in higher eukaryotic cells.     

Effects of UV-B on motility and photobehavior in the green flagellate,Euglena gracilis

UV-B inhibits the motility of the green flagellate, Euglena gracilis, at fluences rates higher than those expected to occur in the natural sunlight even when the stratospheric ozone layer is partially reduced by manmade pollutants. The phototactic orientation of the cells, however, is drastically impaired by only slightly enhanced levels of UV-B irradiation. Since only negative phototaxis (movement away from a strong light source) is impaired while positive phototaxis (movement toward a weak light source) is not, the delicate balance by which the organisms adjust their position in their habitat is disturbed. Under these conditions the cells are unable to retreat from hazardous levels of radiation and are eventually killed not by the UV-B irradiation but by photobleaching of their photosynthetic pigments in the strong daylight at the surface.     

Appearance of a State 1 – State 2 transition during chloroplast development in the wheat leaf: Energetic and structural considerations

The ability of developing chloroplasts to dynamically regulate the distribution of excitation energy between photosystem 1 and photosystem 2, and thus perform a State 1 – State 2 transition, was examined from analyses of chlorophyll fluorescence kinetics in 4- and 8-day-old Triticum aestivum L. cv. Maris Dove leaves grown under a diurnal light regime. Chloroplasts at all stages of development in the two leaf systems could undergo a State 1 – State 2 transition, except those found in the basal 0.5 cm of the 4-day-old leaf. The ability to physiologically modify the excitation energy distribution between the chlorophyll matrices of the two photosystems developed after the development of mature, fully photochemically competent photosystem 2 units and the appearance of excitation energy transfer between photosystem 2 and photosystem 1. Also, changes in the degree of energetic interaction between the two photosystems, in vivo rates of electron transport and the chlorophyll a/b ratio could not be correlated with the appearance of a State 1 – State 2 transition. Ultrastructural studies demonstrated a 32% increase in the degree of thylakoid appression in chloroplasts at the base of the 8-day-old leaf compared to the situation in the basal 0.5 cm of the 4-day-old leaf. This difference in thylakoid stacking can account for the differing abilities of these two tissues to perform a State 1 – State 2 transition when considered in the context of the distribution of the two photosystems within appressed and non-appressed regions of thylakoid membranes.     

Quantitative fluorescence microscopy on dynamic changes of plastid nucleoids during wheat development

Summary Dynamic change of plastid nucleoids (pt nucleoids) was followed by fluorescence microscopy after staining with 46-diamidino-2-phenyl indole (DAPI). The fluorescence image was quantified with a supersensitive photonic microscope system based on photon counting and image analysis. The results showed that small pt nucleoids located in the center of proplastids in the dry seed increased in size after imbibition and formed highly organized ring structures in the dark, which divided into ca. 10 pieces within 3 days. Corresponding to this morphological change, DNA content of a plastid multiplied 7.5 fold. Total increase in DNA content of pt nucleoids per cell was 34 times as that of dry seed, as plastid multiplied 4.6 times in the average during this period. Upon light illumination small pt nucleoids having basic genome size were separated from divided pt nucleoids, suggesting a relationship with the formation of thylakoid system. The significance of the procedure established in this study is discussed in analysing the dynamic changes of intracellular small genomes.On leave from Department of Biology, Faculty of Science, Nagoya University, Furocho, Chikusaku, Nagoya 464, Japan.