Pigment-protein complexes are organized into stable microdomains in cyanobacterial thylakoids.

Thylakoids are the place of the light-photosynthetic reactions. To gain maximal efficiency, these reactions are conditional to proper pigment-pigment and protein-protein interactions. In higher plants thylakoids, the interactions lead to a lateral asymmetry in localization of protein complexes (i.e. granal/stromal thylakoids) that have been defined as a domain-like structures characteristic by different biochemical composition and function (Albertsson P-Å. 2001,Trends Plant Science 6: 349-354). We explored this complex organization of thylakoid pigment-proteins at single cell level in the cyanobacterium Synechocystis sp. PCC 6803. Our 3D confocal images captured heterogeneous distribution of all main photosynthetic pigment-protein complexes (PPCs), Photosystem I (fluorescently tagged by YFP), Photosystem II and Phycobilisomes. The acquired images depicted cyanobacterial thylakoid membrane as a stable, mosaic-like structure formed by microdomains (MDs). These microcompartments are of sub-micrometer in sizes (~0.5-1.5 µm), typical by particular PPCs ratios and importantly without full segregation of observed complexes. The most prevailing MD is represented by MD with high Photosystem I content which allows also partial separation of Photosystems like in higher plants thylakoids. We assume that MDs stability (in minutes) provides optimal conditions for efficient excitation/electron transfer. The cyanobacterial MDs thus define thylakoid membrane organization as a system controlled by co-localization of three main PPCs leading to formation of thylakoid membrane mosaic. This organization might represent evolutional and functional precursor for the granal/stromal spatial heterogeneity in photosystems that is typical for higher plant thylakoids.

Subunit composition of CP43-less photosystem II complexes of Synechocystis sp. PCC 6803: implications for the assembly and repair of photosystem II.

Photosystem II (PSII) mutants are useful experimental tools to trap potential intermediates involved in the assembly of the oxygen-evolving PSII complex. Here, we focus on the subunit composition of the RC47 assembly complex that accumulates in a psbC null mutant of the cyanobacterium Synechocystis sp. PCC 6803 unable to make the CP43 apopolypeptide. By using native gel electrophoresis, we showed that RC47 is heterogeneous and mainly found as a monomer of 220 kDa. RC47 complexes co-purify with small Cab-like proteins (ScpC and/or ScpD) and with Psb28 and its homologue Psb28-2. Analysis of isolated His-tagged RC47 indicated the presence of D1, D2, the CP47 apopolypeptide, plus nine of the 13 low-molecular-mass (LMM) subunits found in the PSII holoenzyme, including PsbL, PsbM and PsbT, which lie at the interface between the two momomers in the dimeric holoenzyme. Not detected were the LMM subunits (PsbK, PsbZ, Psb30 and PsbJ) located in the vicinity of CP43 in the holoenzyme. The photochemical activity of isolated RC47-His complexes, including the rate of reduction of P680(+), was similar to that of PSII complexes lacking the Mn(4)CaO(5) cluster. The implications of our results for the assembly and repair of PSII in vivo are discussed.

[Frangible bullets: wounding capability and clinical aspects of their use]. / Strely frangible: ranivost a klinické aspekty jejich pouzití

The article deals with basic characteristics of the frangible bullets and it documents a very specific behaviour of chosen types of these bullets in testing blocks as a substitute materials of alive tissues. The frangible bullets have several important advantages compared to the classical sorts of firearms bullets. The highest benefit could be seen especially in the limited penetrating capability and very low ricochet hazard connected with the use of these bullets. The absence of poisonous elements in the material of frangible bullets (for instance lead) is highly appreciated from the ecology reasons nowadays as well. The cartridges assembled with frangible bullets are used most of all for the practise reasons by law enforcement units, but can be used very effectively also in combat situations. Results of own shooting experiments confirm that the wound potential of bullet can be changed in a very large extent with the change of the manufacturing technology and the bullets geometry. Newly developed frangible bullets and the already manufactured bullets available on the market are characterized by very specific terminal ballistic features. Some frangible bullets behave in a comparable way to full metal jacketed bullets while penetrating soft tissues. Another frangible bullets of different designs fragment to the pieces in soft tissues and cause very serious wounds with persistent effects. The usage of frangible bullets and a potential risk of misuse of this specific sort of ammunition require also new approaches in the medicine for the treatment of the gunshot wounds.

Degradation of the Photosystem II D1 and D2 proteins in different strains of the cyanobacterium Synechocytis PCC 6803 varying with respect to the type and level of psbA transcript.

The turnover of the D1 and D2 proteins of Photosystem II (PSII) has been investigated by pulse-chase radiolabeling in several strains of the cyanobacterium Synechocystis PCC 6803 containing different types and levels of the psbA transcript. Strains lacking psbA1 and psbA3 gene and containing high levels of the psbA2 transcript showed the selective synthesis of D1 whose degradation could be slowed down by the protein synthesis inhibitor lincomycin. In contrast, in strains containing just the psbA3 gene, the intensity of the D1 protein labeling was lower and labeling of the D2 and CP43 proteins was stimulated in comparison to the psbA2-containing strains. In addition, the rate and selectivity of the D1 degradation and its dependence on the presence of lincomycin was proportional to the level of the psbA3 transcript in the particular strain. Consequently, there was parallel, lincomycin-independent and slowed-down breakdown of the D1 and D2 proteins in strains with the lowest level of psbA3 transcript. These results are discussed in terms of a model in which the rate of D1 and D2 degradation in cyanobacteria is affected not only by the rate of PSII photodamage, but also by the availability of newly synthesized D1 protein. Moreover, the comparison of the non-oxygen-evolving D1 mutants D170A** and Y161F*** differing by the presence of tyrosine Z has indicated a minor role of the oxidized form of this secondary PSII electron donor in the donor side mechanism of D1 and D2 protein breakdown.

Role of two forms of the D1 protein in the recovery from photoinhibition of photosystem II in the cyanobacterium Synechococcus PCC 7942.

The study of turnover of two distinct forms of the photosystem II (PSII) D1 protein in cells of the cyanobacterium Synechococcus PCC 7942 showed that the 'high-light' form D1:2 is degraded significantly faster at 500 microE m(-2) s(-1) as compared with 50 microE m(-2) s(-1) while the degradation rates of the 'low-light' form D1:1 under low and high irradiance are not substantially different. Consequently, the D1:1 turnover does not match photoinactivation of PSII under increased irradiance and therefore the cells containing this D1 form exhibit a decrease in the PSII activity. Monitoring of the content of each D1 form during a recovery from growth-temperature photoinhibition showed a good correlation between the synthesis of D1:2 and restoration of the PSII activity. In contrast, when photoinhibitory treatment was conducted at low temperature, a fast recovery was not accompanied by the D1:2 accumulation. The data suggest that photoinactivation at growth temperature results in a modification of PSII that inhibits insertion of D1:1 and, therefore, for restoration of the photochemical activity in the photoinactivated PSII complexes the D1:2 synthesis is needed. This may represent the primary reason for the requirement of psbAII/psbAIII expression under increased irradiance.

Characterization of processes responsible for the distinct effect of herbicides DCMU and BNT on Photosystem II photoinactivation in cells of the cyanobacterium Synechococcus sp. PCC 7942.

Light-induced modification of Photosystem II (PS II) complex was characterized in the cyanobacterium Synechococcus sp. PCC 7942 treated with either DCMU (a phenylurea PS II inhibitor) or BNT (a phenolic PS II inhibitor). The irradiance response of photoinactivation of PS II oxygen evolution indicated a BNT-specific photoinhibition that saturated at relatively low intensity of light. This BNT-specific process was slowed down under anaerobiosis, was accompanied by the oxygen-dependent formation of a 39 kDa D1 protein adduct, and was not related to stable Q(A) reduction or the ADRY effect. In the BNT-treated cells, the light-induced, oxygen-independent initial drop of PS II electron flow was not affected by formate, an anion modifying properties of the PS II non-heme iron. For DCMU-treated cells, anaerobiosis did not significantly affect PS II photoinactivation, the D1 adduct was not observed and addition of formate induced similar initial decrease of PS II electron flow as in the BNT-treated cells. Our results indicate that reactive oxygen species (most likely singlet oxygen) and modification of the PS II acceptor side are responsible for the fast BNT-induced photoinactivation of PS II.

Photoadaptation of two members of the Chlorophyta (Scenedesmus and Chlorella) in laboratory and outdoor cultures: changes in chlorophyll fluorescence quenching and the xanthophyll cycle.

The role of the xanthophyll cycle in the adaptation of two chlorococcal algae Scenedesmus quadricauda and Chlorella sorokiniana to high irradiance was studied under laboratory and outdoor conditions. We wished to elucidate whether the xanthophyll cycle plays a key role in dissipating the excesses of absorbed light, as in higher plants, and to characterise the relationship between chlorophyll fluorescence parameters and the content of xanthophyll-cycle pigments. The xanthophyll cycle was found to be operative in both species; however, its contribution to overall non-photochemical quenching (NPQ) could only be distinguished in Scenedesmus (15-20% of total NPQ). The Scenedesmus cultures showed a larger pool of xanthophyll-cycle pigments than Chlorella, and lower sensitivity to photoinhibition as judged from the reduction of maximum quantum yield of photosystem II. In general, both algae had a larger xanthophyll-cycle pool when grown outdoors than in laboratory cultures. Comparing the two species, Scenedesmus exhibited a higher capacity to adapt to high irradiance, due to an effective quenching mechanism and high photosynthetic capacity; in contrast, Chlorella represents a species with a larger antennae system, less-efficient quenching and lower photosynthetic performance. Non-photochemical quenching (NPQ) induced through the xanthophyll cycle can, to a limited extent, represent a regulatory factor in diluted algal cultures grown in outdoor solar photobioreactors, as well as in natural algal phytoplankton populations exposed transiently to high irradiance. However, it does not play an appreciable role in dense, well-mixed microalgal suspensions.

A sensitive photosystem II-based biosensor for detection of a class of herbicides.

We have developed a biosensor for the detection of residual triazine-, urea- and phenolic-type herbicides, using isolated photosystem II (PSII) particles from the thermophilic cyanobacterium, Synechococcus elongatus, as biosensing elements. The herbicide detection was based on the fact that, in the presence of artificial electron acceptors, the light-induced electron transfer through isolated PSII particles is accompanied by the release of oxygen, which is inhibited by the herbicide in a concentration-dependent manner. The PSII particles were immobilized between dialysis membrane and the Teflon membrane of the Clark oxygen electrode mounted in a flow cell that was illuminated. Inclusion of the antibiotic chloramphenicol in the reaction mixtures prolonged, by 50%, the lifetime of the biosensor. The use of highly active PSII particles in combination with the flow system resulted in a reusable herbicide biosensor with good stability (50% of initial activity was still remaining after 35-h use at 25 degrees C) and high sensitivity (detection limit for diuron was 5 x 10(-10) M).

Functional and structural changes of the photosystem II complex induced by high irradiance in cyanobacterial cells.

A gradual disintegration of the photosystem II (PSII) complex, initiated by a release of the chlorophyll-protein CP43, was identified during low-temperature illumination of Synechococcus cells. This process was slower compared to the decline of the PSII primary charge separation activity, and much slower than the photoinactivation of oxygen evolution. All three processes were slowed down in the presence of diuron. The results indicate that when the PSII repair was blocked, the inactivation of charge separation activity and the release of CP43 preceded the degradation of the D1 protein. In contrast, a much faster degradation of D1 connected to its rapid exchange was triggered by inactivation of oxygen evolution, and no disassembly of PSII was needed. We propose the existence of two different mechanisms of D1 degradation in the cells of the thermophilic cyanobacterium Synechococcus elongatus.

Comparison of psbO and psbH deletion mutants of Synechocystis PCC 6803 indicates that degradation of D1 protein is regulated by the QB site and dependent on protein synthesis.

Mutants of the cyanobacterium Synechocystis PCC 6803 lacking the psbO or psbH gene are more vulnerable to photoinhibition than the wild type (WT). In the case of the psbO-less mutant, the increased sensitivity to photodamage is also accompanied by accelerated turnover of the D1 protein and a rapid rate of recovery on transfer to non-photoinhibitory conditions. In contrast, in low light the psbH-less mutant has a poor ability to recover after photoinhibition and has a reduced rate of D1 turnover as compared with WT. Since the psbO gene encodes the 33 kDa manganese-stabilizing protein associated with the water-splitting reaction, the increased sensitivity to photoinduced damage is attributed to perturbation of electron transfer processes on the donor side of photosystem II (PSII). In contrast, the absence of H protein, encoded by the psbH gene, affects the acceptor side of PSII with preferential photoinhibitory damage occurring at the QB site. The apparent consequence of this is that the psbH-less mutant, unlike the psbO-less mutant, is not able to regulate the rate of turnover of the D1 protein. In all cases it was shown that chloramphenicol, which blocks protein synthesis, enhances the rate of photoinhibition as judged by a decrease in oxygen evolution but slows down the rate of degradation of D1 protein compared to that observed during normal turnover.(ABSTRACT TRUNCATED AT 250 WORDS)

Deletion of the PEST-like region of photosystem two modifies the QB-binding pocket but does not prevent rapid turnover of D1.

The rapid turn-over of the D1 polypeptide of the photosystem two complex has been suggested to be due to the presence of a "PEST"-like sequence located between putative transmembrane helices IV and V of D1 (Greenberg, B. M., Gaba, V., Mattoo, A. K. and Edelman, M. (1987) EMBO J. 6, 2865-2869). We have tested this hypothesis by constructing a deletion mutant (delta 226-233) of the cyanobacterium Synechocystis sp. PCC 6803 in which residues 226-233 of the D1 polypeptide, containing the PEST-like sequence, have been removed. The resulting mutant, delta PEST, is able to grow photoautotrophically and give light-saturated rates of oxygen at wild type levels. However electron transfer on the acceptor side of the complex is perturbed. Analysis of cells by thermoluminescence and by monitoring the decay in quantum yield of variable fluorescence following saturating flash excitation indicates that Q-B, but not Q-A, is destabilized in this mutant. Electron transfer on the donor side of photosystem two remains largely unchanged in the mutant. Turnover of the D1 polypeptide as examined by pulse-chase experiments using [35S]methionine was enhanced in the delta PEST mutant compared to strain TC31 which is the wild type control. We conclude that the PEST sequence is not absolutely required for turnover of the D1 polypeptide in vivo although deletion of residues 226-233 does have an effect on the redox equilibrium between QA and QB.

Purification of a Photosystem II reaction center from a thermophilic cyanobacterium using immobilized metal affinity chromatography.

Oxygen-evolving PS II particles from the thermophilic cyanobacterium Synechococcus elongatus are partially purified by centrifugation on a sucrose gradient and are bound to a Chelating Sepharose column loaded with Cu(2+) ions. Bound particles are then transformed into PS II RC complexes by two washing steps. First, washing with a phosphate buffer (pH=6.5) containing 0.02% of SB 12 removes the rest of phycobilins and leaves pure PS II core particles on the column. Second, washing with a phosphate buffer (pH=6.2) containing 0.2 M LiClO4 and 0.05% of DM removes CP 47 and CP 43 and leaves bare PS II RC complexes on the column. These are then eluted with a phosphate buffer containing 1% of dodecylmaltoside (DM). The molar ratio of pigments in the eluate changes with the progress of elution but around the middle of the elution period a nearly stable ratio is maintained of Chl a: Pheo a: Car: Cyt b 559 equal to 2.9: 1: 0.9: 0.8. In these fractions the photochemical separation of charges could be demonstrated by accumulation of reduced pheophytin (ΔA of 430-440 nm) and by the flash induced formation of P680(+) (ΔA at 820 nm). The relatively slow relaxation kinetics of the latter signal (t1/2 ≈ 1 ms) may suggest that in a substantial fraction of the RCs QA remains bound to the complex.

[Synthesis of 4-aryl-1-lambda-2, 2-lambda- 4-dithia-3,4-diaza-buta-1,2-dienes, a new dithiadiazabutadiene system]. / Synthese von 4-Aryl-1 lambda 2,2 lambda 4-dithia-3,4-diaza-buta- 1,2-dienen, einem neuen Dithiadiazabutadien-System.

4-Aryl-1 lambda 2, 2 lambda 4-dithia-3,4-diaza-buta-1,2-dienes 1a-12a as representatives of a dithiadiazabutadiene structure in which sulphur occupies a different oxidation number are synthesized by reaction of diazotated acceptor-substituted aryl- and hetarylamines 1-12 with thiosulfates or disodium disulfide in strong acid solution. For some examples a first pharmacological test indicate an immune-stimulating effect. Analytical examination as well as MNDO calculation which give some mechanistical insight, supplements the new synthesis.

Immobilized metal affinity chromatography for the separation of photosystems I and II from the thermophilic cyanobacterium Synechococcus elongatus.

Immobilized metal affinity chromatography (IMAC) of solubilized, photosystem II (PS II) enriched particles from the thermophilic cyanobacterium Synechococcus elongatus was studied. A chelating Sepharose Fast Flow column was charged with various metal ions (Mn2+, Fe2+, Fe3+, Ni2+, Co2+, Ca2+, Sr2+, Zn2+ and Cu2+) and their affinity to photosystem I (PS I) and PS II was examined. Among all the metal ions tested, only copper was able to bind the two protein complexes. For elution of the column, a pH gradient, a pH step gradient and gradients of imidazole, amino acids, organic acids and various other eluents were tested; only the pH step gradient, which selectively eluted PS II at a pH between 6 and 5, was useful for the separation of PS I and PS II. All other gradients proved to be inappropriate for the separation of these two photosystems. Mechanisms of protein elution by these compounds are discussed. Alternatively, a separation of PS I and PS II at pH 7.5 could be achieved when an IMAC column was used on which the free coordination positions of the bound copper ions were occupied by imidazole. When solubilized photosystems were loaded on to this column, PS I replaced imidazole and remained bound on the column, whereas PS II was highly enriched in the effluent.

Three types of Photosystem II photoinactivation : 2. Slow processes.

Oxygen evolving Photosystem II particles were exposed for up to 10 h to 100 W m(-2) white light at 20°C under aerobic, low oxygen, strictly anaerobic and strongly reducing conditions. The fast and slow photoinactivation processes described earlier (Setlík et al. 1989) were observed during the first 120 min. The third and by far the slowest process impaired the primary charge separation P680(+)-Pheo(-). Its half-time was about 2.5 h under aerobic and strongly reducing conditions and about 4 h under anaerobic and low oxygen conditions. In these time intervals there were no changes in the chlorophyll-protein and polypeptide composition of the particles irradiated under anaerobic, low oxygen or strongly reducing conditions while a dramatic degradation of chlorophyll-proteins and polypeptides occurred under aerobic conditions.

Optokinetic nystagmus in progressive retinal degeneration.

Optokinetic nystagmus (OKN) was studied in both normally pigmented and delayed amelanotic (DAM) strains of domestic chicken. The DAM line is characterized by postnatal feather and ocular depigmentation accompanied by progressive retinal degeneration that occurs, initially and most severely, in the central retina. A close association exists between the extent of ocular pigment loss and relative reduction in OKN responsiveness in DAMs. The directional asymmetry of OKN responses, which normally occurs with monocular temporal-to-nasal (T-N) but not to nasal-to-temporal (N-T) stimulation, was altered in relation to the extent of ocular amelanosis among DAMs. In particular, T-N OKN responses were progressively reduced as amelanosis of the central retina increased in severity. In DAMs with moderate to severe reductions in T-N responsiveness, relatively little reduction occurred in N-T responsiveness. The central retina, therefore, appears to play a major role in mediating responses to T-N stimulation, whereas the peripheral retina mediates both directions of response. Optokinetic nystagmus also provides a useful index of the extent of retinal degeneration and the progressive loss of retinal pigment epithelium and photoreceptors which occurs in this mutant strain.