Effects of lipids on the rate-limiting steps in the dark-to-light transition of Photosystem II core complex of Thermostichus vulcanus.

In our earlier works, we have shown that the rate-limiting steps, associated with the dark-to-light transition of Photosystem II (PSII), reflecting the photochemical activity and structural dynamics of the reaction center complex, depend largely on the lipidic environment of the protein matrix. Using chlorophyll-a fluorescence transients (ChlF) elicited by single-turnover saturating flashes, it was shown that the half-waiting time (Δτ 1/2) between consecutive excitations, at which 50% of the fluorescence increment was reached, was considerably larger in isolated PSII complexes of Thermostichus (T.) vulcanus than in the native thylakoid membrane (TM). Further, it was shown that the addition of a TM lipid extract shortened Δτ 1/2 of isolated PSII, indicating that at least a fraction of the 'missing' lipid molecules, replaced by detergent molecules, caused the elongation of Δτ 1/2. Here, we performed systematic experiments to obtain information on the nature of TM lipids that are capable of decreasing Δτ 1/2. Our data show that while all lipid species shorten Δτ 1/2, the negatively charged lipid phosphatidylglycerol appears to be the most efficient species - suggesting its prominent role in determining the structural dynamics of PSII reaction center.

New foundations for the physical mechanism of variable chlorophyll a fluorescence. Quantum efficiency versus the light-adapted state of photosystem II.

Photosystem II (PSII) uses solar energy to oxidize water and delivers electrons to fix CO2. Although the structure at atomic resolution and the basic photophysical and photochemical functions of PSII are well understood, many important questions remain. The activity of PSII in vitro and in vivo is routinely monitored by recording the induction kinetics of chlorophyll a fluorescence (ChlF). According to the 'mainstream' model, the rise from the minimum level (Fo) to the maximum (Fm) of ChlF of dark-adapted PSII reflects the closure of all functionally active reaction centers, and the Fv/Fm ratio is equated with the maximum photochemical quantum yield of PSII (where Fv=Fm-Fo). However, this model has never been free of controversies. Recent experimental data from a number of studies have confirmed that the first single-turnover saturating flash (STSF), which generates the closed state (PSIIC), produces F1

Characterization of the Rate-Limiting Steps in the Dark-To-Light Transitions of Closed Photosystem II: Temperature Dependence and Invariance of Waiting Times during Multiple Light Reactions.

Rate-limiting steps in the dark-to-light transition of Photosystem II (PSII) were discovered by measuring the variable chlorophyll-a fluorescence transients elicited by single-turnover saturating flashes (STSFs). It was shown that in diuron-treated samples: (i) the first STSF, despite fully reducing the QA quinone acceptor molecule, generated only an F1(

Light-adapted charge-separated state of photosystem II: structural and functional dynamics of the closed reaction center.

Photosystem II (PSII) uses solar energy to oxidize water and delivers electrons for life on Earth. The photochemical reaction center of PSII is known to possess two stationary states. In the open state (PSIIO), the absorption of a single photon triggers electron-transfer steps, which convert PSII into the charge-separated closed state (PSIIC). Here, by using steady-state and time-resolved spectroscopic techniques on Spinacia oleracea and Thermosynechococcus vulcanus preparations, we show that additional illumination gradually transforms PSIIC into a light-adapted charge-separated state (PSIIL). The PSIIC-to-PSIIL transition, observed at all temperatures between 80 and 308 K, is responsible for a large part of the variable chlorophyll-a fluorescence (Fv) and is associated with subtle, dark-reversible reorganizations in the core complexes, protein conformational changes at noncryogenic temperatures, and marked variations in the rates of photochemical and photophysical reactions. The build-up of PSIIL requires a series of light-induced events generating rapidly recombining primary radical pairs, spaced by sufficient waiting times between these events-pointing to the roles of local electric-field transients and dielectric relaxation processes. We show that the maximum fluorescence level, Fm, is associated with PSIIL rather than with PSIIC, and thus the Fv/Fm parameter cannot be equated with the quantum efficiency of PSII photochemistry. Our findings resolve the controversies and explain the peculiar features of chlorophyll-a fluorescence kinetics, a tool to monitor the functional activity and the structural-functional plasticity of PSII in different wild-types and mutant organisms and under stress conditions.

Redox transients of P680 associated with the incremental chlorophyll-a fluorescence yield rises elicited by a series of saturating flashes in diuron-treated photosystem II core complex of Thermosynechococcus vulcanus.

Recent chlorophyll-a fluorescence yield measurements, using single-turnover saturating flashes (STSFs), have revealed the involvement of a rate-limiting step in the reactions following the charge separation induced by the first flash. As also shown here, in diuron-inhibited PSII core complexes isolated from Thermosynechococcus vulcanus the fluorescence maximum could only be reached by a train of STSFs. In order to elucidate the origin of the fluorescence yield increments in STSF series, we performed transient absorption measurements at 819 nm, reflecting the photooxidation and re-reduction kinetics of the primary electron donor P680. Upon single flash excitation of the dark-adapted sample, the decay kinetics could be described with lifetimes of 17 ns (∼50%) and 167 ns (∼30%), and a longer-lived component (∼20%). This kinetics are attributed to re-reduction of P680•+ by the donor side of PSII. In contrast, upon second-flash (with Δt between 5 µs and 100 ms) or repetitive excitation, the 819 nm absorption changes decayed with lifetimes of about 2 ns (∼60%) and 10 ns (∼30%), attributed to recombination of the primary radical pair P680•+ Pheo•- , and a small longer-lived component (∼10%). These data confirm that only the first STSF is capable of generating stable charge separation - leading to the reduction of QA ; and thus, the fluorescence yield increments elicited by the consecutive flashes must have a different physical origin. Our double-flash experiments indicate that the rate-limiting steps, detected by chlorophyll-a fluorescence, are not correlated with the turnover of P680.

Rate-limiting steps in the dark-to-light transition of Photosystem II - revealed by chlorophyll-a fluorescence induction.

Photosystem II (PSII) catalyses the photoinduced oxygen evolution and, by producing reducing equivalents drives, in concert with PSI, the conversion of carbon dioxide to sugars. Our knowledge about the architecture of the reaction centre (RC) complex and the mechanisms of charge separation and stabilisation is well advanced. However, our understanding of the processes associated with the functioning of RC is incomplete: the photochemical activity of PSII is routinely monitored by chlorophyll-a fluorescence induction but the presently available data are not free of controversy. In this work, we examined the nature of gradual fluorescence rise of PSII elicited by trains of single-turnover saturating flashes (STSFs) in the presence of a PSII inhibitor, permitting only one stable charge separation. We show that a substantial part of the fluorescence rise originates from light-induced processes that occur after the stabilisation of charge separation, induced by the first STSF; the temperature-dependent relaxation characteristics suggest the involvement of conformational changes in the additional rise. In experiments using double flashes with variable waiting times (∆τ) between them, we found that no rise could be induced with zero or short ∆τ, the value of which depended on the temperature - revealing a previously unknown rate-limiting step in PSII.

Structural and Functional Hierarchy in Photosynthetic Energy Conversion-from Molecules to Nanostructures.

Basic principles of structural and functional requirements of photosynthetic energy conversion in hierarchically organized machineries are reviewed. Blueprints of photosynthesis, the energetic basis of virtually all life on Earth, can serve the basis for constructing artificial light energy-converting molecular devices. In photosynthetic organisms, the conversion of light energy into chemical energy takes places in highly organized fine-tunable systems with structural and functional hierarchy. The incident photons are absorbed by light-harvesting complexes, which funnel the excitation energy into reaction centre (RC) protein complexes containing redox-active chlorophyll molecules; the primary charge separations in the RCs are followed by vectorial transport of charges (electrons and protons) in the photosynthetic membrane. RCs possess properties that make their use in solar energy-converting and integrated optoelectronic systems feasible. Therefore, there is a large interest in many laboratories and in the industry toward their use in molecular devices. RCs have been bound to different carrier matrices, with their photophysical and photochemical activities largely retained in the nano-systems and with electronic connection to conducting surfaces. We show examples of RCs bound to carbon-based materials (functionalized and non-functionalized single- and multiwalled carbon nanotubes), transitional metal oxides (ITO) and conducting polymers and porous silicon and characterize their photochemical activities. Recently, we adapted several physical and chemical methods for binding RCs to different nanomaterials. It is generally found that the P(+)(QAQB)(-) charge pair, which is formed after single saturating light excitation is stabilized after the attachment of the RCs to the nanostructures, which is followed by slow reorganization of the protein structure. Measuring the electric conductivity in a direct contact mode or in electrochemical cell indicates that there is an electronic interaction between the protein and the inorganic carrier matrices. This can be a basis of sensing element of bio-hybrid device for biosensor and/or optoelectronic applications.

Thermal Effects and Structural Changes of Photosynthetic Reaction Centers Characterized by Wide Frequency Band Hydrophone: Effects of Carotenoids and Terbutryn.

Photothermal characteristics and light-induced structural (volume) changes of carotenoid-containing and noncontaining photosynthetic reaction centers (RCs) were investigated by wide frequency band hydrophone. We found that the presence of carotenoid either does not play considerable role in the light-induced conformational movements, or these rearrangements are too slow for inducing a photoacoustic (PA) signal. The kinetic component with a few tens of microseconds, exhibited by the carotenoid-less RCs, appears to be similar to that of triplet state lifetimes, identified by other methods. The binding of terbutryn to the acceptor side is shown to affect the dynamics of the RC. Our results do not confirm large displacements or volume changes induced by the charge movements and by the charge relaxation processes in the RCs in few hundreds of microseconds time scale that accompanies the electron transfer between the primary and secondary electron acceptor quinones.

Structure and binding efficiency relations of QB site inhibitors of photosynthetic reaction centres.

Many herbicides employed in agriculture and also some antibiotics bind to a specific site of the reaction centre protein (RC) blocking the photosynthetic electron transport. Crystal structures showed that all these compounds bind at the secondary ubiquinone (QB) site albeit to slightly different places. Different herbicide molecules have different binding affinities (evaluated as inhibition constants, KI, and binding enthalpy values, ΔHbind). The action of inhibitors depends on the following parameters: (i) herbicide molecular structure; (ii) interactions between herbicide and quinone binding site; (iii) protein environment. In our investigations KI and ΔHbind were determined for several inhibitors. Bound herbicide structures were optimized and their intramolecular charge distributions were calculated. Experimental and calculated data were compared to those available from databank crystal structures. We can state that the herbicide inhibition efficiency depends on steric and electronic, i.e. geometry of binding with the protein and molecular charge distribution, respectively. Apolar bulky groups on N-7 atom of the inhibitor molecule (like t-buthyl in terbutryn) are preferable for establishing stronger interactions with QB site, while such substituents are not recommended on N-8. The N-4,7,8 nitrogen atoms maintain a larger electron density so that more effective H-bonds are formed between the inhibitor and the surrounding amino acids of the protein.

Photosynthetic machineries in nano-systems.

Photosynthetic reaction centres are membrane-spanning proteins, found in several classes of autotroph organisms, where a photoinduced charge separation and stabilization takes place with a quantum efficiency close to unity. The protein remains stable and fully functional also when extracted and purified in detergents thereby biotechnological applications are possible, for example, assembling it in nano-structures or in optoelectronic systems. Several types of bionanocomposite materials have been assembled by using reaction centres and different carrier matrices for different purposes in the field of light energy conversion (e.g., photovoltaics) or biosensing (e.g., for specific detection of pesticides). In this review we will summarize the current status of knowledge, the kinds of applications available and the difficulties to be overcome in the different applications. We will also show possible research directions for the close future in this specific field.

Photosynthetic reaction centers/ITO hybrid nanostructure.

Photosynthetic reaction center proteins purified from Rhodobacter sphaeroides purple bacterium were deposited on the surface of indium tin oxide (ITO), a transparent conductive oxide, and the photochemical/-physical properties of the composite were investigated. The kinetics of the light induced absorption change indicated that the RC was active in the composite and there was an interaction between the protein cofactors and the ITO. The electrochromic response of the bacteriopheophytine absorption at 771 nm showed an increased electric field perturbation around this chromophore on the surface of ITO compared to the one measured in solution. This absorption change is associated with the charge-compensating relaxation events inside the protein. Similar life time, but smaller magnitude of this absorption change was measured on the surface of borosilicate glass. The light induced change in the conductivity of the composite as a function of the concentration showed the typical sigmoid saturation characteristics unlike if the photochemically inactive chlorophyll was layered on the ITO. In this later case the light induced change in the conductivity was oppositely proportional to the chlorophyll concentration due to the thermal dissipation of the excitation energy. The sensitivity of the measurement is very high; few picomole RC can change the light induced resistance of the composite.

[The advantage of Positron Emission Tomography combined with Computer Tomography (PET-CT) in the diagnosis of lung cancer (experience with 408 patients)]. / A számítógépes rétegvizsgálattal kombinált pozitronemissziós tomogrAFIA (PET-CT) diagnosztikus értéke tüdôrákban (Tapasztalataink 408 beteg kapcsán).

The authors analyzed the results of PET-CT scans made with oncological indications among 408 patients. One hundred and fifty-four PET-CTs were done to characterize pulmonary foci, after which in 59 cases lung surgery was performed. The method's sensitivity in respect to malignancy was 100%, specificity was 56%. Staging of affected lymph nodes resulted in 17 mediastinoscopies and 54 thoracotomies. In the former indications PET-CT-positive lymph nodes always need cytologic/histologic verification. M-staging done with PET-CT was performed in 141 cases, mediastinal restaging of patients having received neoadjuvant chemotherapy was done in 24 cases. The latter indications we consider superfluous because of the diagnostic inaccuracy of PET-CT. In 175 cases we analyzed the frequently determining factor of the PET-CT scan in the indication of lung surgery. The authors wish to share their experience for the better use of this method and to accelerate the inclusion of PET-CT into the diagnostic protocol.

[Tumor vasculature as a therapeutic target in non-small cell lung cancer]. / A daganatos érhálózat mint terápiás célpont nem kissejtes tüdôrákban.

Despite developments in conventional (chemo)radiotherapy and surgery, survival of non-small cell lung cancer (NSCLC) patients remains poor. Treatments with targeted molecular drugs offer novel therapeutic strategies. Bevacizumab, a recombinant anti-vascular endothelial growth factor (VEGF) antibody, is the antiangiogenic drug at the most advanced stage of development in the therapy of NSCLC. However, a number of questions and future challenges relating to the use of bevacizumab in NSCLC remain. Furthermore, novel agents targeting the pre-existing NSCLC vasculature (i.e. vascular disrupting agents, VDAs) or multiple tyrosine kinase inhibitors have emerged as unique drug classes delivering promising results in several preclinical and clinical studies. Herein, we review the most recent data using these novel targeted agents either alone or in combination with chemotherapy in NSCLC.

Identification and clinical significance of circulating endothelial progenitor cells in human non-small cell lung cancer.

Until recently, it was generally accepted that vascularization of tumors arises exclusively from endothelial sprouting. Whether circulating bone marrow-derived endothelial progenitor cells (EPC) participate in the progression of non-small cell lung cancer (NSCLC) has not yet been evaluated. EPCs labeled with CD34, CD133, and vascular endothelial growth factor receptor-2 (VEGFR2) antibodies were counted by flow cytometry in the peripheral blood of 53 NSCLC patients. Furthermore, by means of a quantitative reverse transcription-PCR approach, we measured VEGFR2, CD133, CD34, and VE-cadherin mRNA in the peripheral blood samples of the same patient population. EPCs in tumor samples were identified by confocal microscopy using CD31, CD34, CD133, and VEGFR2 antibodies. Although immunofluorescent labeling of microvessels made clear that incorporation of EPCs is a rare phenomenon in NSCLC tissue (9 of 22 cases), circulating EPC levels before therapeutic intervention were increased in NSCLC patients (P < 0.002, versus healthy controls), and high pretreatment circulating EPC numbers correlated with poor overall survival (P < 0.001). Furthermore, in the subgroup of responders to treatment, the posttreatment EPC numbers in the peripheral blood were significantly lower compared with nonresponding patients. Interestingly, pretreatment mRNA levels of CD133, VE-cadherin, and CD34 were not significantly increased in NSCLC patients, whereas VEGFR2 expression was increased by 80-fold. Moreover, posttreatment VEGFR2 mRNA level in the peripheral blood was significantly higher in the subgroup of nonresponding patients when compared with posttreatment level of patients responding to antitumor therapy. Circulating levels of bone marrow-derived EPCs are significantly increased in NSCLC patients and correlate with clinical behavior.