Apparatus and mechanism of photosynthetic oxygen evolution: a personal perspective*

This historical minireview describes basic lines of progress in our understanding of the functional pattern of photosynthetic water oxidation and the structure of the Photosystem II core complex. After a short introduction into the state of the art about 35 years ago, results are reviewed that led to identification of the essential cofactors of this process and the kinetics of their reactions. Special emphasis is paid on the flash induced oxygen measurements performed by Pierre Joliot (in Paris, France) and Bessel Kok (Baltimore, MD) and their coworkers that led to the scheme, known as the Kok-cycle. These findings not only unraveled the reaction pattern of oxidation steps leading from water to molecular oxygen but also provided the essential fingerprint as prerequisite for studying individual redox reactions. Starting with the S. Singer and G. Nicolson model of membrane organization, attempts were made to gain information on the structure of the Photsystem II complex that eventually led to the current stage of knowledge based on the recently published X-ray crystal structure of 3.8 A resolution in Berlin (Germany).With respect to the mechanism of water oxidation, the impact of Gerald T. Babcock's hydrogen abstractor model and all the considerations of electron/proton transfer coupling are outlined. According to my own model cosiderations, the protein matrix is not only a 'cofactor holder' but actively participates by fine tuning via hydrogen bond networks, playing most likely an essential role in water substrate coordination and in oxygen-oxygen bond formation as the key step of the overall process.     

Photoinhibition as a function of the ambient redox potential in Tris-washed PS II membrane fragments

The mode of photoinhibition as a function of the ambient redox potential (E_ambient) in suspensions of Tris-washed PS II membrane fragments has been analyzed by monitoring flash-induced absorption changes at 830 nm. It was found: (a) the detectable initial amplitude, ΔA^total ₈₃₀, as a measure of the capacity to form the `stable' radical pair, P680^+ċ Q^−ċ _A, drastically decreases during a 10 min photoinhibition at E_ambient values below +350 mV; (b) conversely, the normalized extent of the 18 μs relaxation kinetics, ΔA^{18 μ s} ₈₃₀ as a measure of the electron transfer from Y_Z to P680^+ċ becomes highly susceptible to light stress when E_ambient exceeds values of about +350 mV; (c) effects of the ambient redox potentials are highly pronounced during light exposure under anaerobic conditions, while much smaller differences arise under aerobic conditions; (d) the extent of damage does not correlate with the total concentration of K₃[Fe(CN)₆] and K₄[Fe(CN)₆] in the suspension during photoinhibition but rather depends on the E_m-values; (e) qualitatively similar features are observed when the redox buffer system K₃[Fe(CN)₆]/Na₂S₂O₄ is replaced by K₂[IrCl₆]/Na₂S₂O₄; (f) the characteristic E_ambient-dependence of photoinhibition is observed only under anaerobic conditions. The results are discussed with respect to different redox components that might be involved, including brief comments on a possible role of Cyt b559. This revised version was published online in June 2006 with corrections to the Cover Date.     

Elektronenmikroskopische Untersuchungen über die Differenzierung von Insektenmuskeln während der Metamorphose

Zusammenfassung Bildung und Entwicklung der Darmmuskulatur und der dorsoventralen und longitudinalen, indirekten Flugmuskeln der Fleischfliege Phormia regina während der Metamorphose wurden elektronenmikroskopisch untersucht. Dabei wurden in der Anlage der myofibrillären Strukturen zwischen neugebildeten dorsoventralen Flugmuskeln einerseits und transformierten Darm- und longitudinalen Flugmuskeln andererseits deutliche Unterschiede festgestellt.Beim dorsoventralen Flugmuskel lösen sich die Z-Scheibenvorläufer als elektronendichte Bezirke von den beiden Differenzierungsfronten ab, die sich gleichzeitig von der Mitte einer Faser zu deren Ansatzstellen am Außenskelet verschieben. Den transformierten Muskeln fehlen diese Fronten. Hier entstehen Z-Scheibenvorläufer zwar ebenfalls in Form dichter Bezirke, jedoch im Innern der Fasern.Zu Beginn der Wachstumsphase sind in allen drei untersuchten Muskeln Fibrillenvorläufer mit dense-body-ähnlichen Z-Elementen vorhanden. In beiden Flugmuskeltypen vergrößern sie ihren Durchmesser gleichmäßig nach allen Richtungen. Bei der Darmmuskulatur dagegen wachsen die Z-Elemente, indem nur an einer oder zwei sich gegenüberliegenden Stellen des Umfangs zusätzliches Z-Scheibenmaterial aufgelagert wird. Die Differenzierung wird hier durch die Ausbildung einer perforierten Z-Scheibe abgeschlossen, die durch Fusion zahlreicher Z-Elemente entsteht. Die bei dieser Entwicklung auftretenden Zwischenstadien werden mit glatten, schräggestreiften und quergestreiften Muskeln anderer wirbelloser Tiere verglichen und im Hinblick auf die Evolution perforierter und solider Z-Scheiben diskutiert.

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On the differentiation of insect muscles during metamorphosis

Summary The formation and development of three types of imaginai muscle fibres in the blow-fly Phormia regina were investigated by electronmicroscopy. With this technique distinct differences were found in the predisposition (anlage) of myofibrillar structures in newly formed dorsoventral flight muscles on the one side and transformed longitudinal flight muscles and visceral muscles on the other.In dorsoventral flight muscles the precursors of Z-disks were detached as dense bodies from two fronts of differentiation activity (Differenzierungsfronten) which advanced from the centre of the fibres to their ends at insertion spots on the external skeleton. In transformed muscles the precursors of Z-disks developed likewise from dense bodies. These were, however, formed in the interior of the fibre, since differenzierungsfronten were absent.At the beginning of the growth period the three types of muscles contained precursors of fibrils with Z-elements, the latter of which resembled dense bodies. In both types of flight muscles these elements grew uniformly in all directions thus forming solid Z-disks. The Z-elements of visceral muscles, on the other hand, grew by accumulating additional material merely at one spot or at two opposite spots. In these muslces the process of differentiation was completed by the fusion of numerous dense bodies which resulted in the formation of perforated Z-disks.The described intermediate stages in development were compared with the final structure in smooth, obliquely and cross-striated muscles of various invertebrates. This confrontation allows certain conclusions regarding the evolution of perforated and solid Z-disks.

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Für die Anregung zu dieser Arbeit und für wertvolle Ratschläge sowie die Durchsicht des Manuskripts bin ich Herrn Prof. Dr. E. Zebe zu außerordentlichem Dank verpflichtet.

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Auszugsweise vorgetragen auf der 61. Tagung der Deutschen Zoologischen Gesellschaft, Heidelberg 1967.

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Stipendiat der Deutschen Forschungsgemeinschaft.     

Multifunctional Centromere Binding Factor 1 Is Essential for Chromosome Segregation in the Human Pathogenic Yeast Candida glabrata

The CBF1 (centromere binding factor 1) gene of Candida glabrata was cloned by functional complementation of the methionine biosynthesis defect of a Saccharomyces cerevisiae cbf1 deletion mutant. The C. glabrata-coded protein, CgCbf1, contains a basic-helix-loop-helix leucine zipper domain and has features similar to those of other budding yeast Cbf1 proteins. CgCbf1p binds in vitro to the centromere DNA element I (CDEI) sequence GTCACATG with high affinity (0.9 x 10(9) M(-1)). Bandshift experiments revealed a pattern of protein-DNA complexes on CgCEN DNA different from that known for S. cerevisiae. We examined the effect of altering the CDEI binding site on CEN plasmid segregation, using a newly developed colony-sectoring assay. Internal deletion of the CDEI binding site led only to a fivefold increase in rates of plasmid loss, indicating that direct binding of Cbf1p to the centromere DNA is not required for full function. Additional deletion of sequences to the left of CDEI, however, led to a 70-fold increase in plasmid loss rates. Deletion of the CBF1 gene proved to be lethal in C. glabrata. C. glabrata cells containing the CBF1 gene under the influence of a shutdown promoter (tetO-ScHOP) arrested their growth after 5 h of cultivation in the presence of the reactive drug doxycycline. DAPI (4',6'-diamidino-2-phenylindole) staining of the arrested cells revealed a significant increase in the number of large-budded cells with single nuclei, 2C DNA content, and short spindles, indicating a defect in the G(2)/M transition of the cell cycle. Thus, we conclude that Cbf1p is required for chromosome segregation in C. glabrata.     

The co-ordinated regulation of iron homeostasis in murine macrophages limits the availability of iron for intracellular Salmonella typhimurium

In being both, a modifier of cellular immune effector pathways and an essential nutrient for microbes, iron is a critical determinant in host-pathogen interaction. Here, we investigated the metabolic changes of macrophage iron homeostasis and immune function following the infection of RAW264.7 murine macrophages with Salmonella typhimurium. We observed an enhanced expression of the principal iron export protein, ferroportin 1, and a subsequent increase of iron efflux in Salmonella-infected phagocytes. In parallel, the expression of haem oxygenase 1 and of the siderophore-binding peptide lipocalin 2 was markedly enhanced following pathogen entry. Collectively, these modulations reduced both the cytoplasmatic labile iron and the ferritin storage iron pool within macrophages, thus restricting the acquisition of iron by intramacrophage Salmonella. Correspondingly, limitation of macrophage iron decreased microbial survival, whereas iron supplementation impaired immune response pathways in Salmonella-infected macrophages (nitric oxide formation and tumour necrosis factor-alpha production) and promoted intracellular bacterial proliferation. Our findings suggest that the enhancement of ferroportin 1-mediated iron efflux, the upregulation of the haem-degrading enzyme haem oxygenase 1 and the induction of lipocalin 2 following infection concordantly aim at withholding iron from intracellular S. typhimurium and to increase antimicrobial immune effector pathways thus limiting pathogen proliferation.     

Targeting the extracellular membrane-proximal domain of membrane-bound IgE by passive immunization blocks IgE synthesis in vivo

The classical allergic reaction starts seconds or minutes after Ag contact and is committed by Abs produced by a special subset of B lymphocytes. These Abs belong to the IgE subclass and are responsible for Type I hyperreactivity reactions. Treatment of allergic diseases with humanized anti-IgE Abs leads primarily to a decrease of serum IgE levels. As a consequence, the number of high-affinity IgE receptors on mast cells and basophils decreases, leading to a lower excitability of the effector cells. The biological mechanism behind anti-IgE therapy remains partly speculative; however, it is likely that these Abs also interact with membrane IgE (mIgE) on B cells and possibly interfere with IgE production. In the present work, we raised a mouse mAb directed exclusively against the extracellular membrane-proximal domain of mIgE. The interaction between the monoclonal anti-mIgE Ab and mIgE induces receptor-mediated apoptosis in vitro. Passive immunization experiments lead to a block of newly synthesized specific IgEs during a parallel application of recombinant Bet v1a, the major birch pollen allergen. The decrease of allergen-specific serum IgE might be related to tolerance-inducing mechanisms stopping mIgE-displaying B cells in their proliferation and differentiation.     

Combination of highly purified human leukocyte interferon α and 13-cis-retinoic acid for the treatment of metastiatic melanoma

 The effect of 13-cis-retinoic acid and highly purified human leukocyte interferon α (Alphaferon) therapy for metastatic melanoma was studied. A group of 17 patients with disseminated malignant melanoma were treated over a 6-month period. They received 60 mg 13-cis-retinoic acid/day continuously and ten cycles of interferon α (IFNα). IFN was administered by subcutaneous injection, at a daily dose of 6×10⁶ IU Alphaferon. The 5-day treatment period was followed by an IFN-free interval of 2 weeks. We were able to observe an overall response rate of 30% with 12% complete responses (2 out of 17 patients). Sites of response included the skin, lung, liver and lymph nodes. All responses have now lasted over 6 months. Therapy was generally well tolerated and could be performed on an outpatient basis. Side-effects of this combination therapy did not exceed the established side-effects of the two substances. We also studied 2′-5′-oligoadenylate synthetase, β2-microglobulin and neopterin levels during the whole treatment course. All patients were within the normal range before treatment and a sharp rise occurred during each IFN cycle. The maximum being observed 24 h after the third injection. This indicates a high biological activity of IFNα administered cyclicly during the whole treatment course. This finding also corresponds well with the absence of neutralizing antibodies before and after the whole treatment period. Received: 8 December 1994 / Accepted: 10 January 1995     

Endogenous gibberellins and inhibitors in caryopses of rye

Gibberellins A₈, A₁₆, A₂₄, and abscisic acid were identified by GC-MS of derivatized extracts from immature fruits of Secale cereale. Mature caryopses contained ABA and trans-ABA in a ratio 1:1 as well as 4′-dihydrophaseic acid. During milk ripeness a neutral GA conjugate was detected. Free GA, afforded by enzymatic hydrolysis of the conjugate, was chromatographically identified as GA₁₆     

The high Andes, gene flow and a stable hybrid zone shape the genetic structure of a wide-ranging South American parrot

Background

While the gene flow in some organisms is strongly affected by physical barriers and geographical distance, other highly mobile species are able to overcome such constraints. In southern South America, the Andes (here up to 6,900 m) may constitute a formidable barrier to dispersal. In addition, this region was affected by cycles of intercalating arid/moist periods during the Upper/Late Pleistocene and Holocene. These factors may have been crucial in driving the phylogeographic structure of the vertebrate fauna of the region. Here we test these hypotheses in the burrowing parrot Cyanoliseus patagonus (Aves, Psittaciformes) across its wide distributional range in Chile and Argentina.

Results

Our data show a Chilean origin for this species, with a single migration event across the Andes during the Upper/Late Pleistocene, which gave rise to all extant Argentinean mitochondrial lineages. Analyses suggest a complex population structure for burrowing parrots in Argentina, which includes a hybrid zone that has remained stable for several thousand years. Within this zone, introgression by expanding haplotypes has resulted in the evolution of an intermediate phenotype. Multivariate regressions show that present day climatic variables have a strong influence on the distribution of genetic heterogeneity, accounting for almost half of the variation in the data.

Conclusions

Here we show how huge barriers like the Andes and the regional environmental conditions imposed constraints on the ability of a parrot species to colonise new habitats, affecting the way in which populations diverged and thus, genetic structure. When contact between divergent populations was re-established, a stable hybrid zone was formed, functioning as a channel for genetic exchange between populations.     

Studies on the protolytic reactions coupled with water cleavage in photosystem II membrane fragments from spinach

The protolytic reactions of PSII membrane fragments were analyzed by measurements of absorption changes of the water soluble indicator dye bromocresol purple induced by a train of 10 s flashes in dark-adapted samples. It was found that: a) in the first flash a rapid H⁺-release takes place followed by a slower H⁺-uptake. The deprotonation is insensitive to DCMU but is completely eliminated by linolenic acid treatment of the samples; b) the extent of the H⁺-uptake in the first flash depends on the redox potential of the suspension. In this time domain no H⁺-uptake is observed in the subsequent flashes; c) the extent of the H⁺-release as a function of the flash number in the sequence exhibits a characteristic oscillation pattern. Multiphasic release kinetics are observed. The oscillation pattern can be satisfactorily described by a 1, 0, 1, 2 stoichiometry for the redox transitions S_i S_i+1 (i=0, 1, 2, 3) in the water oxidizing enzyme system Y. The H⁺-uptake after the first flash is assumed to be a consequence of the very fast reduction of oxidized Q₄₀₀(Fe³⁺) formed due to dark incubation with K₃[Fe(CN)₆]. The possible participation of component Z in the deprotonation reactions at the PSII donor side is discussed.Abbreviations A protonizable group at the PSII acceptor side - BCP Bromocresol Purple - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - FWHM Full Width at Half Maximum - Q_A, Q_B primary and secondary plastoquinone at PSII acceptor side - Q₄₀₀ redox group at PSII-acceptor side (high spin Fe²⁺) - P680 Photoactive chlorophyll of PSII reaction center - S_i redox states of the catalytic site of water oxidation - Z redox component connecting the catalytic site of water oxidation with the reaction center