Investigations by picosecond polarized fluorescence spectrochronography of structural aspects of energy transfer in living cells of the green bacterium Chlorobium limicola

The orientation of the long-wavelength (Q_y) transition moments of the antenna bacterioviridin (BVr) was examined in living cells of Chlorobium limicola. Previous linear dichroism studies [(1986) FEBS Lett. 199, 234–236] indicated that in each individual chromatophore of C. limicola the Q_y, transition moment vectors of the whole chlorosome BVr are essentially parallel to each other and are practically ideally oriented along the long axis of the chlorosome. We measured the picosecond polarized fluorescence decay kinetics for antenna bacteriochlorophyll (BChl) emissions upon selective excitation with polarized light of the Q_y, transition of BVr. The polarization (p) of the BVr fluorescence is measured to be constant during the BVr excited-state lifetime and to be equal to the limiting value of p achieved in monomeric BChl: P = + 0.42 ± 0.02. The results indicate convincingly that the excitation energy transfer within chlorosomes of C. limicola cells takes place between chromophores (or their coupled associates) with parallel transition moment vectors.     

Chlorophyll fluorescence phenomena in chloroplasts on subnanosecond time-scales probed by picosecond pulse pairs

Fluorescence enhancement phenomena and quenching by exciton-exciton annihilation on subnanosecond and nanosecond time-scales were investigated in spinach chloroplasts utilizing picosecond laser pulse pairs (530 nm, 30 ps wide) of equal intensity, spaced apart in time by variable delays of Δt = 0−6 ns. This new method was devised to study the effect of pulse energies (1·10¹⁰–2·10¹⁵ photons per cm²) on the overall fluorescence yield in order to deduce the degree of correlation between the two pulses as a function of Δt. In the case of open reaction centers (F₀ state) in Photosystem II (PS II), it is shown that the quenching effect of excitons generated by the first pulse on the fluorescence yield of the second pulse diminishes with increasing Δt with a characteristic decorrelation time of 140 ± 60 ps. This effect is attributed to either (1) the decay of mobile excitons in the light-harvesting antenna pigment bed as these excitons migrate towards the PS II reaction centers and the associated smaller core antenna pigment pools, or (2) the decay of a quenching state of the reaction center (and/or core antenna) which appears following a rapid (less than 140 ps) trapping of the excitons initially created in the antenna pigment bed. The absence of a significant decay component of exciton quenchers with a lifetime comparable to the 300–600 ps intermediate phase of fluorescence decay kinetics suggests that this phase, although contributing to more than half of the integrated fluorescence emission signal, is not caused by freely mobile exitons migrating in a lake of pigments, but originates instead from smaller pigment pools to which the excitons have migrated. It is proposed that bimolecular exciton-exciton annihilation in these smaller domains dominates annihilation in the larger antenna pigment bed. In the case of closed reaction centers (F_max state), the decorrelation time between the two pulses is increased to 400 ± 100 ps, which is also attributed to either a mobile exciton component or to the decay of a quenching state of the reaction center. At low pulse intensities (below approx. 2 · 10¹² photons per cm²) anomalous fluorescence enhancement effects are noted, which are clearly linked to the existence of initially open PS II reaction centers. These enhancement effects are different from the well-known fluorescence induction phenomena which occur on longer time-scales, and are tentatively attributed to variations in the quenching efficiencies of transitory photochemical states of PS II reaction centers.     

Asante Calcium Green and Asante Calcium Red—Novel Calcium Indicators for Two-Photon Fluorescence Lifetime Imaging

For a comprehensive understanding of cellular processes and potential dysfunctions therein, an analysis of the ubiquitous intracellular second messenger calcium is of particular interest. This study examined the suitability of the novel Ca²⁺-sensitive fluorescent dyes Asante Calcium Red (ACR) and Asante Calcium Green (ACG) for two-photon (2P)-excited time-resolved fluorescence measurements. Both dyes displayed sufficient 2P fluorescence excitation in a range of 720–900 nm. In vitro, ACR and ACG exhibited a biexponential fluorescence decay behavior and the two decay time components in the ns-range could be attributed to the Ca²⁺-free and Ca²⁺-bound dye species. The amplitude-weighted average fluorescence decay time changed in a Ca²⁺-dependent way, unraveling in vitro dissociation constants K _D of 114 nM and 15 nM for ACR and ACG, respectively. In the presence of bovine serum albumin, the absorption and steady-state fluorescence behavior of ACR was altered and its biexponential fluorescence decay showed about 5-times longer decay time components indicating dye-protein interactions. Since no ester derivative of ACG was commercially available, only ACR was evaluated for 2P-excited fluorescence lifetime imaging microscopy (2P-FLIM) in living cells of American cockroach salivary glands. In living cells, ACR also exhibited a biexponential fluorescence decay with clearly resolvable short (0.56 ns) and long (2.44 ns) decay time components attributable to the Ca²⁺-free and Ca²⁺-bound ACR species. From the amplitude-weighted average fluorescence decay times, an in situ K _D of 180 nM was determined. Thus, quantitative [Ca²⁺]_i recordings were realized, unraveling a reversible dopamine-induced [Ca²⁺]_i elevation from 21 nM to 590 nM in salivary duct cells. It was concluded that ACR is a promising new Ca²⁺ indicator dye for 2P-FLIM recordings applicable in diverse biological systems.     

Application of flow cytometry for estimation of lipid content changes induced by arachidonic acid and methyl-β-cyclodextrin in the lipid bodies of epithelial cells

Lipid bodies (LB) are dynamic inducible organelles with the key roles in cellular lipid metabolism, intracellular trafficking and signaling. These structures have a neutral lipid-rich core which contains mainly triacylglycerides (TAG) and cholesterol esters (CE). With the use of flow cytometry and lipophylic fluorescent dye Nile red (NR) we studied LB biogenesis in nonfixed freshly isolated epithelial cells derived from the frog (Rana temporaria L.) urinary bladder. These cells are characterized by numerous small LB located diffusely in the cytoplasm. To target neutral lipids in LB, we used arachidonic acid (AA), an inducer of LB biogenesis in different cell types, and methyl-β-cyclodextrin (MβCD), non-permeable cholesterol acceptor, widely used to extract cholesterol from the lipid rafts. The cells were incubated with 10–50 μM AA for 1 h or with 400–2000 μM MβCD for 30 min; after that they were stained with NR, and fluorescence was measured by flow cytometer at λ_ex = 488 nm and λ_em = 575 ± 15 nm. In parallel, side scatter (SS) was analyzed. It was found that AA in a dose-dependent manner increased NR fluorescence. At a maximal concentration used, AA increased NR fluorescence and SS by 41 ± 2% and by 15 ± 3% (p < 0.001), respectively. Analysis of lipid composition of cell extracts revealed a significant increase of TAG by 10 μM AA. MβCD starting from 400 μM decreased dose-dependently the NR fluorescence and SS. Its effect was accompanied by a decrease of cellular free cholesterol by 6% (p < 0.01) and cholesterol ester by 21% (p < 0.001). This fact indicates mobilization of cholesterol from cholesterol esters stored in LB in order to restore cholesterol level in the plasma membrane. Taken together, our data demonstrate that flow cytometry in combination with NR staining represents a reliable tool to be used for recording of changes in different neutral lipids class content within LB in living cells non-specialized on the fat storage.     

Fluorescence decay of dyed protozoa: differences between stressed and non‐stressed cysts

Several series of tests have shown that fresh, intact samples of Giardia duodenalis and Cryptosporidium parvum (oo)cysts are not marked by fluorescent probes such as carboxyfluorcein‐succinimidyl‐diacetate‐ester (CFDA‐SE), C12‐resazurin and SYTOX® Green, probably because of their robust cell walls. These dyes fail to indicate the viability of such protozoa and allow negative responses to be recorded from living and infectious samples. Cryptosporidium parvum showed stronger isolation from chemicals, with living oocysts remaining unstained by the probe for up to 90 days after extraction. However, in further fluorescence decay (FD) experiments run with G. duodenalis samples stained using CFDA‐SE (comprising living, non‐stressed but aged cysts, heat‐killed samples and UV‐C‐stressed samples) each showed a different FD decay profile, here studied in seven series of tests of five replicates each. The FD profiles were fitted by double‐exponential decay kinetics, with the decay constant k₂ being five times higher than k₁. This FD procedure is fast and can be easily reproduced in 10 steps, taking ~ 1 h of laboratory work for already purified samples. Copyright © 2015 John Wiley & Sons, Ltd.     

Ratiometric fluorescence imaging of nuclear pH in living cells using Hoechst-tagged fluorescein

Small-molecule fluorescent sensors that allow specific measurement of nuclear pH in living cells will be valuable for biological research. Here we report that Hoechst-tagged fluorescein (hoeFL), which we previously developed as a green fluorescent DNA-staining probe, can be used for this purpose. Upon excitation at 405 nm, the hoeFL–DNA complex displayed two fluorescence bands around 460 nm and 520 nm corresponding to the Hoechst and fluorescein fluorescence, respectively. When pH was changed from 8.3 to 5.5, the fluorescence intensity ratio (F₅₂₀/F₄₆₀) significantly decreased, which allowed reliable pH measurement. Moreover, because hoeFL binds specifically to the genomic DNA in cells, it was applicable to visualize the intranuclear pH of nigericin-treated and intact living human cells by ratiometric fluorescence imaging.     

On the polyphasic quenching kinetics of chlorophyll a fluorescence in algae after light pulses of variable length

This study reports on kinetics of the fluorescence decay in a suspension of the alga Scenedesmus quadricauda after actinic illumination. These are monitored as the variable fluorescence signal in the dark following light pulses of variable intensity and duration. The decay reflects the restoration of chlorophyll fluorescence quenching of the photosystem II (PSII) antennas and shows a polyphasic pattern which suggests the involvement of different processes. The overall quenching curve after a fluorescence-saturating pulse (SP) of 250-ms duration, commonly used in pulse amplitude modulation applications as the tool for estimating the maximal fluorescence (F _m), has been termed P–O, in which P and O have the same meaning as used in the OJIP induction curve in the light. Deconvolution of this signal shows at least three distinguishable exponential phases with reciprocal rate constants of the order of 10, 10², and 10³ ms. The size of the long (>10³ ms) and moderate (~10² ms) lasting components relative to the complete quenching signal after an SP increases with the duration of the actinic pulse concomitantly with an increase in the reciprocal rate constants of the fast (~10 ms) and moderate quenching phases. Fluorescence responses upon single turnover flashes of 30-μs duration (STFs) given at discrete times during the P–O quenching were used as tools for identifying the quencher involved in the P–O quenching phase preceding the STF excitation. Results are difficult to interpret in terms of a single-hit two-state trapping mechanism with distinguishable quenching properties of open and closed reaction centers only. They give support for an earlier hypothesis on a double-hit three-state trapping mechanism in which the so-called semi-closed reaction centers of PSII are considered. In these trapping-competent centers the single reduced acceptor pair [PheQ _A]^1?, depending on the size of photoelectrochemically induced pH effects on the Q _B-binding site, functions as an efficient fluorescence quencher.     

Energy transfer and fluorescence mechanisms in photosynthetic membranes

Energy transfer in photosynthetic membranes involves the migration of excitons from light‐harvesting antenna chlorophyll‐protein complexes to the reaction center complexes. Recent efforts have focused on determining the time of arrival of excitons (trapping times) at the reaction centers following excitation with a single picosecond laser pulse. Three different approaches have been utilized: (1) determination of appearance of separated charges within the reaction centers by differential absorbtion spectroscopy, (2) determination of appearance of separated charges by fast photoemf measurements, and (3) kinetics of decay of fluorescence. The first two methods provide more direct information on exciton trapping by reaction centers than fluorescence methods, but are experimentally difficult to realize. Therefore, much activity has centered around the accurate measurement and analysis of fluorescence‐decay profiles by single‐photon counting methods. In green plants, about three different components with lifetimes of about 100 psec, 200 to 500 psec, and >1 nsec, have been reported. The first two components are believed to be related to trapping rates by reaction centers, while the third component is attributed to a charge recombination (Klimov) mechanism. Results from photoemf and exciton‐exciton annihilation experiments are consistent with the interpretation that the first decay component reflects exciton‐trapping rates. A critical analysis and discussion of these fast energy‐transfer phenomena in photosynthetic membranes of green plants are offered in this review.     

Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells

Diffusion is often an important rate-determining step in chemical reactions or biological processes and plays a role in a wide range of intracellular events. Viscosity is one of the key parameters affecting the diffusion of molecules and proteins, and changes in viscosity have been linked to disease and malfunction at the cellular level.^1-3 While methods to measure the bulk viscosity are well developed, imaging microviscosity remains a challenge. Viscosity maps of microscopic objects, such as single cells, have until recently been hard to obtain. Mapping viscosity with fluorescence techniques is advantageous because, similar to other optical techniques, it is minimally invasive, non-destructive and can be applied to living cells and tissues.Fluorescent molecular rotors exhibit fluorescence lifetimes and quantum yields which are a function of the viscosity of their microenvironment.^4,5 Intramolecular twisting or rotation leads to non-radiative decay from the excited state back to the ground state. A viscous environment slows this rotation or twisting, restricting access to this non-radiative decay pathway. This leads to an increase in the fluorescence quantum yield and the fluorescence lifetime. Fluorescence Lifetime Imaging (FLIM) of modified hydrophobic BODIPY dyes that act as fluorescent molecular rotors show that the fluorescence lifetime of these probes is a function of the microviscosity of their environment.^6-8 A logarithmic plot of the fluorescence lifetime versus the solvent viscosity yields a straight line that obeys the Förster Hoffman equation.⁹ This plot also serves as a calibration graph to convert fluorescence lifetime into viscosity.Following incubation of living cells with the modified BODIPY fluorescent molecular rotor, a punctate dye distribution is observed in the fluorescence images. The viscosity value obtained in the puncta in live cells is around 100 times higher than that of water and of cellular cytoplasm.^6,7 Time-resolved fluorescence anisotropy measurements yield rotational correlation times in agreement with these large microviscosity values. Mapping the fluorescence lifetime is independent of the fluorescence intensity, and thus allows the separation of probe concentration and viscosity effects. In summary, we have developed a practical and versatile approach to map the microviscosity in cells based on FLIM of fluorescent molecular rotors.     

PsbS is required for systemic acquired acclimation and post-excess-light-stress optimization of chlorophyll fluorescence decay times in Arabidopsis

Systemic acquired acclimation (SAA) is an important light acclimatory mechanism that depends on the global adjustments of non-photochemical quenching and chloroplast retrograde signaling. As the exact regulation of these processes is not known, we measured time-resolved fluorescence of chlorophyll a in Arabidopsis thaliana leaves exposed to excess light, in leaves undergoing SAA, and in leaves after excess light episode. We compare the behavior induced in wild-type plants with null mutant of non-photochemical quenching (npq4–1). The wild type rosettes exhibit a small reduction of fluorescence decay times in leaves directly exposed to excess light and in leaves undergoing SAA in ambient low light. However in npq4–1 exposition to excess light results in much faster fluorescence decay, which is insensitive to excitation power. At the same time npq4–1 leaves undergoing SAA displayed intermediate fluorescence decay. The npq4–1 plants also lost the ability to optimize florescence decay, and thus chlorophyll a dynamics up to 2 h after excess light episode. The fluorescence decay dynamics in both WT and npq4–1 can be described by a set of 3 maximum decay times. Based on the results, we concluded that functional PsbS is required for optimization of absorbed photon fate and optimal light acclimatory responses such as SAA or after excess light stress.     

Augmentation of proliferation and generation of specific cytotoxic cells in human mixed lymphocyte culture reactions by colchicine

Cellular proliferation and generation of cytotoxic lymphocytes in mixed lymphocyte culture reactions (MLC) results from cellular interactions initiated by individual differences in cell surface structures determined by the HLA-D locus. Cellular microtubular assemblies (MTA) modulate cell shape and surface architecture and have also been implicated in mediating stimulatory signals from the lymphocyte plasma membrane to intracellular sites. To assess the role of MTA in alloactivation, we tested the effect of colchicine, a microtubule-disrupting alkaloid, on the MLC. Diametrically opposite results were observed depending upon the colchicine treatment protocol. Brief exposure of stimulating cells to 10^?6M colchicine resulted in an increase in [³H]thymidine incorporation from 30, 694 ± 2787 to 47,345 ± 4361 cpm/culture (mean ± SEM) (P < 0.001), exposure of responding cells to 10^?6M colchicine resulted in an increase from 33,054 ± 4012 to 46,790 ± 5458 cpm/culture (P < 0.01), and exposure of both cells to 10^?6M colchicine resulted in an increase from 33,054 ± 4012 to 52,685 ± 6720 cpm/culture (P < 0.01). However, direct addition of colchicine to a final concentration of 10^?6M to MLCs at different times resulted in complete suppression of proliferation when added as late as 96 hr, and 63% suppression when added at 120 hr. Pretreatment of stimulating, responding, or both cells with lumicolchicine did not enhance proliferation. Pretreatment of cells with 10^?6 and 10^?4 M colchicine enhanced proliferation, while pretreatment with 10^?2M colchicine prevented blastogenesis. The potentiation of proliferation induced by colchicine was evident as early as 48 hr after the initiation of the MLC. Generation of specific cytotoxic cells in the MLC was also enhanced by exposing lymphocytes to colchicine prior to the proliferative phase in six out of eight experiments (specific chromium release = 168% of control) despite constant effector:target ratios. These findings indicate that early disruption of microtubules leads to enhanced cellular proliferation and generation of cytotoxic lymphocytes in allogeneic one-way MLCs and suggest that the state of polymerization of the MTA may modulate immune responses involving cell-cell interactions.     

Tubulin and microtubules from bovine kidney: purification, properties, and characterization of ligand binding.

Tubulin was purified from bovine renal medulla by in vitro assembly of microtubules in the presence of dimethyl sulfoxide and glycerol. Light scattering measurements of the polymerization process demonstrate that dimethyl sulfoxide and glycerol decrease the critical concentration of tubulin required for polymerization. The minimum concentration of tubulin from bovine renal medulla is about 1% of the total soluble protein. Assembly occurs in the absence of detectable amounts of high-molecular weight proteins or τ-protein. Microtubules polymerized in the absence and presence of 10% dimethyl sulfoxide and 4 m glycerol are similar morphologically as detected by electron microscopy. Molecular weights of α- and β-tubulin from bovine renal medulla are 54,000 ± 700 and 52,000 ± 800, respectively, as determined by electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate. Colchicine-binding activity of renal medullary tubulin decays in an apparent first-order process which is temperature dependent. The half-time of decay in buffer is 5.1 h and addition of 5 μm vinblastine sulfate increases the half-time of decay to 10.9 h at 37 °C. Calculations based on measurements of the rate of decay of colchicine-binding activity at different temperatures indicates that vinblastine sulfate stabilizes the binding activity by decreasing the entropy of activation of the decay process. Colchicine decreases the rate of decay about 3.5-fold both in the absence and presence of vinblastine sulfate at 37 °C. Values of the apparent colchicine-binding constant, K_A, of bovine renal medullary tubulin are 5.9 × 10⁶ and 7.8 × 10⁶m^?1 at 37 °C in the absence and presence of vinblastine sulfate. Vinblastine sulfate decreases the rate of decay and increases the apparent binding constant of colchicine binding. Lumicolchicine does not affect the binding of colchicine. Podophyllotoxin apparently competitively inhibits the binding of colchicine; the apparent K_i for podophyllotoxin is 4.0 × 10^?7m at 37 °C. Thus, tubulin from bovine renal medulla has ligand-binding characteristics which exhibit differences and similarities to the corresponding characteristics of the brain tubulin. These biochemical properties of the colchicine-binding activity of bovine renal medullary tubulin support previous physiologic studies which demonstrate that microtubules are required for the function of vasopressin in mammalian kidneys.     

Modulation of the fluorescence yield in heliobacterial cells by induction of charge recombination in the photosynthetic reaction center

Heliobacteria contain a very simple photosynthetic apparatus, consisting of a homodimeric type I reaction center (RC) without a peripheral antenna system and using the unique pigment bacteriochlorophyll (BChl) g. They are thought to use a light-driven cyclic electron transport pathway to pump protons, and thereby phosphorylate ADP, although some of the details of this cycle are yet to be worked out. We previously reported that the fluorescence emission from the heliobacterial RC in vivo was increased by exposure to actinic light, although this variable fluorescence phenomenon exhibited very different characteristics to that in oxygenic phototrophs (Collins et al. 2010). Here, we describe the underlying mechanism behind the variable fluorescence in heliobacterial cells. We find that the ability to stably photobleach P₈₀₀, the primary donor of the RC, using brief flashes is inversely correlated to the variable fluorescence. Using pump-probe spectroscopy in the nanosecond timescale, we found that illumination of cells with bright light for a few seconds put them in a state in which a significant fraction of the RCs underwent charge recombination from P₈₀₀ ⁺A₀ ^? with a time constant of ~20 ns. The fraction of RCs in the rapidly back-reacting state correlated very well with the variable fluorescence, indicating that nearly all of the increase in fluorescence could be explained by charge recombination of P₈₀₀ ⁺A₀ ^?, some of which regenerated the singlet excited state. This hypothesis was tested directly by time-resolved fluorescence studies in the ps and ns timescales. The major decay component in whole cells had a 20-ps decay time, representing trapping by the RC. Treatment of cells with dithionite resulted in the appearance of a ~18-ns decay component, which accounted for ~0.6 % of the decay, but was almost undetectable in the untreated cells. We conclude that strong illumination of heliobacterial cells can result in saturation of the electron acceptor pool, leading to reduction of the acceptor side of the RC and the creation of a back-reacting RC state that gives rise to delayed fluorescence.     

Absorption changes of P-700 reversible in milliseconds at low temperature in Triton-solubilized Photosystem I particles

Triton-solubilized Photosystem I particles from spinach chloroplasts exhibit largely reversible P-700 absorption changes over the temperature range from 4.2 K to room temperature. For anaerobic samples treated with dithionite and neutral red at pH 10 and illuminated during cooling, a brief (1 μs) saturating flash produces absorption changes in the long wavelength region that decay in 0.95 ± 0.2 ms from 4.2 to 50 K. Above 80 K a faster (100 ± 30 μs) component dominates in the decay process, but this disappears again above about 180 K. The major decay at temperatures above 200 K occurs in about 1 ms. The difference spectrum of these absorption changes between 500 and 900 nm closely resembles that of P-700. Using ascorbate and 2,6-dichlorophenolindophenol as the reducing system with a sample of Photosystem I particles cooled in darkness to 4.2 K, a fully reversible signal is seen upon both the first and subsequent flashes. The decay time in this case is 0.9 ± 0.3 ms.     

Fluorescence measurement by a streak camera in a single-photon-counting mode

We describe here a recently developed fluorescence measurement system that uses a streak camera to detect fluorescence decay in a single photon-counting mode. This system allows for easy measurements of various samples and provides 2D images of fluorescence in the wavelength and time domains. The great advantage of the system is that the data can be handled with ease; furthermore, the data are amenable to detailed analysis. We describe the picosecond kinetics of fluorescence in spinach Photosystem (PS) II particles at 4–77 K as a typical experimental example. Through the global analysis of the data, we have identified a new fluorescence band (F689) in addition to the already established F680, F685, and F695 emission bands. The blue shift of the steady-state fluorescence spectrum upon cooling below 77 K can be interpreted as an increase of the shorter-wavelength fluorescence, especially F689, due to the slowdown of the excitation energy transfer process. The F685 and F695 bands seem to be thermally equilibrated at 77 K but not at 4 K. The simple and efficient photon accumulation feature of the system allows us to measure fluorescence from leaves, solutions, single colonies, and even single cells. The 2D fluorescence images obtained by this system are presented for isolated spinach PS II particles, intact leaves of Arabidopsis thaliana, the PS I super-complex of a marine centric diatom, Chaetoceros gracilis, isolated membranes of a purple photosynthetic bacterium, Acidiphilium rubrum, which contains Zn-BChl a, and a coral that contains a green fluorescent protein and an algal endosymbiont, Zooxanthella.     

Kinetics of picosecond bacteriochlorophyll luminescence in vivo as a function of the reaction center state

Bacteriochlorophyll (BChl) luminescence lifetimes (τ) were measured in purple bacteria Rhodospirillum rubrum and Rhodopseudomonas sphaeroides at low-excitation pulse energy with the use of a picosecond luminescence spectrochronograph of high sensitivity and high time-resolution. Average high-frequency excitation light density was changed from about 1 · 10¹³ photons · cm⁻² · s⁻¹ up to 1 · 10¹⁷ photons · cm⁻². s⁻¹. Maximal energy density in a single pulse was in the range 10⁻¹⁴–10⁻¹⁰ J/cm², which completely rules out nonlinear exciton interactions. In this range τ increased as a function of excitation light density from about 60 ps to 210 ps. Luminescence yield (ø) for the bacteria investigated measured under continuous or picosecond excitation changed in a similar manner as τ. The luminescence increase was shown to accompany the conversion of the reaction centers to the closed, photooxidized state. Luminescence decay of R. rubrum and Rps. sphaeroides chromatophores without any chemical additions was well approximated by a single exponential component both at low and at saturating intensities of exciting light. The time necessary for the primary charge separation to occur was shown to be 60 ± 10 ps. The pairwise jump-time of excitation-energy transfer, as well as excitation-diffusion characteristics were estimated from these data. On the basis of life-time measurements in the state of active photosynthesis, the quantum yield of the primary charge separation in the reaction centers was estimated to be equal to 0.95 ± 0.02. In intact cells as well as in chromatophores in the presence of reducing agents, a nanosecond component of emission decay was also observed. The relative amplitude of this component, being several percent of the picosecond one at low-excitation intensity levels, increased (2–3)-times with excitation density. Its life-time was estimated to be 3 ± 1 ns. The nanosecond component appeared only under conditions when a part of the reaction centers were converted to the closed state PQ⁻.     

A fluorescence study of Tn10-encoded Tet repressor

Steady-state fluorescence quenching and time-resolved measurements have been performed to resolve the fluorescence contributions of the two tryptophan residues, W43 and W75, in the subunit of the homodimer of the Tet repressor fromEscherichia coli. The W43 residue is localized within the helix-turn-helix structural domain, which is responsible for sequence-specific binding of the Tet repressor to thetet operator. The W75 residue is in the protein matrix near the tetracycline-binding site. The assignment of the two residues has been confirmed by use of single-tryptophan mutants carrying either W43 or W75. The FQRS (fluorescence-quenching-resolved-spectra) method has been used to decompose the total emission spectrum of the wild-type protein into spectral components. The resolved spectra have maxima of fluorescence at 349 and 324 nm for the W43 and W75 residues, respectively. The maxima of the resolved spectra are in excellent agreement with those found using single-tryptophan-containing mutants. The fluorescence decay properties of the wild type as well as of both mutants of Tet repressor have been characterized by carrying out a multitemperature study. The decays of the wild-type Tet repressor and W43-containing mutant can be described as being of double-exponential type. The W75 mutant decay can be described by a Gaussian continuous distribution centered at 5.0 nsec with a bandwidth equal to 1.34 nsec. The quenching experiments have shown the presence of two classes of W43 emission. One of the components, exposed to solvent, has a maximum of fluorescence emission at 355 nm, with the second one at about 334 nm. The red-emitting component can be characterized by bimolecular-quenching rate constant,k _q equal to 2.6×10⁹, 2.8×10⁹, and 2.0×10⁹ M^?1 sec^?1 for acrylamide, iodide, and succinimide, respectively. The bluer component is unquenchable by any of the quenchers used. The W75 residue of the Tet repressor has quenching rate constant equal to 0.85×10⁹ and 0.28 × 10⁹ M^?1 sec^?1 for acrylamide and succinimide, respectively. These values indicate that the W75 is not deeply buried within the protein matrix. Our results indicate that the Tet repressor can exist in its ground state in two distinct conformational states which differ in the microenvironment of the W43 residue.     

Human lymphocyte tubulin: Purification and characterization in normal and leukemic cells

Tubulin has been purified from human blood and tonsil lymphocytes. Using gel filtration, the molecular weight of human lymphocyte tubulin was estimated to be 119 000. The proteins was shown to consist of two subunits, with molecular weights of 61 000 and 58 000 comparable to the α and β polypeptides of human brain tubulin. A partial identity reaction was observed between lymphocyte tubulin and human tubulin when tested by double immunodiffusion against a rabbit anti-human brain tubulin antibody. In the presence of GTP, the purified protein polymerized to form microtubules. Tubulin was localized to the cell's juxtacentriolar region by immunofluorescence and electron microscopy. When assayed by a colchicine-binding assay corrected for time decay, the binding affinity was 1.50 ± 0.86 · 10⁶M^?1 and a level in normal lymphocytes of 1.21 · 10^?2 ± 0.79 g/g of soluble protein was determined. Since chronic lymphocytic leukemia lymphocytes have an anomalous capping behavior as well as an unusual susceptibility to colchicine toxicity, the properties and levels of tubulin were determined in these cells. Similar values were obtained for the level, decay rate, molecular weight, and K_a for colchicine as for normal lymphocytes. Chronic lymphocytic leukemia lymphocyte tubulin polymerized in a normal fashion. It thus appears that a decrease in the quantity or function of tubulin does not account for these anomalies in the chronic lymphocytic leukemia lymphocyte.     

How DASPMI reveals mitochondrial membrane potential: fluorescence decay kinetics and steady-state anisotropy in living cells

Spectroscopic responses of the potentiometric probe 2-(4-(dimethylamino)styryl)-1-methylpyridinium iodide (DASPMI) were investigated in living cells by means of a time- and space-correlated single photon counting technique. Spatially resolved fluorescence decays from single mitochondria or only a very few organelles of XTH2 cells exhibited three-exponential decay kinetics. Based on DASPMI photophysics in a variety of solvents, these lifetimes were attributed to the fluorescence from the locally excited state, intramolecular charge transfer state, and twisted intramolecular charge transfer state. A considerable variation in lifetimes among mitochondria of different morphologies and within single cells was evident, corresponding to high physiological variations within single cells. Considerable shortening of the short lifetime component (τ₁) under a high-membrane-potential condition, such as in the presence of ATP and/or substrate, was similar to quenching and a dramatic decrease of lifetime in polar solvents. Under these conditions τ₂ and τ₃ increased with decreasing contribution. Inhibiting respiration by cyanide resulted in a notable increase in the mean lifetime and a decrease in mitochondrial fluorescence. Increased DASPMI fluorescence under conditions that elevate the mitochondrial membrane potential has been attributed to uptake according to Nernst distributions, delocalization of π-electrons, quenching processes of the methyl pyridinium moiety, and restricted torsional dynamics at the mitochondrial inner membrane. Accordingly, determination of anisotropy in DASPMI-stained mitochondria in living cells revealed a dependence of anisotropy on the membrane potential. The direct influence of the local electric field on the transition dipole moment of the probe and its torsional dynamics monitor changes in mitochondrial energy status within living cells.     

Bauhinia bauhinioides cruzipain inhibitor reduces endothelial proliferation and induces an increase of the intracellular Ca2+ concentration

Proteinase inhibitors, isolated from different types of Bauhinia, have an effect on apoptosis, angiogenesis and inflammation. The Bauhinia bauhinioides cruzipain inhibitor (BbCI) is a Kunitz-type inhibitor and inactivates the cysteine proteinases cruzipain and cruzain from Trypanosoma cruzi. Cruzipain and tissue kallikrein have similar biochemical properties, e.g. the proteolytic cleavage of the kininogen precursor of lys-bradykinin. Tissue kallikrein stimulation in endothelial cells causes migration and capillary tube formation. The aim of this study was to examine whether the antiproliferative effect of BbCI is dependent on changes of the intracellular calcium concentration and membrane hyperpolarization. Endothelial cells were isolated from human umbilical cord veins (HUVEC). For proliferation experiments, HUVEC were incubated with BbCI (10–100 μmol/L) for 48 h. The proliferation was detected by cell counting with a Neubauer chamber. The effect of BbCI (10–100 μM) on the membrane potential was measured with the fluorescence dye DiBAC₄(3) and the effect on [Ca⁺²] _i with the fluorescence probe Fluo-3 AM. The change of the fluorescence intensity was determined with a GENios plate reader (Tecan). The experiments showed that BbCI (10–100 μmol/L) reduces the endothelial cell proliferation significantly in a concentration-dependent manner with a maximum effect at 100 μmol/L (35.1?±?1.8% as compared to control (p?≤?0.05; n?=?45)). As compared to the control, the addition of BbCI (100 μmol/L) caused a significant increase of systolic Ca²⁺ of 28.4?±?5.0% after 30 min incubation. HUVEC treatment with BbCI (100 μmol/L) showed a weak but significant decrease of the membrane potential of 9.5?±?0.9% as compared to control (p?≤?0.05; n?=?80). BbCI influenced significantly the endothelial proliferation, the intracellular Ca²⁺ concentration and the membrane potential.