Pheophytin-mediated energy storage of photosystem II particles detected by photoacoustic spectroscopy

The photoacoustic (PA) characteristics (energy storage and heat dissipation) of photosystem II (PSII) core-enriched particles from barley were studied (i) in conditions where there was electron flow, i.e., in the presence of a combination of the electron acceptor K₃ Fe (CN)₆, referred to as FeCN, and the electron donor diphenylcarbazide (DPC), and (ii) in conditions where electron flow was suppressed, i.e., in the absence of FeCN and DPC. The experimental data show that a decrease of heat dissipation with a minimum at 540 nm can be interpreted as energy storage resulting from the presence of pheophytin (Pheo) in the PSII particles. On account of the capability of the PA method to measure the energy absorbed by the chromophores which is converted to heat, it is suggested that the PA detection of Pheo present in the PSII complex will permit to clarify the function of processes involving non-radiative relaxation of excited states in P680-Pheo-Q_A interactions.Abbreviations -Car -Carotene - Chl Chlorophyll - DPC Diphenylcarbazide - EPR Electron Paramagnetic Resonance - FeCN potassium ferricyanide - HEPES N-2-hydroxyethylenepiperazine-N-2-ethanesulfonate - P680 reaction center of PSII - PA Photoacoustic - Pheo pheophytin - PSI photosystem I - PSII photosystem II - Q_A primary electron acceptor of PSII     

Improvement of four sigma analysis for the investigation of oxygen evolution by Photosystem II

We present here an improvement to the analysis of oxygen evolution with four sigma coefficients (4-S) by computing z, the sum of the S-state probabilities, which was introduced earlier (Delrieu and Rosengard 1987, Biochim Biophys Acta 892: 163–171). We demonstrate that z is equal to the ratio of two consecutive Mean Y (the estimation of the steady state oxygen production based on local properties) found by three sigma analysis. The quantity z is useful for computing double-hits, and for showing the inactivation/activation processes of PS II complexes. Three sigma analysis assumes z=1 exactly; since this is not verified, it is argued that four sigma analysis is closer to the real workings of the water oxidizing complex. Oxygen evolution can then be interpreted in the frame of a modified Kok's model where the sum of the probabilities equals z. We therefore suggest that the closer fitting of four sigma analysis to oxygen production data is not simply due to an extra, unnecessary variable, but to the fact that PS II complexes can be inactivated and reactivated under flashing light. Finally, in order to facilitate the use of four sigma analysis, a computer program is made available upon request.     

Electron Transport-Dependent Chlorophyll-a Fluorescence Quenching by O(2) in Various Algae and Higher Plants

A comparison of chlorophyll-a fluorescence in brown algae (Macrocystis integrifolia, Fucus vesiculosis), green algae (Scenedesmus obliquus, Ulva sp.) and higher plants (bean, corn) show differences in the relative fluorescence intensities and induction time courses which characterize each type of plant. These differences are not reflected in either the maximum fluorescence emission in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (F_max) or the nonvariable fluorescence (F_o). Constancy of F_o and F_max suggests functional similarities of photosystem II and associated antennae pigments in the various classes of plants. The time course differences are observed only in the absence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea and appear, therefore, to be electron transport dependent. During induction, the peak in fluorescence (F_p) is much lower in all of the algae studied than in the higher plants. Exogenous O₂ strongly quenches F_p in all plants studied and our data indicate that the low F_p in the algae can be partially accounted for by endogenous O₂ quenching.     

High fidelity extrachromosomal recombination and gene targeting in plants

The precision of extrachromosomal homologous recombination and gene targeting in plant cells was investigated. Recombination was directed to introns of selectable marker genes where potential changes could persist without affecting the function and therefore the selectability of the genes. Approximately 9 kb of crossover regions was rescued and sequenced. Changes were detected at a frequency below one point mutation per 1000 bp, indicating that extrachromosomal recombination and gene targeting both appear to occur with high fidelity.     

EFFECTS OF LONG TERM EXPOSURE TO LOW TEMPERATURE ON THE PHOTOSYNTHETIC APPARATUS OF DUNALIELLA TERTIOLECTA (CHLOROPHYCEAE)1

The effects of long term exposure to suboptimal growth temperature on the photosynthetic apparatus of Dunaliella tertiolecta Butcher were investigated using carbon fixation rate versus irradiance curves and the variable fluorescence induction method. Carbon fixation rates per unite chlorophyll a at saturating (p^B_m) and subsaturating (α^B) irradiances were 55% and 39% lower, respectively, at 12° C than at 20° C. Chlorophyll a quotas and the spectrally averaged in vivo absorption cross section normalized to chlorophyll a (a^*) were not significantly different at these two temperatures. Analysis of the fluorescence kinetics revealed 1) no significant variations of the amount of PSII photoactive reaction centers per unit chlorophyll a, 2) a 14% decrease of the PSII quantum yield(+) and 3) a 29% decrease of the energy transfer efficiency between the light harvesting chlorophyll a pigment bed and the PSII reaction centers. The decrease in energy transfer efficiency between the antennae and the PSII reaction centers at 12° C was interpreted as a mechanism to avoid photoinhibition.     

1α,25-dihydroxy-24-oxo-16-ene vitamin D3, a metabolite of a synthetic vitamin D3 analog, 1α,25-dihydroxy-16-ene vitamin D3, is equipotent to its parent in modulating growth and differentiation of human leukemic cells

1α,25(OH)₂-16-ene-D₃, a synthetic analog of the steroid hormone, 1α,25(OH)₂D₃, has great potential to become a drug in the treatment of leukemia and other proliferative disorders, because of its minimal in vivo calcemic activity associated with a potent inhibitory effect on cell growth. However, at present, the mechanisms through which 1α,25(OH)₂-16-ene-D₃ expresses its biological activities are still not completely understood. Our previous in vitro study in a perfused rat kidney indicated for the first time that 1α,25(OH)₂-16-ene-D₃ and 1α,25(OH)₂D₃ are metabolized differently. 1α,25(OH)₂-24-oxo-16-ene-D₃, an intermediary metabolite of 1α,25(OH)₂-16-ene-D₃ formed through the C-24 oxidation pathway, accumulated significantly in the perfusate when compared to 1α,25(OH)₂-24-oxo-D₃, the corresponding intermediary metabolite of 1α,25(OH)₂D₃. In a subsequent in vivo study, we also reported that 1α,25(OH)₂-24-oxo-16-ene-D₃ exerted immunosuppressive activity equal to its parent, without causing significant hypercalcemia. In order to establish further the critical role of 1α,25(OH)₂-24-oxo-16-ene-D₃, in generating some of the key biological activities ascribed to its parent, we performed the present in vitro study using a human myeloid leukemic cell line (RWLeu-4) as a model. Comparative target tissue metabolism studies indicated that 1α,25(OH)₂-16-ene-D₃ and 1α,25(OH)₂D₃ are metabolized differently in RWLeu-4 cells, and the differences were similar to the ones we previously observed in the rat kidney. The significant finding was the accumulation of 1α,25(OH)₂-24-oxo-16-ene-D₃ in RWLeu-4 cells because of its resistance to further metabolism. Biological activity studies indicated that both 1α,25(OH)₂-16-ene-D₃ and its 24-oxo metabolite produced growth inhibition and promoted differentiation of RWLeu-4 cells to the same extent, and these activities were several fold higher than those exerted by 1α,25(OH)₂D₃. In addition, the genomic action of each vitamin D compound was assessed in a rat osteosarcoma cell line (ROS 17/2.8) by measuring its ability to transactivate a gene construct containing the vitamin D response element of the osteocalcin gene linked to the growth hormone reporter gene. In these studies, both 1α,25(OH)₂-16-ene-D₃ and its 24-oxo metabolite exerted similar but potent transactivation activity which was several fold greater than that exerted by 1α,25(OH)₂D₃ itself. In summary, our results indicate that the production and slow clearance of the bioactive intermediary metabolite, 1α,25(OH)₂-24-oxo-16-ene-D₃, in RWLeu-4 cells contributes significantly to the final expression of the enhanced biological activities ascribed to its parent analog, 1α,25(OH)₂-16-ene-D₃.     

Dynamic structure of metallothionein

Molecular sieve chromatography of rabbit liver metallothionein at different electrolyte concentrations revealed that this protein undergoes an increase in Stokes radius from 1.50 to 1.78 nm when the ionic strength is lowered from 0.5 to 0.015 indicating a change in molecular shape and/or hydration. The variation in ionic strength also affects the far-UV circular dichroism of metallothionein reflecting a conformational transition in the protein. The effects are attributed to changes in intramolecular repulsion between the strongly negatively charged metal-thiolate clusters of the protein. It is suggested that metallothionein exists in at least two interchangeable conformational states which differ in hydrodynamic properties and whose equilibrium concentrations are determined by the electrostatic free energy of the system.