Efficacy and safety of human mesenchymal stromal cells in healing of critical-size bone defects in immunodeficient rats.

To evaluate the preclinical efficacy and safety of human mesenchymal stem cells (hMSC) rapidly expanded in growth medium for clinical use with human serum and recombinant growth factors, we conducted a controlled, randomized trial of plasma clots with hMSC vs. plasma clots only in critical segmental femoral defects in rnu/rnu immunodeficient rats. X-ray, microCT and histomorphometrical evaluation were performed at 8 and 16 weeks. MSC were obtained from healthy volunteers and patients with lymphoid malignancy. Human MSC survived in the defect for the entire duration of the trial. MSC from healthy volunteers, in contrast to hMSC from cancer patients, significantly improved bone healing at 8, but not 16 weeks. However, at 16 weeks, hMSC significantly improved vasculogenesis in residual defect. We conclude that hMSC from healthy donors significantly contributed to the healing of bone defects at 8 weeks and to the vascularisation of residual connective tissue for up to 16 weeks. We found the administration of hMSC to be safe, as no adverse reaction to human cells at the site of implantation and no evidence of migration of hMSC to distant organs was detected.

Strategies of treatment of chest wall tumors and our experience.

INTRODUCTION: The only curative treatment of tumors of the chest wall (primary or secondary),despite all the progress in oncological therapy, is a surgical radical resection. The goal of the paper is the identification of a complication occurring after chest wall resections for a tumor (evaluation of morbidity and mortality). Furthermore, the tumor type and employed reconstruction method were analyzed. METHODS: A group of patients who underwent resection of the chest wall for primary or secondary tumors at the 1st Dept. Of Surgery, University Hospital Olomouc, was retrospectively analyzed. Age, diagnosis, procedure, histopathology of the tumor, preoperative and postoperative oncological treatment, preoperative co-morbidities, postoperative complications, the use of artificial lung ventilation and recurrences were recorded for all patients. RESULTS: 57 patients aged 16 to 86 years underwent a chest wall resection, 51% for a primary tumor and 49% for a secondary tumor. Resection of at least one rib or partial resections of the sternum were performed in every patient. Reconstruction with a mesh was employed in 22 patients; in 10 patients the mesh was covered with a muscle flap. Postoperative complications occurred in 10 patients (17.5%). CONCLUSION: It is necessary to follow the basic principles of treatment of chest wall tumors; therefore surgery of these tumors should be concentrated to specialized centers. Always before surgery, diagnosis should be established by means of a biopsy and generalization of the disease should be excluded, ideally using PET/CT. Most important for successful treatment is experience and interdisciplinary cooperation of the team. This results in a low mortality and morbidity rate, which was confirmed by our results. KEYWORDS: chest wall tumors chest reconstruction sternum resection - treatment of chest wall tumors chondroma.

Xenogeneic protein-free cultivation of mesenchymal stromal cells - towards clinical applications.

We have studied a rapid cultivation method for human mesenchymal stromal cells based on CellGroTM medium and human serum, supplemented with insulin, ascorbic acid, dexamethasone, epidermal growth factor, platelet-derived growth factor BB, macrophage colony-stimulating factor and fibroblast growth factor 2. This study has shown that rapid expansion of human multipotent mesenchymal stromal cells using human serum could not be achieved without addition of growth factors. Furthermore, we have found that insulin and, quite probably, epidermal growth factor may be omitted from our formula without loss of colony-forming capacity or total cell yield. On the other hand, dexamethasone, ascorbic acid and fibroblast growth factor 2 were necessary for the growth and colony-forming capacity of multipotent mesenchymal stromal cells, while platelet-derived growth factor BB prevented their differentiation into adipogenic lineage. Moreover, multipotent mesenchymal stromal cells cultivated in our system expressed higher levels of bone morphogenetic protein 2, but not bone morphogenetic protein 7, than cells cultivated in α-MEM with foetal bovine serum. This shows that our system promotes differentiation of mesenchymal cells towards osteogenic and chondrogenic lineages, making them more suitable for bone and cartilage engineering than cells grown in conventional media. Furthermore, we have proved that these cells may be conveniently cultivated in a closed system, in vessels certified for clinical use (RoboFlaskTM), making the transfer of our cultivation technology to good clinical practice easier and more convenient.

[Xenogeneic protein free cultivation of mesenchymal multipotent stromal cells]. / Kultivace mezenchymových kmenových bunek bez xenogenních bílkovin.

PURPOSE OF THE STUDY: The aim of this study was to compare the standard laboratory method of cultivation of mesenchymal multipotent stromal cells (MSC) and a novel technique of rapid MSC expansion focused on simple clinical use. MATERIAL AND METHODS: Bone marrow mononuclear cells of donors were cultured for 14 days by the standard and the new cultivation method. The standard method (STD) was based on an alpha MEM medium supplemented with foetal calf serum (FCS). The new animal protein-free method (CLI) was based on the clinical grade medium CellgroTM, pooled human serum and human recombinant growth factors (EGF, PDGF-BB, M-CSF, FGF-2) supplemented with dexamethasone, insulin and ascorbic acid. The cell product was analyzed by flow cytometry. Furthermore, the cell products of STD and CLI methods were differentiated in vitro, and histochemical and immunohistochemical analyses, electron microscopy and elemental analysis were performed. Some cells were seeded on biodegradable scaffolds, in vivo implanted into immunodeficient mice for 6 weeks and evaluated by histological methods. RESULTS: Yields of the CLI method after 14 days of cultivation were 40-fold higher than those obtained by the STD technique (p<0.05). Cell products of both STD and CLI methods fulfilled the criteria of MSC in terms of antigen expression assessed by flow cytometry, as well as osteogenic, chondrogenic and adipogenic in vitro differentiation assays. Moreover, these cells seeded on three-dimensional scaffolds cultured in osteogenic medium produced mineral deposits and a fibrillar extracellular matrix seen with the electron microscope. Deposits examined by element analysis contained calcium and phosphorus at a ratio of 5 to 3, which corresponded to hydroxyapatite. The cell product seeded on biodegradable scaffolds and implanted into immunodeficient mice was able to form a bone-like calcified tissue with blood supply of mouse origin. DISCUSSION: The currently used methods of cultivation have certain disadvantages compared to the CLI technique, such as a longer cultivation period, need of primary expansion and reseeding and use of FCS with all its potential risks. High yields of cells obtained by the CLI method in a very short time make the use of cultured cells potentially suitable for an acute trauma management. Other therapeutic non-orthotopic applications of CLI-cultured cells have to be further investigated. CONCLUSIONS: The CLI method is unique, rapid, simple and lacking the addition of animal proteins. CLI-cultured cells fulfil the criteria of MSC. The CLI method potentially allows for closed system cultivation in good manufacturing practice (GMP) conditions. It seems to be easily transferable to good clinical practice compared to other protocols and should extend the possibilities of cell therapy and tissue engineering of cartilage and bone. The new method is protected by Czech patent 301 148 and by Europian patent EP 1999250 according to Czech and international laws.

In vitro chemoresistance profile and expression/function of MDR associated proteins in resistant cell lines derived from CCRF-CEM, K562, A549 and MDA MB 231 parental cells.

Although cellular experiments have elucidated a number of active principles in the study of the multidrug resistance (MDR) phenomena, most of the drug resistant tumor cells were derived from different parental cell lines. This fact limits generalization of some experimental data and conclusions, and therefore we selected and characterized cell lines resistant to various anti-cancer agents derived from four parental cell lines: CEM (human T-lymphoblastic leukemia), K562 (human myeloid leukemia), A549 (human lung adenocarcinoma) and MDAMB 231 (human breast adenocarcinoma). In total we obtained a set of 42 resistant sublines, which is an excellent tool for the future studies of different aspects of MDR. In this study we report on some basic characteristics of these sublines, namely, cross-resistance to other anti-cancer drugs investigated by in vitro MTT assay, expression of MDR associated proteins (Pgp, MRP1, LRP, GST-pi and Topo IIalpha) as well as the functional activity of Pgp and MRP.

Recruitment of a foreign quinone into the A1 site of photosystem I. In vivo replacement of plastoquinone-9 by media-supplemented naphthoquinones in phylloquinone biosynthetic pathway mutants of Synechocystis sp. PCC 6803.

Interruption of the phylloquinone (PhQ) biosynthetic pathway by interposon mutagenesis of the menA and menB genes in Synechocystis sp. PCC 6803 results in plastoquinone-9 (PQ-9) occupying the A(1) site and functioning in electron transfer from A(0) to the FeS clusters in photosystem (PS) I (Johnson, T. W., Shen, G., Zybailov, B., Kolling, D., Reategui, R., Beauparlant, S., Vassiliev, I. R., Bryant, D. A., Jones, A. D., Golbeck, J. H., and Chitnis, P. R. (2000) J. Biol. Chem. 275, 8523-8530. We report here the isolation of menB26, a strain of the menB mutant that grows in high light by virtue of a higher PS I to PS II ratio. PhQ can be reincorporated into the A(1) site of the menB26 mutant strain by supplementing the growth medium with authentic PhQ. The reincorporation of PhQ also occurs in cells that have been treated with protein synthesis inhibitors, consistent with a displacement of PQ-9 from the A(1) site by mass action. The doubling time of the menB26 mutant cells, but not the menA mutant cells, approaches the wild type when the growth medium is supplemented with naphthoquinone (NQ) derivatives such as 2-CO(2)H-1,4-NQ and 2-CH(3)-1,4-NQ. Since PhQ replaces PQ-9 in the supplemented menB26 mutant cells, but not in the menA mutant cells, the phytyl tail accompanies the incorporation of these quinones into the A(1) site. Studies with menB26 mutant cells and perdeuterated 2-CH(3)-1,4-NQ shows that phytylation occurs at position 3 of the NQ ring because the deuterated 2-methyl group remains intact. Therefore, the specificity of the phytyltransferase enzyme is selective with respect to the group present at ring positions 2 and 3. Supplementing the growth medium of menB26 mutant cells with 1,4-NQ also leads to its incorporation into the A(1) site, but typically without either the phytyl tail or the methyl group. These findings open the possibility of biologically incorporating novel quinones into the A(1) site by supplementing the growth medium of menB26 mutant cells.

Aging mothers and aging daughters: life-long caring and intellectual disability.

While aging and caring are well-discussed in academic literature, the association among aging, caring and intellectual disability is less well documented. This paper draws on a recently completed Australian study which focuses on such mother/daughter relationships and whose narratives form the framework for an argument for a re-imagining of the concept of care for aged people with intellectual disability. Specifically, using a genealogical approach, the paper describes how powerful discourses at the time of the daughter's birth (1940s and 1950s)--associated with eugenics, institutional care and motherhood--are framing the way in which aging mothers are now contemplating the future care for their adult (and also aging) daughters.

Decay kinetics and quantum yields of fluorescence in photosystem I from Synechococcus elongatus with P700 in the reduced and oxidized state: are the kinetics of excited state decay trap-limited or transfer-limited?

Transfer and trapping of excitation energy in photosystem I (PS I) trimers isolated from Synechococcus elongatus have been studied by an approach combining fluorescence induction experiments with picosecond time-resolved fluorescence measurements, both at room temperature (RT) and at low temperature (5 K). Special attention was paid to the influence of the oxidation state of the primary electron donor P700. A fluorescence induction effect has been observed, showing a approximately 12% increase in fluorescence quantum yield upon P700 oxidation at RT, whereas at temperatures below 160 K oxidation of P700 leads to a decrease in fluorescence quantum yield ( approximately 50% at 5 K). The fluorescence quantum yield for open PS I (with P700 reduced) at 5 K is increased by approximately 20-fold and that for closed PS I (with P700 oxidized) is increased by approximately 10-fold, as compared to RT. Picosecond fluorescence decay kinetics at RT reveal a difference in lifetime of the main decay component: 34 +/- 1 ps for open PS I and 37 +/- 1 ps for closed PS I. At 5 K the fluorescence yield is mainly associated with long-lived components (lifetimes of 401 ps and 1.5 ns in closed PS I and of 377 ps, 1.3 ns, and 4.1 ns in samples containing approximately 50% open and 50% closed PS I). The spectra associated with energy transfer and the steady-state emission spectra suggest that the excitation energy is not completely thermally equilibrated over the core-antenna-RC complex before being trapped. Structure-based modeling indicates that the so-called red antenna pigments (A708 and A720, i.e., those with absorption maxima at 708 nm and 720 nm, respectively) play a decisive role in the observed fluorescence kinetics. The A720 are preferentially located at the periphery of the PS I core-antenna-RC complex; the A708 must essentially connect the A720 to the reaction center. The excited-state decay kinetics turn out to be neither purely trap limited nor purely transfer (to the trap) limited, but seem to be rather balanced.

Paramagnetic 1H NMR spectroscopy of the reduced, unbound photosystem I subunit PsaC: sequence-specific assignment of contact-shifted resonances and identification of mixed- and equal-valence Fe-Fe pairs in [4Fe-4S] centers FA- and FB-.

The PsaC subunit of Photosystem I (PS I) is a 9.3-kDa protein that binds two important cofactors in photosynthetic electron transfer: the [4Fe-4S] clusters FA and FB. The g-tensor orientation of FA- and FB- is believed to be correlated to the preferential localization of the mixed-valence and equal-valence (ferrous) iron pairs in each [4Fe-4S]+ cluster. The preferential position of the mixed-valence and equal-valence pairs, in turn. can be inferred from the study of the temperature dependence of contact-shifted resonances by 1H NMR spectroscopy. For this, a sequence-specific assignment of these signals is required. The 1H NMR spectrum of reduced, unbound PsaC from Synechococcus sp. PCC 7002 at 280.4 K in 99% D2O solution shows 18 hyperfine-shifted resonances. The non-solvent-exchangeable, hyperfine-shifted resonances of reduced PsaC are clearly identified as belonging to the cysteines coordinating the clusters FA- and FB- by their downfield chemical shifts, by their temperature dependencies, and by their short T1 relaxation times. The usual fast method of assigning the 1H NMR spectra of reduced [4Fe-4S] proteins through magnetization transfer from the oxidized to the reduced state was not feasible in the case of reduced PsaC. Therefore, a de novo self-consistent sequence-specific assignment of the hyperfine-shifted resonances was obtained based on dipolar connectivities from 1D NOE difference spectra and on longitudinal relaxation times using the X-ray structure of Clostridium acidi urici 2[4Fe-4S] cluster ferredoxin at 0.94 A resolution as a model. The results clearly show the same sequence-specific distribution of Curie and anti-Curie cysteines for unbound, reduced PsaC as established for other [4Fe-4S]-containing proteins; therefore, the mixed-valence and equal-valence (ferrous) Fe-Fe pairs in FA- and FB- have the same preferential positions relative to the protein. The analysis reveals that the magnetic properties of the two [4Fe-4S] clusters are essentially indistinguishable in unbound PsaC, in contrast to the PsaC that is bound as a component of the PS I complex.

Recruitment of a foreign quinone into the A1 site of photosystem I. Altered kinetics of electron transfer in phylloquinone biosynthetic pathway mutants studied by time-resolved optical, EPR, and electrometric techniques.

Interruption of the menA or menB gene in Synechocystis sp. PCC 6803 results in the incorporation of a foreign quinone, termed Q, into the A(1) site of photosystem I with a number of experimental indicators identifying Q as plastoquinone-9. A global multiexponential analysis of time-resolved optical spectra in the blue region shows the following three kinetic components: 1) a 3-ms lifetime in the absence of methyl viologen that represents charge recombination between P700(+) and an FeS(-) cluster; 2) a 750-microseconds lifetime that represents electron donation from an FeS(-) cluster to methyl viologen; and 3) an approximately 15-microseconds lifetime that represents an electrochromic shift of a carotenoid pigment. Room temperature direct detection transient EPR studies of forward electron transfer show a spectrum of P700(+) Q(-) during the lifetime of the spin polarization and give no evidence of a significant population of P700(+) FeS(-) for t

Gender and drought: experiences of Australian women in the drought of the 1990s.

A unique collaborative, sociological study undertaken during 1995-7, explored the social construction of drought as a disaster, looking at farm families in two Australian states: Queensland (beef producers) and New South Wales (sheep/wheat producers). A decision was made to interview the women and men separately to test our hypothesis that there would be gender issues in any analysis of a disaster, but particularly one which has had so much long-term impact on individuals, families and communities, such as drought. Interviews were conducted with over 100 individuals male and female. We conclude that drought as a disaster is a gendered experience. The paper draws on the narratives of some women involved in the study to identify 'themes of difference' which confirm the necessity to maintain gender as a variable in all studies of the social impacts of disaster.

Recruitment of a foreign quinone into the A(1) site of photosystem I. II. Structural and functional characterization of phylloquinone biosynthetic pathway mutants by electron paramagnetic resonance and electron-nuclear double resonance spectroscopy.

Electron paramagnetic resonance (EPR) and electron-nuclear double resonance studies of the photosystem (PS) I quinone acceptor, A(1), in phylloquinone biosynthetic pathway mutants are described. Room temperature continuous wave EPR measurements at X-band of whole cells of menA and menB interruption mutants show a transient reduction and oxidation of an organic radical with a g-value and anisotropy characteristic of a quinone. In PS I complexes, the continuous wave EPR spectrum of the photoaccumulated Q(-) radical, measured at Q-band, and the electron spin-polarized transient EPR spectra of the radical pair P700(+) Q(-), measured at X-, Q-, and W-bands, show three prominent features: (i) Q(-) has a larger g-anisotropy than native phylloquinone, (ii) Q(-) does not display the prominent methyl hyperfine couplings attributed to the 2-methyl group of phylloquinone, and (iii) the orientation of Q(-) in the A(1) site as derived from the spin polarization is that of native phylloquinone in the wild type. Electron spin echo modulation experiments on P700(+) Q(-) show that the dipolar coupling in the radical pair is the same as in native PS I, i.e. the distance between P700(+) and Q(-) (25.3 +/- 0.3 A) is the same as between P700(+) and A(1)(-) in the wild type. Pulsed electron-nuclear double resonance studies show two sets of resolved spectral features with nearly axially symmetric hyperfine couplings. They are tentatively assigned to the two methyl groups of the recruited plastoquinone-9, and their difference indicates a strong inequivalence among the two groups when in the A(1) site. These results show that Q (i) functions in accepting an electron from A(0)(-) and in passing the electron forward to the iron-sulfur clusters, (ii) occupies the A(1) site with an orientation similar to that of phylloquinone in the wild type, and (iii) has spectroscopic properties consistent with its identity as plastoquinone-9.

The role of a surgeon in a palliative treatment of tumours.

The aim of the presented study is to determine a surgeon's part in diagnostics, therapy and dispensary treatment of patients suffering from tumour disease. We carried out an analysis of our own clinical experience and stated the surgeon's participation in the individual stages of diagnostics and treatment of oncological patients with the emphasis on the share in the palliative treatment.

Transient electron paramagnetic resonance spectroscopy on green-sulfur bacteria and heliobacteria at two microwave frequencies.

Spin polarized transient EPR spectra taken at X-band (9 GHz) and K-band (24 GHz) of membrane fragments of Chlorobium tepidum and Heliobacillus mobilis are presented along with the spectra of two fractions obtained in the purification of reaction centers (RC) from C. tepidum. The lifetime of P+. is determined by measuring the decay of the EPR signals following relaxation of the initial spin polarization. All samples except one of the RC fractions show evidence of light induced charge separation and formation of chlorophyll triplet states. The lifetime of P+. is found to be biexponential with components of 1.5 ms and 30 ms for C. tepidum and 1.0 and 4.5 ms for Hc. mobilis at 100 K. In both cases, the rates are assigned to recombination from F-X. The spin polarized radical pair spectra for both species are similar and those from Hc. mobilis at room temperature and 100 K are identical. In all cases, an emission/absorption polarization pattern with a net absorption is observed. A slight narrowing of the spectra and a larger absorptive net polarization is found at K-band. No out-of-phase echo modulation is observed. Taken together, the recombination kinetics, the frequency dependence of the spin polarization and the absence of an out-of-phase echo signal lead to the assignment of the spectra to the contribution from P+. to the state P+.F-X. The origin of the net polarization and its frequency dependence are discussed in terms of singlet-triplet mixing in the precursor. It is shown that the field-dependent polarization expected to develop during the 600-700 ps lifetime of P+.A-.0 is in qualitative agreement with the observed spectra. The identity that the acceptor preceding FX and the conflicting evidence from EPR, optical methods and chemical analyses of the samples are discussed.

The structural organization of the PsaC protein in Photosystem I from single crystal EPR and X-ray crystallographic studies.

In Photosystem I (PS I) the terminal electron acceptors, FA and FB, are iron-sulfur (4Fe-4S) centers, which are bound to the stromal subunit PsaC. The orientation of PsaC is determined relative to the whole PS I complex (see Schubert, W.-D. et al. (1995) in From Light to Biosphere (Mathis, P. ed.), Vol. II, pp. 3-10, Kluwer) from which a molecular model for the structure of PsaC within PS I is derived. Two strategies are followed: (i) PS I single crystal EPR data on the orientation of the g tensors of both FA- and FB- relative to each other and relative to the crystal axes (see preceding paper) are used in conjunction with the central structural part of the bacterial 2 [Fe4S4] ferredoxins, the cysteine binding motifs of which are known to be homologous to those of PsaC; (ii) the same core structure is fitted into the intermediate resolution electron density map of PS I. The PsaC orientation obtained both ways agree well. The local twofold symmetry axis inherent to the ferredoxin model leaves a twofold ambiguity in the structural conclusion. Deviations from this C2-symmetry in the amino acid sequence of PsaC are analyzed with respect to observable properties which would resolve the remaining structural ambiguity. Arguments both for and against FA being the distal iron-sulfur center (to FX) are discussed.

New EPR methods for investigating photoprocesses with paramagnetic intermediates.

Some of the significant advances in time-resolved multifrequency electron paramagnetic resonance (EPR) methods are reviewed, with the explicit focus on studies of light-driven processes and photoreactions in real time. Prominent examples are excited state electron transfer reactions with transient charge-separated radical pairs playing a central role. Paramagnetic intermediates and products are key functional states; thus EPR is the method of choice for their characterization. Photogenerated spin polarization and coherences as process-inherent features add the practical advantage of compensation in the trade-off between sensitivity and time resolution. Additionally, they provide detailed structural and dynamic information on the photoreactive system. Significance and specificity of the results achieved for charge separation in photosynthetic reaction centers and donor-acceptor model complexes indicate highly promising perspectives in photochemical research.

Electron transfer from the acceptor A1 to the iron-sulfur centers in photosystem I as studied by transient EPR spectroscopy.

The electron transfer in photosystem I (PS I) from the secondary acceptor A1 to the iron-sulfur centers is studied by X-band transient EPR with a time resolution of approximately 50 ns. Results are presented for a series of different PS I preparations from the cyanobacterium Synechococcus 6301 ranging from whole cells to core particles in which the iron-sulfur centers have been successively removed. In addition, results from PS I preparations from spinach and the cyanobacterium Synechocystis 6803 are presented. In all samples containing iron-sulfur centers, two consecutive spin-polarized EPR spectra are observed. The two signals have previously been assigned to the charge-separated states P700+.A1-. and P700+.(FeS)-, where (FeS) is one of the three iron-sulfur centers, FX, FA, or FB [Bock, C., van der Est, A., Brettel, K., & Stehlik, D. (1989) FEBS Lett. 247, 91-96]. In agreement with this, the second spectrum is not observed in the sample in which the iron-sulfur centers have been removed. For (P700-FX), core particles which do not contain FA and FB, the second spectrum can unambiguously be assigned to the pair P700+.FX-. In all samples containing FX, the transition from the first to the second spectrum occurs with t1/e approximately 280 ns (t1/2 approximately 190 ns) both in the presence and absence of FA and FB, which strongly suggests that this phase reflects electron transfer from A1-. to FX in intact PS I.(ABSTRACT TRUNCATED AT 250 WORDS)

Transient electron paramagnetic resonance of the triplet state of P700 in photosystem I: evidence for triplet delocalization at room temperature.

Spin-polarized EPR spectra of the triplet state of P700, the primary electron donor in photosystem I (PS I), have been measured for the first time at room temperature. The measurements were performed on intact PS I from Synechococcus sp. after prereduction of all iron-sulfur centers and on vitamin K1 depleted PS I from Synechocystis 6803. The two preparations give similar spectra with a polarization pattern which indicates that the triplet state is formed via recombination of a radical pair. The axial and nonaxial zero-field splitting (zfs) parameters are found to be magnitude of D = (284 +/- 15) x 10(-4) cm-1 and magnitude of E = (22 +/- 3) x 10(-4) cm-1, respectively. The E-value is 42% smaller than in monomeric chlorophyll a, while the D-value is nearly the same. Measurements of the Synechocystis 6803 sample at 4.5 K yielded zfs parameters which are identical with those of the chlorophyll monomer, in agreement with previous results. In order to explain this behavior, it is proposed that the triplet excitation is delocalized over the two halves of a chlorophyll dimer at room temperature but appears localized on one half at low temperature. The observed zfs parameters are obtained if (1) the magnetic z-axes of the two chlorophylls are collinear, (2) the magnetic y-axes (and x-axes) of the two chlorophylls make an angle of approximately 55 degrees with each other, and (3) the admixture of charge-transfer states to 3P700 is negligible.(ABSTRACT TRUNCATED AT 250 WORDS)

Nanosecond electron transfer kinetics in photosystem I following substitution of quinones for vitamin K1 as studied by time resolved EPR.

Room temperature transient EPR spectra of photosystem I (PS I) particles from Synechocystis 6803 are presented. Native PS I samples and preparations depleted in the A1-acceptor site by solvent extraction and then reconstituted with the quinones (Q) vitamin K1 (VK1), duroquinone (DQ and DQd12) and naphthoquinone (NQ) have been studied. Sequential electron transfer to P700+A1- (FeS) and P700+A1 (FeS)- is recovered only with VK1. With DQ and NQ electron transfer is restored to form the radical pair P700+Q- as specified by a characteristic electron spin polarization (ESP)-pattern, but further electron transfer is either slowed down or blocked. A qualitative analysis of the K-band spectrum suggests that the orientation of reconstituted NQ in PS I is different from the native acceptor A1 = VK1.

Comparison of the electron spin polarized spectrum found in plant photosystem I and in iron-depleted bacterial reaction centers with time-resolved K-band EPR; evidence that the photosystem I acceptor A1 is a quinone.

The suggestion that the electron acceptor A1 in plant photosystem I (PSI) is a quinone molecule is tested by comparisons with the bacterial photosystem. The electron spin polarized (ESP) EPR signal due to the oxidized donor and reduced quinone acceptor (P 870 (+) Q(-)) in iron-depleted bacterial reaction centers has similar spectral characteristics as the ESP EPR signal in PSI which is believed to be due to P 700 (+) A 1 (-) , the oxidized PSI donor and reduced A1. This is also true for better resolved spectra obtained at K-band (∼24 GHz). These same spectral characteristics can be simulated using a powder spectrum based on the known g-anisotropy of reduced quinones and with the same parameter set for Q(-) and A1 (-). The best resolution of the ESP EPR signal has been obtained for deuterated PSI particles at K-band. Simulation of the A1 (-) contribution based on g-anisotropy yields the same parameters as for bacterial Q(-) (except for an overall shift in the anisotropic g-factors, which have previously been determined for Q(-)). These results provide evidence that A1 is a quinone molecule. The electron spin polarized signal of P700 (+) is part of the better resolved spectrum from the deuterated PSI particles. The nature of the P700 (+) ESP is not clear; however, it appears that it does not exhibit the polarization pattern required by mechanisms which have been used so far to explain the ESP in PSI.